(526a) Antibody-Immobilized Piezoelectric-Excited Millimeter-Sized Cantilever (Pemc) Sensors Detect Bacillus Anthracis at 300 Spores/ML

Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors consisting of a piezoelectric and a borosilicate glass layer with a sensing area of 2.48 mm2 were fabricated. Antibody specific to Bacillus anthracis (BA, Sterne strain 7702) spores was immobilized on PEMC sensors via carboxylic group to amine groups on cantilever surface. The PEMC was exposed to BA spores (300 to 3x10^6 spores/mL). The resonant frequency decreased at a rate proportional to the spore concentration and reached a steady state frequency change of 0 ± 5 (n=3), 92 ± 5 (n=3), 499 ± 5 (n=3), 1040 ± 5 (n=2), and 2694 ± 5 (n=2) Hz corresponding to 0, 3x10^2, 3x10^3, 3x10^4, and 3x10^6 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 (3x10^6/mL) mixed with spores of Bacillus thuringiensis (BT; 1.5x10^9/mL) in various volume ratios that yielded BA:BT ratios of 1:0, 1:0.008, 1:0.004, 1:0.002 and 0:1. The corresponding resonance frequency decreases were, respectively, 2345, 1980, 1310, 704 and 10 Hz. Sample containing 100% BT spores (1.5x10^9/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 binding rate constant for the pure BA sample was 3.9x10^-8 min-1 (spores/mL)-1, and the BT-containing samples ranged from 4.4x10^-8 to 5.9x10^-8 min-1 (spores/mL)-1. These results show that detection of Bacillus 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.