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Advancing Biomolecular Analysis through Capillary Electrophoresis Techniques
Opallage, Prabhavie Madushanie
Opallage, Prabhavie Madushanie
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Abstract
Capillary electrophoresis (CE) has become an important analytical tool in biomolecular analysis due to its speed, efficiency, low sample consumption and low cost. Recent advancements in CE have broadened its application in bio molecular analysis, making it highly effective for separation and analysis of various molecules including inorganic ions, amino acids, proteins, and aptamers. CE is also used to characterize biomolecular interactions particularly in clinical and biopharmaceutical settings. In this study we developed a new technique, planar capillary electrophoresis (PCE) combined with back-scatter interferometry (BSI) which enables rapid and accurate separation and detection of inorganic ions and underivatized amino acids in both standard solutions and biological samples. Refractive index (RI) detection using BSI offered the universal detection feature to the PCE platform, which broaden its applications with label free detection capability in the analysis of various biomolecules. By using thin- walled capillaries, the method reduces joule heating which facilitates efficient heat dissipation, even at high- field strengths, separating the analytes within seconds. The separation displayed a good repeatability with excellent precision, and also revealed its capability to detect concentrations as low as high nanomolar range. This system has been also applied in close clinical applications like for analyzation of biological samples. It was tested for separation and simultaneous detection of amino acids, creatinine (CR) and inorganic ions in human urine and plasma samples with minimal sample preparation. The separation was achieved in under 100 seconds in both sample types. The results were consistent with physiological levels, providing a powerful tool for diagnosing metabolic disorders. Another significant application of PCE platform is the performance of serum protein electrophoresis (SPE) using universal BSI detection. By integrating the fluorescence detection into the same platform allows simultaneous dual detection capability to detect both serum proteins and immunoassays. This platform uses a single laser source for dual detection, where RI measurements were used for proteins and fluorescence detection was used for immunoassays. The technique achieved a limit of detection of 23 nM for the tested analyte FITC. Additionally, a modified PCE platform has been developed to enhance the separation of fluorescently labeled antibodies, enabling the quantification of III biomarkers like β2-microglobulin (B2M), relevant to diseases such as multiple myeloma, which promises its applicability in future clinical diagnostics. To further enhance the applicability of the PCE platform a significant enhancement of the detection limits was required. Incorporating photothermal excitation with BSI in CE was capable of enhancement of the detection sensitivity by three orders of magnitude. This approach shows a promise in a variety of applications, including enhanced detection of biomolecules in complex biological samples, binding studies, and interaction kinetics. A newly engineered multi-loop CE platform provides the capability of simultaneous calculation of kinetic parameters such as the dissociation constant (Kd), off-rate (koff), and on-rate (kon) in biomolecular interactions. This system was validated with FITC and anti FITC-antibodies and a thrombin-targeting aptamer. The study characterized the real-time monitoring capability of the separation, using multi detection points along the capillary which significantly enhanced the accuracy of calculating the kinetic parameters of the biomolecules. In the future, the multi-looped capillary system could be applied to determine diffusion coefficients and the hydrodynamic radius of molecules through Taylor dispersion analysis (TDA), offering further intuitions into molecular behavior and migration properties. This would expand utility of CE in biophysical characterization of the molecules and pharmaceutical research. These advancements in CE, particularly through the integration of BSI, fluorescence, and photothermal techniques in to the same PCE platform, highlight its growing potential for rapid, sensitive, and cost-effective biomolecular analysis. From clinical diagnostics to therapeutic research, CE continues to be a valuable and innovative tool for advancing biomolecular studies.
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Date
2024-12-31
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University of Kansas
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Keywords
Chemistry, amino acids, antibody, capillary electrophoresis, electro-osmotic flow, proteins, separation