Monolithic integrated microfluidic DNA amplification and capillary electrophoresis analysis system

A monolithic integrated DNA analysis system comprised of microfluidic valves and vents, PCR amplification chambers, and capillary electrophoretic separation channels has been microfabricated in a glass sandwich structure. Valves and hydrophobic vents provide controlled and sensorless sample loading into 280 nl PCR chambers; the low volume reactor and the use of thin film heaters permit thermal cycle times as fast as 30 s. The amplified product is labeled with an intercalating fluorescent dye and directly injected into a microfabricated capillary electrophoresis channel. Analyses with this device have produced and detected PCR products from reactions with as few as 20 starting DNA template copies/μl, (five to six copies/chamber). The extrapolated limit of detection based on data using 20 cycles is two copies/chamber. The optimization of heater placement, thermal anisotropy measurements, and the optimization of thermal profiles are also discussed.