Portable PCR system with point-of-care (POC) monitoring using electrochemical impedance spectroscopy (EIS)

Abstract

Polymerase chain reaction (PCR) is the most widely used method for detecting pathogens such as viruses or bacteria because of its high sensitivity and high selectivity. Conventional PCR systems have limitations in that they can only be used in a laboratory environment because of their large size and high power consumption. However, as the need for rapid detection of pathogens in the field is rapidly increasing, studies on portable PCR systems have been actively carried out. This dissertation reports the portable PCR system with point-of-care (POC) monitoring using electrochemical impedance spectroscopy (EIS). The portable PCR system was developed to consume less power and measure the precise temperature by improving the stability of resistance temperature detectors (RTDs). A thin-film RTD made of non-annealed Pt shows accuracy degradation because the resistance of the RTD tends to decrease during the PCR operation. Thus, the annealing process is applied to the Pt RTD to improve the stability, which is a prerequisite to the accurate measurement of the absolute temperature. The stability of the RTD is experimentally confirmed in terms of resistance variation over repeated PCR operations (four times). The least variation of 0.005%, which corresponds to a negligible temperature variation of 0.038 °C for the PCR, is achieved from the RTD annealed for 5 min at 450 C. The gel-electrophoresis result indicates that the PCR performance of the proposed system using a film-type PCR chip is comparable to that of a conventional system using a vial tube despite its low power consumption. For real-time PCR monitoring, the dissertation proposes a technique for detecting the presence of DNA in real-time PCR using label-free electrochemical impedance spectroscopy. The change in the real and imaginary parts of the impedance with the number of PCR cycles indicates the concentration of amplified DNA. In general, the real part of the impedance tends to increase with the number of PCR cycles over most of the measurement frequency range (100 Hz to 1 MHz), whereas the imaginary part of the impedance increases in the negative direction. The optimal frequency for impedance analysis is determined by the performance index (PI), as one of the figures of merit, which quantitatively indicates how monotonously the impedance changes with the PCR cycle number. The detectability of DNA in the sample is scrutinized by analyzing the impedance at the optimal frequency (3.984 kHz and 20.02 kHz for the real and imaginary parts of the impedance, respectively) obtained from the performance index. The proposed technique can be used to quantitatively analyze PCR by applying simple electrochemical impedance spectroscopy (EIS) technology so that a label-free real-time monitoring system has high potential for a portable PCR system.