Method of Measuring Bacillus anthracis Spores in the Presence of

The observed binding rate constant, based on the Langmuir kinetic model, was determined to be 0.15 min-1. .... Micro- and nano-structure based oligonu...
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Anal. Chem. 2007, 79, 1145-1152

Method of Measuring Bacillus anthracis Spores in the Presence of Copious Amounts of Bacillus thuringiensis and Bacillus cereus Gossett A. Campbell and Raj Mutharasan*

Chemical and Biological Engineering Department, Drexel University, 31st and Market Streets, Philadelphia, Pennsylvania 19104

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 (r) 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 × 105 BT + BC spores/mL, suggesting that r is a strong function of BT and BC concentration. The significance of these results is that antiBA 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. Sensing of both foodborne and airborne pathogens is essential in the fight against bioterrorism. The Bacillus species, Bacillus anthracis (BA, airborne), is the etiology agent of anthrax.1,2 * Corresponding author. Telephone: (215) 895-2236. Fax: (215) 895-5837. E-mail: [email protected]. (1) Campbell, G. A; Mutharasan, R. Biosens. Bioelectron. 2005, 21, 1684-1692. 10.1021/ac060982b CCC: $37.00 Published on Web 12/22/2006

© 2007 American Chemical Society

Bacillus cereus (BC), a foodborne bacterium,3,4 and Bacillus thuringiensis (BT), an insecticide toxin producer5,6 are closely related species. Anthrax spores are biologically dormant structures that are highly resistive to extreme temperatures, physical damage, desiccation, and harsh chemicals. As an airborne pathogen, anthrax spores can cause respiratory infection, as was done in the United States in the fall of 2001. In the case of an anthrax attack, the Centers of Disease Control (CDC) has estimated a cost of $26.2 billion per 100 000 persons exposed,7 and treatment of the disease can only be successful if the antibiotic is administered within 1 day of exposure.8 The threat of a potential biowarfare agent release has created an urgent need for a low pathogen count detector that is selective, sensitive, and robust and gives a quick positive response in real time. In response to the potential biological warfare, several sensing platforms capable of providing reliable identification of airborne and foodborne biowarfare agents are currently under development. Some of these sensor platforms include enzyme-linked immunosorbent assays (ELISA),9 membrane-based online optical analysis system,10 evanescent wave fiber-optic biosensors,11 real-time PCR,12-14 photoluminescence,15 and quartz crystal microbalance (2) La Duc, M. T.; Satomi, M.; Agata, N.; Venkateswaran, K. J. Microbiol. Methods 2004, 56, 383-394. (3) Carretto, E.; Barbarini, D.; Poletti, F.; Marzani, F. C.; Emmi, V.; Marone, P. J. Infect. Dis. 2000, 32, 98-100. (4) Agata, N.; Ohta, M.; Yokoyama, K. Int. J. Food Microbiol. 2002, 73, 2327. (5) Carlson, C. R.; Caugant, D.; Kolstø, A.-B. Appl. Environ. Microbiol. 1994, 60, 1719-1725. (6) Abdel-Hameed, A.; Landen, R. J. Microbiol. Biotechnol. 1994, 10, 406-409. (7) Watson, J.; Koya, V.; Leppla, S. H.; Daniel, H. Vaccine 2004, 22, 43744384. (8) Jernigan, J. A.; Stephens, D. S.; Ashford, D. A.; Omenaca, C.; Topiel, M. S.; Galbraith, M.; Tapper, M.; Fisk, T. L.; Zaki, S.; Popovic, T.; Meyer, R. F.; Quinn, C. P.; Harper, S. A.; Fridkin, S. K.; Sejvar, J. J.; Shepard, C. W.; McConnel, M.; Guanner, J.; Shieh, W. J.; Malecki, J. M.; Gerberding, J. L.; Hughes, J. M.; Perkins, B. A. Emerging Infect. Dis. 2001, 7, 933-944. (9) Morais, S.; Goncalez-Martinez, M. A.; Abad, A.; Montoya, A.; Maquieira, A.; Puchades, R. J. Immunol. Methods 1997, 208, 75-83. (10) Floriano, P. N.; Christodoulides, N.; Romanovicz, D.; Bernard, B.; Simmons, G. W.; Cavell, M.; McDevitt, J. T. Biosens. Bioelectron. 2005, 20, 20792088. (11) Tims, T. B.; Lim, D. V. J. Microbiol. Methods 2004, 59, 127-130. (12) Fasanella, A.; Losito, S.; Trotta, T.; Adone, R.; Massa, S.; Ciuchini, F.; Chiocco, D. Vaccine 2001, 19, 4214-4218. (13) Kim, K.; Seo, J.; Wheeler, K.; Park, C.; Kim, D.; Park, S.; Kim, W.; Chung, S.-I.; Leighton, T. FEMS Immunol. Microbiol. 2005, 43, 301-310. (14) Sperveslage, J.; Stackerbrandt, E.; Lembke, F. W.; Koch, C. J. Microbiol. Methods 1996, 26, 219-224.

Analytical Chemistry, Vol. 79, No. 3, February 1, 2007 1145

(QCM).16 While these sensing platforms have been used in the detection and identification of pathogens, no papers have appeared that show their selectivity and sensitivity (with the exception of PCR) to very low pathogen concentrations in presence of large number of other bacterial species. Recently, we reported the aqueous phase detection of Bacillus anthracis (BA) spores at 300/ mL using a partially immersed anchored PZT/glass cantilever in a batch configuration.1 We also have shown that the detection method using antibody (anti-BA) immobilized cantilever sensor was selective to anthrax spores, by exposing the sensor to various mixtures of BA spore and Bacillus thuringiensis (BT) at very high concentrations. BA at 106 spores/mL and BT at 109 spores/mL, and their mixtures showed an increase in sensor response with increasing BA concentration. The detection of BA at very low concentration in presence of large amounts of other Bacillus species is important and has not been studied using antibody as the recognition element. In this paper, we investigate the binding kinetics of BA spores at very low concentration from samples containing copious amounts of both BT and BC. We recently reported on the PZT-anchored piezoelectric excited millimeter-sized cantilever (PAPEMC) sensors having high sensitivity for biomolecules and pathogens.17 It has a piezoelectric layer (lead zirconate titanate, PZT) as the base sensor platform to which a 1.5 mm2 glass layer is bonded at one end. The composite structure is a few millimeters in length. The sensors are designed so that the bending modes of vibration are the dominate modes. One advantage in using a piezoelectric layer is that it provides both actuation and signal sensing. We have reported piezoelectric actuated cantilevers in which both the PZT and the glass layers are anchored for the detection of Escherichia coli O157:H7,18 proteins,19 and self-assembly monolayer formation at 1 nM.20 Detection of spores requires the specificity of the sensor surface, which is accomplished by using immobilized antibody that is specific to the spore. Binding of spores to antibody on the sensor increases the sensor’s mass and decreases its resonant frequency. Therefore, by tracking the change in resonant frequency the bound species concentration can be determined. CANTILEVER PHYSICS Details on cantilever physics can be found elsewhere.18,19 The resonant frequency ( fn) in air can be expressed as

x

fn ) k n

K Me

(1)

where kn ) 0.1568, 0.9827, 2.7517, and 5.3923 corresponding to the first four eigenvalues for a rectangular cantilever. K is the effective spring constant of the composite structure, which depends on the thickness (t), width (w), length (L), and the Young’s modulus (E) of the cantilever material, namely, both PZT and glass. Me is the effective mass of the cantilever in air and can (15) Rosen, D. L.; Sharpless, C.; McGown, L. B. Anal. Chem. 1997, 69, 10821085. (16) Lee, S.-H.; Stubbs, D. D.; Cairney, J.; Hunt, W. D. IEEE 2003, 1194. (17) Maraldo, D.; Rijal, K.; Campbell, G.; Mutharasan, R. Science 2006, submitted. (18) Campbell, G.; Mutharasan, R. Biosens. Bioelectron. 2005, 21, 462-473. (19) Campbell, G.; Mutharasan, R. Biosens. Bioelectron. 2005, 21, 597-607. (20) Campbell, G.; Mutharasan, R. Langmuir 2005, 21, 11568-11573.

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be estimated as

Me ) 0.236 Fptpw(Lp - Lg) + (Fptp + Fgtg)wLg

(2)

The subscripts p and g refer to PZT and glass, respectively. Upon immersing the sensor in a liquid sample, the fluid adjacent to the cantilever’s surface becomes part of the oscillating mass. Therefore, the resonant frequency response to the added mass of liquid and/or analyte can be written as

f′nf ) kn

x

K Me + mae + ∆m

(3)

where f′nf is the resonant frequency of the nth mode in fluid when analyte of equivalent mass (∆m) binds to the cantilever tip. The term mae is the effective added oscillating liquid mass when immersed, similar to the effective cantilever mass Me. The change in resonant frequency due to antigen attachment is

fnf - f′nf )

1 ∆m f 2 nf Mef

(4)

where fnf and f′nfare the resonant frequencies of the nth mode before and after antigen binding. Mef ) Me + mae is the effective mass of the cantilever under liquid immersion condition. The change in resonant frequency represented by the left-hand side of eq 4 depends linearly on the mass of antigen bound to the sensor. For small mass changes, the above can be rearranged to provide an expression for mass change sensitivity in fluid (σnf):

σnf )

2Mef ∆m ) fnf - f′nf fnf

(5)

A lower numerical value of σnf represents a higher mass change sensitivity. The above indicates that for the same cantilever mass, at higher frequency its sensitivity is higher. MATERIALS AND METHODS Fabrication. PAPEMC sensors are fabricated from a 127 µm thick PZT single sheet (Piezo Systems Inc., Cambridge, MA) and a 160 µm thick quartz cover slip (SPI, West Chester, PA). The PZT layer is the base sensor platform (see Figure 1A). The cantilever free end was designed with the quartz layer, 1.50 ( 0.05 × 1 ( 0.05 mm2 (length × width), bonded at one end of the PZT layer, 4 ( 0.05 × 1 ( 0.05 mm2 (length × width), by a nonconductive adhesive. At the other end, 1.70 ( 0.05 mm length of the PZT layer was epoxied into a glass tube. As a result, the cantilever free end has 0.8 mm of exposed PZT layer on the glass side. Top and bottom electrodes were attached to the PZT layer before it was epoxied using a 30 gauge copper wire soldered to BNC couplers. The PZT layer at the cantilever free end was insulated with 20 µm thick polyurethane coating. The glass layer (1.5 ( 0.05 × 1 ( 0.05 mm2) provides the surface for antibody immobilization. Immobilization and Sample Preparation. BA spores (killed in 4% formaldehyde), the Sterne strain 7702, and protein A purified rabbit polyclonal antibody (anti-BA) in PBS was provided by

Figure 1. (A) Schematic of PAPEMC sensor. (B) Flow circuit of experimental apparatus.

Professor Richard Rest (Drexel University College of Medicine, Philadelphia, PA). BT was purchased from EDVOTEK (West Bethesda, MD), and BC was provided by Dr. Shu-I Tu’s group (USDA, Eastern Regional Research Center, PA). All other chemical reagents were from Sigma-Aldrich. The procedure used to functionalize the glass surface with antibody was reported earlier.1,18 After each detection experiment, the sensor surface was cleaned and fresh antibody was immobilized. A stock sample of BA spore, concentration 2000 BA spores/mL, was prepared in phosphate-buffered saline (PBS) (10 mM, pH 7.4) by serial dilution of a master sample (2 × 105 spores/ mL). BC and BT spores of equal concentrations were mixed to obtain a final concentration of 2 × 103, 2 × 104, 2 × 105, and 2 × 106 (BC + BT) spores/mL. The test samples were prepared by diluting 1 mL of BA sample (2000 BA spores/mL) with 1 mL of

BT + BC mixture. The 2 mL solution was then injected into a flow circuit containing a hold-up volume of 4 mL PBS. Therefore, the concentration of BA in any one experiment was 333 spores/ mL. Selectivity of PAPEMC sensors was investigated using BA and BT + BC mixed samples of various concentration ratios (0:1, 1:0, 1:1, 1:10, 1:100, and 1:1,000 BA:BT + BC), which yield BA spore concentrations labeled A, B, C, D, and E corresponding to 0%, 100%, 50%, 9%, 1%, and 0.01% with total spore count as given in Table 1. Experimental Setup. All experiments were carried out in a flow configuration. The flow circuit is shown in Figure 1B. The setup consisted of five fluid reservoirs, peristaltic pumps, and a sensor flow cell. The fluid reservoirs are as follows: one for PBS buffer pH 7.4, activated antibody solution, 10 mM hydroxylamine solution, antigen sample, and release buffer (PBS/HCl; pH 1.85). Analytical Chemistry, Vol. 79, No. 3, February 1, 2007

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Table 1. Experimentally Determined Mass Sensitivity in Vacuum (60 mTorr) and the Estimated Liquid Sensitivity Using Paraffin Waxa

a

peaks location [kHz]

sensitivity (σnv) in vacuum by wax deposition [fg/Hz]

estd sensitivity in liquid (σnf) [fg/Hz]

102.05 970.05 1808.05

11.01 ( 3.01 1.47 ( 0.47 1.21 ( 0.01

24.33 ( 6.23 2.89 ( 0.83 2.13 ( 0.03

In each case n ) 2 and ( SD are indicated.

The liquid reservoirs were connected to the flow cell via a five entrance port manifold, and the inflow enters the sensor flow cell (SFC) through the bottom. The outlet of the flow cell is connected to a peristaltic pump, which controls the flow of the desired fluid into and out of the SFC. The APTES functionalized PAPEMC sensor was installed vertically into the cell filled with PBS via Swagelok fittings. The SFC has a hold-up capacity of 120 µL after the cantilever is positioned. The cantilever electrodes were connected to an impedance analyzer (Agilent, HP 4192A) interfaced to a data acquisition PC with Labview application for obtaining impedance and phase angle measurements in the frequency range of 0.001-5 MHz with an excitation voltage of 100 mV. The constant temperature bath was set at 35 ( 0.1 °C in order to maintain the cell content at 25 °C. The fluidic system was first primed with PBS to remove any air bubbles. Valves located at the bottom of each fluid reservoir enabled selection of fluid for introduction into the flow cell or for circulation. Switching the outlet line from the peristaltic pump into the desired fluid reservoirs enabled total recirculation. All valves were manipulated manually. All detection experiments were carried out at a flow rate of 1 mL/min. Experimental Determination of Mass Sensitivity in a Vacuum. The mass change sensitivity of PAPEMC sensor was determined by measuring the changes in resonant frequency following the deposition of known amount of mass.18 The approach used the deposition of paraffin wax at 1 pg increments. A mass of 0.23 mg of wax was dissolved in 4 mL of hexane, and 0.5 µL of the solution was dispensed into 10 weighing plates that were already weighed. The plates were placed under a chemical fume wood for 15 min to remove all the solvent. Subsequently, each plate was re-weighed, and the difference in weight between the plates with wax and the empty ones was used to compute the average mass of wax in 0.5 µL of solution. An aliquot of the stock solution was diluted to a final concentration of 1 pg of wax per 0.5 µL of solution. To determine the sensitivity of the cantilever, the sensor was cleaned, dried and placed in a vacuum chamber at 60 mTorr. The resonant frequency of the sensor was monitored and recorded. The cantilever was removed from the chamber, and 0.5 µL of the wax solution was deposited on its glass surface followed by drying under a fume hood for 15 min. Subsequently, the cantilever was placed in vacuum at 60 mTorr, and the resonant frequency was monitored and recorded until it stabilized. This procedure was repeated three times in successive mass addition, and the sensitivity was determined as the slope from a plot of mass change against frequency change. 1148 Analytical Chemistry, Vol. 79, No. 3, February 1, 2007

Figure 2. (A) Resonant spectrum, a plot of phase angle vs excitation frequency, of the PAPEMC sensor investigated. The spectrum is typical of PAPEMC sensors. Resonant peak of 970 kHz was used in all the experiments. (B) Resonant peak of PAPEMC sensor in air (970 kHz, right) and totally submerged in PBS (835.5 kHz, left).

RESULTS AND DISCUSSION Resonance Characterization of PAPEMC Sensors. Several PAPEMC sensors were fabricated and the resonance spectrum shown in Figure 2A is typical. Two of the cantilever sensors were used in the experiments reported here. For consistency and ease of comparison, of the various detection experiments, only the data obtained from one PAPEMC sensor is presented. Each experiment was repeated at least twice, and the data shown are typical of the results obtained. The resonant spectrum, a plot of phase angle versus excitation frequency, in air showed dominant bending mode resonant peaks at 102.15 ( 0.05, 970.05 ( 0.05, and 1810.05 ( 0.05 kHz, respectively. In this study, the 970.05 kHz peak was selected for in-liquid detection experiments as its Q value remained high (Q ∼ 15 in liquid) and exhibited low noise ((30 Hz) under flow conditions. Upon immersion in liquid, the resonant frequency decreased substantially (134.05 kHz), indicating that it is a sensitive peak. Flow initiation, at 1 mL/min, caused a decrease of an additional 450 Hz (see Figure 2B). The change in resonant frequency in going from air to liquid flow is a measure of sensitivity; therefore, the resonance at 970 kHz is a sensitive frequency for conducting detection experiments. Mass Change Sensitivity. Figure 3 shows a plot of mass change versus frequency change of the 970 kHz peak in a vacuum as paraffin wax was sequentially deposited at 1 pg increments. The mass of wax in 0.5 µL of the original stock solution was experimentally determined as 47.8 ( 6.4 (n ) 10) ng. Diluting an aliquot of the stock solution gave an estimated concentration of

Figure 3. Experimental determination of the mass change sensitivity of the 970 kHz peak in vacuum (60 mTorr) using paraffin wax. Error bars represent one standard deviation. The sensitivity was determined as 1.47 fg/Hz.

1 pg of wax per 0.5 mL of solution. The addition of wax (1 pg) onto the cantilever glass surface (1.5 mm2 area at sensor tip) caused the resonant frequency to decrease. The plot in Figure 3 gave a straight line with slope 1.47 ( 0.47 (n ) 2) fg/Hz, and each fit gave a correlation coefficient of 0.998 ( 0.001. Several cantilevers of nearly identical resonance characteristics were used in the sensitivity study. The sensitivity values in vacuum of the dominant bending mode peaks shown in Figure 2A are summarized in Table 1. It is important to note that the sensitivity increased in a nonlinear fashion with the increase in resonant frequency. The mass sensitivity under liquid immersion condition (σnf) can be estimated from the sensitivity value in a vacuum (σnv). Differentiating and rearranging eq 1, one gets the mass change sensitivity in vacuum as

σnv )

dMe -2Me ) dfn fn

(6)

It is clear from eqs 5 and 6 that lower values of Me or Mef and higher values of fn or fnf result in greater mass change sensitivity for a given resonant frequency measurement. The ratio of eq 5 and eq 6 gives

( )

σnf ) σnv

Mef fn Me fnf

(7)

The values calculated for Me and Mef were 2.14 and 3.62 mg, respectively. The estimated values of σnf were computed and are given in Table 1. These values compare favorably with values reported by other investigators, which are in the range of 0.3200 fg/Hz.23-26 Two main differences are (i) the sensors used in (21) Markidou, A.; Shih, W. Y.; Shih, W.-H. Rev. Sci. Instrum. 2005, 76, 0643021-064302-7. (22) Campbell, G.; Mutharasan, R. Biosens. Bioelectron. 2006, 22, 78-85. (23) Teva, J.; Abadal, G.; Torres, F.; Verd, J.; Perez-Murano, F.; Barniol, N. Ultramicroscopy 2006, 106, 800-807. (24) Teva, J.; Abadal, G.; Torres, F.; Verd, J.; Perez-Murano, F.; Barniol, N. Ultramicroscopy 2006, 106, 808-814. (25) Yue, Q.; Song, Z. Microchem. J. 2006, 84, 10-13. (26) Hosaka, S.; Chiyoma, T.; Ikeuchi, A.; Okano, H.; Sone, H.; Izumi, T. Curr. Appl. Phys. 2006, 6, 384-388.

Figure 4. Transient response of PAPEMC sensor to the binding of 333 BA spores/mL from a solution containing various amounts of other Bacillus species. The control response shown is that of the anti-BA functionalized PAPEMC exposed to a mixture of BT and BC spores, at concentrations of 166 BT spores/mL and 166 BC spores/mL, so as to establish the baseline frequency change of the sensor.

this study are millimeter-sized while the reported studies used micron-sized cantilevers; and (ii) in the present study, the mass deposited on the sensor was independently verified using the traditional method, while in refs 23-26 estimates of the deposited mass were made. Sensor Response to BA Spores Binding from Solutions Containing Various Concentrations of Non-antigenic Bacillus Species (BT and BC). In Figure 4, the transient resonant frequency responses to the binding of BA spores at concentrations of 333 BA spores/mL are presented. Each experiment was repeated at least twice, and the data shown are typical of the results obtained. In each experiment, the sensor responded with a rapid decrease in resonant frequency before reaching the same steady-state value at different time periods. For the pure BA sample (333 spores/mL), the resonant frequency decreased most rapidly and reached steady state in 27 min. As the concentration of the non-antigenic Bacillus species (BT and BC) increased as the rate of resonant frequency change decreased and took a longer time to reach steady-state changes. Steady states of 2742 ( 38 (n ) 3), 3053 ( 19 (n ) 2), 2777 ( 26 (n ) 2), 2953 ( 24 (n ) 2), and 3105 ( 27 (n ) 2) Hz were obtained for the 1:0, 1:1, 1:10, 1:100, and 1:1000 BA:BT + BC samples, respectively, in 27, 45, 63, 154, and 219 min correspondingly. We note that the steadystate responses yielded an average frequency decrease of 2926 ( 162 (n ) 11) Hz. The deviation of (162 Hz from 11 separate sensor preparations and detection experiments is small. For all practical purposes, the results indicate that the presence of nonantigenic components in the sample minimally affects the sensor response. We conclude that non-antigenic Bacillus species (BT and BC) hindered the transport of the BA spores to the sensor surface but never completely prevented attachment of antigenic spore. Corresponding to each detection experiment a control consisting of exposing anti-BA functionalized cantilever to a sample of only BT and BC at the same concentration and experimental conditions was carried out. The resonant frequency change fluctuated about zero for the control. The sensor response to the Analytical Chemistry, Vol. 79, No. 3, February 1, 2007

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Table 2. Composition of Mixed Spore Samples Containing Bacillus anthracis, Bacillus thuringiensis, and Bacillus cereus, Sensor Response, and Hindrance Coefficient

Figure 5. Transient frequency response of PAPEMC sensor to the binding of 333 BA spores/mL and the release of the bound spores by exposure to low pH buffer, pH 1.85.

control containing BT + BC presented in Figure 3 is a typical result. Here, the response of the sensor was 14 ( 31 (n ) 11) Hz. We conclude that the control (BT + BC) exhibited no affinity to sensor surface. Also the control response shows the stability of the sensor in the presence of particulates under liquid flow conditions. It is to be noted that without any particulates present in the flowing buffer the sensor had a more stable response of 3 ( 5 Hz. A higher level of sensitivity can be achieved by improving surface concentration of antibody, reducing noise in measurement due to flow, and by using piezoelectric material with improved properties. Using the mass change sensitivity of the 836 kHz peak under liquid (2.89 ( 0.83 (n ) 2) fg/Hz), the average mass of BA spores bound to the sensor surface in any one experiment was 8.46 ( 2.45 pg. If we assume that a single BA spore weighs 1 pg, then approximately 9 spores attached in any one experiment and comparing this with the 2000 BA spores that were available for binding indicated that only 0.5% of the total spores in the sample became attached to the sensor. The amplitude of oscillation of the sensor is estimated from the piezoelectric charge coefficient (d31) of PZT and deflection relationship21 to be 0.12 nm. The residence time of the sample in the SFC is about 7 s, and the volume of the sample that comes in contact with the cantilever oscillation volume is calculated as 0.044% of the total sample volume. If we take the case of the clean BA sample that was recirculated for approximately 50 min, the total sample volume that was contacted with the sensor was ∼22 µL (0.044% × 6 mL × 50 min/6 min per cycle). Given the spore concentration (333 spores/ mL), the number of spores that could potentially attach is 7.33 (0.022 mL × 333 spores/mL). That is, to the first approximation, the number of attached spores predicted from the sensitivity value is in agreement with the number of spores that have access to the sensor surface. The binding strength of antibody to antigen can be nullified by changing either the pH or ionic strength or both. The binding of BA spores to the PAPEMC sensor bearing anti-BA was confirmed by exposure to a low pH (1.85) buffer and is given in Figure 5. The release was signified by a rapid increase of the resonant frequency to a value that was ∼13% lower than the total frequency change observed during the attachment. One notes that after the spores were attached, flowing of PBS through the SFC caused no significant change in the resonant frequency, which 1150

Analytical Chemistry, Vol. 79, No. 3, February 1, 2007

sample designation

BA spores [mL]

BT spores [mL]

BC spores [mL]

∆f∞ [Hz]

R

A B C D E F

333 333 333 333 333 0

0 166 1 665 16 650 166 500 166 500

0 166 1 665 16 650 166 500 166 500

2742 ( 38 3053 ( 19 2777 ( 26 2953 ( 24 3105 ( 27 14 ( 31

1 3.52 4.53 6.13 11.04

suggests that there were no or low weakly bound BA spores to the sensor. Conversely, rinsing of the SFC with PBS after the release step resulted in an increase of the resonant frequency to a value that was present prior to the attachment, which indicates that the antibody surface was now fully recovered. Kinetics of BA Spore Binding. The steady-state frequency response of the sensors give information on the target concentration, while kinetics gives information on the concentration of the contaminating Bacillus species. The binding kinetics of pathogens to PEMC sensors are characterized in the literature by the firstorder Langmuir kinetic model.1 In short, the initial rate analysis was used to analyze the binding process, since at time τ ) 0 there are no concentration gradients, and diffusion effects are absent. The Langmuir model can be expressed as follows:1,18

θ ) 1 - e-kobsτ

(7)

where θ is the fraction of available reactive sites that are occupied (0 e θ e 1), kobs is the observed binding rate constant, which depends possibly on bulk concentration of the binding entity (Cb0), BA spores. In Figure 4, we noted earlier that the total sensor response to BA spore was approximately the same in varying amounts of BT and BC. Since only the time taken to reach the steady-state frequency increased with increasing BT and BC concentrations, it is reasonable to modify the observed binding rate constant in eq 7 to account for the slower kinetics as

θ ) 1 - e-(kobs/R)τ

(8)

where R is a parameter that characterizes the hindrance. Since the sensor response (∆f ) is proportional to mass of antigen attached (eq 4), the above can by written as

θ)

(∆f ) ) (1 - ekobsτ/R) (∆f∞)

(9)

where (∆f ) is the change in resonant frequency at time, τ, and (∆f∞) is the steady-state resonant frequency change. Equation 8 can be rearranged to

ln

(

)

(∆f∞) - (∆f ) (∆f∞)

)-

kobs τ R

(10)

Figure 6. (A) Langmuir kinetic analysis of BA binding on PAPEMC sensor surface. The initial kinetic analysis of the various BA detection experiments with correlation coefficient ranged from 0.95 to 0.99. The slope of each line gives the observed characteristic binding rate constant kobs. (B) Hindrance factor as a function of non-antigenic Bacillus species (BT and BC) concentration to the transport of BA to the cantilever sensing surface.

From the above the characteristic binding rate constant, kobs during initial time (far from equilibrium) can be determined by fitting the data of the pure BA sample, because the hindrance factor (R) by definition is unity. Fitting the experimental data of the pure BA binding presented in Figure 4 to eq 10 gave a straight line with a slope of 0.15 min-1 and correlation coefficient of 0.98 and is shown in Figure 6A. We limit the analysis to the first 1012 min to avoid having to deal with diffusion effects. A summary of the analysis for the various cases is given in Table 2. In Figure 6B we note that non-antigenic bacillus species (BT and BC) concentration increases R in a nearly exponential fashion. At low concentration of BT and BC (333-33 300 BT + BC spores/mL), the hindrance effect seems to increase monotonically with concentration. However, at higher concentration the hindrance factor increased more rapidly, suggesting that the transport of BA spores from the bulk solution to the sensor surface is a strong function of BT and BC concentration. The results we present here

indicate that when BA spores are present in a matrix containing copious amounts of non-antigenic particulate matter the binding kinetics is affected rather strongly, while their effects on steadystate response is relatively small. CONCLUSION In this paper we conclude that selective detection of BA spores in presence of copious amounts of other Bacillus species under liquid immersed flow conditions using a PAPEMC sensor is feasible. The presence of non-antigenic Bacillus species reduced binding kinetics of BA but did not alter the steady-state response of the sensor. ACKNOWLEDGMENT The authors acknowledge the kind contribution of Professor Richard Rest for providing the antibody and formaldehyde-killed Bacillus anthracis spores. We also gratefully acknowledge Dr. Analytical Chemistry, Vol. 79, No. 3, February 1, 2007

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Kevin Scoles for the development of data acquisition program. This work was partially supported by the United States Department of Transportation under Grant PA-26-0017-00 (Federal Transit Administration, in the interest of information exchange), the Environmental Protection Agency Grant R8296041, and the National Institutes of Health Grant 5R01EB000720. The United States Government assumes no liability for the contents or use thereof. The United States Government does not endorse products

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Analytical Chemistry, Vol. 79, No. 3, February 1, 2007

or manufacturers. Trade or manufacturers’ names appear herein solely because they are considered essential to the contents of the paper.

Received for review May 29, 2006. Accepted November 15, 2006. AC060982B