Development of an Integrated Molecular and Immunodiagnostic Paper Chip Technology Ji-Ho Park College of Natural Sciences

Abstract

Point-of-care testing (POCT) technologies that enable rapid, equipment-free diagnostics are essential for improving healthcare accessibility, particularly in resource-limited settings. As the demand for portable, user- friendly, and cost-effective diagnostic platforms continues to rise, the development of integrated POCT systems has gained increasing attention. Among the diagnostic methods considered suitable for POCT, isothermal nucleic acid amplification has emerged as a powerful alternative to conventional PCR. In particular, loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) have garnered significant interest due to their ability to rapidly amplify target nucleic acids under constant temperature conditions, eliminating the need for thermal cyclers. Alongside molecular diagnostics, immunoassays play an essential role in detecting disease-related proteins directly from biological samples. Among various immunoassay formats, lateral flow assays (LFAs) are especially attractive for POCT applications due to their rapid turnaround, equipment-free operation, and low manufacturing cost. To address their inherent sensitivity limitations, automated signal amplification approaches have been explored. To effectively implement both molecular and immunoassays in a compact and low-cost format, paper-based diagnostic platforms have drawn increasing attention. Paper allows for passive fluid transport and patternable surface properties, enabling the integration of complex biochemical workflows in a simplified and scalable structure without requiring external equipment. In Chapter 2, a USB-powered portable diagnostic device integrating LAMP and LFA was developed. The device was designed to carry out a 25-minute LAMP reaction, followed by the opening of a wax valve after approximately 5 minutes to allow fluid flow through the detection strip. A custom-printed circuit board (PCB) and heating module were fabricated to automate the entire process, enabling fully integrated amplification and colorimetric signal detection. Using this system, two major foodborne pathogens were successfully detected: Vibrio vulnificus at concentrations as low as 120 CFU per reaction and Salmonella Typhimurium at concentrations as low as 60 CFU per reaction. In Chapter 3, a portable diagnostic platform integrating recombinase polymerase amplification (RPA) with LFA was developed, powered by a 3.7V lithium-polymer battery. The device incorporates an automatic fluid- switching system based on water-swellable polymers, allowing fully automated assay progression upon two sequential solution injections at the beginning of the test. This enables nucleic acid-specific amplification, on-chip capture of amplified products, and visualization of the signal without intermediate step. Using this platform, SARS-CoV-2 was successfully detected with a sensitivity of 10 copies/µL within 30 minutes. In Chapter 4, to address the limitations of the RPA-based device described in Chapter 3, a LFA-integrated portable device was developed to reproduce benchtop-level performance. The system was designed to support liquid-phase RPA reactions and employed a vertical wax valve to enable automated fluid progression and signal visualization after amplification. The developed device demonstrated sensitive detection of SARS-CoV-2 down to 1 copy/µL. Furthermore, clinical validation was conducted using 20 respiratory infection patient samples. Among them, five healthy samples and five influenza A virus samples yielded negative results, while all ten SARS- CoV-2 patient samples tested positive, confirming high specificity and sensitivity of the system. In Chapter 5, a chemical signal amplification strategy was investigated to overcome the inherent sensitivity limitations of conventional LFA. Among various signal enhancement techniques, the in situ growth of gold nanoparticles via chemical reduction is widely adopted to intensify colorimetric signals. In this study, a novel automation approach was introduced by applying a surfactant-triggered delayed release mechanism using the hydrophobic properties of office paper. This system enabled automated signal enhancement to occur approximately 10 minutes after the initiation of the immunoassay, all triggered by a single solution drop. The developed LFA strip was applied to the detection of cardiac troponin I, a key biomarker for acute myocardial infarction, achieving a detection limit as low as 2.42 pg/mL—representing a 157-fold improvement in sensitivity compared to assays without enhancement. Furthermore, clinical validation using six patient samples demonstrated successful detection even in previously undetectable cases, highlighting the potential of this method to improve diagnostic outcomes in low-concentration clinical scenarios.