In this study, we have demonstrated in situ, all-electrical detection of Bacillus anthracis (BA) spores using lead magnesium niobate-lead titanate/tin (PMN-PT/Sn) piezoelectric microcantilever sensors (PEMS) fabricated from PMN-PT freestanding films and electrically insulated with methyltrimethoxysilane (MTMS) coatings on the tin surface. Antibody specific to BA spore surface antigen was immobilized on the platinum electrode of the PMN-PT layer. In phosphate-buffered saline (PBS) solution, the PMN-PT/Sn PEMS exhibited quality (Q) values ranging from 50 to 75. The detection was carried out in a closed-loop flow cell with a liquid volume of 0.8 ml and a flow rate of 1 ml min(-1). It was shown that one sensor, "PEMS-A" (500 microm long, 800 microm wide, with a 22 microm thick PMN-PT layer, a 20 microm thick tin layer and a 1 +/- 0.5 x 10(-12) g Hz(-1) mass detection sensitivity) exhibited resonance frequency shifts of 2100 +/- 200, 1100 +/- 100 and 700 +/- 100 Hz at concentrations of 20,000, 2000, and 200 spores ml(-1) or 16,000, 1600, and 160 total spores, respectively. Additionally, "PEMS-B" (350 microm long, 800 microm wide, with an 8 microm thick PMN-PT layer, a 6 microm thick tin layer and a 2 +/- 1 x 10(-13) g Hz(-1) mass detection sensitivity) exhibited resonance frequency shifts of 2400 +/- 200, 1500 +/- 200, 500 +/- 150 and 200 +/- 100 Hz at concentrations of 20,000, 2000, 100, and 45 spores ml(-1) or 16,000, 1600, 80, and 36 total spores, respectively.

Differentiation between species of similar biological structure is of critical importance in biosensing applications. Here, we report specific detection of Bacillus anthracis (BA) spores from that of close relatives, such as B. thuringiensis (BT), B. cereus (BC), and B. subtilis (BS) by varying the flow speed of the sampling liquid over the surface of a piezoelectric microcantilever sensor (PEMS). Spore binding to the anti-BA spore IgG coated PEMS surface is determined by monitoring the resonance frequency change in the sensor's impedance vs. frequency spectrum. Flow increases the resonance frequency shift at lower flow rates until the impingement force from the flow overcomes the binding strength of the antigen and decreases the resonance frequency shift at higher flow rates. We showed that the change from increasing to decreasing resonance frequency shift occurred at a lower fluid flow speed for BT, BC, and BS spores than for BA spores. This trend reduces the cross reactivity ratio of BC, BS, and BT to the anti-BA spore IgG immobilized PEMS from around 0.4 at low flow velocities to less than 0.05 at 3.8 mm s −1 . This cross reactivity ratio of 0.05 was essentially negligible considering the experimental uncertainty. The use of the same flow that is used for detection to further distinguish the specific binding (BA to anti-BA spore antibody) from nonspecific binding (BT, BC, and BS to anti-BA spore antibody) is unique and has great potential in the detection of general biological species.

An array of three identical piezoelectric microcantilever sensors ͑PEMSs͒ consisting of a lead zirconate titanate layer bonded to a glass layer was fabricated and examined for simultaneous, in situ, real-time, all-electrical detection of Bacillus anthracis ͑BA͒ spores in an aqueous suspension using the first longitudinal extension mode of resonance. With anti-BA antibody immobilized on the sensor surfaces all three PEMS exhibited identical BA detection resonance frequency shifts at all tested concentrations, 10-10 7 spores/ ml with a standard deviation of less than 10%. The detection concentration limit of 10 spores/ml was about two orders of magnitude lower than would be permitted by flexural peaks. In blinded-sample testing, the array PEMS detected BA in three samples containing BA: ͑1͒ 3.3ϫ 10 3 spores/ ml, ͑2͒ a mixture of 3.3ϫ 10 3 spores/ ml and 3.3 ϫ 10 5 S. aureus ͑SA͒ and P. aeruginosa ͑PA͒ per ml, and ͑3͒ a mixture of 3.3ϫ 10 3 spores/ ml with 3.3ϫ 10 6 SA+ PA/ ml. There was no response to a sample containing only 3.3ϫ 10 6 SA+ PA/ ml. These results illustrate the sensitivity, specificity, reusability, and reliability of array PEMS for in situ, real-time detection of BA spores.

Current methods for analysis of unknown powders in suspicious packages involve sending samples to laboratory facilities where a variety of time-consuming tests are performed. We have developed and investigated the use of a lead zirconate titanate -titanium (PZT-Ti) microcantilever for in situ detection of the common food-and water-born pathogen, Salmonella typhimurium. Using a bifunctional linking molecule to immobilize antibody on the titanium surface of the microcantilever, we can directly detect salmonella cells in suspensions of differing concentration. This novel surface functionalization technique along with the sub-nanogram sensitivity of the cantilever has allowed for direct quantification of S. typhimurium cells in suspension.

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