ONLINE DETERMINATION OF EXTRACELLULAR GLUTATHIONE IN ORGANOTYPIC HIPPOCAMPAL SLICE CULTURES WITH A MCIROFLUIDIC DEVICE AND CONFOCAL LASER-INDUCED FLUORESCENCE DETECTION

摘要

GSH is an essential cellular antioxidant in the brain cells due to its active sulfhydryl group. Measuring the concentration, distribution and metabolic pathways of GSH and related compounds provides a lot of useful information for studying oxidative insult in vivo. Microfluidic chips are ideal platforms for biological samples, because they are capable of analyzing very small amount of analyte in a relative short time. The coupling of microfluidic separation and laser-induced fluorescence provides an effective way for analyzing GSH and related compounds. A confocal LIF detection system has been developed in our laboratory. The key part in building this system is the selection of an optimized set of optical components, which mainly includes a laser, an objective, a front surface mirror, lens, filter sets and a detector. The parameters of these optical components were first optimized according to the preliminary scheme of the system. Meanwhile, an opto-mechanical assembly was designed and made for arranging these components into a whole system. A controlling program in LabVIEW was also developed, which incorporates several major functions, such as data collection and pretreatment, voltage and current monitoring, high voltage power supply (HVPS) manipulation, sampling control, into one program and offers an easy access for user to have a convenient control of the whole system. The procedures of beam alignment for our detection system are discussed in detail in the system testing section. The profiles of the focused laser beam at three different positions were measured by using a simple setup, the results of which were consistent with those from the theoretical calculation. A series of experiments were designed to identify the dominant noise sources in the system for a better signal to noise ratio (SNR). Noise reduction was finally obtained by applying averaging and low pass filter. Experiments with real samples showed that the linear range for GSH labeled with Thioglo 1 is between 0-49.7 µM and the limit of detection (LOD) is around 1.45 nM for our system. Our future work will be focused on connecting electroosmotic sampling with separation on microfluidic device to analyze the extracellular GSH in organotypic hipocampal slice cultures.