Detection of Escherichia coli O157:H7 in Water using an Amperometric Biosensor

Abstract

Escherichia coli O157:H7 contamination is a major hazard in water supplies,causing outbreaks of disease. Conventional methods of E. coli O157:H7 detectionusually involve incubation and require hands-on preparation. Currently, there is a needto develop a rapid, inexpensive means of detecting coliforms in waterbodies. Theamperometric biosensor technology has achieved success in the area of metabolitedetection. In this study, a bench scale amperometric biosensor was investigated torapidly detect Escherichia coli O157:H7. The amperometric biosensor consisted of apower source, Clark electrode, picoammeter, and fabricated polyvinyl chloride (PVC)outer insert with nitrocellulose membrane and attached horseradish peroxidase labeledE. coli antibodies. The interaction of horseradish peroxidase and hydrogen peroxideproduced dissolved oxygen, which is anticipated to be altered by the binding of theantigen to the antibody. After submerging the amperometric biosensor in the samplescontaining various concentrations of heat sterilized E. coli O157:H7 cells, as little as 10cells/ml of E. coli O157:H7 were detected. The time for detection for the final systemwas approximately 20 minutes. Results indicated that there was a need to use acustom conjugated antibody to control and increase the molar concentration ofconjugated HRP. The minimum concentration of HRP needed for this system was 6 X10-8M HRP. The results indicated that change in dissolved oxygen response can beused to distinguish between 0 and 10-5000 cells/ml. Maximum increases in dissolvedoxygen of 3.53mg/L 0.26mg/L when bacterial cells were present and increase in theorder of 6.26 0.64mg/L when no cells were present was observed. Despite satisfactory performance as an indicator method, the amperometric biosensor failed toquantify the E.coli cells. Further optimization experiments of the amperometricbiosensor may be necessary for quantification. The amperometric biosensor with theuse of a sandwich assay evaluated in this study offered a reliable means ofquantification of the organism. Overall, the amperometric biosensor technology offeredan efficient means of detection, primarily due to its ease of use, low cost, rapid detectionand use of simple portable instrumentation. More refined versions of such systems canbe incorporated at various gaging stations to monitor the real-time coliformconcentrations.