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Jan 23, 2007 - PEMC-a, used in batch mode with 1 mL sample, showed limit of detection at 10 cells/mL using the second bending mode at 85.5 kHz in air...
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Environ. Sci. Technol. 2007, 41, 1668-1674

A Method of Measuring Escherichia Coli O157:H7 at 1 Cell/mL in 1 Liter Sample Using Antibody Functionalized Piezoelectric-Excited Millimeter-Sized Cantilever Sensor GOSSETT A. CAMPBELL AND RAJ MUTHARASAN* Department of Chemical and Biological Engineering, Drexel University, 3141 Chestnut Street, Philadelphia, Pennsylvania 19104

Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors immobilized with antibody specific to Escherichia coli (EC) O157:H7 is used to detect EC at 1 cell/mL in 1 mL and 1 L samples in a batch and flow mode, respectively. Two sensor designs were used. The first design (PEMCa) has both the piezoelectric and non-piezoelectric layer anchored, while in the second design (PEMC-b) had only the piezoelectric layer anchored. PEMC-a, used in batch mode with 1 mL sample, showed limit of detection at 10 cells/ mL using the second bending mode at 85.5 kHz in air. PEMC-b exhibited resonant frequencies at 186.5, 883.5, and 1778.5 kHz in air and 162.5, 800.0, and 1725.5 kHz in sample flow conditions. A one-liter sample containing 1000 EC cells was introduced at 1.5, 2.5, 3, and 17 mL/min, and the change in resonant frequency was monitored. The total frequency change observed for the mode at 800 kHz and sample flow rates of 1.5, 2.5, 3, and 17 mL/min were 2230 ( 11, 3069 ( 47, 4686 ( 97, and 7188 ( 52 Hz, respectively. Each detection experiment was confirmed by exposing the sensor to a low pH solution followed by a phosphate buffered saline (PBS) rinse, which caused the release of the attached EC. The final frequency change observed was nearly identical to the value prior to EC attachment. Kinetic analysis showed that the observed binding rate constant at 1.5, 2.5, 3 mL/min were 0.009, 0.015, and 0.021 min-1, respectively. The significance of these results is that very low concentration of pathogens in large sample volumes can be measured in a short time period without the need for filtration or enrichment.

1. Introduction Detecting the presence of pathogens in water at very dilute concentration without a concentrating or an enrichment step is useful in monitoring drinking and source water. Outbreaks of waterborne illness such as the Escherichia coli-induced gastroenteritis in Walkerton, Ontario in 2000 and cryptosporidiosis in Milwaukee in 1993 have necessitated improved analytical methods for detecting microbial pathogens in water and other environmental samples. Detection of the presence * Corresponding author phone: (215) 895-2236; fax: (215) 8955837; e-mail: [email protected]. 1668

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of a single cell has been the focus of several researchers. Of particular interest, is the case of drinking water (2) and food (3, 4). Some of the sensor platforms that have been investigated are the atomic force microbalance (AFM) (5, 6), light-addressable potentiometric sensor (LAPS) (7), and porous polymers (8). The infectious dosage of Escherichia coli (EC) O157:H7 has been reported as 10 cells, and the United States Environmental Protection Agency standard for drinking water is 40 cells per liter (9). At such a low concentration, it is necessary to culture the sample fluid in selective medium and count colonies after a 24- or 48-hour incubation. If a method is developed to detect pathogens in water without an enrichment step, it would save time and enhance our ability to monitor water quality in a near real time fashion. In this paper we extend our earlier approach of using piezoelectric-excited millimeter-sized cantilever (PEMC) sensors for low pathogen concentration samples. In an earlier study, we reported the batch detection of EC O157:H7 at 700 cells per mL with good certainty and repeatability using the PEMC sensors (10, 11). We also showed that they have the sensitivity to monitor viability by measuring specific growth rate of EC (12). Several investigators have reported on E. coli growth characterization and detection using microcantilevers (13-17). Very highly sensitive detection of a single cell was examined by (18). None of these studies examined sample size of one liter or a sample that is to be contacted in a flow configuration. Recently, PEMC sensors of higher sensitivity were developed and used for continuous detection of Bacillus anthracis spores under both batch and flow configuration at 300/mL (19, 20). In this paper, we describe three approaches for the detection of EC at 1 cell/mL using piezoelectric-excited millimeter-sized cantilevers: first a batch configuration, second at modest flow rate of 1 mL/min, and finally a flow and stop measurement done at high flow rate.

2. Cantilever Physics The resonant frequency change of an oscillating rectangular PEMC sensor under liquid submerged conditions during a detection experiment is described in detail in our earlier publication (21). The cantilever’s resonant frequency change is a linear function of its mass change and can be expressed as follows:

fnf - f ′nf )

∆m 1 f 2 nf M′en

(1)

where (fnf - f nf ′ ) is the resonant frequency change of the nth mode in fluid due to the binding of the target antigen of equivalent mass, ∆m. M′en is the effective mass of the cantilever under liquid submerged condition for the nth mode. The number of nodal point increases as the mode increases, and therefore, the effective mass decreases. The term f nf ′ is the resonant frequency of the nth mode in fluid.

3. Materials and Methods 3.1. Sensor Fabrication. Two different PEMC sensor designs were used in this investigation, labeled PEMC-a and PEMCb. Both were fabricated from a 127 microns thick piezoelectric layer (lead zirconante titanate, PZT) bonded to a 160 µm thick fused quartz or borosilicate glass. In PEMC-a, borosilicate glass was used. Both the glass and PZT were anchored at one end and at the other the glass layer was of a longer dimension than the PZT layer. Details of PEMC-a fabrication and a schematic can be found in ref 22. The dimensions of 10.1021/es061947p CCC: $37.00

 2007 American Chemical Society Published on Web 01/23/2007

FIGURE 1. Schematic of PEMC-b sensor. (a) the PZT layer, (b) the adhesive layer, (c) the non-piezoelectric glass layer, and (d) is the nonconductive epoxy used to protect the electrodes on the encapsulated end of the PZT. For a schematic of PEMC-a, reader is referred to ref 1. both the PZT and borosilicate glass were 1 ( 0.05 × 1 ( 0.05 × 0.127 ( 0.005 mm3 (L × W × t) and 3 ( 0.05 × 1 ( 0.05 × 0.160 ( 0.005 mm3, respectively. PEMC-b design has only the PZT anchored at one end and at the free-end a 1.65 × 1.0 × 0.60 mm3 fused quartz layer was attached (Figure 1). The PZT dimensions were 2.54 × 1.0 × 0.127 mm3. The length of the PZT monolayer between the bi-layer and the anchored section was 0.89 mm. The exposed PZT surface was coated with a thin layer of polyurethane for electrical insulation. The top and bottom electrodes of PZT were soldered to 30gauge copper wire that was connected to BNC couplers. 3.2. Sensor Flow Cell (SFC) Design. The sensor flow cell (SFC) was constructed of Plexiglas. The flow cell was 7.0 mm in diameter and had a hold-up volume of 120 µL after the sensor was installed. The inlet and outlets were located at the bottom and on the side of the cell, respectively. The outlet was located 4 mm above the inlet and both were 1.59 mm in diameter. Constant temperature water (35 ( 0.2 °C) was circulated at 17 mL/min through a 4 mm wide shell surrounding the sensor flow cell. At steady state, the temperature within the sensor flow cell was 25 ( 0.2 °C. 3.3. Experimental Setup. For continuous flow experiments the apparatus described earlier in (20) was modified for sample volume. It consisted of a one-liter sample, antibody reservoir, hydroxylamine (Sigma Aldrich) reservoir, phosphate buffered saline (PBS, Sigma-Aldrich) reservoir, peristaltic pumps, and a sensor flow cell (SFC). The flow circuit was setup on a vibration-free table. The reservoirs were connected to the sensor flow cell (SFC) via a four-port manifold and the inflow enters the SFC through the bottom. The EC sample container (1 L) was stirred with a 2 inch magnetic stirrer at 150 rpm to ensure sample homogeneity. Valves located at the bottom of each fluid reservoir enabled selection of fluid that is to be pumped into the SFC. The outlet of the flow cell was connected to a peristaltic pump, which controlled the flow of the desired fluid into and out of the SFC. Batch experiments using PEMC-a sensors was carried out as described in ref 10. In both batch and flow configuration, the sensor was connected to an impedance analyzer (Agilent, HP 4192A) interfaced to a PC running a LabVIEW data acquisition application for obtaining resonant frequencies with time. 3.4. Experimental Procedure. The cantilevers glass surfaces were cleaned and functionalized with 3-aminopropyl-triethoxysilane (APTES, Sigma-Aldrich) and subsequently antibody to EC (affinity purified anti-EC, KPL, Gaithersburg, MD) was immobilized as previously described (20, 23). The immobilization protocol was adapted from ref 23. Working samples of formaldehyde-killed EC (strain B1409, USDA, PA) was prepared from a stock of 1 × 109 EC cells/mL by serial dilution in phosphate buffered saline (PBS; 10 mM, pH 7.4) to a final concentration of 70, 10, and 1 EC/mL. Two types of experiment were carried out in this investigation: batch detection at 70, 10, and 1 EC in 1 mL of PBS buffer using PEMC-a and flow detection of 1 cell/mL in 1 liter of PBS buffer using PEMC-b. The flow experiments were conducted in two modes. In the first, various flow rates were

used and continuous measurements of resonant frequency was performed. In the second, high flow rate was used for a specific time period and the flow was stopped for sufficient time to measure resonant frequency. The latter method was used as the measurements of resonant frequency became noisy at flow rates greater than 5 mL/min. The flow circuit was first primed with PBS to remove any air bubbles. Then, the APTES-functionalized cantilever was installed into the SFC containing PBS (running buffer). The running buffer was recirculated through the SFC at 1.5 mL/min and the sensor’s resonant frequency was monitored until it reached a steady value, which was usually achieved in 10-20 min. Upon stabilization, activated anti-EC solution at 10 µg/mL was recirculated through the SFC at 1.5 mL/min to allow antibody immobilization for 2 h at 25 ( 0.2 °C. Hydroxylamine was flowed through the SFC, and then PBS to flush the system. Upon completion of the rinsing step, the EC sample was flowed once through the SFC. The EC detection experiments were carried out at flow rates of 1.5, 2.5, 3, and 17 mL/min. The experiments carried out at 1.5, 2.5, and 3 mL/min were done under continuous flow conditions. The bound EC was released from the sensor by a low pH solution. For the highest flow rate (17 mL/min) the EC sample was injected into the flow loop for 5 min, stopped, and measurements were made of the three dominant resonant peaks. This procedure was repeated until the entire 1 L sample solution was pumped through the SFC. A control experiment was carried out in each detection experiment using an anti-EC functionalized sensor exposed to PBS solution flowing at a rate identical to the detection experiment.

4. Results and Discussion 4.1. Resonance Spectra. A typical resonant spectrum of PEMC-a sensor in both air and liquid can be found in ref 1. The spectrum, a plot of phase angle versus excitation frequency, showed only two dominant bending modes in the frequency range of 1-100 kHz. For the sensor used in this investigation the first and second bending mode frequencies in air were at 21.5 and 85.5 kHz, respectively. Upon liquid immersion the fundamental mode decreased by ∼7.5 kHz, while the second mode decreased by ∼20 kHz. Dominant high-order resonant modes beyond 100 kHz were absent in PEMC-a design. Modes that exist at high frequency are more sensitive (1, 24, 25). Thus, in this study the second resonant bending mode was used to monitor the detection of EC. The resonant spectrum of PEMC-b in air showed resonance peaks up to 2 MHz, which is an advantage compared to PEMC-a. In Figure 2 the resonant spectra of PEMC-b in both air and under liquid immersion are presented. The fundamental resonant frequency in air was 51.2 kHz, which is 2-3 higher than PEMC-a sensors (1, 10). The resonant frequencies of the dominant modes in air (blue curve) were located at 51.2, 186.5, 883.5, and 1778.5 kHz. As seen in the figure smaller and less useful peaks were observed. These are thought to be due to mixed modes. For example, bending combined with flex or buckling modes which tend to be somewhat weaker than the bending mode alone. Upon fully submerging the sensor in PBS, the resonant frequencies decreased to (red curve) 37.5, 162.5, 800.0, and 1725.5 kHz, respectively. The decrease is due to the added mass effect of the liquid adjacent to the sensor (26). The difference in resonant frequency between air and liquid is a measure of mass change sensitivity. That is, a larger frequency change generally implies a higher sensitivity. For the four dominant modes that are observable, the added mass effect contributed to a decrease of 13.7, 24.0, 83.5, and 53.0 kHz. Since the added mass was the same due to liquid immersion, we infer that the third mode that showed the largest frequency change of 83.5 kHz is likely to be the most sensitive one. We recognize that under liquid immersion, the added mass effect is uniform VOL. 41, NO. 5, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Resonant spectra of, phase angle versus excitation frequencies, PEMC-b sensor in air (blue) and fully immersed in PBS (red). At resonance, the phase angle of the oscillating cantilever increases and exhibits a peak. Away from resonance, the PZT behaves as a capacitor and exhibits a phase angle close to -90°. Under liquid the resonant peaks shifted to the left. The inset is a magnified version of the third dominant resonant peak in both air and under liquid immersion.

FIGURE 3. Frequency response of PEMC-a to the detection of EC O157:H7 in batch at 70, 10, and 1 EC in 1 mL of PBS. No response was observed for the 1 EC/mL sample in seven repeated experiments, suggesting that it was not possible to detect 1 EC in 1 mL of sample buffer. along the cantilever’s length. On the other hand, in a detection experiment uniform attachment of antigen may not occur. The sensor behaves as a capacitor at non-resonant frequencies, and thus its baseline phase angle is close to -90°. At resonance, there is a sharp increase in phase angle as the PZT becomes more resistive due to increased charge accumulation as a result of higher than normal mechanical stress. When immersed in liquid the height of each resonant peak decreased due to mass damping, illustrated in Figure 2 (red curve). The base of a resonant peak is a measure of the dissipative energy of the sensor in the surrounding medium and is defined by the peak’s quality factor (Q value). Q is determined from the ratio of resonant frequency to the peak width at half the peak height. In other words, the Q value gives a measure of peak sharpness. The Q-values of the 1670

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dominant resonant peaks (51.2, 186.5, 883.5, and 1778.5 kHz) of PEMC-b in air were 63.8, 15.3, 52.0, and 18.9, respectively, and in liquid the Q-values decreased to 46.9, 13.6, 32.0, and 11.0, respectively. For PEMC-a, the Q-value in air of the first and second modes were 29 and 38, respectively, and 25 and 32, respectively, in liquid. The non-monotonic change in Q with mode is due to two factors. These are the nonuniform cross section of the cantilever sensor and the use of electrically active PZT layer for sensing. The modes that generate greater stress in PZT provide seemingly higher Q-values. This is contrary to our common experience that higher modes are less dominant. In addition, one notes that the quality factor of the fundamental bending mode was larger in PEMC-b, and furthermore, the fundamental mode is located at a higher frequency, which is an indication that a larger stress is

FIGURE 4. Panel A: Resonant frequency change, of the 800.0 kHz mode under liquid, for the binding of E. coli to PEMC-b. Sample was 1 L at 1 EC/mL. The sample flow rate was 1.5, 2.5, and 3 mL/min. The control was an antibody-functionalized PEMC-b sensor exposed to 1 L of PBS buffer flowing at 3 mL/min. Panel B: Sensor response to the attachment of EC at sample flow rate of 1.5 mL/min and the subsequent release of the bound EC. generated at resonance in PEMC-b. However, a Q value decrease of ∼40% was observed in PEMC-b and only a maximum of ∼20% decrease was seen in PEMC-a design. This is an important advantage of PEMC sensors over microcantilever under liquid conditions. For microcantilevers the Q-value is significantly reduced under liquid immersion. A stable resonant peak with a high Q-value will enable a more accurate determination of resonant frequency. In this study, the response of the 65.5 kHz peak of PEMC-a, and the 162.5, 800.0, and 1725.5 kHz resonant peaks of PEMC-b in liquid were used to detect changes in mass due to EC attachment of sensor surface. We carried out each of the detection experiments at least twice, and the data shown is typical of the results obtained. 4.2. Batch Detection of EC Using PEMC-a. The functionalization of the sensor with antibody was done through chemical immobilization. APTES reacts with the hydroxyl groups on the glass surface leaving an exposed amine group, which binds covalently with the activated carboxyl groups of the anti-EC. One milliliter of PBS buffer containing 70, 10, and 1 EC per mL were prepared and exposed to antibodyfunctionalized PEMC-a for ∼55 min. The frequency response of EC binding to the cantilever is presented in Figure 3. The results showed an increase in the total frequency change

with the increase in EC concentration and correspondingly, the binding rate also increased. The total frequency change observed for 70 and 10 EC/mL was 65 ( 11 (n ) 8) Hz and 19 ( 5 (n ) 8) Hz, respectively. For the 1 cell/mL EC sample no significant change (1 ( 3 Hz) in the resonant frequency was observed. That is the response was similar to the control (0 ( 2 Hz), which suggest one of three things. First, the cantilever was not sensitive enough to detect 1 cell. Second, the target cell did not come in contact with the sensor. Third, there was no cell in the sample. To eliminate the latter, the 1 EC/mL experiment was repeated seven times and no response was observed in any of the experiments. Both the 70 and 10 EC/mL experiments were carried out eight times. The 70 EC/mL sample gave positive detector response six out of the eight time, and the 10 EC/mL results gave positive detection in only two of the eight experiments. These results indicate that the detection of low pathogen concentration in a batch configuration is not an effective modality because the cells may not be in close proximity of the sensor for attachment. In addition, to ensure that the desired cell concentration is measured, we increased the sample volume to one liter, and to overcome target pathogen transport to sensor surface we introduced flow. VOL. 41, NO. 5, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 5. Resonant frequency response of PEMC-b sensor to the binding of EC O157:H7 at concentration of 1 EC/mL (total sample volume was 1 L) for the dominant resonant modes investigated. The control experiments were an anti-EC PEMC-b sensor exposed to only PBS solution in the same fashion as the EC sample. 4.3. Detection of EC Using PEMC-b at 1 EC/mL Under Various Flow Rates. The response of PEMC-b, at 800 kHz under full liquid immersion, due to the binding of EC at various flow rates is presented in Figure 4A. The freshly prepared antibody-functionalized sensor was exposed to a 1 L sample of EC at 1 cell/mL and sample flow rates of 1.5, 2.5, and 3 mL/min. In all the experiments, the sensor responded with an initial rapid decrease in resonant frequency, followed by a slower decrease until the frequency reached a steady-state value. The rate of EC binding and the total resonant frequency change increased with increasing flow rate. The steady-state frequency changes for the binding of EC (1 cell/mL) at 1.5, 2.5, and 3 mL/min were 2230 ( 11, 3069 ( 47, and 4686 ( 97 Hz, respectively. The standard deviation indicated is the variation in resonant frequency at steady state over a period of 10 min. We note that the time to reach steady state is a weak function of the flow rates used. The steady-state frequencies at 1.5, 2.5, and 3 mL/min were achieved in 192, 230, and 290 min, respectively. Since the number of EC cells in each of the sample was the same, we conclude that the increased flow rate improved contact of EC to the sensor. At each flow rate a control experiment was also carried out. The control was an antibody functionalized cantilever exposed to PBS buffer at a flow rate that was the same as in the detection experiment. The control response shown in Figure 4A was carried out at 3 mL/min. The resonant frequency decreased slightly 77 ( 81 Hz, which for practical purposes is within the noise level we observed with cantilevers of this design. The change is also insignificant when compared to the frequency response due to EC binding, which is on the order of 1000 Hz. The data in Figure 4A suggest that PEMC sensors have the sensitivity to detect EC at 1 EC/mL in the flow configuration used. To confirm that the observed sensor response was indeed due to the binding of EC, in each experiment, after completion of the detection segment, the flow circuit was rinsed with PBS followed by exposing the sensor to a pH 2.02 solution, and finally PBS was reintroduced to return the sensor to the environment that was present prior to EC attachment. Figure 4B shows the attachment and release sensor response for the bound EC at 1.5 mL/min. Upon initiation of the sample flow the sensor’s resonant frequencies decreased monotonically by 2230 ( 11 Hz. After reaching steady state, PBS was pumped through the flow-cell for 30 min followed by a pH 1672

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2.02 solution made from PBS and hydrochloric acid (HCl). The PBS-rinse did not show any noticeable change in frequency. In fact, the frequency increased by only 30 Hz, which is lower than the response of the control. On the other hand, exposure of the sensor to the release solution resulted in an immediate increase in frequency and reaching a constant frequency change of 1905 ( 17 Hz. One notes that the total frequency change obtained during the release is less than that of the attachment response; this may be due to the slightly higher density of the release solution. However, flushing the SFC with PBS afterward caused a further increase in the resonant frequency by 392 ( 20 Hz, indicating that the liquid environment prior to the release was re-established. In addition, the final frequency change reached was ∼110 Hz higher than the value before initiation of the EC sample. This difference is small (∼5%) considering the magnitude of EC response and the variation observed in the control studies. 4.4. EC Detection in a Flow and Stop Modality. In this modality we show the use of multiple resonant peaks in the detection of EC. Because the resonance characteristics of PEMC sensors are very unstable under high liquid flow rates (17 mL/min), we carried out the experiment in a flow-stopmeasure modality. We note that the sample volume was 1 liter and the EC sample (1 cell/mL) flow rate was at 17 mL/ min. We also measured the behavior of the sensor’s three higher-order resonant modes by a constant mass. The primary motivation is that the frequency response will give a measure of the sensitivity of the various peaks. In addition, a non-flow condition ensures that the measured resonant frequency changes were not due to flow effects, and only mass changes caused by EC attachment. In Figure 5, the 162.5, 800.0, and 1725.5 kHz resonant peaks, under liquid immersion, were used in detection. The plot shows the frequency response of the sensor versus the sample volume that flowed through the SFC prior to resonant frequency measurement. The sample flowed for 5 min, stopped, and then the resonant frequencies of the three modes were monitored individually until they stabilized, which typically took place in 5-10 min. This step was repeated until the sample volume was completely pumped through the SFC. As shown in the figure, the resonant frequencies of the different modes decreased rapidly after the initial 5 min of sample flow (sample volume of 85 mL) and ultimately reached a steady-state frequency change of 4340 ( 49, 7188 ( 52, and

FIGURE 6. Panel A: Initial kinetic analysis of EC attachment at various flow rates. The analysis is of the experiment data presented in Figure 4A. The correlation coefficient ranged from 0.983 to 0.996. The slope of each line gives the observed characteristic binding rate constant kobs. 25 850 ( 63 Hz corresponding to the 162.5, 800.0, and 1725.5 kHz resonant modes under liquid immersion. The significance of these results is that for the same mass change the various resonant modes responded to different extent. Given that the noise levels were approximately similar, the 1725.5 kHz mode exhibited the highest sensitivity of the modes examined. That is, the frequency change per pathogen detected was ∼26 Hz/pathogen. Along with each detection experiment a control experiment was also conducted in the same fashion. The control experiment conducted with an antibody-functionalized PEMC-b sensor exposed to PBS buffer yielded resonant frequency values that fluctuated at 56 ( 154, 97 ( 225, and 21 ( 328 Hz for the 162.5, 800.0, and 1725.5 kHz modes, respectively. If we compare the frequency response presented in Figure 4, for the 800.0 kHz mode, a larger change in frequency is observed at 17 mL/min, which further confirms that higher flow rate enhances EC attachment. These results suggest that the cantilever is mechanically robust and at the same time very sensitive. 4.5. Kinetics of BA Spore Binding. The binding kinetics of EC has been characterized in our earlier work using Langmuir kinetic model (26). The model is expressed as follows:

(

ln

)

(∆f∞) - (∆f) (∆f∞)

) - kobsτ

(2)

where (∆f) is the change in resonant frequency at time, τ, (∆f∞) is the steady-state frequency change, and kobs is the observed binding rate constant. A plot of the right-hand side against time gives a straight line of slope kobs. A fit of the experimental data presented in Figure 4A to eq 2 yielded straight lines of correlation coefficient ranging from 0.986 to 0.996 and is shown in Figure 6. We limit the analysis to the first 10 min to avoid diffusion effects. The kobs values were determined as 0.021, 0.015, and 0.009 min-1 corresponding to sample flow rates of 3, 2.5, and 1.5 mL/min. These data suggest that flow increases contact with sensor surface and as a result, increase the initial rate of EC attachment.

5. Conclusions In this paper, we have shown the detection of 1 EC/mL at various sample flow rates using antibody functionalized piezoelectric-excited millimeter-sized cantilever sensor. On one hand, detection of 1 EC/mL in a batch configuration did not show a positive response. On the other hand, flow

increased the sensor response and provided a reliable measurement modality. In addition, we observed that binding rate increased with flow rate. We also found that the time to reach a steady-state frequency is a weak function of flow rate. We also demonstrated that higher-order resonant modes are more sensitive than lower-order modes for the same mass change.

Acknowledgments We acknowledge the financial support of the Environmental Protection Agency grant RD-83300701-0. Also, we thank Dr. Kevin Scoles for the development of the data acquisition program and Dan Luu for sensor flow cell fabrication.

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Received for review August 14, 2006. Revised manuscript received December 18, 2006. Accepted December 19, 2006. ES061947P