Biochemical reactions in integrated microfluidic DNA analysis devices.

Abstract

Microfluidics based DNA analyses devices are receiving considerable attention for medical diagnostics and high throughput biochemical analyses. The construction of these devices involves the development of individual miniaturized components and their successful integration into a self-contained system. A polymerase chain reaction and a restriction digest reaction are miniaturized and integrated in a microfabricated device that is capable of performing a variety of genetic analyses. The microfluidic device integrates fluidic and thermal components such as heaters, temperature sensors, and addressable valves to control the PCR and the restriction digest reaction in series followed by an electrophoresis separation. Influenza viral strain (A/LA/1/87 and A/Sydney/5/97) subtyping has been demonstrated in the device. Three thermal isolation techniques have been investigated in a microfabricated DNA analysis device. The thermal conduit technique is based on a selective conduction mechanism, while both the silicon back dicing and back etching techniques are based on a selective insulation mechanism. The performances of the three techniques are compared both numerically and experimentally. Temperature gradients as low as 108°C/cm, 92°C/cm and 158°C/cm can be achieved by the three techniques, respectively. Geometric optimization of the proposed techniques is carried out to further improve their thermal isolation performances. A thermal isolation based microfabricated PCR device that eliminates the use of microvalves for the prevention of evaporative sample loss during thermocycling has been developed. The device uses a diffusion-limited evaporation mechanism to reduce the evaporation rate of the PCR solution during thermocycling. The silicon back etching thermal isolation technique is used to reduce the sample interfacial temperature and facilitate the reduction of evaporation. PCR from Influenza A/LA/1/87 strain has been successfully demonstrated in the device for 18 minutes without microvalves. The evaporative loss has been limited to less than 1% of the reaction volume. A second valveless PCR device that uses diffusion-limited evaporation and on-chip vapor replenishment has been demonstrated. Vapor is generated by temperature-controlled evaporation at pinned water meniscuses and replenished to the diffusion channels to increase local vapor concentration and thus reduce the evaporation rate during thermocycling. PCR from Influenza A/LA/1/87 strain has been successfully demonstrated in the device for 20 minutes without microvalves. The evaporative loss has been limited to less than 5% of the reaction volume.