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

Abstract Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors consisting of a piezoelectric and a borosilicate glass layer with a sensing area of 2.48 mm 2 were fabricated. Antibody specific to Bacillus anthracis (BA, Sterne strain 7702) spores was immobilized on PEMC sensors, and exposed to spores (300 to 3 × 10 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, 3 × 10 2 , 3 × 10 3 , 3 × 10 4 , and 3 × 10 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 (3 × 10 6 /mL) mixed with Bacillus thuringiensis (BT; 1.5 × 10 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 (1.5 × 10 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 3 × 10 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.