Thesis (Ph. D.)--Washington State University, December 2007. / Includes bibliographical references.

abstract: Bioanalytes such as protein, cells, and viruses provide vital information but are inherently challenging to measure with selective and sensitive detection. Gradient separation technologies can provide solutions to these challenges by enabling the selective isolation and pre-concentration of bioanalytes for improved detection and monitoring. Some fundamental aspects of two of these techniques, isoelectric focusing and dielectrophoresis, are examined and novel developments are presented. A reproducible and automatable method for coupling capillary isoelectric focusing (cIEF) and matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) based on syringe pump mobilization is found. Results show high resolution is maintained during mobilization and &beta-lactoglobulin; protein isoforms differing by two amino acids are resolved. Subsequently, the instrumental advantages of this approach are utilized to clarify the microheterogeneity of serum amyloid P component. Comprehensive, quantitative results support a relatively uniform glycoprotein model, contrary to inconsistent and equivocal observations in several gel isoelectric focusing studies. Fundamental studies of MALDI-MS on novel superhydrophobic substrates yield unique insights towards an optimal interface between cIEF and MALDI-MS. Finally, the fundamentals of isoelectric focusing in an open drop are explored. Findings suggest this could be a robust sample preparation technique for droplet-based microfluidic systems. Fundamental advancements in dielectrophoresis are also presented. Microfluidic channels for dielectrophoretic mobility characterization are designed which enable particle standardization, new insights to be deduced, and future devices to be intelligently designed. Dielectrophoretic mobilities are obtained for 1 µm polystyrene particles and red blood cells under select conditions. Employing velocimetry techniques allows models of particle motion to be improved which in turn improves the experimental methodology. Together this work contributes a quantitative framework which improves dielectrophoretic particle separation and analysis. / Dissertation/Thesis / Ph.D. Chemistry 2011

Doctor of Philosophy / Department of Chemistry / Christopher T. Culbertson / In research and industry, almost all chemical analysis methods involve the separation and detection of compounds. Typically, these separations are performed using traditional methods that require volumes in the 10 μL to 10 mL range of sample and in the 200 mL to 2 L range for solvents. These methods are not suitable for low-concentration, volume-limited samples frequently associated with biochemical studies. One way to overcome these limitations is to move the separation and detection to the microscale. The use of the microscale separation technologies enables the study of biological systems that have, until now, been out of reach due to their small volumes or low concentrations. The research presented in this dissertation will discuss two examples of this shift to microscale separation technologies which can solve some small volume sample challenges. These include the detection of protease activity in blood samples for use in cancer detection and the identification of immune system cascade proteins in the mosquito Anopheles gambiae. In Chapter 2 a microfluidic method and device is proposed to monitor protease activities for cancer detection. In this method nanobiosensors are used to measure enzyme activity in biological fluids. These nanobiosensors consist of iron-iron oxide magnetic nanoparticles that are attached to peptide substrates specific for proteases through a disulfide bond. The nanobiosensors are controlled using a neodymium magnet which is attached through a 3D printed adaptor to a rotating motor for mixing and a linear stage to move the nanoparticles between different sections of the device. The separation and detection sections of the device are explained in Chapter 3. Chapter 3 describes the fabrication and optimization of a simple device for microfluidic isoelectric focusing(IEF). IEF is a separation method in which analytes are separated based upon their isoelectric, i.e. neutral charge, points. A reducing agent can be added to the IEF buffer to detach the nanoparticle from the peptide substrate, releasing it for focusing. IEF is also a concentration as well as separation method that will allow the peptide substrates to be focused up to 10⁶ fold. It has a high peak capacity and produces reliable, reproducible separation patterns based on the isoelectric point of the peptide. To meet the detection limits required for cancer detection with proteases, scanning laser induced fluorescence is selected as the method of detection. This scanning system can monitor the separation over time to observe the parameters affecting the separation which cannot be done with typical point or imaging detection systems and allows better separation. This custom automatic detection system can distinguish focused samples of 500 fM from the background with minimal noise from the scanning system. In Chapter 4 the identification of serine protease and inhibitor binding complexes in A. gambiae hemolymph using magnetic bead immunoaffinity chromatography was attempted. These proteases play a key role in the insect innate immunity system and form irreversible complexes. These complexes can be purified from a complex hemolymph sample using an antibody to one of the complex members. To separate the complexes from the hemolymph, Serpin 2 antibodies were attached to protein A coated magnetic beads and then incubated with the hemolymph. Once the purified complexes and Serpin 2 were eluted, the purified proteases were identified on Orbitrap MS. In an attempt to simplify the isolation of the complexes, a magnetic bead mixing rotor column was developed to help reduce the volume of the elution to increase the concentration. This method, however, was not robust and did not improve the concentration.

Microencapsulation of stem cells in hydrogel for use in therapeutic applications has been shown to improve cell retention at the site of injuries due to their mechanical and immunoprotective properties. These microscale droplets (cocoons) can be produced at high throughputs within microfluidic channels. Currently, the ability for cells to egress hydrogel cocoons is under investigation. This egress can correlate with therapeutic efficacy, and so promoting or inhibiting the egress of cells can be a vital component of viable treatments. Previously, a second hydrogel layer was shown to reduce egress, but issues involving cell proliferation were unchanged. We propose a microfluidic process to encapsulate cells in two layers of thermoresponsive hydrogels, in which the inner core melts at physiological temperatures to form hollow cocoons that allow cells free motion inside the immunoprotective shell. We hypothesize that the open volume would increase cell viability and proliferation, without increasing cell egress due to the uninterrupted hydrogel shell. In this project the encapsulation of NIH 3T3 cells in hollow agarose cocoons was achieved. 3T3 cells were first encapsulated in thermoreversible gelatin which were then re-encapsulated in agarose through the use of a flow-focusing microfluidic channel with on-chip mixing of two inlet flows to produce hollow cocoons. The production of these cocoons showed the potential of high throughput, monodisperse samples with future investment. Preliminary investigation in the behavior of the encapsulated cells showed that the cells maintain high viability over the course of 48 hours. There are early indications that the hollow nature of correctly formed cocoons can limit cell egress, and may allow for proliferation in the cocoon.

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Factor VIII is a protein which has therapeutic applications for the treatment of Hemophilia A. Its deficiency, either qualitative or quantitative, results in Hemophilia A, a disorder affecting approximately 1 in 10,000 males. Currently, FVIII replacement therapy uses FVIII derived from plasma or cell culture. The current cost of this therapy is in excess of $150,000 per patient per year. Thus, alternative sources that are more economical are attractive. The present work focuses upon the characterization of recombinant FVIII (rFVIII) made in the milk of transgenic pigs. Two dimensional western analysis of rFVIII obtained from pig whey showed a range of FVIII species having different isoelectric points (pI) consistent with diverse glycosylation patterns. The pI of these diverse FVIII populations were accurately predicted using theoretical calculations based upon primary protein structure as variable biantennary glycosylation patterns having 0, 1, or 2 sialic acid groups present. Kinetic limitations in the adsorption of rFVIII to anion exchange media due to the nature of the complex milk environment were observed. rFVIII was purified quantitatively using batch equilibration of whey with DEAE Sepharose. This material showed proteolytic processing that was very similar to FVIII obtained from human plasma. Based upon these results, it was postulated that a dissociation of the light (A3C1C2) and heavy (A1A2B) chain due to a lack of vWF may be responsible for the low FVIII activity. / Master of Science

Electroporation is an efficient, low-toxic physical method which is used to deliver impermeant macromolecules such as genes and drugs into cells. Genetic modification of the cell is critical for many cell and gene therapy techniques. Common electroporation protocols can only handle small volumes of cell samples. Also, most of the conventional electroporation methods require expensive and sophisticated electro-pulsation equipment. In our lab, we have developed new electroporation methods conducted in microfluidic devices. In microfluidic-base electroporation, exogenous macromolecules can be delivered into cells continuously. Flow-through electroporation systems can overcome the issue of low sample volume limitation. In addition, in our method, electro-pulsation can be done by using a simple dc power supply, without the need for any extra equipment. Furthermore, our microfluidic chips are completely disposable and cheap to produce. We show that electroporation and electroporation-based gene delivery can be conducted employing tapered asymmetric curving channels. The size variation in the channel's cross-sectional area makes it possible to produce electric pulses of various parameters by using a dc power supply. We successfully delivered Enhanced Green Fluorescent Protein, EGFP, plasmid DNA into Chinese Hamster Ovary, CHO-K1, cells in our microfluidic chips. We show that the particles/cells undergo Dean flow in our asymmetric curving channels. We demonstrate that there are three main regimes for particle motion in our channels. At low flow rates (from 0 to ~75μl/min) cells do not focus and they randomly follow stream lines. However, as flow rate increases (~75 to 500μl/min), cells begin to focus into one line and they follow a single path throughout the micro-channel. When flow rate exceeds ~500μl/min, cells do not follow a single line and demonstrate more complex pattern. We show that the electric parameters affect the transfection efficiency and cell viability. Higher electric field intensity results in higher transfection efficiency. This is also true in the cases with longer electroporation duration time. In our experimental work, we executed flow-through electroporation for various duration times (t = 2 ms, 5 ms, and 7 ms), and at various electric field intensities (from 300 to 2200 V/cm) while we utilized different flow rates as well, i. e. 150 μl/min (focused flow) and 600 μl/min (complex flow). To explore the impact of individual electric pulse length and electric pulse number on electroporation results, we designed control channels with straight narrow sections. Cells experience different hydrodynamic forces in straight channels compared to curving channels. Flow pattern and cell focusing were also studied in control channels as well. Also, electroporation on CHO-K1 cells was successfully conducted in control channels. The hydrodynamic forces under the conditions we used do not appear to show substantial impact on transfection efficiency. / Master of Science

Microfluidic devices are ideal for analysis of biological systems. The small dimensions result to controlled handling of the flow profile and the cells in suspension. Implementation of additional forces in the system, such as an electric field, promote further manipulation of the cells. In this dissertation, I show novel, unique microfluidic approaches for manipulation and analysis of mammalian cells by the aid of electrical methods or the architecture of the device. Specifically, for the first time, it is shown, that adoption of electrical methods, using surface electrodes, promotes cell concentration in a microchamber due to isoelectric focusing (IEF). In contrast to conventional IEF techniques for protein separation, a matrix is not required in our system, the presence of which would even block the movement of the bulky cells. Electric field is, also, used to breach the cell membrane and gain access to the cell interior by electroporation (irreversible and reversible). Irreversible electroporation is used in a unique, integrated microfluidic device for cell lysis and reagentless extraction of DNA. The genomic material is subsequently analyzed by on-chip PCR, demonstrating the possible elimination of the purification step. On the other hand, reversible electroporation is used for the delivery of exogenous molecules to cells. For the first time, the effect of shear stress on the electroporation efficiency of both attached and suspended cells is examined. On the second part of my dissertation, I explore the capabilities of the architecture of microfluidic devices for cell analysis. A simple, unique method for compartmentalization of a microchamber in an array of picochambers is presented. The main idea of the device lies on the fabrication of solid supports on the main layer of the device. These features may even hold a dual nature (e.g. for cell trapping, and chamber support), in which case, single cell analysis is possible (such as single cell PCR). On the final chapter of my dissertation, a computational analysis of the flow and concentration profiles of a device with hydrodynamic focusing is conducted. I anticipate, that all these novel techniques will be used on integrated microfluidic systems for cell analysis, towards point-of-care diagnostics. / Ph. D.