Piezoelectric-excited Millimeter-sized Cantilever Research Articles

Mass-change sensitivity of piezoelectric-excited millimeter-sized cantilever (PEMC) sensors: Model and experiments

Abstract We show for the first time that approximate analytical models do not predict mass-change sensitivity of cantilever sensors of non-uniform cross-section. On the other hand, finite element model (FEM) is a better approach for determining the performance of such sensors. Specifically, FEM of piezoelectric excited millimeter-sized cantilever sensors (PEMC) give sensitivity values that are in reasonable agreement with experimentally determined values of 20 pg/Hz, reported earlier. The approximate analytical model predicts resonance frequency of PEMC within 10% error, but it under predicts mass-change sensitivity by three orders of magnitude. FEM analysis of first and second resonance mode frequencies of a sample of ten PEMC sensors of various geometries were found to compare favorably (∼2–4%) with experimental values. Mass sensitivity determined using FEM is within a factor of three of experimental values. Through simulations we discover two important properties of the PEMC sensors: (1) The sensitivity is a strong function of attached mass. Specifically, sensitivity is the highest at low attached mass. (2) PEMC sensors become less sensitive with increasing attached mass, which results in sensors that exhibit wide dynamic range of six logs.

Near real-time detection of Cryptosporidium parvum oocyst by IgM-functionalized piezoelectric-excited millimeter-sized cantilever biosensor

Piezoelectric-excited millimeter-sized cantilever (PEMC) biosensors were fabricated and functionalized with immunoglobulin M (IgM) for the detection of Cryptosporidium parvum oocyst in a flow configuration at 1 mL/min. The detection of 100, 1000, and 10,000 oocysts/mL was achieved with a positive sensor response in less than 1 min. Bovine serum albumin (BSA) was used as a blocking agent in each experiment and was shown to eliminate non-specific binding. The sensor's resonance frequency response correlates with C. parvum oocyst concentration logarithmically. The oocyst attachment rate was found to increase by an order of magnitude in increasing concentration from 100 to 10,000 oocysts/mL. The significance of these results is that IgM-functionalized PEMC sensors are highly selective and sensitive to C. parvum oocyst and therefore, have the potential to accurately identify and quantify C. parvum oocyst in drinking water.

Preparation-Free Method for Detecting Escherichia coli O157:H7 in the Presence of Spinach, Spring Lettuce Mix, and Ground Beef Particulates

We show the detection of 100 cells per ml of Escherichia coli O157:H7 in the presence of spinach, spring lettuce mix, and ground beef washes and particulate matter with piezoelectric-excited millimeter-sized cantilever (PEMC) sensors. The PEMC sensors (sensing area, 2 mm2) were immobilized with polyclonal antibody specific to E. coli O157:H7 (EC) and were exposed to 10 aqueous washes of locally purchased spinach, spring lettuce mix, and ground beef for testing if EC was present. Absence of resonance frequency shift indicated that EC was not present in the 30 samples tested. Following the last sample in each food matrix, 1,000 cells per ml of EC were spiked into the sample container, and resonance frequency change was monitored. The total resonance frequency change was 880 ± 5, 1,875 ± 8, and 1,417 ± 4 Hz for spinach, spring lettuce mix, and ground beef, respectively. A mixture of the three food matrices spiked with 100 cells per ml of EC gave a sensor response of 260 ± 15 Hz. The resonance frequency changes are approximately 40% lower than our previously reported study on ground beef. It is suggested that the reduction in sensitivity is due to differences in pathogen adherence to food matrices, which affects target binding to the sensor surface. We conclude that detection selectivity is conserved in the three food matrices examined and that the magnitude of sensor response is a function of the food matrix.

Detection and Confirmation of Staphylococcal Enterotoxin B in Apple Juice and Milk Using Piezoelectric-Excited Millimeter-Sized Cantilever Sensors at 2.5 fg/mL

A sensitive and reliable method for the detection of a model toxin, staphylococcal enterotoxin B (SEB) in buffer, apple juice, and milk is shown using piezoelectric-excited, millimeter-sized cantilever (PEMC) sensors. Limit of detection in spiked milk and apple juice samples is 10 and 100 fg, respectively. PEMC sensors (2 mm(2)) are prepared by immobilizing a polyclonal antibody specific to SEB, which was exposed to 1 mL of 1% milk and apple juice containing 10 fg-10 ng. Sensor response to 100 fg, 1 pg, and 10 pg of SEB in apple juice resulted in resonance frequency decreases of 113 +/- 18 (n = 4), 308 +/- 24 (n = 4), and 521 +/- 20 (n = 2) Hz, respectively. In milk, 10 fg, 100 fg, 1 pg, and 10 pg of SEB resulted in resonance frequency decreases of 126 +/- 18 (n = 2), 143 +/- 35 (n = 4), 310 +/- 32 (n = 5), and 557 +/- 25 (n = 2) Hz, respectively. Positive detection of SEB in the sample solution was observed within the first 20 min. The responses of the sensor to positive (SEB present, but no antibody on sensor), negative (SEB absent, antibody on sensor), and buffer (SEB absent, antibody on sensor) controls were -17 +/- 10 (n = 3), -9 +/- 5 (n = 3), and -6 +/- 12 (n = 18) Hz, respectively. Positive verification of SEB detection was confirmed by two methods: (1) low-pH buffer release caused increase in resonance frequency, and (2) second antibody binding to SEB attached to sensor that caused further resonance frequency decrease. The significance of these results is that PEMC sensors can reliably detect SEB at 10-100 fg (effective concentration of 2.5 and 25 fg/mL) in complex fluids without sample preparation or the use of labeled reagents.

PEMC-based Method of Measuring DNA Hybridization at Femtomolar Concentration Directly in Human Serum and in the Presence of Copious Noncomplementary Strands

Piezoelectric-excited, millimeter-sized cantilever (PEMC) sensors having high-mode resonance near 1 MHz are shown to exhibit mass change sensitivity of 1-300 ag/Hz. Gold-coated PEMC sensors immobilized with 15-mer single-stranded DNA (ssDNA) were exposed to 10-mer complementary strands at concentrations of 1 fM, 1 pM, and 1 microM, both separately and sequentially at 0.6 mL/min in a sample flow cell housing the sensor. Decrease in resonance frequency occurred as complementary strands hybridized to the immobilized probe DNA on the sensor surface. Hybridization in three background matrixes--buffer, buffer containing 10,000 times higher noncomplementary strands, and 50% human plasma--were successfully tested. Sensor hybridization responses to 1 fM, 1 pM, and 1 microM complementary strand were nearly the same in magnitude in all three matrixes, but the hybridization rates were different. In each case, the sensor detected the presence of 2 amol of complementary 10-mer strand. The extent of hybridization calculated from resonance frequency change did not decrease in serum. The findings suggest ssDNA can be detected at 2 amol without a sample preparation step and without the use of labeled reagents.

Method for Measuring the Self-Assembly of Alkanethiols on Gold at Femtomolar Concentrations

We describe a cantilever-based method for measuring the self-assembly of alkanethiols on a gold surface in a flow system that permits easy step changes in concentration and acquire a continuous in situ measure of the resulting chemisorption through the change in resonance frequency. A gold-coated (2.2 mm2), piezoelectric-excited, millimeter-sized cantilever (PEMC) sensor was exposed to 1-hexadecanethiol (HDT) in ethanol at concentrations ranging from 1 fM to 1 mM, sequentially and separately. A high-order flexural mode at approximately 850 kHz was monitored during the self-assembly. The resonance frequency decreases as a result of increased mass as chemisorption occurs on the surface. We show for the first time that the chemisorption of HDT at 1 fM is readily measurable and gave a response of 220 +/- 13 Hz (n = 4). At higher concentrations (10 and 100 fM; 1, 10, and 100 pM; 1, 10, and 100 nM; 1 microM; and 1 mM), the responses were proportionately, but nonlinearly, higher. At high concentrations (1 mM), the responses to C4, C8, C11, C16, and C18 alkanethiols were linearly proportional and were complete in approximately 25 min. We report for the first time that, once the Au surface is equilibrated at 1 pM, further chemisorption at a lower HDT concentration does not take place, even though over 99% of surface adsorption sites are available. At 1 fM, the overall chemisorption rate did not increase with a 2-fold increase in the HDT flow rate, suggesting that chemisorption at 1 fM is not transport-limited. The measured overall chemisorption rate constant at 1 fM was more rapid than 0.1 min-1.

Viscosity and density values from excitation level response of piezoelectric-excited cantilever sensors

In this paper, we show for the first time that excitation level of piezoelectric-excited millimeter-sized cantilever (PEMC) sensors can be used as an experimental variable for determining both density and viscosity of liquids. PEMC of dimensions 3.8 mm × 2.6 mm × 0.287 mm were fabricated, characterized, and used to determine the densities and viscous damping coefficients of water, 5%, 10%, 15%, and 20% glycerol solutions by resonant frequency responses at applied voltages ranging from 0.01 V to 1 V. The second mode resonant frequency (38.52 kHz in air, 39.02 kHz in vacuum) was used. The resonant frequency decreased with increasing glycerol concentration and applied excitation voltages. The density of the solutions was determined from decrease in resonance frequency and was in good agreement with previously published theory. Dissipative losses due to liquid viscosity were observed in the broadening of the resonant peaks and were characterized by peak shape factor ( Q-value). The viscous damping coefficients determined from the Q-values were used in calculating viscosity. A calibration curve for viscosity determination showed good accuracy of viscosity up to 30% glycerol.

Detection of airborne Bacillus anthracis spores by an integrated system of an air sampler and a cantilever immunosensor

Abstract A near real-time, sensitive and reliable method of detecting airborne Bacillus anthracis spores by air sampling (wet mode) and then exposing the collected sample to an antibody-functionalized piezoelectric-excited millimeter-sized cantilever (PEMC) sensor is presented. Using a commercial air sampler, a 10 min air sample at 267 L/min captured the airborne particulates containing Bacillus anthracis (BA) spores and concentrated them into 5 mL of phosphate buffered saline (PBS). This sample was then injected into a flow cell containing an antibody-functionalized PEMC sensor. The resonant frequency of the PEMC sensor at 925.1 kHz decreased exponentially as the BA spores attached to the sensor surface producing a positive response well beyond the noise level in 2 min and reached a steady state value in 20 min. In liquid phase, the sensor response correlated well (R2 = 0.99) with spore concentration and was shown to follow: [Response in Hz] = (0.0637) × (spore concentration in #/mL). Our results show that detection of 38 BA spores/L of air is achievable in near real-time with an estimated lower limit of detection of ∼5 spores/L of air in the configuration tested.

Detection of Staphylococcus enterotoxin B at picogram levels using piezoelectric-excited millimeter-sized cantilever sensors

We report a highly sensitive and rapid method for the detection of Staphylococcus aureus enterotoxin B (SEB) at picogram levels using a piezoelectric-excited millimeter-sized cantilever (PEMC) sensor. Affinity purified polyclonal antibody to Staphylococcal enterotoxin B (anti-SEB) was immobilized on the aminated cantilever glass surface, followed by exposure of SEB containing samples flowing at a rate of 1 mL/min. The binding of SEB was measured by monitoring the sensor's fundamental resonance frequency. The fundamental frequencies in air and under liquid immersion were 17.50 ± 0.01 kHz and 10.37 ± 0.01 kHz, respectively. Binding of SEB to the sensor surface resulted in an exponential decrease of the resonance frequency and reached a steady state frequency change of 31 ± 1 Hz ( n = 2) and 208 ± 1 Hz ( n = 2) corresponding to SEB concentrations of 50 pg/mL and 12.5 ng/mL, respectively. In each experiment, the bound SEB was released by a pH 2.0 HCl/PBS solution and the sensor response was nearly identical to the frequency change caused by attachment. Full antibody surface regeneration was obtained after the release by rinsing the sensor with PBS pH 7.4. We report the detection limit of PEMC sensors to SEB is between 12.5 pg/mL and 50 pg/mL.

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

Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors immobilized with antibody specific to Escherichia coli (EC) 0157: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 (PEMC-a) 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.

Piezoelectric-excited millimeter-sized cantilever sensors detect density differences of a few micrograms/mL in liquid medium

We show that piezoelectric-excited millimeter-sized cantilever sensors operating in the inviscid region (Reynolds number ∼10 5) detect density differences of a few μg/mL. The self-sensing mechanism of the sensor provides a sensitive method and enables detection of solute concentration differences of 10 ppm. We show detection sensitivity using aqueous solutions of glycerol, sodium chloride and 1-propanol at mass fractions 0–0.2 in an isothermal (20.3 °C) measurement system. While there is good agreement with theory (∼5%) over the entire density range examined (0.9698–1.0786), considerable deviations occur at low mass fraction (∼0.001). Observation of the deviations has been possible because of the high sensitivity of the sensor used in this study and the use of continuous flow measurements. We show for the first time with continuous flow experiments that surface adsorption of solute (glycerol) contributes to the observed deviation.

Method of Measuring Bacillus anthracis Spores in the Presence of Copious Amounts of Bacillus thuringiensis and Bacillus cereus

A sensitive and reliable method for the detection of Bacillus anthracis (BA; Sterne strain 7702) spores in presence of large amounts of Bacillus thuringiensis (BT) and Bacillus cereus (BC) is presented based on a novel PZT-anchored piezoelectric excited millimeter-sized cantilever (PAPEMC) sensor with a sensing area of 1.5 mm2. Antibody (anti-BA) specific to BA spores was immobilized on the sensing area and exposed to various samples of BA, BT, and BC containing the same concentration of BA at 333 spores/mL, and the concentration of BT + BC was varied in concentration ratios of (BA:BT + BC) 0:1, 1:0, 1:1, 1:10, 1:100, and 1:1000. In each case, the sensor responded with an exponential decrease in resonant frequency and the steady-state frequency changes reached were 14 +/- 31 (n = 11), 2742 +/- 38 (n = 3), 3053 +/- 19 (n = 2), 2777 +/- 26 (n = 2), 2953 +/- 24 (n = 2), and 3105 +/- 27 (n = 2) Hz, respectively, in 0, 27, 45, 63, 154, and 219 min. The bound BA spores were released in each experiment, and the sensor response was nearly identical to the frequency change during attachment. These results suggest that the transport of BA spores to the antibody immobilized surface was hindered by the presence of other Bacillus species. The observed binding rate constant, based on the Langmuir kinetic model, was determined to be 0.15 min-1. A hindrance factor (alpha) is defined to describe the reduced attachment rate in the presence of BT + BC and found to increase exponentially with BT and BC concentration. The hindrance factor increased from 3.52 at 333 BT + BC spores/mL to 11.04 at 3.33 x 105 BT + BC spores/mL, suggesting that alpha is a strong function of BT and BC concentration. The significance of these results is that anti-BA functionalized PEMC sensors are highly selective to Bacillus anthracis spores and the presence of other Bacillus species, in large amounts, does not prevent binding but impedes BA transport to the sensor.

Optimization of antibody immobilization for sensing using piezoelectrically excited-millimeter-sized cantilever (PEMC) sensors

The effectiveness of antibody immobilization on sensor performance was evaluated using a piezoelectric-excited millimeter-sized cantilever (PEMC) sensor and a model protein, bovine serum albumin (BSA). The immobilization parameters—antibody and activation reagent concentration and reaction time were systematically varied and the resultant sensor response to 1 pg/mL BSA was measured. The highest frequency shift due to BSA attachment (1931 ± 60 Hz) was obtained when 100 μg/mL antibody solution was activated with EDC (2 mM) and sulfo-NHS (5 mM) for 30 min prior to reaction with primary amine on the sensor surface. Increasing activation time from 30 to 60 min resulted in an 8% decrease in sensor response, while activation at 0.2 mM EDC and 0.5 mM NHS resulted in a 65% decrease. The logarithmic correlation between antibody concentration and sensor response suggests that a lower antibody concentration of 10 μg/mL is sufficient for BSA at 1 pg/mL. With 10 μg/mL antibody, the PEMC sensor response was 725 ± 50 Hz ( n = 2) for 1 pg/mL BSA with a signal to noise ratio of 41.

Detect of Escherichia coli O157:H7 in ground beef samples using piezoelectric excited millimeter-sized cantilever (PEMC) sensors

Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors consisting of a piezoelectric and a borosilicate glass layer with a sensing area of 4 mm2 were fabricated. An antibody specific to Escherichia coli (anti-E. coli) O157:H7 was immobilized on PEMC sensors, and exposed to samples containing E. coli O157:H7 (EC) prepared in various matrices: (1) broth, broth plus raw ground beef, and broth plus sterile ground beef without inoculation of E. coli O157:H7 served as controls, (2) 100 mL of broth inoculated with 25 EC cells, (3) 100 mL of broth containing 25 g of raw ground beef and (4) 100 mL of broth with 25 g of sterile ground beef inoculated with 25 EC cells. The total resonant frequency change obtained for the broth plus EC samples were 16+/-2 Hz (n=2), 30 Hz (n=1), and 54+/-2 Hz (n=2) corresponding to 2, 4, and 6h growth at 37 degrees C, respectively. The response to the broth plus 25 g of sterile ground beef plus EC cells were 21+/-2 Hz (n=2), 37 Hz (n=1), and 70+/-2 Hz (n=2) corresponding to 2, 4, and 6 h, respectively. In all cases, the three different control samples yielded a frequency change of 0+/-2 Hz (n=6). The E. coli O157:H7 concentration in each broth and beef samples was determined by both plating and by pathogen modeling program. The results indicate that the PEMC sensor detects E. coli O157:H7 reliably at 50-100 cells/mL with a 3 mL sample.

Use of Piezoelectric-Excited Millimeter-Sized Cantilever Sensors To Measure Albumin Interaction with Self-Assembled Monolayers of Alkanethiols Having Different Functional Headgroups

In this paper, we describe a new modality of measuring human serum albumin (HSA) adsorption continuously on CH3-, COOH-, and OH-terminated self-assembled monolayers (SAMs) of C11-alkanethiols and the direct quantification of the adsorbed amount. A gold-coated piezoelectric-excited millimeter-sized cantilever (PEMC) sensor of 6-mm2 sensing area was fabricated, where resonant frequency decreases upon mass increase. The resonant frequency in air of the detection peak was 45.5 +/- 0.01 kHz. SAMs of C11-thiols (in absolute ethanol) with different end groups was prepared on the PEMC sensor and then exposed to buffer solution containing HSA at 10 microg/mL. The resonant frequency decreased exponentially and reached a steady-state value within 30 min. The decrease in resonant frequency indicates that the mass of the sensor increased due to HSA adsorption onto the SAM layer. The frequency change obtained for the HSA adsorption on CH3-, COOH-, and OH-terminated SAM were 520.8 +/- 8.6 (n = 3), 290.4 +/- 6.1 (n = 2), and 210.6 +/- 8.1 Hz (n = 3), respectively. These results confirm prior conclusions that albumin adsorption decreased in the order, CH(3) > COOH > OH. Observed binding rate constants were 0.163 +/- 0.003, 0.248 +/- 0.006, and 0.381 +/- 0.001 min(-1), for methyl, carboxylic, and hydroxyl end groups, respectively. The significance of the results reported here is that both the formation of self-assembled monolayers and adsorption of serum protein onto the formed layer can be measured continuously, and quantification of the adsorbed amount can be determined directly.

Detection of Bacillus anthracis spores and a model protein using PEMC sensors in a flow cell at 1mL/min

Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors of 4mm(2) sensing area were immobilized with antibody specific to Bacillus anthracis (anti-BA) spores or bovine serum albumin (anti-BSA). Detection of pathogen (Bacillus anthracis (BA) at 300 spores/mL) and BSA (1 mg/mL) were investigated under both stagnant and flow conditions. Two flow cell designs were evaluated by characterizing flow-induced resonant frequency shifts. One of the flow cells labeled SFC-2 (hold-up volume of 0.3 mL), showed small fluctuations (+/-20 Hz) around a common resonant frequency response of 217 Hz in the flow rate range of 1-17 mL/min. The total resonant frequency change obtained for the binding of 300 spores/mL in 1h was 90+/-5 Hz (n=2), and 162+/-10 Hz (n=2) under stagnant and flow conditions, respectively. Binding of antibodies, anti-BA and anti-BSA, were more rapid under flow than under stagnant conditions. The sensor was repeatedly exposed to BSA with an intermediate release step. The first and second responses to BSA were nearly identical. The total resonant frequency response to BSA was 388+/-10 (n=2) Hz under flow conditions. Kinetic analysis is carried out to quantify the effect of flow rate on antibody immobilization and the two types of detection experiments.

PEMC sensor's mass change sensitivity is 20pg/Hz under liquid immersion

To enhance the mass change sensitivity of the resonating piezoelectric-excited millimeter-sized cantilever (PEMC) sensors, we reduced its length and eliminated one layer of its composite structure. As a result the mass sensitivity of the second flexural mode increased by two orders of magnitude (from 10(-9) to 10(-11)g/Hz) and the resonant frequency increased by more than 5 kHz. We demonstrate the effects of modification by detecting a model pathogen Group A Streptococcus (GAS) at 700 cells/mL. The resonant frequency change of the second mode at concentrations of 700, 7 x 10(3), 7 x 10(5), 7 x 10(6), 7 x 10(7), and 7 x 10(9)cells/mL resulted in, respectively, 3.1+/-0.5, 11.6+/-1, 15.7+/-1, 25.7+/-0.15, 28.5+/-2, and 40.5+/-3 ng (n=3 for all) of pathogen attachment. A kinetic model for the binding is proposed and verified. The observed binding rate constant was found to be in the range of 0.051-0.166 min(-1). The significance of the results we report is that the modified PEMC sensors have high mass sensitivity that pathogens can be detected at very low concentration under liquid immersion conditions.

Rapid assessment of Escherichia coli by growth rate on piezoelectric-excited millimeter-sized cantilever (PEMC) sensors

A piezoelectric-excited millimeter sized glass cantilever with a 1 mm 2 sensing surface was fabricated for the purpose of detecting the growth of Escherichia coli ( E. coli) JM101 in real-time. The resonance characteristic of the fundamental mode in air was 31.08 ± 0.01 kHz. Upon the addition of the agar film to the cantilever sensing surface the fundamental resonance frequency decreased by 2 ± 0.01 kHz. Upon subsequent inoculation of E. coli JM101, the fundamental frequency decreases an additional 0.1 ± 0.01 kHz. The E. coli was grown at 29 °C in a temperature-controlled chamber. The E. coli went through a lag phase of 1 h followed by a continuous decrease in resonant frequency due to growth of the bacteria. The total change in resonant frequency during the growth phase was 5.08 ± 0.01 kHz. After 6 h, the resonant frequency reached a constant value as growth ended. A model was developed to relate resonant frequency to specific growth rate ( μ). The E. coli exhibited exponential growth rate of 1.31 ± 0.05 h −1 and a late exponential growth rate of 0.55 ± 0.05 h −1 which compared favorably with growth rate obtained in submerged culture of 1.28 ± 0.02 h −1 at 29 °C. The significance of the results is that microbial contamination in food processing plants can be assessed rapidly (<4 h) by measuring growth rate on piezoelectric-excited millimeter-sized glass cantilevers. Further, we compare its performance with QCM and find that PEMC is 1000 times more sensitive and 10 times quicker in establishing growth of bacteria.

Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors detect Bacillus anthracis at 300spores/mL

Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors consisting of a piezoelectric and a borosilicate glass layer with a sensing area of 2.48 mm2 were fabricated. Antibody specific to Bacillus anthracis (BA, Sterne strain 7702) spores was immobilized on PEMC sensors, and exposed to spores (300 to 3x10(6) spores/mL). The resonant frequency decreased at a rate proportional to the spore concentration and reached a steady state frequency change of 5+/-5 Hz (n=3), 92+/-7 Hz (n=3), 500+/-10 Hz (n=3), 1030+/-10 Hz (n=2), and 2696+/-6 Hz (n=2) corresponding to 0, 3x10(2), 3x10(3), 3x10(4), and 3x10(6) spores/mL, respectively. The reduction in resonant frequency is proportional to the change in cantilever mass, and thus the observed changes are due to the attachment of spores on the sensor surface. Selectivity of the antibody-functionalized sensor was determined with samples of BA (3x10(6)/mL) mixed with Bacillus thuringiensis (BT; 1.5x10(9)/mL) in various volume ratios that yielded BA:BT ratios of 1:0, 1:125, 1:250, 1:500 and 0:1. The corresponding resonance frequency decreases were, respectively, 2345, 1980, 1310, 704 and 10 Hz. Sample containing 100% BT spores (1.5x10(9)/mL and no BA) gave a steady state frequency decrease of 10 Hz, which is within noise level of the sensor, indicating excellent selectivity. The observed binding rate constant for the pure BA and BT-containing samples ranged from 0.105 to 0.043 min-1 in the spore concentration range 300 to 3x10(6)/mL. These results show that detection of B. anthracis spore at a very low concentration (300 spores/mL) and with high selectivity in presence of another Bacillus spore (BT) can be accomplished using piezoelectric-excited millimeter-sized cantilever sensors.