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

Abstract

An array of three identical piezoelectric microcantilever sensors (PEMSs) 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 (BA) 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: (1) 3.3x10(3) spores/ml, (2) a mixture of 3.3x10(3) spores/ml and 3.3x10(5) S. aureus (SA) and P. aeruginosa (PA) per ml, and (3) a mixture of 3.3x10(3) spores/ml with 3.3x10(6) SA+PA/ml. There was no response to a sample containing only 3.3x10(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.

Figure 1

(a) An optical micrograph of array PEMS and (b) resonance spectra with detection peaks indicated.

Figure 2

In-air and in-water resonance spectra of MPS-insulated PZT∕glass PEMS.

Figure 3

A photograph of the polycarbonate fluidics cartridge with the array PEMS mounted in the center. White arrow indication array PEMS location in the semicirculate flow chamber.

Figure 4

(a) Resonance frequency shift vs time of anti-BA- and BSA-coated PEMS array in BA suspensions of various concentrations, and (b) resonance frequency shift vs time of anti-BA coated PEMS array in BA suspensions in the presence of excess SA and PA cells. Arrows in (a) indicate the times of suspension injection into the flow chamber.

Figure 5

SEM micrograph of PEMS sensor surface after exposure to (a) sample A −3.3×10³ BA spores∕ml, and (b) sample D −3.3×10³ BA spores∕ml with a 3.3×10⁶ cells∕ml of SA and PA mixture. The solid arrows in (b) indicate BA spores which are cigar-shaped as shown in (a) while the dashed arrows indicate SA cells which are spherical.