UC Berkeley

The emergence of micro-Total Analysis Systems has revolutionized the world of molecular diagnostics by enabling sensitive multi-step fluidic processes to be performed and reliably integrated in a single platform. In particular, microfluidic systems now provide the tools and components to enable quantitative detection of biomarkers relevant to pathogen identification and disease characterization. In this thesis, these advances are exploited to develop an integrated microfluidic platform for automated, rapid and sensitive genetic identification of infectious food-borne bacterial and respiratory viral pathogens.

My first goal was the integration of improved sample purification, preconcentration and injection technology with a polymerase chain reaction-capillary electrophoresis (PCR-CE) microdevice. By introducing an in-line affinity capture system utilizing an in situ photopolymerized oligonucleotide capture gel, double-stranded PCR amplicons generated in an integrated PCR reactor were selectively captured, purified and injected with 100% efficiency for high resolution CE separation. The superior performance of this integrated platform was demonstrated in a quantitative genetic analysis of E. coli. This integrated system exhibits a six- fold improvement in resolution of a multiplex analysis of Escherichia coli O157/E. coli K12 and is able to detect E. coli O157 in a 500-fold higher background of E. coli K12.

To enable the parallel detection of multiple infectious pathogens, an improved purification method relying on biotin-streptavidin interaction was developed for universal product capture. This technique has the advantage of eliminating the complications associated with designing sequence-specific oligonucleotide capture probes for multiple targets. This process was integrated into a new 4-unit array PCR-CE microchip designed for automated product amplification, capture, and analysis. Coupled with a portable laser-induced fluorescence rotary scanner, the system can simultaneously detect as few as ten copies per reactor of influenza A & B, human metapneumovirus (hMPV), and coronavirus samples from cloned plasmid standards within 2.5 hours. Furthermore, the ability of the system to process RNA samples was demonstrated by performing RT-PCR analyses of an influenza B/hMPV co-infection model case, with respective detection limits of 50 and 100 copies/reactor.

This thesis concludes with a discussion of proposed methods for nucleic acid isolation from biological samples that will provide a complete sample-in to answer-out diagnostic device and method for pathogen detection. When fully developed, this technology will be a significant advancement in infectious disease detection and surveillance both inside and outside clinical settings.