THE USE OF STREPTAVIDIN COATED MAGNETIC BEADS FOR DETECTING PATHOGENIC BACTERIA BY LIGHT ADDRESSABLE POTENTIOMETRIC SENSOR (LAPS)

A modified procedure for magnetic capture of antibody-conjugated bacteria for light addressable potentiometric sensor (LAPS) detection using the Threshold System was developed. Streptavidin coated magnetic beads, partially labeled with biotinylated anti Escherichia coli O157 antibodies, were used to capture Escherichia coli O157:H7. Captured bacteria were further labeled with fluorescein-conjugated anti -E. coli O157:H7 antibodies and urease-labeled. anti-fluorescein antibody. Magnetically concentrated bacteria-containing complexes were then immobilized through streptavidin-biotin interactions on 0.45 μ biotinylated nitro-cellulose membranes assembled as sample sticks for the Threshold instrument. The rate of pH change associated with the production of NH₃ by the urease in urea-containing solution was measured by a LAPS incorporated in the Threshold instrument. This approach allowed us to detect 10³ to 10⁴ CPU of cultured E. coli O157:H7 in PBS solutions. Furthermore, detectable LAPS signals of the sample sticks remained relatively constant for at least 24 h at 4C. The developed approach was applied to detect the E. coli in beef hamburger spiked with the bacteria. After a 5 to 6-h enrichment at 37C, as low as 1 CFU/g of E. coli O157:H7 in beef hamburger could be detected.