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628b

Droplet-Based PCR In a Valveless Microfluidic Device

Fang Wang and Mark A. Burns. Chemical Engineering, University of Michigan, 2300 Hayward, 3074 H.H.Dow, Ann Arbor, MI 48109

Polymerase chain reaction (PCR) is one of the most versatile molecular biological techniques, playing an indispensable role in many genetic analyses. Relying on microfabrication and microfluidic technologies, miniaturized PCR devices have been developed to achieve rapid amplification with reduced cost of reagents and samples. Certain issues such as evaporative loss and reagent adsorption, which stem from the characteristics of the thermocycled reaction and the micro format itself, have to be considered in designing and constructing the microdevices. Here we present a valveless microfluidic droplet-based PCR device that has no evaporative losses and has amplification efficiencies comparable to benchtop reactions. In this device, aqueous droplets containing the reaction solutions are encapsulated in a continuous oil phase. Single reaction droplets with a maximum volume of 200nL can be formed reproducibly within tens of seconds. With on-chip heaters and temperature sensors, PCR of λDNA has been successfully performed in less than half the time required for PCR in a thermocylcer. By using a FRET probe, the amplification can be detected in situ and in real time using fluorescent microscopy. The fluorescent intensity of the amplified products increases with the increase of template DNA concentration, and increases to a maximum value with the increase of cycle numbers. A higher polymerase concentration is still required in the droplet-based reaction to give on-chip amplifications comparable to benchtop results but the optimum Magnesium ion concentration is the same for both systems.