Integrated Microfluidic Molecular Diagnostics for Point-of-Care

Ideal point-of-care medical diagnostic devices are low cost assays capable of performing quantitative on-site rapid testing with high sensitivity and minimal manual steps./nCurrent mainstream assays have several key limitations. Take, for instance, the common lateral flow assay—e.g. the pregnancy dipstick test. Such assays produce rapid results at low cost; however, they are mostly qualitative tests yielding only positive/negative results rather than quantitative figures. Other standard immunosorbant assays such as ELISA yield quantitative results but require several hours and extensive manual operation. At the other end of the spectrum, nucleic acid amplification techniques such as quantitative real-time PCR can deliver much higher sensitivity and selectivity. Unfortunately, these require costly equipment and several sample preparation steps./nIn this thesis, an integrated low-cost microfluidic chip and peripheral technologies for quantitative molecular diagnostics is described. These technical advances are designed to address the prevailing dilemmas described above./nResearchers have developed and integrated several key components with microfluidic lab-on-chip miniaturization technology. In line with cutting-edge technology, a novel reagent patterning method, termed “digital micro-patterning”, was developed. A very simple method, it can be adopted at low-resource laboratory settings with mainstream equipment. Digital micro-patterning is unique in the sense that it can digitally pattern and concentrate reagents into highly defined micro-patterns. As a proof of concept, it was possible to pattern isothermal amplification reagents in hundreds of microwells and run amplification reactions in these wells./nNext, a next-generation passive microfluidic pumping technology, termed the “vacuum battery system”, has been developed. This system allows for precise passive microfluidic pumping without external pumps, controls, or power sources for up to several hours. It does not require opaque fibers as in capillary systems (e.g. lateral flow assays), thus rendering this pumping method very attractive for optical detection platforms. The vacuum battery system is also significantly more robust compared to previous degas pumping techniques. Due to its portability, excellent optical properties, low cost, and the ability for complete integration with microfluidics, this platform technology opens exciting new opportunities to create a nouveau generation of standalone microfluidic chips readily operable in field settings./nAdditionally, a microfluidic sample preparation technology termed “digital plasma separation” has been developed. This technology uses parallel micro-cliff-like structures and gravity sedimentation to simultaneously separate plasma and compartmentalize samples into hundreds of micro-wells within minutes. Such sample preparation method enables isothermal digital nucleic acid amplification in one step./nAs a proof of concept, these technologies were integrated into a single microfluidic chip, termed the Integrated Molecular Diagnostics Chip (iMDx). This chip is capable of performing one-step quantitative nucleic acid detection directly from human whole blood samples (10~10^5 copies/ μl in 30 minutes). One low-cost disposable chip (~ $10) is designed to integrate and automate sample preparation, quantitative isothermal digital nucleic acid detection, and next generation autonomous microfluidic pumping. This portable integrated chip can acquire template concentration data similar to bench top real-time PCR machines. As the latter can cost three or more orders ($30~80k), this chip opens exciting opportunities for rapid point-of-care diagnostics in resource-low settings./nFinally, in summarizing these cutting-edge methods, a blueprint for next-generation technical development plans is laid out. The key areas of focus are downstream microfluidic integration for advanced functionality such as protein and nucleic acid multiplexed detection on a single chip, telemedicine, mass production, and clinical studies in field settings./nUltimately, the implication of the research in this dissertation is that these platform technologies can be adopted into future medical diagnostic devices to enable rapid on-site quantitative molecular level detection at significantly lower costs. Both the system-level design rationale and component technologies developed herein provide promising building blocks for future point-of-care diagnostic assays.

Advisor: Lee, Luke P