Uropathogen Detection Using DNA Biosensors

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DESCRIPTION (provided by the applicant): Urinary tract infection is the most common urological disease in the United States and is a major cause of patient morbidity and health-care expenditure. This Bioengineering Research Partnership proposal involves development of a microelectromechanical system for the genotypic detection and species-specific identification of uropathogens within a time frame (5-10 minutes from sample collection to readout) that would enable point-of-care diagnosis and treatment. The focus of this proposal is to produce a self-contained microbial pathogen detection device and to examine its performance using clinical urine samples. Research at UCLA has provided two key technological advances that make development of a uropathogen sensor feasible. The first is microfluidics for sample processing. The second is an electrochemical microsensor which allows ultrasensitive detection of specific DNA-RNA or DNA-DNA hybridization events, without the need for target amplification. This project has been in development for over a year involving a multidisciplinary effort including leaders in the fields of microfluidics and microsensor technology, molecular microbiology, pediatrics and biomathematics. Specific Aim 1 describes how microfluidics studies will be applied to development of a crossflow filter for uropathogen concentration, micromixing for processing of uropathogen nucleic acids, and washing of the sensor surface. Specific Aim 2 involves fabrication of the microsensor array, development of a streptavidin self-assembled monolayer, and testing of oligonucleotide probes for electrochemical detection of uropathogen rRNA and mRNA on the microsensor surface. Specific Aim 3 will involve integration of the microfluidics and sensor components and testing of its analytic validity on simulated and actual urine specimens. Specific Aim 4 will involve batch fabrication of the device and clinical examination of the association between urosensor results and clinical correlates of urinary tract infection.