Microfluidic system with open loop control for rapid infrared reverse transcription quantitative PCR (RT-qPCR)

Microfluidic techniques have allowed for fast, sensitive, and low cost applications of the Polymerase Chain Reaction (PCR) through the use of small reaction volumes, rapid amplification speeds, and the on-chip integration of upstream and downstream sample handling processes including purification and electrophoretic separation functionality. While such systems are capable of measuring the expression levels of thousands of genes simultaneously, or in hundreds of cells, or with sample-in and answer-out capability, none of these systems are easily scalable in the time domain. Because of this, the field of gene expression measurement has yet to fully utilize the advantages of microfluidic PCR in developing systems to measure changes in gene expression in increments of hours rather than days. In this project, we developed a microfluidic RT-qPCR system that utilizes infrared heating and open-loop control to reliably reverse transcribe, amplify, and detect samples in a single 1 μl polymer chip. Optimized power profiles were created that allow for fast heating and cooling rates while minimizing undershoot and overshoot from the desired hold temperatures. By utilizing repeatable microfluidic chip manufacturing techniques, and by controlling the environment around the chip, the same open loop program can repeatedly amplify multiple samples without any need for temperature feedback or recalibration between runs. Furthermore, the system was designed to operate on top of a fluorescence microscope to enable real-time fluorescence detection and quantification of starting copy number. By eliminating complicated setup procedures and calibration runs, this system increases the practicality of measuring gene expression at a high temporal frequency.M