Oxygen electrode-based single antibody amperometric biosensor for qualitative detection of E. coli and bacteria in water

Abstract

Design and performance of an amperometric biosensor for E. coli O157:H7 that is based on a common dissolved oxygen probe is discussed. Anti-E. coli O157:H7 antibody was conjugated to horseradish peroxidase and immobilized on a nitrocellulose membrane that was placed over the oxygen probe membrane using a custom-fabricated polyvinyl chloride (PVC) insert. Upon bacterial cell binding, a decrease in enzyme activity resulted in a change in oxygen concentration that was detected with a Clark-type oxygen electrode probe. Validation experiments determined the effect of the outer membrane and insert on the Clarke electrode performance and linearity, and the effects of stirring on sensor response. The mechanism of enzymatic disruption is presumably steric hindrance due to binding of the bacterial cell and conformational change in antibody structure. Sampling various dilutions of heat-sterilized E. coli O157:H7 cells in water, as little as 50 bacterial cells/mL could be detected in approximately 20 minutes of sampling and processing procedures. Bacterial concentrations from 0 to 5000 cells/mL were tested, with 2.52 mg/L ± 0.37 mg/L equivalents of oxygen produced from as few as 50 cells/mL, versus 6.26 ± 0.64 mg/L when no cells were present in solution. Overall, the developed amperometric biosensor technology offered an efficient means of detection primarily due to its ease of use, cost-effectiveness, portability, and amenability to incorporation at existing water quality gaging stations.

Keywords:

• Biosensor
• E. coli
• amperometric
• enzyme-antibody conjugate
• O157:H7

Acknowledgments

The authors wish to acknowledge the assistance offered by two undergraduate students, Susan Losby and April Bernard in the Department of Biological and Agricultural Engineering at LSU. The authors would also like to thank the Biological and Agricultural Engineering Department and the College of Agriculture for a graduate assistantship supporting DDS.