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Rapid Prototyping of a Continuous-Flow Pcr Microchip

Niel Crews, Mechanical Engineering, University of Utah, Salt Lake City, UT 84123, Carl Wittwer, Pathology, University of Utah, Salt Lake City, UT 84123, and Bruce Gale, Department of Mechanical Engineering,, University of Utah, Salt Lake City, UT 84123.

A continuous-flow PCR microfluidic device has been developed using Xurography, a recently published microfluidic rapid prototyping technology. Fabrication of the chips can be performed in less than 45 minutes. Using these PCR chips, it has been shown that amplification cycles (denaturation, annealing, extension) of approximately 10 seconds produce PCR amplicon with high purity and concentration. PCR is now being achieved “from scratch” – which includes both the chip fabrication and a 22-cycle amplification – in less time than a typical PCR process would require on some conventional PCR equipment. In addition to this feature, the temperatures required for the annealing, extension, and denaturing of the PCR sample are generated by a single heating element. This proprietary technology is able to generate a variety of temperatures from a source held at a single steady-state temperature.

PCR is performed by cycling a prepared mixture through a series of temperatures at which specific reactions occur. It is customary to design continuous-flow PCR chips around these three distinct thermal zones. Multiple heaters, either integrated or off-chip, maintain the zones at the appropriate temperatures. Since the device presented here operates with a single heat source, high quality PCR can be performed with greater experimental simplicity.

Each of these new devices consists of a glass chip fitted with fluidic interconnects, a heating element, and a base to hold the heater/chip assembly. Traditionally, an extensive amount of time is required to fabricate glass microfluidic chips. Using microfabrication technology, Lin et al. report that their group was able to fabricate an entire microfluidic chip in just less than 10 hours, which had not previously been achieved. The chips presented as part of this current work are made using the “Xurographic” process, in which channels are cut into ultra-thin polyester (PET) films pre-coated with an adhesive. The patterned films are then sandwiched between glass or plastic blanks and cured for 15 minutes. A single thermal gradient chip can be fabricated in less than 45 minutes (including curing time). Although these chips are considered disposable, more than 20 PCR samples can be amplified before the chips fail.

To show that these devices are capable of amplifying DNA with high efficiency, a 75-bp segment of the Cystic Fibrosis gene exon 11 was amplified on both the PCR chip and an ultra-fast commercial thermocycler (LightCycler®, Roche, Indianapolis, IN). A 22-cycle amplification on the chip was performed in approximately 200 seconds, less than half the time required by the LightCycler®. The cycle time for the chip includes the passing of the sample through a long initial melt, a long final extension, and the dead volumes associated with the inlet and outlet ports. The amplicon obtained was of a high purity and concentration, as was is demonstrated by comparison against an identical sample amplified on the commercial equipment.