Development Of An Ultrasensitive Capillary Electrophores

超灵敏毛细管电泳的开发

• 批准号: 7013039
• 负责人: Terry M. Phillips
• 金额: --
• 依托单位: OFFICE OF THE DIRECTOR, NATIONAL INSTITUTES OF HEALTH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7013039
• 关键词: analytical method; bioengineering /biomedical engineering; biomedical equipment development; capillary electrophoresis; cytoplasm; fluorescent dye /probe; immunoaffinity chromatography; laser spectrometry; method development; protein quantitation /detection; proteomics; single cell analysis

There is a need for the development of very sensitive micro-analytical techniques for measuring proteins in biological fluids and single cells. One approach to such analysis is capillary electrophoresis (CE), which is a powerful analytical tool utilizing only nanoliters of materials. However, conventional detection systems are inadequate for the analysis of very small biological samples and single cells where many analytes are present in pico- and femto-gram quantities. The use of laser-induced fluorescence (LIF) has been demonstrated to be capable of overcoming many of the shortcomings of more conventional detection systems. At DBEPS, we have designed and developed a laboratory-built LIF detector capable of measuring fluorochrome-labeled analytes at approximately 100 femtograms. Although further refinement is required to ensure uniform labeling of multiple analytes pre-analysis, research into more advanced photon detectors is underway. Presently, a complete laboratory-built CE-LIF system has been constructed and is capable of routinely measuring analytes at the 0.5 pg/ml level. Further development will increase this sensitivity to the 1 femtogram/ml level or beyond. Once this is achieved, the instrument will be capable of reliable measurements of single cell secretions and/or cytosol in clinical and research samples. Coupling CE with an immunoaffinity pre-analysis step will further refine the capabilities of this instrument enabling it to analysis samples in the 10-100 pg range. Further development into moving the electrophoresis system into a chip format has progressed due to a interactive arrangement with a commercial micro-fabrication facility in Canada. This has resulted in developments into a chip-based immunoaffinity system with future developments into the design and manufacture of a highly sensitive ?lab-on-a-chip? for clinical analyses.

需要开发用于测量生物流体和单细胞中的蛋白质的非常灵敏的微量分析技术。这种分析的一种方法是毛细管电泳（CE），这是一种仅利用纳升材料的强大分析工具。然而，传统的检测系统不足以分析非常小的生物样品和单细胞，其中许多分析物以皮科和毫微微克的量存在。激光诱导荧光（LIF）的使用已被证明能够克服许多传统检测系统的缺点。在DBEPS，我们设计并开发了一种实验室制造的LIF检测器，能够以大约100毫微微克的速度测量荧光染料标记的分析物。虽然需要进一步改进以确保分析前对多种分析物进行统一标记，但正在研究更先进的光子探测器。目前，已经构建了一个完整的实验室构建的CE-LIF系统，能够在0.5 pg/ml水平上常规测量分析物。进一步的开发将使该灵敏度提高到1毫微微克/毫升或更高的水平。一旦实现这一点，该仪器将能够可靠地测量临床和研究样品中的单细胞分泌物和/或细胞溶质。将CE与免疫亲和预分析步骤结合将进一步完善该仪器的能力，使其能够分析10-100 pg范围内的样品。由于与加拿大的商业微制造设施的互动安排，将电泳系统移动到芯片格式的进一步发展已经取得进展。这导致了发展成为一个基于芯片的免疫亲和系统，未来的发展成为一个高度敏感的？芯片实验室用于临床分析。