Bioassays and Molecular Characterization by Microchip CE

通过 Microchip CE 进行生物测定和分子表征

• 批准号: 6705623
• 负责人: PATRICK A LIMBACH
• 金额: $ 47.51万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6705623
• 关键词: analytical method; bioassay; biological products; capillary electrophoresis; fluorescence; high throughput technology; lasers; mass spectrometry; method development; microprocessor /microchip; nucleic acids; peptides; proteins; rapid diagnosis; small molecule; technology /technique development; trace elements

DESCRIPTION (provided by applicant): The goal of this research is to develop plastic microchip analytical technologies that yield high-throughput approaches for the trace determination and/or physical properties of biological and small molecule therapeutics that are increasingly important to pharmaceutical and medical research. In support of this long-term goal are two specific aims. The first aim is to develop appropriate assays from three categories that would benefit significantly from the advantages of the microchip platform. The research focus will include efforts to significantly improve the determination of trace amounts of biological compounds with respect to speed and selectivity as compared with the standard immunoassay methodology. Aptamer-based assays of proteins will be used as illustrative chemical systems, and capillary electrophoresis on a multilane plastic chip with detection by laser induced fluorescence (LIF) will be the analytical technique. Analysis time will be shortened from hours to minutes by rapid, high-resolution separation of protein-photoaptamer complexes without the need for prior sample preparation. Additionally, a relatively high-throughput non-equilibrium separation-based approach that allows the accurate determination of Kd and IC-50 values from a wide range of ligand-binding systems will be developed. Non-equilibrium affinity capillary electrophoresis performed on a multilane plastic microchip with LIF, indirect LIF or flow-assisted mass spectrometry detection will be used with several model ligand-binding systems. As the final illustration, a universal, low sample consumption, high throughput and low cost separation-based platform to determine two of the key physiochemical properties of small molecules (MW 200-1,000), pKa and log Pow will be developed. Multilane plastic microchips coupled with either indirect laser-induced fluorescence or mass spectrometric detection can provide a rapid means of assessing these key parameters. The second aim will be to develop appropriate plastic microchip platforms from which these assays can be performed. In particular, these platforms must be compatible with the assays developed in the first Specific Aim. Efforts will be directed at developing multilane plastic chips having appropriate properties for use with LIF or indirect LIF as well as the appropriate laser system for LIF-associated methods. Efforts will also focus on appropriate chip designs and plastic substrates for multi-analyte analyses with mass spectrometric detection methods. Upon completion, appropriate microchip-sets will be created for assays of biological and small molecule therapeutics.

描述（由申请人提供）： 这项研究的目的是开发塑料微芯片分析技术，该技术为生物学和小分子疗法的痕量测定和/或物理性质提供高通量方法，这些方法对药物和医学研究越来越重要。为了支持这个长期目标，有两个具体目标。 第一个目的是从三类类别中开发适当的测定，这些测定将从微芯片平台的优势中受益匪浅。与标准免疫测定方法相比，研究重点将包括显着提高痕量生物化合物的确定生物学化合物的努力。基于适体的蛋白质测定方法将用作说明性化学系统，并且在多透明质塑料芯片上通过激光诱导的荧光（LIF）检测的毛细管电泳将是分析技术。分析时间将通过蛋白质 - 光适中复合物的快速高分辨率分离而缩短到几分钟，而无需事先样品制备。 此外，将开发出相对较高的非平衡分离方法，该方法将开发从多种配体结合系统中准确确定KD和IC-50值。在具有LIF，间接LIF或流动辅助质谱检测的多透明塑料微芯片上进行的非平衡亲和力毛细管电泳，将与多种模型配体结合系统一起使用。作为最终的说明，将开发出一种普遍的样本消耗，高吞吐量和低成本分离的平台，以确定小分子的两个关键生理​​化学特性（MW 200-1,000），PKA和LOOD POW。多硅烷塑料微芯片与间接激光诱导的荧光或质谱检测相结合可以提供评估这些关键参数的快速方法。 第二个目的是开发可以从中执行这些测定法的合适塑料微芯片平台。特别是，这些平台必须与第一个特定目的中开发的测定法兼容。努力将致力于开发具有适当特性的多透明塑料芯片，可与LIF或间接LIF一起使用，以及用于LIF相关方法的适当激光系统。努力还将集中在适当的芯片设计和塑料底物上，用于使用质谱检测方法进行多分析物分析。完成后，将创建适当的微芯片组，以测定生物学和小分子疗法。