Discrimination of Escherichia coli Strains using Glycan CantileverArray SensorsAndreas Mader,† Kathrin Gruber,† Riccardo Castelli,§,∥ Bianca A. Hermann,† Peter H. Seeberger,§,∥

Joachim O. Radler,† and Madeleine Leisner*,†,‡

†Center for Nanoscience, Ludwig-Maximilians-Universitat, Fakultat fur Physik, Geschwister-Scholl-Platz 1, 80539 Munchen, Germany‡Arnold Sommerfeld Center for Theoretical Physics and Center for NanoScience, Department of Physics, Ludwig-MaximiliansUniversitat Munchen, Theresienstr. 37, 80333 Munchen, Germany§Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Arnimallee 22, 14195 Berlin, Germany∥Institute for Chemistry and Biochemistry, Freie Universitat Berlin, Arnimallee 22, 14195 Berlin, Germany

*S Supporting Information

ABSTRACT: Carbohydrate-based sensors, that specificallydetect sugar binding molecules or cells, are increasinglyimportant in medical diagnostic and drug screening. Here wedemonstrate that cantilever arrays functionalized with differentmannosides allow the real-time detection of several Escherichiacoli strains in solution. Cantilever deflection is therebydependent on the bacterial strain studied and the glycanused as the sensing molecule. The cantilevers exhibit specific and reproducible deflection with a sensitivity range over four ordersof magnitude.

KEYWORDS: Cantilever array sensors, Escherichia coli, glycomics, biosensors, nanomechanics

In recent years a variety of biosensors have been developedfor the detection of different molecules.1−3 High sensitivities

can be achieved in reduced detection volumes and in parallelformat.4 Cantilever biosensors have been used to study DNA1,5

as well as protein interactions6 and aid the investigation ofcells7 and the analysis of bacterial growth.8,9 This techniqueoffers a low-cost approach to label-free sensing with in situreferencing and fast response times.10 Carbohydrates aresuitable biosensing molecules for medical diagnostics,11 andtheir importance for biological processes, such as cell adhesionor migration,12 was demonstrated using microarray systems.2

Carbohydrate based sensors represent a powerful tool to studyglycan interactions or the detection of pathogens.13 Assequencing technologies for carbohydrate structures mature,14

carbohydrate-based cantilever sensors are developed to analyzebinding properties of a variety of binding partners.15 The fastdetection of bacterial species finds increasing attention:16

Besides classical growth media based methods,17 moresophisticated approaches,18 such as metal oxide-based olfactorysensors,3 to discriminate bacteria have been developed.Recently, Tzeng et al. demonstrated the suitability ofmannose-based cantilever sensors operated in the dynamicmode to recognize formaldehyde killed Escherichia colibacteria.19 However a detailed real-time investigation of livingE. coli cells comprising high selectivity, sensitivity, andreproducibility of bacterial recognition is still missing.Here, we demonstrate that carbohydrate-based cantilever

array biosensors, previously established for the accuraterecognition of antiviral proteins,15 can detect and distinguish

E. coli strains with distinct mannoside binding properties in asensitive and specific manner. Eight parallel, gold-coated topsides of a cantilever array were functionalized individually withself-assembled layers of a trimannoside or a nonamannosidecompound as specific targets and a galactoside as an internalnonspecific reference (see inset in Figure 1). A terminal thiolwas installed on each carbohydrate (see Figure 1, SupportingInformation for detailed structures) as a specific point ofattachment. The E. coli strain ORN 178 presents the idealcandidate to investigate this glycan cantilever array sensor as itcontains type-I pili that specifically bind to mannose-containingstructures20 via the binding protein FimH.21−23 To obtain thefollowing detailed experimental data, we operated the cantileversensor instrument in the static mode,24 measuring in real-timethe cantilever deflection that is caused by the surface stress24

induced upon bacterial adhesion.In a typical experiment with an ORN 178 sample (OD = 0.5)

negative deflections for cantilevers functionalized with all threecarbohydrate structures are observed. The average deflectionsrepresent the combined (averaged) measurements for identi-cally functionalized cantilevers within an array. These averagedeflections for galactose, trimannose, and nonamannose sensorsare plotted against time (upper panel, Figure 1). Whilegalactose sensors, as an internal reference, show the smallestaverage deflection, the trimannose and nonamannose canti-

Received: October 24, 2011Revised: November 29, 2011Published: December 5, 2011

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levers react significantly stronger. Specific binding of ORN 178FimH adhesion protein at the tip of type-I pili to mannosides isresponsible for an increased number of binding events on themannose-covered surfaces. The bound bacteria induce adifference in surface stress between upper and lower side ofthe micrometer thin cantilever. This compressive surface stressis relieved by cantilever bending downward. The largernonamannose signal is assigned to this carbohydrate’s increasedpotential for multisite and multivalent binding, which in turnleads to more binding events on these surfaces. In contrast, thegalactose signal is attributed to nonspecific attachment of thebacteria to galactose or the cantilever surface. It was shown thatORN 178 specifically binds to mannose but not to othercarbohydrates, such as galactose.16 Therefore we used galactosethat resembles mannose structurally as an internal reference.25

To extract nonspecific binding contributions from the totalsignal, while accounting for nonspecific reactions, includingsmall changes of pH, refractive index or reactions occurring onthe underside of the cantilever,25 the in parallel obtainedaveraged galactose deflection is subtracted from both averagedmannoside signals. The resulting differential deflection isplotted in the lower panel in Figure 1 and represents thespecific part of the bacterial recognition. In the following, onlythe differential deflection is given. As nonamannose consis-tently provided larger signals, we focus on these sensors for thefollowing discussion, investigating the critical sensor parametersspecificity, reliability, and sensitivity of the sensor setup forbacterial detection.

To examine the specificity of the ORN 178−nonamannoserecognition using our cantilever array biosensor, a competitiveinhibition assay was carried out.15,22 Free mannose (100 mM)was added abundantly to the running buffer to the second oftwo successive injections with identical concentrations. Thesecond differential deflection is reduced by about 50% (seeFigure 2a) in agreement with other reports of soluble

competitor binding studies.15,22 Since the monosaccharidemannose competes with the mannosides bound to thecantilever surface for binding to E. coli cells, fewer bacteriabind to the sensor surface, and a smaller deflection is observed.Consequently, the mannose sensor coating specificallyrecognizes and detects E. coli ORN 178 cells.In order to examine the accuracy of the recorded sensor

signals on the measured concentration, the cantilever arraysetup was tested with a series of samples with increasingconcentration followed by decreasing sample concentrations.The observed differential deflections demonstrate that thesignal size for repeated concentrations (Figure 2b) are veryreliable with a maximal deviation of only 15%.Besides signal specificity and reliability, the sensitivity of a

sensor is of crucial importance. Testing the sensor responsefrom an OD of 10−1 down to very low E. coli concentrations, wereproducibly detected a significant differential deflection for adilution to an OD of 10−4 (Figure 2, Supporting Information).At this concentration less than ∼800 bacteria bind to each

Figure 1. Detection of ORN 178 with glycan cantilever arrays. Upperpanel: Averaged deflections (Avrg. Deflection) for galactose,trimannose, and nonamannose functionalized cantilevers of an arrayagainst time. Each graph represents an average signal of 2−4identically functionalized cantilevers. Upon E. coli sample injection(OD = 0.5) the mannose cantilevers react significantly stronger (2−3times) than the galactose reference due to increased samplerecognition. Inset: Scheme of a cantilever array functionalized withdifferent carbohydrates. In this example, cantilevers 1 and 2 are coatedwith the internal reference galactose, cantilevers 3, 4, 7, and 8 withnonamannose, and cantilevers 5 and 6 with trimannose, respectively.Lower panel: Differential deflections (Diff. Deflection) representingthe specific binding events for the trimannose and nonamannosesensors derived by subtracting the galactose reference. The largernonamannose deflections indicate increased multisite and multivalentbinding.

Figure 2. Control experiments verifying the specificity andconcentration dependence of the cantilever assay. Differentialdeflection of ORN 178 on nonamannose-coated cantilevers is given.(a) Competitive inhibition: Following a first reference experiment(OD = 0.5), abundant free D(+)-mannose (100 mM) is added to therunning buffer. A second injection with identical bacterial concen-tration (OD = 0.5) shows an about 50% reduced differentialnonamannose deflection. As free mannose successfully competeswith the mannose sensor coatings, this result demonstrates thespecificity of the E. coli−nonamannose recognition. (b) A series ofincreasing and decreasing bacterial concentrations was conducted todemonstrate the sensor’s reproducibility. Identical concentrations(shown in the same color) fit within an acceptable range by a maximaldeviation of 15%. Please note: Error bars represent then standarddeviation obtained for measurements performed with differentcantilever arrays.

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cantilever, as determined by flow cytometry (data not shown).In single experiments we even observed considerabledeflections for ODs of 10−5 and 10−6 with 80 and 8 bacteriaper single cantilever, respectively. This large concentrationrange over five orders of magnitude down to a few cellsdemonstrates the high sensitivity of this sensor setup andcompares well to other reports for cantilever sensors.26 Similarresults for the specificity, concentration reliability, andsensitivity of the bacterial detection were achieved withtrimannose sensors (Figure 3, Supporting Information).To investigate the applicability of the glycan cantilever array

setup, we tested the sensor’s ability to differentiate between E.coli strains with distinct mannoside binding properties. To thisend, the signals of E. coli ORN 178, carrying the fully functionaladhesion protein FimH, were compared to E. coli ORN 208 andORN 206. The strain ORN 208 expresses the truncated proteinFimH*, while strain ORN 206 does not produce any pili. At anidentical concentration of OD = 10−1, measurements wererecorded (Figure 3). Three distinct differential signals were

determined with deflection sizes increasing in the order fromORN 208 to ORN 178 to ORN 206. Comparing the first twostrains, the smaller deflection size for ORN 208 can beattributed to the impaired binding properties of the mannosebinding protein FimH*. Strain ORN 206 with no pili, and thusno specific FimH recognition, surprisingly yields the largestsensor signal. However the missing pili might also explain thehigher number of bound bacteria associated with the largerobserved deflection. Assuming that pili sterically hinder accessto the cell membrane, increased nonspecific binding of thebacterial cell membrane via mannose transporters27 to themannoside-coated cantilever surface could result in the largerdeflection signal.To eliminate the possibility that the larger signal for ORN

206 might be due to excessive growth during samplepreparation, growth curves for the employed E. coli strainswere recorded. A comparison of the results under identicalconditions shows comparable growth for all three strains(Figure 4, Supporting Information). Thus different growthbehavior is excluded, and the observed distinct sensor signalscan be attributed to the different carbohydrate bindingproperties of the three E. coli strains.In this Letter we reported a glycan cantilever array sensor for

the detection and discrimination of E. coli bacteria with

different glycan binding characteristics. We demonstrated thespecificity of the recognition, the signal reproducibility, andhigh sensitivity down to very low sample concentrations ofmannosides binding to type-I pili in E. coli ORN 178. Twoadditional E. coli strains with altered or lacking type-I pili couldbe differentiated on this sensor via their individual deflectionsignals, indicating the usability of this approach to specificallysense bacterial strains in solution. Hence glycan cantileverarrays offer a large potential as a fast, accurate, and differentialscreening tool for future clinical and diagnostic applications.

Experimental Details. The nonamannose, trimannose,and galactose derivates with thiol linker were synthesized asdescribed elsewhere.28 The D(+)-mannose was obtained fromRoth, Germany. Gold-coated cantilever arrays consisting ofeight identical silicon cantilevers (500 × 100 × 1 μm) on asupport were purchased from Concentris GmbH, Switzerland.Following a UV−ozone cleaning cycle, the individual canti-levers were functionalized in parallel by inserting them into anarray of microcapillaries (filled with the respective carbohydratederivate at a concentration of 40 μM in 10 mM of Tris buffer,pH 7.7) for a time between 10 and 12 min, as described inGruber et al.15

E. coli strains ORN 178, ORN 208, and ORN 206 were akind gift from Prof. Orndorff, North Carolina State University,Raleigh, NC. Detailed genetic information of the strains used inthis study can be found in Table 1, Supporting Information. Allbacteria were grown overnight in 5 mL liquid LB medium at37 °C, shaking at 300 rpm. The antibiotic tetracycline wasadded if necessary at a concentration of 12.5 g/L. Overnightcultures were diluted into fresh 5 mL of LB medium to OD =0.1 and grown until an OD = 1 was reached. To record theinteraction between the carbohydrate-coated cantilever and thebacterial strains, bacteria were transferred into the runningbuffer [100 mM of NaCL, 10 mM of Tris, 0.005% Tween 20,and 1 mM of CaCl2 (Roth)]. Bacteria were diluted furtheraccording to the different experiments described in the maintext. To obtain the exact bacterial concentration for eachdilution, cells were counted using a flow cytometer (CyFlowspace, Partec).Measurements were performed on the commercial Cantisens

sensor platform (Concentris GmbH, Switzerland) equippedwith a measurement cell of 5 μL, an automated liquid handlingsystem, an integrated temperature control with samplepreheating stage, and a stability of 0.01 °C. The running bufferwas prepared from 10 mM of Tris, pH 7.7, 100 mM of NaCl, 1mM of CaCl2, and 0.005% Tween 20. Inside the instrument thearray was equilibrated at a constant buffer flow of 0.42 μL/secand a constant temperature of 22 °C for several hours until aconstant drift was achieved.29 Prior to any measurements, allarrays were checked for fabrication variances by subjectingthem to a short heat pulse (heat test).15 Only comparablecantilevers were employed for bacteria sensing. For eachmeasurement 100 μL of bacteria solution, diluted to the desiredconcentrations, was injected into the buffer flow. Thenanomechanical deflection signal is read in real time byemploying an array of eight parallel vertical cavity surfaceemitting lasers (VCSELs). LabView based software wasemployed for instrument control and signal processing. Dataanalysis was performed using Analysis Tools offered byConcentris GmbH, Switzerland. The signal curves of allcantilevers were corrected for constant drift. As indicated inthe main text, signals of identically functionalized cantileverswere averaged. Differential signals representing the specific

Figure 3. Discrimination of E. coli strains with distinct mannosebinding properties (OD = 0.1). The smallest differential signal on thisnonamannose/galactose array is obtained for strain ORN 208 whichcarries an impaired mannose binding protein FimH* at its pili andthus preferentially attaches via mannose transporters. Additionalbinding via mannose-specific binding protein FimH on ORN 178results in a larger differential deflection. The largest signal for strainORN 206 can be explained by the missing pili facilitating access andthe increased nonspecific binding to mannose transporters. See maintext.

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recognition were calculated by subtracting the nonspecificaveraged deflection signal of the internal reference galactosefrom the averaged deflection signal obtained for trimannose- ornonamannose-coated cantilevers.

■ ASSOCIATED CONTENT

*S Supporting InformationFour additional figures and one table: Carbohydrate structures,sensitivity of our approach, control experiments performed forORN 178 and trimannose-coated cantilevers, bacterial growthcurves, and strain descriptions. This material is available free ofcharge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author*E-mail: Madeleine.Leisner@physik.uni-muenchen.de.

■ ACKNOWLEDGMENTS

Financial support is gratefully acknowledged by the DFG ERA-Chemistry program (HE5162/1-1), the excellence clusterNanosystems Initiative Munich, the Walter-Meissner Instituteof the Bavarian academy of sciences and humanities, the Centerfor Nanoscience, the Elite Network of Bavaria, the Max-PlanckSociety (P.H.S.) and The Korber Prize for European Sciences(P.H.S.). We thank Prof. Orndorff for the kind gift of thestrains ORN 178, ORN 206, and ORN 208. For their kindsupport with performance of flow cytometer experiments, wethank Dr. J. Megerle.

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