When considering the measurement of release events from cells, it can be done at levels as small as the single cell and performed in systems increasingly larger and more complex up to in vivo studies. Though in vitro systems lack the physiological relevance of in vivo, their simple and controlled environment is highly advantageous in preliminary mechanistic studies. In spite of this, there exists a serious gap in our ability to perform in vitro measurements on a wide array of analytes within a meaningful time frame. While electrochemical techniques are unparalleled in their ability to temporally resolve minute signals in biological systems, there is only a small class of targets which are suitable for this type of analysis. When analyzing non-electroactive analytes, measurements are often plagued by slow temporal responses (5+ minutes). Fluorescent imaging offers opportunities to monitor faster dynamics of non-electroactive analytes, but the target analyte must be either natively fluorescent or labeled, which can result in nonspecific binding and cytotoxicity. In both of these cases fast dynamics can be observed, but the array of analytes is small and only a few can be monitored simultaneously. In this work, a novel in vitro sampling platform is described which is capable of simultaneously monitoring approximately 15 non-electroactive analytes with 20 second temporal resolution. Cells were cultured on the surface of a microdialysis probe coupled with an analytical system for analysis. Small molecules released from the cells upon stimulation diffuse across the porous membrane because of the close proximity. A high-speed CE, built in house, enabled analysis of the collected dialysate. The ability of our platform to detect basal and stimulated release of amines was confirmed by transferring the probe between artificial cerebrospinal fluid (aCSF) and a potassium-spiked (100 mM K+-aCSF) stimulant solution. A variety of cell models were tested for compatibility with the in vitro-microdialysis platform, both single cell type and co-cultures were initiated. Adherence of viable cells was confirmed by labeling cells with either fluorescein diacetate (FDA) or specific antibody labelling, followed by imaging under a microscope. As a step towards continuously monitoring the change of non-electroactive analytes released from cultured cells, microdialysis was coupled directly to micro free flow electrophoresis (µFFE) device instead of the high-speed CE instrument.
University of Minnesota Ph.D. dissertation. April 2016. Major: Chemistry. Advisor: Michael Bowser. 1 computer file (PDF); xvi, 120 pages.
Development and Applications of In Vitro-Microdialysis: A Sampling Platform for Fast Analysis of Non-Electroactive Analytes.
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