Improvement of immunodetection of bacterial spore antigen by ultrasonic cavitation.

Ultrasonic cavitation was employed to enhance sensitivity of bacterial spore immunoassay detection, specifically, enzyme-linked immunosorbent assay (ELISA) and resonant mirror (RM) sensing. Bacillus spore suspensions were exposed to high-power ultrasound in a tubular sonicator operated at 267 kHz in both batch and flow modes. The sonicator was designed to deliver high output power and is in a form that can be cooled efficiently to avoid thermal denaturation of antigen. The 30-s batch and cooled flow (0.3 mL/min) sonication achieved an approximately 20-fold increase in ELISA sensitivity compared to unsonicated spores by ELISA. RM sensing of sonicated spores achieved detection sensitivity of approximately 10(6) spores/mL, whereas unsonicated spores were undetectable at the highest concentration tested. Improvements in detection were associated with antigen released from the spores. Equilibrium temperature increase in the tubular sonicator was limited to 14 K after 30 min and was maintained for 6 h with cooling and flow (0.3 mL/min). The work described here demonstrates the utility of the tubular sonicator for the improvement in the sensitivity of the detection of spores and its suitability as an in-line component of a rapid detection system.

Spore and micro-particle capture on an immunosensor surface in an ultrasound standing wave system.

The capture of Bacillus subtilis var. niger spores on an antibody-coated surface can be enhanced when that coated surface acts as an acoustic reflector in a quarter wavelength ultrasonic (3 MHz) standing wave resonator. Immunocapture in such a resonator has been characterised here for both spores and 1 microm diameter biotinylated fluorescent microparticles. A mean spatial acoustic pressure amplitude of 460 kPa and a frequency of 2.82 MHz gave high capture efficiencies. It was shown that capture was critically dependent on reflector thickness. The time dependence of particle deposition on a reflector in a batch system was broadly consistent with a calculated time of 35 s to bring 95% of particles to the coated surface. A suspension flow rate of 0.1 ml/min and a reflector thickness of 1.01 mm gave optimal capture in a 2 min assay. The enhancement of particle detection compared with the control (no ultrasound) situation was x 70. The system detects a total of five particles in 15 fields of view in a 2 min assay when the suspending phase concentration was 10(4) particles/ml. A general expression for the dependence of minimum concentration detectable on; number of fields examined, sample volume flowing through the chamber and assay time shows that, for a practical combination of these variables, the threshold detection concentration can be two orders of magnitude lower.