The aim of this proposal is to establish the feasibility of a biosensor for detecting nucleic acid hybridization to be used in conjunction with polymerase chain reaction (PCR) of target nucleic acid base sequences from clinical samples suspected of containing pathogenic organisms or viruses. The biosensor has potentially many advantages over current methods of hybridization, including lack of chemical or radioactive labels, quantitation of product, and rapid hybridization reactions. A disposable sensing element now appear feasible. Phase I goals are to: (a) design and construct an automated hybridization cell for the biosensor; (b) write and test software for control of sensor cell and data collection; (c ) prepare more consistent biosensor surfaces using pulsed plasma polymerization; (d) increase signal to noise ratio by decreasing nonspecific binding; (e) develop a capillary, nested PCR procedure for the Shiga-like toxin genes in E. coli 0157:H7; and (f) evaluate the automated biosensor's potential to provide sensitive and very rapid pathogen detection using E. coli 0157:H7 as an example. The Phase II objectives are to construct a commercial prototype instrument that can accommodate multiple sensors, to explore larger scale production of sensing elements, to target human pathogens where the need for improved tests are greatest, and to begin biosensor evaluation with a medical diagnostic laboratory partner.
