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1 INTRODUCTION

Sensor devices including biosensors are attracting consid-erable interest for molecule-speci c measurements withhigh sensitivity in a variety of applications ranging fromclinical and pharmaceutical analysis to industrial processmonitoring and environmental surveillance applications

A biosensor is composed of two main components (i) asensing interface which speci cally interacts with analytemolecules creating a change in physical or chemical para-meters (eg pH redox state temperature charge refrac-tive index etc) and (ii) a transducer which converts thisinformation into a quanti able read-out signal Most com-monly electrochemical optical mass sensitive and ther-mosensitive transducers are applied for biosensing plat-forms1 Per its de nition the biological recognition elementis in direct contact with the transducer surface Followingtransduction the signal is typically ltered ampli edrecorded or transmitted by a read-out system resulting in a

Scanning Probe Microscopy with Integrated Biosensors

Angelika Kueng Christine Kranz and Boris Mizaikoff

School of Chemistry and Biochemistry Georgia Institute of Technology Atlanta Georgia 30332-0400 USA

(Received 29 July 2003 RevisedAccepted 2 September 2003)

The investigation of complex biological processes and molecular biological interactions (eg at cell sur-faces) demands analytical techniques providing simultaneous specic information on multiple parameterscorrelated in space and time This review highlights different approaches using scanning probe biosen-sors in the micro- and nanometer regime to obtain laterally resolved information on bioactivity at a samplesurface Recent developments in scanning probe microscopy in combination with advances in surfacemodi cation chemistry extend the application of force microscopy to the biological arena by adding corre-lated (bio)chemical information on the investigated surface to the sample topology The focus of this re-view is on cantilever-based biosensors and their application in force recognition imaging and enzymeelectrodes combined with different scanning probe microscopic techniques for surface (bio)activity mea-surements Capabilities limitations and future potential for the investigation of biological system at a cel-lular level are addressed

Keywords Atomic Force Microscopy AFM Scanning Electrochemical Microscopy SECM Micro-biosensor Cantilever Sensor Enzyme Electrode Integrated AFM-SECM Tip IntegratedBiosensor

quantity proportional to the concentration of the target ana-lyte The selectivity of the biosensor is predominantly de-termined by the biological or biochemical sensing elementwhich may recruit from single proteins and enzymes up towhole cells and microorganisms2ndash6 Enzymes are amongthe most commonly applied recognition elements becauseof their high chemical speci city and inherent biocatalyticsignal ampli cation Recent progress in biosensor technol-ogy has mainly been achieved by application of microsys-tems technology and novel biomimetic or biological recog-nition elements (eg recombinant antibody fragmentsmolecularly imprinted polymers etc)

Miniaturization and integration of biosensors utilizingon-chip technology have been successfully demonstrated7

However investigation of complex biological systems andmolecular biological interactions (eg at cell surfaces) re-quires measurement of multiple speci c parameters corre-lated in space and time ideally at a nanometer scale Hencebesides miniaturization of the transducer and sensing inter-face positioning of miniaturized sensors in close proximityto the sample surface and laterally resolved measurements

2 Sensor Lett Vol 1 No 1 2003 copy 2003 by American Scienti c Publishers 1546-198X200301002015$1700+25 doi101166sl2003001

SENSOR LETTERSVol 1 2ndash15 2003

Corresponding author E-mail christinekranzchemistrygatechedu

Copyright copy 2003 American Scienti c PublishersAll rights reservedPrinted in the United States of America

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Sensor Lett Vol 1 No 1 2003 3

are of particular interest Improvements in functionalizingsensor surfaces8 along with advancements in microfabrica-tion9 allow development of highly integrated miniaturizedbiosensors The ability to position and accurately scan thesedevices across a sample surface results in laterally resolvedinformation essential for the monitoring of localized eventsat individual reaction sites10

Scanning probe microscopy (SPM) techniques are gain-ing increasing importance in the understanding of molec-ular biological interactions providing optical topographi-cal and chemical information with high spatial resolutiondependent on the speci c interaction between the samplesurface and the probe scanned in close proximity acrossthe sample11ndash13 Since the introduction of scanning tun-neling microscopy (STM) in 198214 derived techniquessuch as atomic force microscopy (AFM) near- eld scan-ning optical microscopy (NSOM) scanning ion conduc-tance microscopy (SICM) and scanning electrochemicalmicroscopy (SECM) have successfully been applied toprobing of biologically active surfaces Depending on theinvolved tip-sample interaction each technique showslimitations concerning the obtained information As anexample SECM is an attractive technique utilizing micro-or submicroelectrodes for probing bioactivity based on re-dox chemistry However this technique is frequently lim-ited in lateral resolution in comparison with for example

STM which allows imaging of individual atoms and mol-ecules In contrast STM is limited to the investigation ofconducting substrates AFM with a sharp tip mounted on a exible cantilever probes mechanical properties and topo-logical features resulting in surface pro les (topography)with submolecular resolution The lack of information onthe surface chemistry obtained with nonmodi ed probeshas been a major drawback of this technique Since the in-troduction of tapping-mode imaging in 199415 AFM hasattracted remarkable attention for the investigation of softbiological samples Substantial progress in combiningAFM with surface chemistries for tip functionalizationhas added chemical speci city to the topographical infor-mation obtained Hence AFM has matured from a high-resolution imaging instrument to a device capable of de-tecting and quantifying single molecular forces underambient conditions Standard immobilization strategiesinclude the formation of functionalized thiol or silanemonolayers biotin-streptavidin interactions or dextranegel immobilization which are well-established techniquesderived from biosensor technology16 and are readilyadapted for tethering biological recognition elements tothe scanning probe tip Alternative to direct tip modi ca-tion the combination of complementary scanning probetechniques such as AFMNSOM AFMSTM andAFMSECM can provide extended information on the in-

Christine Kranz is a research scientist in the School of Chemistry and Biochemistry at the GeorgiaInstitute of Technology She received her BS degree from Ludwig Maximilian University of Munichand her PhD degree from the Technical University of Munich and was a postdoctoral fellow at theVienna University of Technology Her research interests include conducting polymers microelectro-chemistry atomic force microscopy thin- lm technology scanning electrochemical microscopybiosensors system miniaturization and integration based on micro- and nanofabrication and multi-functional scanning probe techniques

Boris Mizaikoff is an assistant professor in the School of Chemistry and Biochemistry at the GeorgiaInstitute of Technology He received his BS and PhD degrees from Vienna University ofTechnology and was a postdoctoral fellow at the University of Texas at Austin His research interestsinclude mid-infrared chemical sensors biosensors and biomimetic sensors chemical sensing withquantum cascade lasers surface-enhanced spectroscopic techniques deep-sea spectroscopic sensingchemometric data evaluation development of separation materials and sensing membranes based onmolecularly imprinted polymers and sol-gels system miniaturization and integration based on micro-and nanofabrication and multifunctional scanning probe techniques

Angelika Kueng is a postdoctoral fellow in the School of Chemistry and Biochemistry at the GeorgiaInstitute of Technology She received her BS and PhD degrees from the University of Vienna Herresearch interests include bioinorganic chemistry biosensors and multifunctional scanning probetechniques

vestigated sample surface chemistry As an example am-perometric biosensors based on common electrochemicaltransduction principles can be integrated into bifunctionalSECM-AFM probes providing laterally resolved infor-mation on bioactivity simultaneously with submolecularresolution in topography17

This review highlights selected recently developed prin-ciples and examples of cantilever integrated scanning bio-sensors and scanning electrochemical enzyme microelec-trodes

2 CANTILEVER-BASED SCANNINGBIOSENSORS

The atomic force microscope invented in 198618 has devel-oped into a routine tool for biological investigations19 20

The ability to operate in liquid environments and especiallyat physiological conditions makes AFM a versatile tool forstudying the native structure of biological material21ndash23

High lateral resolution at the nanoscale has been achievedduring investigation of topological changes at the surfacesof living cells24ndash26 Furthermore AFM has been used toinvestigate structural27ndash29 and mechanical30ndash32 properties ofindividual proteins and for real-time probing of protein-protein interactions33 Direct observation of enzyme activityhas been described by detecting height uctuations of thecantilever above the protein lysozyme adsorbed to a micasurface34 Excellent reviews on the application of AFM forbiological systems have recently been published19 20

21 Immobilizing Biorecognition Elements at Scanning Probe Tips

Chemical functionalization of AFM tips extends the infor-mation space obtained by conventional force microscopyEnhanced speci city is achieved by controlled modi ca-tion of the scanning probe tip and measurement of the in-teraction forces between the modi ed tip and the samplesurface Successful experiments require controlled tip sur-face modi cation ensuring rmly anchored biomolecularrecognition elements Although strong adhesion is requiredloss of catalytic enzyme activity induced by conforma-tional distortion during the immobilization process has tobe avoided Hence many coating strategies derive fromprocedures well established for transducer surface modi -cations utilized for biosensing devices Thiol chemistry atgold surfaces and silane chemistry at activated silicon sur-faces are common functionalization techniques for chemi-cal modi cation of cantilevers35 Alkyl thiols with func-tional terminal groups form self-assembled monolayers bychemisorption on gold sputtered cantilevers enabling sub-sequent attachment of receptor molecules at the SPM tipsurface Similarly silane monolayers can be formed at thesurface of silicon cantilevers which are activated by UVozone treatment resulting in a high density of reactive sur-face hydroxyl groups Alternatively lipid bilayers or poly-

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4 Sensor Lett Vol 1 No 1 2003

mers grafted to the SPM tip can be used for immobilizationof biomolecular recognition elements Detailed proceduresand strategies for chemical cantilever surface modi cationare reported elsewhere36

22 Force Spectroscopy and Imaging of MolecularRecognition Events

A schematic representation of the tip-substrate modi cationused for the determination of rupture forces of individualmolecules is given in Figure 1A Figure 1B schematicallydepicts the behavior of the resulting force curve in a forcespectroscopic experiment As long as the tip remains farfrom the surface no interaction forces are recorded (1)When the surface is approached in this idealized experimenta single biomolecule at the apex of the surface-modi edAFM tip interacts with a complementary molecule at the

Figure 1 (A) Schematic representation of the tip-substrate con gura-tion used in the initial studies on measurement of rupture forces of indi-vidual molecules The AFM probe is functionalized with a monolayer of themolecule of interest The substrate is modi ed with the counter-molecule(B) Scheme of a simulated single-molecule force-distance curve to deter-mine rupture force (1) The tip is moved toward the sample without inter-action (2) repulsive part of the force curve (3) scan direction is reversed(4) tip-sample contact is ruptured

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Sensor Lett Vol 1 No 1 2003 5

tip Lateral force mapping49ndash51 provides simultaneous infor-mation on topography and ligand-receptor interactionHowever both temporal and lateral resolution of this tech-nique is limited In contrast force-recognition imaging per-mitted by the combination of dynamic force microscopy52

with force spectroscopy allows determination of receptorsites with an accuracy at the nanometer scale and at scanvelocities comparable to conventional topography acquisi-tion modes53 An antibody for lysozyme was tethered to theAFM tip and used for imaging lysozyme molecules immo-bilized at a mica surface with high resolution utilizing amagnetically oscillated AFM mode (ldquoMacModerdquo MolecularImaging Phoenix AZ) The temporal progression of the ex-periment is shown in Figure 2A In Figure 2B the lysozymemolecules are clearly distorted owing to force recognitionWith the addition of excess lysozyme to the solution theantibody at the tip is blocked as indicated in Figure 2C and

sample surface (2) Upon retraction of the tip the restoringforce of the cantilever is initially balanced by the adhesiveforce (3) until the bond of the molecular pair is ruptured (4)The measured force at this point represents the ruptureforce of the bond formed between the recognition ele-ments Ideally localized detection of single molecularbonds between two individual molecules is enabled withthis technique The rst example of direct observation of asingle bond rupture of a distinct streptavidinndashbiotin com-plex was independently reported by the groups of Gaub37

and Colton38 in 1994 Extensive reviews on single-mole-cule force spectroscopy have recently been published39ndash42

Selected examples of single-molecule force spectroscopyusing a variety of receptor-ligand systems are highlightedin Table I

Inasmuch as structure-function relationships play a keyrole in the biosciences their simultaneous detection is apromising approach yielding enhanced insight into the regu-lation of cellular and other biological mechanisms Bindingof ligands to receptors is among the most important mecha-nisms in biological processes frequently triggering signal-ing pathways and cascades In molecular recognition forcemicroscopy (MRFM) the dynamics of the experiments arevaried yielding information on the molecular interactioncharacterizing ligand-receptor binding and dissociation Withthis method af nity constants rate constants and energybarriers have been estimated and the bond widths of bindingpockets have been determined43ndash46 Furthermore the op-portunity of mapping binding energy landscapes is pro-vided47 48 In MRFM receptor binding sites are localizedby molecular interaction with a ligand tethered to the AFM

Figure 2 Force-recognition imaging with (a) an antibody to lysozyme tethered to the AFM tip scanning in liquid over a surface of immobilizedlysozyme molecules A low concentration of lysozyme is added to solution As long as the antibody on the tip has not bound free lysozyme from solution(I) it is reacting with surface-bound lysozyme molecules resulting in distortion of the images due to recognition (visible in (b) and part of (c)) The blackarrow (image c) marks when the antibody on the tip has bound solution lysozyme and the image in part of (c) and all of (d) switches from recognition totopographic imaging Reprinted with permission from Ref 53 A Raab et al Nat Biotechnol 17 902 (1999) copy1999 Nature

Table I Overview of applications of single-molecule force microscopywith different receptor-ligand systems

Ligand-receptor interaction Reference

(Strept-)avidinbiotin 37 38 50 125ndash135Oligonucleotidecomplementary oligonucleotide 136ndash142Antibodyantigen 43ndash45 49 143ndash148Polysaccharides 149ndash154Acetylcholinesteraseacetylcholine 155Proteoglycans 156Selectinligand 157IntegrinRGD-sequences 158Vascular endothelial cathedrin dimers 159

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a clearer image of the lysozyme-decorated surface appearsand is ultimately completely restored in Figure 2D

Another example of molecular recognition of an antibodyby a membrane protein is shown in Figure 354 The antibodyis linked to the tip of the probe by a exible spacer using co-valent bonds Brush border membrane vesicles (BBMV) wereadsorbed to a gold surface and imaged with dynamic forcemicroscopy The AFM tip was modi ed with anti-g-GT anti-body against the antigen g-GT (g-glutamyltranspepidase)present at the BBMV At lateral positions where membranevesicles were identi ed the x-y scan was stopped and forcedistance cycles were recorded as shown in Figure 3A Whenthe antibody at the tip binds to an antigen at the surface an at-tractive force develops during further retraction since the tipis physically connected to the surface The attractive force de-velops in a delayed and non-linear fashion re ecting thestretching and the non-linear spring constant of the exibletether covalently coupling the antibody to the AFM tip Thissignal has a distinct shape and re ects the recognition event ofa single antibody-antigen pair43 44 During the control experi-ment in Figure 3B all g-GT sites at the BBMV surface areblocked by anti-g-GT antibody added to the solution Henceno recognition events are recorded

AFM has been used as a tool to simultaneously studymorphology changes and the presence of extracellularATP at living cells with the use of a biosensing layer im-mobilized on the cantilever55 Myosin-functionalized can-tilevers were used to quantitatively detect ATP in solutionduring AFM tapping-mode imaging of living cells Aschematic view of the measurement principle is given inFigure 4AndashC After ATP binding to the myosin subfrag-ment S1 immobilized at the AFM cantilever ATP hydrol-ysis occurs During hydrolysis a conformational changeof the enzyme occurs (in the dimensional range of approx10 nm)56 This conformational change disturbs the inter-action between the tip and the cell surface and causes ameasurable de ection (Figure 4D) Modi ed cantileverswere used to scan the surface of cultured cells demonstrat-ing that the CFTR1 cell line (S9) had a basal surface ATPconcentration which could be determined with AFM tipintegrated biosensors for the rst time

Very recently chemically modi ed probes were appliedto investigate the surface charges of living cells57 AFMmeasurements utilizing carboxyl-terminated tips were usedto probe the electrostatic properties and more speci callythe isoelectric point of microbial cell surfaces with high spa-tial resolution Functionalization of the surfaces was vali-dated by recording multiple force-distance curves between

Figure 4 (A) Scheme of an enzyme-modi ed (myosin subfragmentS1) AFM tip imaging the extracellular surface of a CFTR-expressing cellCl2 (black dots) and ATP (yellow dots) are released in the area adjacentto the CFTR protein (B) ATP binding and subsequent hydrolysis at thetip resulting in cantilever de ection (C) The tip remains de ectedalthough the point of ATP release is passed due to short contact timescompared with ATPase activity time (D left) One ldquohot spotrsquo of extracel-lular ATP The AFM tip rst dips into a localized ATP pool and thenmoves away from this area as the scan progresses As ATP hydrolysisoccurs a positive de ection of the tip is noted causing a continuous lineof activity (D right) Image recorded after incubation with 1024 Mglybenclamide a known inhibitor of CFTR Reduction in hot spots (ar-rows) indicates reduction of extracellular ATP concentration Reprintedwith permission from Ref 55 S W Schneider et al Proc Natl AcadSci USA 96 12180 (1999) copy1999 National Academy of Sciences of theUnited States of America

Figure 3 Dynamic force imaging of brush border membrane vesicles(BBMV) adsorbed to a gold surface (A) Force distance cycle for single an-tibody-antigen recognition event The modi ed tip is brought toward aBBMV (trace dotted line) and subsequently retracted (retraced solid line)at a constant lateral position Antibody-antigen binding is evident from adelayed and non-linear attractive force that develops during further retrac-tion (unbinding event) Inset shows force-distance cycle comprising tworecognition events separated in space and time (B) Control experiment inthe presence of 1 microM anti-g-GT antibody in solution The recognition sig-nal disappears and the traceretrace looks the same Reprinted with permis-sion from Ref 54 Wielert-Badt el al Biophys J 82 2767 (2002) copy2002The Biophysical Society

modi ed probes and model substrates Furthermore car-boxyl-terminated probes were used to record multipleforce-distance curves at various pHs at the surface ofSaccharomyces cerevisiae cells Adhesion force maps de-pendent on the pH were created by recording multiple (n 5

256) force-distance curves and calculating the adhesionforce for each force curve

To summarize force recognition imaging has developedinto a powerful tool for high-resolution studies of molecularrecognition events and for probing dynamic properties ofbiomolecules at physiological conditions Ongoing progressin instrumental development and improved recording tech-niques has continuously added to the relevance of forcerecognition imaging for in situ investigation of biologicalspecimens

3 SCANNING ELECTROCHEMICALMICROBIOSENSORS

The importance of amperometric biosensors is steadily in-creasing because of the combined advantages offered bythe selectivity of biological recognition elements and thewell-established principle of electrochemical transductionThe key element for the design of any sensor architectureis the immobilization of the speci c biological recognitionelement Common sensor architectures include enzymesimmobilized at the transducer surface In the case of anamperometric biosensor this surface usually resembles anoble metal or carbon electrode

31 Immobilizing Biorecognition Elements atMicroelectrodes

For control of the immobilization process during modi -cation of electrode surfaces reproducible procedures arepreferable especially for the development of miniaturizedbiosensors Most commonly applied electrode surface mod-i cation techniques include screen-printing techniques58 incombination with photoinduced cross-linking59 spin-coatingfollowed by photolithographic structuring and lift-off60 theelectrochemically induced formation of enzyme-containingpolymer lms using electrophoretic accumulation61 62 de-position of conducting polymers63ndash65 and covalent bindingof enzymes through functionalized thiol chemistry66 In par-ticular electrochemical techniques permit speci c modi ca-tion of electrode surfaces with high reproducibility Enzymeentrapment within electrochemically deposited polymer ma-trices is well described in the literature and allows directedmodi cation of electrode surfaces64 67 68 Recently success-ful immobilization based on electrochemically induced lo-calized precipitation of enzyme-containing polymer suspen-sions has been reported69 Most reports of applications ofamperometric biosensors describe accurate and reproducibledetermination of analytes in bulk experiments with only afew examples of scanning microbiosensors

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32 Biosensing with Scanning ElectrochemicalMicroscopy

Laterally resolved information at a submicrometer scalewas added to electroanalytical chemistry with the inventionof scanning electrochemical microscopy (SECM)70ndash72

This method provides spatially resolved electrochemicalinformation on interface processes by combining electro-analytical techniques with the principle of scanning a mi-croelectrode in close vicinity across the sample surfaceSECM is an in situ technique based on surface inducedchanges of a Faraday current which is measured at ascanned and biased ultramicroelectrode (UME) The hemi-spherical diffusion of a redox species in solution toward theUME is disturbed by the sample surface chemistry in closeproximity to the microelectrode Commonly imaging inSECM is achieved in feedback mode operation with redoxspecies arti cially added to the solution

The Faraday current measured at the UME is mainly in- uenced by the reactivity and morphology of the samplethe distance between tip and sample and the size andgeometry of the ultramicroelectrode when the electrode isin close proximity (a few electrode radii) to the sample sur-face In conventional SECM experiments the UME tip isscanned in constant height above the sample surface Inprinciple two modes of SECM are used for imaging appli-cations the feedback mode73 and the generationcollection(GC) mode74 Functional schemes of both SECM operationmodes are shown in Figure 5 The feedback mode is basedon the Faraday current resulting from the oxidation or re-duction of a mediator species at the UME tip with the appli-cation of a potential In bulk solution after reaching steadystate the Faraday current recorded at the UME remainsconstant (Figure 5A a) Approaching an insulating surfacewith the UME results in blocked hemispherical diffusion tothe active electrode surface (negative feedback) as shownin Figure 5A c In contrast approaching a conducting orelectrochemically active surface leads to positive feedbackdue to locally increased mediator concentration induced bysurface recycling effects (Figure 5A b) This technique iswidely used for investigating processes at solidliquid andliquidliquid interfaces75

Most investigations of biological samples are performedin the GC (substrate generationtip collection or tip gener-ationsubstrate collection) mode with the tip signal arisingfrom a species generated at the sample surface (Figure 5B)The tip acts as a sensor establishing concentration maps ofindividual electroactive species near the sample surface

In conventional SECM positioning of the microelectrodeis achieved by recording the Faradaic current measured atthe UME as the tip approaches the surface Usually theoret-ical calculations of approach curves for different electrodegeometries are tted to the experimentally obtained dataand used to determine the distance between the UME andthe sample surface76 However the current signal can beused only for amperometric electrodes Consequently non-

Sensor Lett Vol 1 No 1 2003 7

sitioning of the microelectrode in common operation modesis a major drawback of SECM in comparison with AFM orSTM83 Scanning the electrode in constant height across thesample surface results in a convolution of the electrochemi-cal response and the topographical information especially ifthe sample topography and the surface reactivity are un-known Furthermore risk of damage to the UME andor thesample surface increases with decreasing diameter of theSECM tip to the submicron scale or nanometer scale be-cause of the electrode size-dependent working distanceConsequently any progress toward improved lateral reso-lution and positioning of nonamperometric submicroelec-trodes and nanoelectrodes (eg micro- and nanobiosensors)has to address current independent positioning strategiesensuring precise control of the tip-sample distance

33 Positioning of Electrochemical Microbiosensorswith the Use of Shear Force-Based Constant-Distance Mode

In NSOM shear force-based positioning of optical ber tipswith the use of optical detection systems84 85 or nonopticaldetection based on a tuning fork resonator86 87 has beendescribed The rst successful integration of a shear force-based optical feedback mechanism into SECM using a ex-ible ( ber-shaped) vibrating UME was published in the mid-1990s88 Recently this technology has been adapted to avariety of microelectrodes89 A ber-shaped UME is vi-brated at its resonance frequency with a piezoelectric tubeused for agitation (Figure 6A) A laser beam is focused onthe tip of the UME creating a Fresnel diffraction patternwhich is projected onto a split photodiode The vibrationamplitude of the electrode is monitored by phase-sensitiveampli cation of the difference signal at the split photodiodewith respect to the agitation signal with the use of lock-intechniques Surface shear forces increase because of hydro-dynamic effects in close proximity to the sample90 and leadto amplitude damping and a phase shift of the vibrationWith this information a feedback loop with a de ned setpoint for amplitude damping can be integrated into theSECM software routine thereby maintaining a constant dis-tance between the tip and the sample surface during scan-ning across a sample with variable topography

An alternative method combining the shear force modebased on a tuning fork technique with SECM for currentindependent positioning of ber-shaped electrodes has beendescribed in the literature91ndash94 In these contributions theSECM tip is attached to a tuning fork resonance detectionsystem of a commercially available NSOM The UME is at-tached to the leg of a tuning fork driven by a piezoelectrictube At resonance the movement of the legs of the tuningfork generates an oscillating piezoelectric potential which isproportional to the amplitude of the oscillation Again thesynchronized signal derived with lock-in techniques is usedas the feedback signal by the NSOM controller Recently ahighly sensitive nonoptical shear force distance control for

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amperometric tips such as biosensors require alternative tippositioning strategies during SECM experiments Utilizingoptical microscopy for tip positioning yields only qualita-tive results77 Few approaches have been reported for theapplication of potentiometric UMEs78ndash81 and enzymeUMEs82 Horrocks et al78 used antimony SECM electrodesalso operating in amperometric mode for positioning of thetip prior to pH mapping Wei et al79 introduced positioningtechniques based on dc measurements of the solution resis-tance with AgAgCl micropipette electrodes or potentio-metric measurements of steady-state concentration pro lesusing ion-selective microelectrodes providing the tip-sample distance Furthermore potentiometric UMEs with adual-electrode con guration are reported where one chan-nel is con gured as an ion-selective electrode and the sec-ond channel as an amperometric electrode for distancecontrol during UME approach A hydrogen peroxide micro-biosensor was positioned by the application of a high-frequency alternating potential to the tip and measurementof the solution resistance between the tip and the auxiliaryelectrode82 Although good agreement of the experimentallyobtained data with theory has been achieved this approach islimited to micrometer-sized sensors because the relativecontribution of the solution resistance to the impedance de-creases with decreasing dimensions of the sensor

The lack of lateral resolution due to current dependent po-

Figure 5 Scheme of SECM operation modes (A) Feedback modewith normalized current-distance curve (a) UME in bulk solution(b) positive feedback mode due to mediator recycling at the sample sur-face (c) negative feedback mode due to blocked diffusion of the medi-ator to the UME (B) Generatorcollector mode (a) substrate genera-tiontip collection and (b) tip generationsubstrate collection mode

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Sensor Lett Vol 1 No 1 2003 9

SECM tips based on piezoelectric shear force distancecontrol was developed simplifying shear force-controlledconstant-distance SECM95

Shear force-based constant-distance SECM with opticaldetection has already been applied for positioning of micro-biosensors89 Glass capillaries (inner diam 10 mm) havebeen lled with a polymer hydrogel containing either glu-cose oxidase or glucose dehydrogenase The enzyme elec-trodes were positioned with the use of optical shear forcecontrol and scanned over a 50-mm Pt electrode in the case ofthe glucose oxidase biosensor and over a poly-(methyleneblue)-modi ed microelectrode in the case of the glucose-de-hydrogenase- lled capillary The involved reaction schemes(A) and the obtained SECM images of the reactive spots im-aged with the microbiosensors (B) are shown in Figure 7

To date few results have been published on nanoelec-trodes in SECM experiments High-resolution SECM imag-ing requires reproducible fabrication of nanoelectrodes bymicrofabrication techniques In addition it is still dif cult todetermine the exact distance between the electrode and thesample surface by SECM operation in constant-distance andconstant-height mode Distance calculations are frequentlybased on assumptions such as ideal electrode geometriesHowever these are dif cult to realize for nanoelectrodesfabricated by conventional techniques such as the use oflaser pipette pullers96 In turn quantitative studies on elec-trode processes require precise knowledge of the absolute

distance between tip and surface and demand reproduciblyfabricated electrodes and accurate control of a deliberatelyde ned electrode working distance

4 AFM TIP INTEGRATED ELECTRO-CHEMICAL MICROBIOSENSORS

Recent developments focused on the combination of AFMand SECM enable positioning of the UME independently ofthe current response during simultaneous high-resolutionAFM imaging AFM probes with integrated electrochemicalsensing capabilities provide a versatile tool with inherentcorrelation of structural information with (electro)chemicalsurface activity at excellent lateral resolution97

Figure 8 shows schematics of two different concepts forthe combination of AFM with SECM published by Mac-pherson et al98 99 and Kranz et al100ndash104 The rst approachof combined SECM-AFM imaging in liquid utilizes bentPt-wire nanoelectrodes shaped like AFM cantilevers (Figure8A and B)98 99 Although excellent spatial resolution forthe electrochemical response can be achieved imagingwith these cantilever-shaped electrodes in contact mode islimited to robust nonconducting samples because of thecontact of the cantilever-shaped nanoelectrode and the sam-ple surface Since the electroactive area is located at thevery end of the tip conducting samples have to be investi-gated by lift-mode imaging99 However this requires scan-ning the sample surface twice First the topographical in-formation is recorded in contact mode operation followedby a second scan using the recorded topographical informa-tion to keep the tip at a de ned distance from the surfacetracing the sample contours99

Figure 7 (A) Scheme of the generatorcollector mode detection prin-ciple using enzyme- lled ber-shaped tips (B) Identi cation of electro-active spots with enzyme- lled capillaries (a) Image of a 50-mm-diameterplatinum electrode obtained with a glucose oxidase-modi ed microcapil-lary and (b) image of a 50-mm poly(methylene blue)-modi ed platinumelectrode based on detection of NADH generated at the glucose dehydro-genase-modi ed capillary tip Reprinted with permission from Ref 89A Hengstenberg et al Chem Eur J 6 1547 (2000) copy2000 Wiley

Figure 6 Schematic representation of the different shear force detec-tion principles implemented in SECM for constant distance imaging(A) optical shear-force detection (B and C) Nonoptical shear-force detection

the very end of the original AFM tip Figure 8 shows aschematic cross section of an integrated AFM-SECM tip(C) and FIB images of an integrated frame submicroelec-trode (D E) Because of the resolution of the beam pro-vided by FIB systems integration of submicrometer- andnanoelectrodes can be realized100 103 111 although theelectrode size demonstrated with bent Pt wire electrodescould not yet be achieved So far disc-shaped integratednanoelectrodes with diameters down to 150 nm have beensuccessfully fabricated with the described technology112

Based on the described technology for the combinationof AFM and SECM SECM functionality can be integratedinto any standard AFM with only minor modi cations of(frequently) already existing AFM instrumentation Theelectrochemical response is inherently correlated with thetopographical information obtained by AFM imaging Thuscomplete separation of simultaneously obtained topo-graphical information and electrochemical information isachieved maintaining superior resolution of the topo-graphical information With this technique positioning ofnonamperometric electrodes down to the nanometer regimecan be realized

The application of integrated AFM-SECM tips fortapping-mode AFM operation has recently been demon-strated113 Despite modi cation during tip fabrication theresonance frequency of the unmodi ed silicon nitride can-tilever is not signi cantly shifted which has been con- rmed by comparing frequency spectra of unmodi ed andmodi ed cantilevers The oscillation amplitude of a fewnanometers is small compared with the diameter of theintegrated electrode (usually 300ndash800 nm) It has beenshown that the current response of the integrated electrodein SECM feedback mode is not significantly influencedby the oscillation amplitude of the combined SECM-AFMcantilever Electrochemical images recorded in tappingmode revealed current response and lateral resolutioncomparable to the electrochemical images obtained in con-tact mode113

AFM tip integrated electrodes have been applied forimaging enzyme activity in tapping-mode operation (Fig-ure 9)112 Enzyme activity of glucose oxidase (GOD)entrapped in a soft polymer matrix deposited inside thepores of a periodic micropattern has successfully beenimaged in AFM tapping mode and SECM generation-collection mode In the absence of glucose the currentrecorded at the electrode during AFM tapping-mode imag-ing of the enzyme containing polymer spots is negligible(Figure 9A topography B amplitude image) and revealsno electrochemical features (Figure 9C) In the presence ofglucose an increase in the current recorded at the inte-grated submicroelectrode is observed because of localizedproduction of H2O2 above the GOD-containing polymerspots (Figure 9F) The topography (Figure 9D) and ampli-tude (Figure 9E) images remain unaffected

The presented approach of integrated SECM-AFMprobes provides the opportunity to integrate and position

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10 Sensor Lett Vol 1 No 1 2003

Although several approaches of combined AFM-SECMusing such bent cantilever-shaped nanoelectrodes have beenpublished105ndash107 to date none of these reported measure-ments in AFM tapping mode are due to the variation inspring constant of these hand-made tips AFM tapping modeis a versatile technique for topographical imaging of softsample surfaces as encountered during investigation of forexample biological systems15 During tapping-mode opera-tion the probe gently taps the surface while resonating at ornear its resonance frequency A signi cantly reduced tip-sample contact time results in minimized frictional forcesavoiding damage of soft samples such as biological speciesor polymers108ndash110

An alternative approach combining AFM and SECMhas been described by our research group based on inte-gration of submicro- and nanoelectrodes into atomic forcemicroscopy tips with the use of microfabrication tech-niques (Figure 8CndashE)100ndash104 Electrodes with a variety ofwell-de ned geometries can be integrated into AFM tips atan exactly de ned distance above the apex of the AFM tipsimultaneously obtaining electrochemical information at aprecisely de ned working distance during AFM imagingDuring tip fabrication a conventional silicon nitride AFMcantilever is coated with an electroactive conducting layer(usually gold or platinum) and is subsequently insulatedwith materials such as Si3N4 or parylene C Three-dimen-sional focused ion beam (FIB) milling permits repro-ducible exposure of an electroactive area integrated above

Figure 8 Representation of combined AFM and SECM as publishedby Macpherson et al98 99 (A and B) and Kranz et al100 (CndashE)(A) Schematic of the AFM-SECM tip fabricated by coating etching andbending of Pt microwires Reprinted with permission from Ref 98 J VMacpherson and P R Unwin Anal Chem 72 276 (2000) copy2000American Chemical Society (B) Scanning electron micrograph of a can-tilever-shaped nanoelectrode Reprinted with permission from Ref 99 JV Macpherson and P R Unwin Anal Chem 73 550 (2001) copy2001American Chemical Society (C) Schematic cross section of an integratedAFM-SECM tip fabricated by microfabrication using FIB technology(dimensions are not to scale) (D) FIB image of an integrated frame sub-microelectrode (gold) with Parylene C insulation (electrode edge length770 nm) (E) Side view showing reshaped AFM-tip and tilt correction fora cantilever mounting angle of 10deg in a tapping-mode liquid cell

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micro- and nanobiosensors at a well-de ned and deliber-ately selected distance above the sample surface Standardsurface modi cation processes for biosensors as previ-ously outlined in this review can be used to immobilizebiological recognition elements at the surface of the inte-grated electrode (Figure 10) With the use of electrochemicaltechniques the site of immobilization is con ned to theelectrode surface providing highly localized and repro-ducible integration of a biosensing interface64 65 67 68

With the immobilization of enzymes via self-assembledthiol monolayers with reactive headgroups114 conversion

of electroactive and -inactive species generated at the sam-ple surface is enabled

Figure 11 illustrates simultaneously recorded topo-graphical and electrochemical images obtained with a tip-integrated biosensor The biosensor is based on horse-radish peroxidase immobilized at the electrode surface ofthe integrated SECM-AFM probe (Figure 11A) The per-oxidase activity was imaged in tip generatorsubstrate col-lector mode of SECM (see also Figure 11Bb) duringcontact-mode AFM operation The enzyme was immobi-lized by chemisorption of a functionalized thiol monolayer(cystaminium chloride) to the tip-integrated gold micro-electrode Peroxidase was attached by the addition of amixture of glutaraldehyde and peroxidase resulting in a

Figure 11 (A) Schematic presentation of the tip generationsubstratecollection mode experiment involving simultaneous detection of tipintegrated enzyme activity and AFM topography (B) Simultaneouslyrecorded topographical information and tip integrated peroxidase activityin contact mode The current measured at the microelectrode arrayre ects the enzyme activity integrated in the AFM tip Top view of theheight (a) and (c) and simultaneously recorded current (b) The electro-chemical response with 05 mmol L21 H2O2 in solution Images arerecorded in 2 mmol L21 FMA 01 mol L21 KCl in phosphate buffer (01mol L21 pH 70) (d) In the absence of the enzymatic substrate H2O2The tip was held at a potential of 005 V vs AgAgCl Electrode edgelength 860 nm tip height 410 nm scan rate 1 Hz

Figure 10 Schematic cross section of an AFM tip integrated biosensorEnzymes can be immobilized at the electrode surface integrated in thescanning probe tip via self-assembled thiol monolayers with functional-ized headgroups (A) or via electrochemical deposition of enzyme-containing polymer lms (B)

Figure 9 Simultaneously recorded height and current images of glu-cose oxidase activity in AFM tapping mode and SECM substrate genera-tiontip collection mode Top view of the height (a and d) correspondingamplitude (b and e) and simultaneously recorded current (c and f) imagesrecorded in air-saturated phosphate buffer in absence (andashc) and in thepresence (dndashf) of the substrate glucose in solution Reprinted with per-mission from Ref 112 A Kueng et al Angew Chem Int Ed 42 3237(2003) copy2003 Wiley

Sensor Lett Vol 1 No 1 2003 11

miniaturization of biosensors However only a few ap-proaches have been reported that use scanning micro- ornanobiosensors to obtain laterally resolved (bio)chemicalinformation Using shear force modes in combination withSECM permits positioning of nonamperometric sensorsImprovement of the achievable lateral resolution by de-creasing the dimensions of the applied sensor is the focusof ongoing research in this eld Fiber-based optical micro-biosensors in combination with NSOM techniques mightbe an alternative future concept for obtaining laterally re-solved information on bioactivity AFM tip-integrated bio-sensors provide a promising concept combining advantagesof various techniques in a single microfabricated probeHigh reproducibility of the fabrication process and the po-tential of miniaturization down to the nanometer scalemake them a versatile tool for future applications for prob-ing a wide range of biological substrates and processes

Among the major drawbacks of biosensors in general aresigni cantly increased response times of enzyme-modi edelectrodes compared with direct electrochemical detectionMany biologically relevant processes (eg exocytoticevents) relay signals in intervals of milliseconds Elec-trodes have been fabricated that can detect neurotransmit-ters with response times of 300 ms118 119 However re-sponse times less than 100 ms are needed to accuratelyquantify dynamics at the cellular level and less than 10 msto examine exocytosis of nonelectroactive neurotransmit-ters However it has been demonstrated that the temporalresolution achievable with biosensor systems scales in fa-vor of the proposed technology with device miniaturiza-tion120 121 As an example response times on the order of85 ms have been reported for a glucose-speci c biosensorwith an electrode diameter decreased to the submicronrange122 As recently demonstrated by our research groupdisc electrodes with diameters below 200 nm can be inte-grated into AFM tips by microfabrication techniques112 Itis expected that surface modi cation of such nanoelec-trodes as outlined in this contribution will result in a furtherdecrease in the integrated biosensor response time With arealistic perspective of measurements in the time domain ofmicroseconds suitability for investigation of biologicalprocesses is evident

With the development of multifunctional scanningprobes with tip-integrated biosensors a versatile analyticaltool for the investigation of complex biological systemshas been realized AFM cantilever-based biosensors pro-vide an excellent concept for simultaneous detection oftopographical and chemical information at the surface ofbiological species with high lateral resolution With theintegration of an electroactive area into AFM cantileversenabling subsequent surface modi cation and leading tobiosensor functionality an extension of this concept is pre-sented for detecting electroactive and -inactive speciesgenerated at the sample surface Accurate positioning andscanning of nanobiosensors at a de ned distance above thesample allows quanti cation of kinetic bioprocesses at the

surface-grafted cross-linked protein gel at the aminatedsurface of the self-assembled monolayer covering the inte-grated electrode The electrochemical signal generation isbased on the following enzyme catalyzed reactions(1) H2O2 is reduced to water by the oxidoreductase perox-idase and (2) a metal organic electron donor for the enzy-matic reaction (hydroxyl methyl ferrocene FMA contain-ing Fe21) added to the solution is oxidized during theenzymatic conversion to ferrocinium methylhydroxide(FMA1 containing Fe31) The electroactive by-product ofthe enzymatic reaction FMA1 diffuses to a microstruc-tured electrode array which was used as a model sampleThe microstructured electrode array is biased at a potentialof 50 mV vs AgAgCl At this potential only FMA1 andno other species in solution is reduced Hence informationon the local (electro)chemical activity (Figure 11Bb) withrespect to the position of the tip-integrated biosensor isprovided (Figure 11Ba topography) Comparison with thecontrol experiment in the absence of H2O2 (Figure 11B ctopography B d electrochemical image) demonstratesthe successful integration of a biosensor into a SECM-AFM probe and simultaneous activity imaging during AFMmeasurements17

The presented technique is by no means limited to theintegration of biosensors and can be extended to potentio-metric sensors such as pH microsensors or amalgam elec-trodes Many biological processes including enzymaticreactions biocorrosion and metabolic processes of micro-organisms involve or are driven by pH changes Hencesimultaneous AFM imaging and mapping of local pHmodulations is of substantial interest For example anti-mony and iridiumiridium oxide electrodes have beenreported for electrochemical pH measurements of biolog-ical systems115ndash117 As thin lms of these materials can besputtered the fabrication steps described above for the in-tegration of gold and platinum electrodes can be modi edand optimized to integrate pH-sensitive electrochemicalsensors into AFM tips

5 CONCLUSIONS AND OUTLOOK

Cantilever-based (bio)sensors are versatile analytical sys-tems for the investigation of biological species as they canbe used in solution and are capable of transducing a varietyof different signals Applications in force spectroscopypermit measurements at the level of single moleculesHowever the limiting factor of this technique for imagingof biomolecules is its comparatively poor temporal resolu-tion However ongoing instrumental developments andcombination of AFM with complementary techniques willovercome these limitations in the near future

Using enzymes as recognition elements in biosensorscombines several advantages including high speci city andselectivity Recently advanced immobilization strategiesand mass fabrication based on silicon technology allow

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12 Sensor Lett Vol 1 No 1 2003

25 J H Hoh and P K Hansma Trends Cell Biol 2 208 (1992)26 H Oberleithner G Giebisch and J Geibel P uumlgers Arch 425 506

(1993)27 J Mou S J Sheng R Ho and Z Shao Biophys J 71 2213

(1996)28 J Mou D M Czajkowsky S J Sheng R Ho and Z Shao FEBS

Lett 381 161 (1996)29 D J Muller and A Engel J Mol Biol 285 1347 (1999)30 F Oesterhelt D Oesterhelt M Pfeiffer A Engel H E Gaub and

D Muller Science 288 143 (2000)31 A F Oberhauser P E Marsyale M Carrion-Vazquez and J M

Fernandez Nat Struct Biol 6 1025 (1999)32 M Rief M Gautel F Oesterheit J M Fernandez and H E Gaub

Science 276 1109 (1997)33 M B Viani L I Pietrasanta J B Thompson A Chand I C

Gebeshuber J H Kindt M Richter H G Hansma and P KHansma Nat Struct Biol 7 644 (2000)

34 M Radmacher M Fritz H G Hansma and P K Hansma Science265 1577 (1994)

35 A Noy C D Frisbie L F Rozsnyai M S Wrighton and C MLieber J Am Chem Soc 117 7943 (1995)

36 A M Moulin S J OrsquoShea and M E Welland Ultramicroscopy82 23 (2000)

37 E-L Florin V T Moy and H E Gaub Science 264 415 (1994)38 G U Lee D A Kidwell and R J Colton Langmuir 10 354

(1994)39 A Janshoff M Neitzert Y Oberdoumlrfer and H Fuchs Angew

Chemie Int Ed 39 3212 (2000)40 O H Willemsen M M E Snel A Cambi J Greve B G De

Grooth and C G Figdor Biophys J 79 3267 (2000)41 M Carrion-Vazquez A F Oberhauser T E Fisher P E

Marszalek H Li and J M Fernandez Prog Biophys Mol Biol74 63 (2000)

42 J Zlatanova S M Lindsay and S H Leuba Prog Biophys MolBiol 74 37 (2000)

43 P Hinterdorfer W Baumgartner H J Gruber K Schilcher andH Schindler Proc Natl Acad Sci USA 93 3477 (1996)

44 P Hinterdorfer K Schilcher W Baumgartner H J Gruber andH Schindler Nanobiology 4 39 (1998)

45 R Ros F Schwesinger D Anselmetti M Kubon R SchaeferA Pluumlckthun and L Tiefenauer Proc Natl Acad Sci USA 957402 (1998)

46 F Schwesinger R Ros T Strunz D Anselmetti H-J GuumlntherodtA Honegger L Jermutus L Tiefenauer and A Pluumlckthun ProcNatl Acad Sci USA 97 9972 (2000)

47 R Merkel P Nassoy A Leung A Ritchie and K Evans Nature397 50 (1999)

48 T Strunz K Oroszlan I Schumakovitch H-J Guumlntherodt andM Hegner Biophys J 79 1206 (2000)

49 O H Willemsen M M E Snel K O van der Werf B G DeGrooth J Greve P Hinterdorfer H J Gruber H Schindler Y vanKooyk and C G Figdor Biophys J 75 2220 (1998)

50 M Ludwig W Dettmann and H E Gaub Biophys J 72 445(1997)

51 D P Allison P Hinterdorfer and W Han Curr Opin Biotechnol13 47 (2002)

52 W Han S M Lindsay and T Jing Appl Phys Lett 69 4111(1996)

53 A Raab W Han D Badt S J Smith-Gill S M LindsayH Schindler and P Hinterdorfer Nat Biotechnol 17 902 (1999)

54 G Schuumltz M Sonnleitner P Hinterdorfer and H Schindler MolMembr Biol 17 17 (2000)

55 S W Schneider M E Egan B P Jena W B GugginoH Oberleithner and J P Geibel Proc Natl Acad Sci USA 9612180 (1999)

56 J Finer R M Simmons and J A Spudnich Nature 368 113(1994)

sample surface with high lateral and temporal resolutionGiven the exibility of the described concepts there is con-siderable scope for extended use of multifunctional scan-ning probe systems probing a wide range of biologicalspecimens and processes

Acknowledgments The National Science Foundation(grant 0216368 in the program ldquoBiocomplexity in theEnvironmentrdquo) the National Institutes of Health (grantEB000508) and the ldquoFonds zur Foumlrderung der wissenschaft-lichen Forschungrdquo Austria (grants P14122-CHE and J2230)are greatly acknowledged PCT patents123 124on integratedAFM-SECM are issued

References and Notes

1 A Hulanicki S Glab and F Ingman Pure Appl Chem 63 1247(1991)

2 W Goepel T A Jones M Kleitz I Lundstroem and T SeiyamaEditors Chemical and Biochemical Sensors VCH Weinheim(1992) Vols 2 and 3

3 F W Scheller U Wollenberger A Warsinke and F Lisdat CurrOpin Biotechnol 12 35 (2001)

4 D R Thevenot K Toth R A Durst and G S Wilson BiosensBioelectron 16 121 (2001)

5 M Keusgen Naturwissenschaften 89 433 (2002)6 T H Park and M L Shuler Biotechnol Prog 19 243 (2003)7 K Sato A Hibara M Tokeshi H Hisamoto and T Kitamori Adv

Drug Delivery Rev 55 3791 (2003)8 P N Bartlett Handbook of Chemical and Biological Sensors

edited by R F Taylor Arthur D Little Inc USA J S SchultzUniversity of Pittsburgh (1996) pp 139ff

9 A Hierlemann O Brand C Hagleiter and H Baltes Proc IEEE91 839 (2003)

10 T E Fisher P E Marszalek A F Oberhauser M Carrion-Vazquez and J M Fernandez J Physiol (London) 520 5 (1999)

11 J K Gimzewski and C Joachim Science 283 1683 (1999)12 S O Vansteenkiste M C Davis C J Roberts S J B Tendler

and P M Williams Prog Surf Sci 57 95 (1998)13 M A Poggi L A Bottomley and P T Lillehei Anal Chem 74

2851 (2002)14 G Binnig H Rohrer C Gerber and E Weibel Appl Phys Lett

40 178 (1982)15 P K Hansma J P Cleveland M Radmacher D A Walters P E

Hillner M Bezanilla M Fritz D Vie H G Hansma C B PraterJ Massie L Fukunaga J Gurley and V Elings Appl Phys Lett64 1738 (1994)

16 W H Scouten J H T Luong and S R Brown Trends Biotechnol13 178 (1995)

17 C Kranz A Kueng A Lugstein E Bertagnolli and B Mizaikoffmanuscript in preparation

18 G Binnig C F Quate and C Gerber Phys Rev Lett 56 930 (1986)19 G Charras P Lehenkari and M Horton Methods Cell Biol 68

171 (2002)20 J H Kindt J C Sitko L I Pietrasanta E Oroudjev N Becker

M B Viani and H G Hansma Methods Cell Biol 68 213 (2002)21 B Drake C B Prater A L Weisenhorn S A C Gould T R

Albrecht C F Quate D S Cannell H G Hansma and P KHansma Science 243 1586 (1989)

22 D Rugar and P K Hansma Phys Today 43 23 (1990)23 M Radmacher R W Tillmann M Fritz and H E Gaub Science

157 1900 (1992)24 E Henderson P G Haydon and D S Sakaguchi Science 257

1944 (1992)

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Sensor Lett Vol 1 No 1 2003 13

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Sensor Lett Vol 1 No 1 2003 3

are of particular interest Improvements in functionalizingsensor surfaces8 along with advancements in microfabrica-tion9 allow development of highly integrated miniaturizedbiosensors The ability to position and accurately scan thesedevices across a sample surface results in laterally resolvedinformation essential for the monitoring of localized eventsat individual reaction sites10

Scanning probe microscopy (SPM) techniques are gain-ing increasing importance in the understanding of molec-ular biological interactions providing optical topographi-cal and chemical information with high spatial resolutiondependent on the speci c interaction between the samplesurface and the probe scanned in close proximity acrossthe sample11ndash13 Since the introduction of scanning tun-neling microscopy (STM) in 198214 derived techniquessuch as atomic force microscopy (AFM) near- eld scan-ning optical microscopy (NSOM) scanning ion conduc-tance microscopy (SICM) and scanning electrochemicalmicroscopy (SECM) have successfully been applied toprobing of biologically active surfaces Depending on theinvolved tip-sample interaction each technique showslimitations concerning the obtained information As anexample SECM is an attractive technique utilizing micro-or submicroelectrodes for probing bioactivity based on re-dox chemistry However this technique is frequently lim-ited in lateral resolution in comparison with for example

STM which allows imaging of individual atoms and mol-ecules In contrast STM is limited to the investigation ofconducting substrates AFM with a sharp tip mounted on a exible cantilever probes mechanical properties and topo-logical features resulting in surface pro les (topography)with submolecular resolution The lack of information onthe surface chemistry obtained with nonmodi ed probeshas been a major drawback of this technique Since the in-troduction of tapping-mode imaging in 199415 AFM hasattracted remarkable attention for the investigation of softbiological samples Substantial progress in combiningAFM with surface chemistries for tip functionalizationhas added chemical speci city to the topographical infor-mation obtained Hence AFM has matured from a high-resolution imaging instrument to a device capable of de-tecting and quantifying single molecular forces underambient conditions Standard immobilization strategiesinclude the formation of functionalized thiol or silanemonolayers biotin-streptavidin interactions or dextranegel immobilization which are well-established techniquesderived from biosensor technology16 and are readilyadapted for tethering biological recognition elements tothe scanning probe tip Alternative to direct tip modi ca-tion the combination of complementary scanning probetechniques such as AFMNSOM AFMSTM andAFMSECM can provide extended information on the in-

Christine Kranz is a research scientist in the School of Chemistry and Biochemistry at the GeorgiaInstitute of Technology She received her BS degree from Ludwig Maximilian University of Munichand her PhD degree from the Technical University of Munich and was a postdoctoral fellow at theVienna University of Technology Her research interests include conducting polymers microelectro-chemistry atomic force microscopy thin- lm technology scanning electrochemical microscopybiosensors system miniaturization and integration based on micro- and nanofabrication and multi-functional scanning probe techniques

Boris Mizaikoff is an assistant professor in the School of Chemistry and Biochemistry at the GeorgiaInstitute of Technology He received his BS and PhD degrees from Vienna University ofTechnology and was a postdoctoral fellow at the University of Texas at Austin His research interestsinclude mid-infrared chemical sensors biosensors and biomimetic sensors chemical sensing withquantum cascade lasers surface-enhanced spectroscopic techniques deep-sea spectroscopic sensingchemometric data evaluation development of separation materials and sensing membranes based onmolecularly imprinted polymers and sol-gels system miniaturization and integration based on micro-and nanofabrication and multifunctional scanning probe techniques

Angelika Kueng is a postdoctoral fellow in the School of Chemistry and Biochemistry at the GeorgiaInstitute of Technology She received her BS and PhD degrees from the University of Vienna Herresearch interests include bioinorganic chemistry biosensors and multifunctional scanning probetechniques

vestigated sample surface chemistry As an example am-perometric biosensors based on common electrochemicaltransduction principles can be integrated into bifunctionalSECM-AFM probes providing laterally resolved infor-mation on bioactivity simultaneously with submolecularresolution in topography17

This review highlights selected recently developed prin-ciples and examples of cantilever integrated scanning bio-sensors and scanning electrochemical enzyme microelec-trodes

2 CANTILEVER-BASED SCANNINGBIOSENSORS

The atomic force microscope invented in 198618 has devel-oped into a routine tool for biological investigations19 20

The ability to operate in liquid environments and especiallyat physiological conditions makes AFM a versatile tool forstudying the native structure of biological material21ndash23

High lateral resolution at the nanoscale has been achievedduring investigation of topological changes at the surfacesof living cells24ndash26 Furthermore AFM has been used toinvestigate structural27ndash29 and mechanical30ndash32 properties ofindividual proteins and for real-time probing of protein-protein interactions33 Direct observation of enzyme activityhas been described by detecting height uctuations of thecantilever above the protein lysozyme adsorbed to a micasurface34 Excellent reviews on the application of AFM forbiological systems have recently been published19 20

21 Immobilizing Biorecognition Elements at Scanning Probe Tips

Chemical functionalization of AFM tips extends the infor-mation space obtained by conventional force microscopyEnhanced speci city is achieved by controlled modi ca-tion of the scanning probe tip and measurement of the in-teraction forces between the modi ed tip and the samplesurface Successful experiments require controlled tip sur-face modi cation ensuring rmly anchored biomolecularrecognition elements Although strong adhesion is requiredloss of catalytic enzyme activity induced by conforma-tional distortion during the immobilization process has tobe avoided Hence many coating strategies derive fromprocedures well established for transducer surface modi -cations utilized for biosensing devices Thiol chemistry atgold surfaces and silane chemistry at activated silicon sur-faces are common functionalization techniques for chemi-cal modi cation of cantilevers35 Alkyl thiols with func-tional terminal groups form self-assembled monolayers bychemisorption on gold sputtered cantilevers enabling sub-sequent attachment of receptor molecules at the SPM tipsurface Similarly silane monolayers can be formed at thesurface of silicon cantilevers which are activated by UVozone treatment resulting in a high density of reactive sur-face hydroxyl groups Alternatively lipid bilayers or poly-

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4 Sensor Lett Vol 1 No 1 2003

mers grafted to the SPM tip can be used for immobilizationof biomolecular recognition elements Detailed proceduresand strategies for chemical cantilever surface modi cationare reported elsewhere36

22 Force Spectroscopy and Imaging of MolecularRecognition Events

A schematic representation of the tip-substrate modi cationused for the determination of rupture forces of individualmolecules is given in Figure 1A Figure 1B schematicallydepicts the behavior of the resulting force curve in a forcespectroscopic experiment As long as the tip remains farfrom the surface no interaction forces are recorded (1)When the surface is approached in this idealized experimenta single biomolecule at the apex of the surface-modi edAFM tip interacts with a complementary molecule at the

Figure 1 (A) Schematic representation of the tip-substrate con gura-tion used in the initial studies on measurement of rupture forces of indi-vidual molecules The AFM probe is functionalized with a monolayer of themolecule of interest The substrate is modi ed with the counter-molecule(B) Scheme of a simulated single-molecule force-distance curve to deter-mine rupture force (1) The tip is moved toward the sample without inter-action (2) repulsive part of the force curve (3) scan direction is reversed(4) tip-sample contact is ruptured

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Sensor Lett Vol 1 No 1 2003 5

tip Lateral force mapping49ndash51 provides simultaneous infor-mation on topography and ligand-receptor interactionHowever both temporal and lateral resolution of this tech-nique is limited In contrast force-recognition imaging per-mitted by the combination of dynamic force microscopy52

with force spectroscopy allows determination of receptorsites with an accuracy at the nanometer scale and at scanvelocities comparable to conventional topography acquisi-tion modes53 An antibody for lysozyme was tethered to theAFM tip and used for imaging lysozyme molecules immo-bilized at a mica surface with high resolution utilizing amagnetically oscillated AFM mode (ldquoMacModerdquo MolecularImaging Phoenix AZ) The temporal progression of the ex-periment is shown in Figure 2A In Figure 2B the lysozymemolecules are clearly distorted owing to force recognitionWith the addition of excess lysozyme to the solution theantibody at the tip is blocked as indicated in Figure 2C and

sample surface (2) Upon retraction of the tip the restoringforce of the cantilever is initially balanced by the adhesiveforce (3) until the bond of the molecular pair is ruptured (4)The measured force at this point represents the ruptureforce of the bond formed between the recognition ele-ments Ideally localized detection of single molecularbonds between two individual molecules is enabled withthis technique The rst example of direct observation of asingle bond rupture of a distinct streptavidinndashbiotin com-plex was independently reported by the groups of Gaub37

and Colton38 in 1994 Extensive reviews on single-mole-cule force spectroscopy have recently been published39ndash42

Selected examples of single-molecule force spectroscopyusing a variety of receptor-ligand systems are highlightedin Table I

Inasmuch as structure-function relationships play a keyrole in the biosciences their simultaneous detection is apromising approach yielding enhanced insight into the regu-lation of cellular and other biological mechanisms Bindingof ligands to receptors is among the most important mecha-nisms in biological processes frequently triggering signal-ing pathways and cascades In molecular recognition forcemicroscopy (MRFM) the dynamics of the experiments arevaried yielding information on the molecular interactioncharacterizing ligand-receptor binding and dissociation Withthis method af nity constants rate constants and energybarriers have been estimated and the bond widths of bindingpockets have been determined43ndash46 Furthermore the op-portunity of mapping binding energy landscapes is pro-vided47 48 In MRFM receptor binding sites are localizedby molecular interaction with a ligand tethered to the AFM

Figure 2 Force-recognition imaging with (a) an antibody to lysozyme tethered to the AFM tip scanning in liquid over a surface of immobilizedlysozyme molecules A low concentration of lysozyme is added to solution As long as the antibody on the tip has not bound free lysozyme from solution(I) it is reacting with surface-bound lysozyme molecules resulting in distortion of the images due to recognition (visible in (b) and part of (c)) The blackarrow (image c) marks when the antibody on the tip has bound solution lysozyme and the image in part of (c) and all of (d) switches from recognition totopographic imaging Reprinted with permission from Ref 53 A Raab et al Nat Biotechnol 17 902 (1999) copy1999 Nature

Table I Overview of applications of single-molecule force microscopywith different receptor-ligand systems

Ligand-receptor interaction Reference

(Strept-)avidinbiotin 37 38 50 125ndash135Oligonucleotidecomplementary oligonucleotide 136ndash142Antibodyantigen 43ndash45 49 143ndash148Polysaccharides 149ndash154Acetylcholinesteraseacetylcholine 155Proteoglycans 156Selectinligand 157IntegrinRGD-sequences 158Vascular endothelial cathedrin dimers 159

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6 Sensor Lett Vol 1 No 1 2003

a clearer image of the lysozyme-decorated surface appearsand is ultimately completely restored in Figure 2D

Another example of molecular recognition of an antibodyby a membrane protein is shown in Figure 354 The antibodyis linked to the tip of the probe by a exible spacer using co-valent bonds Brush border membrane vesicles (BBMV) wereadsorbed to a gold surface and imaged with dynamic forcemicroscopy The AFM tip was modi ed with anti-g-GT anti-body against the antigen g-GT (g-glutamyltranspepidase)present at the BBMV At lateral positions where membranevesicles were identi ed the x-y scan was stopped and forcedistance cycles were recorded as shown in Figure 3A Whenthe antibody at the tip binds to an antigen at the surface an at-tractive force develops during further retraction since the tipis physically connected to the surface The attractive force de-velops in a delayed and non-linear fashion re ecting thestretching and the non-linear spring constant of the exibletether covalently coupling the antibody to the AFM tip Thissignal has a distinct shape and re ects the recognition event ofa single antibody-antigen pair43 44 During the control experi-ment in Figure 3B all g-GT sites at the BBMV surface areblocked by anti-g-GT antibody added to the solution Henceno recognition events are recorded

AFM has been used as a tool to simultaneously studymorphology changes and the presence of extracellularATP at living cells with the use of a biosensing layer im-mobilized on the cantilever55 Myosin-functionalized can-tilevers were used to quantitatively detect ATP in solutionduring AFM tapping-mode imaging of living cells Aschematic view of the measurement principle is given inFigure 4AndashC After ATP binding to the myosin subfrag-ment S1 immobilized at the AFM cantilever ATP hydrol-ysis occurs During hydrolysis a conformational changeof the enzyme occurs (in the dimensional range of approx10 nm)56 This conformational change disturbs the inter-action between the tip and the cell surface and causes ameasurable de ection (Figure 4D) Modi ed cantileverswere used to scan the surface of cultured cells demonstrat-ing that the CFTR1 cell line (S9) had a basal surface ATPconcentration which could be determined with AFM tipintegrated biosensors for the rst time

Very recently chemically modi ed probes were appliedto investigate the surface charges of living cells57 AFMmeasurements utilizing carboxyl-terminated tips were usedto probe the electrostatic properties and more speci callythe isoelectric point of microbial cell surfaces with high spa-tial resolution Functionalization of the surfaces was vali-dated by recording multiple force-distance curves between

Figure 4 (A) Scheme of an enzyme-modi ed (myosin subfragmentS1) AFM tip imaging the extracellular surface of a CFTR-expressing cellCl2 (black dots) and ATP (yellow dots) are released in the area adjacentto the CFTR protein (B) ATP binding and subsequent hydrolysis at thetip resulting in cantilever de ection (C) The tip remains de ectedalthough the point of ATP release is passed due to short contact timescompared with ATPase activity time (D left) One ldquohot spotrsquo of extracel-lular ATP The AFM tip rst dips into a localized ATP pool and thenmoves away from this area as the scan progresses As ATP hydrolysisoccurs a positive de ection of the tip is noted causing a continuous lineof activity (D right) Image recorded after incubation with 1024 Mglybenclamide a known inhibitor of CFTR Reduction in hot spots (ar-rows) indicates reduction of extracellular ATP concentration Reprintedwith permission from Ref 55 S W Schneider et al Proc Natl AcadSci USA 96 12180 (1999) copy1999 National Academy of Sciences of theUnited States of America

Figure 3 Dynamic force imaging of brush border membrane vesicles(BBMV) adsorbed to a gold surface (A) Force distance cycle for single an-tibody-antigen recognition event The modi ed tip is brought toward aBBMV (trace dotted line) and subsequently retracted (retraced solid line)at a constant lateral position Antibody-antigen binding is evident from adelayed and non-linear attractive force that develops during further retrac-tion (unbinding event) Inset shows force-distance cycle comprising tworecognition events separated in space and time (B) Control experiment inthe presence of 1 microM anti-g-GT antibody in solution The recognition sig-nal disappears and the traceretrace looks the same Reprinted with permis-sion from Ref 54 Wielert-Badt el al Biophys J 82 2767 (2002) copy2002The Biophysical Society

modi ed probes and model substrates Furthermore car-boxyl-terminated probes were used to record multipleforce-distance curves at various pHs at the surface ofSaccharomyces cerevisiae cells Adhesion force maps de-pendent on the pH were created by recording multiple (n 5

256) force-distance curves and calculating the adhesionforce for each force curve

To summarize force recognition imaging has developedinto a powerful tool for high-resolution studies of molecularrecognition events and for probing dynamic properties ofbiomolecules at physiological conditions Ongoing progressin instrumental development and improved recording tech-niques has continuously added to the relevance of forcerecognition imaging for in situ investigation of biologicalspecimens

3 SCANNING ELECTROCHEMICALMICROBIOSENSORS

The importance of amperometric biosensors is steadily in-creasing because of the combined advantages offered bythe selectivity of biological recognition elements and thewell-established principle of electrochemical transductionThe key element for the design of any sensor architectureis the immobilization of the speci c biological recognitionelement Common sensor architectures include enzymesimmobilized at the transducer surface In the case of anamperometric biosensor this surface usually resembles anoble metal or carbon electrode

31 Immobilizing Biorecognition Elements atMicroelectrodes

For control of the immobilization process during modi -cation of electrode surfaces reproducible procedures arepreferable especially for the development of miniaturizedbiosensors Most commonly applied electrode surface mod-i cation techniques include screen-printing techniques58 incombination with photoinduced cross-linking59 spin-coatingfollowed by photolithographic structuring and lift-off60 theelectrochemically induced formation of enzyme-containingpolymer lms using electrophoretic accumulation61 62 de-position of conducting polymers63ndash65 and covalent bindingof enzymes through functionalized thiol chemistry66 In par-ticular electrochemical techniques permit speci c modi ca-tion of electrode surfaces with high reproducibility Enzymeentrapment within electrochemically deposited polymer ma-trices is well described in the literature and allows directedmodi cation of electrode surfaces64 67 68 Recently success-ful immobilization based on electrochemically induced lo-calized precipitation of enzyme-containing polymer suspen-sions has been reported69 Most reports of applications ofamperometric biosensors describe accurate and reproducibledetermination of analytes in bulk experiments with only afew examples of scanning microbiosensors

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32 Biosensing with Scanning ElectrochemicalMicroscopy

Laterally resolved information at a submicrometer scalewas added to electroanalytical chemistry with the inventionof scanning electrochemical microscopy (SECM)70ndash72

This method provides spatially resolved electrochemicalinformation on interface processes by combining electro-analytical techniques with the principle of scanning a mi-croelectrode in close vicinity across the sample surfaceSECM is an in situ technique based on surface inducedchanges of a Faraday current which is measured at ascanned and biased ultramicroelectrode (UME) The hemi-spherical diffusion of a redox species in solution toward theUME is disturbed by the sample surface chemistry in closeproximity to the microelectrode Commonly imaging inSECM is achieved in feedback mode operation with redoxspecies arti cially added to the solution

The Faraday current measured at the UME is mainly in- uenced by the reactivity and morphology of the samplethe distance between tip and sample and the size andgeometry of the ultramicroelectrode when the electrode isin close proximity (a few electrode radii) to the sample sur-face In conventional SECM experiments the UME tip isscanned in constant height above the sample surface Inprinciple two modes of SECM are used for imaging appli-cations the feedback mode73 and the generationcollection(GC) mode74 Functional schemes of both SECM operationmodes are shown in Figure 5 The feedback mode is basedon the Faraday current resulting from the oxidation or re-duction of a mediator species at the UME tip with the appli-cation of a potential In bulk solution after reaching steadystate the Faraday current recorded at the UME remainsconstant (Figure 5A a) Approaching an insulating surfacewith the UME results in blocked hemispherical diffusion tothe active electrode surface (negative feedback) as shownin Figure 5A c In contrast approaching a conducting orelectrochemically active surface leads to positive feedbackdue to locally increased mediator concentration induced bysurface recycling effects (Figure 5A b) This technique iswidely used for investigating processes at solidliquid andliquidliquid interfaces75

Most investigations of biological samples are performedin the GC (substrate generationtip collection or tip gener-ationsubstrate collection) mode with the tip signal arisingfrom a species generated at the sample surface (Figure 5B)The tip acts as a sensor establishing concentration maps ofindividual electroactive species near the sample surface

In conventional SECM positioning of the microelectrodeis achieved by recording the Faradaic current measured atthe UME as the tip approaches the surface Usually theoret-ical calculations of approach curves for different electrodegeometries are tted to the experimentally obtained dataand used to determine the distance between the UME andthe sample surface76 However the current signal can beused only for amperometric electrodes Consequently non-

Sensor Lett Vol 1 No 1 2003 7

sitioning of the microelectrode in common operation modesis a major drawback of SECM in comparison with AFM orSTM83 Scanning the electrode in constant height across thesample surface results in a convolution of the electrochemi-cal response and the topographical information especially ifthe sample topography and the surface reactivity are un-known Furthermore risk of damage to the UME andor thesample surface increases with decreasing diameter of theSECM tip to the submicron scale or nanometer scale be-cause of the electrode size-dependent working distanceConsequently any progress toward improved lateral reso-lution and positioning of nonamperometric submicroelec-trodes and nanoelectrodes (eg micro- and nanobiosensors)has to address current independent positioning strategiesensuring precise control of the tip-sample distance

33 Positioning of Electrochemical Microbiosensorswith the Use of Shear Force-Based Constant-Distance Mode

In NSOM shear force-based positioning of optical ber tipswith the use of optical detection systems84 85 or nonopticaldetection based on a tuning fork resonator86 87 has beendescribed The rst successful integration of a shear force-based optical feedback mechanism into SECM using a ex-ible ( ber-shaped) vibrating UME was published in the mid-1990s88 Recently this technology has been adapted to avariety of microelectrodes89 A ber-shaped UME is vi-brated at its resonance frequency with a piezoelectric tubeused for agitation (Figure 6A) A laser beam is focused onthe tip of the UME creating a Fresnel diffraction patternwhich is projected onto a split photodiode The vibrationamplitude of the electrode is monitored by phase-sensitiveampli cation of the difference signal at the split photodiodewith respect to the agitation signal with the use of lock-intechniques Surface shear forces increase because of hydro-dynamic effects in close proximity to the sample90 and leadto amplitude damping and a phase shift of the vibrationWith this information a feedback loop with a de ned setpoint for amplitude damping can be integrated into theSECM software routine thereby maintaining a constant dis-tance between the tip and the sample surface during scan-ning across a sample with variable topography

An alternative method combining the shear force modebased on a tuning fork technique with SECM for currentindependent positioning of ber-shaped electrodes has beendescribed in the literature91ndash94 In these contributions theSECM tip is attached to a tuning fork resonance detectionsystem of a commercially available NSOM The UME is at-tached to the leg of a tuning fork driven by a piezoelectrictube At resonance the movement of the legs of the tuningfork generates an oscillating piezoelectric potential which isproportional to the amplitude of the oscillation Again thesynchronized signal derived with lock-in techniques is usedas the feedback signal by the NSOM controller Recently ahighly sensitive nonoptical shear force distance control for

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8 Sensor Lett Vol 1 No 1 2003

amperometric tips such as biosensors require alternative tippositioning strategies during SECM experiments Utilizingoptical microscopy for tip positioning yields only qualita-tive results77 Few approaches have been reported for theapplication of potentiometric UMEs78ndash81 and enzymeUMEs82 Horrocks et al78 used antimony SECM electrodesalso operating in amperometric mode for positioning of thetip prior to pH mapping Wei et al79 introduced positioningtechniques based on dc measurements of the solution resis-tance with AgAgCl micropipette electrodes or potentio-metric measurements of steady-state concentration pro lesusing ion-selective microelectrodes providing the tip-sample distance Furthermore potentiometric UMEs with adual-electrode con guration are reported where one chan-nel is con gured as an ion-selective electrode and the sec-ond channel as an amperometric electrode for distancecontrol during UME approach A hydrogen peroxide micro-biosensor was positioned by the application of a high-frequency alternating potential to the tip and measurementof the solution resistance between the tip and the auxiliaryelectrode82 Although good agreement of the experimentallyobtained data with theory has been achieved this approach islimited to micrometer-sized sensors because the relativecontribution of the solution resistance to the impedance de-creases with decreasing dimensions of the sensor

The lack of lateral resolution due to current dependent po-

Figure 5 Scheme of SECM operation modes (A) Feedback modewith normalized current-distance curve (a) UME in bulk solution(b) positive feedback mode due to mediator recycling at the sample sur-face (c) negative feedback mode due to blocked diffusion of the medi-ator to the UME (B) Generatorcollector mode (a) substrate genera-tiontip collection and (b) tip generationsubstrate collection mode

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Sensor Lett Vol 1 No 1 2003 9

SECM tips based on piezoelectric shear force distancecontrol was developed simplifying shear force-controlledconstant-distance SECM95

Shear force-based constant-distance SECM with opticaldetection has already been applied for positioning of micro-biosensors89 Glass capillaries (inner diam 10 mm) havebeen lled with a polymer hydrogel containing either glu-cose oxidase or glucose dehydrogenase The enzyme elec-trodes were positioned with the use of optical shear forcecontrol and scanned over a 50-mm Pt electrode in the case ofthe glucose oxidase biosensor and over a poly-(methyleneblue)-modi ed microelectrode in the case of the glucose-de-hydrogenase- lled capillary The involved reaction schemes(A) and the obtained SECM images of the reactive spots im-aged with the microbiosensors (B) are shown in Figure 7

To date few results have been published on nanoelec-trodes in SECM experiments High-resolution SECM imag-ing requires reproducible fabrication of nanoelectrodes bymicrofabrication techniques In addition it is still dif cult todetermine the exact distance between the electrode and thesample surface by SECM operation in constant-distance andconstant-height mode Distance calculations are frequentlybased on assumptions such as ideal electrode geometriesHowever these are dif cult to realize for nanoelectrodesfabricated by conventional techniques such as the use oflaser pipette pullers96 In turn quantitative studies on elec-trode processes require precise knowledge of the absolute

distance between tip and surface and demand reproduciblyfabricated electrodes and accurate control of a deliberatelyde ned electrode working distance

4 AFM TIP INTEGRATED ELECTRO-CHEMICAL MICROBIOSENSORS

Recent developments focused on the combination of AFMand SECM enable positioning of the UME independently ofthe current response during simultaneous high-resolutionAFM imaging AFM probes with integrated electrochemicalsensing capabilities provide a versatile tool with inherentcorrelation of structural information with (electro)chemicalsurface activity at excellent lateral resolution97

Figure 8 shows schematics of two different concepts forthe combination of AFM with SECM published by Mac-pherson et al98 99 and Kranz et al100ndash104 The rst approachof combined SECM-AFM imaging in liquid utilizes bentPt-wire nanoelectrodes shaped like AFM cantilevers (Figure8A and B)98 99 Although excellent spatial resolution forthe electrochemical response can be achieved imagingwith these cantilever-shaped electrodes in contact mode islimited to robust nonconducting samples because of thecontact of the cantilever-shaped nanoelectrode and the sam-ple surface Since the electroactive area is located at thevery end of the tip conducting samples have to be investi-gated by lift-mode imaging99 However this requires scan-ning the sample surface twice First the topographical in-formation is recorded in contact mode operation followedby a second scan using the recorded topographical informa-tion to keep the tip at a de ned distance from the surfacetracing the sample contours99

Figure 7 (A) Scheme of the generatorcollector mode detection prin-ciple using enzyme- lled ber-shaped tips (B) Identi cation of electro-active spots with enzyme- lled capillaries (a) Image of a 50-mm-diameterplatinum electrode obtained with a glucose oxidase-modi ed microcapil-lary and (b) image of a 50-mm poly(methylene blue)-modi ed platinumelectrode based on detection of NADH generated at the glucose dehydro-genase-modi ed capillary tip Reprinted with permission from Ref 89A Hengstenberg et al Chem Eur J 6 1547 (2000) copy2000 Wiley

Figure 6 Schematic representation of the different shear force detec-tion principles implemented in SECM for constant distance imaging(A) optical shear-force detection (B and C) Nonoptical shear-force detection

the very end of the original AFM tip Figure 8 shows aschematic cross section of an integrated AFM-SECM tip(C) and FIB images of an integrated frame submicroelec-trode (D E) Because of the resolution of the beam pro-vided by FIB systems integration of submicrometer- andnanoelectrodes can be realized100 103 111 although theelectrode size demonstrated with bent Pt wire electrodescould not yet be achieved So far disc-shaped integratednanoelectrodes with diameters down to 150 nm have beensuccessfully fabricated with the described technology112

Based on the described technology for the combinationof AFM and SECM SECM functionality can be integratedinto any standard AFM with only minor modi cations of(frequently) already existing AFM instrumentation Theelectrochemical response is inherently correlated with thetopographical information obtained by AFM imaging Thuscomplete separation of simultaneously obtained topo-graphical information and electrochemical information isachieved maintaining superior resolution of the topo-graphical information With this technique positioning ofnonamperometric electrodes down to the nanometer regimecan be realized

The application of integrated AFM-SECM tips fortapping-mode AFM operation has recently been demon-strated113 Despite modi cation during tip fabrication theresonance frequency of the unmodi ed silicon nitride can-tilever is not signi cantly shifted which has been con- rmed by comparing frequency spectra of unmodi ed andmodi ed cantilevers The oscillation amplitude of a fewnanometers is small compared with the diameter of theintegrated electrode (usually 300ndash800 nm) It has beenshown that the current response of the integrated electrodein SECM feedback mode is not significantly influencedby the oscillation amplitude of the combined SECM-AFMcantilever Electrochemical images recorded in tappingmode revealed current response and lateral resolutioncomparable to the electrochemical images obtained in con-tact mode113

AFM tip integrated electrodes have been applied forimaging enzyme activity in tapping-mode operation (Fig-ure 9)112 Enzyme activity of glucose oxidase (GOD)entrapped in a soft polymer matrix deposited inside thepores of a periodic micropattern has successfully beenimaged in AFM tapping mode and SECM generation-collection mode In the absence of glucose the currentrecorded at the electrode during AFM tapping-mode imag-ing of the enzyme containing polymer spots is negligible(Figure 9A topography B amplitude image) and revealsno electrochemical features (Figure 9C) In the presence ofglucose an increase in the current recorded at the inte-grated submicroelectrode is observed because of localizedproduction of H2O2 above the GOD-containing polymerspots (Figure 9F) The topography (Figure 9D) and ampli-tude (Figure 9E) images remain unaffected

The presented approach of integrated SECM-AFMprobes provides the opportunity to integrate and position

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10 Sensor Lett Vol 1 No 1 2003

Although several approaches of combined AFM-SECMusing such bent cantilever-shaped nanoelectrodes have beenpublished105ndash107 to date none of these reported measure-ments in AFM tapping mode are due to the variation inspring constant of these hand-made tips AFM tapping modeis a versatile technique for topographical imaging of softsample surfaces as encountered during investigation of forexample biological systems15 During tapping-mode opera-tion the probe gently taps the surface while resonating at ornear its resonance frequency A signi cantly reduced tip-sample contact time results in minimized frictional forcesavoiding damage of soft samples such as biological speciesor polymers108ndash110

An alternative approach combining AFM and SECMhas been described by our research group based on inte-gration of submicro- and nanoelectrodes into atomic forcemicroscopy tips with the use of microfabrication tech-niques (Figure 8CndashE)100ndash104 Electrodes with a variety ofwell-de ned geometries can be integrated into AFM tips atan exactly de ned distance above the apex of the AFM tipsimultaneously obtaining electrochemical information at aprecisely de ned working distance during AFM imagingDuring tip fabrication a conventional silicon nitride AFMcantilever is coated with an electroactive conducting layer(usually gold or platinum) and is subsequently insulatedwith materials such as Si3N4 or parylene C Three-dimen-sional focused ion beam (FIB) milling permits repro-ducible exposure of an electroactive area integrated above

Figure 8 Representation of combined AFM and SECM as publishedby Macpherson et al98 99 (A and B) and Kranz et al100 (CndashE)(A) Schematic of the AFM-SECM tip fabricated by coating etching andbending of Pt microwires Reprinted with permission from Ref 98 J VMacpherson and P R Unwin Anal Chem 72 276 (2000) copy2000American Chemical Society (B) Scanning electron micrograph of a can-tilever-shaped nanoelectrode Reprinted with permission from Ref 99 JV Macpherson and P R Unwin Anal Chem 73 550 (2001) copy2001American Chemical Society (C) Schematic cross section of an integratedAFM-SECM tip fabricated by microfabrication using FIB technology(dimensions are not to scale) (D) FIB image of an integrated frame sub-microelectrode (gold) with Parylene C insulation (electrode edge length770 nm) (E) Side view showing reshaped AFM-tip and tilt correction fora cantilever mounting angle of 10deg in a tapping-mode liquid cell

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micro- and nanobiosensors at a well-de ned and deliber-ately selected distance above the sample surface Standardsurface modi cation processes for biosensors as previ-ously outlined in this review can be used to immobilizebiological recognition elements at the surface of the inte-grated electrode (Figure 10) With the use of electrochemicaltechniques the site of immobilization is con ned to theelectrode surface providing highly localized and repro-ducible integration of a biosensing interface64 65 67 68

With the immobilization of enzymes via self-assembledthiol monolayers with reactive headgroups114 conversion

of electroactive and -inactive species generated at the sam-ple surface is enabled

Figure 11 illustrates simultaneously recorded topo-graphical and electrochemical images obtained with a tip-integrated biosensor The biosensor is based on horse-radish peroxidase immobilized at the electrode surface ofthe integrated SECM-AFM probe (Figure 11A) The per-oxidase activity was imaged in tip generatorsubstrate col-lector mode of SECM (see also Figure 11Bb) duringcontact-mode AFM operation The enzyme was immobi-lized by chemisorption of a functionalized thiol monolayer(cystaminium chloride) to the tip-integrated gold micro-electrode Peroxidase was attached by the addition of amixture of glutaraldehyde and peroxidase resulting in a

Figure 11 (A) Schematic presentation of the tip generationsubstratecollection mode experiment involving simultaneous detection of tipintegrated enzyme activity and AFM topography (B) Simultaneouslyrecorded topographical information and tip integrated peroxidase activityin contact mode The current measured at the microelectrode arrayre ects the enzyme activity integrated in the AFM tip Top view of theheight (a) and (c) and simultaneously recorded current (b) The electro-chemical response with 05 mmol L21 H2O2 in solution Images arerecorded in 2 mmol L21 FMA 01 mol L21 KCl in phosphate buffer (01mol L21 pH 70) (d) In the absence of the enzymatic substrate H2O2The tip was held at a potential of 005 V vs AgAgCl Electrode edgelength 860 nm tip height 410 nm scan rate 1 Hz

Figure 10 Schematic cross section of an AFM tip integrated biosensorEnzymes can be immobilized at the electrode surface integrated in thescanning probe tip via self-assembled thiol monolayers with functional-ized headgroups (A) or via electrochemical deposition of enzyme-containing polymer lms (B)

Figure 9 Simultaneously recorded height and current images of glu-cose oxidase activity in AFM tapping mode and SECM substrate genera-tiontip collection mode Top view of the height (a and d) correspondingamplitude (b and e) and simultaneously recorded current (c and f) imagesrecorded in air-saturated phosphate buffer in absence (andashc) and in thepresence (dndashf) of the substrate glucose in solution Reprinted with per-mission from Ref 112 A Kueng et al Angew Chem Int Ed 42 3237(2003) copy2003 Wiley

Sensor Lett Vol 1 No 1 2003 11

miniaturization of biosensors However only a few ap-proaches have been reported that use scanning micro- ornanobiosensors to obtain laterally resolved (bio)chemicalinformation Using shear force modes in combination withSECM permits positioning of nonamperometric sensorsImprovement of the achievable lateral resolution by de-creasing the dimensions of the applied sensor is the focusof ongoing research in this eld Fiber-based optical micro-biosensors in combination with NSOM techniques mightbe an alternative future concept for obtaining laterally re-solved information on bioactivity AFM tip-integrated bio-sensors provide a promising concept combining advantagesof various techniques in a single microfabricated probeHigh reproducibility of the fabrication process and the po-tential of miniaturization down to the nanometer scalemake them a versatile tool for future applications for prob-ing a wide range of biological substrates and processes

Among the major drawbacks of biosensors in general aresigni cantly increased response times of enzyme-modi edelectrodes compared with direct electrochemical detectionMany biologically relevant processes (eg exocytoticevents) relay signals in intervals of milliseconds Elec-trodes have been fabricated that can detect neurotransmit-ters with response times of 300 ms118 119 However re-sponse times less than 100 ms are needed to accuratelyquantify dynamics at the cellular level and less than 10 msto examine exocytosis of nonelectroactive neurotransmit-ters However it has been demonstrated that the temporalresolution achievable with biosensor systems scales in fa-vor of the proposed technology with device miniaturiza-tion120 121 As an example response times on the order of85 ms have been reported for a glucose-speci c biosensorwith an electrode diameter decreased to the submicronrange122 As recently demonstrated by our research groupdisc electrodes with diameters below 200 nm can be inte-grated into AFM tips by microfabrication techniques112 Itis expected that surface modi cation of such nanoelec-trodes as outlined in this contribution will result in a furtherdecrease in the integrated biosensor response time With arealistic perspective of measurements in the time domain ofmicroseconds suitability for investigation of biologicalprocesses is evident

With the development of multifunctional scanningprobes with tip-integrated biosensors a versatile analyticaltool for the investigation of complex biological systemshas been realized AFM cantilever-based biosensors pro-vide an excellent concept for simultaneous detection oftopographical and chemical information at the surface ofbiological species with high lateral resolution With theintegration of an electroactive area into AFM cantileversenabling subsequent surface modi cation and leading tobiosensor functionality an extension of this concept is pre-sented for detecting electroactive and -inactive speciesgenerated at the sample surface Accurate positioning andscanning of nanobiosensors at a de ned distance above thesample allows quanti cation of kinetic bioprocesses at the

surface-grafted cross-linked protein gel at the aminatedsurface of the self-assembled monolayer covering the inte-grated electrode The electrochemical signal generation isbased on the following enzyme catalyzed reactions(1) H2O2 is reduced to water by the oxidoreductase perox-idase and (2) a metal organic electron donor for the enzy-matic reaction (hydroxyl methyl ferrocene FMA contain-ing Fe21) added to the solution is oxidized during theenzymatic conversion to ferrocinium methylhydroxide(FMA1 containing Fe31) The electroactive by-product ofthe enzymatic reaction FMA1 diffuses to a microstruc-tured electrode array which was used as a model sampleThe microstructured electrode array is biased at a potentialof 50 mV vs AgAgCl At this potential only FMA1 andno other species in solution is reduced Hence informationon the local (electro)chemical activity (Figure 11Bb) withrespect to the position of the tip-integrated biosensor isprovided (Figure 11Ba topography) Comparison with thecontrol experiment in the absence of H2O2 (Figure 11B ctopography B d electrochemical image) demonstratesthe successful integration of a biosensor into a SECM-AFM probe and simultaneous activity imaging during AFMmeasurements17

The presented technique is by no means limited to theintegration of biosensors and can be extended to potentio-metric sensors such as pH microsensors or amalgam elec-trodes Many biological processes including enzymaticreactions biocorrosion and metabolic processes of micro-organisms involve or are driven by pH changes Hencesimultaneous AFM imaging and mapping of local pHmodulations is of substantial interest For example anti-mony and iridiumiridium oxide electrodes have beenreported for electrochemical pH measurements of biolog-ical systems115ndash117 As thin lms of these materials can besputtered the fabrication steps described above for the in-tegration of gold and platinum electrodes can be modi edand optimized to integrate pH-sensitive electrochemicalsensors into AFM tips

5 CONCLUSIONS AND OUTLOOK

Cantilever-based (bio)sensors are versatile analytical sys-tems for the investigation of biological species as they canbe used in solution and are capable of transducing a varietyof different signals Applications in force spectroscopypermit measurements at the level of single moleculesHowever the limiting factor of this technique for imagingof biomolecules is its comparatively poor temporal resolu-tion However ongoing instrumental developments andcombination of AFM with complementary techniques willovercome these limitations in the near future

Using enzymes as recognition elements in biosensorscombines several advantages including high speci city andselectivity Recently advanced immobilization strategiesand mass fabrication based on silicon technology allow

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12 Sensor Lett Vol 1 No 1 2003

25 J H Hoh and P K Hansma Trends Cell Biol 2 208 (1992)26 H Oberleithner G Giebisch and J Geibel P uumlgers Arch 425 506

(1993)27 J Mou S J Sheng R Ho and Z Shao Biophys J 71 2213

(1996)28 J Mou D M Czajkowsky S J Sheng R Ho and Z Shao FEBS

Lett 381 161 (1996)29 D J Muller and A Engel J Mol Biol 285 1347 (1999)30 F Oesterhelt D Oesterhelt M Pfeiffer A Engel H E Gaub and

D Muller Science 288 143 (2000)31 A F Oberhauser P E Marsyale M Carrion-Vazquez and J M

Fernandez Nat Struct Biol 6 1025 (1999)32 M Rief M Gautel F Oesterheit J M Fernandez and H E Gaub

Science 276 1109 (1997)33 M B Viani L I Pietrasanta J B Thompson A Chand I C

Gebeshuber J H Kindt M Richter H G Hansma and P KHansma Nat Struct Biol 7 644 (2000)

34 M Radmacher M Fritz H G Hansma and P K Hansma Science265 1577 (1994)

35 A Noy C D Frisbie L F Rozsnyai M S Wrighton and C MLieber J Am Chem Soc 117 7943 (1995)

36 A M Moulin S J OrsquoShea and M E Welland Ultramicroscopy82 23 (2000)

37 E-L Florin V T Moy and H E Gaub Science 264 415 (1994)38 G U Lee D A Kidwell and R J Colton Langmuir 10 354

(1994)39 A Janshoff M Neitzert Y Oberdoumlrfer and H Fuchs Angew

Chemie Int Ed 39 3212 (2000)40 O H Willemsen M M E Snel A Cambi J Greve B G De

Grooth and C G Figdor Biophys J 79 3267 (2000)41 M Carrion-Vazquez A F Oberhauser T E Fisher P E

Marszalek H Li and J M Fernandez Prog Biophys Mol Biol74 63 (2000)

42 J Zlatanova S M Lindsay and S H Leuba Prog Biophys MolBiol 74 37 (2000)

43 P Hinterdorfer W Baumgartner H J Gruber K Schilcher andH Schindler Proc Natl Acad Sci USA 93 3477 (1996)

44 P Hinterdorfer K Schilcher W Baumgartner H J Gruber andH Schindler Nanobiology 4 39 (1998)

45 R Ros F Schwesinger D Anselmetti M Kubon R SchaeferA Pluumlckthun and L Tiefenauer Proc Natl Acad Sci USA 957402 (1998)

46 F Schwesinger R Ros T Strunz D Anselmetti H-J GuumlntherodtA Honegger L Jermutus L Tiefenauer and A Pluumlckthun ProcNatl Acad Sci USA 97 9972 (2000)

47 R Merkel P Nassoy A Leung A Ritchie and K Evans Nature397 50 (1999)

48 T Strunz K Oroszlan I Schumakovitch H-J Guumlntherodt andM Hegner Biophys J 79 1206 (2000)

49 O H Willemsen M M E Snel K O van der Werf B G DeGrooth J Greve P Hinterdorfer H J Gruber H Schindler Y vanKooyk and C G Figdor Biophys J 75 2220 (1998)

50 M Ludwig W Dettmann and H E Gaub Biophys J 72 445(1997)

51 D P Allison P Hinterdorfer and W Han Curr Opin Biotechnol13 47 (2002)

52 W Han S M Lindsay and T Jing Appl Phys Lett 69 4111(1996)

53 A Raab W Han D Badt S J Smith-Gill S M LindsayH Schindler and P Hinterdorfer Nat Biotechnol 17 902 (1999)

54 G Schuumltz M Sonnleitner P Hinterdorfer and H Schindler MolMembr Biol 17 17 (2000)

55 S W Schneider M E Egan B P Jena W B GugginoH Oberleithner and J P Geibel Proc Natl Acad Sci USA 9612180 (1999)

56 J Finer R M Simmons and J A Spudnich Nature 368 113(1994)

sample surface with high lateral and temporal resolutionGiven the exibility of the described concepts there is con-siderable scope for extended use of multifunctional scan-ning probe systems probing a wide range of biologicalspecimens and processes

Acknowledgments The National Science Foundation(grant 0216368 in the program ldquoBiocomplexity in theEnvironmentrdquo) the National Institutes of Health (grantEB000508) and the ldquoFonds zur Foumlrderung der wissenschaft-lichen Forschungrdquo Austria (grants P14122-CHE and J2230)are greatly acknowledged PCT patents123 124on integratedAFM-SECM are issued

References and Notes

1 A Hulanicki S Glab and F Ingman Pure Appl Chem 63 1247(1991)

2 W Goepel T A Jones M Kleitz I Lundstroem and T SeiyamaEditors Chemical and Biochemical Sensors VCH Weinheim(1992) Vols 2 and 3

3 F W Scheller U Wollenberger A Warsinke and F Lisdat CurrOpin Biotechnol 12 35 (2001)

4 D R Thevenot K Toth R A Durst and G S Wilson BiosensBioelectron 16 121 (2001)

5 M Keusgen Naturwissenschaften 89 433 (2002)6 T H Park and M L Shuler Biotechnol Prog 19 243 (2003)7 K Sato A Hibara M Tokeshi H Hisamoto and T Kitamori Adv

Drug Delivery Rev 55 3791 (2003)8 P N Bartlett Handbook of Chemical and Biological Sensors

edited by R F Taylor Arthur D Little Inc USA J S SchultzUniversity of Pittsburgh (1996) pp 139ff

9 A Hierlemann O Brand C Hagleiter and H Baltes Proc IEEE91 839 (2003)

10 T E Fisher P E Marszalek A F Oberhauser M Carrion-Vazquez and J M Fernandez J Physiol (London) 520 5 (1999)

11 J K Gimzewski and C Joachim Science 283 1683 (1999)12 S O Vansteenkiste M C Davis C J Roberts S J B Tendler

and P M Williams Prog Surf Sci 57 95 (1998)13 M A Poggi L A Bottomley and P T Lillehei Anal Chem 74

2851 (2002)14 G Binnig H Rohrer C Gerber and E Weibel Appl Phys Lett

40 178 (1982)15 P K Hansma J P Cleveland M Radmacher D A Walters P E

Hillner M Bezanilla M Fritz D Vie H G Hansma C B PraterJ Massie L Fukunaga J Gurley and V Elings Appl Phys Lett64 1738 (1994)

16 W H Scouten J H T Luong and S R Brown Trends Biotechnol13 178 (1995)

17 C Kranz A Kueng A Lugstein E Bertagnolli and B Mizaikoffmanuscript in preparation

18 G Binnig C F Quate and C Gerber Phys Rev Lett 56 930 (1986)19 G Charras P Lehenkari and M Horton Methods Cell Biol 68

171 (2002)20 J H Kindt J C Sitko L I Pietrasanta E Oroudjev N Becker

M B Viani and H G Hansma Methods Cell Biol 68 213 (2002)21 B Drake C B Prater A L Weisenhorn S A C Gould T R

Albrecht C F Quate D S Cannell H G Hansma and P KHansma Science 243 1586 (1989)

22 D Rugar and P K Hansma Phys Today 43 23 (1990)23 M Radmacher R W Tillmann M Fritz and H E Gaub Science

157 1900 (1992)24 E Henderson P G Haydon and D S Sakaguchi Science 257

1944 (1992)

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Sensor Lett Vol 1 No 1 2003 13

vestigated sample surface chemistry As an example am-perometric biosensors based on common electrochemicaltransduction principles can be integrated into bifunctionalSECM-AFM probes providing laterally resolved infor-mation on bioactivity simultaneously with submolecularresolution in topography17

This review highlights selected recently developed prin-ciples and examples of cantilever integrated scanning bio-sensors and scanning electrochemical enzyme microelec-trodes

2 CANTILEVER-BASED SCANNINGBIOSENSORS

The atomic force microscope invented in 198618 has devel-oped into a routine tool for biological investigations19 20

The ability to operate in liquid environments and especiallyat physiological conditions makes AFM a versatile tool forstudying the native structure of biological material21ndash23

High lateral resolution at the nanoscale has been achievedduring investigation of topological changes at the surfacesof living cells24ndash26 Furthermore AFM has been used toinvestigate structural27ndash29 and mechanical30ndash32 properties ofindividual proteins and for real-time probing of protein-protein interactions33 Direct observation of enzyme activityhas been described by detecting height uctuations of thecantilever above the protein lysozyme adsorbed to a micasurface34 Excellent reviews on the application of AFM forbiological systems have recently been published19 20

21 Immobilizing Biorecognition Elements at Scanning Probe Tips

Chemical functionalization of AFM tips extends the infor-mation space obtained by conventional force microscopyEnhanced speci city is achieved by controlled modi ca-tion of the scanning probe tip and measurement of the in-teraction forces between the modi ed tip and the samplesurface Successful experiments require controlled tip sur-face modi cation ensuring rmly anchored biomolecularrecognition elements Although strong adhesion is requiredloss of catalytic enzyme activity induced by conforma-tional distortion during the immobilization process has tobe avoided Hence many coating strategies derive fromprocedures well established for transducer surface modi -cations utilized for biosensing devices Thiol chemistry atgold surfaces and silane chemistry at activated silicon sur-faces are common functionalization techniques for chemi-cal modi cation of cantilevers35 Alkyl thiols with func-tional terminal groups form self-assembled monolayers bychemisorption on gold sputtered cantilevers enabling sub-sequent attachment of receptor molecules at the SPM tipsurface Similarly silane monolayers can be formed at thesurface of silicon cantilevers which are activated by UVozone treatment resulting in a high density of reactive sur-face hydroxyl groups Alternatively lipid bilayers or poly-

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4 Sensor Lett Vol 1 No 1 2003

mers grafted to the SPM tip can be used for immobilizationof biomolecular recognition elements Detailed proceduresand strategies for chemical cantilever surface modi cationare reported elsewhere36

22 Force Spectroscopy and Imaging of MolecularRecognition Events

A schematic representation of the tip-substrate modi cationused for the determination of rupture forces of individualmolecules is given in Figure 1A Figure 1B schematicallydepicts the behavior of the resulting force curve in a forcespectroscopic experiment As long as the tip remains farfrom the surface no interaction forces are recorded (1)When the surface is approached in this idealized experimenta single biomolecule at the apex of the surface-modi edAFM tip interacts with a complementary molecule at the

Figure 1 (A) Schematic representation of the tip-substrate con gura-tion used in the initial studies on measurement of rupture forces of indi-vidual molecules The AFM probe is functionalized with a monolayer of themolecule of interest The substrate is modi ed with the counter-molecule(B) Scheme of a simulated single-molecule force-distance curve to deter-mine rupture force (1) The tip is moved toward the sample without inter-action (2) repulsive part of the force curve (3) scan direction is reversed(4) tip-sample contact is ruptured

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Sensor Lett Vol 1 No 1 2003 5

tip Lateral force mapping49ndash51 provides simultaneous infor-mation on topography and ligand-receptor interactionHowever both temporal and lateral resolution of this tech-nique is limited In contrast force-recognition imaging per-mitted by the combination of dynamic force microscopy52

with force spectroscopy allows determination of receptorsites with an accuracy at the nanometer scale and at scanvelocities comparable to conventional topography acquisi-tion modes53 An antibody for lysozyme was tethered to theAFM tip and used for imaging lysozyme molecules immo-bilized at a mica surface with high resolution utilizing amagnetically oscillated AFM mode (ldquoMacModerdquo MolecularImaging Phoenix AZ) The temporal progression of the ex-periment is shown in Figure 2A In Figure 2B the lysozymemolecules are clearly distorted owing to force recognitionWith the addition of excess lysozyme to the solution theantibody at the tip is blocked as indicated in Figure 2C and

sample surface (2) Upon retraction of the tip the restoringforce of the cantilever is initially balanced by the adhesiveforce (3) until the bond of the molecular pair is ruptured (4)The measured force at this point represents the ruptureforce of the bond formed between the recognition ele-ments Ideally localized detection of single molecularbonds between two individual molecules is enabled withthis technique The rst example of direct observation of asingle bond rupture of a distinct streptavidinndashbiotin com-plex was independently reported by the groups of Gaub37

and Colton38 in 1994 Extensive reviews on single-mole-cule force spectroscopy have recently been published39ndash42

Selected examples of single-molecule force spectroscopyusing a variety of receptor-ligand systems are highlightedin Table I

Inasmuch as structure-function relationships play a keyrole in the biosciences their simultaneous detection is apromising approach yielding enhanced insight into the regu-lation of cellular and other biological mechanisms Bindingof ligands to receptors is among the most important mecha-nisms in biological processes frequently triggering signal-ing pathways and cascades In molecular recognition forcemicroscopy (MRFM) the dynamics of the experiments arevaried yielding information on the molecular interactioncharacterizing ligand-receptor binding and dissociation Withthis method af nity constants rate constants and energybarriers have been estimated and the bond widths of bindingpockets have been determined43ndash46 Furthermore the op-portunity of mapping binding energy landscapes is pro-vided47 48 In MRFM receptor binding sites are localizedby molecular interaction with a ligand tethered to the AFM

Figure 2 Force-recognition imaging with (a) an antibody to lysozyme tethered to the AFM tip scanning in liquid over a surface of immobilizedlysozyme molecules A low concentration of lysozyme is added to solution As long as the antibody on the tip has not bound free lysozyme from solution(I) it is reacting with surface-bound lysozyme molecules resulting in distortion of the images due to recognition (visible in (b) and part of (c)) The blackarrow (image c) marks when the antibody on the tip has bound solution lysozyme and the image in part of (c) and all of (d) switches from recognition totopographic imaging Reprinted with permission from Ref 53 A Raab et al Nat Biotechnol 17 902 (1999) copy1999 Nature

Table I Overview of applications of single-molecule force microscopywith different receptor-ligand systems

Ligand-receptor interaction Reference

(Strept-)avidinbiotin 37 38 50 125ndash135Oligonucleotidecomplementary oligonucleotide 136ndash142Antibodyantigen 43ndash45 49 143ndash148Polysaccharides 149ndash154Acetylcholinesteraseacetylcholine 155Proteoglycans 156Selectinligand 157IntegrinRGD-sequences 158Vascular endothelial cathedrin dimers 159

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6 Sensor Lett Vol 1 No 1 2003

a clearer image of the lysozyme-decorated surface appearsand is ultimately completely restored in Figure 2D

Another example of molecular recognition of an antibodyby a membrane protein is shown in Figure 354 The antibodyis linked to the tip of the probe by a exible spacer using co-valent bonds Brush border membrane vesicles (BBMV) wereadsorbed to a gold surface and imaged with dynamic forcemicroscopy The AFM tip was modi ed with anti-g-GT anti-body against the antigen g-GT (g-glutamyltranspepidase)present at the BBMV At lateral positions where membranevesicles were identi ed the x-y scan was stopped and forcedistance cycles were recorded as shown in Figure 3A Whenthe antibody at the tip binds to an antigen at the surface an at-tractive force develops during further retraction since the tipis physically connected to the surface The attractive force de-velops in a delayed and non-linear fashion re ecting thestretching and the non-linear spring constant of the exibletether covalently coupling the antibody to the AFM tip Thissignal has a distinct shape and re ects the recognition event ofa single antibody-antigen pair43 44 During the control experi-ment in Figure 3B all g-GT sites at the BBMV surface areblocked by anti-g-GT antibody added to the solution Henceno recognition events are recorded

AFM has been used as a tool to simultaneously studymorphology changes and the presence of extracellularATP at living cells with the use of a biosensing layer im-mobilized on the cantilever55 Myosin-functionalized can-tilevers were used to quantitatively detect ATP in solutionduring AFM tapping-mode imaging of living cells Aschematic view of the measurement principle is given inFigure 4AndashC After ATP binding to the myosin subfrag-ment S1 immobilized at the AFM cantilever ATP hydrol-ysis occurs During hydrolysis a conformational changeof the enzyme occurs (in the dimensional range of approx10 nm)56 This conformational change disturbs the inter-action between the tip and the cell surface and causes ameasurable de ection (Figure 4D) Modi ed cantileverswere used to scan the surface of cultured cells demonstrat-ing that the CFTR1 cell line (S9) had a basal surface ATPconcentration which could be determined with AFM tipintegrated biosensors for the rst time

Very recently chemically modi ed probes were appliedto investigate the surface charges of living cells57 AFMmeasurements utilizing carboxyl-terminated tips were usedto probe the electrostatic properties and more speci callythe isoelectric point of microbial cell surfaces with high spa-tial resolution Functionalization of the surfaces was vali-dated by recording multiple force-distance curves between

Figure 4 (A) Scheme of an enzyme-modi ed (myosin subfragmentS1) AFM tip imaging the extracellular surface of a CFTR-expressing cellCl2 (black dots) and ATP (yellow dots) are released in the area adjacentto the CFTR protein (B) ATP binding and subsequent hydrolysis at thetip resulting in cantilever de ection (C) The tip remains de ectedalthough the point of ATP release is passed due to short contact timescompared with ATPase activity time (D left) One ldquohot spotrsquo of extracel-lular ATP The AFM tip rst dips into a localized ATP pool and thenmoves away from this area as the scan progresses As ATP hydrolysisoccurs a positive de ection of the tip is noted causing a continuous lineof activity (D right) Image recorded after incubation with 1024 Mglybenclamide a known inhibitor of CFTR Reduction in hot spots (ar-rows) indicates reduction of extracellular ATP concentration Reprintedwith permission from Ref 55 S W Schneider et al Proc Natl AcadSci USA 96 12180 (1999) copy1999 National Academy of Sciences of theUnited States of America

Figure 3 Dynamic force imaging of brush border membrane vesicles(BBMV) adsorbed to a gold surface (A) Force distance cycle for single an-tibody-antigen recognition event The modi ed tip is brought toward aBBMV (trace dotted line) and subsequently retracted (retraced solid line)at a constant lateral position Antibody-antigen binding is evident from adelayed and non-linear attractive force that develops during further retrac-tion (unbinding event) Inset shows force-distance cycle comprising tworecognition events separated in space and time (B) Control experiment inthe presence of 1 microM anti-g-GT antibody in solution The recognition sig-nal disappears and the traceretrace looks the same Reprinted with permis-sion from Ref 54 Wielert-Badt el al Biophys J 82 2767 (2002) copy2002The Biophysical Society

modi ed probes and model substrates Furthermore car-boxyl-terminated probes were used to record multipleforce-distance curves at various pHs at the surface ofSaccharomyces cerevisiae cells Adhesion force maps de-pendent on the pH were created by recording multiple (n 5

256) force-distance curves and calculating the adhesionforce for each force curve

To summarize force recognition imaging has developedinto a powerful tool for high-resolution studies of molecularrecognition events and for probing dynamic properties ofbiomolecules at physiological conditions Ongoing progressin instrumental development and improved recording tech-niques has continuously added to the relevance of forcerecognition imaging for in situ investigation of biologicalspecimens

3 SCANNING ELECTROCHEMICALMICROBIOSENSORS

The importance of amperometric biosensors is steadily in-creasing because of the combined advantages offered bythe selectivity of biological recognition elements and thewell-established principle of electrochemical transductionThe key element for the design of any sensor architectureis the immobilization of the speci c biological recognitionelement Common sensor architectures include enzymesimmobilized at the transducer surface In the case of anamperometric biosensor this surface usually resembles anoble metal or carbon electrode

31 Immobilizing Biorecognition Elements atMicroelectrodes

For control of the immobilization process during modi -cation of electrode surfaces reproducible procedures arepreferable especially for the development of miniaturizedbiosensors Most commonly applied electrode surface mod-i cation techniques include screen-printing techniques58 incombination with photoinduced cross-linking59 spin-coatingfollowed by photolithographic structuring and lift-off60 theelectrochemically induced formation of enzyme-containingpolymer lms using electrophoretic accumulation61 62 de-position of conducting polymers63ndash65 and covalent bindingof enzymes through functionalized thiol chemistry66 In par-ticular electrochemical techniques permit speci c modi ca-tion of electrode surfaces with high reproducibility Enzymeentrapment within electrochemically deposited polymer ma-trices is well described in the literature and allows directedmodi cation of electrode surfaces64 67 68 Recently success-ful immobilization based on electrochemically induced lo-calized precipitation of enzyme-containing polymer suspen-sions has been reported69 Most reports of applications ofamperometric biosensors describe accurate and reproducibledetermination of analytes in bulk experiments with only afew examples of scanning microbiosensors

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32 Biosensing with Scanning ElectrochemicalMicroscopy

Laterally resolved information at a submicrometer scalewas added to electroanalytical chemistry with the inventionof scanning electrochemical microscopy (SECM)70ndash72

This method provides spatially resolved electrochemicalinformation on interface processes by combining electro-analytical techniques with the principle of scanning a mi-croelectrode in close vicinity across the sample surfaceSECM is an in situ technique based on surface inducedchanges of a Faraday current which is measured at ascanned and biased ultramicroelectrode (UME) The hemi-spherical diffusion of a redox species in solution toward theUME is disturbed by the sample surface chemistry in closeproximity to the microelectrode Commonly imaging inSECM is achieved in feedback mode operation with redoxspecies arti cially added to the solution

The Faraday current measured at the UME is mainly in- uenced by the reactivity and morphology of the samplethe distance between tip and sample and the size andgeometry of the ultramicroelectrode when the electrode isin close proximity (a few electrode radii) to the sample sur-face In conventional SECM experiments the UME tip isscanned in constant height above the sample surface Inprinciple two modes of SECM are used for imaging appli-cations the feedback mode73 and the generationcollection(GC) mode74 Functional schemes of both SECM operationmodes are shown in Figure 5 The feedback mode is basedon the Faraday current resulting from the oxidation or re-duction of a mediator species at the UME tip with the appli-cation of a potential In bulk solution after reaching steadystate the Faraday current recorded at the UME remainsconstant (Figure 5A a) Approaching an insulating surfacewith the UME results in blocked hemispherical diffusion tothe active electrode surface (negative feedback) as shownin Figure 5A c In contrast approaching a conducting orelectrochemically active surface leads to positive feedbackdue to locally increased mediator concentration induced bysurface recycling effects (Figure 5A b) This technique iswidely used for investigating processes at solidliquid andliquidliquid interfaces75

Most investigations of biological samples are performedin the GC (substrate generationtip collection or tip gener-ationsubstrate collection) mode with the tip signal arisingfrom a species generated at the sample surface (Figure 5B)The tip acts as a sensor establishing concentration maps ofindividual electroactive species near the sample surface

In conventional SECM positioning of the microelectrodeis achieved by recording the Faradaic current measured atthe UME as the tip approaches the surface Usually theoret-ical calculations of approach curves for different electrodegeometries are tted to the experimentally obtained dataand used to determine the distance between the UME andthe sample surface76 However the current signal can beused only for amperometric electrodes Consequently non-

Sensor Lett Vol 1 No 1 2003 7

sitioning of the microelectrode in common operation modesis a major drawback of SECM in comparison with AFM orSTM83 Scanning the electrode in constant height across thesample surface results in a convolution of the electrochemi-cal response and the topographical information especially ifthe sample topography and the surface reactivity are un-known Furthermore risk of damage to the UME andor thesample surface increases with decreasing diameter of theSECM tip to the submicron scale or nanometer scale be-cause of the electrode size-dependent working distanceConsequently any progress toward improved lateral reso-lution and positioning of nonamperometric submicroelec-trodes and nanoelectrodes (eg micro- and nanobiosensors)has to address current independent positioning strategiesensuring precise control of the tip-sample distance

33 Positioning of Electrochemical Microbiosensorswith the Use of Shear Force-Based Constant-Distance Mode

In NSOM shear force-based positioning of optical ber tipswith the use of optical detection systems84 85 or nonopticaldetection based on a tuning fork resonator86 87 has beendescribed The rst successful integration of a shear force-based optical feedback mechanism into SECM using a ex-ible ( ber-shaped) vibrating UME was published in the mid-1990s88 Recently this technology has been adapted to avariety of microelectrodes89 A ber-shaped UME is vi-brated at its resonance frequency with a piezoelectric tubeused for agitation (Figure 6A) A laser beam is focused onthe tip of the UME creating a Fresnel diffraction patternwhich is projected onto a split photodiode The vibrationamplitude of the electrode is monitored by phase-sensitiveampli cation of the difference signal at the split photodiodewith respect to the agitation signal with the use of lock-intechniques Surface shear forces increase because of hydro-dynamic effects in close proximity to the sample90 and leadto amplitude damping and a phase shift of the vibrationWith this information a feedback loop with a de ned setpoint for amplitude damping can be integrated into theSECM software routine thereby maintaining a constant dis-tance between the tip and the sample surface during scan-ning across a sample with variable topography

An alternative method combining the shear force modebased on a tuning fork technique with SECM for currentindependent positioning of ber-shaped electrodes has beendescribed in the literature91ndash94 In these contributions theSECM tip is attached to a tuning fork resonance detectionsystem of a commercially available NSOM The UME is at-tached to the leg of a tuning fork driven by a piezoelectrictube At resonance the movement of the legs of the tuningfork generates an oscillating piezoelectric potential which isproportional to the amplitude of the oscillation Again thesynchronized signal derived with lock-in techniques is usedas the feedback signal by the NSOM controller Recently ahighly sensitive nonoptical shear force distance control for

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8 Sensor Lett Vol 1 No 1 2003

amperometric tips such as biosensors require alternative tippositioning strategies during SECM experiments Utilizingoptical microscopy for tip positioning yields only qualita-tive results77 Few approaches have been reported for theapplication of potentiometric UMEs78ndash81 and enzymeUMEs82 Horrocks et al78 used antimony SECM electrodesalso operating in amperometric mode for positioning of thetip prior to pH mapping Wei et al79 introduced positioningtechniques based on dc measurements of the solution resis-tance with AgAgCl micropipette electrodes or potentio-metric measurements of steady-state concentration pro lesusing ion-selective microelectrodes providing the tip-sample distance Furthermore potentiometric UMEs with adual-electrode con guration are reported where one chan-nel is con gured as an ion-selective electrode and the sec-ond channel as an amperometric electrode for distancecontrol during UME approach A hydrogen peroxide micro-biosensor was positioned by the application of a high-frequency alternating potential to the tip and measurementof the solution resistance between the tip and the auxiliaryelectrode82 Although good agreement of the experimentallyobtained data with theory has been achieved this approach islimited to micrometer-sized sensors because the relativecontribution of the solution resistance to the impedance de-creases with decreasing dimensions of the sensor

The lack of lateral resolution due to current dependent po-

Figure 5 Scheme of SECM operation modes (A) Feedback modewith normalized current-distance curve (a) UME in bulk solution(b) positive feedback mode due to mediator recycling at the sample sur-face (c) negative feedback mode due to blocked diffusion of the medi-ator to the UME (B) Generatorcollector mode (a) substrate genera-tiontip collection and (b) tip generationsubstrate collection mode

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Sensor Lett Vol 1 No 1 2003 9

SECM tips based on piezoelectric shear force distancecontrol was developed simplifying shear force-controlledconstant-distance SECM95

Shear force-based constant-distance SECM with opticaldetection has already been applied for positioning of micro-biosensors89 Glass capillaries (inner diam 10 mm) havebeen lled with a polymer hydrogel containing either glu-cose oxidase or glucose dehydrogenase The enzyme elec-trodes were positioned with the use of optical shear forcecontrol and scanned over a 50-mm Pt electrode in the case ofthe glucose oxidase biosensor and over a poly-(methyleneblue)-modi ed microelectrode in the case of the glucose-de-hydrogenase- lled capillary The involved reaction schemes(A) and the obtained SECM images of the reactive spots im-aged with the microbiosensors (B) are shown in Figure 7

To date few results have been published on nanoelec-trodes in SECM experiments High-resolution SECM imag-ing requires reproducible fabrication of nanoelectrodes bymicrofabrication techniques In addition it is still dif cult todetermine the exact distance between the electrode and thesample surface by SECM operation in constant-distance andconstant-height mode Distance calculations are frequentlybased on assumptions such as ideal electrode geometriesHowever these are dif cult to realize for nanoelectrodesfabricated by conventional techniques such as the use oflaser pipette pullers96 In turn quantitative studies on elec-trode processes require precise knowledge of the absolute

distance between tip and surface and demand reproduciblyfabricated electrodes and accurate control of a deliberatelyde ned electrode working distance

4 AFM TIP INTEGRATED ELECTRO-CHEMICAL MICROBIOSENSORS

Recent developments focused on the combination of AFMand SECM enable positioning of the UME independently ofthe current response during simultaneous high-resolutionAFM imaging AFM probes with integrated electrochemicalsensing capabilities provide a versatile tool with inherentcorrelation of structural information with (electro)chemicalsurface activity at excellent lateral resolution97

Figure 8 shows schematics of two different concepts forthe combination of AFM with SECM published by Mac-pherson et al98 99 and Kranz et al100ndash104 The rst approachof combined SECM-AFM imaging in liquid utilizes bentPt-wire nanoelectrodes shaped like AFM cantilevers (Figure8A and B)98 99 Although excellent spatial resolution forthe electrochemical response can be achieved imagingwith these cantilever-shaped electrodes in contact mode islimited to robust nonconducting samples because of thecontact of the cantilever-shaped nanoelectrode and the sam-ple surface Since the electroactive area is located at thevery end of the tip conducting samples have to be investi-gated by lift-mode imaging99 However this requires scan-ning the sample surface twice First the topographical in-formation is recorded in contact mode operation followedby a second scan using the recorded topographical informa-tion to keep the tip at a de ned distance from the surfacetracing the sample contours99

Figure 7 (A) Scheme of the generatorcollector mode detection prin-ciple using enzyme- lled ber-shaped tips (B) Identi cation of electro-active spots with enzyme- lled capillaries (a) Image of a 50-mm-diameterplatinum electrode obtained with a glucose oxidase-modi ed microcapil-lary and (b) image of a 50-mm poly(methylene blue)-modi ed platinumelectrode based on detection of NADH generated at the glucose dehydro-genase-modi ed capillary tip Reprinted with permission from Ref 89A Hengstenberg et al Chem Eur J 6 1547 (2000) copy2000 Wiley

Figure 6 Schematic representation of the different shear force detec-tion principles implemented in SECM for constant distance imaging(A) optical shear-force detection (B and C) Nonoptical shear-force detection

the very end of the original AFM tip Figure 8 shows aschematic cross section of an integrated AFM-SECM tip(C) and FIB images of an integrated frame submicroelec-trode (D E) Because of the resolution of the beam pro-vided by FIB systems integration of submicrometer- andnanoelectrodes can be realized100 103 111 although theelectrode size demonstrated with bent Pt wire electrodescould not yet be achieved So far disc-shaped integratednanoelectrodes with diameters down to 150 nm have beensuccessfully fabricated with the described technology112

Based on the described technology for the combinationof AFM and SECM SECM functionality can be integratedinto any standard AFM with only minor modi cations of(frequently) already existing AFM instrumentation Theelectrochemical response is inherently correlated with thetopographical information obtained by AFM imaging Thuscomplete separation of simultaneously obtained topo-graphical information and electrochemical information isachieved maintaining superior resolution of the topo-graphical information With this technique positioning ofnonamperometric electrodes down to the nanometer regimecan be realized

The application of integrated AFM-SECM tips fortapping-mode AFM operation has recently been demon-strated113 Despite modi cation during tip fabrication theresonance frequency of the unmodi ed silicon nitride can-tilever is not signi cantly shifted which has been con- rmed by comparing frequency spectra of unmodi ed andmodi ed cantilevers The oscillation amplitude of a fewnanometers is small compared with the diameter of theintegrated electrode (usually 300ndash800 nm) It has beenshown that the current response of the integrated electrodein SECM feedback mode is not significantly influencedby the oscillation amplitude of the combined SECM-AFMcantilever Electrochemical images recorded in tappingmode revealed current response and lateral resolutioncomparable to the electrochemical images obtained in con-tact mode113

AFM tip integrated electrodes have been applied forimaging enzyme activity in tapping-mode operation (Fig-ure 9)112 Enzyme activity of glucose oxidase (GOD)entrapped in a soft polymer matrix deposited inside thepores of a periodic micropattern has successfully beenimaged in AFM tapping mode and SECM generation-collection mode In the absence of glucose the currentrecorded at the electrode during AFM tapping-mode imag-ing of the enzyme containing polymer spots is negligible(Figure 9A topography B amplitude image) and revealsno electrochemical features (Figure 9C) In the presence ofglucose an increase in the current recorded at the inte-grated submicroelectrode is observed because of localizedproduction of H2O2 above the GOD-containing polymerspots (Figure 9F) The topography (Figure 9D) and ampli-tude (Figure 9E) images remain unaffected

The presented approach of integrated SECM-AFMprobes provides the opportunity to integrate and position

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10 Sensor Lett Vol 1 No 1 2003

Although several approaches of combined AFM-SECMusing such bent cantilever-shaped nanoelectrodes have beenpublished105ndash107 to date none of these reported measure-ments in AFM tapping mode are due to the variation inspring constant of these hand-made tips AFM tapping modeis a versatile technique for topographical imaging of softsample surfaces as encountered during investigation of forexample biological systems15 During tapping-mode opera-tion the probe gently taps the surface while resonating at ornear its resonance frequency A signi cantly reduced tip-sample contact time results in minimized frictional forcesavoiding damage of soft samples such as biological speciesor polymers108ndash110

An alternative approach combining AFM and SECMhas been described by our research group based on inte-gration of submicro- and nanoelectrodes into atomic forcemicroscopy tips with the use of microfabrication tech-niques (Figure 8CndashE)100ndash104 Electrodes with a variety ofwell-de ned geometries can be integrated into AFM tips atan exactly de ned distance above the apex of the AFM tipsimultaneously obtaining electrochemical information at aprecisely de ned working distance during AFM imagingDuring tip fabrication a conventional silicon nitride AFMcantilever is coated with an electroactive conducting layer(usually gold or platinum) and is subsequently insulatedwith materials such as Si3N4 or parylene C Three-dimen-sional focused ion beam (FIB) milling permits repro-ducible exposure of an electroactive area integrated above

Figure 8 Representation of combined AFM and SECM as publishedby Macpherson et al98 99 (A and B) and Kranz et al100 (CndashE)(A) Schematic of the AFM-SECM tip fabricated by coating etching andbending of Pt microwires Reprinted with permission from Ref 98 J VMacpherson and P R Unwin Anal Chem 72 276 (2000) copy2000American Chemical Society (B) Scanning electron micrograph of a can-tilever-shaped nanoelectrode Reprinted with permission from Ref 99 JV Macpherson and P R Unwin Anal Chem 73 550 (2001) copy2001American Chemical Society (C) Schematic cross section of an integratedAFM-SECM tip fabricated by microfabrication using FIB technology(dimensions are not to scale) (D) FIB image of an integrated frame sub-microelectrode (gold) with Parylene C insulation (electrode edge length770 nm) (E) Side view showing reshaped AFM-tip and tilt correction fora cantilever mounting angle of 10deg in a tapping-mode liquid cell

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micro- and nanobiosensors at a well-de ned and deliber-ately selected distance above the sample surface Standardsurface modi cation processes for biosensors as previ-ously outlined in this review can be used to immobilizebiological recognition elements at the surface of the inte-grated electrode (Figure 10) With the use of electrochemicaltechniques the site of immobilization is con ned to theelectrode surface providing highly localized and repro-ducible integration of a biosensing interface64 65 67 68

With the immobilization of enzymes via self-assembledthiol monolayers with reactive headgroups114 conversion

of electroactive and -inactive species generated at the sam-ple surface is enabled

Figure 11 illustrates simultaneously recorded topo-graphical and electrochemical images obtained with a tip-integrated biosensor The biosensor is based on horse-radish peroxidase immobilized at the electrode surface ofthe integrated SECM-AFM probe (Figure 11A) The per-oxidase activity was imaged in tip generatorsubstrate col-lector mode of SECM (see also Figure 11Bb) duringcontact-mode AFM operation The enzyme was immobi-lized by chemisorption of a functionalized thiol monolayer(cystaminium chloride) to the tip-integrated gold micro-electrode Peroxidase was attached by the addition of amixture of glutaraldehyde and peroxidase resulting in a

Figure 11 (A) Schematic presentation of the tip generationsubstratecollection mode experiment involving simultaneous detection of tipintegrated enzyme activity and AFM topography (B) Simultaneouslyrecorded topographical information and tip integrated peroxidase activityin contact mode The current measured at the microelectrode arrayre ects the enzyme activity integrated in the AFM tip Top view of theheight (a) and (c) and simultaneously recorded current (b) The electro-chemical response with 05 mmol L21 H2O2 in solution Images arerecorded in 2 mmol L21 FMA 01 mol L21 KCl in phosphate buffer (01mol L21 pH 70) (d) In the absence of the enzymatic substrate H2O2The tip was held at a potential of 005 V vs AgAgCl Electrode edgelength 860 nm tip height 410 nm scan rate 1 Hz

Figure 10 Schematic cross section of an AFM tip integrated biosensorEnzymes can be immobilized at the electrode surface integrated in thescanning probe tip via self-assembled thiol monolayers with functional-ized headgroups (A) or via electrochemical deposition of enzyme-containing polymer lms (B)

Figure 9 Simultaneously recorded height and current images of glu-cose oxidase activity in AFM tapping mode and SECM substrate genera-tiontip collection mode Top view of the height (a and d) correspondingamplitude (b and e) and simultaneously recorded current (c and f) imagesrecorded in air-saturated phosphate buffer in absence (andashc) and in thepresence (dndashf) of the substrate glucose in solution Reprinted with per-mission from Ref 112 A Kueng et al Angew Chem Int Ed 42 3237(2003) copy2003 Wiley

Sensor Lett Vol 1 No 1 2003 11

miniaturization of biosensors However only a few ap-proaches have been reported that use scanning micro- ornanobiosensors to obtain laterally resolved (bio)chemicalinformation Using shear force modes in combination withSECM permits positioning of nonamperometric sensorsImprovement of the achievable lateral resolution by de-creasing the dimensions of the applied sensor is the focusof ongoing research in this eld Fiber-based optical micro-biosensors in combination with NSOM techniques mightbe an alternative future concept for obtaining laterally re-solved information on bioactivity AFM tip-integrated bio-sensors provide a promising concept combining advantagesof various techniques in a single microfabricated probeHigh reproducibility of the fabrication process and the po-tential of miniaturization down to the nanometer scalemake them a versatile tool for future applications for prob-ing a wide range of biological substrates and processes

Among the major drawbacks of biosensors in general aresigni cantly increased response times of enzyme-modi edelectrodes compared with direct electrochemical detectionMany biologically relevant processes (eg exocytoticevents) relay signals in intervals of milliseconds Elec-trodes have been fabricated that can detect neurotransmit-ters with response times of 300 ms118 119 However re-sponse times less than 100 ms are needed to accuratelyquantify dynamics at the cellular level and less than 10 msto examine exocytosis of nonelectroactive neurotransmit-ters However it has been demonstrated that the temporalresolution achievable with biosensor systems scales in fa-vor of the proposed technology with device miniaturiza-tion120 121 As an example response times on the order of85 ms have been reported for a glucose-speci c biosensorwith an electrode diameter decreased to the submicronrange122 As recently demonstrated by our research groupdisc electrodes with diameters below 200 nm can be inte-grated into AFM tips by microfabrication techniques112 Itis expected that surface modi cation of such nanoelec-trodes as outlined in this contribution will result in a furtherdecrease in the integrated biosensor response time With arealistic perspective of measurements in the time domain ofmicroseconds suitability for investigation of biologicalprocesses is evident

With the development of multifunctional scanningprobes with tip-integrated biosensors a versatile analyticaltool for the investigation of complex biological systemshas been realized AFM cantilever-based biosensors pro-vide an excellent concept for simultaneous detection oftopographical and chemical information at the surface ofbiological species with high lateral resolution With theintegration of an electroactive area into AFM cantileversenabling subsequent surface modi cation and leading tobiosensor functionality an extension of this concept is pre-sented for detecting electroactive and -inactive speciesgenerated at the sample surface Accurate positioning andscanning of nanobiosensors at a de ned distance above thesample allows quanti cation of kinetic bioprocesses at the

surface-grafted cross-linked protein gel at the aminatedsurface of the self-assembled monolayer covering the inte-grated electrode The electrochemical signal generation isbased on the following enzyme catalyzed reactions(1) H2O2 is reduced to water by the oxidoreductase perox-idase and (2) a metal organic electron donor for the enzy-matic reaction (hydroxyl methyl ferrocene FMA contain-ing Fe21) added to the solution is oxidized during theenzymatic conversion to ferrocinium methylhydroxide(FMA1 containing Fe31) The electroactive by-product ofthe enzymatic reaction FMA1 diffuses to a microstruc-tured electrode array which was used as a model sampleThe microstructured electrode array is biased at a potentialof 50 mV vs AgAgCl At this potential only FMA1 andno other species in solution is reduced Hence informationon the local (electro)chemical activity (Figure 11Bb) withrespect to the position of the tip-integrated biosensor isprovided (Figure 11Ba topography) Comparison with thecontrol experiment in the absence of H2O2 (Figure 11B ctopography B d electrochemical image) demonstratesthe successful integration of a biosensor into a SECM-AFM probe and simultaneous activity imaging during AFMmeasurements17

The presented technique is by no means limited to theintegration of biosensors and can be extended to potentio-metric sensors such as pH microsensors or amalgam elec-trodes Many biological processes including enzymaticreactions biocorrosion and metabolic processes of micro-organisms involve or are driven by pH changes Hencesimultaneous AFM imaging and mapping of local pHmodulations is of substantial interest For example anti-mony and iridiumiridium oxide electrodes have beenreported for electrochemical pH measurements of biolog-ical systems115ndash117 As thin lms of these materials can besputtered the fabrication steps described above for the in-tegration of gold and platinum electrodes can be modi edand optimized to integrate pH-sensitive electrochemicalsensors into AFM tips

5 CONCLUSIONS AND OUTLOOK

Cantilever-based (bio)sensors are versatile analytical sys-tems for the investigation of biological species as they canbe used in solution and are capable of transducing a varietyof different signals Applications in force spectroscopypermit measurements at the level of single moleculesHowever the limiting factor of this technique for imagingof biomolecules is its comparatively poor temporal resolu-tion However ongoing instrumental developments andcombination of AFM with complementary techniques willovercome these limitations in the near future

Using enzymes as recognition elements in biosensorscombines several advantages including high speci city andselectivity Recently advanced immobilization strategiesand mass fabrication based on silicon technology allow

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12 Sensor Lett Vol 1 No 1 2003

25 J H Hoh and P K Hansma Trends Cell Biol 2 208 (1992)26 H Oberleithner G Giebisch and J Geibel P uumlgers Arch 425 506

(1993)27 J Mou S J Sheng R Ho and Z Shao Biophys J 71 2213

(1996)28 J Mou D M Czajkowsky S J Sheng R Ho and Z Shao FEBS

Lett 381 161 (1996)29 D J Muller and A Engel J Mol Biol 285 1347 (1999)30 F Oesterhelt D Oesterhelt M Pfeiffer A Engel H E Gaub and

D Muller Science 288 143 (2000)31 A F Oberhauser P E Marsyale M Carrion-Vazquez and J M

Fernandez Nat Struct Biol 6 1025 (1999)32 M Rief M Gautel F Oesterheit J M Fernandez and H E Gaub

Science 276 1109 (1997)33 M B Viani L I Pietrasanta J B Thompson A Chand I C

Gebeshuber J H Kindt M Richter H G Hansma and P KHansma Nat Struct Biol 7 644 (2000)

34 M Radmacher M Fritz H G Hansma and P K Hansma Science265 1577 (1994)

35 A Noy C D Frisbie L F Rozsnyai M S Wrighton and C MLieber J Am Chem Soc 117 7943 (1995)

36 A M Moulin S J OrsquoShea and M E Welland Ultramicroscopy82 23 (2000)

37 E-L Florin V T Moy and H E Gaub Science 264 415 (1994)38 G U Lee D A Kidwell and R J Colton Langmuir 10 354

(1994)39 A Janshoff M Neitzert Y Oberdoumlrfer and H Fuchs Angew

Chemie Int Ed 39 3212 (2000)40 O H Willemsen M M E Snel A Cambi J Greve B G De

Grooth and C G Figdor Biophys J 79 3267 (2000)41 M Carrion-Vazquez A F Oberhauser T E Fisher P E

Marszalek H Li and J M Fernandez Prog Biophys Mol Biol74 63 (2000)

42 J Zlatanova S M Lindsay and S H Leuba Prog Biophys MolBiol 74 37 (2000)

43 P Hinterdorfer W Baumgartner H J Gruber K Schilcher andH Schindler Proc Natl Acad Sci USA 93 3477 (1996)

44 P Hinterdorfer K Schilcher W Baumgartner H J Gruber andH Schindler Nanobiology 4 39 (1998)

45 R Ros F Schwesinger D Anselmetti M Kubon R SchaeferA Pluumlckthun and L Tiefenauer Proc Natl Acad Sci USA 957402 (1998)

46 F Schwesinger R Ros T Strunz D Anselmetti H-J GuumlntherodtA Honegger L Jermutus L Tiefenauer and A Pluumlckthun ProcNatl Acad Sci USA 97 9972 (2000)

47 R Merkel P Nassoy A Leung A Ritchie and K Evans Nature397 50 (1999)

48 T Strunz K Oroszlan I Schumakovitch H-J Guumlntherodt andM Hegner Biophys J 79 1206 (2000)

49 O H Willemsen M M E Snel K O van der Werf B G DeGrooth J Greve P Hinterdorfer H J Gruber H Schindler Y vanKooyk and C G Figdor Biophys J 75 2220 (1998)

50 M Ludwig W Dettmann and H E Gaub Biophys J 72 445(1997)

51 D P Allison P Hinterdorfer and W Han Curr Opin Biotechnol13 47 (2002)

52 W Han S M Lindsay and T Jing Appl Phys Lett 69 4111(1996)

53 A Raab W Han D Badt S J Smith-Gill S M LindsayH Schindler and P Hinterdorfer Nat Biotechnol 17 902 (1999)

54 G Schuumltz M Sonnleitner P Hinterdorfer and H Schindler MolMembr Biol 17 17 (2000)

55 S W Schneider M E Egan B P Jena W B GugginoH Oberleithner and J P Geibel Proc Natl Acad Sci USA 9612180 (1999)

56 J Finer R M Simmons and J A Spudnich Nature 368 113(1994)

sample surface with high lateral and temporal resolutionGiven the exibility of the described concepts there is con-siderable scope for extended use of multifunctional scan-ning probe systems probing a wide range of biologicalspecimens and processes

Acknowledgments The National Science Foundation(grant 0216368 in the program ldquoBiocomplexity in theEnvironmentrdquo) the National Institutes of Health (grantEB000508) and the ldquoFonds zur Foumlrderung der wissenschaft-lichen Forschungrdquo Austria (grants P14122-CHE and J2230)are greatly acknowledged PCT patents123 124on integratedAFM-SECM are issued

References and Notes

1 A Hulanicki S Glab and F Ingman Pure Appl Chem 63 1247(1991)

2 W Goepel T A Jones M Kleitz I Lundstroem and T SeiyamaEditors Chemical and Biochemical Sensors VCH Weinheim(1992) Vols 2 and 3

3 F W Scheller U Wollenberger A Warsinke and F Lisdat CurrOpin Biotechnol 12 35 (2001)

4 D R Thevenot K Toth R A Durst and G S Wilson BiosensBioelectron 16 121 (2001)

5 M Keusgen Naturwissenschaften 89 433 (2002)6 T H Park and M L Shuler Biotechnol Prog 19 243 (2003)7 K Sato A Hibara M Tokeshi H Hisamoto and T Kitamori Adv

Drug Delivery Rev 55 3791 (2003)8 P N Bartlett Handbook of Chemical and Biological Sensors

edited by R F Taylor Arthur D Little Inc USA J S SchultzUniversity of Pittsburgh (1996) pp 139ff

9 A Hierlemann O Brand C Hagleiter and H Baltes Proc IEEE91 839 (2003)

10 T E Fisher P E Marszalek A F Oberhauser M Carrion-Vazquez and J M Fernandez J Physiol (London) 520 5 (1999)

11 J K Gimzewski and C Joachim Science 283 1683 (1999)12 S O Vansteenkiste M C Davis C J Roberts S J B Tendler

and P M Williams Prog Surf Sci 57 95 (1998)13 M A Poggi L A Bottomley and P T Lillehei Anal Chem 74

2851 (2002)14 G Binnig H Rohrer C Gerber and E Weibel Appl Phys Lett

40 178 (1982)15 P K Hansma J P Cleveland M Radmacher D A Walters P E

Hillner M Bezanilla M Fritz D Vie H G Hansma C B PraterJ Massie L Fukunaga J Gurley and V Elings Appl Phys Lett64 1738 (1994)

16 W H Scouten J H T Luong and S R Brown Trends Biotechnol13 178 (1995)

17 C Kranz A Kueng A Lugstein E Bertagnolli and B Mizaikoffmanuscript in preparation

18 G Binnig C F Quate and C Gerber Phys Rev Lett 56 930 (1986)19 G Charras P Lehenkari and M Horton Methods Cell Biol 68

171 (2002)20 J H Kindt J C Sitko L I Pietrasanta E Oroudjev N Becker

M B Viani and H G Hansma Methods Cell Biol 68 213 (2002)21 B Drake C B Prater A L Weisenhorn S A C Gould T R

Albrecht C F Quate D S Cannell H G Hansma and P KHansma Science 243 1586 (1989)

22 D Rugar and P K Hansma Phys Today 43 23 (1990)23 M Radmacher R W Tillmann M Fritz and H E Gaub Science

157 1900 (1992)24 E Henderson P G Haydon and D S Sakaguchi Science 257

1944 (1992)

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Sensor Lett Vol 1 No 1 2003 13

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Sensor Lett Vol 1 No 1 2003 5

tip Lateral force mapping49ndash51 provides simultaneous infor-mation on topography and ligand-receptor interactionHowever both temporal and lateral resolution of this tech-nique is limited In contrast force-recognition imaging per-mitted by the combination of dynamic force microscopy52

with force spectroscopy allows determination of receptorsites with an accuracy at the nanometer scale and at scanvelocities comparable to conventional topography acquisi-tion modes53 An antibody for lysozyme was tethered to theAFM tip and used for imaging lysozyme molecules immo-bilized at a mica surface with high resolution utilizing amagnetically oscillated AFM mode (ldquoMacModerdquo MolecularImaging Phoenix AZ) The temporal progression of the ex-periment is shown in Figure 2A In Figure 2B the lysozymemolecules are clearly distorted owing to force recognitionWith the addition of excess lysozyme to the solution theantibody at the tip is blocked as indicated in Figure 2C and

sample surface (2) Upon retraction of the tip the restoringforce of the cantilever is initially balanced by the adhesiveforce (3) until the bond of the molecular pair is ruptured (4)The measured force at this point represents the ruptureforce of the bond formed between the recognition ele-ments Ideally localized detection of single molecularbonds between two individual molecules is enabled withthis technique The rst example of direct observation of asingle bond rupture of a distinct streptavidinndashbiotin com-plex was independently reported by the groups of Gaub37

and Colton38 in 1994 Extensive reviews on single-mole-cule force spectroscopy have recently been published39ndash42

Selected examples of single-molecule force spectroscopyusing a variety of receptor-ligand systems are highlightedin Table I

Inasmuch as structure-function relationships play a keyrole in the biosciences their simultaneous detection is apromising approach yielding enhanced insight into the regu-lation of cellular and other biological mechanisms Bindingof ligands to receptors is among the most important mecha-nisms in biological processes frequently triggering signal-ing pathways and cascades In molecular recognition forcemicroscopy (MRFM) the dynamics of the experiments arevaried yielding information on the molecular interactioncharacterizing ligand-receptor binding and dissociation Withthis method af nity constants rate constants and energybarriers have been estimated and the bond widths of bindingpockets have been determined43ndash46 Furthermore the op-portunity of mapping binding energy landscapes is pro-vided47 48 In MRFM receptor binding sites are localizedby molecular interaction with a ligand tethered to the AFM

Figure 2 Force-recognition imaging with (a) an antibody to lysozyme tethered to the AFM tip scanning in liquid over a surface of immobilizedlysozyme molecules A low concentration of lysozyme is added to solution As long as the antibody on the tip has not bound free lysozyme from solution(I) it is reacting with surface-bound lysozyme molecules resulting in distortion of the images due to recognition (visible in (b) and part of (c)) The blackarrow (image c) marks when the antibody on the tip has bound solution lysozyme and the image in part of (c) and all of (d) switches from recognition totopographic imaging Reprinted with permission from Ref 53 A Raab et al Nat Biotechnol 17 902 (1999) copy1999 Nature

Table I Overview of applications of single-molecule force microscopywith different receptor-ligand systems

Ligand-receptor interaction Reference

(Strept-)avidinbiotin 37 38 50 125ndash135Oligonucleotidecomplementary oligonucleotide 136ndash142Antibodyantigen 43ndash45 49 143ndash148Polysaccharides 149ndash154Acetylcholinesteraseacetylcholine 155Proteoglycans 156Selectinligand 157IntegrinRGD-sequences 158Vascular endothelial cathedrin dimers 159

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6 Sensor Lett Vol 1 No 1 2003

a clearer image of the lysozyme-decorated surface appearsand is ultimately completely restored in Figure 2D

Another example of molecular recognition of an antibodyby a membrane protein is shown in Figure 354 The antibodyis linked to the tip of the probe by a exible spacer using co-valent bonds Brush border membrane vesicles (BBMV) wereadsorbed to a gold surface and imaged with dynamic forcemicroscopy The AFM tip was modi ed with anti-g-GT anti-body against the antigen g-GT (g-glutamyltranspepidase)present at the BBMV At lateral positions where membranevesicles were identi ed the x-y scan was stopped and forcedistance cycles were recorded as shown in Figure 3A Whenthe antibody at the tip binds to an antigen at the surface an at-tractive force develops during further retraction since the tipis physically connected to the surface The attractive force de-velops in a delayed and non-linear fashion re ecting thestretching and the non-linear spring constant of the exibletether covalently coupling the antibody to the AFM tip Thissignal has a distinct shape and re ects the recognition event ofa single antibody-antigen pair43 44 During the control experi-ment in Figure 3B all g-GT sites at the BBMV surface areblocked by anti-g-GT antibody added to the solution Henceno recognition events are recorded

AFM has been used as a tool to simultaneously studymorphology changes and the presence of extracellularATP at living cells with the use of a biosensing layer im-mobilized on the cantilever55 Myosin-functionalized can-tilevers were used to quantitatively detect ATP in solutionduring AFM tapping-mode imaging of living cells Aschematic view of the measurement principle is given inFigure 4AndashC After ATP binding to the myosin subfrag-ment S1 immobilized at the AFM cantilever ATP hydrol-ysis occurs During hydrolysis a conformational changeof the enzyme occurs (in the dimensional range of approx10 nm)56 This conformational change disturbs the inter-action between the tip and the cell surface and causes ameasurable de ection (Figure 4D) Modi ed cantileverswere used to scan the surface of cultured cells demonstrat-ing that the CFTR1 cell line (S9) had a basal surface ATPconcentration which could be determined with AFM tipintegrated biosensors for the rst time

Very recently chemically modi ed probes were appliedto investigate the surface charges of living cells57 AFMmeasurements utilizing carboxyl-terminated tips were usedto probe the electrostatic properties and more speci callythe isoelectric point of microbial cell surfaces with high spa-tial resolution Functionalization of the surfaces was vali-dated by recording multiple force-distance curves between

Figure 4 (A) Scheme of an enzyme-modi ed (myosin subfragmentS1) AFM tip imaging the extracellular surface of a CFTR-expressing cellCl2 (black dots) and ATP (yellow dots) are released in the area adjacentto the CFTR protein (B) ATP binding and subsequent hydrolysis at thetip resulting in cantilever de ection (C) The tip remains de ectedalthough the point of ATP release is passed due to short contact timescompared with ATPase activity time (D left) One ldquohot spotrsquo of extracel-lular ATP The AFM tip rst dips into a localized ATP pool and thenmoves away from this area as the scan progresses As ATP hydrolysisoccurs a positive de ection of the tip is noted causing a continuous lineof activity (D right) Image recorded after incubation with 1024 Mglybenclamide a known inhibitor of CFTR Reduction in hot spots (ar-rows) indicates reduction of extracellular ATP concentration Reprintedwith permission from Ref 55 S W Schneider et al Proc Natl AcadSci USA 96 12180 (1999) copy1999 National Academy of Sciences of theUnited States of America

Figure 3 Dynamic force imaging of brush border membrane vesicles(BBMV) adsorbed to a gold surface (A) Force distance cycle for single an-tibody-antigen recognition event The modi ed tip is brought toward aBBMV (trace dotted line) and subsequently retracted (retraced solid line)at a constant lateral position Antibody-antigen binding is evident from adelayed and non-linear attractive force that develops during further retrac-tion (unbinding event) Inset shows force-distance cycle comprising tworecognition events separated in space and time (B) Control experiment inthe presence of 1 microM anti-g-GT antibody in solution The recognition sig-nal disappears and the traceretrace looks the same Reprinted with permis-sion from Ref 54 Wielert-Badt el al Biophys J 82 2767 (2002) copy2002The Biophysical Society

modi ed probes and model substrates Furthermore car-boxyl-terminated probes were used to record multipleforce-distance curves at various pHs at the surface ofSaccharomyces cerevisiae cells Adhesion force maps de-pendent on the pH were created by recording multiple (n 5

256) force-distance curves and calculating the adhesionforce for each force curve

To summarize force recognition imaging has developedinto a powerful tool for high-resolution studies of molecularrecognition events and for probing dynamic properties ofbiomolecules at physiological conditions Ongoing progressin instrumental development and improved recording tech-niques has continuously added to the relevance of forcerecognition imaging for in situ investigation of biologicalspecimens

3 SCANNING ELECTROCHEMICALMICROBIOSENSORS

The importance of amperometric biosensors is steadily in-creasing because of the combined advantages offered bythe selectivity of biological recognition elements and thewell-established principle of electrochemical transductionThe key element for the design of any sensor architectureis the immobilization of the speci c biological recognitionelement Common sensor architectures include enzymesimmobilized at the transducer surface In the case of anamperometric biosensor this surface usually resembles anoble metal or carbon electrode

31 Immobilizing Biorecognition Elements atMicroelectrodes

For control of the immobilization process during modi -cation of electrode surfaces reproducible procedures arepreferable especially for the development of miniaturizedbiosensors Most commonly applied electrode surface mod-i cation techniques include screen-printing techniques58 incombination with photoinduced cross-linking59 spin-coatingfollowed by photolithographic structuring and lift-off60 theelectrochemically induced formation of enzyme-containingpolymer lms using electrophoretic accumulation61 62 de-position of conducting polymers63ndash65 and covalent bindingof enzymes through functionalized thiol chemistry66 In par-ticular electrochemical techniques permit speci c modi ca-tion of electrode surfaces with high reproducibility Enzymeentrapment within electrochemically deposited polymer ma-trices is well described in the literature and allows directedmodi cation of electrode surfaces64 67 68 Recently success-ful immobilization based on electrochemically induced lo-calized precipitation of enzyme-containing polymer suspen-sions has been reported69 Most reports of applications ofamperometric biosensors describe accurate and reproducibledetermination of analytes in bulk experiments with only afew examples of scanning microbiosensors

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32 Biosensing with Scanning ElectrochemicalMicroscopy

Laterally resolved information at a submicrometer scalewas added to electroanalytical chemistry with the inventionof scanning electrochemical microscopy (SECM)70ndash72

This method provides spatially resolved electrochemicalinformation on interface processes by combining electro-analytical techniques with the principle of scanning a mi-croelectrode in close vicinity across the sample surfaceSECM is an in situ technique based on surface inducedchanges of a Faraday current which is measured at ascanned and biased ultramicroelectrode (UME) The hemi-spherical diffusion of a redox species in solution toward theUME is disturbed by the sample surface chemistry in closeproximity to the microelectrode Commonly imaging inSECM is achieved in feedback mode operation with redoxspecies arti cially added to the solution

The Faraday current measured at the UME is mainly in- uenced by the reactivity and morphology of the samplethe distance between tip and sample and the size andgeometry of the ultramicroelectrode when the electrode isin close proximity (a few electrode radii) to the sample sur-face In conventional SECM experiments the UME tip isscanned in constant height above the sample surface Inprinciple two modes of SECM are used for imaging appli-cations the feedback mode73 and the generationcollection(GC) mode74 Functional schemes of both SECM operationmodes are shown in Figure 5 The feedback mode is basedon the Faraday current resulting from the oxidation or re-duction of a mediator species at the UME tip with the appli-cation of a potential In bulk solution after reaching steadystate the Faraday current recorded at the UME remainsconstant (Figure 5A a) Approaching an insulating surfacewith the UME results in blocked hemispherical diffusion tothe active electrode surface (negative feedback) as shownin Figure 5A c In contrast approaching a conducting orelectrochemically active surface leads to positive feedbackdue to locally increased mediator concentration induced bysurface recycling effects (Figure 5A b) This technique iswidely used for investigating processes at solidliquid andliquidliquid interfaces75

Most investigations of biological samples are performedin the GC (substrate generationtip collection or tip gener-ationsubstrate collection) mode with the tip signal arisingfrom a species generated at the sample surface (Figure 5B)The tip acts as a sensor establishing concentration maps ofindividual electroactive species near the sample surface

In conventional SECM positioning of the microelectrodeis achieved by recording the Faradaic current measured atthe UME as the tip approaches the surface Usually theoret-ical calculations of approach curves for different electrodegeometries are tted to the experimentally obtained dataand used to determine the distance between the UME andthe sample surface76 However the current signal can beused only for amperometric electrodes Consequently non-

Sensor Lett Vol 1 No 1 2003 7

sitioning of the microelectrode in common operation modesis a major drawback of SECM in comparison with AFM orSTM83 Scanning the electrode in constant height across thesample surface results in a convolution of the electrochemi-cal response and the topographical information especially ifthe sample topography and the surface reactivity are un-known Furthermore risk of damage to the UME andor thesample surface increases with decreasing diameter of theSECM tip to the submicron scale or nanometer scale be-cause of the electrode size-dependent working distanceConsequently any progress toward improved lateral reso-lution and positioning of nonamperometric submicroelec-trodes and nanoelectrodes (eg micro- and nanobiosensors)has to address current independent positioning strategiesensuring precise control of the tip-sample distance

33 Positioning of Electrochemical Microbiosensorswith the Use of Shear Force-Based Constant-Distance Mode

In NSOM shear force-based positioning of optical ber tipswith the use of optical detection systems84 85 or nonopticaldetection based on a tuning fork resonator86 87 has beendescribed The rst successful integration of a shear force-based optical feedback mechanism into SECM using a ex-ible ( ber-shaped) vibrating UME was published in the mid-1990s88 Recently this technology has been adapted to avariety of microelectrodes89 A ber-shaped UME is vi-brated at its resonance frequency with a piezoelectric tubeused for agitation (Figure 6A) A laser beam is focused onthe tip of the UME creating a Fresnel diffraction patternwhich is projected onto a split photodiode The vibrationamplitude of the electrode is monitored by phase-sensitiveampli cation of the difference signal at the split photodiodewith respect to the agitation signal with the use of lock-intechniques Surface shear forces increase because of hydro-dynamic effects in close proximity to the sample90 and leadto amplitude damping and a phase shift of the vibrationWith this information a feedback loop with a de ned setpoint for amplitude damping can be integrated into theSECM software routine thereby maintaining a constant dis-tance between the tip and the sample surface during scan-ning across a sample with variable topography

An alternative method combining the shear force modebased on a tuning fork technique with SECM for currentindependent positioning of ber-shaped electrodes has beendescribed in the literature91ndash94 In these contributions theSECM tip is attached to a tuning fork resonance detectionsystem of a commercially available NSOM The UME is at-tached to the leg of a tuning fork driven by a piezoelectrictube At resonance the movement of the legs of the tuningfork generates an oscillating piezoelectric potential which isproportional to the amplitude of the oscillation Again thesynchronized signal derived with lock-in techniques is usedas the feedback signal by the NSOM controller Recently ahighly sensitive nonoptical shear force distance control for

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8 Sensor Lett Vol 1 No 1 2003

amperometric tips such as biosensors require alternative tippositioning strategies during SECM experiments Utilizingoptical microscopy for tip positioning yields only qualita-tive results77 Few approaches have been reported for theapplication of potentiometric UMEs78ndash81 and enzymeUMEs82 Horrocks et al78 used antimony SECM electrodesalso operating in amperometric mode for positioning of thetip prior to pH mapping Wei et al79 introduced positioningtechniques based on dc measurements of the solution resis-tance with AgAgCl micropipette electrodes or potentio-metric measurements of steady-state concentration pro lesusing ion-selective microelectrodes providing the tip-sample distance Furthermore potentiometric UMEs with adual-electrode con guration are reported where one chan-nel is con gured as an ion-selective electrode and the sec-ond channel as an amperometric electrode for distancecontrol during UME approach A hydrogen peroxide micro-biosensor was positioned by the application of a high-frequency alternating potential to the tip and measurementof the solution resistance between the tip and the auxiliaryelectrode82 Although good agreement of the experimentallyobtained data with theory has been achieved this approach islimited to micrometer-sized sensors because the relativecontribution of the solution resistance to the impedance de-creases with decreasing dimensions of the sensor

The lack of lateral resolution due to current dependent po-

Figure 5 Scheme of SECM operation modes (A) Feedback modewith normalized current-distance curve (a) UME in bulk solution(b) positive feedback mode due to mediator recycling at the sample sur-face (c) negative feedback mode due to blocked diffusion of the medi-ator to the UME (B) Generatorcollector mode (a) substrate genera-tiontip collection and (b) tip generationsubstrate collection mode

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Sensor Lett Vol 1 No 1 2003 9

SECM tips based on piezoelectric shear force distancecontrol was developed simplifying shear force-controlledconstant-distance SECM95

Shear force-based constant-distance SECM with opticaldetection has already been applied for positioning of micro-biosensors89 Glass capillaries (inner diam 10 mm) havebeen lled with a polymer hydrogel containing either glu-cose oxidase or glucose dehydrogenase The enzyme elec-trodes were positioned with the use of optical shear forcecontrol and scanned over a 50-mm Pt electrode in the case ofthe glucose oxidase biosensor and over a poly-(methyleneblue)-modi ed microelectrode in the case of the glucose-de-hydrogenase- lled capillary The involved reaction schemes(A) and the obtained SECM images of the reactive spots im-aged with the microbiosensors (B) are shown in Figure 7

To date few results have been published on nanoelec-trodes in SECM experiments High-resolution SECM imag-ing requires reproducible fabrication of nanoelectrodes bymicrofabrication techniques In addition it is still dif cult todetermine the exact distance between the electrode and thesample surface by SECM operation in constant-distance andconstant-height mode Distance calculations are frequentlybased on assumptions such as ideal electrode geometriesHowever these are dif cult to realize for nanoelectrodesfabricated by conventional techniques such as the use oflaser pipette pullers96 In turn quantitative studies on elec-trode processes require precise knowledge of the absolute

distance between tip and surface and demand reproduciblyfabricated electrodes and accurate control of a deliberatelyde ned electrode working distance

4 AFM TIP INTEGRATED ELECTRO-CHEMICAL MICROBIOSENSORS

Recent developments focused on the combination of AFMand SECM enable positioning of the UME independently ofthe current response during simultaneous high-resolutionAFM imaging AFM probes with integrated electrochemicalsensing capabilities provide a versatile tool with inherentcorrelation of structural information with (electro)chemicalsurface activity at excellent lateral resolution97

Figure 8 shows schematics of two different concepts forthe combination of AFM with SECM published by Mac-pherson et al98 99 and Kranz et al100ndash104 The rst approachof combined SECM-AFM imaging in liquid utilizes bentPt-wire nanoelectrodes shaped like AFM cantilevers (Figure8A and B)98 99 Although excellent spatial resolution forthe electrochemical response can be achieved imagingwith these cantilever-shaped electrodes in contact mode islimited to robust nonconducting samples because of thecontact of the cantilever-shaped nanoelectrode and the sam-ple surface Since the electroactive area is located at thevery end of the tip conducting samples have to be investi-gated by lift-mode imaging99 However this requires scan-ning the sample surface twice First the topographical in-formation is recorded in contact mode operation followedby a second scan using the recorded topographical informa-tion to keep the tip at a de ned distance from the surfacetracing the sample contours99

Figure 7 (A) Scheme of the generatorcollector mode detection prin-ciple using enzyme- lled ber-shaped tips (B) Identi cation of electro-active spots with enzyme- lled capillaries (a) Image of a 50-mm-diameterplatinum electrode obtained with a glucose oxidase-modi ed microcapil-lary and (b) image of a 50-mm poly(methylene blue)-modi ed platinumelectrode based on detection of NADH generated at the glucose dehydro-genase-modi ed capillary tip Reprinted with permission from Ref 89A Hengstenberg et al Chem Eur J 6 1547 (2000) copy2000 Wiley

Figure 6 Schematic representation of the different shear force detec-tion principles implemented in SECM for constant distance imaging(A) optical shear-force detection (B and C) Nonoptical shear-force detection

the very end of the original AFM tip Figure 8 shows aschematic cross section of an integrated AFM-SECM tip(C) and FIB images of an integrated frame submicroelec-trode (D E) Because of the resolution of the beam pro-vided by FIB systems integration of submicrometer- andnanoelectrodes can be realized100 103 111 although theelectrode size demonstrated with bent Pt wire electrodescould not yet be achieved So far disc-shaped integratednanoelectrodes with diameters down to 150 nm have beensuccessfully fabricated with the described technology112

Based on the described technology for the combinationof AFM and SECM SECM functionality can be integratedinto any standard AFM with only minor modi cations of(frequently) already existing AFM instrumentation Theelectrochemical response is inherently correlated with thetopographical information obtained by AFM imaging Thuscomplete separation of simultaneously obtained topo-graphical information and electrochemical information isachieved maintaining superior resolution of the topo-graphical information With this technique positioning ofnonamperometric electrodes down to the nanometer regimecan be realized

The application of integrated AFM-SECM tips fortapping-mode AFM operation has recently been demon-strated113 Despite modi cation during tip fabrication theresonance frequency of the unmodi ed silicon nitride can-tilever is not signi cantly shifted which has been con- rmed by comparing frequency spectra of unmodi ed andmodi ed cantilevers The oscillation amplitude of a fewnanometers is small compared with the diameter of theintegrated electrode (usually 300ndash800 nm) It has beenshown that the current response of the integrated electrodein SECM feedback mode is not significantly influencedby the oscillation amplitude of the combined SECM-AFMcantilever Electrochemical images recorded in tappingmode revealed current response and lateral resolutioncomparable to the electrochemical images obtained in con-tact mode113

AFM tip integrated electrodes have been applied forimaging enzyme activity in tapping-mode operation (Fig-ure 9)112 Enzyme activity of glucose oxidase (GOD)entrapped in a soft polymer matrix deposited inside thepores of a periodic micropattern has successfully beenimaged in AFM tapping mode and SECM generation-collection mode In the absence of glucose the currentrecorded at the electrode during AFM tapping-mode imag-ing of the enzyme containing polymer spots is negligible(Figure 9A topography B amplitude image) and revealsno electrochemical features (Figure 9C) In the presence ofglucose an increase in the current recorded at the inte-grated submicroelectrode is observed because of localizedproduction of H2O2 above the GOD-containing polymerspots (Figure 9F) The topography (Figure 9D) and ampli-tude (Figure 9E) images remain unaffected

The presented approach of integrated SECM-AFMprobes provides the opportunity to integrate and position

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10 Sensor Lett Vol 1 No 1 2003

Although several approaches of combined AFM-SECMusing such bent cantilever-shaped nanoelectrodes have beenpublished105ndash107 to date none of these reported measure-ments in AFM tapping mode are due to the variation inspring constant of these hand-made tips AFM tapping modeis a versatile technique for topographical imaging of softsample surfaces as encountered during investigation of forexample biological systems15 During tapping-mode opera-tion the probe gently taps the surface while resonating at ornear its resonance frequency A signi cantly reduced tip-sample contact time results in minimized frictional forcesavoiding damage of soft samples such as biological speciesor polymers108ndash110

An alternative approach combining AFM and SECMhas been described by our research group based on inte-gration of submicro- and nanoelectrodes into atomic forcemicroscopy tips with the use of microfabrication tech-niques (Figure 8CndashE)100ndash104 Electrodes with a variety ofwell-de ned geometries can be integrated into AFM tips atan exactly de ned distance above the apex of the AFM tipsimultaneously obtaining electrochemical information at aprecisely de ned working distance during AFM imagingDuring tip fabrication a conventional silicon nitride AFMcantilever is coated with an electroactive conducting layer(usually gold or platinum) and is subsequently insulatedwith materials such as Si3N4 or parylene C Three-dimen-sional focused ion beam (FIB) milling permits repro-ducible exposure of an electroactive area integrated above

Figure 8 Representation of combined AFM and SECM as publishedby Macpherson et al98 99 (A and B) and Kranz et al100 (CndashE)(A) Schematic of the AFM-SECM tip fabricated by coating etching andbending of Pt microwires Reprinted with permission from Ref 98 J VMacpherson and P R Unwin Anal Chem 72 276 (2000) copy2000American Chemical Society (B) Scanning electron micrograph of a can-tilever-shaped nanoelectrode Reprinted with permission from Ref 99 JV Macpherson and P R Unwin Anal Chem 73 550 (2001) copy2001American Chemical Society (C) Schematic cross section of an integratedAFM-SECM tip fabricated by microfabrication using FIB technology(dimensions are not to scale) (D) FIB image of an integrated frame sub-microelectrode (gold) with Parylene C insulation (electrode edge length770 nm) (E) Side view showing reshaped AFM-tip and tilt correction fora cantilever mounting angle of 10deg in a tapping-mode liquid cell

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micro- and nanobiosensors at a well-de ned and deliber-ately selected distance above the sample surface Standardsurface modi cation processes for biosensors as previ-ously outlined in this review can be used to immobilizebiological recognition elements at the surface of the inte-grated electrode (Figure 10) With the use of electrochemicaltechniques the site of immobilization is con ned to theelectrode surface providing highly localized and repro-ducible integration of a biosensing interface64 65 67 68

With the immobilization of enzymes via self-assembledthiol monolayers with reactive headgroups114 conversion

of electroactive and -inactive species generated at the sam-ple surface is enabled

Figure 11 illustrates simultaneously recorded topo-graphical and electrochemical images obtained with a tip-integrated biosensor The biosensor is based on horse-radish peroxidase immobilized at the electrode surface ofthe integrated SECM-AFM probe (Figure 11A) The per-oxidase activity was imaged in tip generatorsubstrate col-lector mode of SECM (see also Figure 11Bb) duringcontact-mode AFM operation The enzyme was immobi-lized by chemisorption of a functionalized thiol monolayer(cystaminium chloride) to the tip-integrated gold micro-electrode Peroxidase was attached by the addition of amixture of glutaraldehyde and peroxidase resulting in a

Figure 11 (A) Schematic presentation of the tip generationsubstratecollection mode experiment involving simultaneous detection of tipintegrated enzyme activity and AFM topography (B) Simultaneouslyrecorded topographical information and tip integrated peroxidase activityin contact mode The current measured at the microelectrode arrayre ects the enzyme activity integrated in the AFM tip Top view of theheight (a) and (c) and simultaneously recorded current (b) The electro-chemical response with 05 mmol L21 H2O2 in solution Images arerecorded in 2 mmol L21 FMA 01 mol L21 KCl in phosphate buffer (01mol L21 pH 70) (d) In the absence of the enzymatic substrate H2O2The tip was held at a potential of 005 V vs AgAgCl Electrode edgelength 860 nm tip height 410 nm scan rate 1 Hz

Figure 10 Schematic cross section of an AFM tip integrated biosensorEnzymes can be immobilized at the electrode surface integrated in thescanning probe tip via self-assembled thiol monolayers with functional-ized headgroups (A) or via electrochemical deposition of enzyme-containing polymer lms (B)

Figure 9 Simultaneously recorded height and current images of glu-cose oxidase activity in AFM tapping mode and SECM substrate genera-tiontip collection mode Top view of the height (a and d) correspondingamplitude (b and e) and simultaneously recorded current (c and f) imagesrecorded in air-saturated phosphate buffer in absence (andashc) and in thepresence (dndashf) of the substrate glucose in solution Reprinted with per-mission from Ref 112 A Kueng et al Angew Chem Int Ed 42 3237(2003) copy2003 Wiley

Sensor Lett Vol 1 No 1 2003 11

miniaturization of biosensors However only a few ap-proaches have been reported that use scanning micro- ornanobiosensors to obtain laterally resolved (bio)chemicalinformation Using shear force modes in combination withSECM permits positioning of nonamperometric sensorsImprovement of the achievable lateral resolution by de-creasing the dimensions of the applied sensor is the focusof ongoing research in this eld Fiber-based optical micro-biosensors in combination with NSOM techniques mightbe an alternative future concept for obtaining laterally re-solved information on bioactivity AFM tip-integrated bio-sensors provide a promising concept combining advantagesof various techniques in a single microfabricated probeHigh reproducibility of the fabrication process and the po-tential of miniaturization down to the nanometer scalemake them a versatile tool for future applications for prob-ing a wide range of biological substrates and processes

Among the major drawbacks of biosensors in general aresigni cantly increased response times of enzyme-modi edelectrodes compared with direct electrochemical detectionMany biologically relevant processes (eg exocytoticevents) relay signals in intervals of milliseconds Elec-trodes have been fabricated that can detect neurotransmit-ters with response times of 300 ms118 119 However re-sponse times less than 100 ms are needed to accuratelyquantify dynamics at the cellular level and less than 10 msto examine exocytosis of nonelectroactive neurotransmit-ters However it has been demonstrated that the temporalresolution achievable with biosensor systems scales in fa-vor of the proposed technology with device miniaturiza-tion120 121 As an example response times on the order of85 ms have been reported for a glucose-speci c biosensorwith an electrode diameter decreased to the submicronrange122 As recently demonstrated by our research groupdisc electrodes with diameters below 200 nm can be inte-grated into AFM tips by microfabrication techniques112 Itis expected that surface modi cation of such nanoelec-trodes as outlined in this contribution will result in a furtherdecrease in the integrated biosensor response time With arealistic perspective of measurements in the time domain ofmicroseconds suitability for investigation of biologicalprocesses is evident

With the development of multifunctional scanningprobes with tip-integrated biosensors a versatile analyticaltool for the investigation of complex biological systemshas been realized AFM cantilever-based biosensors pro-vide an excellent concept for simultaneous detection oftopographical and chemical information at the surface ofbiological species with high lateral resolution With theintegration of an electroactive area into AFM cantileversenabling subsequent surface modi cation and leading tobiosensor functionality an extension of this concept is pre-sented for detecting electroactive and -inactive speciesgenerated at the sample surface Accurate positioning andscanning of nanobiosensors at a de ned distance above thesample allows quanti cation of kinetic bioprocesses at the

surface-grafted cross-linked protein gel at the aminatedsurface of the self-assembled monolayer covering the inte-grated electrode The electrochemical signal generation isbased on the following enzyme catalyzed reactions(1) H2O2 is reduced to water by the oxidoreductase perox-idase and (2) a metal organic electron donor for the enzy-matic reaction (hydroxyl methyl ferrocene FMA contain-ing Fe21) added to the solution is oxidized during theenzymatic conversion to ferrocinium methylhydroxide(FMA1 containing Fe31) The electroactive by-product ofthe enzymatic reaction FMA1 diffuses to a microstruc-tured electrode array which was used as a model sampleThe microstructured electrode array is biased at a potentialof 50 mV vs AgAgCl At this potential only FMA1 andno other species in solution is reduced Hence informationon the local (electro)chemical activity (Figure 11Bb) withrespect to the position of the tip-integrated biosensor isprovided (Figure 11Ba topography) Comparison with thecontrol experiment in the absence of H2O2 (Figure 11B ctopography B d electrochemical image) demonstratesthe successful integration of a biosensor into a SECM-AFM probe and simultaneous activity imaging during AFMmeasurements17

The presented technique is by no means limited to theintegration of biosensors and can be extended to potentio-metric sensors such as pH microsensors or amalgam elec-trodes Many biological processes including enzymaticreactions biocorrosion and metabolic processes of micro-organisms involve or are driven by pH changes Hencesimultaneous AFM imaging and mapping of local pHmodulations is of substantial interest For example anti-mony and iridiumiridium oxide electrodes have beenreported for electrochemical pH measurements of biolog-ical systems115ndash117 As thin lms of these materials can besputtered the fabrication steps described above for the in-tegration of gold and platinum electrodes can be modi edand optimized to integrate pH-sensitive electrochemicalsensors into AFM tips

5 CONCLUSIONS AND OUTLOOK

Cantilever-based (bio)sensors are versatile analytical sys-tems for the investigation of biological species as they canbe used in solution and are capable of transducing a varietyof different signals Applications in force spectroscopypermit measurements at the level of single moleculesHowever the limiting factor of this technique for imagingof biomolecules is its comparatively poor temporal resolu-tion However ongoing instrumental developments andcombination of AFM with complementary techniques willovercome these limitations in the near future

Using enzymes as recognition elements in biosensorscombines several advantages including high speci city andselectivity Recently advanced immobilization strategiesand mass fabrication based on silicon technology allow

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12 Sensor Lett Vol 1 No 1 2003

25 J H Hoh and P K Hansma Trends Cell Biol 2 208 (1992)26 H Oberleithner G Giebisch and J Geibel P uumlgers Arch 425 506

(1993)27 J Mou S J Sheng R Ho and Z Shao Biophys J 71 2213

(1996)28 J Mou D M Czajkowsky S J Sheng R Ho and Z Shao FEBS

Lett 381 161 (1996)29 D J Muller and A Engel J Mol Biol 285 1347 (1999)30 F Oesterhelt D Oesterhelt M Pfeiffer A Engel H E Gaub and

D Muller Science 288 143 (2000)31 A F Oberhauser P E Marsyale M Carrion-Vazquez and J M

Fernandez Nat Struct Biol 6 1025 (1999)32 M Rief M Gautel F Oesterheit J M Fernandez and H E Gaub

Science 276 1109 (1997)33 M B Viani L I Pietrasanta J B Thompson A Chand I C

Gebeshuber J H Kindt M Richter H G Hansma and P KHansma Nat Struct Biol 7 644 (2000)

34 M Radmacher M Fritz H G Hansma and P K Hansma Science265 1577 (1994)

35 A Noy C D Frisbie L F Rozsnyai M S Wrighton and C MLieber J Am Chem Soc 117 7943 (1995)

36 A M Moulin S J OrsquoShea and M E Welland Ultramicroscopy82 23 (2000)

37 E-L Florin V T Moy and H E Gaub Science 264 415 (1994)38 G U Lee D A Kidwell and R J Colton Langmuir 10 354

(1994)39 A Janshoff M Neitzert Y Oberdoumlrfer and H Fuchs Angew

Chemie Int Ed 39 3212 (2000)40 O H Willemsen M M E Snel A Cambi J Greve B G De

Grooth and C G Figdor Biophys J 79 3267 (2000)41 M Carrion-Vazquez A F Oberhauser T E Fisher P E

Marszalek H Li and J M Fernandez Prog Biophys Mol Biol74 63 (2000)

42 J Zlatanova S M Lindsay and S H Leuba Prog Biophys MolBiol 74 37 (2000)

43 P Hinterdorfer W Baumgartner H J Gruber K Schilcher andH Schindler Proc Natl Acad Sci USA 93 3477 (1996)

44 P Hinterdorfer K Schilcher W Baumgartner H J Gruber andH Schindler Nanobiology 4 39 (1998)

45 R Ros F Schwesinger D Anselmetti M Kubon R SchaeferA Pluumlckthun and L Tiefenauer Proc Natl Acad Sci USA 957402 (1998)

46 F Schwesinger R Ros T Strunz D Anselmetti H-J GuumlntherodtA Honegger L Jermutus L Tiefenauer and A Pluumlckthun ProcNatl Acad Sci USA 97 9972 (2000)

47 R Merkel P Nassoy A Leung A Ritchie and K Evans Nature397 50 (1999)

48 T Strunz K Oroszlan I Schumakovitch H-J Guumlntherodt andM Hegner Biophys J 79 1206 (2000)

49 O H Willemsen M M E Snel K O van der Werf B G DeGrooth J Greve P Hinterdorfer H J Gruber H Schindler Y vanKooyk and C G Figdor Biophys J 75 2220 (1998)

50 M Ludwig W Dettmann and H E Gaub Biophys J 72 445(1997)

51 D P Allison P Hinterdorfer and W Han Curr Opin Biotechnol13 47 (2002)

52 W Han S M Lindsay and T Jing Appl Phys Lett 69 4111(1996)

53 A Raab W Han D Badt S J Smith-Gill S M LindsayH Schindler and P Hinterdorfer Nat Biotechnol 17 902 (1999)

54 G Schuumltz M Sonnleitner P Hinterdorfer and H Schindler MolMembr Biol 17 17 (2000)

55 S W Schneider M E Egan B P Jena W B GugginoH Oberleithner and J P Geibel Proc Natl Acad Sci USA 9612180 (1999)

56 J Finer R M Simmons and J A Spudnich Nature 368 113(1994)

sample surface with high lateral and temporal resolutionGiven the exibility of the described concepts there is con-siderable scope for extended use of multifunctional scan-ning probe systems probing a wide range of biologicalspecimens and processes

Acknowledgments The National Science Foundation(grant 0216368 in the program ldquoBiocomplexity in theEnvironmentrdquo) the National Institutes of Health (grantEB000508) and the ldquoFonds zur Foumlrderung der wissenschaft-lichen Forschungrdquo Austria (grants P14122-CHE and J2230)are greatly acknowledged PCT patents123 124on integratedAFM-SECM are issued

References and Notes

1 A Hulanicki S Glab and F Ingman Pure Appl Chem 63 1247(1991)

2 W Goepel T A Jones M Kleitz I Lundstroem and T SeiyamaEditors Chemical and Biochemical Sensors VCH Weinheim(1992) Vols 2 and 3

3 F W Scheller U Wollenberger A Warsinke and F Lisdat CurrOpin Biotechnol 12 35 (2001)

4 D R Thevenot K Toth R A Durst and G S Wilson BiosensBioelectron 16 121 (2001)

5 M Keusgen Naturwissenschaften 89 433 (2002)6 T H Park and M L Shuler Biotechnol Prog 19 243 (2003)7 K Sato A Hibara M Tokeshi H Hisamoto and T Kitamori Adv

Drug Delivery Rev 55 3791 (2003)8 P N Bartlett Handbook of Chemical and Biological Sensors

edited by R F Taylor Arthur D Little Inc USA J S SchultzUniversity of Pittsburgh (1996) pp 139ff

9 A Hierlemann O Brand C Hagleiter and H Baltes Proc IEEE91 839 (2003)

10 T E Fisher P E Marszalek A F Oberhauser M Carrion-Vazquez and J M Fernandez J Physiol (London) 520 5 (1999)

11 J K Gimzewski and C Joachim Science 283 1683 (1999)12 S O Vansteenkiste M C Davis C J Roberts S J B Tendler

and P M Williams Prog Surf Sci 57 95 (1998)13 M A Poggi L A Bottomley and P T Lillehei Anal Chem 74

2851 (2002)14 G Binnig H Rohrer C Gerber and E Weibel Appl Phys Lett

40 178 (1982)15 P K Hansma J P Cleveland M Radmacher D A Walters P E

Hillner M Bezanilla M Fritz D Vie H G Hansma C B PraterJ Massie L Fukunaga J Gurley and V Elings Appl Phys Lett64 1738 (1994)

16 W H Scouten J H T Luong and S R Brown Trends Biotechnol13 178 (1995)

17 C Kranz A Kueng A Lugstein E Bertagnolli and B Mizaikoffmanuscript in preparation

18 G Binnig C F Quate and C Gerber Phys Rev Lett 56 930 (1986)19 G Charras P Lehenkari and M Horton Methods Cell Biol 68

171 (2002)20 J H Kindt J C Sitko L I Pietrasanta E Oroudjev N Becker

M B Viani and H G Hansma Methods Cell Biol 68 213 (2002)21 B Drake C B Prater A L Weisenhorn S A C Gould T R

Albrecht C F Quate D S Cannell H G Hansma and P KHansma Science 243 1586 (1989)

22 D Rugar and P K Hansma Phys Today 43 23 (1990)23 M Radmacher R W Tillmann M Fritz and H E Gaub Science

157 1900 (1992)24 E Henderson P G Haydon and D S Sakaguchi Science 257

1944 (1992)

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Sensor Lett Vol 1 No 1 2003 13

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6 Sensor Lett Vol 1 No 1 2003

a clearer image of the lysozyme-decorated surface appearsand is ultimately completely restored in Figure 2D

Another example of molecular recognition of an antibodyby a membrane protein is shown in Figure 354 The antibodyis linked to the tip of the probe by a exible spacer using co-valent bonds Brush border membrane vesicles (BBMV) wereadsorbed to a gold surface and imaged with dynamic forcemicroscopy The AFM tip was modi ed with anti-g-GT anti-body against the antigen g-GT (g-glutamyltranspepidase)present at the BBMV At lateral positions where membranevesicles were identi ed the x-y scan was stopped and forcedistance cycles were recorded as shown in Figure 3A Whenthe antibody at the tip binds to an antigen at the surface an at-tractive force develops during further retraction since the tipis physically connected to the surface The attractive force de-velops in a delayed and non-linear fashion re ecting thestretching and the non-linear spring constant of the exibletether covalently coupling the antibody to the AFM tip Thissignal has a distinct shape and re ects the recognition event ofa single antibody-antigen pair43 44 During the control experi-ment in Figure 3B all g-GT sites at the BBMV surface areblocked by anti-g-GT antibody added to the solution Henceno recognition events are recorded

AFM has been used as a tool to simultaneously studymorphology changes and the presence of extracellularATP at living cells with the use of a biosensing layer im-mobilized on the cantilever55 Myosin-functionalized can-tilevers were used to quantitatively detect ATP in solutionduring AFM tapping-mode imaging of living cells Aschematic view of the measurement principle is given inFigure 4AndashC After ATP binding to the myosin subfrag-ment S1 immobilized at the AFM cantilever ATP hydrol-ysis occurs During hydrolysis a conformational changeof the enzyme occurs (in the dimensional range of approx10 nm)56 This conformational change disturbs the inter-action between the tip and the cell surface and causes ameasurable de ection (Figure 4D) Modi ed cantileverswere used to scan the surface of cultured cells demonstrat-ing that the CFTR1 cell line (S9) had a basal surface ATPconcentration which could be determined with AFM tipintegrated biosensors for the rst time

Very recently chemically modi ed probes were appliedto investigate the surface charges of living cells57 AFMmeasurements utilizing carboxyl-terminated tips were usedto probe the electrostatic properties and more speci callythe isoelectric point of microbial cell surfaces with high spa-tial resolution Functionalization of the surfaces was vali-dated by recording multiple force-distance curves between

Figure 4 (A) Scheme of an enzyme-modi ed (myosin subfragmentS1) AFM tip imaging the extracellular surface of a CFTR-expressing cellCl2 (black dots) and ATP (yellow dots) are released in the area adjacentto the CFTR protein (B) ATP binding and subsequent hydrolysis at thetip resulting in cantilever de ection (C) The tip remains de ectedalthough the point of ATP release is passed due to short contact timescompared with ATPase activity time (D left) One ldquohot spotrsquo of extracel-lular ATP The AFM tip rst dips into a localized ATP pool and thenmoves away from this area as the scan progresses As ATP hydrolysisoccurs a positive de ection of the tip is noted causing a continuous lineof activity (D right) Image recorded after incubation with 1024 Mglybenclamide a known inhibitor of CFTR Reduction in hot spots (ar-rows) indicates reduction of extracellular ATP concentration Reprintedwith permission from Ref 55 S W Schneider et al Proc Natl AcadSci USA 96 12180 (1999) copy1999 National Academy of Sciences of theUnited States of America

Figure 3 Dynamic force imaging of brush border membrane vesicles(BBMV) adsorbed to a gold surface (A) Force distance cycle for single an-tibody-antigen recognition event The modi ed tip is brought toward aBBMV (trace dotted line) and subsequently retracted (retraced solid line)at a constant lateral position Antibody-antigen binding is evident from adelayed and non-linear attractive force that develops during further retrac-tion (unbinding event) Inset shows force-distance cycle comprising tworecognition events separated in space and time (B) Control experiment inthe presence of 1 microM anti-g-GT antibody in solution The recognition sig-nal disappears and the traceretrace looks the same Reprinted with permis-sion from Ref 54 Wielert-Badt el al Biophys J 82 2767 (2002) copy2002The Biophysical Society

modi ed probes and model substrates Furthermore car-boxyl-terminated probes were used to record multipleforce-distance curves at various pHs at the surface ofSaccharomyces cerevisiae cells Adhesion force maps de-pendent on the pH were created by recording multiple (n 5

256) force-distance curves and calculating the adhesionforce for each force curve

To summarize force recognition imaging has developedinto a powerful tool for high-resolution studies of molecularrecognition events and for probing dynamic properties ofbiomolecules at physiological conditions Ongoing progressin instrumental development and improved recording tech-niques has continuously added to the relevance of forcerecognition imaging for in situ investigation of biologicalspecimens

3 SCANNING ELECTROCHEMICALMICROBIOSENSORS

The importance of amperometric biosensors is steadily in-creasing because of the combined advantages offered bythe selectivity of biological recognition elements and thewell-established principle of electrochemical transductionThe key element for the design of any sensor architectureis the immobilization of the speci c biological recognitionelement Common sensor architectures include enzymesimmobilized at the transducer surface In the case of anamperometric biosensor this surface usually resembles anoble metal or carbon electrode

31 Immobilizing Biorecognition Elements atMicroelectrodes

For control of the immobilization process during modi -cation of electrode surfaces reproducible procedures arepreferable especially for the development of miniaturizedbiosensors Most commonly applied electrode surface mod-i cation techniques include screen-printing techniques58 incombination with photoinduced cross-linking59 spin-coatingfollowed by photolithographic structuring and lift-off60 theelectrochemically induced formation of enzyme-containingpolymer lms using electrophoretic accumulation61 62 de-position of conducting polymers63ndash65 and covalent bindingof enzymes through functionalized thiol chemistry66 In par-ticular electrochemical techniques permit speci c modi ca-tion of electrode surfaces with high reproducibility Enzymeentrapment within electrochemically deposited polymer ma-trices is well described in the literature and allows directedmodi cation of electrode surfaces64 67 68 Recently success-ful immobilization based on electrochemically induced lo-calized precipitation of enzyme-containing polymer suspen-sions has been reported69 Most reports of applications ofamperometric biosensors describe accurate and reproducibledetermination of analytes in bulk experiments with only afew examples of scanning microbiosensors

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32 Biosensing with Scanning ElectrochemicalMicroscopy

Laterally resolved information at a submicrometer scalewas added to electroanalytical chemistry with the inventionof scanning electrochemical microscopy (SECM)70ndash72

This method provides spatially resolved electrochemicalinformation on interface processes by combining electro-analytical techniques with the principle of scanning a mi-croelectrode in close vicinity across the sample surfaceSECM is an in situ technique based on surface inducedchanges of a Faraday current which is measured at ascanned and biased ultramicroelectrode (UME) The hemi-spherical diffusion of a redox species in solution toward theUME is disturbed by the sample surface chemistry in closeproximity to the microelectrode Commonly imaging inSECM is achieved in feedback mode operation with redoxspecies arti cially added to the solution

The Faraday current measured at the UME is mainly in- uenced by the reactivity and morphology of the samplethe distance between tip and sample and the size andgeometry of the ultramicroelectrode when the electrode isin close proximity (a few electrode radii) to the sample sur-face In conventional SECM experiments the UME tip isscanned in constant height above the sample surface Inprinciple two modes of SECM are used for imaging appli-cations the feedback mode73 and the generationcollection(GC) mode74 Functional schemes of both SECM operationmodes are shown in Figure 5 The feedback mode is basedon the Faraday current resulting from the oxidation or re-duction of a mediator species at the UME tip with the appli-cation of a potential In bulk solution after reaching steadystate the Faraday current recorded at the UME remainsconstant (Figure 5A a) Approaching an insulating surfacewith the UME results in blocked hemispherical diffusion tothe active electrode surface (negative feedback) as shownin Figure 5A c In contrast approaching a conducting orelectrochemically active surface leads to positive feedbackdue to locally increased mediator concentration induced bysurface recycling effects (Figure 5A b) This technique iswidely used for investigating processes at solidliquid andliquidliquid interfaces75

Most investigations of biological samples are performedin the GC (substrate generationtip collection or tip gener-ationsubstrate collection) mode with the tip signal arisingfrom a species generated at the sample surface (Figure 5B)The tip acts as a sensor establishing concentration maps ofindividual electroactive species near the sample surface

In conventional SECM positioning of the microelectrodeis achieved by recording the Faradaic current measured atthe UME as the tip approaches the surface Usually theoret-ical calculations of approach curves for different electrodegeometries are tted to the experimentally obtained dataand used to determine the distance between the UME andthe sample surface76 However the current signal can beused only for amperometric electrodes Consequently non-

Sensor Lett Vol 1 No 1 2003 7

sitioning of the microelectrode in common operation modesis a major drawback of SECM in comparison with AFM orSTM83 Scanning the electrode in constant height across thesample surface results in a convolution of the electrochemi-cal response and the topographical information especially ifthe sample topography and the surface reactivity are un-known Furthermore risk of damage to the UME andor thesample surface increases with decreasing diameter of theSECM tip to the submicron scale or nanometer scale be-cause of the electrode size-dependent working distanceConsequently any progress toward improved lateral reso-lution and positioning of nonamperometric submicroelec-trodes and nanoelectrodes (eg micro- and nanobiosensors)has to address current independent positioning strategiesensuring precise control of the tip-sample distance

33 Positioning of Electrochemical Microbiosensorswith the Use of Shear Force-Based Constant-Distance Mode

In NSOM shear force-based positioning of optical ber tipswith the use of optical detection systems84 85 or nonopticaldetection based on a tuning fork resonator86 87 has beendescribed The rst successful integration of a shear force-based optical feedback mechanism into SECM using a ex-ible ( ber-shaped) vibrating UME was published in the mid-1990s88 Recently this technology has been adapted to avariety of microelectrodes89 A ber-shaped UME is vi-brated at its resonance frequency with a piezoelectric tubeused for agitation (Figure 6A) A laser beam is focused onthe tip of the UME creating a Fresnel diffraction patternwhich is projected onto a split photodiode The vibrationamplitude of the electrode is monitored by phase-sensitiveampli cation of the difference signal at the split photodiodewith respect to the agitation signal with the use of lock-intechniques Surface shear forces increase because of hydro-dynamic effects in close proximity to the sample90 and leadto amplitude damping and a phase shift of the vibrationWith this information a feedback loop with a de ned setpoint for amplitude damping can be integrated into theSECM software routine thereby maintaining a constant dis-tance between the tip and the sample surface during scan-ning across a sample with variable topography

An alternative method combining the shear force modebased on a tuning fork technique with SECM for currentindependent positioning of ber-shaped electrodes has beendescribed in the literature91ndash94 In these contributions theSECM tip is attached to a tuning fork resonance detectionsystem of a commercially available NSOM The UME is at-tached to the leg of a tuning fork driven by a piezoelectrictube At resonance the movement of the legs of the tuningfork generates an oscillating piezoelectric potential which isproportional to the amplitude of the oscillation Again thesynchronized signal derived with lock-in techniques is usedas the feedback signal by the NSOM controller Recently ahighly sensitive nonoptical shear force distance control for

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8 Sensor Lett Vol 1 No 1 2003

amperometric tips such as biosensors require alternative tippositioning strategies during SECM experiments Utilizingoptical microscopy for tip positioning yields only qualita-tive results77 Few approaches have been reported for theapplication of potentiometric UMEs78ndash81 and enzymeUMEs82 Horrocks et al78 used antimony SECM electrodesalso operating in amperometric mode for positioning of thetip prior to pH mapping Wei et al79 introduced positioningtechniques based on dc measurements of the solution resis-tance with AgAgCl micropipette electrodes or potentio-metric measurements of steady-state concentration pro lesusing ion-selective microelectrodes providing the tip-sample distance Furthermore potentiometric UMEs with adual-electrode con guration are reported where one chan-nel is con gured as an ion-selective electrode and the sec-ond channel as an amperometric electrode for distancecontrol during UME approach A hydrogen peroxide micro-biosensor was positioned by the application of a high-frequency alternating potential to the tip and measurementof the solution resistance between the tip and the auxiliaryelectrode82 Although good agreement of the experimentallyobtained data with theory has been achieved this approach islimited to micrometer-sized sensors because the relativecontribution of the solution resistance to the impedance de-creases with decreasing dimensions of the sensor

The lack of lateral resolution due to current dependent po-

Figure 5 Scheme of SECM operation modes (A) Feedback modewith normalized current-distance curve (a) UME in bulk solution(b) positive feedback mode due to mediator recycling at the sample sur-face (c) negative feedback mode due to blocked diffusion of the medi-ator to the UME (B) Generatorcollector mode (a) substrate genera-tiontip collection and (b) tip generationsubstrate collection mode

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Sensor Lett Vol 1 No 1 2003 9

SECM tips based on piezoelectric shear force distancecontrol was developed simplifying shear force-controlledconstant-distance SECM95

Shear force-based constant-distance SECM with opticaldetection has already been applied for positioning of micro-biosensors89 Glass capillaries (inner diam 10 mm) havebeen lled with a polymer hydrogel containing either glu-cose oxidase or glucose dehydrogenase The enzyme elec-trodes were positioned with the use of optical shear forcecontrol and scanned over a 50-mm Pt electrode in the case ofthe glucose oxidase biosensor and over a poly-(methyleneblue)-modi ed microelectrode in the case of the glucose-de-hydrogenase- lled capillary The involved reaction schemes(A) and the obtained SECM images of the reactive spots im-aged with the microbiosensors (B) are shown in Figure 7

To date few results have been published on nanoelec-trodes in SECM experiments High-resolution SECM imag-ing requires reproducible fabrication of nanoelectrodes bymicrofabrication techniques In addition it is still dif cult todetermine the exact distance between the electrode and thesample surface by SECM operation in constant-distance andconstant-height mode Distance calculations are frequentlybased on assumptions such as ideal electrode geometriesHowever these are dif cult to realize for nanoelectrodesfabricated by conventional techniques such as the use oflaser pipette pullers96 In turn quantitative studies on elec-trode processes require precise knowledge of the absolute

distance between tip and surface and demand reproduciblyfabricated electrodes and accurate control of a deliberatelyde ned electrode working distance

4 AFM TIP INTEGRATED ELECTRO-CHEMICAL MICROBIOSENSORS

Recent developments focused on the combination of AFMand SECM enable positioning of the UME independently ofthe current response during simultaneous high-resolutionAFM imaging AFM probes with integrated electrochemicalsensing capabilities provide a versatile tool with inherentcorrelation of structural information with (electro)chemicalsurface activity at excellent lateral resolution97

Figure 8 shows schematics of two different concepts forthe combination of AFM with SECM published by Mac-pherson et al98 99 and Kranz et al100ndash104 The rst approachof combined SECM-AFM imaging in liquid utilizes bentPt-wire nanoelectrodes shaped like AFM cantilevers (Figure8A and B)98 99 Although excellent spatial resolution forthe electrochemical response can be achieved imagingwith these cantilever-shaped electrodes in contact mode islimited to robust nonconducting samples because of thecontact of the cantilever-shaped nanoelectrode and the sam-ple surface Since the electroactive area is located at thevery end of the tip conducting samples have to be investi-gated by lift-mode imaging99 However this requires scan-ning the sample surface twice First the topographical in-formation is recorded in contact mode operation followedby a second scan using the recorded topographical informa-tion to keep the tip at a de ned distance from the surfacetracing the sample contours99

Figure 7 (A) Scheme of the generatorcollector mode detection prin-ciple using enzyme- lled ber-shaped tips (B) Identi cation of electro-active spots with enzyme- lled capillaries (a) Image of a 50-mm-diameterplatinum electrode obtained with a glucose oxidase-modi ed microcapil-lary and (b) image of a 50-mm poly(methylene blue)-modi ed platinumelectrode based on detection of NADH generated at the glucose dehydro-genase-modi ed capillary tip Reprinted with permission from Ref 89A Hengstenberg et al Chem Eur J 6 1547 (2000) copy2000 Wiley

Figure 6 Schematic representation of the different shear force detec-tion principles implemented in SECM for constant distance imaging(A) optical shear-force detection (B and C) Nonoptical shear-force detection

the very end of the original AFM tip Figure 8 shows aschematic cross section of an integrated AFM-SECM tip(C) and FIB images of an integrated frame submicroelec-trode (D E) Because of the resolution of the beam pro-vided by FIB systems integration of submicrometer- andnanoelectrodes can be realized100 103 111 although theelectrode size demonstrated with bent Pt wire electrodescould not yet be achieved So far disc-shaped integratednanoelectrodes with diameters down to 150 nm have beensuccessfully fabricated with the described technology112

Based on the described technology for the combinationof AFM and SECM SECM functionality can be integratedinto any standard AFM with only minor modi cations of(frequently) already existing AFM instrumentation Theelectrochemical response is inherently correlated with thetopographical information obtained by AFM imaging Thuscomplete separation of simultaneously obtained topo-graphical information and electrochemical information isachieved maintaining superior resolution of the topo-graphical information With this technique positioning ofnonamperometric electrodes down to the nanometer regimecan be realized

The application of integrated AFM-SECM tips fortapping-mode AFM operation has recently been demon-strated113 Despite modi cation during tip fabrication theresonance frequency of the unmodi ed silicon nitride can-tilever is not signi cantly shifted which has been con- rmed by comparing frequency spectra of unmodi ed andmodi ed cantilevers The oscillation amplitude of a fewnanometers is small compared with the diameter of theintegrated electrode (usually 300ndash800 nm) It has beenshown that the current response of the integrated electrodein SECM feedback mode is not significantly influencedby the oscillation amplitude of the combined SECM-AFMcantilever Electrochemical images recorded in tappingmode revealed current response and lateral resolutioncomparable to the electrochemical images obtained in con-tact mode113

AFM tip integrated electrodes have been applied forimaging enzyme activity in tapping-mode operation (Fig-ure 9)112 Enzyme activity of glucose oxidase (GOD)entrapped in a soft polymer matrix deposited inside thepores of a periodic micropattern has successfully beenimaged in AFM tapping mode and SECM generation-collection mode In the absence of glucose the currentrecorded at the electrode during AFM tapping-mode imag-ing of the enzyme containing polymer spots is negligible(Figure 9A topography B amplitude image) and revealsno electrochemical features (Figure 9C) In the presence ofglucose an increase in the current recorded at the inte-grated submicroelectrode is observed because of localizedproduction of H2O2 above the GOD-containing polymerspots (Figure 9F) The topography (Figure 9D) and ampli-tude (Figure 9E) images remain unaffected

The presented approach of integrated SECM-AFMprobes provides the opportunity to integrate and position

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10 Sensor Lett Vol 1 No 1 2003

Although several approaches of combined AFM-SECMusing such bent cantilever-shaped nanoelectrodes have beenpublished105ndash107 to date none of these reported measure-ments in AFM tapping mode are due to the variation inspring constant of these hand-made tips AFM tapping modeis a versatile technique for topographical imaging of softsample surfaces as encountered during investigation of forexample biological systems15 During tapping-mode opera-tion the probe gently taps the surface while resonating at ornear its resonance frequency A signi cantly reduced tip-sample contact time results in minimized frictional forcesavoiding damage of soft samples such as biological speciesor polymers108ndash110

An alternative approach combining AFM and SECMhas been described by our research group based on inte-gration of submicro- and nanoelectrodes into atomic forcemicroscopy tips with the use of microfabrication tech-niques (Figure 8CndashE)100ndash104 Electrodes with a variety ofwell-de ned geometries can be integrated into AFM tips atan exactly de ned distance above the apex of the AFM tipsimultaneously obtaining electrochemical information at aprecisely de ned working distance during AFM imagingDuring tip fabrication a conventional silicon nitride AFMcantilever is coated with an electroactive conducting layer(usually gold or platinum) and is subsequently insulatedwith materials such as Si3N4 or parylene C Three-dimen-sional focused ion beam (FIB) milling permits repro-ducible exposure of an electroactive area integrated above

Figure 8 Representation of combined AFM and SECM as publishedby Macpherson et al98 99 (A and B) and Kranz et al100 (CndashE)(A) Schematic of the AFM-SECM tip fabricated by coating etching andbending of Pt microwires Reprinted with permission from Ref 98 J VMacpherson and P R Unwin Anal Chem 72 276 (2000) copy2000American Chemical Society (B) Scanning electron micrograph of a can-tilever-shaped nanoelectrode Reprinted with permission from Ref 99 JV Macpherson and P R Unwin Anal Chem 73 550 (2001) copy2001American Chemical Society (C) Schematic cross section of an integratedAFM-SECM tip fabricated by microfabrication using FIB technology(dimensions are not to scale) (D) FIB image of an integrated frame sub-microelectrode (gold) with Parylene C insulation (electrode edge length770 nm) (E) Side view showing reshaped AFM-tip and tilt correction fora cantilever mounting angle of 10deg in a tapping-mode liquid cell

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micro- and nanobiosensors at a well-de ned and deliber-ately selected distance above the sample surface Standardsurface modi cation processes for biosensors as previ-ously outlined in this review can be used to immobilizebiological recognition elements at the surface of the inte-grated electrode (Figure 10) With the use of electrochemicaltechniques the site of immobilization is con ned to theelectrode surface providing highly localized and repro-ducible integration of a biosensing interface64 65 67 68

With the immobilization of enzymes via self-assembledthiol monolayers with reactive headgroups114 conversion

of electroactive and -inactive species generated at the sam-ple surface is enabled

Figure 11 illustrates simultaneously recorded topo-graphical and electrochemical images obtained with a tip-integrated biosensor The biosensor is based on horse-radish peroxidase immobilized at the electrode surface ofthe integrated SECM-AFM probe (Figure 11A) The per-oxidase activity was imaged in tip generatorsubstrate col-lector mode of SECM (see also Figure 11Bb) duringcontact-mode AFM operation The enzyme was immobi-lized by chemisorption of a functionalized thiol monolayer(cystaminium chloride) to the tip-integrated gold micro-electrode Peroxidase was attached by the addition of amixture of glutaraldehyde and peroxidase resulting in a

Figure 11 (A) Schematic presentation of the tip generationsubstratecollection mode experiment involving simultaneous detection of tipintegrated enzyme activity and AFM topography (B) Simultaneouslyrecorded topographical information and tip integrated peroxidase activityin contact mode The current measured at the microelectrode arrayre ects the enzyme activity integrated in the AFM tip Top view of theheight (a) and (c) and simultaneously recorded current (b) The electro-chemical response with 05 mmol L21 H2O2 in solution Images arerecorded in 2 mmol L21 FMA 01 mol L21 KCl in phosphate buffer (01mol L21 pH 70) (d) In the absence of the enzymatic substrate H2O2The tip was held at a potential of 005 V vs AgAgCl Electrode edgelength 860 nm tip height 410 nm scan rate 1 Hz

Figure 10 Schematic cross section of an AFM tip integrated biosensorEnzymes can be immobilized at the electrode surface integrated in thescanning probe tip via self-assembled thiol monolayers with functional-ized headgroups (A) or via electrochemical deposition of enzyme-containing polymer lms (B)

Figure 9 Simultaneously recorded height and current images of glu-cose oxidase activity in AFM tapping mode and SECM substrate genera-tiontip collection mode Top view of the height (a and d) correspondingamplitude (b and e) and simultaneously recorded current (c and f) imagesrecorded in air-saturated phosphate buffer in absence (andashc) and in thepresence (dndashf) of the substrate glucose in solution Reprinted with per-mission from Ref 112 A Kueng et al Angew Chem Int Ed 42 3237(2003) copy2003 Wiley

Sensor Lett Vol 1 No 1 2003 11

miniaturization of biosensors However only a few ap-proaches have been reported that use scanning micro- ornanobiosensors to obtain laterally resolved (bio)chemicalinformation Using shear force modes in combination withSECM permits positioning of nonamperometric sensorsImprovement of the achievable lateral resolution by de-creasing the dimensions of the applied sensor is the focusof ongoing research in this eld Fiber-based optical micro-biosensors in combination with NSOM techniques mightbe an alternative future concept for obtaining laterally re-solved information on bioactivity AFM tip-integrated bio-sensors provide a promising concept combining advantagesof various techniques in a single microfabricated probeHigh reproducibility of the fabrication process and the po-tential of miniaturization down to the nanometer scalemake them a versatile tool for future applications for prob-ing a wide range of biological substrates and processes

Among the major drawbacks of biosensors in general aresigni cantly increased response times of enzyme-modi edelectrodes compared with direct electrochemical detectionMany biologically relevant processes (eg exocytoticevents) relay signals in intervals of milliseconds Elec-trodes have been fabricated that can detect neurotransmit-ters with response times of 300 ms118 119 However re-sponse times less than 100 ms are needed to accuratelyquantify dynamics at the cellular level and less than 10 msto examine exocytosis of nonelectroactive neurotransmit-ters However it has been demonstrated that the temporalresolution achievable with biosensor systems scales in fa-vor of the proposed technology with device miniaturiza-tion120 121 As an example response times on the order of85 ms have been reported for a glucose-speci c biosensorwith an electrode diameter decreased to the submicronrange122 As recently demonstrated by our research groupdisc electrodes with diameters below 200 nm can be inte-grated into AFM tips by microfabrication techniques112 Itis expected that surface modi cation of such nanoelec-trodes as outlined in this contribution will result in a furtherdecrease in the integrated biosensor response time With arealistic perspective of measurements in the time domain ofmicroseconds suitability for investigation of biologicalprocesses is evident

With the development of multifunctional scanningprobes with tip-integrated biosensors a versatile analyticaltool for the investigation of complex biological systemshas been realized AFM cantilever-based biosensors pro-vide an excellent concept for simultaneous detection oftopographical and chemical information at the surface ofbiological species with high lateral resolution With theintegration of an electroactive area into AFM cantileversenabling subsequent surface modi cation and leading tobiosensor functionality an extension of this concept is pre-sented for detecting electroactive and -inactive speciesgenerated at the sample surface Accurate positioning andscanning of nanobiosensors at a de ned distance above thesample allows quanti cation of kinetic bioprocesses at the

surface-grafted cross-linked protein gel at the aminatedsurface of the self-assembled monolayer covering the inte-grated electrode The electrochemical signal generation isbased on the following enzyme catalyzed reactions(1) H2O2 is reduced to water by the oxidoreductase perox-idase and (2) a metal organic electron donor for the enzy-matic reaction (hydroxyl methyl ferrocene FMA contain-ing Fe21) added to the solution is oxidized during theenzymatic conversion to ferrocinium methylhydroxide(FMA1 containing Fe31) The electroactive by-product ofthe enzymatic reaction FMA1 diffuses to a microstruc-tured electrode array which was used as a model sampleThe microstructured electrode array is biased at a potentialof 50 mV vs AgAgCl At this potential only FMA1 andno other species in solution is reduced Hence informationon the local (electro)chemical activity (Figure 11Bb) withrespect to the position of the tip-integrated biosensor isprovided (Figure 11Ba topography) Comparison with thecontrol experiment in the absence of H2O2 (Figure 11B ctopography B d electrochemical image) demonstratesthe successful integration of a biosensor into a SECM-AFM probe and simultaneous activity imaging during AFMmeasurements17

The presented technique is by no means limited to theintegration of biosensors and can be extended to potentio-metric sensors such as pH microsensors or amalgam elec-trodes Many biological processes including enzymaticreactions biocorrosion and metabolic processes of micro-organisms involve or are driven by pH changes Hencesimultaneous AFM imaging and mapping of local pHmodulations is of substantial interest For example anti-mony and iridiumiridium oxide electrodes have beenreported for electrochemical pH measurements of biolog-ical systems115ndash117 As thin lms of these materials can besputtered the fabrication steps described above for the in-tegration of gold and platinum electrodes can be modi edand optimized to integrate pH-sensitive electrochemicalsensors into AFM tips

5 CONCLUSIONS AND OUTLOOK

Cantilever-based (bio)sensors are versatile analytical sys-tems for the investigation of biological species as they canbe used in solution and are capable of transducing a varietyof different signals Applications in force spectroscopypermit measurements at the level of single moleculesHowever the limiting factor of this technique for imagingof biomolecules is its comparatively poor temporal resolu-tion However ongoing instrumental developments andcombination of AFM with complementary techniques willovercome these limitations in the near future

Using enzymes as recognition elements in biosensorscombines several advantages including high speci city andselectivity Recently advanced immobilization strategiesand mass fabrication based on silicon technology allow

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12 Sensor Lett Vol 1 No 1 2003

25 J H Hoh and P K Hansma Trends Cell Biol 2 208 (1992)26 H Oberleithner G Giebisch and J Geibel P uumlgers Arch 425 506

(1993)27 J Mou S J Sheng R Ho and Z Shao Biophys J 71 2213

(1996)28 J Mou D M Czajkowsky S J Sheng R Ho and Z Shao FEBS

Lett 381 161 (1996)29 D J Muller and A Engel J Mol Biol 285 1347 (1999)30 F Oesterhelt D Oesterhelt M Pfeiffer A Engel H E Gaub and

D Muller Science 288 143 (2000)31 A F Oberhauser P E Marsyale M Carrion-Vazquez and J M

Fernandez Nat Struct Biol 6 1025 (1999)32 M Rief M Gautel F Oesterheit J M Fernandez and H E Gaub

Science 276 1109 (1997)33 M B Viani L I Pietrasanta J B Thompson A Chand I C

Gebeshuber J H Kindt M Richter H G Hansma and P KHansma Nat Struct Biol 7 644 (2000)

34 M Radmacher M Fritz H G Hansma and P K Hansma Science265 1577 (1994)

35 A Noy C D Frisbie L F Rozsnyai M S Wrighton and C MLieber J Am Chem Soc 117 7943 (1995)

36 A M Moulin S J OrsquoShea and M E Welland Ultramicroscopy82 23 (2000)

37 E-L Florin V T Moy and H E Gaub Science 264 415 (1994)38 G U Lee D A Kidwell and R J Colton Langmuir 10 354

(1994)39 A Janshoff M Neitzert Y Oberdoumlrfer and H Fuchs Angew

Chemie Int Ed 39 3212 (2000)40 O H Willemsen M M E Snel A Cambi J Greve B G De

Grooth and C G Figdor Biophys J 79 3267 (2000)41 M Carrion-Vazquez A F Oberhauser T E Fisher P E

Marszalek H Li and J M Fernandez Prog Biophys Mol Biol74 63 (2000)

42 J Zlatanova S M Lindsay and S H Leuba Prog Biophys MolBiol 74 37 (2000)

43 P Hinterdorfer W Baumgartner H J Gruber K Schilcher andH Schindler Proc Natl Acad Sci USA 93 3477 (1996)

44 P Hinterdorfer K Schilcher W Baumgartner H J Gruber andH Schindler Nanobiology 4 39 (1998)

45 R Ros F Schwesinger D Anselmetti M Kubon R SchaeferA Pluumlckthun and L Tiefenauer Proc Natl Acad Sci USA 957402 (1998)

46 F Schwesinger R Ros T Strunz D Anselmetti H-J GuumlntherodtA Honegger L Jermutus L Tiefenauer and A Pluumlckthun ProcNatl Acad Sci USA 97 9972 (2000)

47 R Merkel P Nassoy A Leung A Ritchie and K Evans Nature397 50 (1999)

48 T Strunz K Oroszlan I Schumakovitch H-J Guumlntherodt andM Hegner Biophys J 79 1206 (2000)

49 O H Willemsen M M E Snel K O van der Werf B G DeGrooth J Greve P Hinterdorfer H J Gruber H Schindler Y vanKooyk and C G Figdor Biophys J 75 2220 (1998)

50 M Ludwig W Dettmann and H E Gaub Biophys J 72 445(1997)

51 D P Allison P Hinterdorfer and W Han Curr Opin Biotechnol13 47 (2002)

52 W Han S M Lindsay and T Jing Appl Phys Lett 69 4111(1996)

53 A Raab W Han D Badt S J Smith-Gill S M LindsayH Schindler and P Hinterdorfer Nat Biotechnol 17 902 (1999)

54 G Schuumltz M Sonnleitner P Hinterdorfer and H Schindler MolMembr Biol 17 17 (2000)

55 S W Schneider M E Egan B P Jena W B GugginoH Oberleithner and J P Geibel Proc Natl Acad Sci USA 9612180 (1999)

56 J Finer R M Simmons and J A Spudnich Nature 368 113(1994)

sample surface with high lateral and temporal resolutionGiven the exibility of the described concepts there is con-siderable scope for extended use of multifunctional scan-ning probe systems probing a wide range of biologicalspecimens and processes

Acknowledgments The National Science Foundation(grant 0216368 in the program ldquoBiocomplexity in theEnvironmentrdquo) the National Institutes of Health (grantEB000508) and the ldquoFonds zur Foumlrderung der wissenschaft-lichen Forschungrdquo Austria (grants P14122-CHE and J2230)are greatly acknowledged PCT patents123 124on integratedAFM-SECM are issued

References and Notes

1 A Hulanicki S Glab and F Ingman Pure Appl Chem 63 1247(1991)

2 W Goepel T A Jones M Kleitz I Lundstroem and T SeiyamaEditors Chemical and Biochemical Sensors VCH Weinheim(1992) Vols 2 and 3

3 F W Scheller U Wollenberger A Warsinke and F Lisdat CurrOpin Biotechnol 12 35 (2001)

4 D R Thevenot K Toth R A Durst and G S Wilson BiosensBioelectron 16 121 (2001)

5 M Keusgen Naturwissenschaften 89 433 (2002)6 T H Park and M L Shuler Biotechnol Prog 19 243 (2003)7 K Sato A Hibara M Tokeshi H Hisamoto and T Kitamori Adv

Drug Delivery Rev 55 3791 (2003)8 P N Bartlett Handbook of Chemical and Biological Sensors

edited by R F Taylor Arthur D Little Inc USA J S SchultzUniversity of Pittsburgh (1996) pp 139ff

9 A Hierlemann O Brand C Hagleiter and H Baltes Proc IEEE91 839 (2003)

10 T E Fisher P E Marszalek A F Oberhauser M Carrion-Vazquez and J M Fernandez J Physiol (London) 520 5 (1999)

11 J K Gimzewski and C Joachim Science 283 1683 (1999)12 S O Vansteenkiste M C Davis C J Roberts S J B Tendler

and P M Williams Prog Surf Sci 57 95 (1998)13 M A Poggi L A Bottomley and P T Lillehei Anal Chem 74

2851 (2002)14 G Binnig H Rohrer C Gerber and E Weibel Appl Phys Lett

40 178 (1982)15 P K Hansma J P Cleveland M Radmacher D A Walters P E

Hillner M Bezanilla M Fritz D Vie H G Hansma C B PraterJ Massie L Fukunaga J Gurley and V Elings Appl Phys Lett64 1738 (1994)

16 W H Scouten J H T Luong and S R Brown Trends Biotechnol13 178 (1995)

17 C Kranz A Kueng A Lugstein E Bertagnolli and B Mizaikoffmanuscript in preparation

18 G Binnig C F Quate and C Gerber Phys Rev Lett 56 930 (1986)19 G Charras P Lehenkari and M Horton Methods Cell Biol 68

171 (2002)20 J H Kindt J C Sitko L I Pietrasanta E Oroudjev N Becker

M B Viani and H G Hansma Methods Cell Biol 68 213 (2002)21 B Drake C B Prater A L Weisenhorn S A C Gould T R

Albrecht C F Quate D S Cannell H G Hansma and P KHansma Science 243 1586 (1989)

22 D Rugar and P K Hansma Phys Today 43 23 (1990)23 M Radmacher R W Tillmann M Fritz and H E Gaub Science

157 1900 (1992)24 E Henderson P G Haydon and D S Sakaguchi Science 257

1944 (1992)

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Sensor Lett Vol 1 No 1 2003 13

modi ed probes and model substrates Furthermore car-boxyl-terminated probes were used to record multipleforce-distance curves at various pHs at the surface ofSaccharomyces cerevisiae cells Adhesion force maps de-pendent on the pH were created by recording multiple (n 5

256) force-distance curves and calculating the adhesionforce for each force curve

To summarize force recognition imaging has developedinto a powerful tool for high-resolution studies of molecularrecognition events and for probing dynamic properties ofbiomolecules at physiological conditions Ongoing progressin instrumental development and improved recording tech-niques has continuously added to the relevance of forcerecognition imaging for in situ investigation of biologicalspecimens

3 SCANNING ELECTROCHEMICALMICROBIOSENSORS

The importance of amperometric biosensors is steadily in-creasing because of the combined advantages offered bythe selectivity of biological recognition elements and thewell-established principle of electrochemical transductionThe key element for the design of any sensor architectureis the immobilization of the speci c biological recognitionelement Common sensor architectures include enzymesimmobilized at the transducer surface In the case of anamperometric biosensor this surface usually resembles anoble metal or carbon electrode

31 Immobilizing Biorecognition Elements atMicroelectrodes

For control of the immobilization process during modi -cation of electrode surfaces reproducible procedures arepreferable especially for the development of miniaturizedbiosensors Most commonly applied electrode surface mod-i cation techniques include screen-printing techniques58 incombination with photoinduced cross-linking59 spin-coatingfollowed by photolithographic structuring and lift-off60 theelectrochemically induced formation of enzyme-containingpolymer lms using electrophoretic accumulation61 62 de-position of conducting polymers63ndash65 and covalent bindingof enzymes through functionalized thiol chemistry66 In par-ticular electrochemical techniques permit speci c modi ca-tion of electrode surfaces with high reproducibility Enzymeentrapment within electrochemically deposited polymer ma-trices is well described in the literature and allows directedmodi cation of electrode surfaces64 67 68 Recently success-ful immobilization based on electrochemically induced lo-calized precipitation of enzyme-containing polymer suspen-sions has been reported69 Most reports of applications ofamperometric biosensors describe accurate and reproducibledetermination of analytes in bulk experiments with only afew examples of scanning microbiosensors

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32 Biosensing with Scanning ElectrochemicalMicroscopy

Laterally resolved information at a submicrometer scalewas added to electroanalytical chemistry with the inventionof scanning electrochemical microscopy (SECM)70ndash72

This method provides spatially resolved electrochemicalinformation on interface processes by combining electro-analytical techniques with the principle of scanning a mi-croelectrode in close vicinity across the sample surfaceSECM is an in situ technique based on surface inducedchanges of a Faraday current which is measured at ascanned and biased ultramicroelectrode (UME) The hemi-spherical diffusion of a redox species in solution toward theUME is disturbed by the sample surface chemistry in closeproximity to the microelectrode Commonly imaging inSECM is achieved in feedback mode operation with redoxspecies arti cially added to the solution

The Faraday current measured at the UME is mainly in- uenced by the reactivity and morphology of the samplethe distance between tip and sample and the size andgeometry of the ultramicroelectrode when the electrode isin close proximity (a few electrode radii) to the sample sur-face In conventional SECM experiments the UME tip isscanned in constant height above the sample surface Inprinciple two modes of SECM are used for imaging appli-cations the feedback mode73 and the generationcollection(GC) mode74 Functional schemes of both SECM operationmodes are shown in Figure 5 The feedback mode is basedon the Faraday current resulting from the oxidation or re-duction of a mediator species at the UME tip with the appli-cation of a potential In bulk solution after reaching steadystate the Faraday current recorded at the UME remainsconstant (Figure 5A a) Approaching an insulating surfacewith the UME results in blocked hemispherical diffusion tothe active electrode surface (negative feedback) as shownin Figure 5A c In contrast approaching a conducting orelectrochemically active surface leads to positive feedbackdue to locally increased mediator concentration induced bysurface recycling effects (Figure 5A b) This technique iswidely used for investigating processes at solidliquid andliquidliquid interfaces75

Most investigations of biological samples are performedin the GC (substrate generationtip collection or tip gener-ationsubstrate collection) mode with the tip signal arisingfrom a species generated at the sample surface (Figure 5B)The tip acts as a sensor establishing concentration maps ofindividual electroactive species near the sample surface

In conventional SECM positioning of the microelectrodeis achieved by recording the Faradaic current measured atthe UME as the tip approaches the surface Usually theoret-ical calculations of approach curves for different electrodegeometries are tted to the experimentally obtained dataand used to determine the distance between the UME andthe sample surface76 However the current signal can beused only for amperometric electrodes Consequently non-

Sensor Lett Vol 1 No 1 2003 7

sitioning of the microelectrode in common operation modesis a major drawback of SECM in comparison with AFM orSTM83 Scanning the electrode in constant height across thesample surface results in a convolution of the electrochemi-cal response and the topographical information especially ifthe sample topography and the surface reactivity are un-known Furthermore risk of damage to the UME andor thesample surface increases with decreasing diameter of theSECM tip to the submicron scale or nanometer scale be-cause of the electrode size-dependent working distanceConsequently any progress toward improved lateral reso-lution and positioning of nonamperometric submicroelec-trodes and nanoelectrodes (eg micro- and nanobiosensors)has to address current independent positioning strategiesensuring precise control of the tip-sample distance

33 Positioning of Electrochemical Microbiosensorswith the Use of Shear Force-Based Constant-Distance Mode

In NSOM shear force-based positioning of optical ber tipswith the use of optical detection systems84 85 or nonopticaldetection based on a tuning fork resonator86 87 has beendescribed The rst successful integration of a shear force-based optical feedback mechanism into SECM using a ex-ible ( ber-shaped) vibrating UME was published in the mid-1990s88 Recently this technology has been adapted to avariety of microelectrodes89 A ber-shaped UME is vi-brated at its resonance frequency with a piezoelectric tubeused for agitation (Figure 6A) A laser beam is focused onthe tip of the UME creating a Fresnel diffraction patternwhich is projected onto a split photodiode The vibrationamplitude of the electrode is monitored by phase-sensitiveampli cation of the difference signal at the split photodiodewith respect to the agitation signal with the use of lock-intechniques Surface shear forces increase because of hydro-dynamic effects in close proximity to the sample90 and leadto amplitude damping and a phase shift of the vibrationWith this information a feedback loop with a de ned setpoint for amplitude damping can be integrated into theSECM software routine thereby maintaining a constant dis-tance between the tip and the sample surface during scan-ning across a sample with variable topography

An alternative method combining the shear force modebased on a tuning fork technique with SECM for currentindependent positioning of ber-shaped electrodes has beendescribed in the literature91ndash94 In these contributions theSECM tip is attached to a tuning fork resonance detectionsystem of a commercially available NSOM The UME is at-tached to the leg of a tuning fork driven by a piezoelectrictube At resonance the movement of the legs of the tuningfork generates an oscillating piezoelectric potential which isproportional to the amplitude of the oscillation Again thesynchronized signal derived with lock-in techniques is usedas the feedback signal by the NSOM controller Recently ahighly sensitive nonoptical shear force distance control for

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8 Sensor Lett Vol 1 No 1 2003

amperometric tips such as biosensors require alternative tippositioning strategies during SECM experiments Utilizingoptical microscopy for tip positioning yields only qualita-tive results77 Few approaches have been reported for theapplication of potentiometric UMEs78ndash81 and enzymeUMEs82 Horrocks et al78 used antimony SECM electrodesalso operating in amperometric mode for positioning of thetip prior to pH mapping Wei et al79 introduced positioningtechniques based on dc measurements of the solution resis-tance with AgAgCl micropipette electrodes or potentio-metric measurements of steady-state concentration pro lesusing ion-selective microelectrodes providing the tip-sample distance Furthermore potentiometric UMEs with adual-electrode con guration are reported where one chan-nel is con gured as an ion-selective electrode and the sec-ond channel as an amperometric electrode for distancecontrol during UME approach A hydrogen peroxide micro-biosensor was positioned by the application of a high-frequency alternating potential to the tip and measurementof the solution resistance between the tip and the auxiliaryelectrode82 Although good agreement of the experimentallyobtained data with theory has been achieved this approach islimited to micrometer-sized sensors because the relativecontribution of the solution resistance to the impedance de-creases with decreasing dimensions of the sensor

The lack of lateral resolution due to current dependent po-

Figure 5 Scheme of SECM operation modes (A) Feedback modewith normalized current-distance curve (a) UME in bulk solution(b) positive feedback mode due to mediator recycling at the sample sur-face (c) negative feedback mode due to blocked diffusion of the medi-ator to the UME (B) Generatorcollector mode (a) substrate genera-tiontip collection and (b) tip generationsubstrate collection mode

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Sensor Lett Vol 1 No 1 2003 9

SECM tips based on piezoelectric shear force distancecontrol was developed simplifying shear force-controlledconstant-distance SECM95

Shear force-based constant-distance SECM with opticaldetection has already been applied for positioning of micro-biosensors89 Glass capillaries (inner diam 10 mm) havebeen lled with a polymer hydrogel containing either glu-cose oxidase or glucose dehydrogenase The enzyme elec-trodes were positioned with the use of optical shear forcecontrol and scanned over a 50-mm Pt electrode in the case ofthe glucose oxidase biosensor and over a poly-(methyleneblue)-modi ed microelectrode in the case of the glucose-de-hydrogenase- lled capillary The involved reaction schemes(A) and the obtained SECM images of the reactive spots im-aged with the microbiosensors (B) are shown in Figure 7

To date few results have been published on nanoelec-trodes in SECM experiments High-resolution SECM imag-ing requires reproducible fabrication of nanoelectrodes bymicrofabrication techniques In addition it is still dif cult todetermine the exact distance between the electrode and thesample surface by SECM operation in constant-distance andconstant-height mode Distance calculations are frequentlybased on assumptions such as ideal electrode geometriesHowever these are dif cult to realize for nanoelectrodesfabricated by conventional techniques such as the use oflaser pipette pullers96 In turn quantitative studies on elec-trode processes require precise knowledge of the absolute

distance between tip and surface and demand reproduciblyfabricated electrodes and accurate control of a deliberatelyde ned electrode working distance

4 AFM TIP INTEGRATED ELECTRO-CHEMICAL MICROBIOSENSORS

Recent developments focused on the combination of AFMand SECM enable positioning of the UME independently ofthe current response during simultaneous high-resolutionAFM imaging AFM probes with integrated electrochemicalsensing capabilities provide a versatile tool with inherentcorrelation of structural information with (electro)chemicalsurface activity at excellent lateral resolution97

Figure 8 shows schematics of two different concepts forthe combination of AFM with SECM published by Mac-pherson et al98 99 and Kranz et al100ndash104 The rst approachof combined SECM-AFM imaging in liquid utilizes bentPt-wire nanoelectrodes shaped like AFM cantilevers (Figure8A and B)98 99 Although excellent spatial resolution forthe electrochemical response can be achieved imagingwith these cantilever-shaped electrodes in contact mode islimited to robust nonconducting samples because of thecontact of the cantilever-shaped nanoelectrode and the sam-ple surface Since the electroactive area is located at thevery end of the tip conducting samples have to be investi-gated by lift-mode imaging99 However this requires scan-ning the sample surface twice First the topographical in-formation is recorded in contact mode operation followedby a second scan using the recorded topographical informa-tion to keep the tip at a de ned distance from the surfacetracing the sample contours99

Figure 7 (A) Scheme of the generatorcollector mode detection prin-ciple using enzyme- lled ber-shaped tips (B) Identi cation of electro-active spots with enzyme- lled capillaries (a) Image of a 50-mm-diameterplatinum electrode obtained with a glucose oxidase-modi ed microcapil-lary and (b) image of a 50-mm poly(methylene blue)-modi ed platinumelectrode based on detection of NADH generated at the glucose dehydro-genase-modi ed capillary tip Reprinted with permission from Ref 89A Hengstenberg et al Chem Eur J 6 1547 (2000) copy2000 Wiley

Figure 6 Schematic representation of the different shear force detec-tion principles implemented in SECM for constant distance imaging(A) optical shear-force detection (B and C) Nonoptical shear-force detection

the very end of the original AFM tip Figure 8 shows aschematic cross section of an integrated AFM-SECM tip(C) and FIB images of an integrated frame submicroelec-trode (D E) Because of the resolution of the beam pro-vided by FIB systems integration of submicrometer- andnanoelectrodes can be realized100 103 111 although theelectrode size demonstrated with bent Pt wire electrodescould not yet be achieved So far disc-shaped integratednanoelectrodes with diameters down to 150 nm have beensuccessfully fabricated with the described technology112

Based on the described technology for the combinationof AFM and SECM SECM functionality can be integratedinto any standard AFM with only minor modi cations of(frequently) already existing AFM instrumentation Theelectrochemical response is inherently correlated with thetopographical information obtained by AFM imaging Thuscomplete separation of simultaneously obtained topo-graphical information and electrochemical information isachieved maintaining superior resolution of the topo-graphical information With this technique positioning ofnonamperometric electrodes down to the nanometer regimecan be realized

The application of integrated AFM-SECM tips fortapping-mode AFM operation has recently been demon-strated113 Despite modi cation during tip fabrication theresonance frequency of the unmodi ed silicon nitride can-tilever is not signi cantly shifted which has been con- rmed by comparing frequency spectra of unmodi ed andmodi ed cantilevers The oscillation amplitude of a fewnanometers is small compared with the diameter of theintegrated electrode (usually 300ndash800 nm) It has beenshown that the current response of the integrated electrodein SECM feedback mode is not significantly influencedby the oscillation amplitude of the combined SECM-AFMcantilever Electrochemical images recorded in tappingmode revealed current response and lateral resolutioncomparable to the electrochemical images obtained in con-tact mode113

AFM tip integrated electrodes have been applied forimaging enzyme activity in tapping-mode operation (Fig-ure 9)112 Enzyme activity of glucose oxidase (GOD)entrapped in a soft polymer matrix deposited inside thepores of a periodic micropattern has successfully beenimaged in AFM tapping mode and SECM generation-collection mode In the absence of glucose the currentrecorded at the electrode during AFM tapping-mode imag-ing of the enzyme containing polymer spots is negligible(Figure 9A topography B amplitude image) and revealsno electrochemical features (Figure 9C) In the presence ofglucose an increase in the current recorded at the inte-grated submicroelectrode is observed because of localizedproduction of H2O2 above the GOD-containing polymerspots (Figure 9F) The topography (Figure 9D) and ampli-tude (Figure 9E) images remain unaffected

The presented approach of integrated SECM-AFMprobes provides the opportunity to integrate and position

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10 Sensor Lett Vol 1 No 1 2003

Although several approaches of combined AFM-SECMusing such bent cantilever-shaped nanoelectrodes have beenpublished105ndash107 to date none of these reported measure-ments in AFM tapping mode are due to the variation inspring constant of these hand-made tips AFM tapping modeis a versatile technique for topographical imaging of softsample surfaces as encountered during investigation of forexample biological systems15 During tapping-mode opera-tion the probe gently taps the surface while resonating at ornear its resonance frequency A signi cantly reduced tip-sample contact time results in minimized frictional forcesavoiding damage of soft samples such as biological speciesor polymers108ndash110

An alternative approach combining AFM and SECMhas been described by our research group based on inte-gration of submicro- and nanoelectrodes into atomic forcemicroscopy tips with the use of microfabrication tech-niques (Figure 8CndashE)100ndash104 Electrodes with a variety ofwell-de ned geometries can be integrated into AFM tips atan exactly de ned distance above the apex of the AFM tipsimultaneously obtaining electrochemical information at aprecisely de ned working distance during AFM imagingDuring tip fabrication a conventional silicon nitride AFMcantilever is coated with an electroactive conducting layer(usually gold or platinum) and is subsequently insulatedwith materials such as Si3N4 or parylene C Three-dimen-sional focused ion beam (FIB) milling permits repro-ducible exposure of an electroactive area integrated above

Figure 8 Representation of combined AFM and SECM as publishedby Macpherson et al98 99 (A and B) and Kranz et al100 (CndashE)(A) Schematic of the AFM-SECM tip fabricated by coating etching andbending of Pt microwires Reprinted with permission from Ref 98 J VMacpherson and P R Unwin Anal Chem 72 276 (2000) copy2000American Chemical Society (B) Scanning electron micrograph of a can-tilever-shaped nanoelectrode Reprinted with permission from Ref 99 JV Macpherson and P R Unwin Anal Chem 73 550 (2001) copy2001American Chemical Society (C) Schematic cross section of an integratedAFM-SECM tip fabricated by microfabrication using FIB technology(dimensions are not to scale) (D) FIB image of an integrated frame sub-microelectrode (gold) with Parylene C insulation (electrode edge length770 nm) (E) Side view showing reshaped AFM-tip and tilt correction fora cantilever mounting angle of 10deg in a tapping-mode liquid cell

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micro- and nanobiosensors at a well-de ned and deliber-ately selected distance above the sample surface Standardsurface modi cation processes for biosensors as previ-ously outlined in this review can be used to immobilizebiological recognition elements at the surface of the inte-grated electrode (Figure 10) With the use of electrochemicaltechniques the site of immobilization is con ned to theelectrode surface providing highly localized and repro-ducible integration of a biosensing interface64 65 67 68

With the immobilization of enzymes via self-assembledthiol monolayers with reactive headgroups114 conversion

of electroactive and -inactive species generated at the sam-ple surface is enabled

Figure 11 illustrates simultaneously recorded topo-graphical and electrochemical images obtained with a tip-integrated biosensor The biosensor is based on horse-radish peroxidase immobilized at the electrode surface ofthe integrated SECM-AFM probe (Figure 11A) The per-oxidase activity was imaged in tip generatorsubstrate col-lector mode of SECM (see also Figure 11Bb) duringcontact-mode AFM operation The enzyme was immobi-lized by chemisorption of a functionalized thiol monolayer(cystaminium chloride) to the tip-integrated gold micro-electrode Peroxidase was attached by the addition of amixture of glutaraldehyde and peroxidase resulting in a

Figure 11 (A) Schematic presentation of the tip generationsubstratecollection mode experiment involving simultaneous detection of tipintegrated enzyme activity and AFM topography (B) Simultaneouslyrecorded topographical information and tip integrated peroxidase activityin contact mode The current measured at the microelectrode arrayre ects the enzyme activity integrated in the AFM tip Top view of theheight (a) and (c) and simultaneously recorded current (b) The electro-chemical response with 05 mmol L21 H2O2 in solution Images arerecorded in 2 mmol L21 FMA 01 mol L21 KCl in phosphate buffer (01mol L21 pH 70) (d) In the absence of the enzymatic substrate H2O2The tip was held at a potential of 005 V vs AgAgCl Electrode edgelength 860 nm tip height 410 nm scan rate 1 Hz

Figure 10 Schematic cross section of an AFM tip integrated biosensorEnzymes can be immobilized at the electrode surface integrated in thescanning probe tip via self-assembled thiol monolayers with functional-ized headgroups (A) or via electrochemical deposition of enzyme-containing polymer lms (B)

Figure 9 Simultaneously recorded height and current images of glu-cose oxidase activity in AFM tapping mode and SECM substrate genera-tiontip collection mode Top view of the height (a and d) correspondingamplitude (b and e) and simultaneously recorded current (c and f) imagesrecorded in air-saturated phosphate buffer in absence (andashc) and in thepresence (dndashf) of the substrate glucose in solution Reprinted with per-mission from Ref 112 A Kueng et al Angew Chem Int Ed 42 3237(2003) copy2003 Wiley

Sensor Lett Vol 1 No 1 2003 11

miniaturization of biosensors However only a few ap-proaches have been reported that use scanning micro- ornanobiosensors to obtain laterally resolved (bio)chemicalinformation Using shear force modes in combination withSECM permits positioning of nonamperometric sensorsImprovement of the achievable lateral resolution by de-creasing the dimensions of the applied sensor is the focusof ongoing research in this eld Fiber-based optical micro-biosensors in combination with NSOM techniques mightbe an alternative future concept for obtaining laterally re-solved information on bioactivity AFM tip-integrated bio-sensors provide a promising concept combining advantagesof various techniques in a single microfabricated probeHigh reproducibility of the fabrication process and the po-tential of miniaturization down to the nanometer scalemake them a versatile tool for future applications for prob-ing a wide range of biological substrates and processes

Among the major drawbacks of biosensors in general aresigni cantly increased response times of enzyme-modi edelectrodes compared with direct electrochemical detectionMany biologically relevant processes (eg exocytoticevents) relay signals in intervals of milliseconds Elec-trodes have been fabricated that can detect neurotransmit-ters with response times of 300 ms118 119 However re-sponse times less than 100 ms are needed to accuratelyquantify dynamics at the cellular level and less than 10 msto examine exocytosis of nonelectroactive neurotransmit-ters However it has been demonstrated that the temporalresolution achievable with biosensor systems scales in fa-vor of the proposed technology with device miniaturiza-tion120 121 As an example response times on the order of85 ms have been reported for a glucose-speci c biosensorwith an electrode diameter decreased to the submicronrange122 As recently demonstrated by our research groupdisc electrodes with diameters below 200 nm can be inte-grated into AFM tips by microfabrication techniques112 Itis expected that surface modi cation of such nanoelec-trodes as outlined in this contribution will result in a furtherdecrease in the integrated biosensor response time With arealistic perspective of measurements in the time domain ofmicroseconds suitability for investigation of biologicalprocesses is evident

With the development of multifunctional scanningprobes with tip-integrated biosensors a versatile analyticaltool for the investigation of complex biological systemshas been realized AFM cantilever-based biosensors pro-vide an excellent concept for simultaneous detection oftopographical and chemical information at the surface ofbiological species with high lateral resolution With theintegration of an electroactive area into AFM cantileversenabling subsequent surface modi cation and leading tobiosensor functionality an extension of this concept is pre-sented for detecting electroactive and -inactive speciesgenerated at the sample surface Accurate positioning andscanning of nanobiosensors at a de ned distance above thesample allows quanti cation of kinetic bioprocesses at the

surface-grafted cross-linked protein gel at the aminatedsurface of the self-assembled monolayer covering the inte-grated electrode The electrochemical signal generation isbased on the following enzyme catalyzed reactions(1) H2O2 is reduced to water by the oxidoreductase perox-idase and (2) a metal organic electron donor for the enzy-matic reaction (hydroxyl methyl ferrocene FMA contain-ing Fe21) added to the solution is oxidized during theenzymatic conversion to ferrocinium methylhydroxide(FMA1 containing Fe31) The electroactive by-product ofthe enzymatic reaction FMA1 diffuses to a microstruc-tured electrode array which was used as a model sampleThe microstructured electrode array is biased at a potentialof 50 mV vs AgAgCl At this potential only FMA1 andno other species in solution is reduced Hence informationon the local (electro)chemical activity (Figure 11Bb) withrespect to the position of the tip-integrated biosensor isprovided (Figure 11Ba topography) Comparison with thecontrol experiment in the absence of H2O2 (Figure 11B ctopography B d electrochemical image) demonstratesthe successful integration of a biosensor into a SECM-AFM probe and simultaneous activity imaging during AFMmeasurements17

The presented technique is by no means limited to theintegration of biosensors and can be extended to potentio-metric sensors such as pH microsensors or amalgam elec-trodes Many biological processes including enzymaticreactions biocorrosion and metabolic processes of micro-organisms involve or are driven by pH changes Hencesimultaneous AFM imaging and mapping of local pHmodulations is of substantial interest For example anti-mony and iridiumiridium oxide electrodes have beenreported for electrochemical pH measurements of biolog-ical systems115ndash117 As thin lms of these materials can besputtered the fabrication steps described above for the in-tegration of gold and platinum electrodes can be modi edand optimized to integrate pH-sensitive electrochemicalsensors into AFM tips

5 CONCLUSIONS AND OUTLOOK

Cantilever-based (bio)sensors are versatile analytical sys-tems for the investigation of biological species as they canbe used in solution and are capable of transducing a varietyof different signals Applications in force spectroscopypermit measurements at the level of single moleculesHowever the limiting factor of this technique for imagingof biomolecules is its comparatively poor temporal resolu-tion However ongoing instrumental developments andcombination of AFM with complementary techniques willovercome these limitations in the near future

Using enzymes as recognition elements in biosensorscombines several advantages including high speci city andselectivity Recently advanced immobilization strategiesand mass fabrication based on silicon technology allow

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12 Sensor Lett Vol 1 No 1 2003

25 J H Hoh and P K Hansma Trends Cell Biol 2 208 (1992)26 H Oberleithner G Giebisch and J Geibel P uumlgers Arch 425 506

(1993)27 J Mou S J Sheng R Ho and Z Shao Biophys J 71 2213

(1996)28 J Mou D M Czajkowsky S J Sheng R Ho and Z Shao FEBS

Lett 381 161 (1996)29 D J Muller and A Engel J Mol Biol 285 1347 (1999)30 F Oesterhelt D Oesterhelt M Pfeiffer A Engel H E Gaub and

D Muller Science 288 143 (2000)31 A F Oberhauser P E Marsyale M Carrion-Vazquez and J M

Fernandez Nat Struct Biol 6 1025 (1999)32 M Rief M Gautel F Oesterheit J M Fernandez and H E Gaub

Science 276 1109 (1997)33 M B Viani L I Pietrasanta J B Thompson A Chand I C

Gebeshuber J H Kindt M Richter H G Hansma and P KHansma Nat Struct Biol 7 644 (2000)

34 M Radmacher M Fritz H G Hansma and P K Hansma Science265 1577 (1994)

35 A Noy C D Frisbie L F Rozsnyai M S Wrighton and C MLieber J Am Chem Soc 117 7943 (1995)

36 A M Moulin S J OrsquoShea and M E Welland Ultramicroscopy82 23 (2000)

37 E-L Florin V T Moy and H E Gaub Science 264 415 (1994)38 G U Lee D A Kidwell and R J Colton Langmuir 10 354

(1994)39 A Janshoff M Neitzert Y Oberdoumlrfer and H Fuchs Angew

Chemie Int Ed 39 3212 (2000)40 O H Willemsen M M E Snel A Cambi J Greve B G De

Grooth and C G Figdor Biophys J 79 3267 (2000)41 M Carrion-Vazquez A F Oberhauser T E Fisher P E

Marszalek H Li and J M Fernandez Prog Biophys Mol Biol74 63 (2000)

42 J Zlatanova S M Lindsay and S H Leuba Prog Biophys MolBiol 74 37 (2000)

43 P Hinterdorfer W Baumgartner H J Gruber K Schilcher andH Schindler Proc Natl Acad Sci USA 93 3477 (1996)

44 P Hinterdorfer K Schilcher W Baumgartner H J Gruber andH Schindler Nanobiology 4 39 (1998)

45 R Ros F Schwesinger D Anselmetti M Kubon R SchaeferA Pluumlckthun and L Tiefenauer Proc Natl Acad Sci USA 957402 (1998)

46 F Schwesinger R Ros T Strunz D Anselmetti H-J GuumlntherodtA Honegger L Jermutus L Tiefenauer and A Pluumlckthun ProcNatl Acad Sci USA 97 9972 (2000)

47 R Merkel P Nassoy A Leung A Ritchie and K Evans Nature397 50 (1999)

48 T Strunz K Oroszlan I Schumakovitch H-J Guumlntherodt andM Hegner Biophys J 79 1206 (2000)

49 O H Willemsen M M E Snel K O van der Werf B G DeGrooth J Greve P Hinterdorfer H J Gruber H Schindler Y vanKooyk and C G Figdor Biophys J 75 2220 (1998)

50 M Ludwig W Dettmann and H E Gaub Biophys J 72 445(1997)

51 D P Allison P Hinterdorfer and W Han Curr Opin Biotechnol13 47 (2002)

52 W Han S M Lindsay and T Jing Appl Phys Lett 69 4111(1996)

53 A Raab W Han D Badt S J Smith-Gill S M LindsayH Schindler and P Hinterdorfer Nat Biotechnol 17 902 (1999)

54 G Schuumltz M Sonnleitner P Hinterdorfer and H Schindler MolMembr Biol 17 17 (2000)

55 S W Schneider M E Egan B P Jena W B GugginoH Oberleithner and J P Geibel Proc Natl Acad Sci USA 9612180 (1999)

56 J Finer R M Simmons and J A Spudnich Nature 368 113(1994)

sample surface with high lateral and temporal resolutionGiven the exibility of the described concepts there is con-siderable scope for extended use of multifunctional scan-ning probe systems probing a wide range of biologicalspecimens and processes

Acknowledgments The National Science Foundation(grant 0216368 in the program ldquoBiocomplexity in theEnvironmentrdquo) the National Institutes of Health (grantEB000508) and the ldquoFonds zur Foumlrderung der wissenschaft-lichen Forschungrdquo Austria (grants P14122-CHE and J2230)are greatly acknowledged PCT patents123 124on integratedAFM-SECM are issued

References and Notes

1 A Hulanicki S Glab and F Ingman Pure Appl Chem 63 1247(1991)

2 W Goepel T A Jones M Kleitz I Lundstroem and T SeiyamaEditors Chemical and Biochemical Sensors VCH Weinheim(1992) Vols 2 and 3

3 F W Scheller U Wollenberger A Warsinke and F Lisdat CurrOpin Biotechnol 12 35 (2001)

4 D R Thevenot K Toth R A Durst and G S Wilson BiosensBioelectron 16 121 (2001)

5 M Keusgen Naturwissenschaften 89 433 (2002)6 T H Park and M L Shuler Biotechnol Prog 19 243 (2003)7 K Sato A Hibara M Tokeshi H Hisamoto and T Kitamori Adv

Drug Delivery Rev 55 3791 (2003)8 P N Bartlett Handbook of Chemical and Biological Sensors

edited by R F Taylor Arthur D Little Inc USA J S SchultzUniversity of Pittsburgh (1996) pp 139ff

9 A Hierlemann O Brand C Hagleiter and H Baltes Proc IEEE91 839 (2003)

10 T E Fisher P E Marszalek A F Oberhauser M Carrion-Vazquez and J M Fernandez J Physiol (London) 520 5 (1999)

11 J K Gimzewski and C Joachim Science 283 1683 (1999)12 S O Vansteenkiste M C Davis C J Roberts S J B Tendler

and P M Williams Prog Surf Sci 57 95 (1998)13 M A Poggi L A Bottomley and P T Lillehei Anal Chem 74

2851 (2002)14 G Binnig H Rohrer C Gerber and E Weibel Appl Phys Lett

40 178 (1982)15 P K Hansma J P Cleveland M Radmacher D A Walters P E

Hillner M Bezanilla M Fritz D Vie H G Hansma C B PraterJ Massie L Fukunaga J Gurley and V Elings Appl Phys Lett64 1738 (1994)

16 W H Scouten J H T Luong and S R Brown Trends Biotechnol13 178 (1995)

17 C Kranz A Kueng A Lugstein E Bertagnolli and B Mizaikoffmanuscript in preparation

18 G Binnig C F Quate and C Gerber Phys Rev Lett 56 930 (1986)19 G Charras P Lehenkari and M Horton Methods Cell Biol 68

171 (2002)20 J H Kindt J C Sitko L I Pietrasanta E Oroudjev N Becker

M B Viani and H G Hansma Methods Cell Biol 68 213 (2002)21 B Drake C B Prater A L Weisenhorn S A C Gould T R

Albrecht C F Quate D S Cannell H G Hansma and P KHansma Science 243 1586 (1989)

22 D Rugar and P K Hansma Phys Today 43 23 (1990)23 M Radmacher R W Tillmann M Fritz and H E Gaub Science

157 1900 (1992)24 E Henderson P G Haydon and D S Sakaguchi Science 257

1944 (1992)

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Sensor Lett Vol 1 No 1 2003 13

sitioning of the microelectrode in common operation modesis a major drawback of SECM in comparison with AFM orSTM83 Scanning the electrode in constant height across thesample surface results in a convolution of the electrochemi-cal response and the topographical information especially ifthe sample topography and the surface reactivity are un-known Furthermore risk of damage to the UME andor thesample surface increases with decreasing diameter of theSECM tip to the submicron scale or nanometer scale be-cause of the electrode size-dependent working distanceConsequently any progress toward improved lateral reso-lution and positioning of nonamperometric submicroelec-trodes and nanoelectrodes (eg micro- and nanobiosensors)has to address current independent positioning strategiesensuring precise control of the tip-sample distance

33 Positioning of Electrochemical Microbiosensorswith the Use of Shear Force-Based Constant-Distance Mode

In NSOM shear force-based positioning of optical ber tipswith the use of optical detection systems84 85 or nonopticaldetection based on a tuning fork resonator86 87 has beendescribed The rst successful integration of a shear force-based optical feedback mechanism into SECM using a ex-ible ( ber-shaped) vibrating UME was published in the mid-1990s88 Recently this technology has been adapted to avariety of microelectrodes89 A ber-shaped UME is vi-brated at its resonance frequency with a piezoelectric tubeused for agitation (Figure 6A) A laser beam is focused onthe tip of the UME creating a Fresnel diffraction patternwhich is projected onto a split photodiode The vibrationamplitude of the electrode is monitored by phase-sensitiveampli cation of the difference signal at the split photodiodewith respect to the agitation signal with the use of lock-intechniques Surface shear forces increase because of hydro-dynamic effects in close proximity to the sample90 and leadto amplitude damping and a phase shift of the vibrationWith this information a feedback loop with a de ned setpoint for amplitude damping can be integrated into theSECM software routine thereby maintaining a constant dis-tance between the tip and the sample surface during scan-ning across a sample with variable topography

An alternative method combining the shear force modebased on a tuning fork technique with SECM for currentindependent positioning of ber-shaped electrodes has beendescribed in the literature91ndash94 In these contributions theSECM tip is attached to a tuning fork resonance detectionsystem of a commercially available NSOM The UME is at-tached to the leg of a tuning fork driven by a piezoelectrictube At resonance the movement of the legs of the tuningfork generates an oscillating piezoelectric potential which isproportional to the amplitude of the oscillation Again thesynchronized signal derived with lock-in techniques is usedas the feedback signal by the NSOM controller Recently ahighly sensitive nonoptical shear force distance control for

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8 Sensor Lett Vol 1 No 1 2003

amperometric tips such as biosensors require alternative tippositioning strategies during SECM experiments Utilizingoptical microscopy for tip positioning yields only qualita-tive results77 Few approaches have been reported for theapplication of potentiometric UMEs78ndash81 and enzymeUMEs82 Horrocks et al78 used antimony SECM electrodesalso operating in amperometric mode for positioning of thetip prior to pH mapping Wei et al79 introduced positioningtechniques based on dc measurements of the solution resis-tance with AgAgCl micropipette electrodes or potentio-metric measurements of steady-state concentration pro lesusing ion-selective microelectrodes providing the tip-sample distance Furthermore potentiometric UMEs with adual-electrode con guration are reported where one chan-nel is con gured as an ion-selective electrode and the sec-ond channel as an amperometric electrode for distancecontrol during UME approach A hydrogen peroxide micro-biosensor was positioned by the application of a high-frequency alternating potential to the tip and measurementof the solution resistance between the tip and the auxiliaryelectrode82 Although good agreement of the experimentallyobtained data with theory has been achieved this approach islimited to micrometer-sized sensors because the relativecontribution of the solution resistance to the impedance de-creases with decreasing dimensions of the sensor

The lack of lateral resolution due to current dependent po-

Figure 5 Scheme of SECM operation modes (A) Feedback modewith normalized current-distance curve (a) UME in bulk solution(b) positive feedback mode due to mediator recycling at the sample sur-face (c) negative feedback mode due to blocked diffusion of the medi-ator to the UME (B) Generatorcollector mode (a) substrate genera-tiontip collection and (b) tip generationsubstrate collection mode

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Sensor Lett Vol 1 No 1 2003 9

SECM tips based on piezoelectric shear force distancecontrol was developed simplifying shear force-controlledconstant-distance SECM95

Shear force-based constant-distance SECM with opticaldetection has already been applied for positioning of micro-biosensors89 Glass capillaries (inner diam 10 mm) havebeen lled with a polymer hydrogel containing either glu-cose oxidase or glucose dehydrogenase The enzyme elec-trodes were positioned with the use of optical shear forcecontrol and scanned over a 50-mm Pt electrode in the case ofthe glucose oxidase biosensor and over a poly-(methyleneblue)-modi ed microelectrode in the case of the glucose-de-hydrogenase- lled capillary The involved reaction schemes(A) and the obtained SECM images of the reactive spots im-aged with the microbiosensors (B) are shown in Figure 7

To date few results have been published on nanoelec-trodes in SECM experiments High-resolution SECM imag-ing requires reproducible fabrication of nanoelectrodes bymicrofabrication techniques In addition it is still dif cult todetermine the exact distance between the electrode and thesample surface by SECM operation in constant-distance andconstant-height mode Distance calculations are frequentlybased on assumptions such as ideal electrode geometriesHowever these are dif cult to realize for nanoelectrodesfabricated by conventional techniques such as the use oflaser pipette pullers96 In turn quantitative studies on elec-trode processes require precise knowledge of the absolute

distance between tip and surface and demand reproduciblyfabricated electrodes and accurate control of a deliberatelyde ned electrode working distance

4 AFM TIP INTEGRATED ELECTRO-CHEMICAL MICROBIOSENSORS

Recent developments focused on the combination of AFMand SECM enable positioning of the UME independently ofthe current response during simultaneous high-resolutionAFM imaging AFM probes with integrated electrochemicalsensing capabilities provide a versatile tool with inherentcorrelation of structural information with (electro)chemicalsurface activity at excellent lateral resolution97

Figure 8 shows schematics of two different concepts forthe combination of AFM with SECM published by Mac-pherson et al98 99 and Kranz et al100ndash104 The rst approachof combined SECM-AFM imaging in liquid utilizes bentPt-wire nanoelectrodes shaped like AFM cantilevers (Figure8A and B)98 99 Although excellent spatial resolution forthe electrochemical response can be achieved imagingwith these cantilever-shaped electrodes in contact mode islimited to robust nonconducting samples because of thecontact of the cantilever-shaped nanoelectrode and the sam-ple surface Since the electroactive area is located at thevery end of the tip conducting samples have to be investi-gated by lift-mode imaging99 However this requires scan-ning the sample surface twice First the topographical in-formation is recorded in contact mode operation followedby a second scan using the recorded topographical informa-tion to keep the tip at a de ned distance from the surfacetracing the sample contours99

Figure 7 (A) Scheme of the generatorcollector mode detection prin-ciple using enzyme- lled ber-shaped tips (B) Identi cation of electro-active spots with enzyme- lled capillaries (a) Image of a 50-mm-diameterplatinum electrode obtained with a glucose oxidase-modi ed microcapil-lary and (b) image of a 50-mm poly(methylene blue)-modi ed platinumelectrode based on detection of NADH generated at the glucose dehydro-genase-modi ed capillary tip Reprinted with permission from Ref 89A Hengstenberg et al Chem Eur J 6 1547 (2000) copy2000 Wiley

Figure 6 Schematic representation of the different shear force detec-tion principles implemented in SECM for constant distance imaging(A) optical shear-force detection (B and C) Nonoptical shear-force detection

the very end of the original AFM tip Figure 8 shows aschematic cross section of an integrated AFM-SECM tip(C) and FIB images of an integrated frame submicroelec-trode (D E) Because of the resolution of the beam pro-vided by FIB systems integration of submicrometer- andnanoelectrodes can be realized100 103 111 although theelectrode size demonstrated with bent Pt wire electrodescould not yet be achieved So far disc-shaped integratednanoelectrodes with diameters down to 150 nm have beensuccessfully fabricated with the described technology112

Based on the described technology for the combinationof AFM and SECM SECM functionality can be integratedinto any standard AFM with only minor modi cations of(frequently) already existing AFM instrumentation Theelectrochemical response is inherently correlated with thetopographical information obtained by AFM imaging Thuscomplete separation of simultaneously obtained topo-graphical information and electrochemical information isachieved maintaining superior resolution of the topo-graphical information With this technique positioning ofnonamperometric electrodes down to the nanometer regimecan be realized

The application of integrated AFM-SECM tips fortapping-mode AFM operation has recently been demon-strated113 Despite modi cation during tip fabrication theresonance frequency of the unmodi ed silicon nitride can-tilever is not signi cantly shifted which has been con- rmed by comparing frequency spectra of unmodi ed andmodi ed cantilevers The oscillation amplitude of a fewnanometers is small compared with the diameter of theintegrated electrode (usually 300ndash800 nm) It has beenshown that the current response of the integrated electrodein SECM feedback mode is not significantly influencedby the oscillation amplitude of the combined SECM-AFMcantilever Electrochemical images recorded in tappingmode revealed current response and lateral resolutioncomparable to the electrochemical images obtained in con-tact mode113

AFM tip integrated electrodes have been applied forimaging enzyme activity in tapping-mode operation (Fig-ure 9)112 Enzyme activity of glucose oxidase (GOD)entrapped in a soft polymer matrix deposited inside thepores of a periodic micropattern has successfully beenimaged in AFM tapping mode and SECM generation-collection mode In the absence of glucose the currentrecorded at the electrode during AFM tapping-mode imag-ing of the enzyme containing polymer spots is negligible(Figure 9A topography B amplitude image) and revealsno electrochemical features (Figure 9C) In the presence ofglucose an increase in the current recorded at the inte-grated submicroelectrode is observed because of localizedproduction of H2O2 above the GOD-containing polymerspots (Figure 9F) The topography (Figure 9D) and ampli-tude (Figure 9E) images remain unaffected

The presented approach of integrated SECM-AFMprobes provides the opportunity to integrate and position

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10 Sensor Lett Vol 1 No 1 2003

Although several approaches of combined AFM-SECMusing such bent cantilever-shaped nanoelectrodes have beenpublished105ndash107 to date none of these reported measure-ments in AFM tapping mode are due to the variation inspring constant of these hand-made tips AFM tapping modeis a versatile technique for topographical imaging of softsample surfaces as encountered during investigation of forexample biological systems15 During tapping-mode opera-tion the probe gently taps the surface while resonating at ornear its resonance frequency A signi cantly reduced tip-sample contact time results in minimized frictional forcesavoiding damage of soft samples such as biological speciesor polymers108ndash110

An alternative approach combining AFM and SECMhas been described by our research group based on inte-gration of submicro- and nanoelectrodes into atomic forcemicroscopy tips with the use of microfabrication tech-niques (Figure 8CndashE)100ndash104 Electrodes with a variety ofwell-de ned geometries can be integrated into AFM tips atan exactly de ned distance above the apex of the AFM tipsimultaneously obtaining electrochemical information at aprecisely de ned working distance during AFM imagingDuring tip fabrication a conventional silicon nitride AFMcantilever is coated with an electroactive conducting layer(usually gold or platinum) and is subsequently insulatedwith materials such as Si3N4 or parylene C Three-dimen-sional focused ion beam (FIB) milling permits repro-ducible exposure of an electroactive area integrated above

Figure 8 Representation of combined AFM and SECM as publishedby Macpherson et al98 99 (A and B) and Kranz et al100 (CndashE)(A) Schematic of the AFM-SECM tip fabricated by coating etching andbending of Pt microwires Reprinted with permission from Ref 98 J VMacpherson and P R Unwin Anal Chem 72 276 (2000) copy2000American Chemical Society (B) Scanning electron micrograph of a can-tilever-shaped nanoelectrode Reprinted with permission from Ref 99 JV Macpherson and P R Unwin Anal Chem 73 550 (2001) copy2001American Chemical Society (C) Schematic cross section of an integratedAFM-SECM tip fabricated by microfabrication using FIB technology(dimensions are not to scale) (D) FIB image of an integrated frame sub-microelectrode (gold) with Parylene C insulation (electrode edge length770 nm) (E) Side view showing reshaped AFM-tip and tilt correction fora cantilever mounting angle of 10deg in a tapping-mode liquid cell

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micro- and nanobiosensors at a well-de ned and deliber-ately selected distance above the sample surface Standardsurface modi cation processes for biosensors as previ-ously outlined in this review can be used to immobilizebiological recognition elements at the surface of the inte-grated electrode (Figure 10) With the use of electrochemicaltechniques the site of immobilization is con ned to theelectrode surface providing highly localized and repro-ducible integration of a biosensing interface64 65 67 68

With the immobilization of enzymes via self-assembledthiol monolayers with reactive headgroups114 conversion

of electroactive and -inactive species generated at the sam-ple surface is enabled

Figure 11 illustrates simultaneously recorded topo-graphical and electrochemical images obtained with a tip-integrated biosensor The biosensor is based on horse-radish peroxidase immobilized at the electrode surface ofthe integrated SECM-AFM probe (Figure 11A) The per-oxidase activity was imaged in tip generatorsubstrate col-lector mode of SECM (see also Figure 11Bb) duringcontact-mode AFM operation The enzyme was immobi-lized by chemisorption of a functionalized thiol monolayer(cystaminium chloride) to the tip-integrated gold micro-electrode Peroxidase was attached by the addition of amixture of glutaraldehyde and peroxidase resulting in a

Figure 11 (A) Schematic presentation of the tip generationsubstratecollection mode experiment involving simultaneous detection of tipintegrated enzyme activity and AFM topography (B) Simultaneouslyrecorded topographical information and tip integrated peroxidase activityin contact mode The current measured at the microelectrode arrayre ects the enzyme activity integrated in the AFM tip Top view of theheight (a) and (c) and simultaneously recorded current (b) The electro-chemical response with 05 mmol L21 H2O2 in solution Images arerecorded in 2 mmol L21 FMA 01 mol L21 KCl in phosphate buffer (01mol L21 pH 70) (d) In the absence of the enzymatic substrate H2O2The tip was held at a potential of 005 V vs AgAgCl Electrode edgelength 860 nm tip height 410 nm scan rate 1 Hz

Figure 10 Schematic cross section of an AFM tip integrated biosensorEnzymes can be immobilized at the electrode surface integrated in thescanning probe tip via self-assembled thiol monolayers with functional-ized headgroups (A) or via electrochemical deposition of enzyme-containing polymer lms (B)

Figure 9 Simultaneously recorded height and current images of glu-cose oxidase activity in AFM tapping mode and SECM substrate genera-tiontip collection mode Top view of the height (a and d) correspondingamplitude (b and e) and simultaneously recorded current (c and f) imagesrecorded in air-saturated phosphate buffer in absence (andashc) and in thepresence (dndashf) of the substrate glucose in solution Reprinted with per-mission from Ref 112 A Kueng et al Angew Chem Int Ed 42 3237(2003) copy2003 Wiley

Sensor Lett Vol 1 No 1 2003 11

miniaturization of biosensors However only a few ap-proaches have been reported that use scanning micro- ornanobiosensors to obtain laterally resolved (bio)chemicalinformation Using shear force modes in combination withSECM permits positioning of nonamperometric sensorsImprovement of the achievable lateral resolution by de-creasing the dimensions of the applied sensor is the focusof ongoing research in this eld Fiber-based optical micro-biosensors in combination with NSOM techniques mightbe an alternative future concept for obtaining laterally re-solved information on bioactivity AFM tip-integrated bio-sensors provide a promising concept combining advantagesof various techniques in a single microfabricated probeHigh reproducibility of the fabrication process and the po-tential of miniaturization down to the nanometer scalemake them a versatile tool for future applications for prob-ing a wide range of biological substrates and processes

Among the major drawbacks of biosensors in general aresigni cantly increased response times of enzyme-modi edelectrodes compared with direct electrochemical detectionMany biologically relevant processes (eg exocytoticevents) relay signals in intervals of milliseconds Elec-trodes have been fabricated that can detect neurotransmit-ters with response times of 300 ms118 119 However re-sponse times less than 100 ms are needed to accuratelyquantify dynamics at the cellular level and less than 10 msto examine exocytosis of nonelectroactive neurotransmit-ters However it has been demonstrated that the temporalresolution achievable with biosensor systems scales in fa-vor of the proposed technology with device miniaturiza-tion120 121 As an example response times on the order of85 ms have been reported for a glucose-speci c biosensorwith an electrode diameter decreased to the submicronrange122 As recently demonstrated by our research groupdisc electrodes with diameters below 200 nm can be inte-grated into AFM tips by microfabrication techniques112 Itis expected that surface modi cation of such nanoelec-trodes as outlined in this contribution will result in a furtherdecrease in the integrated biosensor response time With arealistic perspective of measurements in the time domain ofmicroseconds suitability for investigation of biologicalprocesses is evident

With the development of multifunctional scanningprobes with tip-integrated biosensors a versatile analyticaltool for the investigation of complex biological systemshas been realized AFM cantilever-based biosensors pro-vide an excellent concept for simultaneous detection oftopographical and chemical information at the surface ofbiological species with high lateral resolution With theintegration of an electroactive area into AFM cantileversenabling subsequent surface modi cation and leading tobiosensor functionality an extension of this concept is pre-sented for detecting electroactive and -inactive speciesgenerated at the sample surface Accurate positioning andscanning of nanobiosensors at a de ned distance above thesample allows quanti cation of kinetic bioprocesses at the

surface-grafted cross-linked protein gel at the aminatedsurface of the self-assembled monolayer covering the inte-grated electrode The electrochemical signal generation isbased on the following enzyme catalyzed reactions(1) H2O2 is reduced to water by the oxidoreductase perox-idase and (2) a metal organic electron donor for the enzy-matic reaction (hydroxyl methyl ferrocene FMA contain-ing Fe21) added to the solution is oxidized during theenzymatic conversion to ferrocinium methylhydroxide(FMA1 containing Fe31) The electroactive by-product ofthe enzymatic reaction FMA1 diffuses to a microstruc-tured electrode array which was used as a model sampleThe microstructured electrode array is biased at a potentialof 50 mV vs AgAgCl At this potential only FMA1 andno other species in solution is reduced Hence informationon the local (electro)chemical activity (Figure 11Bb) withrespect to the position of the tip-integrated biosensor isprovided (Figure 11Ba topography) Comparison with thecontrol experiment in the absence of H2O2 (Figure 11B ctopography B d electrochemical image) demonstratesthe successful integration of a biosensor into a SECM-AFM probe and simultaneous activity imaging during AFMmeasurements17

The presented technique is by no means limited to theintegration of biosensors and can be extended to potentio-metric sensors such as pH microsensors or amalgam elec-trodes Many biological processes including enzymaticreactions biocorrosion and metabolic processes of micro-organisms involve or are driven by pH changes Hencesimultaneous AFM imaging and mapping of local pHmodulations is of substantial interest For example anti-mony and iridiumiridium oxide electrodes have beenreported for electrochemical pH measurements of biolog-ical systems115ndash117 As thin lms of these materials can besputtered the fabrication steps described above for the in-tegration of gold and platinum electrodes can be modi edand optimized to integrate pH-sensitive electrochemicalsensors into AFM tips

5 CONCLUSIONS AND OUTLOOK

Cantilever-based (bio)sensors are versatile analytical sys-tems for the investigation of biological species as they canbe used in solution and are capable of transducing a varietyof different signals Applications in force spectroscopypermit measurements at the level of single moleculesHowever the limiting factor of this technique for imagingof biomolecules is its comparatively poor temporal resolu-tion However ongoing instrumental developments andcombination of AFM with complementary techniques willovercome these limitations in the near future

Using enzymes as recognition elements in biosensorscombines several advantages including high speci city andselectivity Recently advanced immobilization strategiesand mass fabrication based on silicon technology allow

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12 Sensor Lett Vol 1 No 1 2003

25 J H Hoh and P K Hansma Trends Cell Biol 2 208 (1992)26 H Oberleithner G Giebisch and J Geibel P uumlgers Arch 425 506

(1993)27 J Mou S J Sheng R Ho and Z Shao Biophys J 71 2213

(1996)28 J Mou D M Czajkowsky S J Sheng R Ho and Z Shao FEBS

Lett 381 161 (1996)29 D J Muller and A Engel J Mol Biol 285 1347 (1999)30 F Oesterhelt D Oesterhelt M Pfeiffer A Engel H E Gaub and

D Muller Science 288 143 (2000)31 A F Oberhauser P E Marsyale M Carrion-Vazquez and J M

Fernandez Nat Struct Biol 6 1025 (1999)32 M Rief M Gautel F Oesterheit J M Fernandez and H E Gaub

Science 276 1109 (1997)33 M B Viani L I Pietrasanta J B Thompson A Chand I C

Gebeshuber J H Kindt M Richter H G Hansma and P KHansma Nat Struct Biol 7 644 (2000)

34 M Radmacher M Fritz H G Hansma and P K Hansma Science265 1577 (1994)

35 A Noy C D Frisbie L F Rozsnyai M S Wrighton and C MLieber J Am Chem Soc 117 7943 (1995)

36 A M Moulin S J OrsquoShea and M E Welland Ultramicroscopy82 23 (2000)

37 E-L Florin V T Moy and H E Gaub Science 264 415 (1994)38 G U Lee D A Kidwell and R J Colton Langmuir 10 354

(1994)39 A Janshoff M Neitzert Y Oberdoumlrfer and H Fuchs Angew

Chemie Int Ed 39 3212 (2000)40 O H Willemsen M M E Snel A Cambi J Greve B G De

Grooth and C G Figdor Biophys J 79 3267 (2000)41 M Carrion-Vazquez A F Oberhauser T E Fisher P E

Marszalek H Li and J M Fernandez Prog Biophys Mol Biol74 63 (2000)

42 J Zlatanova S M Lindsay and S H Leuba Prog Biophys MolBiol 74 37 (2000)

43 P Hinterdorfer W Baumgartner H J Gruber K Schilcher andH Schindler Proc Natl Acad Sci USA 93 3477 (1996)

44 P Hinterdorfer K Schilcher W Baumgartner H J Gruber andH Schindler Nanobiology 4 39 (1998)

45 R Ros F Schwesinger D Anselmetti M Kubon R SchaeferA Pluumlckthun and L Tiefenauer Proc Natl Acad Sci USA 957402 (1998)

46 F Schwesinger R Ros T Strunz D Anselmetti H-J GuumlntherodtA Honegger L Jermutus L Tiefenauer and A Pluumlckthun ProcNatl Acad Sci USA 97 9972 (2000)

47 R Merkel P Nassoy A Leung A Ritchie and K Evans Nature397 50 (1999)

48 T Strunz K Oroszlan I Schumakovitch H-J Guumlntherodt andM Hegner Biophys J 79 1206 (2000)

49 O H Willemsen M M E Snel K O van der Werf B G DeGrooth J Greve P Hinterdorfer H J Gruber H Schindler Y vanKooyk and C G Figdor Biophys J 75 2220 (1998)

50 M Ludwig W Dettmann and H E Gaub Biophys J 72 445(1997)

51 D P Allison P Hinterdorfer and W Han Curr Opin Biotechnol13 47 (2002)

52 W Han S M Lindsay and T Jing Appl Phys Lett 69 4111(1996)

53 A Raab W Han D Badt S J Smith-Gill S M LindsayH Schindler and P Hinterdorfer Nat Biotechnol 17 902 (1999)

54 G Schuumltz M Sonnleitner P Hinterdorfer and H Schindler MolMembr Biol 17 17 (2000)

55 S W Schneider M E Egan B P Jena W B GugginoH Oberleithner and J P Geibel Proc Natl Acad Sci USA 9612180 (1999)

56 J Finer R M Simmons and J A Spudnich Nature 368 113(1994)

sample surface with high lateral and temporal resolutionGiven the exibility of the described concepts there is con-siderable scope for extended use of multifunctional scan-ning probe systems probing a wide range of biologicalspecimens and processes

Acknowledgments The National Science Foundation(grant 0216368 in the program ldquoBiocomplexity in theEnvironmentrdquo) the National Institutes of Health (grantEB000508) and the ldquoFonds zur Foumlrderung der wissenschaft-lichen Forschungrdquo Austria (grants P14122-CHE and J2230)are greatly acknowledged PCT patents123 124on integratedAFM-SECM are issued

References and Notes

1 A Hulanicki S Glab and F Ingman Pure Appl Chem 63 1247(1991)

2 W Goepel T A Jones M Kleitz I Lundstroem and T SeiyamaEditors Chemical and Biochemical Sensors VCH Weinheim(1992) Vols 2 and 3

3 F W Scheller U Wollenberger A Warsinke and F Lisdat CurrOpin Biotechnol 12 35 (2001)

4 D R Thevenot K Toth R A Durst and G S Wilson BiosensBioelectron 16 121 (2001)

5 M Keusgen Naturwissenschaften 89 433 (2002)6 T H Park and M L Shuler Biotechnol Prog 19 243 (2003)7 K Sato A Hibara M Tokeshi H Hisamoto and T Kitamori Adv

Drug Delivery Rev 55 3791 (2003)8 P N Bartlett Handbook of Chemical and Biological Sensors

edited by R F Taylor Arthur D Little Inc USA J S SchultzUniversity of Pittsburgh (1996) pp 139ff

9 A Hierlemann O Brand C Hagleiter and H Baltes Proc IEEE91 839 (2003)

10 T E Fisher P E Marszalek A F Oberhauser M Carrion-Vazquez and J M Fernandez J Physiol (London) 520 5 (1999)

11 J K Gimzewski and C Joachim Science 283 1683 (1999)12 S O Vansteenkiste M C Davis C J Roberts S J B Tendler

and P M Williams Prog Surf Sci 57 95 (1998)13 M A Poggi L A Bottomley and P T Lillehei Anal Chem 74

2851 (2002)14 G Binnig H Rohrer C Gerber and E Weibel Appl Phys Lett

40 178 (1982)15 P K Hansma J P Cleveland M Radmacher D A Walters P E

Hillner M Bezanilla M Fritz D Vie H G Hansma C B PraterJ Massie L Fukunaga J Gurley and V Elings Appl Phys Lett64 1738 (1994)

16 W H Scouten J H T Luong and S R Brown Trends Biotechnol13 178 (1995)

17 C Kranz A Kueng A Lugstein E Bertagnolli and B Mizaikoffmanuscript in preparation

18 G Binnig C F Quate and C Gerber Phys Rev Lett 56 930 (1986)19 G Charras P Lehenkari and M Horton Methods Cell Biol 68

171 (2002)20 J H Kindt J C Sitko L I Pietrasanta E Oroudjev N Becker

M B Viani and H G Hansma Methods Cell Biol 68 213 (2002)21 B Drake C B Prater A L Weisenhorn S A C Gould T R

Albrecht C F Quate D S Cannell H G Hansma and P KHansma Science 243 1586 (1989)

22 D Rugar and P K Hansma Phys Today 43 23 (1990)23 M Radmacher R W Tillmann M Fritz and H E Gaub Science

157 1900 (1992)24 E Henderson P G Haydon and D S Sakaguchi Science 257

1944 (1992)

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Sensor Lett Vol 1 No 1 2003 13

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Sensor Lett Vol 1 No 1 2003 9

SECM tips based on piezoelectric shear force distancecontrol was developed simplifying shear force-controlledconstant-distance SECM95

Shear force-based constant-distance SECM with opticaldetection has already been applied for positioning of micro-biosensors89 Glass capillaries (inner diam 10 mm) havebeen lled with a polymer hydrogel containing either glu-cose oxidase or glucose dehydrogenase The enzyme elec-trodes were positioned with the use of optical shear forcecontrol and scanned over a 50-mm Pt electrode in the case ofthe glucose oxidase biosensor and over a poly-(methyleneblue)-modi ed microelectrode in the case of the glucose-de-hydrogenase- lled capillary The involved reaction schemes(A) and the obtained SECM images of the reactive spots im-aged with the microbiosensors (B) are shown in Figure 7

To date few results have been published on nanoelec-trodes in SECM experiments High-resolution SECM imag-ing requires reproducible fabrication of nanoelectrodes bymicrofabrication techniques In addition it is still dif cult todetermine the exact distance between the electrode and thesample surface by SECM operation in constant-distance andconstant-height mode Distance calculations are frequentlybased on assumptions such as ideal electrode geometriesHowever these are dif cult to realize for nanoelectrodesfabricated by conventional techniques such as the use oflaser pipette pullers96 In turn quantitative studies on elec-trode processes require precise knowledge of the absolute

distance between tip and surface and demand reproduciblyfabricated electrodes and accurate control of a deliberatelyde ned electrode working distance

4 AFM TIP INTEGRATED ELECTRO-CHEMICAL MICROBIOSENSORS

Recent developments focused on the combination of AFMand SECM enable positioning of the UME independently ofthe current response during simultaneous high-resolutionAFM imaging AFM probes with integrated electrochemicalsensing capabilities provide a versatile tool with inherentcorrelation of structural information with (electro)chemicalsurface activity at excellent lateral resolution97

Figure 8 shows schematics of two different concepts forthe combination of AFM with SECM published by Mac-pherson et al98 99 and Kranz et al100ndash104 The rst approachof combined SECM-AFM imaging in liquid utilizes bentPt-wire nanoelectrodes shaped like AFM cantilevers (Figure8A and B)98 99 Although excellent spatial resolution forthe electrochemical response can be achieved imagingwith these cantilever-shaped electrodes in contact mode islimited to robust nonconducting samples because of thecontact of the cantilever-shaped nanoelectrode and the sam-ple surface Since the electroactive area is located at thevery end of the tip conducting samples have to be investi-gated by lift-mode imaging99 However this requires scan-ning the sample surface twice First the topographical in-formation is recorded in contact mode operation followedby a second scan using the recorded topographical informa-tion to keep the tip at a de ned distance from the surfacetracing the sample contours99

Figure 7 (A) Scheme of the generatorcollector mode detection prin-ciple using enzyme- lled ber-shaped tips (B) Identi cation of electro-active spots with enzyme- lled capillaries (a) Image of a 50-mm-diameterplatinum electrode obtained with a glucose oxidase-modi ed microcapil-lary and (b) image of a 50-mm poly(methylene blue)-modi ed platinumelectrode based on detection of NADH generated at the glucose dehydro-genase-modi ed capillary tip Reprinted with permission from Ref 89A Hengstenberg et al Chem Eur J 6 1547 (2000) copy2000 Wiley

Figure 6 Schematic representation of the different shear force detec-tion principles implemented in SECM for constant distance imaging(A) optical shear-force detection (B and C) Nonoptical shear-force detection

the very end of the original AFM tip Figure 8 shows aschematic cross section of an integrated AFM-SECM tip(C) and FIB images of an integrated frame submicroelec-trode (D E) Because of the resolution of the beam pro-vided by FIB systems integration of submicrometer- andnanoelectrodes can be realized100 103 111 although theelectrode size demonstrated with bent Pt wire electrodescould not yet be achieved So far disc-shaped integratednanoelectrodes with diameters down to 150 nm have beensuccessfully fabricated with the described technology112

Based on the described technology for the combinationof AFM and SECM SECM functionality can be integratedinto any standard AFM with only minor modi cations of(frequently) already existing AFM instrumentation Theelectrochemical response is inherently correlated with thetopographical information obtained by AFM imaging Thuscomplete separation of simultaneously obtained topo-graphical information and electrochemical information isachieved maintaining superior resolution of the topo-graphical information With this technique positioning ofnonamperometric electrodes down to the nanometer regimecan be realized

The application of integrated AFM-SECM tips fortapping-mode AFM operation has recently been demon-strated113 Despite modi cation during tip fabrication theresonance frequency of the unmodi ed silicon nitride can-tilever is not signi cantly shifted which has been con- rmed by comparing frequency spectra of unmodi ed andmodi ed cantilevers The oscillation amplitude of a fewnanometers is small compared with the diameter of theintegrated electrode (usually 300ndash800 nm) It has beenshown that the current response of the integrated electrodein SECM feedback mode is not significantly influencedby the oscillation amplitude of the combined SECM-AFMcantilever Electrochemical images recorded in tappingmode revealed current response and lateral resolutioncomparable to the electrochemical images obtained in con-tact mode113

AFM tip integrated electrodes have been applied forimaging enzyme activity in tapping-mode operation (Fig-ure 9)112 Enzyme activity of glucose oxidase (GOD)entrapped in a soft polymer matrix deposited inside thepores of a periodic micropattern has successfully beenimaged in AFM tapping mode and SECM generation-collection mode In the absence of glucose the currentrecorded at the electrode during AFM tapping-mode imag-ing of the enzyme containing polymer spots is negligible(Figure 9A topography B amplitude image) and revealsno electrochemical features (Figure 9C) In the presence ofglucose an increase in the current recorded at the inte-grated submicroelectrode is observed because of localizedproduction of H2O2 above the GOD-containing polymerspots (Figure 9F) The topography (Figure 9D) and ampli-tude (Figure 9E) images remain unaffected

The presented approach of integrated SECM-AFMprobes provides the opportunity to integrate and position

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10 Sensor Lett Vol 1 No 1 2003

Although several approaches of combined AFM-SECMusing such bent cantilever-shaped nanoelectrodes have beenpublished105ndash107 to date none of these reported measure-ments in AFM tapping mode are due to the variation inspring constant of these hand-made tips AFM tapping modeis a versatile technique for topographical imaging of softsample surfaces as encountered during investigation of forexample biological systems15 During tapping-mode opera-tion the probe gently taps the surface while resonating at ornear its resonance frequency A signi cantly reduced tip-sample contact time results in minimized frictional forcesavoiding damage of soft samples such as biological speciesor polymers108ndash110

An alternative approach combining AFM and SECMhas been described by our research group based on inte-gration of submicro- and nanoelectrodes into atomic forcemicroscopy tips with the use of microfabrication tech-niques (Figure 8CndashE)100ndash104 Electrodes with a variety ofwell-de ned geometries can be integrated into AFM tips atan exactly de ned distance above the apex of the AFM tipsimultaneously obtaining electrochemical information at aprecisely de ned working distance during AFM imagingDuring tip fabrication a conventional silicon nitride AFMcantilever is coated with an electroactive conducting layer(usually gold or platinum) and is subsequently insulatedwith materials such as Si3N4 or parylene C Three-dimen-sional focused ion beam (FIB) milling permits repro-ducible exposure of an electroactive area integrated above

Figure 8 Representation of combined AFM and SECM as publishedby Macpherson et al98 99 (A and B) and Kranz et al100 (CndashE)(A) Schematic of the AFM-SECM tip fabricated by coating etching andbending of Pt microwires Reprinted with permission from Ref 98 J VMacpherson and P R Unwin Anal Chem 72 276 (2000) copy2000American Chemical Society (B) Scanning electron micrograph of a can-tilever-shaped nanoelectrode Reprinted with permission from Ref 99 JV Macpherson and P R Unwin Anal Chem 73 550 (2001) copy2001American Chemical Society (C) Schematic cross section of an integratedAFM-SECM tip fabricated by microfabrication using FIB technology(dimensions are not to scale) (D) FIB image of an integrated frame sub-microelectrode (gold) with Parylene C insulation (electrode edge length770 nm) (E) Side view showing reshaped AFM-tip and tilt correction fora cantilever mounting angle of 10deg in a tapping-mode liquid cell

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micro- and nanobiosensors at a well-de ned and deliber-ately selected distance above the sample surface Standardsurface modi cation processes for biosensors as previ-ously outlined in this review can be used to immobilizebiological recognition elements at the surface of the inte-grated electrode (Figure 10) With the use of electrochemicaltechniques the site of immobilization is con ned to theelectrode surface providing highly localized and repro-ducible integration of a biosensing interface64 65 67 68

With the immobilization of enzymes via self-assembledthiol monolayers with reactive headgroups114 conversion

of electroactive and -inactive species generated at the sam-ple surface is enabled

Figure 11 illustrates simultaneously recorded topo-graphical and electrochemical images obtained with a tip-integrated biosensor The biosensor is based on horse-radish peroxidase immobilized at the electrode surface ofthe integrated SECM-AFM probe (Figure 11A) The per-oxidase activity was imaged in tip generatorsubstrate col-lector mode of SECM (see also Figure 11Bb) duringcontact-mode AFM operation The enzyme was immobi-lized by chemisorption of a functionalized thiol monolayer(cystaminium chloride) to the tip-integrated gold micro-electrode Peroxidase was attached by the addition of amixture of glutaraldehyde and peroxidase resulting in a

Figure 11 (A) Schematic presentation of the tip generationsubstratecollection mode experiment involving simultaneous detection of tipintegrated enzyme activity and AFM topography (B) Simultaneouslyrecorded topographical information and tip integrated peroxidase activityin contact mode The current measured at the microelectrode arrayre ects the enzyme activity integrated in the AFM tip Top view of theheight (a) and (c) and simultaneously recorded current (b) The electro-chemical response with 05 mmol L21 H2O2 in solution Images arerecorded in 2 mmol L21 FMA 01 mol L21 KCl in phosphate buffer (01mol L21 pH 70) (d) In the absence of the enzymatic substrate H2O2The tip was held at a potential of 005 V vs AgAgCl Electrode edgelength 860 nm tip height 410 nm scan rate 1 Hz

Figure 10 Schematic cross section of an AFM tip integrated biosensorEnzymes can be immobilized at the electrode surface integrated in thescanning probe tip via self-assembled thiol monolayers with functional-ized headgroups (A) or via electrochemical deposition of enzyme-containing polymer lms (B)

Figure 9 Simultaneously recorded height and current images of glu-cose oxidase activity in AFM tapping mode and SECM substrate genera-tiontip collection mode Top view of the height (a and d) correspondingamplitude (b and e) and simultaneously recorded current (c and f) imagesrecorded in air-saturated phosphate buffer in absence (andashc) and in thepresence (dndashf) of the substrate glucose in solution Reprinted with per-mission from Ref 112 A Kueng et al Angew Chem Int Ed 42 3237(2003) copy2003 Wiley

Sensor Lett Vol 1 No 1 2003 11

miniaturization of biosensors However only a few ap-proaches have been reported that use scanning micro- ornanobiosensors to obtain laterally resolved (bio)chemicalinformation Using shear force modes in combination withSECM permits positioning of nonamperometric sensorsImprovement of the achievable lateral resolution by de-creasing the dimensions of the applied sensor is the focusof ongoing research in this eld Fiber-based optical micro-biosensors in combination with NSOM techniques mightbe an alternative future concept for obtaining laterally re-solved information on bioactivity AFM tip-integrated bio-sensors provide a promising concept combining advantagesof various techniques in a single microfabricated probeHigh reproducibility of the fabrication process and the po-tential of miniaturization down to the nanometer scalemake them a versatile tool for future applications for prob-ing a wide range of biological substrates and processes

Among the major drawbacks of biosensors in general aresigni cantly increased response times of enzyme-modi edelectrodes compared with direct electrochemical detectionMany biologically relevant processes (eg exocytoticevents) relay signals in intervals of milliseconds Elec-trodes have been fabricated that can detect neurotransmit-ters with response times of 300 ms118 119 However re-sponse times less than 100 ms are needed to accuratelyquantify dynamics at the cellular level and less than 10 msto examine exocytosis of nonelectroactive neurotransmit-ters However it has been demonstrated that the temporalresolution achievable with biosensor systems scales in fa-vor of the proposed technology with device miniaturiza-tion120 121 As an example response times on the order of85 ms have been reported for a glucose-speci c biosensorwith an electrode diameter decreased to the submicronrange122 As recently demonstrated by our research groupdisc electrodes with diameters below 200 nm can be inte-grated into AFM tips by microfabrication techniques112 Itis expected that surface modi cation of such nanoelec-trodes as outlined in this contribution will result in a furtherdecrease in the integrated biosensor response time With arealistic perspective of measurements in the time domain ofmicroseconds suitability for investigation of biologicalprocesses is evident

With the development of multifunctional scanningprobes with tip-integrated biosensors a versatile analyticaltool for the investigation of complex biological systemshas been realized AFM cantilever-based biosensors pro-vide an excellent concept for simultaneous detection oftopographical and chemical information at the surface ofbiological species with high lateral resolution With theintegration of an electroactive area into AFM cantileversenabling subsequent surface modi cation and leading tobiosensor functionality an extension of this concept is pre-sented for detecting electroactive and -inactive speciesgenerated at the sample surface Accurate positioning andscanning of nanobiosensors at a de ned distance above thesample allows quanti cation of kinetic bioprocesses at the

surface-grafted cross-linked protein gel at the aminatedsurface of the self-assembled monolayer covering the inte-grated electrode The electrochemical signal generation isbased on the following enzyme catalyzed reactions(1) H2O2 is reduced to water by the oxidoreductase perox-idase and (2) a metal organic electron donor for the enzy-matic reaction (hydroxyl methyl ferrocene FMA contain-ing Fe21) added to the solution is oxidized during theenzymatic conversion to ferrocinium methylhydroxide(FMA1 containing Fe31) The electroactive by-product ofthe enzymatic reaction FMA1 diffuses to a microstruc-tured electrode array which was used as a model sampleThe microstructured electrode array is biased at a potentialof 50 mV vs AgAgCl At this potential only FMA1 andno other species in solution is reduced Hence informationon the local (electro)chemical activity (Figure 11Bb) withrespect to the position of the tip-integrated biosensor isprovided (Figure 11Ba topography) Comparison with thecontrol experiment in the absence of H2O2 (Figure 11B ctopography B d electrochemical image) demonstratesthe successful integration of a biosensor into a SECM-AFM probe and simultaneous activity imaging during AFMmeasurements17

The presented technique is by no means limited to theintegration of biosensors and can be extended to potentio-metric sensors such as pH microsensors or amalgam elec-trodes Many biological processes including enzymaticreactions biocorrosion and metabolic processes of micro-organisms involve or are driven by pH changes Hencesimultaneous AFM imaging and mapping of local pHmodulations is of substantial interest For example anti-mony and iridiumiridium oxide electrodes have beenreported for electrochemical pH measurements of biolog-ical systems115ndash117 As thin lms of these materials can besputtered the fabrication steps described above for the in-tegration of gold and platinum electrodes can be modi edand optimized to integrate pH-sensitive electrochemicalsensors into AFM tips

5 CONCLUSIONS AND OUTLOOK

Cantilever-based (bio)sensors are versatile analytical sys-tems for the investigation of biological species as they canbe used in solution and are capable of transducing a varietyof different signals Applications in force spectroscopypermit measurements at the level of single moleculesHowever the limiting factor of this technique for imagingof biomolecules is its comparatively poor temporal resolu-tion However ongoing instrumental developments andcombination of AFM with complementary techniques willovercome these limitations in the near future

Using enzymes as recognition elements in biosensorscombines several advantages including high speci city andselectivity Recently advanced immobilization strategiesand mass fabrication based on silicon technology allow

RE

VIE

W

12 Sensor Lett Vol 1 No 1 2003

25 J H Hoh and P K Hansma Trends Cell Biol 2 208 (1992)26 H Oberleithner G Giebisch and J Geibel P uumlgers Arch 425 506

(1993)27 J Mou S J Sheng R Ho and Z Shao Biophys J 71 2213

(1996)28 J Mou D M Czajkowsky S J Sheng R Ho and Z Shao FEBS

Lett 381 161 (1996)29 D J Muller and A Engel J Mol Biol 285 1347 (1999)30 F Oesterhelt D Oesterhelt M Pfeiffer A Engel H E Gaub and

D Muller Science 288 143 (2000)31 A F Oberhauser P E Marsyale M Carrion-Vazquez and J M

Fernandez Nat Struct Biol 6 1025 (1999)32 M Rief M Gautel F Oesterheit J M Fernandez and H E Gaub

Science 276 1109 (1997)33 M B Viani L I Pietrasanta J B Thompson A Chand I C

Gebeshuber J H Kindt M Richter H G Hansma and P KHansma Nat Struct Biol 7 644 (2000)

34 M Radmacher M Fritz H G Hansma and P K Hansma Science265 1577 (1994)

35 A Noy C D Frisbie L F Rozsnyai M S Wrighton and C MLieber J Am Chem Soc 117 7943 (1995)

36 A M Moulin S J OrsquoShea and M E Welland Ultramicroscopy82 23 (2000)

37 E-L Florin V T Moy and H E Gaub Science 264 415 (1994)38 G U Lee D A Kidwell and R J Colton Langmuir 10 354

(1994)39 A Janshoff M Neitzert Y Oberdoumlrfer and H Fuchs Angew

Chemie Int Ed 39 3212 (2000)40 O H Willemsen M M E Snel A Cambi J Greve B G De

Grooth and C G Figdor Biophys J 79 3267 (2000)41 M Carrion-Vazquez A F Oberhauser T E Fisher P E

Marszalek H Li and J M Fernandez Prog Biophys Mol Biol74 63 (2000)

42 J Zlatanova S M Lindsay and S H Leuba Prog Biophys MolBiol 74 37 (2000)

43 P Hinterdorfer W Baumgartner H J Gruber K Schilcher andH Schindler Proc Natl Acad Sci USA 93 3477 (1996)

44 P Hinterdorfer K Schilcher W Baumgartner H J Gruber andH Schindler Nanobiology 4 39 (1998)

45 R Ros F Schwesinger D Anselmetti M Kubon R SchaeferA Pluumlckthun and L Tiefenauer Proc Natl Acad Sci USA 957402 (1998)

46 F Schwesinger R Ros T Strunz D Anselmetti H-J GuumlntherodtA Honegger L Jermutus L Tiefenauer and A Pluumlckthun ProcNatl Acad Sci USA 97 9972 (2000)

47 R Merkel P Nassoy A Leung A Ritchie and K Evans Nature397 50 (1999)

48 T Strunz K Oroszlan I Schumakovitch H-J Guumlntherodt andM Hegner Biophys J 79 1206 (2000)

49 O H Willemsen M M E Snel K O van der Werf B G DeGrooth J Greve P Hinterdorfer H J Gruber H Schindler Y vanKooyk and C G Figdor Biophys J 75 2220 (1998)

50 M Ludwig W Dettmann and H E Gaub Biophys J 72 445(1997)

51 D P Allison P Hinterdorfer and W Han Curr Opin Biotechnol13 47 (2002)

52 W Han S M Lindsay and T Jing Appl Phys Lett 69 4111(1996)

53 A Raab W Han D Badt S J Smith-Gill S M LindsayH Schindler and P Hinterdorfer Nat Biotechnol 17 902 (1999)

54 G Schuumltz M Sonnleitner P Hinterdorfer and H Schindler MolMembr Biol 17 17 (2000)

55 S W Schneider M E Egan B P Jena W B GugginoH Oberleithner and J P Geibel Proc Natl Acad Sci USA 9612180 (1999)

56 J Finer R M Simmons and J A Spudnich Nature 368 113(1994)

sample surface with high lateral and temporal resolutionGiven the exibility of the described concepts there is con-siderable scope for extended use of multifunctional scan-ning probe systems probing a wide range of biologicalspecimens and processes

Acknowledgments The National Science Foundation(grant 0216368 in the program ldquoBiocomplexity in theEnvironmentrdquo) the National Institutes of Health (grantEB000508) and the ldquoFonds zur Foumlrderung der wissenschaft-lichen Forschungrdquo Austria (grants P14122-CHE and J2230)are greatly acknowledged PCT patents123 124on integratedAFM-SECM are issued

References and Notes

1 A Hulanicki S Glab and F Ingman Pure Appl Chem 63 1247(1991)

2 W Goepel T A Jones M Kleitz I Lundstroem and T SeiyamaEditors Chemical and Biochemical Sensors VCH Weinheim(1992) Vols 2 and 3

3 F W Scheller U Wollenberger A Warsinke and F Lisdat CurrOpin Biotechnol 12 35 (2001)

4 D R Thevenot K Toth R A Durst and G S Wilson BiosensBioelectron 16 121 (2001)

5 M Keusgen Naturwissenschaften 89 433 (2002)6 T H Park and M L Shuler Biotechnol Prog 19 243 (2003)7 K Sato A Hibara M Tokeshi H Hisamoto and T Kitamori Adv

Drug Delivery Rev 55 3791 (2003)8 P N Bartlett Handbook of Chemical and Biological Sensors

edited by R F Taylor Arthur D Little Inc USA J S SchultzUniversity of Pittsburgh (1996) pp 139ff

9 A Hierlemann O Brand C Hagleiter and H Baltes Proc IEEE91 839 (2003)

10 T E Fisher P E Marszalek A F Oberhauser M Carrion-Vazquez and J M Fernandez J Physiol (London) 520 5 (1999)

11 J K Gimzewski and C Joachim Science 283 1683 (1999)12 S O Vansteenkiste M C Davis C J Roberts S J B Tendler

and P M Williams Prog Surf Sci 57 95 (1998)13 M A Poggi L A Bottomley and P T Lillehei Anal Chem 74

2851 (2002)14 G Binnig H Rohrer C Gerber and E Weibel Appl Phys Lett

40 178 (1982)15 P K Hansma J P Cleveland M Radmacher D A Walters P E

Hillner M Bezanilla M Fritz D Vie H G Hansma C B PraterJ Massie L Fukunaga J Gurley and V Elings Appl Phys Lett64 1738 (1994)

16 W H Scouten J H T Luong and S R Brown Trends Biotechnol13 178 (1995)

17 C Kranz A Kueng A Lugstein E Bertagnolli and B Mizaikoffmanuscript in preparation

18 G Binnig C F Quate and C Gerber Phys Rev Lett 56 930 (1986)19 G Charras P Lehenkari and M Horton Methods Cell Biol 68

171 (2002)20 J H Kindt J C Sitko L I Pietrasanta E Oroudjev N Becker

M B Viani and H G Hansma Methods Cell Biol 68 213 (2002)21 B Drake C B Prater A L Weisenhorn S A C Gould T R

Albrecht C F Quate D S Cannell H G Hansma and P KHansma Science 243 1586 (1989)

22 D Rugar and P K Hansma Phys Today 43 23 (1990)23 M Radmacher R W Tillmann M Fritz and H E Gaub Science

157 1900 (1992)24 E Henderson P G Haydon and D S Sakaguchi Science 257

1944 (1992)

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Sensor Lett Vol 1 No 1 2003 13

the very end of the original AFM tip Figure 8 shows aschematic cross section of an integrated AFM-SECM tip(C) and FIB images of an integrated frame submicroelec-trode (D E) Because of the resolution of the beam pro-vided by FIB systems integration of submicrometer- andnanoelectrodes can be realized100 103 111 although theelectrode size demonstrated with bent Pt wire electrodescould not yet be achieved So far disc-shaped integratednanoelectrodes with diameters down to 150 nm have beensuccessfully fabricated with the described technology112

Based on the described technology for the combinationof AFM and SECM SECM functionality can be integratedinto any standard AFM with only minor modi cations of(frequently) already existing AFM instrumentation Theelectrochemical response is inherently correlated with thetopographical information obtained by AFM imaging Thuscomplete separation of simultaneously obtained topo-graphical information and electrochemical information isachieved maintaining superior resolution of the topo-graphical information With this technique positioning ofnonamperometric electrodes down to the nanometer regimecan be realized

The application of integrated AFM-SECM tips fortapping-mode AFM operation has recently been demon-strated113 Despite modi cation during tip fabrication theresonance frequency of the unmodi ed silicon nitride can-tilever is not signi cantly shifted which has been con- rmed by comparing frequency spectra of unmodi ed andmodi ed cantilevers The oscillation amplitude of a fewnanometers is small compared with the diameter of theintegrated electrode (usually 300ndash800 nm) It has beenshown that the current response of the integrated electrodein SECM feedback mode is not significantly influencedby the oscillation amplitude of the combined SECM-AFMcantilever Electrochemical images recorded in tappingmode revealed current response and lateral resolutioncomparable to the electrochemical images obtained in con-tact mode113

AFM tip integrated electrodes have been applied forimaging enzyme activity in tapping-mode operation (Fig-ure 9)112 Enzyme activity of glucose oxidase (GOD)entrapped in a soft polymer matrix deposited inside thepores of a periodic micropattern has successfully beenimaged in AFM tapping mode and SECM generation-collection mode In the absence of glucose the currentrecorded at the electrode during AFM tapping-mode imag-ing of the enzyme containing polymer spots is negligible(Figure 9A topography B amplitude image) and revealsno electrochemical features (Figure 9C) In the presence ofglucose an increase in the current recorded at the inte-grated submicroelectrode is observed because of localizedproduction of H2O2 above the GOD-containing polymerspots (Figure 9F) The topography (Figure 9D) and ampli-tude (Figure 9E) images remain unaffected

The presented approach of integrated SECM-AFMprobes provides the opportunity to integrate and position

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10 Sensor Lett Vol 1 No 1 2003

Although several approaches of combined AFM-SECMusing such bent cantilever-shaped nanoelectrodes have beenpublished105ndash107 to date none of these reported measure-ments in AFM tapping mode are due to the variation inspring constant of these hand-made tips AFM tapping modeis a versatile technique for topographical imaging of softsample surfaces as encountered during investigation of forexample biological systems15 During tapping-mode opera-tion the probe gently taps the surface while resonating at ornear its resonance frequency A signi cantly reduced tip-sample contact time results in minimized frictional forcesavoiding damage of soft samples such as biological speciesor polymers108ndash110

An alternative approach combining AFM and SECMhas been described by our research group based on inte-gration of submicro- and nanoelectrodes into atomic forcemicroscopy tips with the use of microfabrication tech-niques (Figure 8CndashE)100ndash104 Electrodes with a variety ofwell-de ned geometries can be integrated into AFM tips atan exactly de ned distance above the apex of the AFM tipsimultaneously obtaining electrochemical information at aprecisely de ned working distance during AFM imagingDuring tip fabrication a conventional silicon nitride AFMcantilever is coated with an electroactive conducting layer(usually gold or platinum) and is subsequently insulatedwith materials such as Si3N4 or parylene C Three-dimen-sional focused ion beam (FIB) milling permits repro-ducible exposure of an electroactive area integrated above

Figure 8 Representation of combined AFM and SECM as publishedby Macpherson et al98 99 (A and B) and Kranz et al100 (CndashE)(A) Schematic of the AFM-SECM tip fabricated by coating etching andbending of Pt microwires Reprinted with permission from Ref 98 J VMacpherson and P R Unwin Anal Chem 72 276 (2000) copy2000American Chemical Society (B) Scanning electron micrograph of a can-tilever-shaped nanoelectrode Reprinted with permission from Ref 99 JV Macpherson and P R Unwin Anal Chem 73 550 (2001) copy2001American Chemical Society (C) Schematic cross section of an integratedAFM-SECM tip fabricated by microfabrication using FIB technology(dimensions are not to scale) (D) FIB image of an integrated frame sub-microelectrode (gold) with Parylene C insulation (electrode edge length770 nm) (E) Side view showing reshaped AFM-tip and tilt correction fora cantilever mounting angle of 10deg in a tapping-mode liquid cell

RE

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micro- and nanobiosensors at a well-de ned and deliber-ately selected distance above the sample surface Standardsurface modi cation processes for biosensors as previ-ously outlined in this review can be used to immobilizebiological recognition elements at the surface of the inte-grated electrode (Figure 10) With the use of electrochemicaltechniques the site of immobilization is con ned to theelectrode surface providing highly localized and repro-ducible integration of a biosensing interface64 65 67 68

With the immobilization of enzymes via self-assembledthiol monolayers with reactive headgroups114 conversion

of electroactive and -inactive species generated at the sam-ple surface is enabled

Figure 11 illustrates simultaneously recorded topo-graphical and electrochemical images obtained with a tip-integrated biosensor The biosensor is based on horse-radish peroxidase immobilized at the electrode surface ofthe integrated SECM-AFM probe (Figure 11A) The per-oxidase activity was imaged in tip generatorsubstrate col-lector mode of SECM (see also Figure 11Bb) duringcontact-mode AFM operation The enzyme was immobi-lized by chemisorption of a functionalized thiol monolayer(cystaminium chloride) to the tip-integrated gold micro-electrode Peroxidase was attached by the addition of amixture of glutaraldehyde and peroxidase resulting in a

Figure 11 (A) Schematic presentation of the tip generationsubstratecollection mode experiment involving simultaneous detection of tipintegrated enzyme activity and AFM topography (B) Simultaneouslyrecorded topographical information and tip integrated peroxidase activityin contact mode The current measured at the microelectrode arrayre ects the enzyme activity integrated in the AFM tip Top view of theheight (a) and (c) and simultaneously recorded current (b) The electro-chemical response with 05 mmol L21 H2O2 in solution Images arerecorded in 2 mmol L21 FMA 01 mol L21 KCl in phosphate buffer (01mol L21 pH 70) (d) In the absence of the enzymatic substrate H2O2The tip was held at a potential of 005 V vs AgAgCl Electrode edgelength 860 nm tip height 410 nm scan rate 1 Hz

Figure 10 Schematic cross section of an AFM tip integrated biosensorEnzymes can be immobilized at the electrode surface integrated in thescanning probe tip via self-assembled thiol monolayers with functional-ized headgroups (A) or via electrochemical deposition of enzyme-containing polymer lms (B)

Figure 9 Simultaneously recorded height and current images of glu-cose oxidase activity in AFM tapping mode and SECM substrate genera-tiontip collection mode Top view of the height (a and d) correspondingamplitude (b and e) and simultaneously recorded current (c and f) imagesrecorded in air-saturated phosphate buffer in absence (andashc) and in thepresence (dndashf) of the substrate glucose in solution Reprinted with per-mission from Ref 112 A Kueng et al Angew Chem Int Ed 42 3237(2003) copy2003 Wiley

Sensor Lett Vol 1 No 1 2003 11

miniaturization of biosensors However only a few ap-proaches have been reported that use scanning micro- ornanobiosensors to obtain laterally resolved (bio)chemicalinformation Using shear force modes in combination withSECM permits positioning of nonamperometric sensorsImprovement of the achievable lateral resolution by de-creasing the dimensions of the applied sensor is the focusof ongoing research in this eld Fiber-based optical micro-biosensors in combination with NSOM techniques mightbe an alternative future concept for obtaining laterally re-solved information on bioactivity AFM tip-integrated bio-sensors provide a promising concept combining advantagesof various techniques in a single microfabricated probeHigh reproducibility of the fabrication process and the po-tential of miniaturization down to the nanometer scalemake them a versatile tool for future applications for prob-ing a wide range of biological substrates and processes

Among the major drawbacks of biosensors in general aresigni cantly increased response times of enzyme-modi edelectrodes compared with direct electrochemical detectionMany biologically relevant processes (eg exocytoticevents) relay signals in intervals of milliseconds Elec-trodes have been fabricated that can detect neurotransmit-ters with response times of 300 ms118 119 However re-sponse times less than 100 ms are needed to accuratelyquantify dynamics at the cellular level and less than 10 msto examine exocytosis of nonelectroactive neurotransmit-ters However it has been demonstrated that the temporalresolution achievable with biosensor systems scales in fa-vor of the proposed technology with device miniaturiza-tion120 121 As an example response times on the order of85 ms have been reported for a glucose-speci c biosensorwith an electrode diameter decreased to the submicronrange122 As recently demonstrated by our research groupdisc electrodes with diameters below 200 nm can be inte-grated into AFM tips by microfabrication techniques112 Itis expected that surface modi cation of such nanoelec-trodes as outlined in this contribution will result in a furtherdecrease in the integrated biosensor response time With arealistic perspective of measurements in the time domain ofmicroseconds suitability for investigation of biologicalprocesses is evident

With the development of multifunctional scanningprobes with tip-integrated biosensors a versatile analyticaltool for the investigation of complex biological systemshas been realized AFM cantilever-based biosensors pro-vide an excellent concept for simultaneous detection oftopographical and chemical information at the surface ofbiological species with high lateral resolution With theintegration of an electroactive area into AFM cantileversenabling subsequent surface modi cation and leading tobiosensor functionality an extension of this concept is pre-sented for detecting electroactive and -inactive speciesgenerated at the sample surface Accurate positioning andscanning of nanobiosensors at a de ned distance above thesample allows quanti cation of kinetic bioprocesses at the

surface-grafted cross-linked protein gel at the aminatedsurface of the self-assembled monolayer covering the inte-grated electrode The electrochemical signal generation isbased on the following enzyme catalyzed reactions(1) H2O2 is reduced to water by the oxidoreductase perox-idase and (2) a metal organic electron donor for the enzy-matic reaction (hydroxyl methyl ferrocene FMA contain-ing Fe21) added to the solution is oxidized during theenzymatic conversion to ferrocinium methylhydroxide(FMA1 containing Fe31) The electroactive by-product ofthe enzymatic reaction FMA1 diffuses to a microstruc-tured electrode array which was used as a model sampleThe microstructured electrode array is biased at a potentialof 50 mV vs AgAgCl At this potential only FMA1 andno other species in solution is reduced Hence informationon the local (electro)chemical activity (Figure 11Bb) withrespect to the position of the tip-integrated biosensor isprovided (Figure 11Ba topography) Comparison with thecontrol experiment in the absence of H2O2 (Figure 11B ctopography B d electrochemical image) demonstratesthe successful integration of a biosensor into a SECM-AFM probe and simultaneous activity imaging during AFMmeasurements17

The presented technique is by no means limited to theintegration of biosensors and can be extended to potentio-metric sensors such as pH microsensors or amalgam elec-trodes Many biological processes including enzymaticreactions biocorrosion and metabolic processes of micro-organisms involve or are driven by pH changes Hencesimultaneous AFM imaging and mapping of local pHmodulations is of substantial interest For example anti-mony and iridiumiridium oxide electrodes have beenreported for electrochemical pH measurements of biolog-ical systems115ndash117 As thin lms of these materials can besputtered the fabrication steps described above for the in-tegration of gold and platinum electrodes can be modi edand optimized to integrate pH-sensitive electrochemicalsensors into AFM tips

5 CONCLUSIONS AND OUTLOOK

Cantilever-based (bio)sensors are versatile analytical sys-tems for the investigation of biological species as they canbe used in solution and are capable of transducing a varietyof different signals Applications in force spectroscopypermit measurements at the level of single moleculesHowever the limiting factor of this technique for imagingof biomolecules is its comparatively poor temporal resolu-tion However ongoing instrumental developments andcombination of AFM with complementary techniques willovercome these limitations in the near future

Using enzymes as recognition elements in biosensorscombines several advantages including high speci city andselectivity Recently advanced immobilization strategiesand mass fabrication based on silicon technology allow

RE

VIE

W

12 Sensor Lett Vol 1 No 1 2003

25 J H Hoh and P K Hansma Trends Cell Biol 2 208 (1992)26 H Oberleithner G Giebisch and J Geibel P uumlgers Arch 425 506

(1993)27 J Mou S J Sheng R Ho and Z Shao Biophys J 71 2213

(1996)28 J Mou D M Czajkowsky S J Sheng R Ho and Z Shao FEBS

Lett 381 161 (1996)29 D J Muller and A Engel J Mol Biol 285 1347 (1999)30 F Oesterhelt D Oesterhelt M Pfeiffer A Engel H E Gaub and

D Muller Science 288 143 (2000)31 A F Oberhauser P E Marsyale M Carrion-Vazquez and J M

Fernandez Nat Struct Biol 6 1025 (1999)32 M Rief M Gautel F Oesterheit J M Fernandez and H E Gaub

Science 276 1109 (1997)33 M B Viani L I Pietrasanta J B Thompson A Chand I C

Gebeshuber J H Kindt M Richter H G Hansma and P KHansma Nat Struct Biol 7 644 (2000)

34 M Radmacher M Fritz H G Hansma and P K Hansma Science265 1577 (1994)

35 A Noy C D Frisbie L F Rozsnyai M S Wrighton and C MLieber J Am Chem Soc 117 7943 (1995)

36 A M Moulin S J OrsquoShea and M E Welland Ultramicroscopy82 23 (2000)

37 E-L Florin V T Moy and H E Gaub Science 264 415 (1994)38 G U Lee D A Kidwell and R J Colton Langmuir 10 354

(1994)39 A Janshoff M Neitzert Y Oberdoumlrfer and H Fuchs Angew

Chemie Int Ed 39 3212 (2000)40 O H Willemsen M M E Snel A Cambi J Greve B G De

Grooth and C G Figdor Biophys J 79 3267 (2000)41 M Carrion-Vazquez A F Oberhauser T E Fisher P E

Marszalek H Li and J M Fernandez Prog Biophys Mol Biol74 63 (2000)

42 J Zlatanova S M Lindsay and S H Leuba Prog Biophys MolBiol 74 37 (2000)

43 P Hinterdorfer W Baumgartner H J Gruber K Schilcher andH Schindler Proc Natl Acad Sci USA 93 3477 (1996)

44 P Hinterdorfer K Schilcher W Baumgartner H J Gruber andH Schindler Nanobiology 4 39 (1998)

45 R Ros F Schwesinger D Anselmetti M Kubon R SchaeferA Pluumlckthun and L Tiefenauer Proc Natl Acad Sci USA 957402 (1998)

46 F Schwesinger R Ros T Strunz D Anselmetti H-J GuumlntherodtA Honegger L Jermutus L Tiefenauer and A Pluumlckthun ProcNatl Acad Sci USA 97 9972 (2000)

47 R Merkel P Nassoy A Leung A Ritchie and K Evans Nature397 50 (1999)

48 T Strunz K Oroszlan I Schumakovitch H-J Guumlntherodt andM Hegner Biophys J 79 1206 (2000)

49 O H Willemsen M M E Snel K O van der Werf B G DeGrooth J Greve P Hinterdorfer H J Gruber H Schindler Y vanKooyk and C G Figdor Biophys J 75 2220 (1998)

50 M Ludwig W Dettmann and H E Gaub Biophys J 72 445(1997)

51 D P Allison P Hinterdorfer and W Han Curr Opin Biotechnol13 47 (2002)

52 W Han S M Lindsay and T Jing Appl Phys Lett 69 4111(1996)

53 A Raab W Han D Badt S J Smith-Gill S M LindsayH Schindler and P Hinterdorfer Nat Biotechnol 17 902 (1999)

54 G Schuumltz M Sonnleitner P Hinterdorfer and H Schindler MolMembr Biol 17 17 (2000)

55 S W Schneider M E Egan B P Jena W B GugginoH Oberleithner and J P Geibel Proc Natl Acad Sci USA 9612180 (1999)

56 J Finer R M Simmons and J A Spudnich Nature 368 113(1994)

sample surface with high lateral and temporal resolutionGiven the exibility of the described concepts there is con-siderable scope for extended use of multifunctional scan-ning probe systems probing a wide range of biologicalspecimens and processes

Acknowledgments The National Science Foundation(grant 0216368 in the program ldquoBiocomplexity in theEnvironmentrdquo) the National Institutes of Health (grantEB000508) and the ldquoFonds zur Foumlrderung der wissenschaft-lichen Forschungrdquo Austria (grants P14122-CHE and J2230)are greatly acknowledged PCT patents123 124on integratedAFM-SECM are issued

References and Notes

1 A Hulanicki S Glab and F Ingman Pure Appl Chem 63 1247(1991)

2 W Goepel T A Jones M Kleitz I Lundstroem and T SeiyamaEditors Chemical and Biochemical Sensors VCH Weinheim(1992) Vols 2 and 3

3 F W Scheller U Wollenberger A Warsinke and F Lisdat CurrOpin Biotechnol 12 35 (2001)

4 D R Thevenot K Toth R A Durst and G S Wilson BiosensBioelectron 16 121 (2001)

5 M Keusgen Naturwissenschaften 89 433 (2002)6 T H Park and M L Shuler Biotechnol Prog 19 243 (2003)7 K Sato A Hibara M Tokeshi H Hisamoto and T Kitamori Adv

Drug Delivery Rev 55 3791 (2003)8 P N Bartlett Handbook of Chemical and Biological Sensors

edited by R F Taylor Arthur D Little Inc USA J S SchultzUniversity of Pittsburgh (1996) pp 139ff

9 A Hierlemann O Brand C Hagleiter and H Baltes Proc IEEE91 839 (2003)

10 T E Fisher P E Marszalek A F Oberhauser M Carrion-Vazquez and J M Fernandez J Physiol (London) 520 5 (1999)

11 J K Gimzewski and C Joachim Science 283 1683 (1999)12 S O Vansteenkiste M C Davis C J Roberts S J B Tendler

and P M Williams Prog Surf Sci 57 95 (1998)13 M A Poggi L A Bottomley and P T Lillehei Anal Chem 74

2851 (2002)14 G Binnig H Rohrer C Gerber and E Weibel Appl Phys Lett

40 178 (1982)15 P K Hansma J P Cleveland M Radmacher D A Walters P E

Hillner M Bezanilla M Fritz D Vie H G Hansma C B PraterJ Massie L Fukunaga J Gurley and V Elings Appl Phys Lett64 1738 (1994)

16 W H Scouten J H T Luong and S R Brown Trends Biotechnol13 178 (1995)

17 C Kranz A Kueng A Lugstein E Bertagnolli and B Mizaikoffmanuscript in preparation

18 G Binnig C F Quate and C Gerber Phys Rev Lett 56 930 (1986)19 G Charras P Lehenkari and M Horton Methods Cell Biol 68

171 (2002)20 J H Kindt J C Sitko L I Pietrasanta E Oroudjev N Becker

M B Viani and H G Hansma Methods Cell Biol 68 213 (2002)21 B Drake C B Prater A L Weisenhorn S A C Gould T R

Albrecht C F Quate D S Cannell H G Hansma and P KHansma Science 243 1586 (1989)

22 D Rugar and P K Hansma Phys Today 43 23 (1990)23 M Radmacher R W Tillmann M Fritz and H E Gaub Science

157 1900 (1992)24 E Henderson P G Haydon and D S Sakaguchi Science 257

1944 (1992)

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VIE

W

Sensor Lett Vol 1 No 1 2003 13

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VIE

W

micro- and nanobiosensors at a well-de ned and deliber-ately selected distance above the sample surface Standardsurface modi cation processes for biosensors as previ-ously outlined in this review can be used to immobilizebiological recognition elements at the surface of the inte-grated electrode (Figure 10) With the use of electrochemicaltechniques the site of immobilization is con ned to theelectrode surface providing highly localized and repro-ducible integration of a biosensing interface64 65 67 68

With the immobilization of enzymes via self-assembledthiol monolayers with reactive headgroups114 conversion

of electroactive and -inactive species generated at the sam-ple surface is enabled

Figure 11 illustrates simultaneously recorded topo-graphical and electrochemical images obtained with a tip-integrated biosensor The biosensor is based on horse-radish peroxidase immobilized at the electrode surface ofthe integrated SECM-AFM probe (Figure 11A) The per-oxidase activity was imaged in tip generatorsubstrate col-lector mode of SECM (see also Figure 11Bb) duringcontact-mode AFM operation The enzyme was immobi-lized by chemisorption of a functionalized thiol monolayer(cystaminium chloride) to the tip-integrated gold micro-electrode Peroxidase was attached by the addition of amixture of glutaraldehyde and peroxidase resulting in a

Figure 11 (A) Schematic presentation of the tip generationsubstratecollection mode experiment involving simultaneous detection of tipintegrated enzyme activity and AFM topography (B) Simultaneouslyrecorded topographical information and tip integrated peroxidase activityin contact mode The current measured at the microelectrode arrayre ects the enzyme activity integrated in the AFM tip Top view of theheight (a) and (c) and simultaneously recorded current (b) The electro-chemical response with 05 mmol L21 H2O2 in solution Images arerecorded in 2 mmol L21 FMA 01 mol L21 KCl in phosphate buffer (01mol L21 pH 70) (d) In the absence of the enzymatic substrate H2O2The tip was held at a potential of 005 V vs AgAgCl Electrode edgelength 860 nm tip height 410 nm scan rate 1 Hz

Figure 10 Schematic cross section of an AFM tip integrated biosensorEnzymes can be immobilized at the electrode surface integrated in thescanning probe tip via self-assembled thiol monolayers with functional-ized headgroups (A) or via electrochemical deposition of enzyme-containing polymer lms (B)

Figure 9 Simultaneously recorded height and current images of glu-cose oxidase activity in AFM tapping mode and SECM substrate genera-tiontip collection mode Top view of the height (a and d) correspondingamplitude (b and e) and simultaneously recorded current (c and f) imagesrecorded in air-saturated phosphate buffer in absence (andashc) and in thepresence (dndashf) of the substrate glucose in solution Reprinted with per-mission from Ref 112 A Kueng et al Angew Chem Int Ed 42 3237(2003) copy2003 Wiley

Sensor Lett Vol 1 No 1 2003 11

miniaturization of biosensors However only a few ap-proaches have been reported that use scanning micro- ornanobiosensors to obtain laterally resolved (bio)chemicalinformation Using shear force modes in combination withSECM permits positioning of nonamperometric sensorsImprovement of the achievable lateral resolution by de-creasing the dimensions of the applied sensor is the focusof ongoing research in this eld Fiber-based optical micro-biosensors in combination with NSOM techniques mightbe an alternative future concept for obtaining laterally re-solved information on bioactivity AFM tip-integrated bio-sensors provide a promising concept combining advantagesof various techniques in a single microfabricated probeHigh reproducibility of the fabrication process and the po-tential of miniaturization down to the nanometer scalemake them a versatile tool for future applications for prob-ing a wide range of biological substrates and processes

Among the major drawbacks of biosensors in general aresigni cantly increased response times of enzyme-modi edelectrodes compared with direct electrochemical detectionMany biologically relevant processes (eg exocytoticevents) relay signals in intervals of milliseconds Elec-trodes have been fabricated that can detect neurotransmit-ters with response times of 300 ms118 119 However re-sponse times less than 100 ms are needed to accuratelyquantify dynamics at the cellular level and less than 10 msto examine exocytosis of nonelectroactive neurotransmit-ters However it has been demonstrated that the temporalresolution achievable with biosensor systems scales in fa-vor of the proposed technology with device miniaturiza-tion120 121 As an example response times on the order of85 ms have been reported for a glucose-speci c biosensorwith an electrode diameter decreased to the submicronrange122 As recently demonstrated by our research groupdisc electrodes with diameters below 200 nm can be inte-grated into AFM tips by microfabrication techniques112 Itis expected that surface modi cation of such nanoelec-trodes as outlined in this contribution will result in a furtherdecrease in the integrated biosensor response time With arealistic perspective of measurements in the time domain ofmicroseconds suitability for investigation of biologicalprocesses is evident

With the development of multifunctional scanningprobes with tip-integrated biosensors a versatile analyticaltool for the investigation of complex biological systemshas been realized AFM cantilever-based biosensors pro-vide an excellent concept for simultaneous detection oftopographical and chemical information at the surface ofbiological species with high lateral resolution With theintegration of an electroactive area into AFM cantileversenabling subsequent surface modi cation and leading tobiosensor functionality an extension of this concept is pre-sented for detecting electroactive and -inactive speciesgenerated at the sample surface Accurate positioning andscanning of nanobiosensors at a de ned distance above thesample allows quanti cation of kinetic bioprocesses at the

surface-grafted cross-linked protein gel at the aminatedsurface of the self-assembled monolayer covering the inte-grated electrode The electrochemical signal generation isbased on the following enzyme catalyzed reactions(1) H2O2 is reduced to water by the oxidoreductase perox-idase and (2) a metal organic electron donor for the enzy-matic reaction (hydroxyl methyl ferrocene FMA contain-ing Fe21) added to the solution is oxidized during theenzymatic conversion to ferrocinium methylhydroxide(FMA1 containing Fe31) The electroactive by-product ofthe enzymatic reaction FMA1 diffuses to a microstruc-tured electrode array which was used as a model sampleThe microstructured electrode array is biased at a potentialof 50 mV vs AgAgCl At this potential only FMA1 andno other species in solution is reduced Hence informationon the local (electro)chemical activity (Figure 11Bb) withrespect to the position of the tip-integrated biosensor isprovided (Figure 11Ba topography) Comparison with thecontrol experiment in the absence of H2O2 (Figure 11B ctopography B d electrochemical image) demonstratesthe successful integration of a biosensor into a SECM-AFM probe and simultaneous activity imaging during AFMmeasurements17

The presented technique is by no means limited to theintegration of biosensors and can be extended to potentio-metric sensors such as pH microsensors or amalgam elec-trodes Many biological processes including enzymaticreactions biocorrosion and metabolic processes of micro-organisms involve or are driven by pH changes Hencesimultaneous AFM imaging and mapping of local pHmodulations is of substantial interest For example anti-mony and iridiumiridium oxide electrodes have beenreported for electrochemical pH measurements of biolog-ical systems115ndash117 As thin lms of these materials can besputtered the fabrication steps described above for the in-tegration of gold and platinum electrodes can be modi edand optimized to integrate pH-sensitive electrochemicalsensors into AFM tips

5 CONCLUSIONS AND OUTLOOK

Cantilever-based (bio)sensors are versatile analytical sys-tems for the investigation of biological species as they canbe used in solution and are capable of transducing a varietyof different signals Applications in force spectroscopypermit measurements at the level of single moleculesHowever the limiting factor of this technique for imagingof biomolecules is its comparatively poor temporal resolu-tion However ongoing instrumental developments andcombination of AFM with complementary techniques willovercome these limitations in the near future

Using enzymes as recognition elements in biosensorscombines several advantages including high speci city andselectivity Recently advanced immobilization strategiesand mass fabrication based on silicon technology allow

RE

VIE

W

12 Sensor Lett Vol 1 No 1 2003

25 J H Hoh and P K Hansma Trends Cell Biol 2 208 (1992)26 H Oberleithner G Giebisch and J Geibel P uumlgers Arch 425 506

(1993)27 J Mou S J Sheng R Ho and Z Shao Biophys J 71 2213

(1996)28 J Mou D M Czajkowsky S J Sheng R Ho and Z Shao FEBS

Lett 381 161 (1996)29 D J Muller and A Engel J Mol Biol 285 1347 (1999)30 F Oesterhelt D Oesterhelt M Pfeiffer A Engel H E Gaub and

D Muller Science 288 143 (2000)31 A F Oberhauser P E Marsyale M Carrion-Vazquez and J M

Fernandez Nat Struct Biol 6 1025 (1999)32 M Rief M Gautel F Oesterheit J M Fernandez and H E Gaub

Science 276 1109 (1997)33 M B Viani L I Pietrasanta J B Thompson A Chand I C

Gebeshuber J H Kindt M Richter H G Hansma and P KHansma Nat Struct Biol 7 644 (2000)

34 M Radmacher M Fritz H G Hansma and P K Hansma Science265 1577 (1994)

35 A Noy C D Frisbie L F Rozsnyai M S Wrighton and C MLieber J Am Chem Soc 117 7943 (1995)

36 A M Moulin S J OrsquoShea and M E Welland Ultramicroscopy82 23 (2000)

37 E-L Florin V T Moy and H E Gaub Science 264 415 (1994)38 G U Lee D A Kidwell and R J Colton Langmuir 10 354

(1994)39 A Janshoff M Neitzert Y Oberdoumlrfer and H Fuchs Angew

Chemie Int Ed 39 3212 (2000)40 O H Willemsen M M E Snel A Cambi J Greve B G De

Grooth and C G Figdor Biophys J 79 3267 (2000)41 M Carrion-Vazquez A F Oberhauser T E Fisher P E

Marszalek H Li and J M Fernandez Prog Biophys Mol Biol74 63 (2000)

42 J Zlatanova S M Lindsay and S H Leuba Prog Biophys MolBiol 74 37 (2000)

43 P Hinterdorfer W Baumgartner H J Gruber K Schilcher andH Schindler Proc Natl Acad Sci USA 93 3477 (1996)

44 P Hinterdorfer K Schilcher W Baumgartner H J Gruber andH Schindler Nanobiology 4 39 (1998)

45 R Ros F Schwesinger D Anselmetti M Kubon R SchaeferA Pluumlckthun and L Tiefenauer Proc Natl Acad Sci USA 957402 (1998)

46 F Schwesinger R Ros T Strunz D Anselmetti H-J GuumlntherodtA Honegger L Jermutus L Tiefenauer and A Pluumlckthun ProcNatl Acad Sci USA 97 9972 (2000)

47 R Merkel P Nassoy A Leung A Ritchie and K Evans Nature397 50 (1999)

48 T Strunz K Oroszlan I Schumakovitch H-J Guumlntherodt andM Hegner Biophys J 79 1206 (2000)

49 O H Willemsen M M E Snel K O van der Werf B G DeGrooth J Greve P Hinterdorfer H J Gruber H Schindler Y vanKooyk and C G Figdor Biophys J 75 2220 (1998)

50 M Ludwig W Dettmann and H E Gaub Biophys J 72 445(1997)

51 D P Allison P Hinterdorfer and W Han Curr Opin Biotechnol13 47 (2002)

52 W Han S M Lindsay and T Jing Appl Phys Lett 69 4111(1996)

53 A Raab W Han D Badt S J Smith-Gill S M LindsayH Schindler and P Hinterdorfer Nat Biotechnol 17 902 (1999)

54 G Schuumltz M Sonnleitner P Hinterdorfer and H Schindler MolMembr Biol 17 17 (2000)

55 S W Schneider M E Egan B P Jena W B GugginoH Oberleithner and J P Geibel Proc Natl Acad Sci USA 9612180 (1999)

56 J Finer R M Simmons and J A Spudnich Nature 368 113(1994)

sample surface with high lateral and temporal resolutionGiven the exibility of the described concepts there is con-siderable scope for extended use of multifunctional scan-ning probe systems probing a wide range of biologicalspecimens and processes

Acknowledgments The National Science Foundation(grant 0216368 in the program ldquoBiocomplexity in theEnvironmentrdquo) the National Institutes of Health (grantEB000508) and the ldquoFonds zur Foumlrderung der wissenschaft-lichen Forschungrdquo Austria (grants P14122-CHE and J2230)are greatly acknowledged PCT patents123 124on integratedAFM-SECM are issued

References and Notes

1 A Hulanicki S Glab and F Ingman Pure Appl Chem 63 1247(1991)

2 W Goepel T A Jones M Kleitz I Lundstroem and T SeiyamaEditors Chemical and Biochemical Sensors VCH Weinheim(1992) Vols 2 and 3

3 F W Scheller U Wollenberger A Warsinke and F Lisdat CurrOpin Biotechnol 12 35 (2001)

4 D R Thevenot K Toth R A Durst and G S Wilson BiosensBioelectron 16 121 (2001)

5 M Keusgen Naturwissenschaften 89 433 (2002)6 T H Park and M L Shuler Biotechnol Prog 19 243 (2003)7 K Sato A Hibara M Tokeshi H Hisamoto and T Kitamori Adv

Drug Delivery Rev 55 3791 (2003)8 P N Bartlett Handbook of Chemical and Biological Sensors

edited by R F Taylor Arthur D Little Inc USA J S SchultzUniversity of Pittsburgh (1996) pp 139ff

9 A Hierlemann O Brand C Hagleiter and H Baltes Proc IEEE91 839 (2003)

10 T E Fisher P E Marszalek A F Oberhauser M Carrion-Vazquez and J M Fernandez J Physiol (London) 520 5 (1999)

11 J K Gimzewski and C Joachim Science 283 1683 (1999)12 S O Vansteenkiste M C Davis C J Roberts S J B Tendler

and P M Williams Prog Surf Sci 57 95 (1998)13 M A Poggi L A Bottomley and P T Lillehei Anal Chem 74

2851 (2002)14 G Binnig H Rohrer C Gerber and E Weibel Appl Phys Lett

40 178 (1982)15 P K Hansma J P Cleveland M Radmacher D A Walters P E

Hillner M Bezanilla M Fritz D Vie H G Hansma C B PraterJ Massie L Fukunaga J Gurley and V Elings Appl Phys Lett64 1738 (1994)

16 W H Scouten J H T Luong and S R Brown Trends Biotechnol13 178 (1995)

17 C Kranz A Kueng A Lugstein E Bertagnolli and B Mizaikoffmanuscript in preparation

18 G Binnig C F Quate and C Gerber Phys Rev Lett 56 930 (1986)19 G Charras P Lehenkari and M Horton Methods Cell Biol 68

171 (2002)20 J H Kindt J C Sitko L I Pietrasanta E Oroudjev N Becker

M B Viani and H G Hansma Methods Cell Biol 68 213 (2002)21 B Drake C B Prater A L Weisenhorn S A C Gould T R

Albrecht C F Quate D S Cannell H G Hansma and P KHansma Science 243 1586 (1989)

22 D Rugar and P K Hansma Phys Today 43 23 (1990)23 M Radmacher R W Tillmann M Fritz and H E Gaub Science

157 1900 (1992)24 E Henderson P G Haydon and D S Sakaguchi Science 257

1944 (1992)

RE

VIE

W

Sensor Lett Vol 1 No 1 2003 13

miniaturization of biosensors However only a few ap-proaches have been reported that use scanning micro- ornanobiosensors to obtain laterally resolved (bio)chemicalinformation Using shear force modes in combination withSECM permits positioning of nonamperometric sensorsImprovement of the achievable lateral resolution by de-creasing the dimensions of the applied sensor is the focusof ongoing research in this eld Fiber-based optical micro-biosensors in combination with NSOM techniques mightbe an alternative future concept for obtaining laterally re-solved information on bioactivity AFM tip-integrated bio-sensors provide a promising concept combining advantagesof various techniques in a single microfabricated probeHigh reproducibility of the fabrication process and the po-tential of miniaturization down to the nanometer scalemake them a versatile tool for future applications for prob-ing a wide range of biological substrates and processes

Among the major drawbacks of biosensors in general aresigni cantly increased response times of enzyme-modi edelectrodes compared with direct electrochemical detectionMany biologically relevant processes (eg exocytoticevents) relay signals in intervals of milliseconds Elec-trodes have been fabricated that can detect neurotransmit-ters with response times of 300 ms118 119 However re-sponse times less than 100 ms are needed to accuratelyquantify dynamics at the cellular level and less than 10 msto examine exocytosis of nonelectroactive neurotransmit-ters However it has been demonstrated that the temporalresolution achievable with biosensor systems scales in fa-vor of the proposed technology with device miniaturiza-tion120 121 As an example response times on the order of85 ms have been reported for a glucose-speci c biosensorwith an electrode diameter decreased to the submicronrange122 As recently demonstrated by our research groupdisc electrodes with diameters below 200 nm can be inte-grated into AFM tips by microfabrication techniques112 Itis expected that surface modi cation of such nanoelec-trodes as outlined in this contribution will result in a furtherdecrease in the integrated biosensor response time With arealistic perspective of measurements in the time domain ofmicroseconds suitability for investigation of biologicalprocesses is evident

With the development of multifunctional scanningprobes with tip-integrated biosensors a versatile analyticaltool for the investigation of complex biological systemshas been realized AFM cantilever-based biosensors pro-vide an excellent concept for simultaneous detection oftopographical and chemical information at the surface ofbiological species with high lateral resolution With theintegration of an electroactive area into AFM cantileversenabling subsequent surface modi cation and leading tobiosensor functionality an extension of this concept is pre-sented for detecting electroactive and -inactive speciesgenerated at the sample surface Accurate positioning andscanning of nanobiosensors at a de ned distance above thesample allows quanti cation of kinetic bioprocesses at the

surface-grafted cross-linked protein gel at the aminatedsurface of the self-assembled monolayer covering the inte-grated electrode The electrochemical signal generation isbased on the following enzyme catalyzed reactions(1) H2O2 is reduced to water by the oxidoreductase perox-idase and (2) a metal organic electron donor for the enzy-matic reaction (hydroxyl methyl ferrocene FMA contain-ing Fe21) added to the solution is oxidized during theenzymatic conversion to ferrocinium methylhydroxide(FMA1 containing Fe31) The electroactive by-product ofthe enzymatic reaction FMA1 diffuses to a microstruc-tured electrode array which was used as a model sampleThe microstructured electrode array is biased at a potentialof 50 mV vs AgAgCl At this potential only FMA1 andno other species in solution is reduced Hence informationon the local (electro)chemical activity (Figure 11Bb) withrespect to the position of the tip-integrated biosensor isprovided (Figure 11Ba topography) Comparison with thecontrol experiment in the absence of H2O2 (Figure 11B ctopography B d electrochemical image) demonstratesthe successful integration of a biosensor into a SECM-AFM probe and simultaneous activity imaging during AFMmeasurements17

The presented technique is by no means limited to theintegration of biosensors and can be extended to potentio-metric sensors such as pH microsensors or amalgam elec-trodes Many biological processes including enzymaticreactions biocorrosion and metabolic processes of micro-organisms involve or are driven by pH changes Hencesimultaneous AFM imaging and mapping of local pHmodulations is of substantial interest For example anti-mony and iridiumiridium oxide electrodes have beenreported for electrochemical pH measurements of biolog-ical systems115ndash117 As thin lms of these materials can besputtered the fabrication steps described above for the in-tegration of gold and platinum electrodes can be modi edand optimized to integrate pH-sensitive electrochemicalsensors into AFM tips

5 CONCLUSIONS AND OUTLOOK

Cantilever-based (bio)sensors are versatile analytical sys-tems for the investigation of biological species as they canbe used in solution and are capable of transducing a varietyof different signals Applications in force spectroscopypermit measurements at the level of single moleculesHowever the limiting factor of this technique for imagingof biomolecules is its comparatively poor temporal resolu-tion However ongoing instrumental developments andcombination of AFM with complementary techniques willovercome these limitations in the near future

Using enzymes as recognition elements in biosensorscombines several advantages including high speci city andselectivity Recently advanced immobilization strategiesand mass fabrication based on silicon technology allow

RE

VIE

W

12 Sensor Lett Vol 1 No 1 2003

25 J H Hoh and P K Hansma Trends Cell Biol 2 208 (1992)26 H Oberleithner G Giebisch and J Geibel P uumlgers Arch 425 506

(1993)27 J Mou S J Sheng R Ho and Z Shao Biophys J 71 2213

(1996)28 J Mou D M Czajkowsky S J Sheng R Ho and Z Shao FEBS

Lett 381 161 (1996)29 D J Muller and A Engel J Mol Biol 285 1347 (1999)30 F Oesterhelt D Oesterhelt M Pfeiffer A Engel H E Gaub and

D Muller Science 288 143 (2000)31 A F Oberhauser P E Marsyale M Carrion-Vazquez and J M

Fernandez Nat Struct Biol 6 1025 (1999)32 M Rief M Gautel F Oesterheit J M Fernandez and H E Gaub

Science 276 1109 (1997)33 M B Viani L I Pietrasanta J B Thompson A Chand I C

Gebeshuber J H Kindt M Richter H G Hansma and P KHansma Nat Struct Biol 7 644 (2000)

34 M Radmacher M Fritz H G Hansma and P K Hansma Science265 1577 (1994)

35 A Noy C D Frisbie L F Rozsnyai M S Wrighton and C MLieber J Am Chem Soc 117 7943 (1995)

36 A M Moulin S J OrsquoShea and M E Welland Ultramicroscopy82 23 (2000)

37 E-L Florin V T Moy and H E Gaub Science 264 415 (1994)38 G U Lee D A Kidwell and R J Colton Langmuir 10 354

(1994)39 A Janshoff M Neitzert Y Oberdoumlrfer and H Fuchs Angew

Chemie Int Ed 39 3212 (2000)40 O H Willemsen M M E Snel A Cambi J Greve B G De

Grooth and C G Figdor Biophys J 79 3267 (2000)41 M Carrion-Vazquez A F Oberhauser T E Fisher P E

Marszalek H Li and J M Fernandez Prog Biophys Mol Biol74 63 (2000)

42 J Zlatanova S M Lindsay and S H Leuba Prog Biophys MolBiol 74 37 (2000)

43 P Hinterdorfer W Baumgartner H J Gruber K Schilcher andH Schindler Proc Natl Acad Sci USA 93 3477 (1996)

44 P Hinterdorfer K Schilcher W Baumgartner H J Gruber andH Schindler Nanobiology 4 39 (1998)

45 R Ros F Schwesinger D Anselmetti M Kubon R SchaeferA Pluumlckthun and L Tiefenauer Proc Natl Acad Sci USA 957402 (1998)

46 F Schwesinger R Ros T Strunz D Anselmetti H-J GuumlntherodtA Honegger L Jermutus L Tiefenauer and A Pluumlckthun ProcNatl Acad Sci USA 97 9972 (2000)

47 R Merkel P Nassoy A Leung A Ritchie and K Evans Nature397 50 (1999)

48 T Strunz K Oroszlan I Schumakovitch H-J Guumlntherodt andM Hegner Biophys J 79 1206 (2000)

49 O H Willemsen M M E Snel K O van der Werf B G DeGrooth J Greve P Hinterdorfer H J Gruber H Schindler Y vanKooyk and C G Figdor Biophys J 75 2220 (1998)

50 M Ludwig W Dettmann and H E Gaub Biophys J 72 445(1997)

51 D P Allison P Hinterdorfer and W Han Curr Opin Biotechnol13 47 (2002)

52 W Han S M Lindsay and T Jing Appl Phys Lett 69 4111(1996)

53 A Raab W Han D Badt S J Smith-Gill S M LindsayH Schindler and P Hinterdorfer Nat Biotechnol 17 902 (1999)

54 G Schuumltz M Sonnleitner P Hinterdorfer and H Schindler MolMembr Biol 17 17 (2000)

55 S W Schneider M E Egan B P Jena W B GugginoH Oberleithner and J P Geibel Proc Natl Acad Sci USA 9612180 (1999)

56 J Finer R M Simmons and J A Spudnich Nature 368 113(1994)

sample surface with high lateral and temporal resolutionGiven the exibility of the described concepts there is con-siderable scope for extended use of multifunctional scan-ning probe systems probing a wide range of biologicalspecimens and processes

Acknowledgments The National Science Foundation(grant 0216368 in the program ldquoBiocomplexity in theEnvironmentrdquo) the National Institutes of Health (grantEB000508) and the ldquoFonds zur Foumlrderung der wissenschaft-lichen Forschungrdquo Austria (grants P14122-CHE and J2230)are greatly acknowledged PCT patents123 124on integratedAFM-SECM are issued

References and Notes

1 A Hulanicki S Glab and F Ingman Pure Appl Chem 63 1247(1991)

2 W Goepel T A Jones M Kleitz I Lundstroem and T SeiyamaEditors Chemical and Biochemical Sensors VCH Weinheim(1992) Vols 2 and 3

3 F W Scheller U Wollenberger A Warsinke and F Lisdat CurrOpin Biotechnol 12 35 (2001)

4 D R Thevenot K Toth R A Durst and G S Wilson BiosensBioelectron 16 121 (2001)

5 M Keusgen Naturwissenschaften 89 433 (2002)6 T H Park and M L Shuler Biotechnol Prog 19 243 (2003)7 K Sato A Hibara M Tokeshi H Hisamoto and T Kitamori Adv

Drug Delivery Rev 55 3791 (2003)8 P N Bartlett Handbook of Chemical and Biological Sensors

edited by R F Taylor Arthur D Little Inc USA J S SchultzUniversity of Pittsburgh (1996) pp 139ff

9 A Hierlemann O Brand C Hagleiter and H Baltes Proc IEEE91 839 (2003)

10 T E Fisher P E Marszalek A F Oberhauser M Carrion-Vazquez and J M Fernandez J Physiol (London) 520 5 (1999)

11 J K Gimzewski and C Joachim Science 283 1683 (1999)12 S O Vansteenkiste M C Davis C J Roberts S J B Tendler

and P M Williams Prog Surf Sci 57 95 (1998)13 M A Poggi L A Bottomley and P T Lillehei Anal Chem 74

2851 (2002)14 G Binnig H Rohrer C Gerber and E Weibel Appl Phys Lett

40 178 (1982)15 P K Hansma J P Cleveland M Radmacher D A Walters P E

Hillner M Bezanilla M Fritz D Vie H G Hansma C B PraterJ Massie L Fukunaga J Gurley and V Elings Appl Phys Lett64 1738 (1994)

16 W H Scouten J H T Luong and S R Brown Trends Biotechnol13 178 (1995)

17 C Kranz A Kueng A Lugstein E Bertagnolli and B Mizaikoffmanuscript in preparation

18 G Binnig C F Quate and C Gerber Phys Rev Lett 56 930 (1986)19 G Charras P Lehenkari and M Horton Methods Cell Biol 68

171 (2002)20 J H Kindt J C Sitko L I Pietrasanta E Oroudjev N Becker

M B Viani and H G Hansma Methods Cell Biol 68 213 (2002)21 B Drake C B Prater A L Weisenhorn S A C Gould T R

Albrecht C F Quate D S Cannell H G Hansma and P KHansma Science 243 1586 (1989)

22 D Rugar and P K Hansma Phys Today 43 23 (1990)23 M Radmacher R W Tillmann M Fritz and H E Gaub Science

157 1900 (1992)24 E Henderson P G Haydon and D S Sakaguchi Science 257

1944 (1992)

RE

VIE

W

Sensor Lett Vol 1 No 1 2003 13

25 J H Hoh and P K Hansma Trends Cell Biol 2 208 (1992)26 H Oberleithner G Giebisch and J Geibel P uumlgers Arch 425 506

(1993)27 J Mou S J Sheng R Ho and Z Shao Biophys J 71 2213

(1996)28 J Mou D M Czajkowsky S J Sheng R Ho and Z Shao FEBS

Lett 381 161 (1996)29 D J Muller and A Engel J Mol Biol 285 1347 (1999)30 F Oesterhelt D Oesterhelt M Pfeiffer A Engel H E Gaub and

D Muller Science 288 143 (2000)31 A F Oberhauser P E Marsyale M Carrion-Vazquez and J M

Fernandez Nat Struct Biol 6 1025 (1999)32 M Rief M Gautel F Oesterheit J M Fernandez and H E Gaub

Science 276 1109 (1997)33 M B Viani L I Pietrasanta J B Thompson A Chand I C

Gebeshuber J H Kindt M Richter H G Hansma and P KHansma Nat Struct Biol 7 644 (2000)

34 M Radmacher M Fritz H G Hansma and P K Hansma Science265 1577 (1994)

35 A Noy C D Frisbie L F Rozsnyai M S Wrighton and C MLieber J Am Chem Soc 117 7943 (1995)

36 A M Moulin S J OrsquoShea and M E Welland Ultramicroscopy82 23 (2000)

37 E-L Florin V T Moy and H E Gaub Science 264 415 (1994)38 G U Lee D A Kidwell and R J Colton Langmuir 10 354

(1994)39 A Janshoff M Neitzert Y Oberdoumlrfer and H Fuchs Angew

Chemie Int Ed 39 3212 (2000)40 O H Willemsen M M E Snel A Cambi J Greve B G De

Grooth and C G Figdor Biophys J 79 3267 (2000)41 M Carrion-Vazquez A F Oberhauser T E Fisher P E

Marszalek H Li and J M Fernandez Prog Biophys Mol Biol74 63 (2000)

42 J Zlatanova S M Lindsay and S H Leuba Prog Biophys MolBiol 74 37 (2000)

43 P Hinterdorfer W Baumgartner H J Gruber K Schilcher andH Schindler Proc Natl Acad Sci USA 93 3477 (1996)

44 P Hinterdorfer K Schilcher W Baumgartner H J Gruber andH Schindler Nanobiology 4 39 (1998)

45 R Ros F Schwesinger D Anselmetti M Kubon R SchaeferA Pluumlckthun and L Tiefenauer Proc Natl Acad Sci USA 957402 (1998)

46 F Schwesinger R Ros T Strunz D Anselmetti H-J GuumlntherodtA Honegger L Jermutus L Tiefenauer and A Pluumlckthun ProcNatl Acad Sci USA 97 9972 (2000)

47 R Merkel P Nassoy A Leung A Ritchie and K Evans Nature397 50 (1999)

48 T Strunz K Oroszlan I Schumakovitch H-J Guumlntherodt andM Hegner Biophys J 79 1206 (2000)

49 O H Willemsen M M E Snel K O van der Werf B G DeGrooth J Greve P Hinterdorfer H J Gruber H Schindler Y vanKooyk and C G Figdor Biophys J 75 2220 (1998)

50 M Ludwig W Dettmann and H E Gaub Biophys J 72 445(1997)

51 D P Allison P Hinterdorfer and W Han Curr Opin Biotechnol13 47 (2002)

52 W Han S M Lindsay and T Jing Appl Phys Lett 69 4111(1996)

53 A Raab W Han D Badt S J Smith-Gill S M LindsayH Schindler and P Hinterdorfer Nat Biotechnol 17 902 (1999)

54 G Schuumltz M Sonnleitner P Hinterdorfer and H Schindler MolMembr Biol 17 17 (2000)

55 S W Schneider M E Egan B P Jena W B GugginoH Oberleithner and J P Geibel Proc Natl Acad Sci USA 9612180 (1999)

56 J Finer R M Simmons and J A Spudnich Nature 368 113(1994)

sample surface with high lateral and temporal resolutionGiven the exibility of the described concepts there is con-siderable scope for extended use of multifunctional scan-ning probe systems probing a wide range of biologicalspecimens and processes

Acknowledgments The National Science Foundation(grant 0216368 in the program ldquoBiocomplexity in theEnvironmentrdquo) the National Institutes of Health (grantEB000508) and the ldquoFonds zur Foumlrderung der wissenschaft-lichen Forschungrdquo Austria (grants P14122-CHE and J2230)are greatly acknowledged PCT patents123 124on integratedAFM-SECM are issued

References and Notes

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