Sequence-specific electrochemical DNA detection and its implementation in integrated PCR-electrochemical microdevices

The miniaturization of DNA analytical systems onto silicon, glass, and plastic microdevices offers a number of advantages over the conventional bench-top instruments. These microdevices feature small sample and reagent consumption, short assay time, high analytical performance, low cost, and automation. Of particular significance is the possibility of integrating all DNA analytical steps onto self-contained microdevices. Potential applications of these portable systems include point-of-care genetic diagnostics. One obstacle to the realization of such a hand-held biosensor is on-chip sequence-specific DNA detection. In this regard, electrochemical method would be an ideal candidate. This thesis describes a systematic approach to develop an integrated microdevice for simultaneous DNA amplification and detection.; The approach starts with the sequence-specific (hybridization-based) detection of polymerase chain reaction (PCR, an enzymatic DNA amplification technique) product with a redox-active DNA intercalator. Moreover, the use of other indicators such as redox enzymes and gold nanoparticles for sensitive and selective hybridization transduction is demonstrated. Of particular significance is capability to perform these assays on microchip-compatible electrode surfaces.; Finally, the established detection schemes are implemented onto integrated PCR-electrochemical microdevices for trace DNA analysis. The device has a reaction chamber volume of 8 μL formed in a silicon substrate, which is sealed by bonding to a glass substrate. The glass substrate also functions as a detection platform where electrodes are patterned on its surface. The reaction chamber is thermal cycled by the heaters and temperature sensors integrated on top of the silicon substrate to amplify the target sequence by PCR. After the amplification, the patterned electrodes are utilized for the electrochemical sequence-specific detection of the amplicon. The ability to perform DNA amplification and detection simultaneously in a single reaction chamber is a great leap towards the realization of a truly integrated DNA analysis system.