期刊
JOURNAL OF MICROMECHANICS AND MICROENGINEERING
卷 20, 期 5, 页码 -出版社
IOP PUBLISHING LTD
DOI: 10.1088/0960-1317/20/5/055003
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资金
- National Science Foundation (NSF) [EPS-0346411, EPS 0701491/LEQSF(2007-10)-CyberRII-02]
- State of Louisiana from the Board of Regents Support Fund, Industrial Ties Program [LEQSF(2005-08)-RD-B-04]
A high throughput, multi-well (96) polymerase chain reaction (PCR) platform, based on a continuous flow (CF) mode of operation, was developed. Each CFPCR device was confined to a footprint of 8 x 8 mm(2), matching the footprint of a well on a standard micro-titer plate. While several CFPCR devices have been demonstrated, this is the first example of a high-throughput multi-well continuous flow thermal reactor configuration. Verification of the feasibility of the multi-well CFPCR device was carried out at each stage of development from manufacturing to demonstrating sample amplification. The multi-well CFPCR devices were fabricated by micro-replication in polymers, polycarbonate to accommodate the peak temperatures during thermal cycling in this case, using double-sided hot embossing. One side of the substrate contained the thermal reactors and the opposite side was patterned with structures to enhance thermal isolation of the closely packed constant temperature zones. A 99 bp target from a lambda-DNA template was successfully amplified in a prototype multi-well CFPCR device with a total reaction time as low as similar to 5 min at a flow velocity of 3 mm s(-1) (15.3 s cycle(-1)) and a relatively low amplification efficiency compared to a bench-top thermal cycler for a 20-cycle device; reducing the flow velocity to 1 mm s(-1) (46.2 s cycle(-1)) gave a seven-fold improvement in amplification efficiency. Amplification efficiencies increased at all flow velocities for 25-cycle devices with the same configuration.
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