We have developed a high-throughput microfabricated, reusable glass chip for the functional integration of reverse transcription (RT) and polymerase chain reaction (PCR) in a continuous-flow mode. The chip allows for selection of the number of amplification cycles. A single microchannel network was etched that defines four distinct zones, one for RT and three for PCR (denaturation, annealing, extension). The zone temperatures were controlled by placing the chip over four heating blocks. Samples and reagents for RT and PCR were pumped continuously through appropriate access holes. Outlet channels were etched after cycles 20, 25, 30, 35, and 40 for product collection. The surface-to-volume ratio for the PCR channel is 57 mm(-1) and the channel depth is 55 microm, both of which allow very rapid heat transfer. As a result, we were able to collect PCR product after 30 amplification cycles in only 6 min. Products were collected in 0.2-mL tubes and analyzed by agarose gel electrophoresis and ethidium bromide staining. We studied DNA and RNA amplification as a function of cycle number. The effect of the number of the initial DNA and RNA input molecules was studied in the range of 2.5 x 10(6) - 1.6 x 10(8) and 6.2 x 10(6) - 2 x 10(8), respectively. Successful amplification of a single-copy gene (beta-globin) from human genomic DNA was carried out. Furthermore, PCR was performed on three samples of DNA of different lengths (each of 2-microL reaction volume) flowing simultaneously in the chip, and the products were collected after various numbers of cycles. Reverse transcription was also carried out on four RNA samples (0.7-microL reaction volume) flowing simultaneously in the chip, followed by PCR amplification. Finally, we have demonstrated the concept of manually pumped injection and transport of the reaction mixture in continuous-flow PCR for the rapid generation of amplification products with minimal instrumentation. To our knowledge, this is the first report of a monolithic microdevice that integrates continuous-flow RT and PCR with cycle number selection.

While performing routine electroosmotically driven CE separations on microfluidic chips, we have observed peak shape, migration time, and baseline drift anomalies. Pressure-driven backflow (opposing electroosmotic flow (EOF)) has been observed and characterized, and meniscus surface tension (Laplace pressure) is cited as the likely cause. However, there are a number of interdependent factors that affect bulk flow in a microchip environment, including evaporation, buffer depletion due to hydrolysis, EOF pumping, siphoning, viscosity changes due to Joule heating, and Laplace pressure. Given the complexity of such a system, pressure effects were isolated from EOF, and to some extent, siphoning effects were isolated from suspected meniscus effects. Pressure flow observed in the absence of an applied field ranged from 0.4 to 0.8 mm/s, which was on the order of the EOF generated experimentally, 0.6 mm/s at a field of 150 V/cm, and was some 10-20 times larger than what would be predicted merely from a difference in liquid levels (siphoning). Furthermore, experiments were performed without an electric field and with the chip tilted so that meniscus flow ran "uphill" against a siphoning backflow and showed siphoning flow to have a negligible effect upon meniscus flow under the microchip conditions studied. These findings are relevant to the profusion of microfluidic and array-based technology that also use microliter liquid volumes in like-sized reservoirs with similar menisci.