Abstract
We report the successful application of low-power (approximately 30 mW) laser radiation as an optical heating source for high-speed real-time polymerase chain reaction (PCR) amplification of DNA in nanoliter droplets dispersed in an oil phase. Light provides the heating, temperature measurement, and Taqman real-time readout in nanoliter droplets on a disposable plastic substrate. A selective heating scheme using an infrared laser appears ideal for driving PCR because it heats only the droplet, not the oil or plastic substrate, providing fast heating and completing the 40 cycles of PCR in 370 seconds. No microheaters or microfluidic circuitry were deposited on the substrate, and PCR was performed in one droplet without affecting neighboring droplets. The assay performance was quantitative and its amplification efficiency was comparable to that of a commercial instrument.
Highlights
Recent advances in optical microfluidics have demonstrated the practical applications of how light can control a liquid sample in the same way as the fluidic circuitry [1,2,3,4]
A cylindrical well 6 mm in height and 20 mm in diameter machined in a plastic substrate was placed on top of a Hybrislip (HS22, Grace Bio-labs), which in turn rested on the conductive glass slide. (The Hybrislip, a singleuse hydrophobic cover that replaces the cover slip, is a 0.25-mm-thick square plastic piece made of polycarbonate for rigidity and temperature resistance.) A silicon O-ring was inserted, forming the wall of the chamber to keep the oil phase optically flat by geometrically forcing a smooth interface between the O-ring and the oil phase
A simple computer code was written to determine the cycle of threshold (Ct) when the increase of the averaged fluorescence intensity surpassed the standard deviation computed from all the previous cycles
Summary
Recent advances in optical microfluidics have demonstrated the practical applications of how light can control a liquid sample in the same way as the fluidic circuitry [1,2,3,4]. With laser heating operating on a droplet, a convenient reaction chamber unit for miniaturized assay systems [5,6,7], many optical fluidic control blocks have been simulated. The same infrared laser that we used previously for droplet movement and mixing [3, 8] can be used to drive the heating cycles of PCR on the same platform. Another complementary advantage of the droplet approach is that the size of the droplet is matched to the short extinction length of the infrared laser in water, naturally following the trend of miniaturization while optimizing efficiency. The optical heating method seems to be one of the promising candidates for dramatic improvement of the scalability of this technology
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