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首页> 外文学位 >New approaches to stabilize black lipid membranes--Towards ion channel functionalized detectors for capillary separations.
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New approaches to stabilize black lipid membranes--Towards ion channel functionalized detectors for capillary separations.

机译：稳定黑色脂质膜的新方法-走向离子通道功能化的毛细管分离检测器。

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摘要

Capillary electrophoresis (CE) is an excellent analytical separation method with promising features such as small sample volumes (microL 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.
机译：毛细管电泳（CE）是一种出色的分析分离方法，具有诸如小样品体积（微升至pL），快速分析时间，高选择性和高效率以及与生物样品的优异兼容性等有前途的功能。然而，常规的CE检测器不能感测光学和电化学无活性的生物活性化合物，限制了它们在生物传感和药物筛选中的用途。我们已经开发了一个高度稳定的电生理检测平台，该平台由在合成双层膜（也称为黑脂质膜（BLM））中重构的离子通道（IC）组成，该双层膜悬浮在功能化的微孔上，与高分辨率毛细管分离通道耦合。改性剂用于大大改善BLM的电气，机械和时间稳定性。由于具有两亲性质（H2O /拒油性），使用十三氟1,1,2,2-四氢二甲基氯硅烷改性的玻璃微孔促进了高稳定BLM的快速形成。此外，化学修饰的孔表面和脂质膜内的化学交联的组合被用来显着提高BLM的稳定性。双层内的部分交联可保持流动性，从而允许离子通道蛋白重建，同时保持BLM-IC平台的稳定性。;跨玻璃移液器孔的稳定BLM-IC与微芯片电泳偶联，显示可承受电场强度（> 250 V / cm）。此外，在SU8中制造的平面微孔用于形成稳定的BLM-IC平台，以构建集成设备。 SU8的化学性质支持在可聚合脂质或掺有可聚合甲基丙烯酸酯单体的脂质双层中的形成和交联。此外，我们在HEK 293细胞中表达了离子通道偶联受体融合蛋白，以开发宽范围的离子通道传感器BLM传感器平台中配体检测的过程。使用基于膜片钳电生理的基于细胞的测定法，用毒蕈碱型乙酰胆碱（M2-K）受体功能化的IC的药理学研究显示，乙酰胆碱可活化，阿托品抑制。因此，该平台作为下一代综合分析系统具有广阔的前景，该系统可用于快速筛选复杂基质（例如全血和尿液）中的生物活性化合物（例如胰高血糖素），以诊断和管理慢性疾病（例如糖尿病）。

著录项

  • 作者

    Bright, Leonard Kofi.;

  • 作者单位

    The University of Arizona.;

  • 授予单位 The University of Arizona.;
  • 学科 Analytical chemistry.;Biochemistry.
  • 学位 Ph.D.
  • 年度 2015
  • 页码 257 p.
  • 总页数 257
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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