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

Abstract

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.

Array lead zirconate titanate/glass piezoelectric microcantilevers for real-time detection of Bacillus anthracis with 10 spores/ml sensitivity and 1/1000 selectivity in bacterial mixtures

“…From our earlier studies, a typical saturation time ranges from a few minutes at higher concentrations, e.g., 10 5 spores/ ml to 15-30 min at lower concentrations, e.g., 10 1 spores/ ml. [20][21][22][23]26 The present detection scheme allowed us to detect BA over a wide range of concentrations, 10 1 -10 7 spores/ ml over a short time. While these experiments did not yield a linear dose response of the PEMS to these concentrations of BA, which would require preparing the sensor surface after detection at each concentration, they nonetheless amply demonstrated the "repeatability" of the detection from sensor to sensor, which is one of the main goals of this study.…”

“…Likewise, anti-BA spore antiserum was prepared as described in Ref. 22. The anti-BA antibody was immobilized on the MPS insulation coating of PEMS by means of a bifunctional linker, sulfosuccinimidyl-4-͑N-maleimidomethyl͒cyclohexane-1-carboxylate ͑sulfo-smcc͒ as described in Ref.…”

“…However, we have shown that the binding of anti-BA-spore IgG to BT, BC, and BS is much weaker than the binding of anti-BA-spore IgG to BA. 22 Binding of close relatives such as BS, BT, BC to anti-BA IgG coated PEMS can be effectively prevented by selecting a flow speed larger than 3.8 mm/s. 22 The anti-BA-spore IgG should be selective for BA detection against most other species.…”

“…Real-time, label-free antigen detection is achieved by electrically monitoring the PEMS flexural resonance frequency shift. [19][20][21][22][23][24][25][26] PEMS flexural-mode detection sensitivity is related to the thickness and the size of the PEMS. Generally, a PEMS flexural-mode detection sensitivity increases with a reduced PEMS size and thickness.…”

Enhancing detection sensitivity of piezoelectric plate sensor by increasing transverse electromechanical coupling constant

“…Piezoelectric microcantilever sensors (PEMSs) [24][25][26][27] are a relatively new type of sensor with high sensitivity, consisting of a highly piezoelectric layer made of lead zirconate-lead titanate (PZT) 27 or lead magnesium niobate-lead titanate (PMN-PT) [24][25][26] bonded to a nonpiezoelectric layer. More recently, the piezoelectric plate sensor (PEPS) 28 was developed and used solely as a highly piezoelectric plate without the nonpiezoelectric layer.…”