Polymer-Based Microfluidic Devices for High Throughput Single Molecule Detection: Applications in Biological and Drug Discovery
The realization of high throughput sample processing has become a primary ambition in many research applications with an example being high throughput screening (HTS), which represents the first step in the drug discovery pipeline. Microfluidics is a viable platform for parallel processing of biochemical reactions to increase data production rates due to its ability to generate fluidic networks with a high number of processors over small footprints suitable for optical imaging. Single-molecule detection (SMD) is another technology which has emerged to facilitate the realization of high throughput data processing afforded by its ability to eliminate sample processing steps and generate results with high statistical accuracy. A combination of microfluidics and SMD with wide-field fluorescence detection provides the ability to monitor biochemical reactions in a high throughput format with single-molecule sensitivity. In this dissertation, the integration of these techniques was presented for HTS applications in drug discovery. An ultra-sensitive fluorescence detection system with a wide field-of-view (FoV) was constructed to transduce fluorescence signatures from single chromophores that were electrokinetically transported through a series of tightly packed fluidic channels poised on poly(methylmethacrylate), PMMA and contained within the FoV of a laser detection system. The system was used to monitor biochemical reactions at the single-molecule level in a continuous-flow format. Enhancement in sampling-throughput was demonstrated by constructing a high density fluidic network for parallel analysis of multiple biochemical assays. In another development, the ability to enhance single-molecule sensitivity in a flow-based biochemical assay was investigated using a novel cyclic olefin copolymer (COC) planar waveguide embedded in PMMA and situated orthogonal to multiple fluidic channels. This design allowed for fluorescence detection from multiple fluidic channels using evanescent excitation and a wide FoV fluorescence detection system for parallel readout. Results from these technologies were presented as well as their applications in drug discovery for increasing data production rates and quality. An approach toward monitoring the efficacy of therapeutic agents, which is important in clinical evaluation of drug potency in the drug discovery process, was also considered, by designing a microfluidic system with integrated conductivity sensor for label-free enumeration of isolated tumor cells from clinical samples.
High Throughput Screening, Wide-Field Illumination, Ultrasensitive Flourescence Detection, Single-Molecule Detection, CCD, Microfluidics
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