Development Of An Ultrasensitive Capillary Electrophoresis Detection System

有必要开发非常敏感的微分析技术来测量生物液体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），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，其中许多分析物以微克和飞克的数量存在。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。我们设计并开发了一种实验室建造的LIF检测器，能够测量约100飞图的荧光染料标记分析物。虽然需要进一步改进以确保多种分析物的统一分析前标记，但更先进的光子探测器的研究正在进行中。目前，已经建立了一个完整的实验室构建的CE-LIF系统，能够常规测量0.5 pg/ml水平的分析物。一个附加的检测器已经开发，结合两种不同的激光系统，从而允许同时检测未知的分析物和内部标准。该探测器极大地提高了CE系统的实用性。为了提高对1飞图/ml或更高水平的灵敏度，进一步的发展仍在进行中，目标是开发一种能够可靠地测量临床和研究样品中的单细胞分泌物和/或细胞质的仪器。耦合CE与免疫亲和预分析步骤进一步完善了该仪器的能力，使样品的分析在0.5 - 100 pg范围内。

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. 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 pre-analysis labeling of multiple analytes, research into more advanced photon detectors is underway. At present, a complete laboratory-built CE-LIF system has been constructed and is capable of routinely measuring analytes at the 0.5 pg/ml level. An additional detector has been developed that incorporates two different laser systems, thus allowing the simultaneous detection of both unknown analytes and internal standards. This detector has greatly enhanced the usefulness of the CE system. Further developments to increase sensitivity to the 1 femtogram/ml level or beyond are still progressing with the goal of developing an instrument capable of reliable measurements of single cell secretions and/or cytosol in clinical and research samples. Coupling CE with an immunoaffinity pre-analysis step has further refined the capabilities of this instrument, enabling the analysis of samples in the 0.5 - 100 pg range. Development toward moving the electrophoresis system into a chip format has progressed due to an interactive arrangement with commercial micro-fabrication facilities in Canada and Holland. This has resulted in development of both a chip-based immunoaffinity and a highly sensitive lab-on-a-chip. In collaboration with investigators from the NIH Clinical Center we have designed and implemented a micro-electrophoresis system for measuring inflammatory mediators in pediatric patients and a rapid immunoassay for the measurement of hormones in real-time. This electro-kinetically-driven chip performs liquid-phase immunoassays in under five minutes on approximately half-microliter samples of biological fluids. The chip enables rapid patient assessment with minimal intervention during other clinical or surgical procedures. There is a growing need for the development of techniques capable of measuring biological markers as they occur in the human body, in real-time. Although this ideal may never be achieved, there are very short measurement times that once achieved can revolutionize some clinical and surgical practices. The development of techniques to assess injury rapidly in emergency rooms or to assess the efficiency of a surgical procedure during the surgery could greatly change outcomes. The chip-based system is capable of performing such analyses within only two minutes. Although this is far from real-time, it does provide emergency room staff with a portable instrument that can quickly and efficiently triage trauma patients using the presence of inflammatory cytokines as markers of injury outcome. The simple chip-based system is being further developed to perform multiple assays on ultra-small biological samples (circa 0.1 microliters).