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Open Dmo Dissertation Final.Pdf The Pennsylvania State University The Graduate School Department of Chemistry ELECTROCHEMICAL CYTOMETRY: A NOVEL TECHNIQUE TO QUANTITATIVELY PROBE INDIVIDUAL NEUROSECRETORY AND ARTIFICIAL VESICLES A Dissertation in Chemistry by Donna M. Omiatek © 2010 Donna M. Omiatek Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2010 The dissertation of Donna M. Omiatek was reviewed and approved* by the following: Andrew G. Ewing Professor of Chemistry Professor of Neural and Behavioral Chemistry J. Lloyd Huck Chair in Natural Sciences Dissertation Advisor Chair of Committee Christine D. Keating Associate Professor of Chemistry Scott T. Phillips Assistant Professor of Chemistry Michael L. Heien Assistant Professor of Chemistry Peter J. Butler Associate Professor of Bioengineering Barbara J. Garrison Shapiro Professor of Chemistry Head of the Department of Chemistry *Signatures are on file in the Graduate School ii ABSTRACT This thesis details the development of a novel analytical method, electrochemical cytometry, which has been used to probe the contents of individual biological vesicles extracted from the cell environment in a high-throughput format. This experimental approach is based on technology that I have developed to electrochemically interrogate individual vesicles using a hybrid capillary-microfluidic device. In this format, a vesicle suspension can be injected onto a fused-silica capillary and subsequently isolated to individual components in an applied electric field by capillary electrophoresis. The separation capillary terminates into a PDMS-based microfluidic device that continuously delivers surfactant from microchannels to the detection zone in a sheath-flow format. As individual intact vesicles exit the separation capillary, they are chemically lysed and their contents subsequently detected at a carbon-fiber microelectrode positioned at the outlet. Electrooxidation of vesicular contents by constant potential amperometry allows for the mole amount encapsulant to be quantified on a per vesicle basis. The electrochemical cytometry method design and characterization studies are outlined in Chapter 2. Here, I investigated how sheath flow of the lysis buffer affected analyte dispersion in the detection zone of the hybrid capillary-microfluidic platform using confocal fluorescence microscopy, computational fluid dynamics simulations, and end-column electrochemical detection to monitor the eluent flow profile. Then a proof- of-concept study was performed to investigate artificial vesicles (nanoliposomes) both containing and lacking an electroactive analyte (dopamine) by electrochemical cytometry to highlight both the sensitivity and selectivity of the detection scheme. By modeling the amperometric peak characteristics with the theoretical flux of dopamine from of a lysed iii vesicle at the detector, I was able to determine the coulometric efficiency of the electrochemical cytometry detection scheme to be 87% (which was increased to > 95% via less signal filtering for investigations in Chapters 3 and 4). An interesting application of this novel technique is discussed in Chapter 3, where electrochemical data recorded from stimulus-coupled secretion experiments at single PC12 cells were compared to cell-free measurements of vesicular dopamine content using electrochemical cytometry. Although standard methods used to measure stimulus- coupled release from single cells have classically been thought to assess the entire content of vesicles, there is evidence in the literature that suggests the total transmitter stored in vesicles is not expelled during exocytosis. This hypothesis was directly interrogated using electrochemical cytometry which allows for the quantification of total vesicular neurotransmitter in a manner that circumvents biophysical release processes of the cell associated with exocytosis. By comparing total dopamine content from electrochemical cytometry measurements at individual isolated vesicles with the vesicular amount released at single PC12 cells, it was determined that during full exocytosis only a fraction (~40%) of total transmitter load is released from a typical vesicle. The data from these experiments support the intriguing hypothesis that the average vesicle does not open all the way during exocytosis, resulting in incomplete distention of the neurotransmitter contents. The implications of these results to neuroscience are large; namely: even during full exocytosis vesicular neurotransmitter release is not necessarily all-or-none. This suggests that transmitter secretion can be regulated within a single exocytosis event, imparting a potential molecular basis for synaptic plasticity at the subcellular level. Upon iv establishing that release in exocytotic processes proceeded in an incomplete manner, electrochemical data quantified from both single cell release experiments and electrochemical cytometry of vesicles were related to vesicular volume from electron microscopy measurements to investigate the location of intravesicular catecholamine stores (e.g., halo or dense core) in PC12 cells retained post-fusion. In Chapter 4, the electrochemical cytometry method was expanded to investigate vesicular content from the midbrain neurons of a mammalian animal model. Vesicles were isolated from mice striatal tissue and individually probed for endogenous dopamine content in a manner that was independent of release. This brain region is of great interest in neuroscience research since dopamine pathways that terminate into the striatum have been directly linked to a variety of neurobiological phenomena including motor function, reward, addiction, cognition, and neurological dysfunction, including Parkinson’s and Huntington’s disease. In addition, electrochemical cytometry was applied to monitor the effects of synaptic vesicle neurotransmitter loading and depletion from mice injected with various pharmacological agents. It was demonstrated that vesicular neurotransmitter levels were altered and variances observed from these treatments resolved from single synaptic vesicles in a high-throughput manner, thus providing an efficient methodology to screen for the effects of neurological therapeutics in the subcellular domain. Moreover, the effect of the psychostimulant, amphetamine was investigated and shown to significantly deplete dopamine in the average striatal synaptic vesicle. In Chapter 5, the development and characterization of a reagentless modified carbon-fiber microelectrode sensor capable of monitoring pH in biological v microenvironments is presented. The voltammetric carbon-fiber sensor was modified using a simple and reproducible procedure that involved electrochemically grafting a commercially available diazonium salt (Fast Blue RR) onto the microelectrode surface. Fast-scan cyclic voltammetry was used to probe redox activity of a quinone-moiety on the surface bound diazonium. A quantifiable oxidative wave was observed to yield a linear pH-dependent voltammetric response by flow injection analyses. Then, the sensor was used to measure fluctuations of pH in vivo that were evoked by optogenetic stimulus- coupled secretion in the central nervous system of a mutant fruit fly. The work in Chapter 6 outlines several future bioanalytical applications of electrochemical cytometry. The first few involve elements that apply to the technical aspects of the measurement to expand upon the separation and detection capabilities of the method. The latter describe specific applications in liposome research and neuroscience that can be investigated for the quantitative characterization of volume- limited submicron vesicles via electrochemical cytometry. vi TABLE OF CONTENTS List of Figures .............................................................................................................. ix List of Tables ............................................................................................................... xii List of Schemes ............................................................................................................ xiii Preface ........................................................................................................................ xiv Acknowledgements .................................................................................................... xv Chapter 1. Subcellular Analyses for Vesicular Content Quantification ............ 1 Introduction ................................................................................................................ 1 Imaging Exocytosis at Single Cells ........................................................................... 3 Electrochemical Measurements of Vesicular Transmitter Content from Single Cells ................................................................................................................ 11 Separation Techniques Used to Quantify Vesicular Transmitter Content ................. 18 Scope of the Thesis .................................................................................................... 22 References .................................................................................................................. 26 Chapter 2. Development and Characterization of a Hybrid Capillary- Microfluidic Device for the Electrochemical Cytometry of Vesicles ................... 31 Introduction ................................................................................................................ 31 Materials and Methods ..............................................................................................
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