Label-free flow-enhanced specific detection of Bacillus anthracis using a piezoelectric microcantilever sensor

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.