This thesis presents the development and evaluation of innovative methods and materials for real-time polymerase chain reaction (real-time PCR) in microfluidic chips, aiming to enhance nucleic acid testing in both hospitals and laboratories usage. The research is structured around three interconnected studies that address different aspects of real-time PCR technology.

The first study tackles the challenge of PCR reagent delivery and storage by developing the formulations for lyophilizing mix containing all necessary components for real-time PCR, so called all-in-one real-time PCR reagent, eliminating the need for cold chain during storage and transportation. The lyophilized mix was formulated with polyhydroxy compounds to protect enzyme and primer’s function and provide the dried-cake s...[ Read more ]

This thesis presents the development and evaluation of innovative methods and materials for real-time polymerase chain reaction (real-time PCR) in microfluidic chips, aiming to enhance nucleic acid testing in both hospitals and laboratories usage. The research is structured around three interconnected studies that address different aspects of real-time PCR technology.

The first study tackles the challenge of PCR reagent delivery and storage by developing the formulations for lyophilizing mix containing all necessary components for real-time PCR, so called all-in-one real-time PCR reagent, eliminating the need for cold chain during storage and transportation. The lyophilized mix was formulated with polyhydroxy compounds to protect enzyme and primer’s function and provide the dried-cake structure. The optimal formulation, consisting of trehalose, Ficoll 400, and type-B gelatin, demonstrated remarkable stability at various temperatures. The final product maintained its functionality and sensitivity after 300 days of storage at 45 °C, suggesting that it could be fully functional at room temperature (22.5−25.5 °C) for approximately two years.

In the second study, after developing a room-temperature-stable reagent before, I turned my attention to the portable testing chips that can integrated with the promising reagent. An aluminum-based microfluidic chip was developed for real-time PCR testing. The reaction chamber was coated with a silicone-modified epoxy resin to isolate the reaction system from metal surfaces, preventing metal ions from interfering with the reaction process. The patterned and coated aluminum substrate was bonded with a hydroxylated glass mask using silicone sealant, addressing the thermal expansion issue with the elasticity of cured silicone simultaneously. Real-time PCR testing in reaction chambers yielded quantitative cycle (Cq) values similar to traditional test sets. Surface characterizations, including SEM, AFM, EDS, XPS, ICP-OES, and FTIR analyses, confirmed the effective coating, isolation, and unreacted antifouling surfaces. The limit of detection (LOD) of at least 2 copies can be obtained in this chip.

The third study developed the portable device based on a more traditional substrate. I developed a silicon-based polydimethylsiloxane-polyethylene-glycol (PDMS-PEG) copolymer microfluidic chip, the PDMS-PEG copolymer silicon chip (PPc-Si chip), for biomolecular diagnosis. By chemically modifying the PDMS with a hydrophilic PEG, a rapid hydrophilic switch was achieved within 15 seconds after contact with water, resulting in only a 0.8% reduction in transmittance. By assessing the transmittance across an extensive wavelength range from 200 nm to 1000 nm, this study offers valuable insights into the optical properties of the material and serves as a reference for potential applications of the modified material in optical-related devices. The improved hydrophilicity was achieved by introducing numerous hydroxyl groups, which also contributed to the excellent bonding strength of PPc-Si chips. Real-time PCR tests conducted on the chip demonstrated higher efficiency and lower non-specific absorption.

Collectively, these studies described in this thesis contribute to the advancement of real-time PCR technologies by addressing critical challenges in reagent storage, chip material, and surface properties. The developed methods and materials offer significant potential for a wide range of applications in point-of-care tests (POCT) and rapid disease diagnosis, ultimately improving patient outcomes and healthcare efficiency.