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 a commercial micro-fabrication facilities in Canada and Holland. This has resulted in developments 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 the outcome. 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).
