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: 7002691
• 负责人: PATRICK A LIMBACH
• 金额: $ 32.11万
• 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/7002691
• 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，产生高通量的方法，用于痕量测定和/或生物和小分子治疗剂的物理性质，这对制药和医学研究越来越重要。为支持这一长期目标，有两个具体目标。 第一个目标是从三个类别中开发适当的检测方法，这些方法将显著受益于微芯片平台的优势。研究重点将包括努力在速度和选择性方面与标准免疫测定方法相比，显著提高痕量生物化合物的测定。将使用基于适体的蛋白质测定作为说明性化学系统，并使用激光诱导荧光（LIF）检测的多通道塑料芯片上的毛细管电泳作为分析技术。通过快速、高分辨率地分离蛋白质-光适体复合物，无需预先制备样品，分析时间将从数小时缩短到数分钟。 此外，将开发一种相对高通量的基于非平衡分离的方法，该方法允许从广泛的配体结合系统中准确测定Kd和IC-50值。将在具有LIF、间接LIF或流动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，将开发一个通用的、低样品消耗、高通量和低成本的基于分离的平台，以确定小分子（MW 200- 1，000）的两个关键理化性质，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，可以提供一种快速评估这些关键参数的方法。 第二个目标将是开发适当的塑料微芯片平台，从这些分析可以进行。特别是，这些平台必须与第一个特定目标中开发的测定兼容。努力将致力于开发具有适当性能的多通道塑料芯片，用于LIF或间接LIF以及适当的激光系统，用于LIF相关方法。工作还将集中在适当的芯片设计和塑料基板的多分析物分析与质谱检测方法。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。