Capillary Electrophoresis: Bioanalytical Applications

描述（由申请人提供）：本研究的主要目标是发展毛细管电泳（CE）作为一种分析技术来研究万古霉素（Van）、瑞斯托司汀（Ris）和替可普兰宁（Tel）等抗生素的理化参数。其重点是展示原理和开发有用的生物分析程序，可应用于分析受体-配体相互作用和微尺度反应。抗生素耐药菌的进化使van类抗生素的研究和开发成为一个重要的研究热点。因此，通过化学修饰来探测这些抗生素的理化参数是非常必要的。两种特定的CE技术将在这项工作中使用：柱上微反应器技术和亲和毛细管电泳（ACE）。本研究中使用的技术包括化学修饰和表征，化学分离和小分子的CE鉴定，利用激光诱导荧光（LIF）和紫外/可见（UV/VIS）检测方案，高效液相色谱（HPLC）和酶动力学。这项研究将开发新的生物分析技术，并将重点研究与疾病预防有关的小生物分子，从而涉及公共卫生问题。这种理解将加强CE的设计、制备和应用于一系列健康相关问题的科学基础。

DESCRIPTION (provided by applicant): The broad goals of this research are to develop capillary electrophoresis (CE) as an analytical technique to study the physicochemical parameters of the antibiotics vancomycin (Van), ristocetin (Ris), and teicoplanin (Tel). Its focus is on demonstrating principles and on developing useful bioanalytical procedures that can be applied to the analysis of receptor-ligand interactions and microscale reactions. The evolution of antibiotic resistant bacteria has made the study and development of Van-like antibiotics a critical area of research focus. Hence, studies probing the physicochemical parameters of these antibiotics via chemical modification are highly warranted. Two specific CE techniques will be utilized in this work: on-column microreactor techniques and affinity capillary electrophoresis (ACE). Techniques utilized in this study include chemical modification and characterization, chemical separation and identification of small molecules by CE utilizing both laser-induced fluorescence (LIF) and ultraviolet/visible (UV/VIS) detection schemes and high performance liquid chromatography (HPLC), and enzyme kinetics. The research will develop new bioanalytical techniques and will focus on examining small biomolecules involved in the prevention of disease and, hence, to the issue of public health. This understanding will strengthen the scientific base underlying the design, preparation, and application of CE towards a host of health-related problems. The specific aims of the research are to I. Utilize On-Column Microreactor Techniques and ACE in the Estimation of Binding Constants Between Antibiotics and Ligands. II. Determine the Charge and pl of Antibiotics Using On-Column Microreactor Techniques. III. Examine the Kinetics of Derivatization of Antibiotics Using Reactive Chemical Reagents. IV. Estimate Binding Constants of Linked Antibiotics to Peptides. V. Conduct Experimental Studies on D-Ala-D-Lac Terminus Peptide Binding to Van-Group Antibiotics. The proposed research will: Demonstrate the versatility of CE in determining physicochemical parameters of antibiotics; Demonstrate high-throughput derivatization of receptors and ligands coupled to ACE; Provide for rapid synthesis and accurate analysis of multiple drug targets simultaneously; Require reduced sample volumes compared to complementary analytical techniques, and; Reduce sample waste and disposal.