document

This journal is c The Royal Society of Chemistry 2013 Chem. Soc. Rev.

Cite this: DOI: 10.1039/c3cs60207a

Breaking the concentration limit of opticalsingle-molecule detection

Phil Holzmeister, Guillermo P. Acuna, Dina Grohmann and Philip Tinnefeld*

Over the last decade, single-molecule detection has been successfully utilized in the life sciences and

materials science. Yet, single-molecule measurements only yield meaningful results when working in a

suitable, narrow concentration range. On the one hand, diffraction limits the minimal size of the

observation volume in optical single-molecule measurements and consequently a sample must be

adequately diluted so that only one molecule resides within the observation volume. On the other

hand, at ultra-low concentrations relevant for sensing, the detection volume has to be increased in

order to detect molecules in a reasonable timespan. This in turn results in the loss of an optimal signal-

to-noise ratio necessary for single-molecule detection. This review discusses the requirements for

effective single-molecule fluorescence applications, reflects on the motivation for the extension of the

dynamic concentration range of single-molecule measurements and reviews various approaches that

have been introduced recently to solve these issues. For the high-concentration limit, we identify four

promising strategies including molecular confinement, optical observation volume reduction, temporal

separation of signals and well-conceived experimental designs that specifically circumvent the high

concentration limit. The low concentration limit is addressed by increasing the measurement speed,

parallelization, signal amplification and preconcentration. The further development of these ideas will

expand our possibilities to interrogate research questions with the clarity and precision provided only

by the single-molecule approach.

Key learning points(1) Optical single-molecule measurements with diffraction-limited optics can only be performed in a limited concentration range from roughly 1 pM to 1 nM.(2) The majority of enzymatic reactions require higher concentrations (mM–mM), whereas diagnostics and biosensing require significantly lower concentrations(opM).(3) Approaches aiming to overcome the upper concentration barrier include the reduction of the observation volume, confinement of molecules, temporalseparation of fluorescent signals, and smart experimental designs that avoid the high concentration of labeled species.(4) Physical confinement of molecules and the reduction of the observation volume by nanophotonic devices are generally applicable strategies.(5) The low concentration barrier is tackled by increasing measurement speed often involving micro- and nanofluidics, optimizing the signal-to-noise ratio andpreconcentration.

1. Introduction

The field of single-molecule fluorescence spectroscopy hasgrown rapidly over the last two decades mainly because techni-cal and methodological developments increased the sensitivity andgave access to biological processes not observable before. Forexample, live observation of the catalytic reaction of individualenzymes, memory effects in enzymatic activity, the heterogeneous

behavior of subpopulations no longer masked by ensemble mea-surements, observation of short-lived intermediates or sensing ofpicomolar concentrations have been achieved. Stochastic multi-step processes like protein folding, the choreography of molecularmotors and machines and complex processes like prokaryotictranslation became amenable to single-molecule inquiry circum-venting the need for synchronization. The single-moleculeapproach was extended to complex intracellular environmentswhere the significance of stochastic effects in gene expressioncould be studied. Single-molecule detection also enabled super-resolution microscopy delivering crisp images through improvedresolution by more than one order of magnitude.

Braunschweig University of Technology, Institute for Physical & Theoretical

Chemistry, Hans-Sommer-Str. 10, 38106 Braunschweig, Germany.

E-mail: [email protected]; Fax: +49 531 391 5334; Tel: +49 531 391 5330

Received 17th June 2013

DOI: 10.1039/c3cs60207a

www.rsc.org/csr

Chem Soc Rev

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Chem. Soc. Rev. This journal is c The Royal Society of Chemistry 2013

The advantages of the single-molecule approach, namely thepossibility to detect static and dynamic heterogeneity in apopulation of molecules, are well illustrated considering theinformation gathered from single-enzyme studies. The activityof a single enzyme can be monitored by optical means when anunambiguous fluorescent readout such as a fluorogenic sub-strate is available. The non-fluorescent substrate becomesfluorescent upon turnover by the enzyme. This enables a oneby one observation of the reaction by monitoring the emissionof fluorescent bursts originating from the freshly convertedsubstrate. Such experiments revealed heterogeneity of activitybetween individual enzymes (static heterogeneity) as well asdynamic changes of enzyme activity of the same molecule overtime (dynamic heterogeneity).1–3

Given that the advantages of single-molecule techniques aregenerally established and that many technological barriers havebeen eliminated the question arises why single-molecule techniqueshave not dramatically impacted commercial applications e.g. in thefields of drug screening, quantitative biology, molecular diagnostics,bio-and chemical sensing, or even information processing. What arethe key limitations of optical single-molecule techniques that haveto be overcome to exploit the advantages offered by single-moleculetechniques for a broader range of applications?

In the case of enzymatic reactions, the availability of fluoro-genic substrates that are completely non-fluorescent before areaction and become sufficiently bright for single-moleculedetection after the turnover is unfortunately limited and notavailable for most enzymes.

Phil Holzmeister

Phil Holzmeister studied physicsat the University of Wurzburg andreceived his master’s degreein Physics from the Universityof Texas at Austin in 2010 forhis work on the interactions ofmolecular motors. He is currentlya PhD student in the group ofProf. Philip Tinnefeld atBraunschweig University of Tech-nology. His research interestsinclude single-molecule fluores-cence spectroscopy, photophysicsand plasmonics.

Guillermo P. Acuna

Guillermo P. Acuna (BuenosAires, Argentina, 1980) studiedPhysics at the Buenos AiresUniversity. In 2010 he obtainedhis PhD from the Ludwig-Maximilians-Universitat (Munich,Germany) under the supervision ofProf. Roland Kersting. Currentlyhe is a Research Assistant inthe NanoBioSciences group ofProf. Philip Tinnefeld at theTU Braunschweig (Braunschweig,Germany). His research interestsinclude single-molecule fluores-cence, plasmonics and nano-photonics.

Dina Grohmann

Dina Grohmann joined the Nano-BioSciences group at TechnischeUniversitat Braunschweig as aJunior Research Group Leader in2011. She studied biology atthe Heinrich-Heine UniversityDusseldorf and received herPhD in physical biochemistry in2006 working under thesupervision of Prof. Tobias Restleat the Max Planck Instituteof Molecular Physiology(Dortmund) and the Institute ofMolecular Medicine (University

of Lubeck). After graduating, she joined the group of Prof. FinnWerner at the Institute of Structural and Molecular Biology atUniversity College London as a postdoctoral researcher.Combining biophysics (single-molecule FRET) and biochemistry,her research aims to gain a deeper understanding of molecularmachineries central to biology including the transcriptionalapparatus.

Philip Tinnefeld

Philip Tinnefeld studied chemistryin Munster, Montpellier, andHeidelberg. In 2002, he receivedhis PhD from the Universityof Heidelberg in the group ofProf. J. Wolfrum. He carriedout postdoctoral research withS. Weiss (UCLA), F. C. deSchryver (KU Leuven) and M.Sauer (Bielefeld) on thedevelopment of single-moleculefluorescence techniques. Afterthree years as an associateprofessor of biophysics at the

Ludwig-Maximilians-Universitat Munchen he was appointedfull professor of biophysical chemistry at Braunschweig Universityof Technology in 2010. His research interests include thedevelopment of microscopy techniques and applications of DNAnanotechnology to study problems at the interface of physics,chemistry and biology.

Tutorial Review Chem Soc Rev

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Instead, a general strategy to observe single-molecule inter-actions is required. This general strategy is challenged by thelimited accessible dynamic concentration range of opticalsingle-molecule detection (see Fig. 1). To detect a single mole-cule the microscopic observation volume must only host asingle fluorescent molecule of interest. If diffraction-limited,a situation typically found for confocal microscopes and far-field optics, optimal focusing allows the illumination of a spotwith a diameter of approximately 250 nm leading to an excitedvolume of about 1 femtoliter. This corresponds to a concen-tration of 2 nM of fluorescently labelled molecules. Accordingly,at higher concentrations more than one molecule resides inthe observation volume prohibiting the detection of static anddynamic heterogeneity.

The problem with the high concentration limit is aggravatedby the nature of biology that commonly requires higher con-centrations. Many biomolecular interactions like protein–protein and protein–DNA interactions are transient and exhibitdissociation constants (KD) in the micromolar range. Similarly,the majority of enzyme substrate interactions as characterizedby the Michaelis–Menten constant KM take place in the micro-to millimolar range (see Fig. 2 for a histogram of typical KD andKM values). Similarly, for a bimolecular interaction, the con-centrations of the interacting molecules have to be in the nano-to millimolar range to obtain a significant fraction of themolecular complex. This is easily rationalized by the law ofmass action.5 The complex formation between two labeledreactants, A and B, with a dissociation constant of 1 mM willonly result in picomolar concentrations of the complex if thereactants are present at nanomolar concentrations. Thus thesignal of the complex AB is masked by the excess of A and Bonly. However, if the interaction was to be studied usingmicromolar concentrations, the complex AB would be equallyconcentrated as the reactants and could easily be detected.5

This discrepancy between affinities found in biologicalsystems and the limited dynamic concentration range forsingle-molecule detection has seriously hampered spreadingof single-molecule techniques because systems for single-moleculemeasurements have to be carefully selected.

The upper concentration barrier can in many respects alsobe translated into a temporal barrier. At equilibrium, inter-actions that occur at high concentrations are unlikely to beformed at low concentrations. Away from equilibrium, e.g. byrapid dilution and a quick measurement, the lifetime of thecomplex might be long enough to carry out a relevant single-molecule measurement. Assuming diffusion limited associa-tion rates of the order of 5 � 105 s�1 M�1 (ref. 6 and referencestherein), the lifetime of a biomolecular complex with a micro-molar binding constant is B1 s. This means that a techniquehas to be fast enough to characterize the complex quickly afterdilution. Therefore, we additionally depict estimated complexlifetimes together with the complex strength in Fig. 2. Further-more, the concentration as well as the temporal resolution ofthe most commonly used single-molecule techniques, i.e. con-focal and total internal reflection microscopy, are indicated.

Finally, the time scale of biomolecular interactions has to betaken into account, e.g. if binding and unbinding of interaction

Fig. 1 Single-molecule experiments are feasible in a narrow concentrationrange between pM and low nM. At very low concentrations ({pM) interestingfor sensing, the detection of the small number of molecules is time-consumingand generally impractical. Protein interactions and enzymatic activity oftenrequire concentrations larger than mM which renders the observation of anindividual molecule with diffraction-limited optics impossible.

Fig. 2 Comparison of the concentration range of optical single-molecule detec-tion with biologically relevant binding strengths and complex lifetimes. The toppanel shows normalized histograms of B113 000 KM and B4000 KD valuesextracted from BRENDA (white, http://www.brenda-enzymes.org/) and PDBbind(grey, http://www.pdbbind.org.cn/) in May 2013. In the right panel, the dis-sociation constants are converted to complex lifetimes assuming a diffusionlimited association rate constant of 5 � 105 M�1 s�1. Black dots in the centerindicate binding affinities of 29 randomly chosen antibody–antigen affinitiesmeasured with ensemble methods.4 Boxes indicate the concentration range aswell as the temporal resolution of two common single-molecule microscopytechniques. Temporal resolution is mainly limited by the frame rate for EMCCD-camera based measurements and by setup stability, respectively. Intersectionsbetween the boxes and the blue line represent accessible protein interactions.Association rates can significantly deviate from this estimation because theydepend on the size and shape of individual proteins and not all biologicalinteractions are diffusion controlled. For interactions limited by conformationalrearrangements slower association rates are observed whereas long rangeintermolecular forces, predominately electrostatic interactions, can lead to orderof magnitudes faster association rates. This results in longer or shorter complexlifetimes t at the same Kd value t = 1/koff = 1/(Kd � kass).

Chem Soc Rev Tutorial Review

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partners are to be visualized. Consider one reaction partnerattached to the surface while the other transiently binds to it. Inorder to analyze the binding and unbinding kinetics, sufficientstatistics of at least 10–20 binding events per molecule need tobe detected. Visualizing either very weak or very strong bindingcan both be challenging. In the case of weak interactions,binding events might be too short or happen too rarely to bedetected. For strong binders, the lifetime of a complex caneasily reach minutes or even hours requiring very stable setupsand long measurement times (see complex lifetimes in Fig. 2).7

At the other end of the concentration range, i.e. very smallconcentrations, one might wonder why the ultimate single-molecule sensitivity has not become the golden standard forsensing, trace analysis, environmental monitoring and molec-ular diagnostics. Many biomarkers for cancer, neurodegenera-tive diseases, and viral loads are present in body fluids atsmaller concentrations than what can conventionally bedetected with clinical assays. If single-molecule detection issimple, should it not be possible to detect them at ultra-lowconcentrations? Here, the size of the observation volume hasthe opposite effect: it is too small to be sensitive enough todetect molecules at lower than typically picomolar concentra-tions because molecules just do not diffuse through the smallobservation volume of single-molecule setups on reasonabletime scales.

As mentioned above, to achieve an average occupancy of onemolecule per diffraction-limited observation volume a concen-tration of approximately 2 nM is required. In the case ofbiomarker sensing, where the analyte concentration often liesin the femtomolar range, the observation volume has to beincreased by a factor of B1000 000 (i.e. 1 nanoliter) to reach acomparable occupancy. Such an increase of the observationvolume is, however, detrimental to the signal-to-noise ratio(SNR) so that single-molecule detection is obstructed. Histori-cally, the reduction of the probe volume had been a leitmotiv toachieve single-molecule detection in the first place. This isbecause the signal of a single molecule is in first approximationindependent of the size of the excitation volume whereasthe background interference, which may arise from Rayleigh-or Raman-scattering or fluorescent impurities scales linearlywith volume. Therefore, it is the loss of the SNR that preventssingle-molecule detection at ultra-low concentration. The keychallenge here is comparable to "finding the needle in thehaystack" since it takes a molecule a considerable amount oftime to reach a smaller detection volume by diffusion.

In summary, we have outlined that common single-moleculedetection schemes suffer from a narrow dynamic concentrationrange from roughly picomolar to lower nanomolar. This smalldynamic concentration range compromises the application ofinformation-rich single-molecule techniques to key problemsof biomolecular interactions and enzymatic activity on the onehand and to ultrasensitive sensing on the other hand.

In this review, we outline how the field started addressingthese issues more than a decade ago. We discuss currentstrategies employed for specific experimental challenges. Wewill focus on the upper concentration barrier first and then

extend our discussion to the low concentration SNR problem.Interestingly, as we will see, both concentration barriers aresimultaneously addressed by experimental techniques thatincrease the SNR in single-molecule experiments.

2. Experimental strategies aiming toovercome the high concentration limit

Experimentally, the problem of high concentrations of fluor-escent molecules in a diffraction-limited volume is that thesemolecules can no longer be identified and recorded as indivi-dual molecules. The most logical solution is to reduce theobservation volume to an even smaller volume which is justpopulated by a single molecule. This is, however, not simple aslight cannot be focused on an infinitely small volume. One wayto solve this problem is to reduce the efficient observationvolume by saturable optical transitions (as realized in stimu-lated emission depletion microscopy, for example)8 or to con-trol the wave nature of light e.g. using near-field optics as innear-field scanning optical microscopy. Nanophotonic struc-tures that exploit near-field optical effects can create many non-diffraction limited observation volumes in parallel and areadvantageous for single-molecule detection.

Instead of reducing the optical observation volume, highconcentrations can be created in locally defined volumes byconfining the molecules into small reaction containers. Here,instead of reducing the observation volume of the measure-ment, the space accessible to the molecules is restricted to sub-wavelength dimensions by enclosing them in nanovesicles orother nanostructures thus enabling measurements at high localconcentrations with diffraction-limited optics.

An alternative approach to circumvent the need for an ultra-small volume is to transfer the problem into the time domaincarrying out successive single-molecule localizations. Molec-ular complexes could, for example, be immobilized at highconcentrations, the supernatant be washed away and theinteraction be studied on the single-molecule level.9 The read-out is then carried out before the system has reached its lowconcentration equilibrium binding state.

Moreover, there are many clever experimental strategiesthat avoid the necessity to decrease the observation volume.A common approach is to use partly labelled populations of amolecule of interest. Here, labelled species are used at lowconcentration and are supplemented to reach high concentra-tions by an excess of unlabelled molecules. Binding of the twospecies has to be deduced indirectly by a physical parameterother than direct two-color colocalization. This sensing can forexample be a change of a fluorescent property of the labelledspecies. A simple example is the detection of transcriptionfactor binding to a doubly labelled DNA detected by a changeof fluorescence resonance energy transfer from a donor to anacceptor dye (FRET).10 In this specific case, change in FRETis the result of protein-induced bending of the DNA and hasbeen used to characterize binding and unbinding of transcrip-tion factors.


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In the following we elaborate on these four differentapproaches based on the reduction of observation volumes,confinement of molecules, temporal separation of fluorescentsignals, and well-conceived experimental designs that avoid thehigh concentration of labeled species.

2.1 Well-conceived experimental strategies and probe design

A particular outstanding achievement of the single-moleculeapproach was the discovery of a fluctuating rate constant forthe rate-limiting step in enzymatic reactions, a phenomenontermed dynamic disorder. Such measurements have changedour microscopic view of enzymatic reactions in that enzymemolecules are structurally heterogeneous and interconvertbetween different states on a broad range of time scales.

A very high temporal resolution is required to study a singleenzyme and single-turnover events. The turnover can com-monly not be followed by visualizing the interaction betweenthe enzyme and a fluorescently labelled substrate because ofthe relatively large Michaelis–Menten constants of enzymaticreactions (compare with Fig. 2). If the enzyme is immobilizedfor example, high concentrations of substrates are required. Inorder to avoid high concentration of fluorescently labelledsubstrates a well-conceived readout of the enzyme activity hasto be found. Some enzymes such as cholesterol oxidase arefluorescent themselves and alterations of the cofactor fluores-cence could be used to detect the catalytic turnover cycle in oneof the earliest single-enzyme studies.11 Certainly, the cofactoradenine dinucleotide (FAD) is not a favorable single-moleculefluorophore and only few enzymes exhibit sufficient fluores-cence that can be correlated with enzymatic activity.

A very elegant method to detect enzyme turnover is to labelthe enzyme with two fluorescent dyes for single-molecule FRET.FRET serves as a spectroscopic ruler by which the conforma-tional changes during the catalytic cycles can be directlydeduced from the changes in energy transfer.12,13 A relatedapproach is very useful for enzymes with an associated metal inthe active center that changes its redox state during thecatalytic cycle. The change of the redox state of the metal isoften accompanied by a change of the absorption spectrum. Byattaching a fluorescent dye to the enzyme, the change ofabsorption can be translated into a change of fluorescencevia FRET. Here, the fluorescent dye acts as a donor thattransfers its energy to the metal center depending on thespectral overlap.

Kuznetsova et al. used this approach to study the molecularmechanism of the enzyme nitrite reductase that contains acopper center. The copper acts as a FRET acceptor in theoxidized form but has no spectral overlap with an ATTO655donor in the reduced form. As is often found in such experi-ments, a distribution of electron transfer rates could be relatedto disorder in the catalytic site.14

One drawback however is the difficulty to site-specificallycouple two fluorescent moieties to the enzyme without affectingits function. Even worse, photobleaching of the donor oracceptor limits the length of the time transients and numbersof turnover events that can be recorded. Long transients and

many turnovers are required to detect dynamic heterogeneity aswell as to obtain statistically significant precision.

Long transients can be obtained if the fluorescence readoutis not related to the immobilized enzyme but to the substrateitself. Since the substrate is renewed for every turnover photo-bleaching is not a limiting factor and the stability of the setupand the fatigue of the enzyme become the critical factors forthe observation time. In order to avoid a high background oflabeled substrates, fluorogenic substrates (substrates thatbecome fluorescent upon enzymatic turnover) had been intro-duced a long time ago. With fluorogenic enzyme substrates forb-D-galactosidase, the earliest single-molecule experiments hadbeen carried out by Rotman.15 45 years later English et al.studied the same enzyme with single turnover resolution(Fig. 3).2 While such studies confirmed the classic Michaelis–Menten mechanism they have also revealed a new microscopic

Fig. 3 (a) Illustration of single-enzyme immobilization with a streptavidincoated polystyrene bead. b-galactosidase is attached to the bead through aflexible PEG linker and converts the nonfluorescent substrate resorufin-b-D-galactopyranoside (RGP) to the fluorescent resorufin (R). (b) The turnover ofindividual substrate molecules by a single enzyme in the presence of 20 mM RGPis visualized by fluorescent bursts (left, 0.5 ms per bin) and stops after addition of200 mM of an inhibitor (middle). Without the presence of an enzyme and aninhibitor, no bursts are detected. Adapted by permission from Macmillan PublishersLtd: Nature,2 Copyright 2006.


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picture of enzyme function including fluctuating enzymaticactivity ("a molecular memory") that indicates dynamic molec-ular heterogeneity on a rugged energy landscape.2,16 The resultsalso indicate possible mechanisms to control enzyme activity inthe biological context by reshaping this energy landscapethrough environmental conditions and molecular interactions.Similar substrates are available for enzymes including lipase B(CALB),3 for a-chymotrypsin and some other enzymes and wereused to monitor the activity of the same enzyme over longerperiods of time.

Still, the approach is not general enough to study the myriad ofenzymatic processes. There are many reactions that involve achange in fluorescence of a substrate but the change is not digitalfrom 0% quantum yield to high intensity. Substrates used forenzymes mentioned above are also not always simple to handle.Some substrates such as resorufin derivatives exhibit competingautohydrolysis so that a fluorescent background emerges duringthe course of the reaction that was eliminated by an additionalbleaching laser beam.2 Other substrates such as a rhodamine 110derivative require a two-step reaction by a-chymotrypsin to yieldthe high quantum yield rhodamine. Fluorescence lifetime mea-surements revealed that only the intermediate singly hydrolysedform with a quantum yield of 31% is detected in the single-molecule experiment.17 There are many probes that can detectenzyme turnover by a change of quantum yield or anotherfluorescence parameter. The action of nucleases or proteases is,for example, commonly reported by probes that are partiallyquenched before cleavage of a short oligonucleotide or peptide.Upon cleavage of the substrate, the fluorophore is spatially sepa-rated from the quencher (acceptor) and its fluorescence is released(e.g. ref. 18). In contrast to other fluorogenic enzyme substratesthat exhibit fluorescence enhancements from essentially 0% to100% relative quantum yield, these substrates typically exhibit a5–10 fold fluorescence increase upon cleavage. At the concentra-tions required, the non-zero fluorescence of the uncleaved sub-strate will create an unacceptable background for single-moleculestudies in common diffraction-limited detection volumes.

Even more generally, typical substrates such as ATP or othernucleotides are available in different fluorophore labeled versionsbut do not change their fluorescent state at all during enzymaticturnover. Such substrates can still be very useful to followbiomolecular reactions, e.g. if a sufficient signal-to-noise ratiocan be achieved and if substrate binding lasts long enough. Suchexperiments are especially informative when the binding can becorrelated with conformational changes such as revealed for theheat-shock protein Hsp90. In an elegant three-color experimentRatzke et al. revealed the stochastic nature of conformationaltransitions and nucleotide binding to this abundant molecularchaperone.19 Generally, however, it is difficult to use fluorescentlylabeled substrates in diffraction limited volumes and the correla-tion of enzyme activity and fuel consumption in motor proteinshas to be deduced indirectly.20

2.2 Temporal separation of high concentration and readout

An important approach to monitor weak interactions at highconcentration is to work under conditions before equilibrium

is reached by quickly diluting the sample that is too concen-trated for single-molecule interrogation. The dilution can forexample be carried out by binding the biomolecular complex toa surface until a desired density is reached. A washing stepremoves the excess of labeled components. This strategy isfor example realized in a single-molecule pull-down assay(SiMPull) that complements classical co-immunoprecipitationassays for protein–protein interactions.9 Here, protein com-plexes extracted from a comparable small number of lysed cellsare immobilized on a coverslip using specific antibodies. Aftera washing step, single-molecule analysis is directly carried outwithout any further time-consuming purification steps (Fig. 4).Stoichiometry, complex composition or even enzymatic actioncan be directly deduced using e.g. bleaching steps of fluores-cent proteins or other reporters such as FRET to convey therequired information about the biomolecular complex and itsactivity. The kind of interaction accessible to this techniquestrongly depends on the lifetime of the complex. The issue ofshort complex lifetimes of weak biomolecular bonds has beenaddressed in a derivate of the SiMPull technique.21 The groupof Yoon demonstrated real-time imaging of a single-moleculeco-IP preparation. Instead of washing the immobilized complexto remove unbound molecules, total cell extracts are main-tained in the flow chamber and reversible single-moleculeinteractions are recorded in real time (Fig. 4). This single-molecule co-IP technique enabled the visualization of weakprotein–protein interactions and revealed that oncogenic Rasmutations selectively increase the active-Ras fraction withoutan increased total Ras level.21 The technique can be applied ifthe signal-to-noise ratio is sufficient to visualize single-moleculebinding events in the presence of total cell extracts.

Generally, the time lapse between incubation and interroga-tion is the critical parameter that determines what kind of weakinteraction can be studied. To run such experiments repeti-tively, Loveland et al. have deployed a technique, termed PhADE(PhotoActivation, Diffusion and Excitation), that demonstrateshow temporal separation can be used for weak binding stu-dies22 (see Fig. 4d and e). For PhADE, a protein of interest istagged by a photoactivatable fluorescent protein, in this casemKikGR, and is added to its surface-immobilized substrate. Inorder to observe individual molecules at appropriate density,the authors illuminated a small area in total internal reflectiongeometry with the wavelength for photoactivation. After 500 ms,when activated but non-interacting proteins have diffused away,fluorescence imaging of the remaining activated molecules onthe surface is carried out. This approach shows great potentialfor measuring at high concentrations as found in total cellextract if the interaction is strong enough to remain stable forthe time between activation and readout. Whereas the currenttime resolution is B500 ms, the method could be extended toshorter complex lifetimes e.g. by speeding up the ‘diffusion’ stepwith a continuous flow through the measurement chamber. Themethod does, however, not directly allow the determination ofassociation rates and observation of the binding process. Evenfaster kinetics can be followed in free solution with microfluidicmixers. Several fast single-molecule mixers have been developed


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that can change the environment and follow rapid changes inthe new environment with a time resolution of down to 0.2 ms.23

Temporal separation was also used for single-moleculesequencing by the synthesis approach.24 A DNA polymeraseprolongs surface-immobilized primer-template duplexes usinglabeled nucleotides in a stepwise fashion. The four possiblenucleotides are sequentially added to the template and theposition where nucleotides were incorporated are registered.A washing step between incorporation and readout enablesthe localization of the single-molecule incorporation eventsalthough the incorporation itself was carried out at highlabeled nucleotide concentration. Over time, the DNA sequencefor each primer-template is obtained. Thus the replication of280 000 DNA molecules was monitored in parallel.24

The idea of temporal separation can even be applied in morecomplex environments such as a living cell. Madl et al.25

combined the fluorescence recovery after photobleaching(FRAP) approach with a brightness analysis of the individualdiffusing molecules. Orai1, the essential pore forming subunitof T-cells is fluorescently labeled, but is initially present at highconcentrations thereby preventing the single-molecule analysis.

After a small part of the membrane is actively photobleachedindividual pore complexes can be observed and analyzedas they diffuse into the bleached area. Moreover, the brightnessof these individual complexes was used to determine theoligomerization state.

2.3 Confinement of molecules

The ability to detect single molecules at higher concentrationscan be achieved by reducing the effective observation volume inthe sample. One way to realize this strategy is to confine themolecules of interest into volumes that are considerably smal-ler than diffraction-limited volumes. Lipid vesicles of typically100 nm diameter can for example be used to create high localconcentrations of molecules. With a volume of about 5� 10�19 l,single proteins inside the nanovesicles have an effective concen-tration of B3 mM.26 Benitez and co-workers26 used this approachto observe transient interactions of the copper chaperone Hah1and the Wilson disease protein (Fig. 5a). The study revealeddistinct protein interaction dynamics and captured interconver-sion dynamics with an intermediate complex that could noteasily be resolved by ensemble methods. The nanovesicle offers

Fig. 4 Strategies for temporal separation of the high concentration. (a) A protein of interest is specifically selected from a total cell lysate by a surface-immobilizedantibody. If the protein is fused to a fluorescent protein, the density of molecules on the surface can be controlled. (b) In order to probe comparably stable protein–protein interactions, the cell lysate can be removed and the second interaction partner can be identified by a fluorescently labeled bait-specific antibody.9 (c) For lessstable complexes, repetitive transient binding events of a fluorescently labeled interaction partner can be observed even in the presence of the total cell lysate.21

(d) Photoactivatable fluorescent proteins offer a solution in case significantly higher concentrations are required. Proteins in a small area are photoactivated with anactivating laser wavelength. (e) The photoactivated interaction partner diffuses out of the observation area leaving only a low concentration of activated andinteracting proteins behind for interrogation.


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the additional advantage that non-specific protein–glass surfaceinteractions are minimized. Unspecific surface interactions are acommon issue at the large surface to volume ratios encounteredat the nanoscale. Initially, the nanovesicle projects were moti-vated by the possibility to immobilize the vesicles rather than thebiomolecule under study on a surface preventing the directinteraction between the biomolecule and the surface therebyprotecting the biological function. This experimental strategyhas been used extensively to study for example protein foldingon the single-molecule level.29 In the following, vesicles werefurther developed to allow solute exchange, e.g. by incorporatingthe bacterial pore-forming toxin a-hemolysin.27 This overcamethe intrinsic non-permeability of a lipid bilayer membrane andrendered these biomimetic nanocontainers amenable to bio-chemical manipulation (Fig. 5b). Buffer exchange is for examplenecessary to induce reactions, biochemical change or to replen-ish enzymatic fuels such as ATP. The repetitive shuttling of Rephelicase on a DNA construct equipped with a FRET pair fueled byATP could thus be monitored in the nanocontainers.27

Even simpler, the molecules in the sample can be confinedby physical reduction of the accessible volume. This has beenrealized by a conventional TIRF-type microscope that isextended by lowering a plano-convex lens into the sample fluid,creating a confined area between the convex surface of the lensand the cover glass28 (Fig. 5c). With this technique, single-molecule measurements at concentrations up to 2 mM wereperformed. The approach not only allows a tuning of the size of

the observation volume but also increases the diffusion-limitedobservation time. Biomolecules such as fluorescently labeledproteins (bovine serum albumin) and a pure dye (Alexa647) canbe separated by size and the molecule size can be determined.Like all methods employing TIRF, many molecules can bemeasured in parallel. The method was further advanced bydistorting the top coverslip of a flow chamber rather thandirectly repressing the solution with the lens.30 This approachadds the advantage that the lens does not have to be cleanedbetween measurements. The technique was used to monitorthe motion of myosin V and VI along actin filaments on thesingle-molecule level.30 Further approaches to confine mole-cules employ nanofluidics,31 nanocapillaries or nanopipettes.32

Recently developed methods trap molecules in free solutionwithout surface interference by an electrokinetic feedback tocounter Brownian motion33 or geometry-induced electrostatictrapping techniques.34

2.4 Reduction of the observation volume

The challenge of single-molecule measurements at high con-centrations is directly connected to the diffraction limit of light.Therefore it seems appropriate to tackle this issue with techni-ques that are used for fluorescence microscopy beyond thediffraction limit like the recently introduced superresolutionmicroscopy techniques. Here, a similar barrier has beenencountered and it has been broken by different approaches.Two of the first superresolution techniques are near-fieldscanning optical microscopy (NSOM) and stimulated emissiondepletion (STED) microscopy. STED microscopy providespotentially unlimited resolution by restricting the excitationvolume of a confocal microscope with a second, doughnutshaped laser beam. While it is used in combination withfluorescence correlation spectroscopy8 STED is not a commontool to study individual molecules at elevated concentrationsdue to the high laser intensities involved. In NSOM, theexcitation light is guided through a fiber with an openingsignificantly smaller than the wavelength of light, which createsan evanescent field in a small, sub-diffraction volume outsidethe fiber that can excite fluorescent molecules. The principlehas been picked up and led to the development of nanopho-tonic structures that allow the fabrication and observation ofultra-small volumes in a parallel fashion.

The arguably most prominent example are the so-called zeromode waveguides (ZMWs). These nanophotonic devices arebasically holes in a metal film deposited on a glass surface witha diameter smaller than the diffraction limit of light (Fig. 6a).

Fig. 5 Examples for molecular confinement strategies. (a) Weakly interactingproteins can be trapped in a vesicle and attached to the surface so that multipleshort interaction events of the same proteins can be observed. In this case thecopper chaperone Hah1 and the Wilson disease protein are labeled with theFRET-pair Cy3-Cy5. The single pair FRET analysis (right panel, green, red: fluores-cence of Cy3 and Cy5 respectively, grey: FRET efficiency) unravels the presence ofone distinct intermediate interaction state (E1) between separation (E0) andinteraction (E2) of the proteins. (b) Incorporation of the bacterial pore-formingtoxin a-hemolysin (blue channels) makes the vesicle permeable for small mole-cules such as ions or ATP. (c) Camera images of individual Alexa 647 moleculesconfined between a coverslip and a convex lens (CLIC) at concentrations of200 nM (left) and 2 mM (right). The black circle corresponds to a distance of 2 nmbetween the two surfaces and this distance increases from the center to theperiphery. While at the edge single-molecule sensitivity is lost due to the highconcentration, single molecules can still be monitored in the center. Scale bar:10 mm. Adapted with permission from ref. 26–28. Copyright 2008, 2009, 2010American Chemical Society.

Fig. 6 (a) Sketch of an aluminum ZMW with a diameter of 50 nm and a width of100 nm on a glass surface. (b) Numerical simulation of the electric field intensityin the ZMW structure at a wavelength of 500 nm.


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They confine the volume for excitation and detection laterally bythe dimension of the aperture (typically 50–200 nm in diameter)and axially by their inability to guide light (see numericalsimulation of the electric field intensity included in Fig. 6b).This leads to an evanescent excitation field on the bottom ofthe aperture and consequently only molecules from that regioncan emit photons into the objective. The reduced observationvolume provides single-molecule sensitivity even at 10 mMconcentration of fluorescent dyes.35 ZMWs are produced in largearrays, allowing the parallel imaging of hundreds of aperturessimultaneously.

Because the ZMW design allows measurements at concen-trations found in living cells they have been widely employed inbiological sciences. The enormous potential of the ZMWs isimpressively demonstrated in their usage for high throughputfluorescence-based single-molecule sequencing.36 Here, a sin-gle polymerase molecule is immobilized in the nanoapertureenabling sequencing-by-synthesis in real-time.36 Because of theconfined volume the polymerase captures a sample DNA andincorporates fluorescently-labeled nucleotides in the newlysynthesized DNA strand. Only the fluorescence bursts of thenucleotides bound by the constrained polymerase close to thesurface of the ZMW are detected excluding the backgroundfluorescence of the highly concentrated nucleotide solution(10 mM). Each nucleotide is labeled with a different color sothat they can be distinguished at each incorporation step. Oneadvantage of this single-molecule sequencing technique is thatthe long sequence reads up to thousands of bases, much longerthan achieved with conventional sequencing systems.

The possibility to measure at high concentrations in theZMW enabled the single-molecule analysis of protein–proteininteractions and enzymatic reactions under physiologicallyrelevant conditions. Complex stoichiometries, interaction path-ways and kinetic data could be obtained for individual mole-cules for the first time. Miyake et al. presented one of the firstexamples of a protein–protein interaction study carried out in aZMW.37 Individual binding-release events could be monitoredfor the interaction of the chaperonin GroEL with its co-chaperoninGroES which is strictly dependent on the presence of ATP. GroESwas used at a concentration of 5 mM (4.5 mM unlabeled and 0.5 mMCy3-labeled protein), equivalent to the intracellular concentrationwhile IC5-labeled GroEL was immobilized in the ZMW. Here, thecomplex lifetime and the kinetic rate constants for GroEL andGroES interaction could be determined showing the feasibility ofthe ZMW system for biological applications.

The ZMW technique shows its real strength in a series ofstudies carried out on the bacterial translation machinery. Thebacterial translational apparatus is a complex multicomponentsystem composed of the 30S and 50S subunits of the ribosome,the mRNA template and tRNA substrates as well as numerousinitiation and elongation factors that modulate the activity ofthe ribosome. Analogous to real-time sequencing, translationcould be monitored in real-time making use of fluorescently-labeled tRNAs at submicromolar concentrations.39 The ribo-some has been captured via an immobilized mRNA strand andthe transit of the color-coded tRNA through the ribosome could

be monitored. These studies revealed that ribosomes are rarelyoccupied by two tRNA simultaneously even though the ribo-some harbors three tRNA binding sites. In order to delineatethe multistep process of translation these studies wereextended to include labeled translation initiation factors,labeled ribosomal subunits and initiator tRNA at micromolarconcentrations (Fig. 7). The full spectrum of color-coding wasexploited using Cy3.5, Cy5, Cy3 and Alexa488 as identifiers forthe constituents of the translation machinery that ultimatelyallowed the monitoring of the initiation complex assembly inreal-time.38 Interestingly, the assembly of the initiationcomplex is not restricted to a linear assembly line but exhibitedinnate heterogeneity indicative of the compositional dynamicsof the system.

The range of applications has also been extended to challen-ging biological systems like membrane-embedded receptors. Ina study carried out by Richards et al., the subunit stoichiometryof the pentameric neuronal nicotinic acetylcholine receptors(nAChRs) was determined using living cells.40 The ZMWapproach has been instrumental for the experimental designas the ZMW allows a spatial isolation of a single receptorand concomitantly suppresses the autofluorescence of the cell.

Fig. 7 Molecular choreography during translation initiation analyzed on thesingle-molecule level using a zero-mode waveguide. (a) During translationinitiation three protein factors (IF1, IF2 and IF3) support the formation of thepre-initiation complex (PIC) composed of the ribosome (30S–50S) and theinitiating tRNA (fMet-tRNAMet) to the mRNA. The individual components ofthe PIC can be distinguished on the basis of the spectral properties of theattached organic dyes (ribosomal subunit 30S: Alexa488-labelled; fMet-tRNAMet:Cy3-labelled, ribosomal subunit 50S: CY3.5-labelled, IF2: Cy5-labelled). Single-labeled 30S is immobilized on the bottom of the ZMW via a biotinylated mRNAstrand. (b) Factor arrival and departure during translation initiation can bemonitored over time after all components have been flushed in at time-pointt = 7 s. The order of arrival was determined by the sequence of fluorescent pulsesand departure of factor leads to the disappearance of a signal. In this example,the formation of productive initiation complexes indicated by 50S joining ispreceded by IF2 and tRNA association. After GTP hydrolysis IF2 leaves the PIC.Monitoring tens to hundreds of initiation events revealed the heterogeneity ofthe PIC formation pathway. Adapted by permission from Macmillan PublishersLtd: Nature,38 Copyright 2012.


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Neuronal cells that are able to form filopodia have been directlycultured on the ZMW arrays. Interestingly, the filopodia pro-truded into the cavity of the ZMW making it possible to isolatesmall patches of the membrane containing a single receptor forthe single-molecule study. The chosen cell type does not con-tain endogenous nAChRs and therefore the receptor densitycould be adjusted via the expression level of the GFP-taggednAChR subunits. At low expression levels the ZMW most likelycontains just a single receptor and as the evanescent fieldpenetrates about 50 nm into the ZMW the background fluores-cence of the cell and non-assembled subunits in the cytoplasmis suppressed. 80% of the molecules showed three bleachingsteps while 20% showed just two steps corresponding to a(a4)3(b4)2 and a (a4)2(b4)3 stoichiometry, respectively, demon-strating the heterogeneous composition of this receptor type.

Although membrane patches of a complete cell could beimaged using ZMWs the movement of an isolated molecularmotor like myosin along its filamentous track could not befollowed in a nanoaperture. Here, the small dimensions of theZMWs that form the basis for measurements in confinedvolumes pose a major hurdle as actin filaments easily reach alength of several micrometers and do not fit into the cavity of aZMW. In a recent report, this issue has been successfullybypassed using linear rather than cylindrical ZMWs whichallowed the direct polymerization of actin filaments in theZMW.30 These filaments formed functional tracks for a molec-ular motor as demonstrated with YFP-tagged myosin V. Myosinruns of about one micrometer were recorded which correlate to30 steps along actin considering the step-length of 35 nm formyosin V.

The membrane receptor studies and linear ZMW nicelyillustrate the versatility of the ZMW technique but also demon-strate that there is still room for further development to adaptthe ZMW technique to a specific biological problem.

While the ZMW approach significantly advanced the opticalsingle-molecule field, it faces some challenges when moleculesneed to be selectively immobilized at the center of the ZMW.The occupation of the apertures is subject to Poissonianstatistics, leaving about 2/3 of the apertures futile for single-molecule measurements due to the presence of zero or morethan one molecule. Furthermore, the position of the labelledmolecule within the nanoaperture can strongly affect theintensity of the fluorescence signal and results in undesirableheterogeneity that complicates data analysis. A significantfraction of ZMWs will not be usable because molecules arefound in close proximity to the metallic sidewalls, where astrong quenching of fluorescence occurs. These problems havemotivated recent works in which single molecules are placed atthe center of nanoapertures using an atomic force microscopeas a nanoscopic robotic arm.41 Molecules placed in the centerof nanoapertures revealed a drastically reduced heterogeneityof fluorescent properties compared to stochastically immobi-lized molecules indicating that paralleled ways of targetedplacing of molecules might constitute a key advancement.41

Interestingly, metal nanostructures can not only quenchthe fluorescence but can also act as nanoantennas strongly

enhancing the fluorescence of molecules placed in close proxi-mity (see ref. 42 and references therein). Upon illumination, thefree electrons of metal nanostructures with sub-wavelengthdimensions are forced into collective oscillations termed loca-lized surface plasmon (LSP). This electron displacement leadsto a polarization of the metallic elements "focusing" the electricfield to a hotspot in the vicinity of the nanostructure andtherefore fluorescent molecules placed in such a hotspot canexperience an enhancement in the fluorescence.43

A prominent example of a metal nanoantenna developed bythe group of W. E. Moerner using electron-beam lithography issketched in Fig. 8a.44 It consists of two triangular elementsforming a "bowtie" antenna. With a gap of 10 nm, the hotspot isreduced to a volume in the zeptoliter range leading to anexcitation rate enhancement of more than 2 orders of magni-tude. Additionally, when dyes with a low intrinsic quantumyield (2.5%) are placed at the hotspot, the bowtie nanoantennamediates the emission enhancing the quantum yield by a factorof 10. Thus, with these antennas a fluorescence intensityenhancement of more than 3 orders of magnitude was demon-strated and single molecule measurements at concentrations inthe mM range were realized using fluorescence correlationspectroscopy.

Strikingly, the concept of a nanoantenna brings along twosolutions for the high concentration problem: firstly, theantenna is able to focus the light to a spot of roughly 10 nmdiameter and as a result the illuminated volume is greatly reducedto the zeptoliter range. Secondly, the fluorescence signal of the dyecan be greatly enhanced by some orders of magnitude. Thisenhancement can be compared to a spotlight directed to thefluorophore dwelling in the hotspot between the nanoparticles.

Fig. 8 Sketch of different dimer nanoantennas (left) together with their corre-sponding numerical simulation for the electric field intensity enhancement (right)for a gold bowtie antenna (a), an antenna-in-box (b) and 80 nm gold nano-spheres mounted on a DNA self-assembled (origami) structure (sketched in blue)(c). In every case the structures are mounted on a glass surface (represented ingreen). The scale bar represents 50 nm. The numerical simulations were per-formed with an electric field polarization along the dimer orientation assumingincidence from the glass surface at a wavelength of 780, 632 and 640 nmrespectively.


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A combination of these two beneficiary properties would in theoryenable single-molecule measurements at high concentrations offluorescent molecules. However, a pre-requisite for an optimalsignal enhancement is a defined geometry and requires a site-specific arrangement of metallic particles and the fluorescent dye.

Very recently, a synergistic combination of ZMWs andnanoantennas termed "nanoantenna-in-box" was introduced byWenger and co-workers.46 It consists of a nanoantenna dimerformed by two semi-spheres placed within a rectangular aperture(Fig. 8b). While the nanoantenna is mostly responsible forenhancing the fluorescence signal at the hotspot, the rectangularnanoaperture reduces the signal from dyes diffusing outside thehotspot due to the light confinement to a volume surroundingthe nanoantenna. FCS measurements revealed that single mole-cules can be detected at concentrations higher than 15 mM at thehotspot of a nanoantenna-in-box.

The aforementioned examples together with the ZMWs have incommon that fabrication is implemented using top-down litho-graphic techniques. ZMWs have even become commercially avail-able. While convenient for FCS measurements, the immobilizationof molecules, such as for example a polymerase at the hotspot ofthese nanoantennas or at a determined position within the ZMWsis particularly challenging. An alternative bottom-up strategy hasbeen realized making use of a DNA-based self-assembled structure(so-called DNA origami) as a scaffold on which two sphericalnanoparticles could be placed to form dimer antennas(Fig. 8c).47 This structure also offers handles to place a biomole-cular assay in the hot-spot. For gold nanoparticles of 100 nmdiameter with a surface to surface spacing of 23 nm, fluorescenceenhancement of more than two orders of magnitude was demon-strated together with single-molecule measurements at 500 nM.

3. Experimental strategies aiming to breakthe low concentration limit

Biomolecules often serve as markers for diseases. Biomarkerdetection at ultralow concentration can enable early diagnosisand post-therapeutic monitoring of viral infections, cancer,diabetes and many other pathophysiological conditions. Cur-rently, the protein levels of interleukins, cytokines or growthfactors are often used as diagnostic biomarkers48 but many ofthese proteins are categorized as low-abundant proteins (LAPs)with an abundance of less than 1 pg ml�1 (o10–100 fM)present in extracellular body fluids.49 Protein biomarkers areclassically detected using immunoassays like the enzyme-linked immunosorbent assay (ELISA) but this method doesnot provide the sensitivity required to sense LAPs. Nucleic acidsthat freely circulate in body fluids constitute a second class ofbiomarkers. For diagnostic purposes nucleic acids are prefer-entially amplified via polymerase chain reaction (PCR) as thismethod allows detection of extremely low concentrations.However, PCR is also sensitive to contamination and bias.Hence, alternative ways to detect ultra-small biomarker con-centrations are desirable but necessitate an extremely highspecificity as well as sensitivity.

Could single-molecule measurements advance the field ofbiomarker recognition and detection? In order to find ananswer to this question consider a cuvette with 10 targetmolecules that can be recognized by an antibody coupled to abright fluorophore. 10 molecules will not give rise to a signalthat allows the researcher to recognize it as the background ofbillions of scattering solvent molecules is simultaneouslydetected in using a conventional fluorescence spectrometer. Ifthese 10 molecules, however, diffused through a single-moleculedetection device each of the molecules would give rise to a brightsignal that could easily be discriminated against the backgroundcaused by the solvent molecules that transit the detectionvolume in the meantime given that the analytes transit one byone separated by an adequate time gap. Thus, the concentrationof molecules could be determined in the most fundamental way,i.e. by counting individual molecules. The evaluation parameterused to discriminate between the signal and noise is notrestricted to fluorescence intensity but can be any parameterused in single-molecule spectroscopy, including the spectrum,fluorescence lifetime, polarization anisotropy, diffusion timeand so on. This is a crucial advantage as impurities often giverise to comparable fluorescence intensities but can be clearlydistinguished from a fluorophore according to their differentfluorescence properties. Single-molecule detection thereforeallows for the evaluation of each signal individually and enablesmolecules sorting into populations. Hence, the sensitivity andquality of a signal can be critically increased even supportingthreshold based diagnostic approaches that require quantitativeinformation about a native and mutated form of a biomolecule.

When single-molecule detection at low concentrations hasthe potential to considerably advance the diagnostic toolboxwhy is it not widely implemented? The challenge here is thelimited volume screened in a given period of time. For example,the observation volume in a typical single-molecule setup is ofthe order of 1 fl. Considering diffusion coefficients of single-molecules in the range of 1–5 � 10�10 m2 s�1, the average timebetween two molecules reaching the detection volume at lowfemtomolar concentrations is of the order of several minutes tohours.45 Current attempts to overcome the low concentrationbarrier seek to decrease the measurement time until a suffi-cient number of molecules is detected. One possibility toreduce the measurement time is to increase the observationvolume. Since bigger observation volumes are detrimental tothe optimal SNR essential for optical single-molecule detectionthis approach is not practicable. Instead, single-molecule detec-tion at ultra-low concentrations can be accelerated by increas-ing the speed of diffusion, by parallelized detection,preconcentration of molecules or by a signal amplificationmechanism. These techniques are often implemented in nano-fluidic devices that distinguish themselves by their excellentlevel of control.

3.1 Mixing for faster detection

Haas et al.45 introduced a simple yet effective experimentalsetup that drastically increases the rate of detecting molecules.Here, the rate of detecting single molecules at low concentrations


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was enhanced by the flux through the observation volumeinduced by turbulent mixing of the sample and led to a collec-tion of significant amounts of data within 60 seconds at con-centrations as low as 1 fM (see Fig. 9). The strategy is intriguingbecause of its simplicity, but so far relies on a clever design of theprobe. In order to increase the SNR, a FRET-based signal waschosen as a readout. This allowed the use of a spectral filter thatremoves the background luminescence and the fluorescenceoriginating from impurities present in the sample.

3.2 Single molecule equivalents of ensemble assays

Preconcentration is an alternative solution to accelerate detec-tion of single-molecules at ultra-low concentrations51 and is forexample the first step of a commercial platform by Singulex.52

The target biomarkers are specifically captured on magneticbeads and later recognized by a fluorescently labeled antibody.Subsequently, the sample is thoroughly washed while the mag-netic beads are kept in place and the antibodies are released in asmall elution volume containing urea. The eluate contains anequal number of biomarkers and labeled antibodies at increasedconcentrations. Next, the number of antibodies is opticallycounted when the sample is flushed through a laser focus in acapillary. One of the major advantages of this technique is thatthere is no need to modify or label the biomarker as the finalsingle-molecule counting is based on the signal of the fluorescentantibody. The limit of detection for this assay is 10–100 fg ml�1.

Duffy and colleagues extended this approach and combinedit with enzymatic amplification of the signal and paralleleddetection establishing a digital ELISA50 (Fig. 10). The targetmolecule is captured by 3 mm diameter beads carrying a specificantibody. In order to capture just one analyte per bead thenumber of beads significantly exceeds the number of analytesin solution. On the surface of the bead the analyte finds over100 000 capturing antibodies and the high number of beads inthe solution results in a reduced bead-to-bead distance so thateach molecule encounters a microsphere in less than 30 minutes.After washing, the beads are loaded into microwells of a size thatallows only one bead to enter. A detection antibody links theprotein to an enzyme that converts a substrate to its fluorescent

product and thereby enables single-molecule detection withoutthe actual need for single-fluorophore sensitivity. This way adigital discrimination between wells that produce fluorescentmolecules and those that do not is possible. The sensitivityachieved in this type of assay is remarkable with 10–20 enzymecomplexes of a prostate-specific antigen detected in 100 ml of thesample (B10�19 M).50

An interesting aspect of this work is that the enzymaticamplification of the signal occurs after the isolation of individualmolecules. This preserves the single-molecule advantage since thesignal is read out one by one and the introduction of additionalnoise caused by an amplification step is prevented. It is generallyimportant to consider which amplification scheme is in accor-dance with the single-molecule idea. Usually, signal amplificationand polymerase chain reaction (PCR) for nucleic acids are prone tobias and contaminations. This is also due to the fact that thres-holds have to be carefully selected to distinguish the signal fromthe noise equivalent to a rather analog way of signal recognition.

In analogy to the concept of the digital ELISA, purelyphysical amplification schemes could directly improve single-molecule assays. If a DNA nanoantenna with docking sites, forexample, is used to capture a target, the signal amplification bythe nanoantenna directly contributes to an improved singlemolecule assay that can be read out one by one.47 With ananoantenna with docking sites, fluorescence enhancement bya factor of 100 can be achieved improving the SNR accordingly.Ultimately, this facilitates single-molecule detection in muchbigger detection volumes. Thus the lower concentration barriercould be shifted by the factor of fluorescence enhancement.47

3.3 Micro- and Nanofluidics

From an experimental perspective, microfluidic and nano-fluidic structures can provide a number of features that are

Fig. 9 Left: fluorescence time trace of diffusing double-stranded DNA at 10 pMconcentration illustrates the drastic effect of mixing on the measurement time.During the first 20 s of the measurement no molecule is detected, but after therotor is turned on the detection is no longer diffusion limited and the measure-ment is significantly accelerated. Right: histograms of the first (red) and second(black) part of the measurement further illustrate the difference. Adapted withpermission from ref. 45. Copyright 2010 American Chemical Society.

Fig. 10 (a) Capturing of single protein molecules on beads using the standardELISA reagent and labeling with the enzyme b-galactosidase that reports on thepresence of the protein by converting a nonfluorescent substrate to its fluorescentform. (b) Single 2.7 mm diameter beads are loaded in well arrays with 4.5 mmdiameter and 3.25 mm depth as shown by a SEM image. (c) Fluorescence imagingreveals that only a small fraction of these beads carried a protein of interest and thepercentage of fluorescent wells reports on the concentration. Adapted by permis-sion from Macmillan Publishers Ltd: Nature Biotechnology,50 Copyright 2010.


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beneficial for single-molecule experiments over a broadconcentration range.54 Nanofluidic devices can, for example,reduce the volume accessible to molecules to some ten attoli-ters, which supports measurements at elevated concentrations.But there are also considerable benefits for detection of lowconcentrations. The sample flow can be controlled, manychannels can be observed in parallel,32,54 there is just a smallsample volume needed and the structures can be produced inlarge numbers. Moreover, nanofluidic devices can ensure thatevery single molecule is detected when passing the detectionvolume as the detector completely covers the cross-section ofthe nanofluidic channel.32,54 The idea of an experimental setupthat allows the detection of the sample in its entirety as itpasses through a small but paralleled bottleneck has beenrealized with an array of nanopores in combination with TIRFimaging. DNA molecules are electrokinetically driven throughthe nanopores once a voltage is applied and the completesample is guided through this nanopore array. Because thewhole array can be monitored in parallel, this setup guarantees100% detection efficiency as each target molecule that trans-locates through the array is observed.55

Furthermore, nano- and microfluidics are tools that can becombined with most of the previously presented techniqueslike zero-mode waveguides or nanoantennas and multiple stepscan be carried out on the same chip. Puleo and Wang, forexample, used a combination of micro-evaporators, molecularbeacons and confocal detection to identify DNA molecules atconcentrations down to 50 aM.53 The initial sample volume of25 ml is reduced by the micro-evaporator to B4 nl, whichincreases the concentration by a factor of more than 5000(Fig. 11). The sample is then hybridized to and detected via amolecular beacon, a fluorescently labeled DNA hairpin whoseemission is suppressed by a quencher until the target DNAbinds to it – a concept similar to the previously discussedfluorogenic substrates. After the hybridization process, fluores-cence bursts of individual target DNA – molecular beaconcomplexes are directly registered on the chip with a confocalmicroscope (Fig. 11). In this specific example, the time limitingstep is the duration of the evaporation process.

Their outstanding features make nanofluidic devices pro-mising candidates for high-throughput tests in drug screeningand DNA analysis. The reduction of the channel cross-section ishowever limited by the desired throughput and the risk ofclogging channels.

4. Conclusion

The concentration barrier of optical single-molecule detectionis defined by the dynamic concentration range of single-moleculedetection with common diffraction-limited optics. For the lowconcentration side, single-molecule detection is limited by theloss of SNR with increasing observation volume. On the otherhand, the concentration when a single molecule resides in adiffraction limited observation volume defines the upperconcentration barrier. The concentration barrier is one of themain limitations for the broad applicability of optical single-molecule detection in biology and medicine. Its significanceis well exemplified by the two killer applications of single-molecule detection, single-molecule DNA sequencing and super-resolution microscopy based on successive single-moleculelocalizations. Both of these outstanding examples demonstratethe benefits of single-molecule detection and are capable ofovercoming the concentration barrier although in very differentways. Single-molecule sequencing was achieved by temporalseparation of single-molecule signals in combination withmicrofluidics24 (Helicos BioSciences) or by real-time sequen-cing in nanophotonic structures36 (zero-mode waveguides,Pacific BioSciences). The single-molecule approach to super-resolution microscopy stretches the ensemble signal out intime in order to localize one molecule after the other andreconstruct fluorescence images.

The second decade of single-molecule detection has gener-ated a multitude of ideas how to overcome the concentrationbarrier and numerous solutions have been presented. Gener-ally, we classified four approaches to overcome the upperconcentration barrier. It could be circumvented by well-conceivedsingle-molecule visualization approaches with the classic exampleof fluorogenic probes to monitor substrate turnover by a singleenzyme. Single-molecule detection of weak binding proteins couldbe visualized at high concentrations by temporal separation e.g.using photoactivatable proteins. Alternatively, molecules of inter-est could be confined in nanocontainers or nanostructures muchsmaller than a diffraction-limited observation volume. Finally, thediffraction limit could be overcome using nanophotonic struc-tures such as zero-mode waveguides or optical antennas. All theseapproaches have beautifully contributed to the expansion of thesingle-molecule toolbox and have revealed new insights intobiological functions and the dynamics of interactions. Weconsider the nanophotonic approach to be the one that canbe applied most generally even though issues due to the largesurface to volume ratio remain. The nanophotonic strategydirectly reduces the observation volume and has few principallimitations. It still has to unlock its full potential since opticalantennas are just beginning to emerge and to be applied tobiological problems.

Fig. 11 Microfluidic device for sensing of ultra-low concentrations after pre-concentration of the sample. The target sample is injected at (inlet i). And thesolvent is removed through an evaporation membrane (yellow, ii). After thesample is enriched at the accumulation zone, it is mixed in the rotary chamberwith the hybridization buffer (inlet iii). Three valves (red) act as a rotary pump thatcircles the sample through a confocal detection volume (blue). Adapted fromref. 53 with permission from The Royal Society of Chemistry.


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Similarly, several approaches have been developed to trans-fer the advantages of single-molecule detection (e.g. the sortingof signals) to the low concentration regime where single-molecule biosensing can significantly aid ultra-sensitive detection.

Altogether, an important step towards the implementationof single-molecule detection as a standard-method in biologicalresearch will be the widespread dissemination and availabilityof new tools such as the nanophotonic and nanofluidic devices.New types of coverslips that integrate these nanotechnologiesthereby combining multiple high-sensitivity assays might repre-sent an important step in the development of next-generationsingle-molecule devices and microscopes.

Acknowledgements

The authors thank Frank Demming from CST (www.cst.com) forhelp with the FDTD simulations. We are grateful to all membersof the Tinnefeld lab for their input. P.H. is grateful for support bythe Studienstiftung des Deutschen Volkes. This work was sup-ported by a starting grant of the European Research Council(SiMBA, ERC-2010-StG-20091118) and the Volkswagen Founda-tion to P.T. and a DFG grant (GR 3840/2-1) as well as a German-Israel Foundation grant (2292-2264.13/2011) to D.G.

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