electrochemical biosensor array for the identification of microorganisms based on...

Electrochemical Biosensor Array for theIdentification of Microorganisms Based onLectin-Lipopolysaccharide Recognition

Peter Ertl and Susan R. Mikkelsen*

Department of Chemistry, University of Waterloo, Waterloo, Ontario, N2L 3G1 Canada

Rapid identification of bacterial strains remains a well-known problem in applied medicine and, for viablepathogens, is an important diagnostic goal. We haveinvestigated an electrochemical biosensor array, in whichtransduction is based on respiratory cycle activity mea-surements, where the microorganism’s native respiratorychain is interrupted with non-native external oxidants. Theselective biochemical recognition agents employed in thisstudy are lectins that, once immobilized, recognize andbind to cell surface lipopolysaccharides. Porous mem-branes with different surface properties were examinedas potential immobilization supports for these lectins.Optimizations performed using concanavalin A and E. coliJM105 show that immobilization methods involving pre-activated membranes significantly reduce the time re-quired to create a functional lectin layer on the membranesurface. Overall, we found general agreement betweenagglutination test results and the electrochemical assess-ment of lectin-cell binding. Chronocoulometric measure-ments were made for cells captured on lectin-modifiedImmunodyne ABC membranes physically affixed to Ptworking electrodes. This lectin-based sensor array wasexposed to viable cells of Gram-negative and Gram-positive bacteria as well as yeast, and chronocoulometricmeasurements were used to generate a pattern of re-sponses for each organism toward each lectin. Principalcomponent analysis was used to classify the chronocou-lometric results for the different microbial strains. Withthis new method, six microbial species (Baccilus cereus,Staphylococcus aureus, Proteus vulgaris, Escheri-chia coli, Enterobacter aerogenes, Saccharomycescerevisiae) were readily distinguished.

The detection, identification, and quantitation of microorgan-isms play a vital role in fermentation technology, medical practice,and environmental monitoring. Bacterial pathogens are distributedin soil, marine waters, water contaminated with fecal matter, andthe intestinal tracts of animals.1 Many bacterial species have beenidentified as significant food and waterborne pathogens that haveprofound effects on humans, and the incidence of pathogen-related

diseases among humans has not yet decreased.2 Generally, nosingle test provides definitive identification of an unknownbacterium. Traditional bacterial detection methods involve a pre-enrichment step and a selective enrichment step, followed bybiochemical screening and serological confirmations;3 this com-plex series of tests can last up to 72 h. Thus a sensitive, reliable,and more rapid identification method is required for the detectionand early treatment of infections.

Many instrumental methods have been reported for microbialdetection, including surface plasmon resonance, fiber optics, andflow cytometry.4 Infrared spectroscopy and the gas chromato-graphic mass spectrometric detection of fatty acids have also beenused for the detection and identification of microorganisms.2 Othermethods use piezoelectric crystals, impedimetry, calorimetry, andselective detection of cellular compounds such as ATP, DNA,protein, and lipid derivatives, as well as the detection of metabolicprocesses such as redox reactions.5-7

Lectins are structurally diverse proteins or glycoproteins thatselectively and reversibly associate with mono- and oligosaccha-ride components of polysaccharide structures.8 Lectins are foundin most organisms, ranging from microorganisms to plants andmammals, and are invaluable tools for the structural and functionalinvestigation of complex carbohydrates.9,10

The lectin concanavalin A (Con A) has been used in areversible immobilization strategy for glycoenzymes such asglucose oxidase and peroxidase in a FET-based biosensor forglucose.11 A competitive sensor for glucose has also been reportedin which a fluorescent Con A derivative and a quenching dextranderivative are trapped in a hydrogel at the distal tip of an opticalfiber, to allow free entry of glucose from the external solution.12

* Corresponding author: (voice) (519)888-4567 ext 6871; (fax) (519) 746-0435;(e-mail) [email protected].(1) Atlas, R. M. Principles of Microbiology, 2nd ed.; Wm. C. Braun Publishers:

Dubuque, IA, 1997; Chapter 12.

(2) Swaminathan, B.; Feng, P. Annu. Rev. Microbiol. 1994, 48, 401-426.(3) Hobsen, N. S.; Tothill, I. E.; Turner, A. F. P. Biosens. Bioelectron. 1996,

11, 455-477.(4) Cunningham, A. J. Introduction to Bioanalytical Sensors; John Wiley &

Sons: New York, 1998.(5) Ivinski, D.; Abdel-Hamid, I.; Atanasov, P.; Wilkins, E. Biosens. Bioelectron.

1999, 14, 599-624.(6) Wood, K. V.; Gruber, M. G. Biosens. Bioelectron. 1996, 11, 207-214.(7) Mouritsen, C. L.; Hillyard, D. R. Anal. Chem. 1999, 71, 293R- 366R.(8) Sharon, N.; Lis, H. Lectins; Chapman and Hall: New York, 1984.(9) Liener, I. E., Sharon, N., Goldstein, I. J., Eds. The Lectins: Properties,

Functions and Applications in Biology and Medicine, Academic Press:Orlando, FL, 1986.

(10) Dwek, R. A. Chem. Rev. 1996, 96, 683-721.(11) Koeneke, R.; Menzel, C.; Ulber, R.; Schuegerl, K.; Scheper, T. Biosens.

Bioelectron. 1996, 11, 1229-1236.(12) Russell, R. J.; Pishko, M. V.; Gefrides, C. C.; McShane, M. J.; Cote, G. L.

Anal. Chem. 1999, 71, 1, 3126-32

Anal. Chem. 2001, 73, 4241-4248

10.1021/ac010324l CCC: $20.00 © 2001 American Chemical Society Analytical Chemistry, Vol. 73, No. 17, September 1, 2001 4241Published on Web 07/24/2001

Bacteria and fungi have chemically distinct surface polysac-charide carbohydrate structures that can be recognized by lectinsin agglutination studies.13 A recent report shows that Helicobacterpylori isolates can be differentiated based on their lectin agglutina-tion patterns; 36 strains of this organism were grouped into 8 lectinreaction patterns, using an optimized panel of 5 lectins.14 Lectinshave also been suggested for bacterial purification purposes dueto their ability to selectively bind cell wall oligosaccharides andto retain microbial cells in affinity chromatography.13,15-17 Im-mobilized lectins have been incorporated into affinity surfaces usedto isolate broad classes of bacterial samples for MALDI massspectrometric analysis,18 and the same group has comparedimmobilized lectins with immobilized polysaccharides for affinitypurification of microorganisms based on recognition of bacterialpolysaccharides and surface-expressed lectins, respectively.19

In recent years, intensive research has been undertaken todevelop portable, rapid, and sensitive biosensors capable ofdetecting microbes with high specificity and sensitivity. Bacterialdetection strategies with biosensors have used biological recogni-tion components such as receptors, nucleic acids, or antibodies,in intimate contact with an appropriate transducer.3,5,20 Althoughthe concept of using lectins to recognize bacteria is not new,21,22

the application of lectins to bacterial identification in a biosensorarray has never been reported. Furthermore, lectins are readilyavailable and inexpensive recognition agents, well suited toexploitation in a sensor array targeted toward mass production.

We have recently shown that the reduction of ferricyanide byviable Escherichia coli cells is particularly sensitive to growthconditions due to variations in the expression levels of enzymessuch as the terminal cytochrome oxidases and the primarysuccinate dehydrogenase in the bacterial cell wall. In the presenceof the respiratory substrates succinate and formate as well as theredox mediator ferricyanide, the respiratory cycle activity of a cellsuspension can be measured electrochemically by chronocou-lometry.23 We have also found that brief incubations of microor-ganisms with effective antibiotic compounds cause dramaticdecreases in measured respiratory cycle activities, allowing rapidassessment of antibiotic drug susceptibility.24

In this study, lectins are employed as selective recognitionelements in a sensor array that uses electrochemical signals fromrespiratory cycle activity to identify bacterial strains. Lectins wereimmobilized onto various membrane surfaces by adsorption withand without intermolecular cross-linking, through avidin-biotin

anchors using biotinylated lectins and by covalent coupling toactivated membrane surfaces. Lectin-modified membranes wereincubated in pure suspensions of microorganisms to allow selec-tive cell attachment and were then rinsed and fixed at the surfaceof a Pt electrode. Chronocoulometric measurements were madein an assay buffer containing succinate, formate, and the redoxmediators ferricyanide and menadione, for an array of 10 lectin-modified membranes, with each of 6 microorganisms. Factor-based principal component analysis (PCA)25-28 was used to analyzethe chronocoulometric data. The results show clear groupings ofreplicate measurements for the six microorganisms in principalcomponent plots, suggesting that rapid bacterial identification ispossible using an array of lectin-modified electrodes.

EXPERIMENTAL SECTIONMaterials and Instrumentation. Sigma supplied lectins from

the species Artocarpus integrifolia, Arachis hypogaea, Galanthusnivalis, Phytolacca americana, Lens culinaris, Helix pomatia,Triticum vulgaris, and Codium fragile as well as Con A andbiotinylated Con A (97% biotinylated). Bovine serum albumin(BSA, 99%), streptavidin (14 units/mg of protein), avidin (12.9units/mg of protein), biotinamidocaproyl-labeled peroxidase (240units/mg of protein), 2′,2′-azinobis(3-ethylbenzthioxoloine-6 sul-furic acid) (ABTS), citric acid, sodium cyanoborohydride, N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) buffer,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), menadione(2-methyl-1,4-naphthoquinone sodium bisulfite), and glutaralde-hyde (25% aqueous solution) were also obtained from Sigma.Hydrogen peroxide (30% solution) was supplied by BDH andstored at 4 °C. Pierce supplied the cross-linking agents maleimi-dobenzyl-N-hydroxysulfosuccinimide ester (sulfo-MBS), dimethyladipimidate-2 hydrochloride (DMA), and bis(sulfosuccinimidyl)suberate (BS3). Dialysis membrane (6-8000 kDa MWCO) wasobtained from Fisher Scientific. Gibco BRL supplied nitrocellulose,supported nitrocellulose, neutral nylon (Biodyne A), and thepositively charged nylon (Biodyne B) membranes. Pall SpecialtyMaterials supplied the preactivated membranes ImmunodyneABCand UltraBind; both of these membranes were specified with apore diameter of 0.45 µm. Bioanalytical Systems supplied theplatinum disk working electrodes (1.8-mm diameter) and Ag/AgClreference electrodes. Silver wire (1.0-mm diameter, 99.99%) waspurchased from Aldrich. The microbial strains Saccharomycescerevisiae ATCC9896, Bacillus cereus, Staphylococcus aureusATCC6538P, Enterobacter aerogenes ATCC13048e, and Proteusvulgaris ATCC6380 were obtained from the strain collection ofthe Department of Biology, University of Waterloo. E. coli JM105was obtained from Prof. G. Guillemette, Department of Chemistry,University of Waterloo.

METHODSCultivation of Microorganisms. All strains were cultivated

under the same conditions using a growth medium with the

(13) Mirelman, D.; Ofek, I. In Microbial Lectins and Agglutinins, Properties andBiological Activity; Mirelman, D., Ed.; John Wiley & Sons: New York, 1986.

(14) Hynes, S. O.; Hirmo, S.; Wadstroem, T.; Moran, A. P. J. Clin. Microbiol.1999, 37, 1994-1998.

(15) Patchett, R. A.; Kelly, A. F.; Kroll, R. G. J. Appl. Bacteriol. 1991, 71, 277-284.

(16) Lis, H.; Sharon, N. Chem. Rev. 1998, 98, 637-675.(17) Oldenberg, K. R.; Loganathan, D.; Goldstein, I. J.; Schultz, P. G.; Gallop, M.

A. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 5393.(18) Bundy, J. L.; Fenselau, C. Anal. Chem. 1999, 71, 1460-1463(19) Bundy, J. L.; Fenselau, C. Anal. Chem. 2001, 73, 751-757.(20) Zhou, C.; Pivarnik, P.; Rand, G.; Letcher, S. V. Biosens. Bioelectron. 1998,

13, 495-500.(21) Payne, M. J.; Campell, S.; Kroll, R. G. J. Appl. Bacteriol. 1993, 74, 276-

283.(22) Pistole, T. G. Annu. Rev. Microbiol. 1981, 35, 85-112.(23) Ertl, P.; Unterladstaetter, B.; Bayer, K.; Mikkelsen, S. R. Anal. Chem. 2000,

72, 4949-4956.(24) Ertl, P.; Robello, E.; Battaglini, F.; Mikkelsen, S. R. Anal. Chem. 2000, 72,

4957-4964.

(25) Jurs, P. C.; Bakken, G. A.; McClelland, H. E. Chem. Rev. 2000, 100, 2694-2678.

(26) Kramer, R. Chemometric Techniques for Quantitative Analysis; MarcelDekker: New York, 1998.

(27) Schonkopf, S. Am. Lab. 1999, 31, 32-36.(28) Di Natale, C.; Davide, F. A. M.; D’Amico, A.; Sberveglieri, G.; Nelli, P.; Faglia,

G.; Perego, C. Sens. Actuators, B 1995, 24-25, 801-805.

4242 Analytical Chemistry, Vol. 73, No. 17, September 1, 2001

following components: KH2PO4 (2.88 g/L), K2HPO4‚3H2O (5.76g/L), tryptone (2.4 g/L), yeast extract (1.2 g/L), trisodium citratedihydrate (1.2 g/L), MgSO4‚7H2O (0.48 g/L), CaCl2‚2H2O (0.048g/L), (NH4)2SO4 (1.63 g/L), NH4Cl (1.34 g/L), glucose (13.2 g/L),and 240 µL of the trace element stock solution per liter of medium.The trace element stock solution was prepared in 5 N HCl (Merck)and contained the following compounds: FeSO4‚7H2O (40 g/L),MnSO4‚H2O (10 g/L), AlCl3‚6H2O (10 g/L), CoCl2‚6H2O (4 g/L),ZnSO4‚7H2O (2 g/L), Na2MoO4‚2H2O (10 g/L), CuCl2‚2H2O (1g/L), and H3BO3 (0.5 g/L). Glucose was prepared as a concen-trated aqueous solution, sterilized separately, and added to thegrowth medium prior to inoculation. Cultivations were performedby adding 1 mL of 1:1 glycerol/exponential-phase cell culturemixture (previously stored at -80 °C) to 50 mL of growth mediumin a shake flask. Growth proceeded for a maximum of 10 h in anincubator-shaker (200 rpm, 37 °C). Cell culture samples werecentrifuged (5000 rpm, 5 min), resuspended in buffer consistingof growth medium without glucose, proteins, or trace elementsbut containing 10 mM succinate, and stored on ice.

Agglutination Tests. An aliquot (50 µL) of the centrifugedand resuspended cell sample was combined with 150 µL of thebuffered lectin stock solution containing 200 µg of protein/mL ofbuffer. Different microtiter wells were used to compare rowscontaining lectin/bacterial suspension samples to controls, wherebuffer was combined with the cell suspension. All lectins wereinvestigated for their ability to form visible agglutinin after 6-hincubation at room temperature. The extent of the visible forma-tion of agglutinin was used to classify binding patterns.

Immobilization Methods. Adsorption. Lectin, avidin, andstreptavidin solutions (100 µg/mL) were prepared in a bufferconsisting of growth medium, as above, without glucose, proteins,or trace elements. Precut membrane disks (0.28 cm2) wereimmersed in 300 µL of these solutions and were incubated for 1h at room temperature (22 ( 2 °C). Just prior to use, disks wereremoved from the protein solutions and rinsed with buffer.

Cross-Linking. Unless otherwise noted, cross-linking agentswere dissolved in 50 mM HEPES (pH 7.5-8.5) containing 5 mMMgSO4 to final concentrations of 5 (MBS and BS3) or 10 mM(DMA and EDC). Glutaraldehyde solutions (25% aqueous) wereused as received. Membrane disks with adsorbed proteins wereimmersed in 300 µL of the cross-linking solution and allowed toreact at room temperature (22 °C) for 30 min. An additional 15min was used for glutaraldehyde cross-linking; this was donefollowing addition of sodium cyanoborohydride to a final concen-tration of 10 mM. The disks were then removed and rinsed withbuffer consisting of growth medium, as above, without glucose,proteins, or trace elements.

Preactivated Membranes. Precut disks (0.28 cm2) of Immuno-dyne ABC and UltraBind membranes, featuring nucleophile-selective and aldehyde-activated surfaces, respectively, wereimmersed in 100 µg/mL lectin, avidin, or streptavidin solutionsand allowed to react for 30 min at room temperature. The diskswere removed from the protein solutions, rinsed with buffer, andstored in buffer in an ice/water bath.

Peroxidase Activity Assay. Biotinylated peroxidase was usedas a label to detect membrane-bound avidin and streptavidinfollowing adsorptive immobilization. Modified membranes weretransferred to aliquots of a biotinylated peroxidase stock solution

(300 µL, 20 µg/mL) and incubated for 1 h at room temperature.Excess unbound peroxidase was washed away with buffer andthe modified membrane was transferred into the ABTS reagentsolution. The enzymatic reaction was then started by adding 3µL of 30% H2O2 to the assay solution. The accumulation of oxidizeddye over time (2-6 min) was measured spectrophotometricallyat 412 nm using buffer as a blank. The calculation of ABTSconversion (µM/min) was performed using Beer’s law (A ) εbC)with a molar absorptivity (ε) of 32 400 M-1 cm-1.29

Cell Capture on Lectin-Modified Membranes. Exponential-phase bacteria were harvested, centrifuged at 5000 rpm (3000g),resuspended in buffer containing 10 mM succinate, and storedon ice. The lectin-modified membranes were added to the bacterialsuspension and incubated for 100 min on ice (unless otherwiseindicated). Membrane disks were then removed from the cellsuspensions, rinsed with buffer, and affixed to the surfaces of thePt working electrodes using nylon netting and rubber O-rings.

Chronocoulometry. Unless otherwise noted, membrane-modified electrodes and Ag/AgCl counter electrodes were im-mersed into an electrochemical cell containing 200 µL of thereagent solution (50 mM ferricyanide, 10 mM formate, 10 mMsuccinate, and 0.1 mM menadione in growth medium lackingglucose, proteins, or trace elements). After an incubation periodof 2 min at 37 °C, the accumulation of ferrocyanide was detectedby means of chronocoulometry. The potential of the Pt workingelectrode was set at + 0.50 V versus Ag/AgCl and the resultingcurrent was integrated over 800 s to yield a plot of total chargeagainst time. The difference in total charge between 200 and 800s was recorded as the analytical signal, unless otherwise indicated.

Chemometric Data Analysis. Normalized chronocoulometricdata obtained for three replicate runs with each cell culture wereused to generate a matrix for (PCA. One column in the data matrixconsisted of the chronocoulometric signals for each of the 10lectin-modified membranes, plus controls (membranes modifiedwith BSA and unmodified membranes). Thus, three columns weregenerated for each microorganism. The matrix was converted intospreadsheet format using Microsoft Excel and was then convertedinto a Lotus file for incorporation into MATLAB programs. Factoranalysis was performed using the Chemometrics Toolbox ofMATLAB (Version 2.3, The MathWorks, Natick, MA, 1998) andinvolved the generation of reduced eigenvectors to determine theoptimal number of factors, examination of the resulting residualsplots for randomness, and the generation of scores for the firstthree principal components. These scores were obtained for eachof the three replicate trials for each microorganism and wereplotted to determine whether qualitative groupings of microorgan-isms existed in the lectin-binding/chronocoulometric responses.

RESULTS AND DISCUSSIONAgglutination tests were performed using 10 lectins, to

determine whether they interact selectively enough with outermembrane lipopolysaccharides of the six microorganism speciesto allow discrimination between cultures based on the formationof visible agglutinin after a 6-h incubation. Lectins for this studywere chosen based on their differing known mono- and oligosac-charide binding selectivities.9 Negative controls (without lectin)were run alongside the lectin tests for each species, to ensure

(29) Makinen, K. K.; Tenovuo, J. Anal. Biochem. 1982, 126, 100-108.

Analytical Chemistry, Vol. 73, No. 17, September 1, 2001 4243

the absence of autoagglutination.14 The results, shown in Table1, indicate a range of selectivities, with each lectin causing noagglutination (-) of some cultures to strong agglutination (+++)of others. No identical agglutination patterns were observed withthis group of lectins; however, similar patterns exist for S.tuberosum and C. fragile, A. integrifolia and H. pomatia, H. pomatiaand C. fragile, and P. americana and G. nivalis, with only twominor differences in agglutination abilities noted for each pair.Similarities in the agglutination patterns of Solanum tuberosum,A. integrifolia, H. pomatia, and C. fragile lectins may be attributedto their known selectivities toward galactose or N-acetyl moieties,but the similar pattern observed for P. americana and G. nivalislectins would not be predicted from their selectivities towardN-acetylglucosamine and nonreducing mannose, respectively.Possibly these two lipopolysaccharide components are coex-pressed in the species studied.

Table 1 also shows that two of the microorganisms studied,B. cereus and S. cerevisiae, yield only weak agglutination with abouthalf of the lectins in this group. Despite the lack of strongagglutination, the agglutination patterns are sufficiently differentto allow discrimination. One species, E. aerogenes, stronglyagglutinates most of the lectins studied, suggesting the presenceof diverse oligosaccharide structures in its outer membrane.

Lectin immobilization studies were carried out using Con A,since it strongly agglutinates E. coli JM105, as shown in Table 1.Prior to Con A immobilization, preliminary experiments wereconducted to select conditions for the detection of viable cellstrapped at Pt electrode surfaces, using a dilution series of aresuspended, exponential phase culture of E. coli (OD600 ) 3.51).Previous calibration studies with this organism23 allowed correla-tion of optical density at 600 nm with viable cell counts, or cfuvalues. Aliquots of cell suspension (5 µL) were placed onunmodified nitrocellulose membrane disks, and these were fixedon the electrode surface using dialysis membrane and a rubberO-ring. The difference in chronocoulometric charge between 200and 800 s is plotted against viable cell number in Figure 1, wherean approximately linear increase in signal can be seen with anincreasing numbers of trapped cells. On the basis of the area ofthe membrane disks used in immobilization studies (0.28 cm2),and an estimated requirement of 1 µm2 for each surface-boundcell, a close-packed monolayer of viable E. coli on one side of themembrane would be expected to involve 2.8 × 107 cfu. Figure 1shows that 2 orders of magnitude fewer cells are readily detected

by mediated chronocoulometric measurement of respiratory cycleactivity, when the cells are trapped at the electrode surface ratherthan bound to a membrane.

Initial immobilization studies involved avidin and streptavi-din,4,30,38 which were adsorbed onto nitrocellulose, supportednitrocellulose, Biodyne A and Biodyne B membranes, exposedto cross-linking agents to stabilize the immobilized proteinlayer30-37 and then incubated with biotinylated peroxidase. Thisenzyme was used as a label to compare the quantities of activeavidin or streptavidin on the membranes: ABTS conversion rateswere measured, and these values (Table 2) indicate the relativequantities of peroxidase attached to the membranes.29,39,40

(30) Tatsuma, T.; Watanabe, T. Anal. Chem. 1992, 64, 625-630.(31) Staros, J.; Wright, W.; Swingle, D. Anal. Biochem. 1986, 156, 220-222.(32) Braun, B.; Klein, E.; Lopez, J. L. Biotechnol. Bioeng., 1996, 51, 327-342.(33) Oswald, P. R.; Evans, R. A.; Henderson, W.; Daniel, R. M.; Fee, C. J. Enzymol.

Microb. Technol. 1998, 23, 14-20.(34) Cochrane, F. C.; Petach, H. H.; Henderson, W. Enzymol. Microb. Technol.

1996, 18, 373-379.(35) Yoshitake, S.; Imagawa, M.; Ishikava, E. Anal. Lett. 1982, 15, 147.(36) Saurina, J.; Hernandez-Cassou, S.; Fabregas, E.; Alegret, S. Anal. Chim. Acta

1998, 371, 49-57.(37) Moody, J.; Sanghera, G. S.; Thomas, J. D. R. Analyst 1986, 111, 605-704.(38) Madras, M. B.; Buck, R. P. Anal. Chem. 1998, 98, 637-641.(39) Childs, R. E.; Bardsley, W. G. Biochem. J. 1975, 145, 93-103.(40) Shindler, J. S.; Childs, R. E.; Bardsley, W. G. Eur. J. Biochem. 1976, 64,

325-331.

Table 1. Agglutination Test Results for the Reactions of Lectins with Microorganismsa

agglutination observedb

lectin sugar specificity E. coli E. aerogenes P. vulgaris B. cereus S. aureus S. cerevisiae

Con A R-man, R-GLC +++ - ++ - ++ -S. tuberosum (glcNAc)3 + +++ - - + +L. culinaris R-man + +++ ++ - + -A.integrifolia R-gal + ++ +++ + + -H. pomatia galNAc + ++ +++ + ++ +T. vulgaris (glcNAc)3, NeuNAc ++ + - - + +A. hypogaea â-gal, galNAc - +++ ++ + +++ -C. fragile galNAc + +++ - + ++ +P. americana (glcNAc)3 - +++ ++ - +++ +G. nivalis nonred R-man - +++ + - ++ +

a Conditions: 50-µL aliquots of exponential-phase bacterial samples were combined with 150 µL of lectin solution (200 µg/mL) and incubatedfor 6 h at room temperature. b Key: (+++) strong, (++) medium, (+) low, and (-) no agglutination

Figure 1. Chronocoulometric signal as a function of the number ofcells (cfu) trapped at the working electrode surface. Modified elec-trodes were incubated at 37 °C for 10 min in 200 µL of a reagentsolution containing 50 mM ferricyanide, 0.10 mM menadione, 10 mMsuccinate, and 10 mM formate; chronocoulometry was then performedfor 800 s at + 0.5 V (Ag/AgCl).


With adsorbed avidin, the highest ABTS conversion rates(Table 2) were found following cross-linking with either glutaral-dehyde or BS3. Signals obtained with streptavidin-glutaraldehydemembranes are significantly lower. Results obtained with the twonitrocellulose membranes suggest that avidin adsorption occursbut that deactivation results from cross-linking, especially withglutaraldehyde and BS3. With these membranes, EDC and DMAcross-linking procedures provide the highest remaining biotin-binding activity. Since supported nitrocellulose yielded the lowestABTS conversion rates, it was not used in further experiments.

Chronocoulometric measurements were used to determinewhich combination of membrane and immobilization chemistryyields the greatest signals following capture of E. coli JM105 bysurface-bound Con A. In these experiments, two preactivatedmembrane materials were studied (Immunodyne ABC and Ultra-Bind) in addition to Biodyne A, Biodyne B, and nitrocellulose.Following immobilization of Con A, avidin, or streptavidin as wellas cross-linking and incubation with biotinylated Con A (wherenecessary), membranes were incubated in resuspended, expo-nential-phase E. coli at 0 °C for 60 min. Membranes were thenrinsed and fixed to the surfaces of Pt working electrodes andincubated 10 min with reagent solution (as in Figure 1). Table 3shows the results of these experiments, where the signal corre-sponds to the total charge accumulated between 200 and 800 sduring the chronocoulometric run. With this 10-min integrationperiod, results ranged from 1.2 to 44.6 µC and clearly illustratethe effect of immobilization chemistry on surface activity.

The results shown in Table 3 mainly represent experimentswhere Con A was bound directly to the membrane surfaces, unlikethose shown in Table 2, where avidin or streptavidin wasimmobilized. However, for comparison, Biodyne B was used toimmobilize avidin and was then cross-linked with glutaraldehydeprior to biotin-Con A and E. coli exposure, since this methodyielded the highest ABTS conversion rate for biotinylated per-oxidase binding, as shown in Table 2. This immobilization methodyielded a large chronocoulometric signal (30.6 µC), indicatinggood Con A immobilization and good E. coli binding, but stilllarger signals were obtained using the preactivated membranes,particularly Immunodyne ABC with direct immobilization of ConA (44.6 µC) and UltraBind with the streptavidin-biotin-Con Aconfiguration (37.0 µC). Further experiments involved the directbinding of lectins to Immunodyne ABC membranes.

Con A concentration was varied (5-500 µg/mL) during the30-min Immunodyne immobilization step, and chronocoulometricsignals obtained over 600 s were largest using 100 µg/mL ConA. These traces are shown in Figure 2, where a background trace

obtained with an unmodified membrane that was not exposed toE. coli is also shown. Subsequent immobilizations were performedusing 100 µg/mL protein.

The effect of E. coli incubation time was studied usingImmunodyne ABC membranes that had been modified with eachof the 10 lectins as well as BSA, which was included as a controlto deactivate reactive groups on the membrane surface (agglutina-tion tests with BSA and E. coli were negative). Using thesemembranes, chronocoulometric measurements were made as afunction of their time of exposure to resuspended, exponential-phase E. coli, for a minimum of 3 h. Results for three representa-tive lectin-modified and control (unmodified) membranes areshown in Figure 3. Six of the 10 lectins (Con A, S. tuberosum, L.culinaris, A. hypogaea, G. nivalis and P. americana) as well asBSA caused increasing chronocoulometric signals as a functionof E. coli capture time; the L. culinaris response is shown in Figure3. Three lectins (H. pomatia, T. vulgaris, C. fragile) producedsignals that increased to a maximum and then declined at longer

Table 2. ABTS Conversion Rates for Modified Membranes Labeled with Biotinylated Peroxidase

ABTS conversion rate, µM/min, 412 nmacross-linking

agentspecies

adsorbed Biodyne A Biodyne B nitrocellulose supported NC

glutaraldehyde avidin 7.6 10.7 3.7 4.6EDC avidin 7.3 3.2 8.8 5.6MBS avidin 2.8 1.2 6.8 1.7DMA avidin 8.2 6.4 8.4 6.7BS3 avidin 9.8 9.1 0.0 0.0

glutaraldehyde streptavidin 5.8 6.4 1.2 0.6

a Average of three replicate measurements, RSD < 10%.

Table 3. Chronocoulometric Signals ObtainedFollowing E. coli JM105 Binding to Con A-ModifiedMembranes

membraneimmobilized

speciesadditional membrane

treatmentcharge,

µCa

nitrocellulose Con A none 9.9 ( 4.3EDC 4.3 ( 0.8BS3 2.4 ( 0.1MBS 17.7 ( 0.4DMA 17.3 ( 1.5

Biodyne A Con A none 4.6 ( 0.8EDC 1.2 ( 0.4BS3 2.2 ( 0.1MBS 17.4 ( 2.4DMA 7.8 ( 2.0

Biodyne B Con A none 9.3 ( 3.3EDC 5.3 ( 1.9BS3 3.4 ( 0.2MBS 16.7 ( 0.8DMA 13.0 ( 3.0

avidin glutaraldehyde,biotinylated Con A

30.6 ( 3.5

ImmunodyneABC

Con A none 44.6 ( 6.5

avidin biotinylated Con A 17.4 ( 2.6streptavidin biotinylated Con A 16.7 ( 1.6

UltraBind Con A none 21.0 ( 5.0avidin biotinylated Con A 18.0 ( 4.8streptavidin biotinylated Con A 37.0 ( 4.5

a Values represent the average difference in charge measuredbetween 200 and 800 s of each chronocoulometric run. Uncertainty isone standard deviation.


E. coli incubation times; the H. pomatia response is shown inFigure 3. One lectin (A. integrifolia) yielded a slowly decreasingchronocoulometric signal. The unmodified membrane yieldedreasonably large signals (40 µC), but these did not vary signifi-cantly over more than 2-h incubation with E. coli. From theseresults, an incubation time of 100 min was selected for furtherstudies.

Finally, the 10 lectin-modified Immunodyne membranes alongwith the BSA-modified and an unmodified membrane were usedin a model 12-element array with each of the 6 microorganisms(E. coli, E. aerogenes, P. vulgaris, S. aureus, B. cereus, S. cerevisiae).These were grown under identical conditions, harvested in theexponential phase (OD600 between 2.5 and 3.5), centrifuged andresuspended in buffer, and stored on ice. Membranes wereincubated individually in aliquots (300 µL) of the cell suspensionin microtiter wells for 100 min at 0 °C. Membranes were thenfixed onto Pt electrode surfaces, and the modified electrodes wereincubated at 37 °C for 30 min in 200 µL of the assay solution priorto an 800-s chronocoulometric measurement at +0.5 V (Ag/AgCl).Three replicate measurements of respiratory cycle activity weremade with each microorganism/membrane combination.

Table 4 shows the background-subtracted chronocoulometricresults, as the difference in total charge consumed between 200and 800 s, for each replicate of the 72 microorganism-membranecombinations. The range of the chronocoulometric signals islarge: after background subtraction, values ranged from 0 to 389µC. Within most of the replicate groupings, significant variationbetween individual measurements can be seen. However, overallagreement was observed between these results and the resultsof agglutination tests (Table 1). With this group of lectins, thestrongest agglutination was observed with E. aerogenes, P. vulgaris,and S. aureus (see Table 1), and these organisms also yieldedthe highest chronocoulometric signals at the electrochemicallectin array (Table 4). Similarly, the organisms that showed onlyweak agglutination with these lectins, B. cereus and S. cerevisiae,gave the lowest electrochemical signals.

Occasional noncorrespondence of agglutination and electro-chemical results occurred. For example, the very large electro-chemical signals observed with P. vulgaris at a C. fragile lectin-modified membrane (Table 4) does not correspond to theagglutination test result (Table 1), which was negative for thiscombination of microorganism and lectin. Similarly, with E.aerogenes, reasonably strong signals were observed with ConA-modified membranes, while weak signals were seen with S.tuberosum (Table 4); in contrast, agglutination test results werenegative for this organism with Con A and strongly positive withS. tuberosum (Table 1). We have attributed these discrepanciesto changes in lectin structure that may be expected to occur uponcovalent immobilization on the Immunodyne ABC membranes.

The large chronocoulometric signals obtained with some ofthe organisms at unmodified and BSA-modified Immunodyne ABCmembranes were unexpected, since these membranes wereincluded as controls for which significant cell binding wasexpected to be minimal. It is possible that unmodified membranes,possessing aldehyde functional groups, react with componentsof the outer membranes of the cells to affect covalent attachmentof cells to the membrane surfaces. Although this was not evidentwith S. cerevisiae, large chronocoulometric signals were observedwith E. aerogenes, P. vulgaris, and S. aureus (Table 4). BSA-modified membranes, which were intended as deactivated con-trols, showed large chronocoulometric signals for E. coli, P.vulgaris, and S. aureus; this has been attributed to hydrophobic,noncovalent interaction of the cells with the immobilized protein.

Chemometric PCA was applied to the chronocoulometricresults shown in Table 4, in an attempt to apply pattern recognitionmethods41 to classify the response patterns according to species.Individual measurements were incorporated as a 12-elementcolumn into the data matrix, so that each column contained onemeasurement with each of the 12 membranes for one microorgan-ism. Thus, three replicate columns existed for each microorgan-ism, with each column corresponding to one complete arraymeasurement. PCA yielded three factors containing more than90% of the total variance across the matrix: PC 1 (76%), PC 2(6%), and PC 3 (5%). Scores for each column were computed forthe first two principal components, and these are plotted in Figure4. In this scores plot, each point represents 12 chronocoulometricexperiments (10 different lectins, BSA, and control) made with

(41) Malinkowski, E. R. Factor Analysis in Chemistry, 2nd ed.; Wiley-Inter-science: New York, 1991.

Figure 2. Chronocoulometric traces obtained for E. coli JM105captured on Immunodyne ABC membranes modified using Con Aconcentrations of (a) 0, (b) 50, (c) 100, (d) 300, and (e) 500 µg/mL.

Figure 3. Chronocoulometric signals as a function of cell capturetime for E. coli JM105 on unmodified (b) and lectin-modified Immu-nodyne ABC membranes. Lectins are (∆) L. culinaris, ([) H. pomatia,and (1) A. integrifolia. Chronocoulometry was performed at 37 oC anda run time of 800 s.


each microorganism. In Figure 4, replicate array measurementshave been given the same symbol and have been circled to showthe groupings of replicate measurements that resulted from PCA.Figure 4 shows that the array results for all six microbial strainsform subpopulations that can be distinguished from one another.

Variances between microbial strains appear to be high enoughwith this two-dimensional plot to allow classification, whileindividual replicates are similar enough to cluster together. Thethird principal component, which contains variance similar to thesecond, can be used in a three-dimensional plot (not shown),which allows further discrimination of E. coli, B. cereus, and S.cerevisiae, which are the three organisms grouped on the left sideof Figure 4.

This work demonstrates the feasibility of lectin arrays used inconjunction with electrochemical methods for microorganismidentification in pure cultures. Development of the arrays forpractical identification would require the application of computa-tional methods, such as the calculation of Euclidean distances andthe determination of confidence intervals in these values, andwould require a much larger data set to clearly identify the volumeoccupied by each organism on the three-dimensional plot.42

Applications of lectin arrays to mixtures of microorganisms mayalso be possible, if large lectin arrays are used in conjunction withcomputational methods such as the generalized rank annihilation

(42) Massart, B.; Guo, Q.; Questier, F.; Massart, D.; Boucon, C.; de Jong, S.;Vandeginste, B. G. M. Trends Anal. Chem. 2001, 20, 35-41.

Table 4. Chronocoulometric Signals Obtained after Selective Cell Capture on Lectin-Modified Electrodes with SixMicrobial Strains

charge, µCamodified

electrodes E. coli E. aerogenes P. vulgaris B. cererus S. aureus S. cerevisiae

Con A 33 188 203 0 139 630 109 124 0 248 331 101 148 0 193 0

S. tuberosum 16 34 213 0 151 513 37 95 0 69 415 36 204 0 177 0

L. culinaris 29 139 182 16 72 046 151 253 23 98 025 106 163 18 102 0

A. integrifolia 32 116 210 40 35 313 144 257 73 97 08 223 102 23 86 0

H. pomatia 25 139 132 17 128 1913 176 49 0 130 919 159 65 20 143 7

T. vulgaris 52 116 68 6 100 2732 230 46 1 113 442 173 57 2 120 0

A. hypogaea 19 160 209 23 193 012 113 118 52 169 215 104 310 38 140 1

C. fragile 27 77 268 16 58 314 61 389 37 198 020 110 253 20 130 6

P. americana 32 92 177 0 187 4426 157 196 1 239 026 125 105 0 213 0

G. nivalis 15 44 128 11 144 5819 159 237 8 114 1611 83 197 3 140 11

control 77 116 172 16 144 08 133 113 16 131 0

74 143 110 10 105 0BSA 138 35 243 8 118 0

271 35 66 18 100 318 35 122 15 135 2

a Values are corrected for background observed with unmodified membranes that had not been exposed to microorganisms and represent thedifference in charge consumed between 200 and 800 s of the chronocoulometric run.

Figure 4. Pattern recognition plot obtained using data shown inTable 4. Each point represents a column of 12 individual measure-ments (10 lectins, BSA and control membranes) with (X) E. coli, (b)S. aureus, (0) S. cerevisiae, (2) B. cereus, (+) P. vulgaris, and (∇)E. aerogenes.


method (GRAM)43 to distinguish (and remove) signals fromindividual species. Further work is underway to achieve this goal.

CONCLUSIONSMembrane-bound lectins were used as recognition agents in

a sensor array for the identification of microorganisms. Theselective, but not specific, binding of lectins to oligosaccharideresidues present on the exterior surface of the microorganismswas exploited to generate characteristic patterns of binding of themicroorganisms to the different lectin-modified membranes.Bound microorganisms were detected electrochemically, by medi-ated chronocoulometric measurement of respiratory cycle activity,so that only viable cells bound to the membranes were detected.

Principal component analysis of the array measurements showedclear groupings of six microorganisms and will be pursued as anovel method of microorganism species identification.

ACKNOWLEDGMENT

Financial support from NSERC (Canada) and an InternationalScholarship from the Austrian Ministry of Science (P.E.) aregratefully acknowledged.

Received for review March 19, 2001. Accepted June 19,2001.

AC010324L(43) Sanchez, E.; Kowalski, B. R. Anal. Chem. 1986, 58, 496-499.


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