David Maraldo

10-minute assay for detecting Escherichia coli O157:H7 in ground beef samples using piezoelectric-excited millimeter-size cantilever sensors

We detected Escherichia coli O157:H7 (EC) at approximately 10 cells per ml in spiked ground beef samples in 10 min using piezoelectric-excited millimeter-size cantilever (PEMC) sensors. The composite PEMC sensors have a sensing area of 2 mm2 and are prepared by immobilizing a polyclonal antibody specific to EC on the sensing surface. Ground beef (2.5 g) was spiked with EC at 10 to 10,000 cells per ml in phosphate-buffered saline (PBS). One milliliter of supernatant was removed from the blended samples and used to perform the detection experiments. The total resonant frequency change obtained for the inoculated samples was 138 +/- 9, 735 +/- 23, 2,603 +/- 51, and 7,184 +/- 606 Hz, corresponding to EC concentrations of 10, 100, 1,000, and 10,000 cells per ml, respectively. EC was detected in the sample solution within the first 10 min. The responses of the sensor to positive, negative, and buffer controls were 36 +/- 6, 27 +/- 2, and 2 +/- 7 Hz, respectively. Verification of EC attachment was confirmed by low-pH buffer release (PBS-HCl, pH 2.2), microscopy, and second antibody EC binding postdetection. The results indicate that PEMC sensors can reliably detect EC at less than 10 cells per ml in 10 min without sample preparation and with label-free reagents.

Preparation-Free Method for Detecting Escherichia coli O157:H7 in the Presence of Spinach, Spring Lettuce Mix, and Ground Beef Particulates

We show the detection of 100 cells per ml of Escherichia coli O157:H7 in the presence of spinach, spring lettuce mix, and ground beef washes and particulate matter with piezoelectric-excited millimeter-sized cantilever (PEMC) sensors. The PEMC sensors (sensing area, 2 mm2) were immobilized with polyclonal antibody specific to E. coli O157:H7 (EC) and were exposed to 10 aqueous washes of locally purchased spinach, spring lettuce mix, and ground beef for testing if EC was present. Absence of resonance frequency shift indicated that EC was not present in the 30 samples tested. Following the last sample in each food matrix, 1,000 cells per ml of EC were spiked into the sample container, and resonance frequency change was monitored. The total resonance frequency change was 880 +/- 5, 1,875 +/- 8, and 1,417 +/- 4 Hz for spinach, spring lettuce mix, and ground beef, respectively. A mixture of the three food matrices spiked with 100 cells per ml of EC gave a sensor response of 260 +/- 15 Hz. The resonance frequency changes are approximately 40% lower than our previously reported study on ground beef. It is suggested that the reduction in sensitivity is due to differences in pathogen adherence to food matrices, which affects target binding to the sensor surface. We conclude that detection selectivity is conserved in the three food matrices examined and that the magnitude of sensor response is a function of the food matrix.