A BIENZYME ELECTROCHEMICAL BIOSENSOR COUPLED WITH IMMUNOMAGNETIC SEPARATION FOR RAPID DETECTION OF ESCHERICHIA COLI O157:H7 IN FOOD SAMPLES

Abstract

A biosensing system, including immunomagnetic separation (IMS), flow injection, and a bienzyme electrode, wasdeveloped for rapid detection of Escherichia coli O157:H7 in food samples. Samples inoculated with E. coli O157:H7 weremixed simultaneously with magnetic beads coated with antiE. coli antibodies and alkaline phosphatase labeled antiE. coli(APLAE) antibodies to form beadsE. coliAPLAE conjugates by antibodyantigen reaction. The conjugates were separatedby a magnetic field and then incubated with phenyl phosphate to produce phenol. An amperometric tyrosinasehorseradishperoxidase biosensor in a flow injection system was used to detect the phenol concentration that is proportional to the cellnumber of E. coli O157:H7. The biosensor was evaluated using samples of chicken carcass wash water, ground beef, andfreshcut broccoli. This biosensor was able to detect as few as 6
10 2 cells/ml of heatkilled E. coli O157:H7 under optimizedconditions (1 mM MgCl2, 0.4
g/ml APLAE, and 1 mM phenylphosphate in 25 mM tris buffer solution pH 10.0). The totaldetection time from separating target bacteria with immunomagnetic beads to analyzing flow injection electrochemicaldetection was approximately 2 h.