Development of paper-based rapid PCR technology for point-of-care molecular diagnosis

Abstract

The worldwide incidence of deadly infectious diseases has increased in recent years. Rapid and accurate nucleic acid diagnostic systems are in high demand for early diagnosis and treatment to prevent the spread of infectious diseases. Polymerase chain reaction (PCR) is the standard method for amplifying and quantifying nucleic acid biomarkers for disease detection. Conventional PCR techniques require strict laboratory conditions that need to be relaxed for PCR-based molecular diagnostic devices used in point-of-care testing (POCT). Here, I demonstrated a fast and sensitive paper-based PCR technique that satisfies many POCT conditions, such as inexpensiveness, disposability, and simplicity. In chapter 2, by focusing on high-speed PCR detection, a rapid membrane-based photothermal PCR that enables quantitative fluorescence detection without the need for post-PCR procedures such as gel electrophoresis was developed. Gold (Au) nanostructures were deposited on the reaction pad that served as a light-to-heat converter for rapid PCR thermocycling. The automatically controlled light source heated the solution in the reaction pad with a photothermal effect. The temperature of the reaction solution, which was absorbed in a membrane, was cycled 25 times between 63–95 °C within 6 min using a photothermal cycler. The amplified DNA samples on the membrane were then quantitatively analyzed by measuring the fluorescence signal of the intercalating SYBR dye within 12 min. DNA samples extracted from Staphylococcus aureus and unpurified cells were successfully amplified and analyzed. In Chapter 3, I demonstrated a lateral flow paper chip integrated with a thin-film Joule heater for a POC PCR chip that enables fast heating and cooling cycles with minimal equipment and low power consumption and detects amplified products. A nichrome thin film was used as a Joule heater to achieve stable thermocycling by applying various voltages. After PCR, the amplified products with biotin and Cy5 labels at each end were directed to a detection membrane and captured at streptavidin spots. As a result, cDNA of severe acute respiratory syndrome β coronavirus (SARS-CoV-2) can be detected by measuring fluorescence intensities within 20 min after PCR solution injection, whereas conventional and real-time PCR techniques with bulky equipment took 105 min and 115 min, respectively. Finally, the PCR chip successfully detected SARS-CoV-2 RNA. This membrane-based PCR approach can provide easy connectivity with various membrane-based sample pre-treatment processes; therefore, the proposed procedure could be used as the basis for an ideal all-in-one molecular diagnosis platform. This inexpensive, disposable, simple, rapid, and sensitive PCR device could also serve as the basis for advanced molecular diagnostics suitable for POCT in both developing and developed countries.