Laboratory test results are important in making decisions regarding a patient's diagnosis and response to treatment. These tests often measure the biomarkers found in biological fluids such blood, urine, and saliva. Immunoassay is one type of laboratory test used to measure the level of biomarkers using specific antibodies. Microfluidics offer several advantages such as speed, small sample volume requirement, portability, integration, and automation. These advantages are motivating to develop microfluidic platforms of conventional laboratory tests. I have fabricated polymer microfluidic devices and developed immunoassays on-chip for potential cancer markers. Silicon template devices were fabricated using standard photolithographic techniques. The template design was transferred to a poly(methyl methacrylate) (PMMA) piece by hot embossing and subsequently bonded to another PMMA piece with holes for reservoirs. I used these devices to perform microchip immunoaffinity electrophoresis to detect purified recombinant thymidine kinase 1 (TK1). Buffer with 1% methylcellulose acted as a dynamic coating that minimized nonspecific adsorption of protein and as sieving matrix that enabled separation of free antibody from antibody-TK1 complexes. Using this technique, I was able to detect TK1 concentration >80 nM and obtained separation results within 1 minute using a 5 mm effective separation length. Detection of endogenous TK1 in serum is difficult because TK1 is present at the pM range. I compared three different depletion methods to eliminate high abundance immunoglobulin and human serum albumin. Cibacron blue columns depleted abundant protein but also nonspecifically bound TK1. I found that ammonium sulfate precipitation and IgG/albumin immunoaffinity columns effectively depleted high abundance proteins. TK1 was salted out of the serum with saturated ammonium sulfate and still maintained activity. To integrate affinity columns in microfluidic devices, I have developed a fast and easy strategy for initial optimization of monolith affinity columns using bulk polymerization of multiple monolith solutions. The morphology, surface area, and porosity, were qualitatively assessed using scanning electron microscopy. This method decreased the time, effort, and resources compared to in situ optimization of monoliths in microfluidic devices. This strategy could be used when designing novel formulations of monolith columns. I have also integrated poly(ethylene glycol dimethacrylate-glycidyl methacrylate) monolith affinity columns in polymer microfluidic devices to demonstrate the feasibility of extracting human interleukin 8 (IL8), a cancer biomarker, from saliva. Initial results have shown that the affinity column (~3 mm) was successfully integrated into the devices without prior surface modification. Furthermore, anti-IL8 was immobilized on the surface of the monolith. Electrochromatograms showed that 1 ng/mL of IL8 can be detected when in buffer while 10 ng/mL was detected when IL8 was spiked in saliva. Overall, these findings can be used to further develop immunoassays in microfluidic platforms, especially for analyzing biological fluids.

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The need for miniaturized, portable devices to separate and detect unknown compounds has greatly multiplied, leading to an increased interest in microfluidics. Total integration of the detector and pump are necessary to decrease the overall size of the microfluidic device. Using previously developed thin film technologies, an electroosmotic flow (EOF) pump was incorporated in a microfluidic liquid chromatography device. An EOF pump was chosen because of its simple design and small size. EOF pumps fabricated on silicon and glass substrates were evaluated. The experimental flow rates were 0.19-2.30 microliters/minute for 40-400 V. The theoretical pump efficiency was calculated along with the generated mechanical power by various pump shapes to elucidate more efficient pump designs. To better understand the EOF on plasma enhanced chemical vapor deposition (PECVD) silicon dioxide, the zeta potential was investigated. PECVD oxide is amorphous and less dense than thermal silicon dioxide, which slightly changes the zeta potential. Zeta potentials were found for pH values from 2.6 to 8.3. Also, surface defects that affect the zeta potential were observed, and procedures to detect and prevent such defects were proposed. Finally, surface modifications to the microfluidic device were attempted to demonstrate that thin film EOF pumps can be used in the liquid chromatographic separation of mixtures. The microfluidic separation channel was coated with chlorodimethyloctadecylsilane, however, due to problems with channel filling and reservoir adhesives, separation was not achieved. The use of new adhesives and external pumps were proposed to resolve these problems for future testing. Also new methods to combine EOF pumps with microfluidic channels and on-chip detectors were suggested.

In this dissertation, several microfabricated devices are introduced to develop new applications in the area of chemical analysis. Electrochemical micropumps, chip-based liquid chromatography systems and multilayer capillary electrophoresis microdevices with crossover channels were fabricated using various substrates such as poly(dimethylsiloxane) (PDMS), glass, and poly(methyl methacrylate) (PMMA). I have demonstrated pressure-driven pumping of liquids in microfabricated channels using electrochemical actuation. PDMS-based micropumps were integrated easily with channel-containing PMMA substrates. Flow rates on the order of ~10 µL/min were achieved using low voltages (10 V). The potential of electrolysis-based pumping in microchannels was further evaluated for pressure driven microchip liquid chromatography (LC). Two micropumps were connected with reservoirs for sample and mobile phase, situated at the ends of microchannels for sample injection and separation, respectively. Columns micromachined in glass were coated covalently with an organic stationary phase to provide a separation medium. A pressure-balanced sample injection method was developed and allowed the injection of picoliter sample volumes into the separation channel. Fast (<40 s) separation of three fluorescently tagged amino acids was performed in a 2.5-cm-long microchip column with an efficiency of 3300 theoretical plates. Improved electrode designs that eliminate the stochastic formation of bubbles on the electrode surface will enhance pumping reproducibility. Multilayer polymeric microdevices having fluidically and electrically independent crossover channels were made using phase-changing sacrificial layers (PCSLs). High-performance electrophoretic separations of fluorescently labeled amino acids were carried out in multilayer PMMA microchips. Neither pressure nor voltage applied in a crossover channel resulted in negative effects on the separation quality in the main fluidic path. A fifty-fold reduction in crossover volumes was achieved in next-generation multilayered microchips. The ability to make minimal dead volume crossover channels facilitated the design and operation of multichannel array microdevices with a minimum number of electrical and fluidic inputs. Replicate electrophoretic separation of two peptides was performed in parallel for three independent microchannels connected to a single sample reservoir. My work demonstrates the value of PCSLs in making complex microfluidic structures that should expand the application of micro-total analysis systems.

Polymeric microchips have received increasing attention in chemical analysis because polymers have attractive properties, such as low cost, ease of fabrication, biocompatibility and high flexibility. However, commercial polymers usually exhibit analyte adsorption on their surfaces, which can interfere with microfluidic transport in, for example, chemical separations such as chromatography or electrophoresis. Usually, surface modification is required to eliminate this problem. To perform stable and durable surface modification, a new polymer, poly(methyl methacrylate-co-glycidyl methacrylate) (PGMAMMA) was prepared for microchip fabrication, which provides epoxy groups on the surface. Whole surface atom transfer radical polymerization (ATRP) and in-channel ATRP approaches were employed to create uniform and dense poly(ethylene glycol) (PEG)-functionalized polymer brush channel surfaces for capillary electrophoresis (CE) separation of biomolecules, such as peptides and proteins. In addition, a novel microchip material was developed for bioanalysis, which does not require surface modification, made from a PEG-functionalized copolymer. The fabrication is easy and fast, and the bonding is strong. Microchips fabricated from this material have been applied for CE separation of small molecules, peptides, proteins and enantiomers. Electric field gradient focusing (EFGF) is an attractive technique, which depends on an electric field gradient and a counter-flow to focus, concentrate and separate charged analytes, such as peptides and proteins. I used the PEG-functionalized copolymer to fabricate EFGF substrates. The separation channel was formed in an ionically conductive and protein resistant PEG-functionalized hydrogel, which was cast in a changing cross-sectional cavity in the plastic substrate. The hydrogel shape was designed to create linear or non-linear gradients. These EFGF devices were successfully used for protein focusing, and their performance was optimized. Use of buffers containing small electrolyte ions promoted rapid ion transport in the hydrogel for achieving the designed gradients. A PEG-functionalized monolith was incorporated in the EFGF separation channel to reduce dispersion and improve focusing performance. Improvement in peak capacity was proposed using a bilinear EFGF device. Protein concentration exceeding 10,000-fold was demonstrated using such devices.

Biomarkers present a non-invasive means of detecting cancer because they can be obtained from body fluids. They can also be used for prognosis and assessing response to treatment. To limit interferences it is essential to pretreat biological samples before analysis. Sample preparation methods include extraction of analyte from an unsuitable matrix, purification, concentration or dilution and labeling. The many advantages offered by microfluidics include portability, speed, automation and integration. Because of the difficulties encountered in integrating this step in microfluidic devices most sample preparation methods are often carried out off-chip. In the fabrication of micro-total analysis systems it is important that all steps be integrated in a single platform. To fabricate polymeric microdevices, I prepared templates from silicon wafers by the process of photolithography. The design on the template was transferred to a polymer piece by hot embossing, and a complete device was formed by bonding the imprinted piece with a cover plate. I prepared affinity columns in these devices and used them for protein extraction. The affinity monolith was prepared from reactive monomers to facilitate immobilization of antibodies. Extraction and concentration of biomarkers on this column showed specificity to the target molecule. This shows that biomarkers could be extracted, purified and concentrated with the use of microfluidic affinity columns.I prepared negatively charged ion-permeable membranes in poly(methyl methacrylate) microchips by in situ polymerization just beyond the injection intersection. Cancer marker proteins were electrophoretically concentrated at the intersection by exclusion from this membrane on the basis of both size and charge, prior to microchip capillary electrophoresis. I optimized separation conditions to achieve baseline separation of the proteins. Band broadening and peak tailing were limited by controlling the preconcentration time. Under my optimized conditions a 40-fold enrichment of bovine serum albumin was achieved with 4 min of preconcentration while >10-fold enrichment was obtained for cancer biomarker proteins with just 1 min of preconcentration. I have also demonstrated that the processes of sample enrichment, on-chip fluorescence labeling and purification could be automated in a single voltage-driven platform. This required the preparation of a reversed-phase monolithic column, polymerized from butyl methacrylate monomers, in cyclic olefin copolymer microdevices. Samples enriched through solid phase extraction were labeled on the column, and much of the unreacted dye was rinsed off before elution. The retention and elution characteristics of fluorophores, amino acids and proteins on these columns were investigated. A linear relationship between eluted peak areas and protein concentration demonstrated that this technique could be used to quantify on-chip labeled samples. This approach could also be used to simultaneously concentrate, label and separate multiple proteins.

Using silicon based microfabrication and materials, a photonic platform, capable of single bioparticle analysis, has been developed. This platform combines liquid and hollow core waveguides on the micron-scale (5 µm x 12 µm) to isolate femtoliter sized sample volumes. Fluorescence excitation and signals in the visible range are directed into and out of the sample volume at an orthogonal angle to maximize signal-to-noise. In order to guide light in a low-index material antiresonant reflecting optical waveguides (ARROWs) were incorporated into the platform. This thesis reveals the development path of these structures over several device generations including innovations in material, geometries, and fabrication techniques to increase detection sensitivity. As a result of these developments, this photonic platform has shown to successfully detect virus samples and other particles. This thesis also presents a new idea for increasing the signal to noise ratio (SNR) by incorporating Y-splitter devices into the design. Specifically, the 1 x 2 and 1 x 4 splitter structures can be used as orthogonal excitation points to the liquid core waveguide. When fluorescently tagged particles are introduced into the hollow core, these points create an optical signal that is correlated in time and space. The data collected by a photodetector can then be processed by an algorithm to increase SNR. Such advancements have shown to increase the SNR by 175 times.

This thesis presents an analysis of the signal-to-noise ratio in liquid core anti-resonant reflecting optical waveguides (ARROWs) and the application of hydrodynamic focusing to the waveguides. These concepts are presented as a method to improve the detection capabilities of the ARROW platform. The improvements are specifically targeted at achieving single molecule detection (SMD) with the devices. To analyze the SNR of the waveguides a test platform was designed and fabricated. This test platform was then used to examine relationship between the SNR and the location of the excitation region. It was determined that the excitation region should be moved closer to the solid-core. By moving the excitation region closer to the solid-core the distance the signal was required to travel in the hollow-core was reduced. This reduction led to a decrease in optical signal loss and resulted in a more than 2x increase in the SNR. Hydrodynamic focusing in the waveguides was developed as a method to increase the consistency of detection of the devices. In hydrodynamic focusing particles in the sample are forced towards the center of the waveguide with a buffer solution. With the particles focused to the center of the channel the percentage that passed through the excitation region can be increased improving the detection consistency of the device. ARROW chips designed for hydrodynamic focusing were simulated, fabricated, and preliminary testing was performed. Initial results have shown a more than 30% increase in particle focusing.

Rapid determination of the concentrations of molecules related to diseases can provide timely information for treatment options. However, most biomarker quantitation methods require costly and complex equipment. On the other hand, point-of-care systems have less complex instrumentation needs than laboratory-based equipment, but often provide less information; for example, biomarker presence or absence instead of concentration. A complete analysis setup addressing key limitations of both laboratory-based and portable systems is highly desirable. I developed microfluidic devices with visual inspection readout of a target’s concentration from microliter volumes of solution flowed into a microchannel. Microchannels are formed within polydimethylsiloxane (PDMS), and the surfaces are coated with receptors. Capillary flow of target solution in the channel crosslinks the top and bottom surfaces, which constricts the channel and stops flow. The flow distance of the target solution in the channel before flow stops indicates the target’s concentration, enabling simple visual inspection readout without complex detection instrumentation. Because of its easy readout and portability, my system has great potential for use in point-of-care diagnostics. I initially demonstrated a proof-of-concept assay using biotin-streptavidin. Solution capillary flow distances scaled linearly with the negative logarithm of streptavidin concentration over a 100,000-fold range. I measured streptavidin concentrations as low as 1 ng/mL using these microsystems, demonstrating low detection limits. I also characterized the mechanism wherein time-dependent channel constriction in the first few millimeters leads to concentration-dependent flow distances. I demonstrated the visual detection and quantification capability of my system to determine an antigen target, thymidine kinase 1 (TK1). I developed surface modification methods for carrying out flow assays and verified receptor attachment on channel surfaces using fluorescence imaging. I obtained a 1 ng/mL TK1 detection limit in flow assays. I also demonstrated nucleic acid quantitation in my flow devices. I detected specific DNA targets in buffer and synthetic urine at 10 pg/mL levels. A dynamic range of 106 was obtained with single-base mismatch specificity. DNA analogues of two miRNA biomarkers were measured near clinically significant levels, showing great promise for future medical application. The promising results demonstrate that this diagnostic tool offers a simple route to analyte quantitation in microliter volumes, with excellent potential for point-of-care application.

Detection of disease specific biomarkers is of great importance in diagnosis and treatment of diseases. Modern bioanalytical techniques, such as liquid chromatography with mass spectrometry (LC-MS), have the ability to identify biomarkers, but their cost and scalability are two main drawbacks. Enzyme-linked immunosorbent assay (ELISA) is another potential tool, but it works best for proteins, rather than peptide biomarkers. Recently, microfluidics has emerged as a promising technique due to its small fluid volume consumption, rapidness, low fabrication cost, portability and versatility. Therefore, it shows prominent potential in the analysis of disease specific biomarkers. In this thesis, microfluidic systems that integrate monolith columns for preconcentration and on-chip labeling are developed to analyze several protein biomarkers. I have successfully fabricated cyclic olefin copolymer (COC) microfluidic devices with standard micromachining techniques. Monoliths are prepared in situ in microchannels via photopolymerization, and the physical properties of monoliths are optimized by varying the composition and concentration of monomers to achieve better flow and extraction. On-chip labeling of protein biomarkers is achieved by driving solution through the monolith using voltage and incubating fluorescent dye with protein retained in the monolith. Subsequently, the labeled proteins are eluted by applying voltages to reservoirs on the microdevice and detected by laser-induced fluorescence. Finally, automation of on-chip preconcentration and labeling is successfully demonstrated.