Design and experimental validation of a miniature real-time polymerase chain reaction device using disposable microfluidic chips

Abstract

This thesis evaluates the use of inexpensive, disposable, microfluidic chips to detect methicillin-resistant Staphylococcus aureus (MRSA) that has undergone three forms of sample preparation: one containing purified genomic DNA, another containing the supernatant of a crude preparation using simple reagents, and a third through boiled culture preparation without any additional reagents. Polydimethylsiloxane (PDMS) microfluidic chips were fabricated using soft lithography and then bonded to a 22 ~ 22 ~ 0.1 mm glass cover slip. A lid fabricated in a similar manner was used during compression to prevent bubble formation and evaporation in the stationary well-based chip. A miniature thermal cycler based on a thin resistive heater and a small fan were used to cycle through desired temperatures for polymerase chain reaction (PCR) and fluorescent intensity measurements were taken at each cycle. Each form of template provided positive results utilizing the developed micro-PCR system (verified with gel electrophoresis). A serial dilution of the purified genomic DNA provided a standard curve with an efficiency of 1.77. The lowest concentration that provided clear positive results came from a 3 ƒÊl sample containing 11.2 pg of DNA. This is a significant step towards the development of a point-of-care detection system capable of identifying infectious organisms such as MRSA with little or no pre-PCR manipulations.