@article{de alwis_denison_shah_mccarty_sombers_2023, title={Exploiting Microelectrode Geometry for Comprehensive Detection of Individual Exocytosis Events at Single Cells}, volume={8}, ISSN={["2379-3694"]}, DOI={10.1021/acssensors.3c00884}, abstractNote={Carbon fiber microelectrodes are commonly used for real-time monitoring of individual exocytosis events at single cells. Since the nature of an electrochemical signal is fundamentally governed by mass transport to the electrode surface, microelectrode geometry can be exploited to achieve precise and accurate measurements. Researchers traditionally pair amperometric measurements of exocytosis with a ∼10-μm diameter, disk microelectrode in an "artificial synapse" configuration to directly monitor individual release events from single cells. Exocytosis is triggered, and released molecules diffuse to the "post-synaptic" electrode for oxidation. This results in a series of distinct current spikes corresponding to individual exocytosis events. However, it remains unclear how much of the material escapes detection. In this work, the performance of 10- and 34-μm diameter carbon fiber disk microelectrodes was directly compared in monitoring exocytosis at single chromaffin cells. The 34-μm diameter electrode was more sensitive to catecholamines and enkephalins than its traditional, 10-μm diameter counterpart, and it more effectively covered the entire cell. As such, the larger sensor detected more exocytosis events overall, as well as a larger quantal size, suggesting that the traditional tools underestimate the above measurements. Both sensors reliably measured l-DOPA-evoked changes in quantal size, and both exhibited diffusional loss upon adjustment of cell-electrode spacing. Finite element simulations using COMSOL support the improved collection efficiency observed using the larger sensor. Overall, this work demonstrates how electrode geometry can be exploited for improved detection of exocytosis events by addressing diffusional loss─an often-overlooked source of inaccuracy in single-cell measurements.}, journal={ACS SENSORS}, author={De Alwis, A. Chathuri and Denison, J. Dylan and Shah, Ruby and McCarty, Gregory S. and Sombers, Leslie A.}, year={2023}, month={Aug} }
 @article{denison_de alwis_shah_mccarty_sombers_2023, title={Untapped Potential: Real-Time Measurements of Opioid Exocytosis at Single Cells}, volume={145}, ISSN={["1520-5126"]}, DOI={10.1021/jacs.3c07487}, abstractNote={The endogenous opioid system is commonly targeted in pain treatment, but the fundamental nature of neuropeptide release remains poorly understood due to a lack of methods for direct detection of specific opioid neuropeptides in situ. These peptides are concentrated in, and released from, large dense-core vesicles in chromaffin cells. Although catecholamine release from these neuroendocrine cells is well characterized, the direct quantification of opioid peptide exocytosis events has not previously been achieved. In this work, a planar carbon-fiber microelectrode served as a "postsynaptic" sensor for probing catecholamine and neuropeptide release dynamics via amperometric monitoring. A constant potential of 500 mV was employed for quantification of catecholamine release, and a higher potential of 1000 mV was used to drive oxidation of tyrosine, the N-terminal amino acid in the opioid neuropeptides released from chromaffin cells. By discriminating the results collected at the two potentials, the data reveal unique kinetics for these two neurochemical classes at the single-vesicle level. The amplitude of the peptidergic signals decreased with repeat stimulation, as the halfwidth of these signals simultaneously increased. By contrast, the amplitude of catecholamine release events increased with repeat stimulation, but the halfwidth of each event did not vary. The chromogranin dense core was identified as an important mechanistic handle by which separate classes of transmitter can be kinetically modulated when released from the same population of vesicles. Overall, the data provide unprecedented insight into key differences between catecholamine and opioid neuropeptide release from isolated chromaffin cells.}, number={44}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, author={Denison, J. Dylan and De Alwis, A. Chathuri and Shah, Ruby and Mccarty, Gregory S. and Sombers, Leslie A.}, year={2023}, month={Oct}, pages={24071–24080} }
 @article{mccarty_dunaway_denison_sombers_2022, title={Neurotransmitter Readily Escapes Detection at the Opposing Microelectrode Surface in Typical Amperometric Measurements of Exocytosis at Single Cells}, volume={6}, ISSN={["1520-6882"]}, DOI={10.1021/acs.analchem.2c00060}, abstractNote={For decades, carbon-fiber microelectrodes have been used in amperometric measurements of neurotransmitter release at a wide variety of cell types, providing a tremendous amount of valuable information on the mechanisms involved in dense-core vesicle fusion. The electroactive molecules that are released can be detected at the opposing microelectrode surface, allowing for precise quantification as well as detailed kinetic information on the stages of neurotransmitter release. However, it remains unclear how much of the catecholamine that is released into the artificial synapse escapes detection. This work examines two separate mechanisms by which released neurotransmitter goes undetected in a typical amperometric measurement. First, diffusional loss is assessed by monitoring exocytosis at single bovine chromaffin cells using carbon-fiber microelectrodes fabricated in a recessed (cavity) geometry. This creates a microsampling vial that minimizes diffusional loss of analyte prior to detection. More molecules were detected per exocytotic release event when using a recessed cavity sensor as compared to the conventional configuration. In addition, pharmacological inhibition of the norepinephrine transporter (NET), which serves to remove catecholamine from the extracellular space, increased both the size and the time course of individual amperometric events. Overall, this study characterizes distinct physical and biological mechanisms by which released neurotransmitter escapes detection at the opposing microelectrode surface, while also revealing an important role for the NET in “presynaptic” modulation of neurotransmitter release.}, journal={ANALYTICAL CHEMISTRY}, author={McCarty, Gregory S. and Dunaway, Lars E. and Denison, J. Dylan and Sombers, Leslie A.}, year={2022}, month={Jun} }
 @article{meunier_denison_mccarty_sombers_2020, title={Interpreting Dynamic Interfacial Changes at Carbon Fiber Microelectrodes Using Electrochemical Impedance Spectroscopy}, volume={36}, ISSN={0743-7463 1520-5827}, url={http://dx.doi.org/10.1021/acs.langmuir.9b03941}, DOI={10.1021/acs.langmuir.9b03941}, abstractNote={Carbon-fiber microelectrodes are an instrumental tool in neuroscience, used for electroanalysis of neurochemical dynamics and recordings of neural activity. However, performance is variable and dependent on fabrication strategies, the biological response to implantation, and the physical and chemical composition of the recording environment. This presents an analytical challenge, as electrode performance is difficult to quantitatively assess in situ, especially when electrodes are permanently implanted or cemented in place. We previously reported that electrode impedance directly impacts electrochemical performance for molecular sensing. In this work, we investigate the impact of individual components of the electrochemical system on impedance. Equivalent circuit models for glass- and silica-insulated carbon-fiber microelectrodes were determined using electrochemical impedance spectroscopy (EIS). The models were validated based on the ability to assign individual circuit elements to physical properties of the electrochemical system. Investigations were performed to evaluate the utility of the models in providing feedback on how changes in ionic strength and carbon fiber material alter impedance properties. Finally, EIS measurements were used to investigate the electrode/solution interface prior to, during, and following implantation in live brain tissue. A significant increase in impedance and decrease in capacitance occur during tissue exposure and persist following implantation. Electrochemical conditioning, which occurs continually during fast-scan cyclic voltammetry recordings, etches and renews the carbon surface, mitigating these effects. Overall, the results establish EIS as a powerful method for characterization of carbon-fiber microelectrodes, providing unprecedented insight into how real-world factors affect the electrode/solution interface.}, number={15}, journal={Langmuir}, publisher={American Chemical Society (ACS)}, author={Meunier, Carl J. and Denison, J. Dylan and McCarty, Gregory S. and Sombers, Leslie A.}, year={2020}, month={Mar}, pages={4214–4223} }