Master of Science / Department of Chemistry / Christopher T. Culbertson / Mixing is an essential process to many areas of science for example it is important in studying chemical reaction kinetics, chemical synthesis, DNA hybridization and PCR amplification. Mixing on the macroscale level is readily achieved through convection. Rapid mixing on microchips however, is problematic as the low Reynolds numbers and high Peclet numbers indicate that fluid flow is in the laminar regime and limits mixing on microchips to diffusion. Because of these limitations mixing on microchips is often relegated to diffusional mixing which requires long channels and long time periods. Several methods have been developed to increase the speed and efficiency of mixing on microfluidic devices. A variety of techniques have been employed to overcome these obstacles including for example 1) 3 dimensional channel designs to split up and recombine flows 2) employing sophisticated lithographic techniques to make grooves within a channel to generate transverse flows and 3) using lateral flow created by using spiral channels. Other groups have used outside energy sources to achieve mixing by changing of the zeta potential within the channel, using induced charge electroosmosis, and also by modifying the electrokinetic flow. We propose using dielectric elastomers (DEs) to modulate flow as a means to achieve rapid and active mixing on the microchip format. Electroactive polymers such as poly(dimethylsiloxane) function as DEs and are capable of converting electrical energy into mechanical energy. The application of an electrical potential across the PDMS results in a change in the dimensions of the PDMS dielectric layer between the two actuating electrodes creating an actuator. When employed in microfluidic devices this actuator can be used to change the volumes of the microfluidic channels on the PDMS. If the actuators are placed near a T-intersection where two components are entering the intersection the actuators can serve to improve mixing on microfluidic devices. Studies were conducted on how on the magnitude of the actuation, the frequency of actuation, the field strength, the electrode design and position relative to the T intersection, the channel dimensions and the overall channel design impacted mixing efficiency. Mixing results showed promise but further development of technology is necessary to achieve adequate mixing in microfluidic channels using DEs.

Doctor of Philosophy / Department of Chemistry / Takashi Ito / This research was focused on understanding how external stimuli affect the permeability of the chemically modified electrodes, and how the materials used in modifying the working electrodes respond to the changes in the surface charge. We adopted a voltammetric type electrochemical sensor to investigate the permeability effects induced by pH and organic solvents. The working electrodes used in this research were chemically modified with thioctic acid self assembled monolayer (TA SAM), track etched polycarbonate membranes (TEPCM) and PS-b-PMMA nanoporous films (polystyrene-block-polymethylmethacrylate). We studied the permeability behavior of each of the material upon application of external stimuli. In chapter 3, the permeability changes induced by change in surface charge of thioctic acid SAM was investigated. The surface charge of the monolayer was tuned by changing pH of the medium, which resulted in decrease of redox current of a negatively charged marker due to deprotonation of the surface –COOH groups of TA SAM. Decrease in redox current reflected a decrease in the reaction rate, and by using closed form equations the effective rate constants at several pKa values were extracted. In chapter 4, permeability changes induced by pH in TEPCM were investigated. We assessed the surface charge of these membranes via cyclic voltammetry generated for neutral and charged redox molecules. Limiting current of charged markers were affected by the surface charge induced by pH, where as the redox current for the neutral marker was not affected. Experimental redox currents were larger than the theoretical current, indicating that redox molecules preferentially distributed in a surface layer on the nanopore. Organic solvent induced permeability changes of PS-b-PMMA nanoporous films were investigated via electrochemical impedance spectroscopy and AFM. Higher response of pore resistance in the presence of organic solvents indicated either swelling of the nanoporous film or partitioning of organic solvents in the pores. However AFM data revealed that the permeability changes are due to partitioning of the solvents rather than swelling of the porous film, since there was no appreciable change if the pore diameter in the presence of solvents.

Doctor of Philosophy / Department of Chemistry / Daniel A. Higgins / In this dissertation, state-of-the-art fluorescence microscopy techniques are employed to probe unique nanoscale phenomena in poly(dimethylsiloxane) (PDMS) films and on single carbon nanofibers. In one study, the mobility and physical entrapment of single dye molecules in dry and solvent-loaded PDMS films is explored. Experiments are performed under dry nitrogen and at various levels of isopropyl alcohol (IPA) loading from the vapor phase, as monitored by a PDMS-coated quartz-crystal microbalance. Single molecules are shown to be predominantly immobile under dry conditions and mostly mobile under IPA-saturated conditions. FCS is used to measure the apparent diffusion coefficient, yielding a mean that is virtually independent of IPA loading and sample class. An increase in the population of mobile molecules under high IPA conditions is attributed to the filling of film micropores with solvent, rather than by incorporation of molecularly dispersed solvent into the PDMS. In a second study, the molecular mobility of both neutral and cationic molecules in cured PDMS films is studied as a function of oligomer extraction. Cross correlation and Bayesian burst analysis methods were used to quantify the populations of fixed and total molecules, respectively. The results show that the total concentration of dye increases with increased oligomer extraction, while the relative populations of fixed and mobile molecules decrease and increase, respectively. These results are relevant to the use of PDMS in microfluidics, nanofiltration and pervaporation membranes and solid phase microextraction fibers. In a final study, molecular beacons (MBs) were immobilized onto the ends of single, sol-gel encapsulated vertically-alligned carbon nanofibers (VACNFs) attached to a silicon electrode. MB fluorescence was monitored as a function of the potential applied to the VACNF in a three-electrode electrochemical cell. Application of positive potentials attracts the negatively charged backbone of the MB, causing hybridization of the stem and a reduction in beacon fluorescence. Negative potentials cause dehybridization of the stem, and an increase in MB fluorescence. This study presents the first measurement of potential-dependent dehybridization/rehybridization of MBs attached directly to the end of a single VACNF. These studies will help to characterize the mechanism by which future lab-on-a-chip devices will detect harmful bio-organisms.

Doctor of Philosophy / Department of Chemistry / Peter M.A. Sherwood / Thin films can be created on the surface of a metal, protecting it from oxidation and corrosion. Phosphate films have historically been a common choice for these corrosion resistant films. In this dissertation, the oxidation of vanadium metal by water and atmosphere is studied. Also, a series of phosphate films on the surface of vanadium metal were created and are studied as potential corrosion resistant films. Lastly, an independent study identifying the oxidation state of copper in a biological sample is carried out. To characterize these thin films, X-ray Photoelectron Spectroscopy (XPS) is employed. The reaction of vanadium metal with the atmosphere and distilled, de-ionized, water is studied. The core level and valence band results are explored and compared to calculated valence band spectra for some vanadium oxides. The etching of vanadium metal and reaction of the etched metal with a phosphoric acid solution are studied. Synthesized vanadium phosphate compounds serve as model compounds for the analysis of a phosphate coating created on the surface of vanadium metal by the reaction of vanadium metal with phosphoric acid by a newly developed bench top method. The core level and valence band regions for the compounds and coating are discussed along with cluster and band structure calculations for interpretation. The variation in the coating on vanadium metal by biasing the metal at different potentials during reaction is also studied. Coatings are also created on vanadium metal using different forms of phosphorus oxy-acid. An analysis of the various coatings is performed by XPS and accompanied by predictive calculations. In an additional study, the oxidation state of copper in a biological compound is identified. The analysis makes use of satellite features commonly seen in XPS to make the determination. A discussion of the origin of these features and the energy of the shifts is given, along with the results for the other core level XPS regions for the compound.

Doctor of Philosophy / Department of Chemistry / Christopher T. Culbertson / My doctoral dissertation consists of three fundamental studies: 1) synthesis of biocompatible materials that can be used as microfluidic substrates, 2) characterizing these materials with respect to properties important to microfluidic fabrication, biochemical separations and concentration enrichment, and 3) employing these novel devices for real world applications in bioanalytical chemistry. The surface properties of a substrate will dramatically affect the resolution and efficiency that can be obtained for a specific CE separation. Thus, the ability to modify the surface is very useful in tailoring a microfluidic chip to a specific separation mode. The substrates we have synthesized for microfluidic devices include metal oxide modified poly(dimethylsiloxane) (PDMS), poly(ethyleneoxide)-PDMS (PEO-PDMS) coblock polymers, and surfactant coated PDMS. The metal oxide modified PDMS materials we synthesized include silica-PDMS, titania-PDMS, vanadia-PDMS and zirconia-PDMS. The surfaces of these materials were characterized using contact angle, X-ray photoelectron spectroscopy (XPS), Raman, transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM) and electroosmotic mobility (EOM) measurements. All of the metal oxide modified PDMS surfaces were significantly more hydrophilic than native PDMS, suggesting potential application in separations of biopolymers. In addition to being more hydrophilic the EOF and zeta potential of the channels were stable and quite durable with aging. Well characterized silane chemistry was used to derivitize the surface of the PDMS metal oxide surfaces allowing a number of different functionalities to be placed on the surface. This method has the potential for wide applicability in many different fields, but specifically for the fabrication of microstructures that need specific surface chemistries. We have also made a number of advancements using sol-gel chemistry and laminar flow within microfluidic channels to fabricate nanoporous membranes. Sol-gel patterned membranes are a simple and facile method of incorporating nanoscale diameter channels within a microfluidic manifold. These membranes have been used to perform preconcentration of amino acids, proteins and small particles for further analysis and separation using CE. We are also using these membranes for further study in desilanization and protein recrystallization studies.

Master of Science / Department of Chemistry / Christopher T. Culbertson / A microfluidic device integrated with valves and a peristaltic pump was fabricated using multilayer soft lithography to analyze single cells. Fluid flow was generated and mammalian cells were transported through the channel manifold using the peristaltic pump. A laser beam was focused at the cross-section of the channels so fluorescence of individual labeled intact cells could be detected. Triggered by the fluorescence signals of intact cells, valves could be actuated so fluid flow was stopped and a single cell was trapped at the intersection. The cell was then rapidly lysed through the application of large electric fields and injected into a separation channel. Various conditions such as channel geometry, pumping frequency, control channel size, and pump location were optimized for cell transport. A Labview program was developed to control the actuation of the trapping valves and a control device was fabricated for operation of the peristaltic pump. Cells were labeled with a cytosolic dye, Calcein AM or Oregon Green, and cell transport and lysis were visualized using epi-fluorescent microscope. The cells were transported at rates of [simular to] 1mm/s. This rate was optimized to obtain both high throughput and single cell trapping. An electric field of 850-900 V/cm was applied so cells could be efficiently lysed and cell lysate could be electrophoretically separated. Calcein AM and Oregon Green released from single cells were separated and detected by laser-induced fluorescence. The fluorescence signals were collected by PMT and sampled with a multi-function I/O card. This analyzing method using microchip may be applied to explore other cellular contents from single cells in the future.

Doctor of Philosophy / Department of Chemistry / Christopher T. Culbertson / Microfluidic devices have a wide variety of biological applications. My Ph.D. dissertation focuses on three major projects. A) culturing a non-adherent immortal cell line within a microfluidic device under static and dynamic media flow conditions; B) designing and fabricating novel microfluidic devices for electrokinetic injecting analytes from a hydrodynamic fluid; and C) using this novel injection method to lyse single non-adherent cells by applying a high electric field across the cell at a microfluidic channel intersection. There are several potential advantages to the use of microfluidic devices for the analysis of single cells: First, cells can be handled with care and precision while being transported in the microfluidic channels. Second, cell culturing, handling, and analysis can be integrated together in a single, compact microfluidic device. Third, cell culturing and analysis in microfluidic devices uses only extremely small volumes of culturing media and analysis buffer. In this dissertation a non-adherent immortal cell line was studied under static media flow conditions inside a CO[subscript]2 incubator and under dynamic media flow conditions in a novel portable cell culture chamber. To culture cells they must first be trapped on a microfluidic device. To attempt to successfully trap cells, three different types of cellular traps were designed, fabricated and tested in polydimethylsiloxane (PDMS)-based microfluidic devices. In the first generation device, cubic-shaped traps were used. After 48 h of culturing in these devices the cell viability of 79 [plus or minus] 6 % (n = 3). In the second generation device, circular wells with narrow connecting channels were employed. However, after 12 h of culturing, no viable cells were found. While the second generation device was not capable of successfully culturing cells, it did demonstrate the importance of culturing under dynamic conditions which lead to next design. The third generation microfluidic device consisted of hydrodynamic shaped traps that were used to culture the cells in a less confined environment. The cell viability after 12 h in this design was 29 [plus or minus] 41% (n = 3). In addition to cell trapping, a novel electrokinetic injection method was developed for injecting analytes from a hydrodynamic flow into a separation channel that was followed by an electrokinetic separation. As the hydrodynamic flow could introduce some excess band broadening in the separation, the actual band broadening of an analyte was measured for different channel depths and hydrodynamic fluid flow rates. The results consistently showed that the separations performed on these devices were diffusion limited. Finally, using this novel injection method, single cell lysis was performed by applying a high voltage at the microfluidic channel intersection. The results of these studies may eventually be applied to help answer some fundamental questions in the areas of biochemistry and pharmaceutical science.

Master of Science / Department of Chemistry / Christopher T. Culbertson / The study of aphid salivary secretome has practical importance on understanding interactions of aphids and their host plants. Around 250 species of aphids out of the identified 4000 aphid species are considered as serious pests. The experiments were performed with pea aphids (Acyrthosiphon pisum) that were feeding on bean plants (Vivia fabe). Pea aphids feed on plant phloem sap by probing their stylet into the sieve elements of the plant and secreting saliva for external digestion. In order to collect aphid salivary proteins from the secreted saliva, small scale and large scale sample collection methods were carried out. The small scale sample method was performed in microfluidic devices using 10-25 aphids. Aphids were able to feed on the artificial diet by probing through a stretched ParafilmTM and survived for 2-3 days in the microfluidic devices. The experiments proved that the aphid survival and feeding rate could be improved with the factors such as ventilation, light intensity and increasing diet volume. However it was difficult to collect sufficient amounts of aphid saliva for detection using small scale devices. The large scale sample collection method was performed by feeding 8000 aphids in large screened chamber for 24/48h. The collected salivary samples after undergone a concentration process was capable of collecting detectable aphid salivary secretions. The experimental conditions were adjusted to obtain optimized HPLC separations. Finally, LC/MS/MS followed by peptide sequence database searching were able to identify potential aphid salivary proteins.

Doctor of Philosophy / Department of Chemistry / Christopher T. Culbertson / Chemical separations are a necessary component in many scientific analyses. Microfluidics, the use of micron-sized fluidic channels defined in glass or polymer blends, is a powerful branch of separation science that is developing rapidly. Miniaturized analytical devices offer important advantages compared to traditional bench-top techniques, most notably capillary electrophoresis (CE). This dissertation was focused on developing several novel methods to improve microfluidic based separations and techniques. The electrophoretic separation of small similarly charged analytes can be very difficult. Chapter 2 discusses a new buffer that has been developed for fast, high efficiency separations of amino acids by micellar electrokinetic chromatography (MEKC). This buffer is more environmentally friendly than the most commonly used surfactant containing buffers for MEKC separations. It uses a commercially available dish washing soap by Seventh Generation™ Inc. that contains three micelle forming agents; sodium lauryl ether sulfate (anionic), cocamidopropyl betaine (zwitterionic), and cocamide monoethanolamine (MEA) (non-ionic), and is completely void of organic solvents. Many biological samples contain analytes below the limit of detection of traditional detection systems; therefore, chapter 3 reports the fabrication of nanoporous membranes on microfluidic devices that are capable of analyte concentration enrichment. Donnan exclusion is responsible for the preconcentration of fluorescent dyes near a charged, porous titania membrane. The level of analyte enrichment was monitored, and enrichment factors greater than 4000 in 400 s were obtained for 2,7-Dichlorofluorescein. Chapter 4 describes the fabrication and characterization of paper based microfluidic devices. Mixtures of acrylate modified photocurable polymers were used to photolithographically define channels on multiple paper substrates. Flow characteristics are described and their use for monitoring complications associated with type 1 diabetes is demonstrated. Finally in Chapter 5, Sol-gel modified gold surfaces for preventing protein adsorption during surface plasmon resonance (SPR) detection are also presented.

Doctor of Philosophy / Department of Chemistry / Christopher T. Culbertson / During the last five years, great advancements in microfluidics have been achieved with the development of “sample-in-answer-out” systems. Such systems have begun to realize the true potential of analytical miniaturization since the concept of the “micro-Total Analysis System” was first envisioned. These systems are characterized by the elegant integration of multiple fluid-handling channel architectures that enable serial execution of sample preparation, separation and detection techniques on a single device. While miniaturization and portability are often identified as key advantages for microfluidics, these highly integrated systems are heavily reliant upon large off-chip equipment, i.e. the microchip is often tethered to the laboratory via multiple syringe pumps, vacuum pumps, solenoid valves, gas cylinders and high voltage power supplies. In this dissertation, a procedure for the facile integration of dielectric elastomer (DE) actuators (called IDEAs) onto microfluidic devices is described. Poly(dimethylsiloxane) (PDMS) is commonly used as a microchip substrate because it is cheap and easy to fabricate, mechanically robust and optically transparent. The operation of an IDEA exploits the ability of PDMS to behave as a smart material and deform in the presence of an electric field. In Chapter 2, the fabrication of IDEA units on a standard microchip electrophoresis device is described. IDEA-derived injections were used to evaluate the physical performance of this novel actuator configuration. In Chapter 3, the analytical merits of IDEA-derived injections were evaluated. Sampling bias caused by electokinetic injection techniques has been problematic for conventional microchip electrophoresis systems due to the lack of fluid access. The hydrodynamic injections created by IDEA operation were found to be highly reproducible, efficient, and possess a negligible degree of sampling bias. In Chapter 4, the spatial characteristics of microchannel deformation due to IDEA actuation have been investigated using fluorescence microscopy. It was determined that the DE compresses more along the edge of the channel than in the middle of the channel. This information can be used to design a new generation of more efficient IDEAs.