IVIEC, a recently developed in the Ewing group, is a nano-electrochemical technique for in-situ quantification of vesicular content. VIEC is also a similar technique to IVIEC, but instead of in-situ quantification of vesicular content, vesicles are isolated from the cell machinery and then the content is measured. However, in both methods, the vesicles rupture on an electrode surface and it was not clear that if they open towards the electrode or to the other sides. We provided a microwell array electrode to trap the chromaffin vesicles and subsequently their neurotransmitters after rupturing by applying a potential. Our finding showed that the number of neurotransmitter molecules measured in these microwell-arrays was in good agreement with the previously reported results on the regular electrodes. It shows that the entire vesicle content is measured in IVIEC and VIEC; either the vesicle is ruptured towards the electrode or other sides. Thus, these results verify that IVIEC is a robust tool for observing the cellular response of a drug or stimulus on the vesicular content. The obtained result was published as a scientific article in an American Chemical Society publication:
Ranjbari, E., Taleat, Z., Mapar, M., Aref, M., Dunevall, J., & Ewing, A. (2020). Direct Measurement of Total Vesicular Catecholamine Content with Electrochemical Microwell Arrays. Analytical Chemistry, 92(16), 11325-11331.
Intracellular lipid injection, as a promising strategy, was developed to in-situ manipulate the lipidic structure of not only the cell membrane inner leaflet but the outer leaflet of the vesicles. We injected/delivered different phospholipids possessing different intrinsic curvature into chromaffin single cells using a nanopipette and monitored the effect of so-called intracellular incubation of phospholipids on the exocytosis release process and also the vesicular content using SCA and IVIEC techniques, respectively. This work provides new insights into the mechanism of synaptic plasticity. Our exocytotic analysis reveals that the intracellular nano-injection of phosphatidylcholine and lysophosphatidylcholine decreases the number of released catecholamines, whereas phosphatidylethanolamine shows the opposite effect. These observations support the emerging hypothesis that lipid curvature results in membrane remodeling through secretory pathways and also provides new evidence for a critical role of the lipid localization in modulating the release process. The obtained result was published as a scientific article in the Royal Society of Chemistry
Aref, M., Ranjbari, E., Romiani, A., & Ewing, A. G. (2020). Intracellular injection of phospholipids directly alters exocytosis and the fraction of chemical release in chromaffin cells as measured by nano-electrochemistry. Chemical Science, 11(43), 11869-11876.
A new methodology to study zinc’s effect on exocytosis and memory formation was proposed. In this part of the project, two electrochemical techniques (potentiometry and amperometry at single cell level) were considered to combine. To develop and apply the potentiometric sensor for in-vivo zinc measurement and study the role of zinc in STM formation, fabrication of a model potentiometric sensor, the pH sensor, was necessary. This model sensor has been successfully developed. However, before moving to the zinc sensor development, the project reached its 24th month. The initial results of this sensor for intracellular monitoring will be reported in a manuscript.
