High sensitivity PCR assay in plastic micro reactors

Abstract

Small volume operation and rapid thermal cycling have been subjects of numerous reports in micro reactor chip development. Sensitivity aspects of the micro PCR reactor have not been studied in detail, however, despite the fact that detection of rare targets or trace genomic material from clinical and/or environmental samples has been a great challenge for microfluidic devices. In this study, a serpentine shaped thin (0.75 mm) polycarbonate plastic PCR micro reactor was designed, constructed, and tested for not only its rapid operation and efficiency, but also its detection sensitivity and specificity, in amplification of Escherichia coli (E. coli) K12-specific gene fragment. At a template concentration as low as 10 E. coli cells (equivalent to 50 fg genomic DNA), a K12-specific gene product (221 bp) was adequately amplified with a total of 30 cycles in 30 min. Sensitivity of the PCR micro reactor was demonstrated with its ability to amplify K12-specific gene from 10 cells in the presence of 2% blood. Specificity of the polycarbonate PCR micro reactor was also proven through multiplex PCR and/or amplification of different pathogen-specific genes. This is, to our knowledge, the first systematic study of assay sensitivity and specificity performed in plastic, disposable micro PCR devices.

A disposable microfluidic cassette for DNA amplification and detection

“…The detection of the nucleic acid material is either facilitated by capillary electrophoresis of the PCR products 3,10,11 or by hybridization of the PCR products with a fluorescent dye. 8,12,13 For example, Lagally et al 11 reported an amplification and electrophoresis device comprised of an integrated glass structure that achieved detection of 2 copies of a target DNA template. Yang et al 13 described a plasticbased microfluidic device integrating PCR and fluorescence detection in which DNA template, derived from samples of 10 E. coli cells, were detected.…”

“…8,12,13 For example, Lagally et al 11 reported an amplification and electrophoresis device comprised of an integrated glass structure that achieved detection of 2 copies of a target DNA template. Yang et al 13 described a plasticbased microfluidic device integrating PCR and fluorescence detection in which DNA template, derived from samples of 10 E. coli cells, were detected. More recently, developments in nanotechnology have led to several novel, particle-based, quantitative, high sensitivity bioassays.…”

“…[1][2][3][4][5][6][7][8][9][10][11][12][13] Microfluidic devices, which include miniaturized PCR thermal cyclers, conduits, chambers, and microvalves, have been fabricated in PDMS, 1 silicon, 3,4,12 glass, 5,10,11 silicon-glass combinations, 6,12 ceramics, 7 and various plastic materials. 2,8,9,13 Low-cost, disposable, plasticbased devices are of particular interest. A few of these devices include a means of detecting the nucleic acid amplicons directly integrated with the PCR thermal cycler reaction chamber.…”

A Practical Liquid Plug Flow-through Polymerase Chain-Reaction System Based on a Heat-Resistant Resin Chip

“…In contrast, polymer resins are inexpensive, and thus disposable. [14][15][16][17] The use of polymers as PCR materials requires that they a) have high thermal stability, with a glass-transition temperature (Tg) above 95 C; b) be resistant to the chemicals used in PCR; c) be able to contain the fluids and prevent evaporation; d) have good dimensional stability (not undergo expansion or contraction); and e) inhibit nonspecific adsorption of PCR reagents on the surface of the microchannels. Furthermore, it is important that the enclosed structure of the microchannels can be made by throughput production and fabrication precision for practical applications.…”

Electrochemical Immunosensing of Cortisol in an Automated Microfluidic System Towards Point-of-Care Applications

“…With the need for point of care systems gaining ground, microreactor technology has found good application in biosensing and PCR based systems, which require highly controlled environments in a microchamber [163]. Microreactors require the integration of multiple sensing and actuation components such as micromixers, microextractors, microheaters, microsensors for temperature, pH, humidity and redox measurements and many more depending on the demands of the application [164].…”