Rapid real-time PCR and high resolution melt analysis in a self-filling thermoplastic chip

Abstract

A microfluidic platform designed for point-of-care PCR-based nucleic acid diagnostics is described. Compared to established microfluidic PCR technologies, the system is unique in its ability to achieve exceptionally rapid PCR amplification in a low cost thermoplastic format, together with high temperature accuracy enabling effective validation of reaction product by high resolution melt analysis performed in the same chamber as PCR. In addition, the system employs capillary pumping for automated loading of sample into the reaction chamber, combined with an integrated hydrophilic valve for precise self-metering of sample volumes into the device. Using the microfluidic system to target a mutation in the G6PC gene, efficient PCR from human genomic DNA template is achieved with cycle times as low as 14 s, full amplification in 8.5 min, and final melt analysis accurately identifying the desired amplicon.

Capillary microfluidics in microchannels: from microfluidic networks to capillaric circuits

“…These heating steps were carried out either with Peltier heaters placed underneath the microchip 116,117 or with thin film heaters integrated directly into the CC. 119 CCs used for DNA amplification have so far been fabricated in hydrophobic materials like PDMS and cyclic olefin copolymer (COC). As an alternative to surface treatment to make the microchip hydrophilic, surfactant was added to the reagents to lower the surface tension, the contact angle, and resulting in capillary-driven flow of PCR reagents in the CC.…”

“…CCs were also used to perform on-chip DNA amplification by PCR. [116][117][118][119] PCR typically requires incubation of samples at three separate temperatures, ≈95°C for DNA denaturation, ≈55°C for primer annealing to denote which DNA segments to amplify, and ≈72°C for enzymatic DNA extension. These heating steps were carried out either with Peltier heaters placed underneath the microchip 116,117 or with thin film heaters integrated directly into the CC.…”

“…As an alternative to surface treatment to make the microchip hydrophilic, surfactant was added to the reagents to lower the surface tension, the contact angle, and resulting in capillary-driven flow of PCR reagents in the CC. 116,118,119 Numerical models were also developed to characterize the effect of surfactant addition and the effect of heating on capillary driven flow of the PCR mixture 120 in the CC. 117 These numerical models guided the implementation of PCR in CCs.…”

“…Meanwhile, the DNA amplicon can be identified after PCR by performing a melting curve analysis. 119 PCR with real time monitoring of DNA amplification, using a fluorescent intercalating dye, was completed in 8.5 min. Most of the demonstrations of PCR in CCs so far use relatively simple channel designs, and do not yet leverage advanced liquid handling capabilities available with capillaric elements.…”

A rapid microfluidic platform with real-time fluorescence detection system for molecular diagnosis

“…Meanwhile, the inherent characteristics of microfluidic chips, such as usability, affordability and portability, can replace the conventional laboratory testing [13,14]. Particularly, when compared to macro-scale testing, it holds great preponderance in molecular diagnosis, such as a lower reagent consumption and shorter analysis time, all of which enable the fluorescence PCR technique to undergo a greater development in future [15,16].…”