Phospholipid Giant Unilamellar Vesicles (GUVs) are usually prepared by electroformation in water, that is in a low-conductivity solution. We developed a protocol allowing their electroformation in the most common physiological buffer, phosphate-buffered saline (PBS). This was achieved based on a specific sequence of increasing electrical fields and for the two usual electrode types for electroformation, namely Indium Tin oxide-coated glass slides and Pt electrodes. These GUVs are stable over time (hour time-scale) and they can be isolated or micro-injected. The membrane composition was modified by adding cholesterol in order to adjust its mechanical properties. The optimal proportion of cholesterol vs. total phospholipid concentration was a ratio of 20 mol%, which increases membrane rigidity and facilitates vesicle microinjection.

Amplex Red (AR) is a very useful chemical probe that is employed in biochemical assays. In these assays, the non-fluorescent AR is converted to resorufin (RS), which strongly absorbs in the visible region (l abs = 572 nm) and yields strong fluorescence (l fluo = 583 nm). Even if AR is commonly used to report on enzymatic oxidase activities, an increasing number of possible interferences have been reported, thus lowering the accuracy of the so-called AR assay. As a redox-based reaction, we propose here to directly promote the conversion of AR to RS by means of electrochemistry. The process was first assessed by classic electrochemical and spectroelectrochemical investigations. In addition, we imaged the electrochemical conversion of AR to RS at the electrode surface by in situ confocal microscopy. The coupling of methodologies allowed to demonstrate that RS is directly formed from AR by an oxidation step, unlike what was previously reported. This gives a new insight in the deciphering of AR assays' mechanism and about their observed discrepancy.