Mechanistic Aspects of Vesicle Opening during Analysis with Vesicle Impact Electrochemical Cytometry

Abstract

Vesicle impact electrochemical cytometry (VIEC) has been used to quantify the vesicular transmitter content in mammalian vesicles. In the present study, we studied the mechanism of VIEC by quantifying the catecholamine content in single vesicles isolated from pheochromocytoma (PC12) cells. These vesicles contain about one tenth of the catecholamine compared with adrenal chromaffin vesicles. The existence of a prespike foot for many events suggests the formation of an initial transiently stable pore at the beginning of vesicle rupture. Increasing the detection temperature from 6 to 30 °C increases the possibility of vesicle rupture on the electrode, implying that there is a temperature-dependent process that facilitates electroporation. Natively larger vesicles are shown to rupture earlier and more frequently than smaller ones in VIEC. Likewise, manipulating vesicle content and size with drugs leads to similar trends. These data support the hypothesis that electroporation is the primary force for pore opening in VIEC. We further hypothesize that a critical step for initiating vesicle opening by electroporation is diffusion of membrane proteins away from the membrane region of contact with the electrode to allow closer contact, increasing the lateral potential field and thus facilitating electroporation.

Lipid Membrane Permeability of Synthetic Redox DMPC Liposomes Investigated by Single Electrochemical Collisions

“…[12][13][14]16,17 According to Ewing and co-workers, the so-called vesicle impact electrochemical amperometry is mainly driven by an electroporation process of the vesicle membrane on polarized carbon UMEs which leads to the vesicle rupture and the electrolysis of its content. [18][19][20][21] Vesicle membrane opening by electroporation is strongly dependent on lipid membrane properties, liposome content, vesicle size, temperature, electrode potential, the nature of the electrode and probably the concentration of redox species inside and outside the liposome. [18][19][20][21][22] For example, to observe the current spikes corresponding to the oxidation of ferrocyanide encapsulated inside phospholipid vesicles when they collide with a Pt UME, the presence of an appropriate concentration of surfactant in solution is required.…”

“…[18][19][20][21] Vesicle membrane opening by electroporation is strongly dependent on lipid membrane properties, liposome content, vesicle size, temperature, electrode potential, the nature of the electrode and probably the concentration of redox species inside and outside the liposome. [18][19][20][21][22] For example, to observe the current spikes corresponding to the oxidation of ferrocyanide encapsulated inside phospholipid vesicles when they collide with a Pt UME, the presence of an appropriate concentration of surfactant in solution is required. 14 In the absence of surfactant, we found that collision and adhesion of vesicles at the Pt UME does not allow the electrolysis of their ferrocyanide content.…”

Effects of Instrumental Filters on Electrochemical Measurement of Single‐Nanoparticle Collision Dynamics

“…Although we chose nanoparticle collision experiments as the system of interest for this study, the basic concepts of filters discussed throughout this report are generally applicable to all single‐entity experiments. For example, the instrument bandwidth could affect the measurement of the shortest lifetimes associated with single‐nucleotide base‐flipping states in a single DNA molecule, or the timescale for electrochemical conversion of neurotransmitters in vesicles …”

Counting and Sizing of Single Vesicles/Liposomes by Electrochemical Events

“…They compared the results from intracellular vesicle electrochemical cytometry with those from chemically stimulated exocytosis. [90] Furthermore, they found that the probability of vesicle rupture was related to vesicle size, temperature, and preexposure to drugs that manipulate vesicular content. [89] Recently, pretransient or prespike feet in events under certain conditions were observed, thus supporting the mechanism of electroporation for pore opening.…”

“…[89] Recently, pretransient or prespike feet in events under certain conditions were observed, thus supporting the mechanism of electroporation for pore opening. [90] Furthermore, they found that the probability of vesicle rupture was related to vesicle size, temperature, and preexposure to drugs that manipulate vesicular content. A recent study showed closely agreeing catecholamine distributions obtained by measuring the vesicular catecholamine content in extracted and intracellular vesicles by employing disk and nanotip conical microelectrodes.…”