On the mechanism of electrochemical vesicle cytometry: chromaffin cell vesicles and liposomes

Abstract

The mechanism of mammalian vesicle rupture onto the surface of a polarized carbon fiber microelectrode during electrochemical vesicle cytometry is investigated. It appears that following adsorption to the surface of the polarized electrode, electroporation leads to the formation of a pore at the interface between a vesicle and the electrode and this is shown to be potential dependent. The chemical cargo is then released through this pore to be oxidized at the electrode surface. This makes it possible to quantify the contents as it restricts diffusion away from the electrode and coulometric oxidation takes place. Using a bottom up approach, lipid-only transmitter-loaded liposomes were used to mimic native vesicles and the rupture events occurred much faster in comparison with native vesicles. Liposomes with added peptide in the membrane result in rupture events with a lower duration than that of liposomes and faster in comparison to native vesicles. Diffusional models have been developed and suggest that the trend in pore size is dependent on soft nanoparticle size and diffusion of the content in the nanometer vesicle. In addition, it appears that proteins form a barrier for the membrane to reach the electrode and need to move out of the way to allow close contact and electroporation. The protein dense core in vesicles matrixes is also important in the dynamics of the events in that it significantly slows diffusion through the vesicle.

Vesicle impact electrochemical cytometry compared to amperometric exocytosis measurements

“…Despite a small applied potential, if the vesicle membrane is within the double layer at the electrode surface, then the potential field across the membrane will be in the range for electroporation to occur (1.4 × 10 6 V/cm). The most evident proof of electroporation as the cause of membrane rupture is the potential dependence of the probability of detecting vesicles (Figure 2A and B) [18•]. The number of molecules detected does not seem to change with increased potential, suggesting that electroporation is facilitated but the kinetics of the oxidation reaction is not increased.…”

“…This is clearly demonstrated when using liposomes as simplified models of vesicles. Liposomes filled with dopamine appear to open as the contact the electrode surface [18•]. They have significantly narrower pore opening and faster oxidation of the loaded transmitters.…”

“…Liposomes decorated with polyarginine polypeptides have slower pore opening than the non-decorated liposomes, but are still faster than biologically derived vesicles (Figure 2C). This suggests that proteins, or polypeptides, indeed need to diffuse away for the membrane to reach the electrode and for electroporation to occur [18•]. …”

“…A, Differences in potential does not change the amount of molecules detected significantly [18]. B, The number of events, however, increase with increasing applied potential, indicating that electroporation might be the mechanism of membrane opening [18]. C, The presence of peptides and proteins seem to determine the dimensions of the pore formed through electroporation [18].…”

“…The Ewing group pioneered this area with a method called flow vesicle electrochemical cytometry (FVEC) [15,16]. The FVEC has later been replaced with two related methods called vesicle impact electrochemical cytometry (VIEC) and intracellular vesicle impact electrochemical cytometry (IVIEC) [17••,18•]. The combination of VIEC and IVIEC with SCA and, to a lesser degree, imaging techniques like NanoSIMS is the topic of this review.…”