Phage-coupled Piezoelectric Biodetector for Salmonella typhimurium
Type of DegreeDissertation
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Salmonella typhimurium is a leading cause of foodborne illness and a critical threat agent for potential bioterrorism. Current rapid detection initiatives include biosensors that routinely incorporate antibodies for biorecognition. However, antibodies are costly and may degrade under unfavorable environmental conditions. A stable, inexpensive substitute may be filamentous bacteriophage affinity selected from a phage display library for specificity to S. typhimurium. We immobilized affinity-selected phage to a quartz crystal microbalance for detection of S.typhimurium in solution. An ELISA procedure, precipitation assay, and flow cytometry were employed to confirm phage specificity and selectivity. The phage was up to 22,000 times more specific for S. typhimurium than controls and up to 1,000 times more selective in comparison to other bacteria. For recognition of the phage targeted bacterial outer membrane structure, biotinylated S. typhimurium surface proteins from lysate were reacted with phage crosslinked to water-soluble resin to prepare a protein eluate for Western blot, which revealed a single 60-70 kD band. Three immobilization methods (physical adsorption, biotinstreptavidin-phage self-assembly, and Langmuir-Blodgett) using two phage forms(filamentous and phage coat proteins) were evaluated for proof of concept sensor preparation. Specific binding between phage and target on the biosensor resulted in concentration dependent resonance frequency changes. Best results were obtained when 10-10 to 10-11 filamentous phage particles converted to spherical forms (spheroids) by chloroform denaturation were immobilized as phage coat proteins using Langmuir-Blodgett technique. The sensors had an average rapid response time of <180 s, lower threshold of 10 to 10-2 cells/ml, linear response from 10 to 10-7 cells/ml, and a sensitivity of 2 Hz/mV per order of magnitude of bacterial concentration. Fluorescent, optical, and scanning electron microscopy confirmed binding of bacteria to filamentous phage-coated sensors, while transmission electron microscopy verified spheroid-bacteria binding in solution. In summary, filamentous phage selected from a phage library can be used for the preparation of rapid, specific, and selective biodetectors that may ultimately be suitable for continuous food and environmental monitoring devices, diagnostic assays, and biosorbents.