Rapid Prototyping of a Continuous-Flow PCR Microchip

Niel Crews**, Carl Wittwer*, Luming Zhou*, and Bruce Gale**
** Department of Mechanical Engineering, University of Utah, UT
* Department of Pathology, University of Utah, UT

 Keywords:  DNA, PCR, continuous-flow, rapid prototyping, thermal cycling

 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 1 hour.  Using these PCR chips, it has been shown that thermal cycles of approximately 12 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 conventional PCR equipment. 

 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 [4], which had not previously been achieved.  The chips presented as part of this current work are made using the “Xurographic” process [5], in which channels are cut into ultra-thin polyester (PET) films pre-coated with an adhesive.  The patterned films are then sandwiched between glass blanks and cured for 15 minutes. Although these chips are considered disposable, more than 20 PCR samples can be amplified on a chip before failure. 

 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 400 seconds, almost 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.  Figure 1 shows an approximation of the thermal cycling through which the sample passes.  The amplicon obtained was of a high purity and concentration in comparison against an identical sample amplified on the commercial equipment.  Figure 2 shows a fluorescent image of a gel electrophoresis in which samples from both were analyzed simultaneously. 

 Reference

 [1]        Kopp, M.U., de Mello, A.J., and Manz, A., 1998, Science, 280 (15 May), pp. 1046-1048.

 [2]        Obeid, P.J., et al., 2003, Analytical Chemistry, 75 (2), pp. 288-295

 [3]        Hashimoto, M., et al., 2004, Lab on a Chip, 4, pp. 638-645

 [4]        Lin, C.H., et al., 2001, Journal of Micromechanical Microengineering, 11, pp. 726-732.

 [5]        Bartholomeusz, D. A.; Boutte, R. W.; Andrade, J. D., 2005, JMEMS, 14 (6), pp. 1364-1374.