Single-isomer sulfated cyclodextrins (SISCDs) have proven to be reliable, effective, robust means for separation of enantiomers by capillary electrophoresis (CE). SISCD derivatives used as chiral resolving agents in CE can carry the sulfo groups either at the C2, C3 or C6 positions of the glucopyranose subunits which provides varied intermolecular interactions to bring about favorable enantioselectivities. The first single-isomer, sulfated β-CD that carries the sulfo group at the C2 position, the sodium salt of heptakis(2-O-sulfo-3-O-methyl-6-Oacetyl) cyclomaltoheptaose (HAMS) has been synthesized. The purity of each synthetic intermediate and of the final product was determined by HILIC and reversed phase HPLC. The structural identity of each intermediate and the final product was verified by 1D, and 2D NMR, and MALDI-TOF mass spectrometry. HAMS has been used as chiral resolving agent for the CE separation of a set of nonionic, weak base and strong acid enantiomers in pH 2.5 background electrolytes. Rapid separations with satisfactory peak resolution values were obtained for the enantiomers of most of the nonionic and weak base analytes. Typically, low concentrations of HAMS were required to effect good enantiomer resolution. The trends in the effective mobilities and separation selectivities as a function of HAMS concentrations followed the predictions of the ionic strength-corrected charged resolving agent migration model (CHARM model). HAMS showed poor complexation with the anionic strong electrolyte enantiomers for which no peak resolution was observed. The separation patterns observed with HAMS as chiral resolving agent were compared with those of other β-cyclodextrin analogues, including heptakis(2-O-methyl- 3-O-acetyl-6-O-sulfo)-b-cyclodextrin (HMAS), heptakis(2-O-methyl-3,6-di-O-sulfo)-b- cyclodextrin (HMdiSu), heptakis(2,3-di-O-acetyl-6-O-sulfo)-b-cyclodextrin (HDAS) and heptakis(2,3-di-O-methyl-6-O-sulfo)-b-cyclodextrin (HDMS).

This thesis reports design, fabrication, and implementation of two different micro electrophoresis system architectures for DNA analyses. The first architecture is traditional single channel layout with several design alternatives for size-based separation of DNA fragments. The second one is novel double channel architecture specialized for rapid mutation detection using heteroduplex analysis (HDA) method with an application of a newly designed injection technique. Besides achieving high resolution separations within the length of 1 mm with single channel arrangement, HDA was successfully applied for 590 base pair (bp) long PCR sample with 3 bp mutations in a separation length of 50 &micro / m in less than 3 minutes with double channel structure. Microchannels were formed using parylene-C due to its conformal deposition, no surface treatment requirement, transparency, biocompatibility, low background fluorescence, etc. Using the advantage of parylene in fabrication, the microchannels were fabricated with an only three-mask process. New double channel architecture is obtained by dividing the 200 &micro / m-wide separation channel into two parts by a 20 &micro / m-thick wall between them. For sample injection, various techniques, such as traditional cross, double-T, and double-L were investigated and optimized for single channel architecture assisting with pullback injection method. For double channel architecture, a novel, u-turn injection technique was applied. Precise control of sample amount by adjusting the injection time was accomplished by this new technique. Using high resolution cross-linked polyacrylamide gel as sieving material, separations were achieved in a very short length and time. Electrophoresis was performed in both channels of the double channel microchips simultaneously under the same conditions. This gives the chance of having a control channel in microchip format, which is very critical for the accuracy and reliability of the results in genetic analyses.

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The first three, pure, single-isomer, 6-O-sulfo a-cyclodextrins, the sodium salts of hexakis(6-O-sulfo)-a-CD (HxS), hexakis(2,3-di-O-methyl-6-O-sulfo)-a-cyclodextrin (HxDMS) and hexakis(2,3-di-O-acetyl-6-O-sulfo)-a-cyclodextrin (HxDAS) have been synthesized, analytically characterized and utilized as chiral resolving agents in capillary electrophoresis. The purity of each synthetic intermediate and of the final product was determined by HPLC-ELSD and indirect UV-detection capillary electrophoresis. The structural identity of each intermediate and final product was verified by 1D and 2D NMR, and mass spectrometry.HxS, HxDMS and HxDAS have been used to separate a series of neutral, basic, ampholytic and acidic enantiomers in pH 2.5 and pH 9.5 aqueous and acidic methanol background electrolytes using capillary electrophoresis. Rapid separations with satisfactory peak resolution values were obtained for most of the analytes, indicating that HxS, HxDAS and HxDMS can serve as chiral resolving agent for a wide range of analytes. The observed separation patterns follow the predictions of the CHArged Resolving agent Migration (CHARM) model. The separation patterns observed with HxS, HxDAS and HxDMS as chiral resolving agent were compared with those of (1) b-cyclodextrin analogues, such as, heptakis(6-O-sulfo)-b-cyclodextrin (HS), heptakis(2,3-di-O-acetyl-6-O-sulfo)-b-cyclodextrin (HDAS) and heptakis(2,3-di-O-methyl-6-O-sulfo)-b-cyclodextrin (HDMS); (2) g-cyclodextrin analogues, such as, octakis(6-O-sulfo)-g-cyclodextrin (OS), octakis(2,3-di-O-acetyl-6-Osulfo)- g-cyclodextrin (ODAS) and octakis(2,3-di-O-methyl-6-O-sulfo)-g-cyclodextrin (ODMS). The effects of the structure of the analytes, and those of the pH and the solvent of the background electrolyte were also studied.

Thesis (Ph. D.)--Brigham Young University. Dept. of Chemistry and Biochemistry, 2004. / Includes bibliographical references.

Thesis (Ph. D.)--University of Hong Kong, 2001. / Includes bibliographical references.

published_or_final_version / abstract / toc / Chemistry / Master / Master of Philosophy

Capillary electrophoresis (CE) is an excellent analytical separation method with promising features such as small sample volumes (µL to pL), fast analysis times (s), high selectivity and efficiency, and excellent compatibility with biological samples. However, the inability of conventional CE detectors to sense biologically active compounds that are optically and electrochemically inactive limits their use for biosensing and drug screening. We have developed a highly stable electrophysiological detection platform consisting of ion channel (IC) reconstituted in synthetic bilayer membrane also known as black lipid membranes (BLM) suspended across a functionalized microaperture to be coupled to a high resolution capillary separation channel. Low energy surface modifiers were used to drastically improve the electrical, mechanical, and temporal stability of BLMs. Glass microapertures modified using tridecafluoro 1, 1, 2, 2-tetrahydrodimethylchlorosilane facilitated the rapid formation of highly stable BLMs due to the amphiphobic property (H₂O/oil repellency). Furthermore, a combination of chemically modified aperture surfaces and chemical cross-linking within the lipid membrane were used to dramatically improve BLM stability. Partial cross-linking within the bilayer maintained fluidity which allowed reconstitution of ion channel proteins while maintaining the stability of BLM-IC platform. The stable BLM-IC across glass pipette aperture was coupled to microchip electrophoresis and was shown to withstand field strength (>250 V/cm) from separation channel. Additionally, planar microapertures fabricated in SU8 were used for the formation of stable BLM-IC platform towards the construction of an integrated device. The chemical properties of the SU8 supported the formation and cross-linking within polymerizable lipid or lipid bilayer doped with polymerizable methacrylate monomers. Additionally, we expressed ion channel coupled receptor fusion protein in HEK 293 cells towards the development of ion channel sensors for wide range of ligand detection in BLM sensor platforms. The pharmacology of IC functionalized with muscarinic acetyl choline (M2-K) receptor using cell based assay by patch clamp electrophysiology showed activation by acetylcholine and inhibition by atropine. Thus this platform holds a great promise as the next-generation integrated analysis system for rapid screening of biologically active compounds (eg. glucagon) in complex matrix such as whole blood and urine for the diagnosis and management of chronic disease such as diabetes.

Neurological and neuroendocrine disorders result from signaling dysregulation at the molecular, cellular, and multi-cellular levels. This dissertation presents the development of separation methods, using capillary zone electrophoresis (CZE) and capillary liquid chromatography (CLC), for detecting and quantifying small molecules, peptides, and proteins involved in cellular signaling. CZE is a rapid separation technique, making it ideal for monitoring cellular dynamics with high temporal resolution. An ultraviolet - light emitting diode was used for photolytic optical gating of caged fluorophore-labeled biogenic amines, common functional groups in neurotransmitters. Additionally, a novel caged fluorophore with faster reaction kinetics than commercially available dyes was used to label reduced thiols and primary amines in the presence of o-phthalaldehyde. Together this light source and novel caged dye illustrate the utility of these methods for monitoring chemical dynamics during continuous sampling. Many cellular second messengers, including inositol phosphates, are known to exist within the cell, but their dynamics and intermolecular interactions are poorly understood since they lack chromophores or electroactive functional groups making direct detection difficult. Utilizing CZE with capacitive coupled contactless conductivity detection (C4D), biological phosphates were separated and detected based on their high anionic charge, suggesting the utility of C4D in label-free detection of biological molecules. The techniques described above require higher sensitivity to monitor physiologically relevant analyte concentrations; therefore, Hadamard transform capillary electrophoresis (HTCE) was used as a multiplexing method in which multiple separations were performed simultaneously. HTCE resulted in increased sensitivity by decreasing the random background noise. Peptides and proteins propagate signals within or between cells; yet, they are difficult to separate and detect by CZE since their highly charged surfaces result in non-specific adsorption to the capillary wall. To minimize these interactions, stable hybrid phospholipid bilayers were prepared as capillary coatings for CZE separations of cationic proteins. Additionally, stabilized phospholipid bilayer coatings were formed on silica particles through redox polymerization of synthetic, polymerizable lipids. These bilayers were stable after exposure to surfactant, organic solvents, and after storage for one month, suggesting their value as lipid chromatography stationary phases for future incorporation of transmembrane proteins to analyze binding interactions with small molecules.

Analysis of M-components is an important part of the diagnosis of monoclonal gammopathies and for the evaluation of disease response during treatment. In this project, two widely used electrophoresis methods and their corresponding immunotyping method were compared to evaluate the sensitivity of each method for the detection of small M-components. The project included 30 plasma samples from patients with identified M-components; 10 samples containing each IgG, IgA and IgM, respectively. All samples were diluted with normal EDTA plasma to achieve M-components of 5,00g/L. The samples were then serially diluted to achieve M-component concentrations of; 5,00, 2,50, 1,25, 0,63, 0,31 and 0,16g/L. All 180 samples were analysed with agarose gel electrophoresis and capillary electrophoresis. The dilutions above and below the detection level of each method were then analysed with immunofixation and immunosubtraction. The results showed good agreement between agarose gel electrophoresis and capillary electrophoresis in the highest concentrations of IgG and IgM. With agarose gel electrophoresis, IgA was detected in the same location as transferrin and the lowest concentration detected were therefore 1,25g/L. Besides the samples containing IgG, immunofixation was the most sensitive method.