Improvement of fluorescence-based microfluidic DNA analyzers

A tremendous effort continues in the development o f micro-total-analysissystems; in support o f this, many chemical passivation methods have been developed to enhance the biocompatibility o f such microfluidic systems. However, the suitability of these passivation techniques to many fluorescence-based assays still remains inconsistent. This part o f this work is focused on the performance of a third generation intercalating DNA dye when used within microfluidic devices treated with a select variety of passivating coatings. The results of these tests indicate that passivation coatings which are intended to shed DNA based on electrostatic repulsion will in fact imbibe the fluorescent DNA intercalating dye by the same mechanism. Blocking this charge-based dye adsorption, such as with bovine serum albumen (BSA), has yielded mixed results in the literature. As an alternative, this present work indicates that preloading the bio­ passivated microchannel with a small amount of this dye will prevent both the DNA and the DNA dye from being adsorbed from solution onto the channel walls. By characterizing the saturating behavior o f this preloaded dye, a protocol is here suggested to optimize dye performance in passivated microfluidics. Furthermore, the intent and achievement o f this work has been to design a BSA-free treatment method, thereby eliminating common fluorescent artifacts. The amount of dye preloading required is found to be proportional to the microchannel surface area, and can be predicted by a new material property defined for each chemical coating processes. Theoretical and experimental results indicate that this is independent of operating temperature, flow rate, and channel aspect ratio. Thus this is a property o f the material, and not just a product of the several operational parameters. This property has been measured for four coatings as part of this work. Improvements in passivation are crucial to the development o f lab-on-a-chip devices, important in solving current medical and healthcare problems. Challenging topics include the need for fast pathogen detection (i.e. ebola epidemic, HIV, water-borne diseases typhoid, cholera, dysentery) and the need for personalized medicine (i.e. cancer genomics, drug susceptibility). A novel microfluidic DNA analysis device was developed, incorporating helicase dependent amplification (HDA) and sequence-specific fluorescence based detection. These are incorporated into a glass/polymer platform that is hand operated and powered by a laptop computer. Thermal modeling sets operation at less than 3 Watts and fabrication consistency testing ensures samples volumes of 17pL. The heating element and optical components are powered via 3 USB ports. The heating element consists o f a thin film heater and thermistor controlled in a feedback loop with Matlab and Arduino interfacing. Using fuzzy logic control, the temperature of the PCR chamber has been controlled by varying the heater voltage. On-chip amplification has been verified using a commercial LightScanner32 device and HDA detection and DNA melting analysis were performed on-chip. APPROVAL FOR SCHOLARLY DISSEMINATION The author grants to the Prescott Memorial Library of Louisiana Tech University the right to reproduce, by appropriate methods, upon request, any or all portions o f this Dissertation. It is understood that “proper request” consists o f the agreement, on the part of the requesting party, that said reproduction is for his personal use and that subsequent reproduction will not occur without written approval o f the author o f this Dissertation. Further, any portions o f the Dissertation used in books, papers, and other works must be appropriately referenced to this Dissertation. Finally, the author o f this Dissertation reserves the right to publish freely, in the literature, at any time, any or all portions o f this Dissertation.