Development of a resistance based biosensor utilizing conducting microfibers for microbial pathogen detection.

摘要

Escherichia coli O157:H7 (E. coli O157:H7) is one of the U.S. military's top pathogens of interest for the development of rapid diagnostic systems. The enteric pathogen can cause severe gastroenteritis and is spread through the consumption of contaminated food and water. This is of concern to the U.S. military and warfighter because an outbreak of diarrheal disease in the field has the ability to rapidly render a large number of warfighters ineffective in performing their duties. Current field "portable" detection technologies can be cumbersome and require generous quantities of chemicals to operate. In addition, the current FDA gold standard for identification of this pathogen from food matrices takes up to 3 days to generate a confirmed positive result. The objective of this dissertation research was to develop a rapid, novel electrochemical biosensor based on the use of polypropylene microfiber membranes coated with a conductive polypyrrole and antibody functionalized for the biological capture and detection of E. coli O157:H7. In this dissertation research, an electrotextile composed of conductive polymer coated microfibers containing functional attachment sites for biorecognition elements was developed. The electrotextiles were optically and electrically assessed based on the polymerization chemicals and reaction time to determine how these factors affected the resistance of the fibers. Based on these experiments, a mathematical model was developed, optimized, and validated. Various methods of antibody immobilization and surface blocking on the fibers were also assessed. Using glutaraldehyde, pathogen specific antibodies were covalently attached to the conductive microfiber electrotextiles which were then blocked using a 5% bovine serum albumin solution. The functionalized membranes were exposed to E. coli O157:H7 cells, washed in Butterfield's phosphate buffer and added to a phosphate buffer electrolyte solution. When a voltage was applied to the system, the presence of the captured pathogen on the fiber surface resulted in an increase in resistance at the electrotextile electrode surface, indicating a positive result. It was found that the conductivity of the components of the system, other than the electrotextile fibers, was not statistically significant. Proof-of-concept experiments were conducted and it was determined that the electrotextile electrode was able to differentiate between positive and negative samples using the pathogen E. coli O157:H7 cells as the target over a concentration range of 100 -- 109 colony forming units per milliliter (CFU/mL). The reproducibility of the sensor results was tested and it was found that the trends in the biosensor results were reproducible. By testing the significance of the biosensor response it was determined that the biosensor can successfully function as a yes / no screening system. The results show that the biosensor has an experimental lower limit of detection of 3.23 x 100 CFU/mL for the detection of E. coli O157:H7 in pure culture.