Engineering a microfluidic DNA biosensor on nanostructured Ag surfaces

Thesis (Ph. D.)--University of Rochester. Dept. of Biomedical Engineering, 2011.The development of rapid and sensitive DNA biosensors continues to be an urgent endeavor in the biomedical research community, particularly since DNA molecules have become a prime target for medical diagnostics and biological studies. The success of these devices greatly hinges not only on their detection accuracy, but also on their inherent efficiency and multiplexing capability. My thesis begins by demonstrating the utility of a "transparent" nanostructured Ag substrate for the NanoLantern, which is a unique "switch-on" biosensor that lights up when a target DNA is detected and stays dark otherwise. As expected, Ag nanoparticles provided fluorescence quenching in the absence of target DNA, and moreover, fluorescence enhancement after probe-target hybridization, a result of nanoplasmonic effect. The systematic study of Ag nanoparticles for DNA detection allowed us to examine Ag nanoparticle stability over time and potential consequences for nanoparticle-enhanced DNA biosensing. Controverting the widely held perception that Ag aging under ambient conditions will degrade a device's performance, our studies showed that Ag aging actually improved our detection readout. Thirty days of Ag nanoparticle aging on a glass substrate yielded a dramatic morphological change. This change led to a 17-fold fluorescence enhancement. The final portion of my thesis work involved the implementation of DNA detection on nanostructured Ag substrates in a microfluidic system. This integration permitted a real-time and rapid detection with a dynamic range spanning from 25 nM to 25 µM. A detection time as low as 8 min was sufficient to produce a significant detection signal. Our subsequent efforts further established this device's capability for multiplex detection by arraying multiple probe sequences on the same substrate. Though this microfluidic DNA detection system is still at its early developmental stage, its inherent multiplexing capability and the rapid detection time shows great promise for high-throughput label-free DNA detection. Moreover, we believe the findings described in this thesis will be beneficial for the design of microfluidic DNA biosensors in the biomedical research community and lead to great advances in point-of-care (POC) diagnostics