A bienzyme electrochemical biosensor coupled with immunomagnetic separation for rapid detection of Escherichia coli O15 : H7 in food samples

摘要

A biosensing system, including immunomagnetic separation (IMS), flow injection, and a bienzyme electrode, was developed for rapid detection of Escherichia coli O157:H7 in food samples. Samples inoculated with E. coli O157:H7 were mixed simultaneously with magnetic beads coated with anti-E. coli antibodies and alkaline phosphatase labeled anti-E. coli (APLAE) antibodies to form beads-E. coli-APLAE conjugates by antibody-antigen reaction. The conjugates were separated by a magnetic field and then incubated with phenyl phosphate to produce phenol. An amperometric tyrosinase-horseradish peroxidase biosensor in a flow injection system was used to detect the phenol concentration that is proportional to the cell number of E. coli O157:H7. The biosensor was evaluated using samples of chicken carcass wash water ground beef, and fresh-cut broccoli. This biosensor was able to detect as few as 6 x 10(2) cells/ml of heat-killed E. coli 0157:H7 tinder optimized conditions (1 muM MgCl2, 0.4 mug/ml APL4E, and 1 mM phenylphosphate in 25 mM tris buffer solution pH 10.0). The total detection time from separating target bacteria with immunomagnetic beads to analyzing flow injection electrochemical detection was approximately 2 h.