Microfluidics miniaturizes many benchtop processes and provides advantages of low cost, reduced reagent usage, process integration, and faster analyses. Microfluidic devices have been fabricated from a wide variety of materials and methods for many applications. This dissertation describes four such examples, each employing different features and fabrication methods or materials in order to achieve their respective goals. In the first example of microfluidic applications in this dissertation, thermoplastics are hot embossed to form t-shaped channels for microchip electrophoresis. These devices are used to separate six preterm birth (PTB) biomarkers and establish a limit of detection for each. The next chapter describes 3D printed devices with reversed-phase monoliths for solid-phase extraction and on-chip fluorescent labeling of PTB biomarkers. I demonstrate the optimization of the monolith and selective retention of nine PTB biomarkers, the first microchip study to perform an analysis on this entire panel. The third project describes the iterative design and fabrication of glass/polydimethylsiloxane (PDMS) devices with gold and nickel electrodes for the self-assembly of DNA nanotubes for site-selective placement of nanowires. Simple flow channels and “patch electrode” devices were successfully used, and DNA seeding was achieved on gold electrodes. Finally, a 3D printed device for cancer drug screening was developed as a replacement for one previously fabricated in PDMS. Devices of increasing complexity were fabricated, and those tested found to give good control over fluid flow for multiple inlets and valves. Although the applications and methods of these projects are varied, the work in this dissertation demonstrates the potential of microfluidics in several fields, particularly for diagnostics, therapeutics, and nanoelectronics. Furthermore, it demonstrates the importance of applying appropriate tools to each problem to gain specific advantages. Each of the described devices has the potential for increased complexity and integration, which further emphasizes the advantages of miniaturized analyses and the potential for microfluidics for analytical testing in years to come.

Antimicrobial resistance in bacteria is a global health challenge with a projected fallout of 10 million deaths annually and cumulative costs of over 1 trillion dollars by 2050. The currently available tools exploited in the detection of bacteria or their DNA can be expensive, time inefficient, or lack multiplex capabilities among others. The research work highlighted in this dissertation advances techniques employed in the phenotypic or genotypic detection of bacteria and their DNA. In this dissertation, I present polymethyl methacrylate-pressure sensitive adhesive microfluidic platforms developed using a time-efficient, inexpensive fabrication technique. Microfluidic devices were then equipped with functionalized monoliths and utilized for sequence-specific capture and detection of picomolar concentrations of bacterial plasmid DNA harvested from cultured bacteria. I then showed multiplex detection of multiple bacteria gene targets in these devices with an improved monolith column. Finally, I demonstrated a genotypic approach to studying single bacteria growth in water-in-oil droplets with nanomolar concentrations of a fluorescence reporter, and detection via laser-induced fluorescence after convenient room temperature 2-h incubation conditions. The systems and methods described herein show potential to advance tools needed to address the surging problems and effects of drug-resistant bacteria.

Microfluidics can take laboratory processes and miniaturize them, which led to the term lab-on-a-chip. Microfluidic devices are fabricated with a variety of materials and methods, each offering distinct advantages for bioanalysis. This dissertation describes two methods to create these devices, with the use of four different materials to achieve different assay needs. In the first application in this dissertation, hot-embossed cyclic olefin copolymer was used to create microdevices to electrophoretically separate seven preterm birth biomarkers. One biomarker, thrombin-antithrombin III, cannot be purchased commercially so I developed methods for its assembly in the lab. Dot blots and mass spectrometry were used to evaluate the synthesis of thrombin-antithrombin III. The next application evaluated digital-light processing stereolithography 3D printing resins. A new optically clear resin was developed and compared to two previously described resins. The physical characteristics (i.e., hardness and Young's modulus), biocompatibility, and electrophoretic separation capabilities were compared. Lastly, 3D printing was used to create microfluidic devices with embedded affinity columns to extract, fluorescently label, and detect chikungunya virus RNA. Conditions for detecting RNA were optimized using oligonucleotides, and a linear relationship was determined for concentration of RNA loaded and fluorescent signal detected. The specificity of the column was tested with a genetically similar virus; viral RNA from both viruses was loaded to demonstrate ability to extract and detect only chikungunya virus. These applications show microfluidic devices' ability to analyze various biomolecules. This work also exhibits multiple tools that can be used in microfluidics. Using these methods provides better characterization of diseases, drugs, and wellness.

Background: Separation science is heavily reliant on materials to fulfill ever more complicated demands raised by other areas of science, notably the rapidly expanding molecular biosciences and environmental monitoring. The key to successful separations lies in a combination of physical properties and surface chemistry of stationary phases used in liquid chromatographic separation, and this thesis address both aspects of novel separation materials. Methods: The thesis accounts for several approaches taken during the course of my graduate studies, and the main approaches have been i) to test a wild-grown variety of published methods for surface treatment of fused silica capillaries, to ascertain firm attachment of polymeric monoliths to the wall of microcolumns prepared in silica conduits; ii) developing a novel porogen scheme for organic monoliths including polymeric porogens and macromonomers; iii) evaluating a mesoporous styrenic monolith for characterization of telomers intended for use in surface modification schemes and; iv) to critically assess the validity of a common shortcut used for estimating the porosity of monoliths prepared in microconduits; and finally v) employing plasma chemistry for activating and subsequently modifying the surface of rigid, monodisperse particles prepared from divinylbenzene. Results: The efforts accounted for above have resulted in i) better knowledge of the etching and functionalization parameters that determine attachment of organic monoliths prepared by radical polymerization to the surface of silica; ii) polar methacrylic monoliths with a designed macroporosity that approaches the desired "connected rod" macropore morphology; iii) estab¬lishing the usefulness of monoliths prepared via nitroxide mediated polymerization in gradient polymer elution chromatography; iv) proving that scanning electron microscopy images are of limited value for assessing the macroporous properties of organic monoliths, and that pore measurements on externally polymerized monolith cocktails do not represent the porous properties of the same cocktail polymerized in narrow confinements; and v) showing that plasma bromination can be used as an activation step for rigid divinylbenzene particles to act as grafting handles for epoxy-containing telomers, that can be attached in a sufficiently dense layer and converted into carboxylate cation exchange layer that allows protein separations in fully aqueous eluents.

Preterm birth (PTB) results in over 15 million early births annually and is the leading cause of neonatal deaths. There are no clinical methods currently available to evaluate risk of PTB at early stages in pregnancy; thus, a rapid diagnostic to analyze PTB risk would be beneficial. Microfluidic immunoaffinity extraction is a promising platform for preparing complex samples, such as maternal serum with PTB risk biomarkers. 3D printed microfluidic devices have advantages over conventional microfluidic systems including simple fabrication and potential for iterative optimization to improve designs. In this work, I developed immunoaffinity monoliths in 3D printed microfluidic devices modified with antibodies to enrich PTB biomarkers from human blood serum. I retained and eluted a peptide PTB biomarker in both buffer and blood serum using an immunoaffinity column. An additional three PTB biomarkers were also successfully extracted either from buffer or blood serum on single-antibody columns. Both polyclonal and monoclonal antibodies to PTB biomarkers were characterized by dot blots, biolayer interferometry, and surface plasmon resonance to determine their specificity and dissociation constants. I created multiplexed immunoaffinity columns to simultaneously enrich three PTB biomarkers from depleted human blood serum in a single extraction. This is the first demonstration of multiplexed immunoaffinity columns for PTB biomarkers in a 3D printed microfluidic device. My work is a key step towards the future development of 3D printed microfluidic devices for rapid PTB testing.