Nanopore Opening at Flat and Nanotip Conical Electrodes during Vesicle Impact Electrochemical Cytometry

Artikel i vetenskaplig tidskrift, 2018

The oxidation of catecholamine at a microelectrode, following its release from individual vesicles, allows interrogation of the content of single nanometer vesicles with vesicle impact electrochemical cytometry (VIEC). Previous to this development, there were no methods available to quantify the chemical load of single vesicles. However, accurate quantification of the content is hampered by uncertainty in the proportion of substituent molecules reaching the electrode surface (collection efficiency). In this work, we use quantitative modeling to calculate this collection efficiency. For all vesicles except those at the very edge of the electrode, modeling shows that ∼100% oxidation efficiency is achieved when employing a 33 μm diameter disk microelectrode for VIEC, independent of the location of the vesicle release pore. We use this to experimentally determine a precise distribution of catecholamine in individual vesicles extracted from PC12 cells. In contrast, we calculate that when a nanotip conical electrode (∼4 μm length, ∼1.5 μm diameter at the base) is employed, as in intracellular VIEC (IVIEC), the current-time response depends strongly on the position of the catecholamine-releasing pore in the vesicle membrane. When vesicle release occurs with the pore opening occurring far from the electrode, lower currents and partial oxidation (∼75%) of the catecholamine are predicted, as compared to higher currents and ∼100% oxidation, when the pore is close to/at the electrode surface. As close agreement is observed between the experimentally measured vesicular content in intracellular and extracted vesicles from the same cell line using nanotip and disk electrodes, respectively, we conclude that pores open at the electrode surface. Not only does this suggest that electroporation of the vesicle membrane is the primary driving force for catecholamine release from vesicles at polarized electrodes, but it also indicates that IVIEC with nanotip electrodes can directly assess vesicular content without correction.