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）： 这项研究的目标是开发塑料微芯片分析技术，为生物和小分子疗法的痕量测定和/或物理特性提供高通量方法，这对制药和医学研究越来越重要。支持这一长期目标的是两个具体目标。 第一个目标是从三个类别中开发适当的检测方法，这些检测方法将显着受益于微芯片平台的优势。研究重点将包括努力显着提高痕量生物化合物的测定速度和选择性，与标准免疫测定方法相比。基于适体的蛋白质测定将用作说明性化学系统，多通道塑料芯片上的毛细管电泳和激光诱导荧光 (LIF) 检测将成为分析技术。通过快速、高分辨率地分离蛋白质-光适体复合物，无需事先制备样品，分析时间将从数小时缩短至数分钟。 此外，还将开发一种相对高通量的非平衡分离方法，可以从各种配体结合系统中准确测定 Kd 和 IC-50 值。在具有 LIF、间接 LIF 或流辅助质谱检测的多通道塑料微芯片上进行的非平衡亲和毛细管电泳将与几种模型配体结合系统一起使用。作为最后的说明，将开发一个通用、低样品消耗、高通量和低成本的基于分离的平台，用于确定小分子（MW 200-1,000）的两个关键理化特性：pKa 和 log Pow。多通道塑料微芯片与间接激光诱导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。 第二个目标是开发合适的塑料微芯片平台来进行这些分析。特别是，这些平台必须与第一个特定目标中开发的检测兼容。将致力于开发具有适当特性的多通道塑料芯片，用于 LIF 或间接 LIF 以及用于 LIF 相关方法的适当激光系统。工作重点还将集中在适当的芯片设计和塑料基板上，以利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，用于生物和小分子疗法的测定。