Use of light addressable potentiometric sensor (LAPS) to detect magnetically captured Escherichia coli O157:H7 in ground beef

An improved process using magnetic capture of antibody-conjugated bacteria for light addressable potentiometric sensor detection by the Threshold instrument was developed. Cells of Escherichia coli 0157:H7 were captured by the biotinylated anti-E. coli 0157 antibodies conjugated to streptavidin coated magnetic beads. Magnetically concentrated bacteria were further labeled with by fluorescein-conjugated anti-E. coli 0157 antibodies that were bound to urease-conjugated anti-fluorescein antibody. The whole bacteria-containing complex was then immobilized on 0.45 μ biotinylated nitro-cellulose membranes via streptavidin-biotin interactions. The rates of pH change associated with the production of NH 3 by conjugated urease were measured by a LAPS technique incorporated in the Threshold instrument. This approach allowed us to detect -10 4 CFU of cultured E. coli 0157:H7 in tris-buffered saline (TBS). The same approach was applied to detect the E. coli in beef hamburger spiked with the bacteria. After a 5 to 6-hour enrichment, as low as 1 CFU/g of E. coli 0157:H7 in the hamburger could be detected. In addition, the immobilized bacterial complexes on the nitro-cellulose membranes exhibited a stability longer than 48 h at 4 and 22 °C in TBS allowing the possibility of conveniently shipping collected samples rather than raw beef to testing laboratories.