A polymer lab-on-a-chip for reverse transcription (RT)-PCR for point-of-care clinical diagnostics.

摘要

In this dissertation, a new polymer Reverse Transcription (RT)-Polymerase Chain Reaction (PCR) lab-on-a-chip (LOC) with embedded micro pinch-valves has been designed, fabricated and characterized for point-of-care testing (POCT) clinical diagnostics. In addition, a portable analyzer, which consists of an optical detection system and a non-contact infrared (IR) based temperature control system for RT-PCR process, has been developed and characterized as the analyzer monitoring for the developed RT-PCR LOC. The developed LOC and analyzer have been interfaced and optimized for performing the RT-PCR procedure and chemiluminescence assay in sequence, and then as a demonstration of clinical diagnostics Human Immunodeficiency Virus (HIV) for the early diagnostics of Acquired Immune Deficiency Syndrome (AIDS) has been successfully analyzed using the developed LOC and analyzer.;In the RT-PCR LOC, a new concept of the embedded micro pinch-valve has been proposed, fabricated and characterized as one of the key microfluidic components desired for the RT-PCR procedure. In the development of the embedded micro pinch-valve, a new sealing method using UV adhesive has been developed, which provides enough sealing required for the prevention of leakage at the high pressure developed during the PCR procedure. The developed embedded micro pinch-valve can possibly have numerous applications for the functional lab-on-a-chips which need to be integrated with on-chip microvalves.;Also, in the portable analyzer, a temperature control system and an optical detection system have been developed, assembled and fully characterized for the procedure control and optical detection of the RT-PCR LOC. In order to minimize the process time of RT-PCR, a non-contact IR heating method has been adopted for controlling the temperature on the RT-PCR chip. For the optical detection system, an extremely high sensitive photodiode with a high gain amplifier circuit with low noise has been used for detecting the chemiluminescence assay.;AIDS comes from infecting HIV (Human Immunodeficiency Virus), a syndrome where the immune system begins to fail, leading to many life-threatening infections. Since early diagnostics of the disease by identifying HIV is essential for the treatment of the disease, the development of POCT clinical diagnostics near to the patients is very desirable. So, in this work, the developed RT-PCR LOC and analyzer has been applied for the detection of HIV in a POCT platform.;The developed RT-PCR LOC has been characterized first for both fluorescence and chemiluminescence assays in terms of sensitivity and adoptability with the portable analyzer. From the characterization and comparison, the chemiluminescence assay has been chosen better for the RT-PCR LOC and the analyzer, and the assay has been performed and characterized for both conventional scanner and the developed portable analyzer. The analysis results obtained from the developed portable analyzer have shown an excellent performance comparable to those obtained from the conventional scanner, which ensures the developed RT-PCR LOC and portable analyzer as a promising POCT for clinical diagnostics.;In conclusion, in this research work, a new RT-PCR polymer lab-on-a-chip and its portable analyzer has been successfully developed and fully characterized for the POCT of clinical diagnostics, and then the developed RT-PCR LOC and analyzer has successfully performed the whole assay of HIV, envisaging the POCT of early diagnostics of AIDS.