Letter pubs.acs.org/NanoLett
Discrimination of Escherichia coli Strains using Glycan Cantilever Array Sensors Andreas Mader,† Kathrin Gruber,† Riccardo Castelli,§,∥ Bianca A. Hermann,† Peter H. Seeberger,§,∥ Joachim O. Rad̈ ler,† and Madeleine Leisner*,†,‡ †
Center for Nanoscience, Ludwig-Maximilians-Universität, Fakultät für Physik, Geschwister-Scholl-Platz 1, 80539 München, Germany Arnold Sommerfeld Center for Theoretical Physics and Center for NanoScience, Department of Physics, Ludwig-Maximilians Universität München, Theresienstr. 37, 80333 München, Germany § Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Arnimallee 22, 14195 Berlin, Germany ∥ Institute for Chemistry and Biochemistry, Freie Universität Berlin, Arnimallee 22, 14195 Berlin, Germany ‡
S Supporting Information *
ABSTRACT: Carbohydrate-based sensors, that specifically detect sugar binding molecules or cells, are increasingly important in medical diagnostic and drug screening. Here we demonstrate that cantilever arrays functionalized with different mannosides allow the real-time detection of several Escherichia coli strains in solution. Cantilever deflection is thereby dependent on the bacterial strain studied and the glycan used as the sensing molecule. The cantilevers exhibit specific and reproducible deflection with a sensitivity range over four orders of magnitude. KEYWORDS: Cantilever array sensors, Escherichia coli, glycomics, biosensors, nanomechanics
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E. coli strains with distinct mannoside binding properties in a sensitive and specific manner. Eight parallel, gold-coated top sides of a cantilever array were functionalized individually with self-assembled layers of a trimannoside or a nonamannoside compound as specific targets and a galactoside as an internal nonspecific reference (see inset in Figure 1). A terminal thiol was installed on each carbohydrate (see Figure 1, Supporting Information for detailed structures) as a specific point of attachment. The E. coli strain ORN 178 presents the ideal candidate to investigate this glycan cantilever array sensor as it contains type-I pili that specifically bind to mannose-containing structures20 via the binding protein FimH.21−23 To obtain the following detailed experimental data, we operated the cantilever sensor instrument in the static mode,24 measuring in real-time the 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 three carbohydrate structures are observed. The average deflections represent the combined (averaged) measurements for identically functionalized cantilevers within an array. These average deflections for galactose, trimannose, and nonamannose sensors are plotted against time (upper panel, Figure 1). While galactose sensors, as an internal reference, show the smallest average deflection, the trimannose and nonamannose canti-
n recent years a variety of biosensors have been developed for the detection of different molecules.1−3 High sensitivities can be achieved in reduced detection volumes and in parallel format.4 Cantilever biosensors have been used to study DNA1,5 as well as protein interactions6 and aid the investigation of cells7 and the analysis of bacterial growth.8,9 This technique offers a low-cost approach to label-free sensing with in situ referencing and fast response times.10 Carbohydrates are suitable biosensing molecules for medical diagnostics,11 and their importance for biological processes, such as cell adhesion or migration,12 was demonstrated using microarray systems.2 Carbohydrate based sensors represent a powerful tool to study glycan interactions or the detection of pathogens.13 As sequencing technologies for carbohydrate structures mature,14 carbohydrate-based cantilever sensors are developed to analyze binding properties of a variety of binding partners.15 The fast detection of bacterial species finds increasing attention:16 Besides classical growth media based methods,17 more sophisticated approaches,18 such as metal oxide-based olfactory sensors,3 to discriminate bacteria have been developed. Recently, Tzeng et al. demonstrated the suitability of mannose-based cantilever sensors operated in the dynamic mode to recognize formaldehyde killed Escherichia coli bacteria.19 However a detailed real-time investigation of living E. coli cells comprising high selectivity, sensitivity, and reproducibility of bacterial recognition is still missing. Here, we demonstrate that carbohydrate-based cantilever array biosensors, previously established for the accurate recognition of antiviral proteins,15 can detect and distinguish © 2011 American Chemical Society
Received: October 24, 2011 Revised: November 29, 2011 Published: December 5, 2011 420
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To examine the specificity of the ORN 178−nonamannose recognition using our cantilever array biosensor, a competitive inhibition assay was carried out.15,22 Free mannose (100 mM) was added abundantly to the running buffer to the second of two successive injections with identical concentrations. The second differential deflection is reduced by about 50% (see Figure 2a) in agreement with other reports of soluble
Figure 1. Detection of ORN 178 with glycan cantilever arrays. Upper panel: Averaged deflections (Avrg. Deflection) for galactose, trimannose, and nonamannose functionalized cantilevers of an array against time. Each graph represents an average signal of 2−4 identically functionalized cantilevers. Upon E. coli sample injection (OD = 0.5) the mannose cantilevers react significantly stronger (2−3 times) than the galactose reference due to increased sample recognition. Inset: Scheme of a cantilever array functionalized with different carbohydrates. In this example, cantilevers 1 and 2 are coated with the internal reference galactose, cantilevers 3, 4, 7, and 8 with nonamannose, and cantilevers 5 and 6 with trimannose, respectively. Lower panel: Differential deflections (Diff. Deflection) representing the specific binding events for the trimannose and nonamannose sensors derived by subtracting the galactose reference. The larger nonamannose deflections indicate increased multisite and multivalent binding.
Figure 2. Control experiments verifying the specificity and concentration dependence of the cantilever assay. Differential deflection 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 the running buffer. A second injection with identical bacterial concentration (OD = 0.5) shows an about 50% reduced differential nonamannose deflection. As free mannose successfully competes with the mannose sensor coatings, this result demonstrates the specificity of the E. coli−nonamannose recognition. (b) A series of increasing and decreasing bacterial concentrations was conducted to demonstrate the sensor’s reproducibility. Identical concentrations (shown in the same color) fit within an acceptable range by a maximal deviation of 15%. Please note: Error bars represent then standard deviation obtained for measurements performed with different cantilever arrays.
levers react significantly stronger. Specific binding of ORN 178 FimH adhesion protein at the tip of type-I pili to mannosides is responsible for an increased number of binding events on the mannose-covered surfaces. The bound bacteria induce a difference in surface stress between upper and lower side of the micrometer thin cantilever. This compressive surface stress is relieved by cantilever bending downward. The larger nonamannose signal is assigned to this carbohydrate’s increased potential for multisite and multivalent binding, which in turn leads to more binding events on these surfaces. In contrast, the galactose signal is attributed to nonspecific attachment of the bacteria to galactose or the cantilever surface. It was shown that ORN 178 specifically binds to mannose but not to other carbohydrates, such as galactose.16 Therefore we used galactose that resembles mannose structurally as an internal reference.25 To extract nonspecific binding contributions from the total signal, while accounting for nonspecific reactions, including small changes of pH, refractive index or reactions occurring on the underside of the cantilever,25 the in parallel obtained averaged galactose deflection is subtracted from both averaged mannoside signals. The resulting differential deflection is plotted in the lower panel in Figure 1 and represents the specific part of the bacterial recognition. In the following, only the differential deflection is given. As nonamannose consistently provided larger signals, we focus on these sensors for the following discussion, investigating the critical sensor parameters specificity, reliability, and sensitivity of the sensor setup for bacterial detection.
competitor binding studies.15,22 Since the monosaccharide mannose competes with the mannosides bound to the cantilever surface for binding to E. coli cells, fewer bacteria bind to the sensor surface, and a smaller deflection is observed. Consequently, the mannose sensor coating specifically recognizes and detects E. coli ORN 178 cells. In order to examine the accuracy of the recorded sensor signals on the measured concentration, the cantilever array setup was tested with a series of samples with increasing concentration followed by decreasing sample concentrations. The observed differential deflections demonstrate that the signal size for repeated concentrations (Figure 2b) are very reliable with a maximal deviation of only 15%. Besides signal specificity and reliability, the sensitivity of a sensor is of crucial importance. Testing the sensor response from an OD of 10−1 down to very low E. coli concentrations, we reproducibly detected a significant differential deflection for a dilution to an OD of 10−4 (Figure 2, Supporting Information). At this concentration less than ∼800 bacteria bind to each 421
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different glycan binding characteristics. We demonstrated the specificity of the recognition, the signal reproducibility, and high sensitivity down to very low sample concentrations of mannosides binding to type-I pili in E. coli ORN 178. Two additional E. coli strains with altered or lacking type-I pili could be differentiated on this sensor via their individual deflection signals, indicating the usability of this approach to specifically sense bacterial strains in solution. Hence glycan cantilever arrays offer a large potential as a fast, accurate, and differential screening tool for future clinical and diagnostic applications. Experimental Details. The nonamannose, trimannose, and galactose derivates with thiol linker were synthesized as described elsewhere.28 The D(+)-mannose was obtained from Roth, Germany. Gold-coated cantilever arrays consisting of eight identical silicon cantilevers (500 × 100 × 1 μm) on a support were purchased from Concentris GmbH, Switzerland. Following a UV−ozone cleaning cycle, the individual cantilevers were functionalized in parallel by inserting them into an array of microcapillaries (filled with the respective carbohydrate derivate 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 in Gruber et al.15 E. coli strains ORN 178, ORN 208, and ORN 206 were a kind gift from Prof. Orndorff, North Carolina State University, Raleigh, NC. Detailed genetic information of the strains used in this study can be found in Table 1, Supporting Information. All bacteria were grown overnight in 5 mL liquid LB medium at 37 °C, shaking at 300 rpm. The antibiotic tetracycline was added if necessary at a concentration of 12.5 g/L. Overnight cultures were diluted into fresh 5 mL of LB medium to OD = 0.1 and grown until an OD = 1 was reached. To record the interaction between the carbohydrate-coated cantilever and the bacterial strains, bacteria were transferred into the running buffer [100 mM of NaCL, 10 mM of Tris, 0.005% Tween 20, and 1 mM of CaCl2 (Roth)]. Bacteria were diluted further according to the different experiments described in the main text. To obtain the exact bacterial concentration for each dilution, cells were counted using a flow cytometer (CyFlow space, Partec). Measurements were performed on the commercial Cantisens sensor platform (Concentris GmbH, Switzerland) equipped with a measurement cell of 5 μL, an automated liquid handling system, an integrated temperature control with sample preheating stage, and a stability of 0.01 °C. The running buffer was prepared from 10 mM of Tris, pH 7.7, 100 mM of NaCl, 1 mM of CaCl2, and 0.005% Tween 20. Inside the instrument the array was equilibrated at a constant buffer flow of 0.42 μL/sec and a constant temperature of 22 °C for several hours until a constant drift was achieved.29 Prior to any measurements, all arrays were checked for fabrication variances by subjecting them to a short heat pulse (heat test).15 Only comparable cantilevers were employed for bacteria sensing. For each measurement 100 μL of bacteria solution, diluted to the desired concentrations, was injected into the buffer flow. The nanomechanical deflection signal is read in real time by employing an array of eight parallel vertical cavity surface emitting lasers (VCSELs). LabView based software was employed for instrument control and signal processing. Data analysis was performed using Analysis Tools offered by Concentris GmbH, Switzerland. The signal curves of all cantilevers were corrected for constant drift. As indicated in the main text, signals of identically functionalized cantilevers were averaged. Differential signals representing the specific
cantilever, as determined by flow cytometry (data not shown). In single experiments we even observed considerable deflections for ODs of 10−5 and 10−6 with 80 and 8 bacteria per single cantilever, respectively. This large concentration range over five orders of magnitude down to a few cells demonstrates the high sensitivity of this sensor setup and compares well to other reports for cantilever sensors.26 Similar results for the specificity, concentration reliability, and sensitivity of the bacterial detection were achieved with trimannose 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 this end, the signals of E. coli ORN 178, carrying the fully functional adhesion protein FimH, were compared to E. coli ORN 208 and ORN 206. The strain ORN 208 expresses the truncated protein FimH*, while strain ORN 206 does not produce any pili. At an identical concentration of OD = 10−1, measurements were recorded (Figure 3). Three distinct differential signals were
Figure 3. Discrimination of E. coli strains with distinct mannose binding properties (OD = 0.1). The smallest differential signal on this nonamannose/galactose array is obtained for strain ORN 208 which carries an impaired mannose binding protein FimH* at its pili and thus preferentially attaches via mannose transporters. Additional binding via mannose-specific binding protein FimH on ORN 178 results in a larger differential deflection. The largest signal for strain ORN 206 can be explained by the missing pili facilitating access and the increased nonspecific binding to mannose transporters. See main text.
determined with deflection sizes increasing in the order from ORN 208 to ORN 178 to ORN 206. Comparing the first two strains, the smaller deflection size for ORN 208 can be attributed to the impaired binding properties of the mannose binding protein FimH*. Strain ORN 206 with no pili, and thus no specific FimH recognition, surprisingly yields the largest sensor signal. However the missing pili might also explain the higher number of bound bacteria associated with the larger observed deflection. Assuming that pili sterically hinder access to the cell membrane, increased nonspecific binding of the bacterial cell membrane via mannose transporters27 to the mannoside-coated cantilever surface could result in the larger deflection signal. To eliminate the possibility that the larger signal for ORN 206 might be due to excessive growth during sample preparation, growth curves for the employed E. coli strains were recorded. A comparison of the results under identical conditions shows comparable growth for all three strains (Figure 4, Supporting Information). Thus different growth behavior is excluded, and the observed distinct sensor signals can be attributed to the different carbohydrate binding properties 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 422
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recognition were calculated by subtracting the nonspecific averaged deflection signal of the internal reference galactose from the averaged deflection signal obtained for trimannose- or nonamannose-coated cantilevers.
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ASSOCIATED CONTENT
S Supporting Information *
Four additional figures and one table: Carbohydrate structures, sensitivity of our approach, control experiments performed for ORN 178 and trimannose-coated cantilevers, bacterial growth curves, and strain descriptions. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION Corresponding Author *E-mail:
[email protected].
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ACKNOWLEDGMENTS Financial support is gratefully acknowledged by the DFG ERAChemistry program (HE5162/1-1), the excellence cluster Nanosystems Initiative Munich, the Walter-Meissner Institute of the Bavarian academy of sciences and humanities, the Center for Nanoscience, the Elite Network of Bavaria, the Max-Planck Society (P.H.S.) and The Körber Prize for European Sciences (P.H.S.). We thank Prof. Orndorff for the kind gift of the strains ORN 178, ORN 206, and ORN 208. For their kind support with performance of flow cytometer experiments, we thank Dr. J. Megerle.
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