Indian Journal of Science and Technology

Abstract

Conventional Polymerase Chain Reaction (PCR) has been used widely in the clinical diagnostic and environmental monitoring where it still possesses larger volume and thermal mass leading to a slower PCR amplification for ultimate target detection. Microchamber stationary PCR is another alternative of conventional bench-top PCR in principle where the reaction chamber is cycled between two to three temperatures and a critical optimization of the system thermal mass is obvious to attain optimum reaction times and power consumption. Whereas our continuous flow through PCR system is independent of system thermal mass but depends mostly on the flow rates of PCR mixture in a microchannel for optimum target amplification along with the prevention of sample evaporation. In this report, a Polydimethylsiloxane (PDMS)- glass based two temperatures PCR microfluidic module was fabricated by soft-lithography and the Bacillus Anthracis target specific amplicon was detected using a disposable electrode instantly within 23 min with the detection limit of 106 copies/mL. Electrochemical sensing was done by Linear Sweep Voltammetry (LSV) and Hoechst 33258 [H33258, 2'-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi(1H-benzimidazole)] - a DNA minor groove aggregating, redox active species - was used as electrochemical reporter. LSV was performed directly on microfluidics post-PCR products without further purification or immbolization within 60s of PCR completion. DNA-Hoechst 33258 aggregation due tominor groove binding led to a significant drop in the peak oxidation current intensity of redox molecule. We believe our sensor has the potential to integrate into the multiplexed, real-time microfluidics reusable PCR electrochemcial platform with further improvement in the future.

Keywords: Biosensor, Hoechst 33258, PCR