In the past several decades, tremendous progress has been made in drug discovery, treatment of diseases and overall patient care. But clinical diagnostics is still lagging behind primarily due to its dependence on central labs which often result in delayed or missed diagnosis of diseases. Point-of-care testing (POCT) holds the promise of revolutionizing diagnostics by enabling clinical tests at near-patient settings and in resource-poor environments.; The objective of this thesis work has been to develop hand-held devices with microfluidic addressability to enable point-of-care immunodiagnostics and nucleic-acids based testing. The microfluidic devices were fabricated in thermoplastics using micro-hot-embossing and thermal bonding techniques in an effort to make them cost-efficient for disposable applications. The plastic microfluidic platform was then developed into a disposable immunosensor for testing of disease-related protein biomarkers in clinical samples. The system was able to detect femtomolar concentrations of C-reactive protein using a chemiluminescent ELISA technique. The detection limit of the microfluidic chemiluminescent assay was significantly better than both conventional colorimetric assays and microfluidic fluorescence immunoassays. The assay results were read via an on-board instant photographic film, which obviates the need for any dedicated bench-top analyzer, and therefore makes the device self-sufficient for point-of-care diagnostics when simple positive/negative results are sought.; The microfluidic analytical platform was also applied to on-chip sample preparation to facilitate microchip-based molecular diagnostics (nucleic acid testing) The availability of robust, cost-effective and disposable sample preparation platforms is the much needed catalyst that can accelerate the penetration of nucleic acid-based tests into the in vitro diagnostics market. On-chip cell lysis was achieved by diffusive mixing of the sample with lysis reagents in serpentine mixing channels. The isolation and purification of nucleic acids from the lysates was performed via solid-phase extraction in the microfluidic channels. The solid-phase consisted of a microporous polymer monolith embedded with functional microparticles and covalently attached to the channel walls via photoinitiated grafting. The in situ photografting process and the porous polymer monolith chemistry used here are very versatile and can be used to entrap any micro- or nanoparticles to form functionalized solid-phases within UV transparent thermoplastic microchips. For DNA and total RNA purification, the microporous polymer monolith was impregnated with silica particles, and the extraction was achieved due to the preferential binding of nucleic acids to the silica particles in the presence of chaotropic salts. The sample preparation system was shown to purify and concentrate phage lambda DNA, human genomic DNA from whole blood, and viral RNA from infected mammalian cells. The extraction efficiency of the system was 70% +/- 3% and the nucleic acid binding capacity of the solid-phase was found to be approximately 4.0 ng. The micro solid-phase system was also used for the extraction of mRNA by embedding oligo(dT) beads in the porous monolith. The system was able to extract mRNAs of both abundant and rare genes from total RNA as well as from whole cell lysates. The micro solid-phase extraction technology presented lays the groundwork for a high-throughput nucleic acid sample preparation platform that can eventually be coupled with a downstream amplification/detection module to form an integrated molecular diagnostic device.
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