A bienzyme electrochemical biosensor coupled with immunomagnetic separation for rapid detection of Escherichia coli O157:H7 in food samples

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
10 2 cells/ml of heat–killed E. coli O157:H7 under optimized conditions (1 mM MgCl2, 0.4
g/ml APLAE, 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.