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.

需要开发非常敏感的微分析技术来测量生物液和单细胞中的蛋白质。这种分析的一种方法是毛细管电泳（CE），它是仅利用材料纳米含量的强大分析工具。但是，常规检测系统不足以分析非常小的生物样品和单个细胞，在这些样品中，在pico和femto-gram中存在许多分析物。已证明使用激光诱导的荧光（LIF）能够克服更常规检测系统的许多缺点。在DBEPS，我们设计并开发了一个实验室建造的LIF探测器，能够在约100张fermtograpon上测量荧光体标记的分析物。尽管需要进一步的细化以确保多个分析物预先分析的均匀标记，但仍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CE-LIF系统，并能够以0.5 pg/mL的水平定期测量分析物。进一步的发展将提高对1 femtogram/ml水平或更高水平的敏感性。一旦实现了这一目标，该仪器将能够在临床和研究样品中可靠地测量单细胞分泌物和/或细胞质。与免疫亲和力预动力步骤的耦合CE将进一步完善该仪器的能力，从而使其能够在10-100 pg范围内进行分析样品。由于与加拿大的商业微型制作设施的互动布置，进一步发展到电泳系统转化为芯片格式。这导致了发展为基于芯片的免疫亲和力系统的发展，并将未来的发展发展为设计和制造高度敏感的？用于临床分析。