Microfluidic chips have gained wide applications in various fields, including medicine, environmental sciences and forensic investigations. They are used for the separation of proteins, blood, bacterial cell suspensions, antibody solutions, and drugs. Microfluidic chips display significant advantages, which include faster analysis time, reduced amounts of samples and reagents volumes, flexibility in design and increased separation efficiency. Whole column imaging detection (WCID) exhibits significant advantages compared to other detection methods that are widely used for detecting analytes after the separation of these analytes using isoelectric focusing. With these other methods, there is a need to mobilize the focused sample bands past the detector after separation but with WCID, there is no need for mobilization step. The aim of this research is further development of WCID by characterizing microfluidic chips fabricated for the detection system, to enhance its detection so that high efficiency can be obtained. The chips were fabricated using soft lithography technology at the Microfluidic laboratory, University of Waterloo and they were used to perform isoelectric focusing of various proteins in our laboratory. The fabricated chips with straight channel design were used to carry out isoelectric focusing of some proteins and the results obtained were compared with the results obtained using commercial cartridges. The chips with tapered channel design were used to carry out isoelectric focusing of proteins in which thermally generated pH gradient principle was employed. The samples after separation were sprayed into a mass spectrometer using nano-electrospray interface to obtain their molecular masses. Compatible cartridges for nano-electrospray mass spectrometer were developed and these cartridges were used to carry out capillary isoelectric focusing of low molecular pI markers and proteins. These cartridges were also connected to the nano-electrospray mass spectrometer to obtain the mass to charge ratios of some proteins. The fabricated microfluidic chips with straight channel design were also used to investigate the interaction between drugs and protein.

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Proteomics is an increasingly important area of biological research and has gathered much attention over recent years. Major challenges that make a proteomic analysis difficult are sample complexity, diversity and dynamic range. Progress in the area of proteomics relies heavily on new analytical tools for the sensitive, selective, and high-throughput studies of target analytes. It is estimated that there are several hundred thousand proteins in a human cell. In order to be able to analyze such a complex sample, an analytical method must be capable of separating and detecting many different sample peaks. The complexity of such samples indicates that a single separation method will not be able to provide the needed resolution. If two methods that are orthogonal are combined, then the peak capacity of the combined system is the product of the two individual peak capacities. Development of such systems would cater to the current demands of proteomics studies. Matrix assisted laser desorption/ionization (MALDI) mass spectrometry has evolved into a primary analytical tool for proteomics research. MALDI is fast and efficient and has a high tolerance to non-volatile buffers and impurities. The samples for MALDI are typically applied to solid supports after having been subjected to off-line liquid or gel separations. Several methods have been reported involving various chromatographic or electrophoretic separation methods. However, the current methods often require highly sophisticated sample handling systems, which are often expensive and in need of skilled human resources. The current demands of proteomic analyses require fast, efficient and inexpensive methods for separation to fully harness the capability of MALDI mass spectrometry. In this work a microfluidic device has been designed to perform dynamic isoelectric focusing (DIEF) based protein separation with digital sample deposition directly on a MALDI target for offline analysis. DIEF is related to capillary isoelectric focusing which and can facilitate the interface without the loss of the separation resolution. Compared to traditional capillary isoelectric focusing (cIEF) DIEF uses additional high-voltage power supplies to control the pH gradient by manipulating the electric field. The proteins can be focused at a desired sampling position according to their isoelectric point, to be collected for further analysis by MALDI mass spectrometry. DIEF has a peak capacity of over a thousand and offers an ease of interfacing to other techniques making it a preferred separation method for the interface with mass spectrometric techniques such as MALDI. The design of the microfluidic device is based on a digital droplet fractionation. Multiple fractions of the sample solution from DIEF are generated to retain the resolution and to act as an additional separation mode. The microfluidic device is controlled by actuating pneumatic valves built into the device. The DIEF operational parameters were optimized according to the surface functionality and the design of the microfluidic device. A suitable MALDI sample preparation method was found by studying different existing methods. The methods were studied using test proteins prepared in solutions having the additives used in the experiment. A simple mixture of three proteins was used to demonstrate the application of the developed method. The separation between the proteins insulin, hemoglobin and the myoglobin was demonstrated by varying the separation resolution in three experiments.

Small heat shock proteins (hsp) have been implicated in mediation of classic preconditioning in the rabbit. Hsp27 is a terminal substrate of the p38 MAPK cascade. One and 2D gel electrophoresis and immunoblotting of cell fractions was used to determine p38 MAPK and hsp27 phosphorylation levels, respectively, during in vitro ischemia in control, calyculin A (Cal A)-treated (protein phosphatase inhibitor), SB203580-treated (p38MAPK inhibitor) and preconditioned (IPC) isolated adult rabbit cardiomyocytes. The dual phosphorylation of p38 MAPK was increased by early ischemia (30-60 min), after which there was a loss of total cytosolic p38 MAPK. The ischemic increase of p38 MAPK dual phosphorylation was enhanced by IPC. Cal A strongly activated dual phosphorylation of p38 MAPK in oxygenated cells and this was maintained into early ischemia. SB203580 inhibited the dual phosphorylation of p38 MAPK and attenuated the loss of total cytosolic p38 MAPK. In each protocol, ischemia translocated hsp27 from the cytosolic fraction to the cytoskeletal fraction at similar rates and extents. Hsp27 phosphorylation was quantitated as the fraction of diphosphorylated hsp27, based on IEF mobility shifts of hsp27 phosphorylation isoforms. In oxygenated control cells, cytosolic and cytoskeletal hsp27 was highly phosphorylated. After 90 min ischemia, cytoskeletal hsp27 was markedly dephosphorylaled. Cal A slightly increased control cytoskeletal hsp27 phosphorylation. During ischemic incubation, Cal A blocked ischemic dephosphorylation. SB203580 accelerated ischemic hsp27 dephosphorylation and injury. IPC insignificantly decreased the initial rate of ischemic dephosphorylation of hsp27, but not the extent of dephosphorylation in later ischemia. Phosphorylation is regulated by both kinase and phosphatase activities. IPC protection was not correlated with a significant increase in cytosolic or cytoskeletal hsp27 phosphorylation levels during prolonged (> 60-90 min) ischemia.

Thesis (MSc (Biochemistry))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: This study describes: a) the isolation and purification of horseradish peroxidase isoenzymes from horseradish roots, b) the characterization of various forms and components of the enzyme by cation-exchange and reversed-phase high performance liquid chromatography, c) the preparation of antibodies against horseradish peroxidase isoenzymes, d) immunological studies for the development of an isoenzyme quantification method and e) the formation of an enzyme-melamine conjugate for use in a melamine quantification immunoassay. / AFRIKAANSE OPSOMMING: Hierdie studie beskryf: a) die isolering en suiwering van peperwortel-peroksidase-isoënsieme vanuit die peperwortel, b) die karakterisering van verskillende vorme en komponente van dié ensiem deur katioonuitruilings en omgekeerde-fase HPLC c) die voorbereiding van teenliggaampies vir peperwortel-peroksidase-isoënsieme, d) immunologiese studies vir die ontwikkeling van 'n isoënsiem-kwantifiseringsmetode; en e) die vorming van 'n ensiem-melamien-konjugaat vir gebruik in 'n melamienkwantifiseringsmetode.

Two types of fluorescent molecules had been designed and synthesized to improve the analytical infrastructure of capillary electrophoretic separations. First, a hydrophilic version of the permanently cationic acridine-based fluorophore, HEG2Me2-DAA was synthesized. HEG2Me2-DAA has a lambda^ex max of 490 nm which matches the 488 nm line of the commonly used argon ion laser. The emission spectra of HEG2Me2-DAA are pH-independent. HEG2Me2-DAA was used in capillary electrophoresis with an aqueous background electrolyte and was found to be free of the detrimental peak tailing of the acridine orange-based fluorophore that was caused by adsorption on the inner wall of the fused silica capillary. Bovine serum albumin was labeled with excess of the designed amine reactive reagent and the lowest concentration at which the tagged bovine serum albumin was tested was 15 nM. Chicken ovalbumin was also labeled with FL-CA-PFP and analyzed by capillary isoelectric focusing (cIEF) with LIF detection. The pI values of the tagged proteins shifted in the alkaline direction by about 0.02 compared to the pI values of the non-tagged proteins. A tri-functional probe intended to enable selective enrichment and selective detection of a variety of molecules (e.g., natural products, pharmaceuticals, inhibitors, etc.) was also designed and synthetized by combining FL-CA with biotin and an azide group in a "proof-of-principle" level experiment. In cIEF, the profile of the pH gradient can only be determined with the help of pI markers. A large set of pyrene-based fluorescent pI markers was rationally designed to cover the pI range 3 to 10. To prove the feasibility of the proposed synthetic approach, the subgroup of the pI markers having the greatest structural complexity was synthesized and characterized. The classical zone electrophoretic pI determination methods failed due to severe chromatographic retention of the APTS based pI markers on the capillary wall. Exploratory work was done to design a new pI value determination method that combines the advantages of the immobilized pH gradient technology of the OFFGEL instrument and the carrier-ampholyte-based IEF technology. The method aspects of cIEF have also been improved in this work. The new segmented loading method yielded a more linear pH gradient than the previously known cIEF methods. To exploit a unique property of the newly developed fluorescent pI markers, we used them as pyrene-based ampholytic carbohydrate derivatizing reagents. The pI4 carbohydrate derivatization reagent proved advantageous over 8-aminopyrene-1,3,6-trisulfonic acid (APTS): the pI4 conjugates have higher molar absorbance at 488 nm than the APTS conjugates and become detectable in positive ion mode of MS affording better detection sensitivity.

Microfluidics is gaining in importance due to its wide ranging benefits and applicability in chemical and biological analysis. Although traditional microfluidic devices are created with glass or silicon based fabrication technologies, polymer based devices are gaining in popularity. Soft lithography and replica molding are techniques for the rapid prototyping of such devices, utilizing Polydimethylsiloxane (PDMS) as the dominant material. Other benefits include its low costs and ease of fabrication. Even though soft lithography is a well researched and developed fabrication process, new applications have been discovered in which the technology can be applied. Often, changes in the fabrication process are necessary for their application in other areas of research. This thesis will address several microfluidic applications using soft lithography. These areas of research include microfluidic thermometry, isoelectric focusing (IEF), and micromixers. In microfluidic thermometry, a novel thin film PDMS/Rhodamine B has been developed allowing whole-chip temperature measurements. In addition, compatibility problems between Rhodamine B and PDMS microfluidic devices were resolved. The thin film fabrication process, experimental results, and issues with its use are discussed. Future work and attempts at improving the thin film performance are also provided. IEF involves applications in which samples are separated according to its electrostatic charge. Two types of IEF applications are shown in which soft lithography has been shown to be beneficial to its development and performance. In isoelectric focusing with the use of thermally generated pH gradients, soft lithography allows for the rapid design, production and testing of different channel layouts. In general, due to PDMS insulation and overall low heat transfer rates, the temperatures detected are more gradual than those previously reported in literature. IEF using carrier ampholytes are also discussed, with preliminary results in which devices fabricated using soft lithography are compared to commercially available IEF cartridges. Its fabrication issues are discussed in detail. In micromixers, soft lithography fabrication issues and overall integration with flow mechanisms is discussed. In general it is difficult to perform mixing in the microscale due to the predominantly laminar flow and flow rate restrictions. Channel geometry is insignificant, as can be seen through numerical simulations.

Microfluidics is gaining in importance due to its wide ranging benefits and applicability in chemical and biological analysis. Although traditional microfluidic devices are created with glass or silicon based fabrication technologies, polymer based devices are gaining in popularity. Soft lithography and replica molding are techniques for the rapid prototyping of such devices, utilizing Polydimethylsiloxane (PDMS) as the dominant material. Other benefits include its low costs and ease of fabrication. Even though soft lithography is a well researched and developed fabrication process, new applications have been discovered in which the technology can be applied. Often, changes in the fabrication process are necessary for their application in other areas of research. This thesis will address several microfluidic applications using soft lithography. These areas of research include microfluidic thermometry, isoelectric focusing (IEF), and micromixers. In microfluidic thermometry, a novel thin film PDMS/Rhodamine B has been developed allowing whole-chip temperature measurements. In addition, compatibility problems between Rhodamine B and PDMS microfluidic devices were resolved. The thin film fabrication process, experimental results, and issues with its use are discussed. Future work and attempts at improving the thin film performance are also provided. IEF involves applications in which samples are separated according to its electrostatic charge. Two types of IEF applications are shown in which soft lithography has been shown to be beneficial to its development and performance. In isoelectric focusing with the use of thermally generated pH gradients, soft lithography allows for the rapid design, production and testing of different channel layouts. In general, due to PDMS insulation and overall low heat transfer rates, the temperatures detected are more gradual than those previously reported in literature. IEF using carrier ampholytes are also discussed, with preliminary results in which devices fabricated using soft lithography are compared to commercially available IEF cartridges. Its fabrication issues are discussed in detail. In micromixers, soft lithography fabrication issues and overall integration with flow mechanisms is discussed. In general it is difficult to perform mixing in the microscale due to the predominantly laminar flow and flow rate restrictions. Channel geometry is insignificant, as can be seen through numerical simulations.

Thesis (PhD)--Macquarie University, Division of Environmental & Life Sciences, Dept. of Chemistry & Biomolecular Sciences, 2009. / Bibliography: p. 200-212.