More transparency in bioanalysis of exocytosis : application of fluorescent false neurotransmitters in coupling methodology of electrochemistry with fluorescence microscopy at ITO microelectrodes

Abstract : Vesicular exocytosis is a ubiquitous way for intercellular communications. TIRFM (total internal reflection fluorescence microscopy) and amperometry are nowadays the two most frequently used analytical methods with complementary features for its investigation. The combination of these two analytical techniques to track exocytotic secretions was firstly achieved by our group in 2011 and this new technique was demonstrated to show both high temporal and spatial resolutions by simultaneously recording the fluorescent and amperometric signals. The major disadvantage of this former work was the independent loading of optical and electrochemical probes to the secretory vesicles, which resulted in 'sightless' amperometric or optical signals as well as low coupling efficiency. Therefore, in this thesis, we attempted to develop a unique probe with dual fluorescent/electrochemical characteristics to track exocytotic process by TIRFM/amperometry coupling technique. This is why an analog of biogenic monoamine neurotransmitters, 4-(2-aminoethyl)-6-chloro-7-hydroxy-2H-1-benzopyran-2-one hydrochloride (named as 1 in this work) was synthesized. 1 exhibited bright, stable, pH-dependent fluorescence. When excited at 405 nm, its fluorescence intensity was almost doubled with the increase of pH values from 5 (similar to that in the vesicular lumen) to 7 (similar to the extracellular medium). Furthermore, in voltammetry, 1 was demonstrated to be easily electrooxidized on GCE (glassy carbon electrode), CFE (carbon fiber electrode) and ITO (indium tin oxide) electrodes surfaces, showing good electroactivity. 1 was also shown to penetrate easily into the vesicles of BON N13 cells within 1 hour incubation, testifying its specific affinity with these VMAT-equipped (vesicular monoamine transporter) vesicles. The applications of 1 as optical and electrochemical probes for exocytosis monitoring were then separately validated in BON N13 cells by TIRFM and amperometry measurements, respectively. Simultaneous recording of fluorescent and amperometric information by using 1 dual probes loaded cells was subsequently acquired in a microfabricated device constituted by conductive and transparent ITO electrodes. Our results based on the unique probe 1 for electrochemical and fluorescent detection of exocytotic release seemed more adapted than all the previous works involving independent probes. The high spatial and temporal resolutions of this combined method also allowed analyzing consecutive exocytotic secretions as well as overlapped events in 1-stained cells. Further analysis of these two signals with complementary information will shed more light on the correlation of the fusion pore behavior (opening/closure dynamics, stability…) measured by amperometry and the motion of a secretory vesicle in three dimensions (tethering, docking, fusion and retrieval) detected by TIRFM.
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Xiaoqing Liu. More transparency in bioanalysis of exocytosis : application of fluorescent false neurotransmitters in coupling methodology of electrochemistry with fluorescence microscopy at ITO microelectrodes. Chemical Physics [physics.chem-ph]. Université Pierre et Marie Curie - Paris VI, 2016. English. ⟨NNT : 2016PA066415⟩. ⟨tel-02295570⟩

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