Spinning Disk Platform for Microfluidic Digital Polymerase Chain Reaction

University of Utah, Rm 5R441, 1795 E South Campus Dr., Salt Lake City, Utah 84112, USA.
Analytical Chemistry (Impact Factor: 5.64). 02/2010; 82(4):1546-50. DOI: 10.1021/ac902398c
Source: PubMed


An inexpensive plastic disk disposable was designed for digital polymerase chain reaction (PCR) applications with a microfluidic architecture that passively compartmentalizes a sample into 1000 nanoliter-sized wells by centrifugation. Well volumes of 33 nL were attained with a 16% volume coefficient of variation (CV). A rapid air thermocycler with aggregate real-time fluorescence detection was used, achieving PCR cycle times of 33 s and 94% PCR efficiency, with a melting curve to validate product specificity. A CCD camera acquired a fluorescent image of the disk following PCR, and the well intensity frequency distribution and Poisson distribution statistics were used to count the positive wells on the disk to determine the number of template molecules amplified. A 300 bp plasmid DNA product was amplified within the disk and analyzed in 50 min with 58-1000 wells containing plasmid template. Target concentrations measured by the spinning disk platform were 3 times less than that predicted by absorbance measurements. The spinning disk platform reduces disposable cost, instrument complexity, and thermocycling time compared to other current digital PCR platforms.

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    • "Implementing thin polymeric discs enable the researchers to design PCR systems with high temperature ramping rates. Various polymers such as polycarbonate (PC) [10], Poly methyl methacrylate (PMMA) [11], and Cyclic Olefin Copolymer (COP) [12] have been utilized for fabrication of such discs using different micro-fabrication technologies such as CNC milling and soft lithography. "

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    • "Therefore, the utility of U-dHRM in diagnostic and research applications will be improved by future work to increase the number and reduce the volumes of digital reactions, leading to improved resolution in the presence of contaminants, higher content, higher throughput and reduced reagent costs. High-throughput microfluidic digital droplet technologies (8,43–46) that incorporate simultaneous highly controlled heating and sensitive fluorescence detection for millions of reactions are needed. In a real heterogeneous sample where unknown sequences are expected and starting concentrations of targets may be unknown, millions of broad-based U-dHRM reactions will ensure enough dynamic range for successful single molecule detections (Table 3). "
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