• Bateson, Simon
• Ali, Zulfiqur
• Carney, Helen
• Salman, Abbas

Abstract

Polymerase chain reaction (PCR) is commonly used for the analysis of nucleic acids in a variety of applications including clinical. There is, however, a need for a low cost portable PCR device that allows rapid identification of pathogenic bacteria. We report a shunting PCR microfluidic device comprising: polycarbonate microfluidic PCR chip; shunting thermal cycler and fluorescence detector. The microfluidic PCR chip – fabricated using micro-milling and thermal fusion bonding for sealing of the cover – was shunted between three double side temperature zones for thermal cycling. Rapid amplification was observed with heating and cooling rates of 1.8 °C/s and 2 °C/s respectively. Lock-in photodetector for fluorescence detection of the microfluidic PCR chip achieved at 95% confidence an LOD of 75pM FITC and 0.7 ng μl−1 of dsDNA using a QuantiFluor assay kit. The device was validated using universal primers - based on chromosomal DNA extracted from non-pathogenic K-12 subtype of Escherichia coli (E. coli) – for amplification of fragments of 250, 552 and 1500 bp. PCR amplification was demonstrated, with annealing temperatures ranging between 54 °C and 68 °C, and confirmed using gel electrophoresis. The developed shunting PCR microfluidic device will allow for low cost and portable nucleic acid amplification for the detection of infectious diseases.

Topics

• impedance spectroscopy
• grinding
• milling
• annealing