Microfluidics nucleic acid testing for point-of-care liver assessment
Lim, Geok Soon
Date of Issue2016-05-30
School of Electrical and Electronic Engineering
Liver assessment is currently one of the most important medical diagnostics. The inherent complexities of current gold standards of liver assessment such as histopathology and Liver Function Tests are generally incompatible with point-of-care testing (POCT). Hence, there is a real need for a truly practical Lab-on-a-Chip (LOC) system POCT liver assessment. The embodiment of such a system should feature rapid, full-automation, ease-of-use, and sample-in-answer-out diagnostic capabilities. To date, reported microfluidic devices for POCT liver assessment remain largely rudimentary as they embody semi-integrated, semi-quantitative and single gene detection capabilities. In this thesis, we propose the use of microfluidics Nucleic Acid Testing (NAT) to demonstrate the feasibility for a practical and clinically pertinent POCT liver assessment device. In our research, we propose and realize two different integrated LOC systems for multiplex gene expression studies in POCT hepatotoxicity assessment. For our first integrated LOC system, we propose and describe an LOC system (stationary PCR approach) which encompasses the entire NAT process for sample-in-answer-out capabilities. Our first integrated LOC system embodies the integration and semi-automation of a tissue sample preparation module, a polymeric chip-compatible thermal cycler for multiplex RT-PCR, and a real-time fully-quantitative fluorescent detection methodology. To evaluate the feasibility of our first integrated LOC system in POCT hepatotoxicity assessment, we select and validate two different liver-critical genes of interest (GOIs) – namely, alanine transaminase and aspartate transaminase, based on critical liver protein biomarkers used in Liver Function Tests. To validate our integrated LOC system, we perform a preclinical drug toxicity animal study via the administration of cyclophosphamide, and subsequent microfluidic gene expression analysis depicts up-regulations in the GOIs’ expression levels – consistent with results obtained from conventional methodology. For our second integrated LOC system, we propose and describe an LOC system (continuous-flow PCR approach) which features a small equipment footprint and embodies an easy-to-assemble fluorescent detection module. With a contact-based isothermal heating setup, a polymeric biochip embodying a segmented flow approach, and a fully-quantitative coordinate-based detection methodology, we describe a method for multiplex gene expression analysis on a continuous-flow PCR platform. We demonstrate the rapidness of our second LOC system using the same drug-treated mice tissue samples, and illustrate significant reductions in RT-qPCR completion, wherein the entire process can be completed in <20mins – 3.9-fold faster than a conventional thermal cycler and ~3.15-fold faster than our first integrated LOC system embodying the stationary PCR approach. In short, both of our integrated LOC systems are designed to be somewhat close to a practical POCT liver assessment device, and feature semi-automation, relative ease-of-use, and sample-in-answer-out capabilities for multiplex gene expression analysis. Collectively, the contributions from both of our integrated LOC systems are pertinent towards the realization of a truly practical POCT liver assessment device.
DRNTU::Engineering::Electrical and electronic engineering::Microelectromechanical systems