Selective Electrochemical Bleaching of the Outer Leaflet of Fluorescently Labeled Giant Liposomes

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Selective Electrochemical Bleaching of the Outer Leaflet of Fluorescently Labeled Giant Liposomes ,Chem. Eur. J., 23,1–8, 2017


The combination of electrochemistry with confocal or wide field fluorescence microscopy techniques is of high interest in the fields of analytical chemistry, biophysics, and chemical physics. On the one hand, fluorescence microscopy allows imaging and fluorescence quantification. On the other hand, electrochemistry can be used to switch fluorescence on and off.



We have established that electrochemistry is a powerful tool to selectively bleach the fluorescence emitted by redox-active fluorophores localized on the outer leaflet of a lipid bilayer. Compared to photobleaching techniques such as FRAP and FLIP, the electrochemical bleaching process does not affect the fluorescent probes localized inside the vesicles thus providing an exploration tool allowing discrimination between the outer and the inner membrane leaflets. This electrochemical process also discards the use of chemical reducing agents that may undergo slow internalization within vesicles. Furthermore, the FLIE procedure appeared reliable to explore the reorganization dynamics of a single leaflet in real time. The capacity to totally bleach the fluorophores located in the outer leaflet also enables the possibility to monitor their passage between inner and outer leaflets (e.g. flip-flop of lipids) with confocal microscopy. This versatile approach appears very promising in the exploration of biological and pharmacological issues related to the internalization or release of analytes across lipid membranes.


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Chem. Eur. J., 23,1–8, 2017


Electrochemistry and confocal fluorescence microscopy were successfully combined to selectively bleach and monitor the fluorescence of NBD (7-nitrobenz-2-oxa-1,3- diazole)-labeled phospholipids of giant liposomes. Three types of giant unilamellar vesicles have been investigated, the fluorescent phospholipids being localized either mainly on their outer-, inner-, or both inner/outer leaflets. We established that only the fluorescent lipids incorporated in the outer leaflet of the vesicles underwent electrochemical bleaching upon reduction. The relative fluorescence intensity decay was quantified all along the electrochemical extinction through an original fluorescence loss in electrobleaching (FLIE) assay. As expected, the reorganization of the fluorescent phospholipids followed diffusion-driven dynamics. This was also evidenced by comparison with fluorescence loss in photobleaching (FLIP) and the corresponding numerical model. The value of the lateral diffusion coefficient of phospholipids was found to be similar to that obtained by other methods reported in the literature. This versatile and selective bleaching procedure appears reliable to explore important biological and pharmacological issues.


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Selective Electrochemical Bleaching of the Outer Leaflet of Fluorescently Labeled Giant Liposomes


Ana Isabel Perez Jimenez, Lylian Challier, Eric Aït-Yahiatène, Jérôme Delacotte, Eric Labbé, and Olivier Buriez


Chem. Eur. J., 23,1–8, 2017


DOI: 10.1002/chem.201605786