Development Of An Ultrasensitive Capillary Electrophoresis Detection System

需要开发非常灵敏的微量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法(CE)，这是一种仅使用纳米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，在这些样品中，许多分析物以皮克和毫微克的数量存在。激光诱导荧光(LIF)的使用已被证明能够克服更传统检测系统的许多缺点。我们设计并开发了一种实验室建造的激光诱导荧光检测器，能够在大约100毫微克的频率下测量荧光标记的分析物。尽管需要进一步改进以确保对多个分析物进行统一的分析前标记，但更先进的光子探测器的研究正在进行中。目前，一套完整的实验室构建的CE-LIF系统已经建成，能够对0.5pg/ml的分析物进行常规测量。另外开发了一种检测器，它结合了两种不同的激光系统，从而允许同时检测未知分析物和内标。该检测器大大提高了毛细管电泳法的实用性。提高对1毫微克/毫升或更高水平的灵敏度的进一步发展仍在取得进展，目标是开发一种能够可靠地测量临床和研究样本中的单细胞分泌物和/或胞浆的仪器。将CE与免疫亲和预分析步骤相结合，进一步完善了该仪器的功能，使样品分析能够在0.5-100 pg的范围内进行。 由于与加拿大和荷兰的商业微制造设施进行了互动安排，将电泳系统转移到芯片格式的开发取得了进展。这导致了基于芯片的免疫亲和力和高度敏感的芯片上实验室的发展。我们与美国国立卫生研究院临床中心的研究人员合作，设计并实现了一套用于检测儿科患者炎症介质的微电泳系统和一种用于实时检测激素的快速免疫分析系统。这种电动驱动的芯片在不到五分钟的时间内对大约半微升的生物液体样本进行液体免疫分析。该芯片能够在其他临床或外科手术过程中以最小的干预快速对患者进行评估。人们越来越需要开发能够在生物标记出现在人体内时实时测量它们的技术。尽管这一理想可能永远不会实现，但一旦实现，测量时间非常短，可能会给一些临床和外科实践带来革命性的变化。在急诊室中快速评估损伤或在手术中评估外科手术的效率的技术的发展可能会极大地改变结果。这个基于芯片的系统能够在两分钟内进行这样的分析。尽管这远不是实时的，但它确实为急诊室工作人员提供了一种便携式仪器，可以使用炎症细胞因子的存在作为损伤结果的标志，快速有效地对创伤患者进行分诊。这种基于芯片的简单系统正在进一步开发，以对超小生物样本(约0.1微升)进行多种检测。

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).