| Microchip electrophoresis and microchip-based sample preparation are growing fields of study. Multi-step genomic analysis on a single device, with a sample in-answer out functionality, will soon be a reality. However, before such analyses become commonplace, issues such as surface passivation, choice of microchip substrate, integration of chemistries and functionalities, and reduction of total analysis time need to be addressed. Towards this end, our laboratory has demonstrated the first integrated DNA purification, amplification, and separation microchip, for the analysis of diagnostically-significant samples. The primary focus of this work has been in developing infrared (IR)-mediated thermocycling for the polymerase chain reaction (PCR)-based amplification of DNA. Infrared light is used to excite vibrational bands of water to heat nanoliter volumes of reaction mixtures on glass and plastic chips. With on-chip IR-mediated thermocycling achieved, incorporating DNA size-based separations on the same device increased microchip complexity further, while reducing the total analysis time significantly over conventional amplification and separation methods.; Efforts were not limited to the analysis of DNA, but also addressed a growing concern in the microchip field---the on-chip detection of proteins. In the absence of sensitive and reliable ultraviolet-absorbance detection for microchips, it is necessary to covalently-modify proteins with dyes for laser-induced fluorescent (LIF) detection---with conventional methods, this is time consuming and unreliable. Initially, a dynamic labeling mechanism was developed, where a fluorescent dye was included in the separation buffer for protein sizing. During electrophoresis, dye bound to protein-SDS complexes and underwent a conformational change, which allowed it to fluoresce maximally at 590 nm when excited by 488 nm laser light. The second mechanism took advantage of conductivity differences between a sample zone and the background electrolyte to generate localized elevated temperatures within the capillary. In these "heated zones" proteins unfold, exposing more surface area for dye binding, and enhancing detection limits by nearly an order-of-magnitude when heating was not used. |
