Membrane chromatography is a promising process for the isolation, purification, and recovery of proteins, enzymes, and nuclear acids. Comparing with traditional beads column chromatography, membrane chromatography can faster, easier and cheaper to mass-produce. And also, it is easy to set up and scale up. In this thesis, we are trying to study the performance of membrane chromatography, and the mixture of HSA and chicken egg white is used as an example. We are investigating the purification of Human serum albumin (HSA) from chicken egg white in terms of precondition, dilution, purification method, product recovery, product purity and product cost. HSA, is a very important clinical protein. In order to obtain low cost, high efficiency and less risk HSA, recombinant DNA technology is used. Many kinds of host organism have been used to produce recombinant HSA (rHSA). In this thesis, a kind of ion-exchange membrane (Mustang Q membrane capsule) chromatography was used. The membrane capsule is disposable because it is designed for use in pharmaceutical production. For this project, a cleaning method was used which made the membrane capsule reusable. Washing with 4 mL 1 M NaCl and 4 mL NaOH was sufficient for this purpose. Since the egg white protein solution was very viscous, it needs to be diluted before loaded on FPLC. Dilute experiment was done to find the best dilution level. In this thesis, we found that 5 times dilution was best not only for high efficiency but also for FPLC operation. After getting the basic conditions, some purification experiments were done to find the optimal operation condition to purify HSA form chicken egg white protein solution by changing buffer pH, salt concentration in elution buffer and gradient used to elute proteins. The best purification condition for loading buffer is Tris-HCl buffer A (4.75g/L, pH 9.5) and the elution buffer is Tris-HCl buffer A + 0.2M NaCl. The purity of HSA recovered was 93% on the Mustang Q membrane capsule at 1 ml/min when the mixture of HSA and chicken egg white was diluted 10 times. And the yield was 85%. The impurity is probably ovoglobulin as suggested by the result of SDS-PAGE, whose molecular weight is close to 40kd. To characterize the separation capability of the Mustang Q membrane capsules, equilibrium adsorption and breakthrough curve studies were made using bovine serum albumin (BSA). 1mg/mL BSA solution was used to get the breakthrough curve with different flow rate ranging from 1 to 4 ml/min. With a flow rate is 1 ml/min, breakthrough curve were obtained with different concentrations of BSA ranging from 1 to 16 mg/mL. The dynamic binding capacity was found to be from 9.1 to 119.1 mg/mL. The equilibrium adsorption isotherm showed Langmuir isotherm behavior with dissociation constant and a maximum adsorption capability. According to the result of isotherm adsorption, a multi-plate mathematical model was used to get the theoretical breakthrough curve. By fitting the theoretical breakthrough curve to the experimental breakthrough curve, constants in the multi-plate model were obtained and were used to estimate the axial dispersion coefficient of the membrane capsule. The estimated axial dispersion coefficient of 2.45*10-6 cm2/s is very small which means that the axial ispersion is not significant. The adsorption process is therefore controlled by radial radius dispersion or film dispersion.

Electrophoresis separation corresponds to the motion and separation of dispersed particles under the influence of a constant electric field. In molecular biology, electrophoresis separation plays a major role in identifying, quantifying and studying different biological samples such as proteins, peptides, RNA acids, and DNA. In electrophoresis separation, different characteristics of particles, such as charge to mass ratio, size, and pI, can be used to separate and isolate those particles. For very complex samples, two or more characteristics can be combined to form a multi-dimensional electrophoresis separation system, significantly improving separation efficiency. Much effort has been devoted in recent years to performing electrophoresis separations in microfluidic format. Employing microfluidic technology for this purpose provides several benefits, such as improved transport control, reduced sample volumes, simplicity of operation, portability, greater accessibility, and reduced cost. The aim of this study is to develop microfluidic systems for high-performance separation of biochemical samples using electrophoresis methods. The first part of the thesis concerns the development of a fully integrated microfluidic chip for isoelectric focusing separation of proteins with whole-channel imaging detection. All the challenges posed in fabricating and integrating the chip were addressed. The chip was tested by performing protein and pI marker separations, and the separation results obtained from the chip were compared with those obtained from commercial cartridges. Side-by-side comparison of the results validated the developed chip and fabrication techniques. The research also focuses on improving the peak capacity and separation resolution of two counter-flow gradient electrofocusing methods: Temperature Gradient Focusing (TGF) and Micellar Affinity Gradient Focusing (MAGF). In these techniques, a temperature gradient across a microchannel or capillary is used to separate analytes. With an appropriate buffer, the temperature gradient creates a gradient in the electrophoretic velocity (TGF) or affinity (MAGF) of analytes and, if combined with a bulk counter-flow, ionic species concentrate at unique points where their total velocity is zero, and separate from each other. A bilinear temperature gradient is used along the separation channel to improve both peak capacity and separation resolution simultaneously. The temperature profile along the channel consists of a very sharp gradient used to pre-concentrate the sample, followed by a shallow gradient that increases separation resolution. A simple numerical model was applied to predict the improvement in resolution when using a bilinear gradient. A hybrid PDMS/glass chip integrated with planar micro-heaters for generating bilinear temperature gradients was fabricated using conventional sputtering and soft lithography techniques. A specialized design was developed for the heaters to achieve the desired bilinear profiles using both analytical and numerical modeling. To confirm the temperature profile along the channel, a two-color thermometry technique was also developed for measuring the temperature inside the chip. Separation performance was characterized by separating several different dyes, amino acids and peptides. Experiments showed a dramatic improvement in peak capacity and resolution of both techniques over the standard linear temperature gradients. Next, an analytical model was developed to investigate the effect of bilinear gradients in counter-flow gradient electrofocusing methods. The model provides a general equation for calculating the resolution for different gradients, diffusion coefficients and bulk flow scan rates. The results indicate that a bilinear gradient provides up to 100% improvement in separation resolution over the linear case. Additionally, for some scanning rates, an optimum bilinear gradient can be found that maximizes separation resolution. Numerical modeling was also developed to validate some of the results. The final part of the thesis describes the development of a two-dimensional separation system for protein separation, combining temperature gradient focusing (TGF) and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) in a PDMS/glass microfluidic chip. An experimental study was performed on separating a mixture of proteins using two characteristics: charge to mass ratio, and size. Experimental results showed a dramatic improvement in peak capacity over each of the one-dimensional separation techniques.

Electrophoresis separation corresponds to the motion and separation of dispersed particles under the influence of a constant electric field. In molecular biology, electrophoresis separation plays a major role in identifying, quantifying and studying different biological samples such as proteins, peptides, RNA acids, and DNA. In electrophoresis separation, different characteristics of particles, such as charge to mass ratio, size, and pI, can be used to separate and isolate those particles. For very complex samples, two or more characteristics can be combined to form a multi-dimensional electrophoresis separation system, significantly improving separation efficiency. Much effort has been devoted in recent years to performing electrophoresis separations in microfluidic format. Employing microfluidic technology for this purpose provides several benefits, such as improved transport control, reduced sample volumes, simplicity of operation, portability, greater accessibility, and reduced cost. The aim of this study is to develop microfluidic systems for high-performance separation of biochemical samples using electrophoresis methods. The first part of the thesis concerns the development of a fully integrated microfluidic chip for isoelectric focusing separation of proteins with whole-channel imaging detection. All the challenges posed in fabricating and integrating the chip were addressed. The chip was tested by performing protein and pI marker separations, and the separation results obtained from the chip were compared with those obtained from commercial cartridges. Side-by-side comparison of the results validated the developed chip and fabrication techniques. The research also focuses on improving the peak capacity and separation resolution of two counter-flow gradient electrofocusing methods: Temperature Gradient Focusing (TGF) and Micellar Affinity Gradient Focusing (MAGF). In these techniques, a temperature gradient across a microchannel or capillary is used to separate analytes. With an appropriate buffer, the temperature gradient creates a gradient in the electrophoretic velocity (TGF) or affinity (MAGF) of analytes and, if combined with a bulk counter-flow, ionic species concentrate at unique points where their total velocity is zero, and separate from each other. A bilinear temperature gradient is used along the separation channel to improve both peak capacity and separation resolution simultaneously. The temperature profile along the channel consists of a very sharp gradient used to pre-concentrate the sample, followed by a shallow gradient that increases separation resolution. A simple numerical model was applied to predict the improvement in resolution when using a bilinear gradient. A hybrid PDMS/glass chip integrated with planar micro-heaters for generating bilinear temperature gradients was fabricated using conventional sputtering and soft lithography techniques. A specialized design was developed for the heaters to achieve the desired bilinear profiles using both analytical and numerical modeling. To confirm the temperature profile along the channel, a two-color thermometry technique was also developed for measuring the temperature inside the chip. Separation performance was characterized by separating several different dyes, amino acids and peptides. Experiments showed a dramatic improvement in peak capacity and resolution of both techniques over the standard linear temperature gradients. Next, an analytical model was developed to investigate the effect of bilinear gradients in counter-flow gradient electrofocusing methods. The model provides a general equation for calculating the resolution for different gradients, diffusion coefficients and bulk flow scan rates. The results indicate that a bilinear gradient provides up to 100% improvement in separation resolution over the linear case. Additionally, for some scanning rates, an optimum bilinear gradient can be found that maximizes separation resolution. Numerical modeling was also developed to validate some of the results. The final part of the thesis describes the development of a two-dimensional separation system for protein separation, combining temperature gradient focusing (TGF) and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) in a PDMS/glass microfluidic chip. An experimental study was performed on separating a mixture of proteins using two characteristics: charge to mass ratio, and size. Experimental results showed a dramatic improvement in peak capacity over each of the one-dimensional separation techniques.

Whey protein fractionation is an important industrial process that requires effective large-scale processes. Although packed bed chromatography has been used extensively, it suffers from low processing rates due to high back-pressures generated at high flow rates. Batch chromatography has been applied but generally has a low efficiency. More recently, adsorptive membranes have shown great promise for large-scale protein purification, particularly from large-volume dilute feedstocks. A new method for producing versatile adsorptive membranes by combining membrane and chromatographic resin matrices has been developed but not previously applied to whey protein fractionation. In this work, a series of mixed matrix membranes (MMMs) were developed for membrane chromatography using ethylene vinyl alcohol (EVAL) based membranes and various types of adsorbent resin. The feasibility of MMM was tested in bovine whey protein fractionation processes. Flat sheet anion exchange MMMs were cast using EVAL and crushed Lewatit® MP500 (Lanxess, Leverkusen, Germany) anion resin, expected to bind the acidic whey proteins β-lactoglobulin (β-Lac), α-lactalbumin (α-Lac) and bovine serum albumin (BSA). The MMM showed a static binding capacity of 120 mg β-Lac g⁻¹ membrane (36 mg β-Lac mL⁻¹ membrane) and 90 mg α-Lac g⁻¹ membrane (27 mg α-Lac mL⁻¹ membrane). It had a selective binding towards β-Lac in whey with a binding preference order of β-Lac > BSA > α-Lac. In batch whey fractionation, average binding capacities of 75.6 mg β-Lac g⁻¹ membrane, 3.5 mg α-Lac g⁻¹ membrane and 0.5 mg BSA g⁻¹ membrane were achieved with a β-Lac elution recovery of around 80%. Crushed SP Sepharose™ Fast Flow (GE Healthcare Technologies, Uppsala, Sweden) resin was used as an adsorbent particle in preparing cation exchange MMMs for lactoferrin (LF) recovery from whey. The static binding capacity of the cationic MMM was 384 mg LF g⁻¹membrane or 155 mg LF mL⁻¹ membrane, exceeding the capacity of several commercial adsorptive membranes. Adsorption of lysozyme onto the embedded ion exchange resin was visualized by confocal laser scanning microscopy. In LF isolation from whey, cross-flow operation was used to minimize membrane fouling and to enhance the protein binding capacity. LF recovery as high as of 91% with a high purity (as judged by the presence of a single band in gel electrophoresis) was achieved from 150 mL feed whey. The MMM preparation concept was extended, for the first time, to produce a hydrophobic interaction membrane using crushed Phenyl Sepharose™ (GE Healthcare Technologies, Uppsala, Sweden) resin and tested for the feasibility in whey protein fractionation. Phenyl Sepharose MMM showed binding capacities of 20.54 mg mL⁻¹ of β-Lac, 45.58 mg mL⁻¹ of α-Lac, 38.65 mg mL⁻¹ of BSA and 42.05 mg mL⁻¹ of LF for a pure protein solution (binding capacity values given on a membrane volume basis). In flow through whey fractionation, the adsorption performance of the Phenyl Sepharose MMM was similar to the HiTrap™ Phenyl hydrophobic interaction chromatography column. However, in terms of processing speed and low pressure drop across the column, the benefits of using MMM over a packed bed column were clear. A novel mixed mode interaction membrane was synthesized in a single membrane by incorporating a certain ratio of SP Sepharose cation resin and Lewatit MP500 anion resin into an EVAL base polymer solution. The mixed mode cation and anion membrane chromatography developed was able to bind basic and acidic proteins simultaneously from a solution. Furthermore, the ratio of the different types of adsorptive resin incorporated into the membrane matrix could be customised for protein recovery from a specific feedstream. The customized mixed mode MMM consisting of 42.5 wt% of MP500 anionic resin and 7.5 wt% SP Sepharose cationic resin showed a binding capacity of 7.16 mg α-Lac g⁻¹ membrane, 11.40 mg LF g⁻¹ membrane, 59.21 mg β-Lac g⁻¹ membrane and 6.79 mg IgG g⁻¹ membrane from batch fractionation of 1 mL LF-spiked whey. A tangential flow process using this membrane was predicted to be able to produce 125 g total whey protein per L membrane per h.

Sepsis is a “life-threatening organ dysfunction caused by a dysregulated host response to infection” that has a widespread impact on human life around the world. It affects more than 1.5 million people, killing at least 250,000 each year in the US alone and affects 90,000 people annually, with estimated mortality rates of up to 30% in Canada. Our understanding of the different biochemical pathways that in the progression of sepsis has improved patient care for sepsis patients. One part of patient care is the use of biomarkers for patient prognosis that draws on the full range of relevant and available information to model the possible outcomes for an individual. Numerous biomarkers have been studied for patient prognosis that includes Procalcitonin (PCT), C-reactive protein (CRP), TNF-α, cfDNA, protein C and PAI 1. Using a panel of multiple biomarkers provided more accuracy in patient prognosis than using individual biomarkers and one such panel that was proposed used cfDNA, protein C, platelet count, creatinine, Glasgow Coma Scale [GCS] score, and lactate. Commercial, low cost POC techniques were available for the measurement of all biomarkers besides cfDNA and protein C. The objective of this doctoral thesis was chosen to develop low cost, microfluidic devices for the measurement of protein C and cfDNA using nonspecific fluorescence dyes that would enable the eventual integration of the systems and improve patient prognosis. The measurement of protein C in plasma required the separation of protein C from interfering proteins in plasma. This was done through the development of a two-stage separation process that included the development of tunable agarose isoelectric gates for separating proteins using their isoelectric point and the miniaturization of immobilized metal affinity chromatography and its extension to Barium for the selective binding of proteins using their chemical affinity. This was performed in a xurographically fabricated chip to reduce costs and enable the use of geometric focusing of the electric field to enable the operation of the device at a lower applied voltage. The challenges faced with cfDNA were different due to the different characteristics of the material and less interference from plasma. The requirement was to measure the total cfDNA content with minimal cost in comparison to currently available techniques. This was achieved through the development of thread microfluidic devices that showed the use of thread for automated aliquoting of samples by controlling length and twists of the thread. Preconcentration and use of external apparatus was avoided by showing that thread could be used to amplify fluorescence response to a range that was sufficient for the measurement of cfDNA in sepsis patients. A portable fluorescence imaging setup was developed for this purpose and was used in demonstration for the measurement of cfDNA in plasma with sufficient resolution. In conclusion, we developed technologies for rapid and low-cost measurement of protein C and cfDNA using xurographic and thread-based microfluidics that may serve as valuable in improving patient prognosis. / Thesis / Doctor of Philosophy (PhD) / Sepsis is a major reason for hospitalization and cause of death in hospitals worldwide. Its treatment is highly time sensitive with each hour of delay in diagnosis causing a significant increase in chances of death. Due to the wide range of symptoms that can be caused by sepsis, its diagnosis uses a scoring method that relies on the expertise of the onsite doctors and nurses increasing their workload. A more objective system for detection requires the measurement of the quantities of different biomarkers in blood. Biomarkers are proteins present in plasma that change in quantity due to the body’s reaction to sepsis. Several of these biomarkers have been identified and studied for their use in both diagnosing the presence of sepsis and in predicting the outcome with the current treatment plan. In this PhD study, we chose two of these biomarkers – circulating free DNA (cfDNA) and protein C and developed low-cost techniques for rapidly measuring their concentration in blood plasma. To do this, we made microfluidic devices with techniques that use low-cost materials such as plastic sheets and threads.The device for the measurement of protein C required separating it from many other proteins in plasma. We showed that a device fabricated from stacked plastic sheets and integrated with agarose gels could be used for the measurement of protein C in plasma with sufficient resolution to help with treating septic patients at a cost of less $5 per device. Similarly, we showed that a device that integrated threads with plastic sheets could be used for measuring the quantity of cfDNA in plasma in a portable format within 15 minutes. Overall, we developed tools for rapid measurement of two biomarkers of sepsis using low cost device that cost under $5 to run and could led to improving the quality of care for sepsis patients.

Chickpea grain contains a high amount of starch and valuable protein. Many grain legumes (pulses) can be processed by pin milling and air classification with high separation efficiency. However, chickpea exhibits low separation efficiency because it has a relatively high fat content compared to other pulses. Therefore, the main goal of this research was to improve the starch-protein separation from chickpea flour in order to increase the economic value of chickpea grain.<p>The chemical composition of pin-milled chickpea flour was determined. The functional and physical properties of chickpea flour affecting starch-protein separation were determined. No chemical interactive force was detected between starch granules and protein particles. Therefore, a physical separation technique, i.e. applying centrifugal force in a hydrocyclone, was employed to separate starch granules from protein particles. <p>Using a hydrocyclone, centrifugal force was applied to chickpea flour particles. Chickpea flour was suspended in two different media, isopropyl alcohol or deionized water. In both media, high inlet pressure resulted in smaller geometric mean diameter of particles collected in the overflow and underflow. Isopropyl alcohol as a medium resulted in particles with smaller geometric mean diameter than did deionized water. Starch and protein separation efficiencies were higher at greater inlet pressures. The application of a double-pass hydrocyclone process increased the purity of starch in the underflow and of protein in the overflow, although this process reduced separation efficiencies. Starch granules and protein particles were separated at higher purities in deionized water than in isopropyl alcohol. Separation in deionized water resulted in higher starch separation efficiency and lower protein separation efficiency than did separation in isopropyl alcohol. This difference was due to the difference in density and viscosity of the two media. The higher viscosity of isopropyl alcohol reduced the likelihood of starch granules reaching the inner hydrocyclone wall. Thus, some starch granules were retained in the overflow instead of in the underflow. Additionally, the centrifugal force and drag force applied to the chickpea flour particles differed between the two different media. Hydrocyclone operation resulted in higher centrifugal force and lower drag force in deionized water than in isopropyl alcohol. Since the drag force in isopropyl alcohol was higher than that in deionized water, some small starch granules were diverted to the overflow which caused reduction of protein purity. <p>The use of pH 9.0 and defatting of chickpea flour improved both starch and protein separation efficiencies. Chickpea flour in deionized water at a feed concentration of 5% yielded a pumpable slurry which was delivered efficiently to the hydrocyclone at an inlet pressure of 827 kPa Fractionation of starch and protein from chickpea flour in deionized water using an integrated separation process resulted in starch and protein fractions containing 75.0 and 81.9% (d.b.) starch and protein, respectively. This process resulted in starch and protein separation efficiencies of 99.7 and 89.3%, respectively. <p>Experiments were also conducted to determine the physical and functional properties of chickpea flour and starch and protein fractions. Thermal conductivity, specific heat, and thermal diffusivity were determined and the polynomial linear models were fitted very well to experimental data. Internal and external friction properties of chickpea flour and starch and protein fractions were determined. Samples were subjected to uniaxial compression testing to determine force-time relationships. The samples particles underwent rearrangement rather than deformation during compression. The asymptotic modulus of samples was also computed, and it was linearly related to maximum compressive pressure. The functional properties of fractionated products were highly affected by the separation process. The water hydration capacity of starch fraction increased, whereas the emulsion capacity and foaming capacity of starch and protein fractions were reduced, compared to that of chickpea flour.

Chickpea grain contains a high amount of starch and valuable protein. Many grain legumes (pulses) can be processed by pin milling and air classification with high separation efficiency. However, chickpea exhibits low separation efficiency because it has a relatively high fat content compared to other pulses. Therefore, the main goal of this research was to improve the starch-protein separation from chickpea flour in order to increase the economic value of chickpea grain.<p>The chemical composition of pin-milled chickpea flour was determined. The functional and physical properties of chickpea flour affecting starch-protein separation were determined. No chemical interactive force was detected between starch granules and protein particles. Therefore, a physical separation technique, i.e. applying centrifugal force in a hydrocyclone, was employed to separate starch granules from protein particles. <p>Using a hydrocyclone, centrifugal force was applied to chickpea flour particles. Chickpea flour was suspended in two different media, isopropyl alcohol or deionized water. In both media, high inlet pressure resulted in smaller geometric mean diameter of particles collected in the overflow and underflow. Isopropyl alcohol as a medium resulted in particles with smaller geometric mean diameter than did deionized water. Starch and protein separation efficiencies were higher at greater inlet pressures. The application of a double-pass hydrocyclone process increased the purity of starch in the underflow and of protein in the overflow, although this process reduced separation efficiencies. Starch granules and protein particles were separated at higher purities in deionized water than in isopropyl alcohol. Separation in deionized water resulted in higher starch separation efficiency and lower protein separation efficiency than did separation in isopropyl alcohol. This difference was due to the difference in density and viscosity of the two media. The higher viscosity of isopropyl alcohol reduced the likelihood of starch granules reaching the inner hydrocyclone wall. Thus, some starch granules were retained in the overflow instead of in the underflow. Additionally, the centrifugal force and drag force applied to the chickpea flour particles differed between the two different media. Hydrocyclone operation resulted in higher centrifugal force and lower drag force in deionized water than in isopropyl alcohol. Since the drag force in isopropyl alcohol was higher than that in deionized water, some small starch granules were diverted to the overflow which caused reduction of protein purity. <p>The use of pH 9.0 and defatting of chickpea flour improved both starch and protein separation efficiencies. Chickpea flour in deionized water at a feed concentration of 5% yielded a pumpable slurry which was delivered efficiently to the hydrocyclone at an inlet pressure of 827 kPa Fractionation of starch and protein from chickpea flour in deionized water using an integrated separation process resulted in starch and protein fractions containing 75.0 and 81.9% (d.b.) starch and protein, respectively. This process resulted in starch and protein separation efficiencies of 99.7 and 89.3%, respectively. <p>Experiments were also conducted to determine the physical and functional properties of chickpea flour and starch and protein fractions. Thermal conductivity, specific heat, and thermal diffusivity were determined and the polynomial linear models were fitted very well to experimental data. Internal and external friction properties of chickpea flour and starch and protein fractions were determined. Samples were subjected to uniaxial compression testing to determine force-time relationships. The samples particles underwent rearrangement rather than deformation during compression. The asymptotic modulus of samples was also computed, and it was linearly related to maximum compressive pressure. The functional properties of fractionated products were highly affected by the separation process. The water hydration capacity of starch fraction increased, whereas the emulsion capacity and foaming capacity of starch and protein fractions were reduced, compared to that of chickpea flour.

Automated and continuous processes are the future trends in downstream protein purification. A functionalized nanometer-scale membrane electrode system, mimicking the function of cell wall transporters, can selectively capture genetically modified proteins and subsequently pump them through the system under programmed voltage pulses. Numerical study of the two-step pulse pumping cycles coupled with experimental His-GFP releasing study reveals the optimal 14s/1s pumping/repel pulse pumping condition at 10 mM bulk imidazole concentration in the permeate side. A separation factor for GFP: BSA of 9.7 was achieved with observed GFP electrophoretic mobility of 3.1×10-6 cm2 s-1 V-1 at 10 mM bulk imidazole concentration and 14 s/1 s pumping/repel duration. The purification of His6-OleD Loki variant directly from crude E. coli extracts expression broth was demonstrated using the pulse pumping process, simplifying the separation process as well as reducing biopharmaceutical production costs. The enzymatic reactions showed that His6-OleD Loki was still active after purification. A nanoporous membrane/electrode system with directed flow carrying reagents to sequentially attached enzymes to mimic nature’s enzymes-complex system was demonstrated. The substrates residence time on the immobilized enzyme can be precisely controlled by changing the pumping rate and thereby prevent a secondary hydrolysis reaction. Immobilized enzyme showed long term storage longevity with activity half-life of 50 days at 4℃ and the ability to be regenerated. One-step immobilization and purification of His-tagged OleD Loki variant directly from expression broth, yielded 98% Uridine Diphosphate glycosylation and 80% 4-methylumbelliferone glycosylation conversion efficiency for the sequential reaction. A flow-through electroporation system, based on a novel membrane/electrode design, for the delivery of membrane-impermeant molecules into Model Leukocyte cells was demonstrated. The ability to apply low voltage between two short distance electrodes contributes to high cell viability. The flow-through system can be easily scaled-up by varying the micro-fluidic channel geometry and/or the applied voltage pulse frequency. More importantly, the system allows the electrophoretical pumping of molecules from the reservoir across the membrane/electrode system to the micro-fluidic channel for transfection, which reduces large amount of reagents used.

Membrane-bound proteins and charged lipids are separated based on their charge-to-size ratio by electrophoretic-electroosmotic focusing (EEF) method on supported lipid bilayers (SLBs). EEF uses opposing electrophoretic and electroosmotic forces to focus and separate proteins and lipids into narrow bands from an initially homogeneous mixture. Membrane-associated species were focused into specific positions within the SLB in a highly repeatable fashion. The steady-state focusing positions of the proteins could be predicted and controlled by tuning experimental conditions, such as buffer pH, ionic strength, electric field and temperature. Careful tuning of the variables should enable one to separate mixtures of membrane proteins with only subtle differences. The EEF technique was found to be an effective way to separate protein mixtures with low initial concentrations and it overcame diffusive peak broadening problem. A "SLB differentiation" post-separation SLB treatment method was also developed by using magnetic particles to rapidly slice the whole SLB into many small patches after electrophoretic separation, while keeping the majority of materials on surface and avoiding the use of chemical reactions. Label-free detection techniques were also developed based on EEF on SLBs. First, a new separation based label-free detection method was developed based on the change of focusing position of fluorescently labeled ligands. This technique is capable of simultaneous detecting multiple protein competitive binding on the same ligand on SLBs. Low concentration protein can be detected in the presence of interfering proteins and high concentration of BSA. The fluorescent ligands were moved to different focusing positions in a charged SLB patch by different binding proteins. Both free ligand and protein bound ligand concentrations were obtained. Therefore, both protein identity and quantity information were obtained simultaneously. Second, the focusing position of fluorescent biomarkers on SLB was used to monitor the phospholipase D catalyzed hydrolysis of phosphatidylcholine (PC) to form phosphatidic acid (PA), which is involved with the change of charge on the phospholipids. The focusing position of fluorescent membrane-bound biomarker in the EEF experiment is directly determined by the negative charge density on SLB. Other enzyme reactions involved with the change of phospholipids charge can be monitored in a label-free fashion in a similar way.

The primary aim of this work is to explore the potential applications of isoporous block copolymer membranes. Block copolymers (BCPs) have demonstrated their versatility in the formation of isoporous membranes. However, application spectrum of these isoporous membranes can be further broadened by exploring the technical aspects, such as desired surface chemistry, well-defined pore size, appropriate pore density, stimuli responsive behavior, and by imparting desired functionalities through chemical modifications. We believe, by exploring these possibilities, isoporous membranes hold tremendous potential as high performance next generation separation membranes. Motivated by these attractive prospects we systematically investigated novel routes for modification of isoporous membranes and their implications on properties and performance of the membranes for various applications. In this work, polystyrene-block-poly(4-vinyl pyridine) (PS-b-P4VP) has been selected to fabricate isoporous membranes using non-solvent induced phase separation (NIPS). We selected PS-b-P4VP since its well-defined isoporous morphology is studied in detail and it is extensively characterized. In order to further widen the application bandwidth of BCP membranes, it is desirable to integrate different functionalities in the BCP architecture through a straightforward approach like post-membrane-modification or fabrication of composite membranes to impart anticipated functionalities. The most critical challenge in this approach is to retain the well-defined nanoporous morphology of BCP membranes. We focused on exploring new routes for chemical functionalization of isoporous PS-b-P4VP membranes via various in-situ and post-membrane fabrication approaches. To date, most of the work reported in the literature on PS-b-P4VP presented different routes to fabricate isoporous membranes and their conventional performance in liquid separations. Few efforts have been dedicated to alter the chemistry of PS-b-P4VP membranes by tuning the reactivity of the chemically active P4VP block or the surface chemistry to enhance the membrane performance for desired applications. During the Ph.D. study, we primarily focused on: (i) post modification approach, (ii) surface modification and (iii) in-situ membrane modification approach for fabrication of the mixed-matrix nanoporous membranes without altering the isoporous morphology of the membrane. The membranes fabricated using the mentioned above routes were tested for different applications like stimuli-responsive separations, self-cleaning membranes, protein separations and high-performance humidity sensors.