Fast-scan Cyclic Voltammetry Detection Research Articles

Surface-Roughened Graphene Oxide Microfibers Enhance Electrochemical Reversibility.

Here, we provide an optimized method for fabricating surface-roughened graphene oxide disk microelectrodes (GFMEs) with enhanced defect density to generate a more suitable electrode surface for dopamine detection with fast-scan cyclic voltammetry (FSCV). FSCV detection, which is often influenced by adsorption-based surface interactions, is commonly impacted by the chemical and geometric structure of the electrode's surface, and graphene oxide is a tunable carbon-based nanomaterial capable of enhancing these two key characteristics. Synthesized GFMEs possess exquisite electronic and mechanical properties. We have optimized an applied inert argon (Ar) plasma treatment to increase defect density, with minimal changes in chemical functionality, for enhanced surface crevices to momentarily trap dopamine during detection. Optimal Ar plasma treatment (100 sccm, 60 s, 100 W) generates crevice depths of 33.4 ± 2.3 nm with high edge plane character enhancing dopamine interfacial interactions. Increases in GFME surface roughness improve electron transfer rates and limit diffusional rates out of the crevices to create nearly reversible dopamine electrochemical redox interactions. The utility of surface-roughened disk GFMEs provides comparable detection sensitivities to traditional cylindrical carbon fiber microelectrodes while improving temporal resolution ten-fold with amplified oxidation current due to dopamine cyclization. Overall, surface-roughened GFMEs enable improved adsorption interactions, momentary trapping, and current amplification, expanding the utility of GO microelectrodes for FSCV detection.

Microfluidic-Assisted Alignment of Graphene Nanosheets for Microfiber Formation

Recently, our lab developed graphene oxide microfibers for ultrasensitive neurochemical detection with fast-scan cyclic voltammetry (FSCV). Graphene-fiber microelectrodes provide a significant advance over traditional carbon-fiber microelectrodes due to their resistance to detrimental fouling, fast electron transfer kinetics, and frequency independent behavior. Despite our recent advancement, there still exists a gap in our understanding of how specific changes in the carbon chemical structure influences adsorption and interaction of various neurochemicals at the surface. Understanding the extent to which the carbon chemical structure influences electrochemical detection is critical for designing tailor-made electrodes for ultrasensitive analyte detection. From a molecular level point-of-view, the graphene sheet consists of two regions with different structure: i) the basal plane, consisting of conjugated sp2 hybridized carbon atoms, and characterized by an atomic flatness and low defect density; and ii) the edge plane, that forms sp3 sites with a high level of defects and a variety of functional groups and dangling bonds. It is generally believed that the electrochemical properties of the basal and edge plane are significantly different, however, there has been debate concerning the electrochemical activity at the basal and edge plane. The conventional consensus is that electron transfer kinetics is faster on the edge plane in comparison with the basal plane sites, while the opposite barrier of this debate provides evidence for fast electron transfer at pristine basal plane. Moreover, how these factors influence neurochemical detection, particularly at rapid scan rates, is still lacking due to the necessity of using microelectrodes with FSCV. To date, most studies analyzing the influence of basal vs. edge plane surfaces on electrochemical detection have used macroelectrodes like highly ordered pyrolytic graphite (HOPG) which are not amenable to FSCV detection due to the large capacitive currents obtained at macroelectrodes at scan rates consisting of hundreds of volts per second. Therefore, to address this existing knowledge gap in understanding how edge vs. basal planes influence subsecond neurochemical detection, the ability to develop microelectrodes with controlled surfaces is crucial. Here, I will discuss our labs latest effort in developing graphene oxide microfibers with controllable nanosheet alignment to study the influence of basal vs. edge plane availability on neurochemical detection at rapid scan rates. To assist in nanosheet alignment, we use microfluidic channels to assist in fluid-driven control of nanosheet orientation. Using microfluidics to help develop highly controllable electrode materials provides a critical advance in our ability to develop novel carbon surfaces for detection.

MPCVD-Grown Nanodiamond Microelectrodes with Oxygen Plasma Activation for Neurochemical Applications.

Nanodiamonds (NDs) are a carbon nanomaterial that has a diamond core with heteroatoms and defects at the surface. The large surface area, defect sites, and functional groups on NDs make them a promising material for electrochemical sensing. Previously, we dip-coated ND onto carbon-fiber microelectrodes (CFMEs) and found increases in sensitivity, but the coating was sparse. Here, we directly grew thin films of ND on niobium wires using microwave plasma chemical vapor deposition (MP-CVD) to provide full surface coverage. ND microelectrodes show a reliable performance in neurotransmitter detection with good antifouling properties. To improve sensitivity, we oxygen plasma etched ND films to activate the surface and intentionally add defects and oxygen surface functional groups. For fast-scan cyclic voltammetry detection of dopamine, oxygen plasma-etching increases the sensitivity from 21 nA/μM to 90 nA/μM after treatment. Fouling was tested by repeated injections of serotonin or tyramine, and both ND and plasma oxidized nanodiamond (NDO) microelectrodes maintain their currents better compared to CFMEs and therefore are more antifouling. A biofouling test in brain slices shows that ND microelectrodes barely have any current drop, while the more hydrophilic NDO microelectrodes decrease more, but still not as much as CFMEs. Overall, grown ND microelectrodes are promising in neurotransmitter detection with excellent fouling resistance, whereas oxygen plasma etching slightly lowers the fouling resistance but dramatically increases sensitivity.

Graphene-Fiber Microelectrodes for Ultrasensitive Neurochemical Detection

Here, we have synthesized and characterized graphene-fiber microelectrodes (GFME's) for subsecond detection of neurochemicals with fast-scan cyclic voltammetry (FSCV) for the first time. GFME's exhibited extraordinary properties including faster electron transfer kinetics, significantly improved sensitivity, and ease of tunability that we anticipate will have major impacts on neurochemical detection for years to come. GF's have been used in the literature for various applications; however, scaling their size down to microelectrodes and implementing them as neurochemical microsensors is significantly less developed. The GF's developed in this paper were on average 20-30 μm in diameter and both graphene oxide (GO) and reduced graphene oxide (rGO) fibers were characterized with FSCV. Neat GF's were synthesized using a one-step dimension-confined hydrothermal strategy. FSCV detection has traditionally used carbon-fiber microelectrodes (CFME's) and more recently carbon nanotube fiber electrodes; however, uniform functionalization and direct control of the 3D surface structure of these materials remain limited. The expansion to GFME's will certainly open new avenues for fine-tuning the electrode surface for specific electrochemical detection. When comparing to traditional CFME's, our GFME's exhibited significant increases in electron transfer, redox cycling, fouling resistance, higher sensitivity, and frequency independent behavior which demonstrates their incredible utility as biological sensors.

Extended sawhorse waveform for stable zinc detection with fast-scan cyclic voltammetry.

Zinc (Zn(II)) is a divalent cation involved in regulating intracellular signal transduction and gene expression through transcription factor activity, and can act as a metal neurotransmitter by modulating synaptic activity and neuronal plasticity. Previous research has demonstrated spatial heterogeneity of Zn(II) in the brain, has estimated extracellular concentrations of Zn(II) across various brain regions, and has measured rapid intracellular changes in Zn(II) concentration during glutamate flux. Despite this work, quantification of rapid extracellular Zn(II) release from neurons, on a millisecond time scale, in real time has remained difficult with existing technologies. Here, we have developed an electrochemical waveform, called the "extended sawhorse waveform (ESW)," for fast-scan cyclic voltammetry detection at carbon-fiber microelectrodes which enabled rapid and stable Zn(II) monitoring over time. This waveform was developed to overcome existing challenges in monitoring metallotransmitters stably over time electrochemically by introducing a brief cleaning step to facilitate rapid cleaning of the electrode surface in between scans. The ESW scans from 0.5 V down to -1.0 V, up to 1.45 V for 3 ms (cleaning step), and back to 0.5 V at a scan rate of 400 V/s. Repeated introductions of Zn(II) at the electrode using a traditional waveform cause plating which ultimately deteriorates the sensitivity over time; however, using the ESW, significant improvements in stability were observed. Overall, we provide a unique approach to monitor and quantitate rapid Zn(II) signaling in the brain at carbon electrodes which will impact our ability to advance fundamental knowledge of Zn(II) involvement in extracellular signaling pathways in the brain.

Real-Time Fast Scan Cyclic Voltammetry Detection and Quantification of Exogenously Administered Melatonin in Mice Brain.

Melatonin (MT) has been recently considered an excellent candidate for the treatment of sleep disorders, neural injuries, and neurological diseases. To better investigate the actions of MT in various brain functions, real-time detection of MT concentrations in specific brain regions is much desired. Previously, we have demonstrated detection of exogenously administered MT in anesthetized mouse brain using square wave voltammetry (SWV). Here, for the first time, we show successful detection of exogenous MT in the brain using fast scan cyclic voltammetry (FSCV) on electrochemically pre-activated carbon fiber microelectrodes (CFEs). In vitro evaluation showed the highest sensitivity (28.1 nA/μM) and lowest detection limit (20.2 ± 4.8 nM) ever reported for MT detection at carbon surface. Additionally, an extensive CFE stability and fouling assessment demonstrated that a prolonged CFE pre-conditioning stabilizes the background, in vitro and in vivo, and provides consistent CFE sensitivity over time even in the presence of a high MT concentration. Finally, the stable in vivo background, with minimized CFE fouling, allows us to achieve a drift-free FSCV detection of exogenous administered MT in mouse brain over a period of 3 min, which is significantly longer than the duration limit (usually < 90 s) for traditional in vivo FSCV acquisition. The MT concentration and dynamics measured by FSCV are in good agreement with SWV, while microdialysis further validated the concentration range. These results demonstrated reliable MT detection using FSCV that has the potential to monitor MT in the brain over long periods of time.

Enhancement of fast scan cyclic voltammetry detection of dopamine with tryptophan-modified electrodes.

Fast scan cyclic voltammetry (FSCV) allows for real -time analysis of phasic neurotransmitter levels. Tryptophan (TRP) is an aromatic amino acid responsible for facilitating electron transfer kinetics in oxidoreductase enzymes. Previous work with TRP-modified electrodes showed increased sensitivity for cyclic voltammetry detection of dopamine (DA) when used with slower scan rates (0.05 V/s). Here, we outline an in vitro proof of concept for TRP-modified electrodes in FSCV detection of DA, and decreased sensitivity for ascorbic acid (AA). TRP-modified electrodes had a limit of detection (LOD) for DA of 2.480 ± 0.343 nM compared to 8.348 ± 0.405 nM for an uncoated electrode. Selectivity for DA/ascorbic acid (AA) was 1.107 ± 0.3643 for uncoated and 15.57 ± 4.184 for TRP-modified electrodes. Additionally, these TRP-modified electrodes demonstrated reproducibility when exposed to extended cycling. TRP-modified electrodes will provide an effective modification to increase sensitivity for DA.

Improving serotonin fast-scan cyclic voltammetry detection: new waveforms to reduce electrode fouling.

Serotonin is a neuromodulator implicated in depression that is often measured in real-time by fast-scan cyclic voltammetry (FSCV). A specialized "Jackson" waveform (JW, 0.2, 1.0 V, -0.1 V, 0.2 V, 1000 V s-1) was developed to reduce serotonin fouling, but the 1.0 V switching potential limits sensitivity and electrodes still foul. The goal of this study was to test the effects of extending the FSCV switching potential to increase serotonin sensitivity and decrease fouling. We compared the Jackson waveform, the dopamine waveform (DA, -0.4 V, 1.3 V, 400 V s-1), and two new waveforms: the extended serotonin waveform (ESW, 0.2, 1.3, -0.1, 0.2, 1000 V s-1) and extended hold serotonin waveform (EHSW, 0.2, 1.3 (hold 1 ms), -0.1, 0.2, 400 V s-1). The EHSW was the most sensitive (LOD = 0.6 nM), and the JW the least sensitive (LOD = 2.4 nM). With the Jackson waveform, electrode fouling was significant with repeated injections of serotonin or exposure to its metabolite, 5-hydroxyindoleacetic acid (5-HIAA). Using the extended waveforms, electrodes fouled 50% less than with the Jackson waveform for both analytes. No electrode fouling was observed with the dopamine waveform because of the negative holding potential. The Jackson waveform was the most selective for serotonin over dopamine (800×), and the ESW was also highly selective. All waveforms were useful for measuring serotonin with optogenetic stimulation in Drosophila larvae. These results provide new FSCV waveforms to measure dynamic serotonin changes with different experimental requirements, like high sensitivity (EHSW), high selectivity (ESW, JW), or eliminating electrode fouling (DA).

Nanodiamond Coating Improves the Sensitivity and Antifouling Properties of Carbon Fiber Microelectrodes.

Nanodiamonds (NDs) are carbon nanomaterials with a core diamond crystalline structure and crystal defects, such as graphitic carbon and heteroatoms, on their surface. For electrochemistry, NDs are promising to increase active sites and decrease fouling, but NDs have not been studied for neurotransmitter electrochemistry. Here, we optimized ND coatings on microelectrodes and found that ND increases the sensitivity for neurotransmitters with fast-scan cyclic voltammetry detection and decreases electrochemical and biofouling. Different sizes and functionalizations of NDs were tested, and ND suspensions were drop-casted onto carbon-fiber microelectrodes (CFMEs). The 5 nm ND-H and 5 nm ND-COOH formed thick coatings, while the 15 and 60 nm ND-COOH formed more sparse coatings. With electrochemical impedance spectroscopy, 5 nm ND-H and 5 nm ND-COOH had high charge-transfer resistance, while 15 and 60 nm ND-COOH had low charge-transfer resistance. ND-COOH (15 nm) was optimal, with the best electrocatalytic properties and current for dopamine. Sensitivity was enhanced 2.1 ± 0.2 times and the limit of detection for dopamine improved to 3 ± 1 nM. ND coating increased current for other cations such as serotonin, norepinephrine, and epinephrine, but not for the anion ascorbic acid. Moreover, NDs decreased electrochemical fouling from serotonin and 5-hydroxyindoleacetic acid, and they also decreased biofouling in brain slice tissue by 50%. The current at biofouled ND-coated electrodes is similar to the signal of pristine, unfouled CFMEs. The carboxylated ND-modified CFMEs are beneficial for neurotransmitter detection because of easy fabrication, improved limit of detection, and antifouling properties.

Mechanism of Histamine Oxidation and Electropolymerization at Carbon Electrodes.

Histamine plays an important role in neuromodulation and the biological immune response. Although many electrochemical methods have been developed for histamine detection, the mechanism of its redox reaction has not been directly investigated. Here, we studied the mechanism of histamine oxidation at carbon electrodes and used that mechanistic information to design better fast-scan cyclic voltammetry (FSCV) methods for histamine. Using amperometry, cyclic voltammetry (CV), and X-ray photoelectron spectroscopy (XPS), we demonstrate that histamine oxidation requires a potential of at least +1.1 V vs Ag/AgCl. We propose that histamine undergoes one-electron oxidation on an imidazole nitrogen that produces a radical. The radical species dimerize and continue to undergo oxidation, leading to electropolymerization, which fouls the electrode. CV shows a peak at 1.3 V that is pH dependent, consistent with a one-proton, one-electron oxidation reaction. This mechanism is confirmed using 1- and 3-methylhistamine, which do not electropolymerize, compared to Nα-methylhistamine, which does. XPS also revealed a nitrogen-containing product adsorbed on the electrode surface after histamine oxidation. For FSCV detection of histamine at carbon-fiber microelectrodes, histamine oxidation was adsorption-controlled, and the anodic peak was observed at +1.2 V on the backward scan because of the rapid scan rate. However, the oxidation fouled the electrode and convoluted the FSCV temporal response; therefore, we implemented Nafion coating to alleviate the electrode fouling and preserve the time response of FSCV. Knowing the mechanism of histamine oxidation will facilitate design of better electrochemical methods for real-time monitoring of histamine.

Evaluation of carbon nanotube fiber microelectrodes for neurotransmitter detection: Correlation of electrochemical performance and surface properties

Fibers made of CNTs are attractive microelectrode sensors because they can be directly fabricated into microelectrodes. Different protocols for making CNT fibers have been developed, but differences in surface structure and therefore electrochemical properties that result have not been studied. In this study, we correlated the surface and electrochemical properties for neurochemical detection at 3 types of materials: CNT fibers produced by wet spinning with (1) polyethylenimine (PEI/CNT) or (2) chlorosulfonic acid (CA/CNT), and (3) CNT yarns made by solid-based CNT drawing. CNT yarns had well-aligned, high purity CNTs, abundant oxygen functional groups, and moderate surface roughness which led to the highest dopamine current density (290 ± 65 pA/cm2) and fastest electron transfer kinetics. The crevices of the CNT yarn and PEI/CNT fiber microelectrodes allow dopamine to be momentarily trapped during fast-scan cyclic voltammetry detection, leading to thin-layer cell conditions and a response that was independent of applied waveform frequency. The larger crevices on the PEI/CNT fibers led to a slower time response, showing too much roughness is detrimental to fast detection. CA/CNT fibers have a smoother surface and lower currents, but their negative surface charge results in high selectivity for dopamine over uric acid or ascorbic acid. Overall, small crevices, high conductivity, and abundant oxygen groups led to high sensitivity for amine neurotransmitters, such as dopamine and serotonin. Thus, different surfaces of CNT fibers result in altered electrochemical properties and could be used in the future to predict and control electrochemical performance.

Quantitation of dopamine, serotonin and adenosine content in a tissue punch from a brain slice using capillary electrophoresis with fast-scan cyclic voltammetry detection

Methods to determine neurochemical concentrations in small samples of tissue are needed to map interactions among neurotransmitters. In particular, correlating physiological measurements of neurotransmitter release and the tissue content in a small region would be valuable. HPLC is the standard method for tissue content analysis but it requires microliter samples and the detector often varies by the class of compound being quantified; thus detecting molecules from different classes can be difficult. In this paper, we develop capillary electrophoresis with fast-scan cyclic voltammetry detection (CE-FSCV) for analysis of dopamine, serotonin, and adenosine content in tissue punches from rat brain slices. Using field-amplified sample stacking, the limit of detection was 5 nM for dopamine, 10 nM for serotonin, and 50 nM for adenosine. Neurotransmitters could be measured from a tissue punch as small as 7 µg (7 nL) of tissue, three orders of magnitude smaller than a typical HPLC sample. Tissue content analysis of punches in successive slices through the striatum revealed higher dopamine but lower adenosine content in the anterior striatum. Stimulated dopamine release was measured in a brain slice, then a tissue punch collected from the recording region. Dopamine content and release had a correlation coefficient of 0.71, which indicates much of the variance in stimulated release is due to variance in tissue content. CE-FSCV should facilitate measurements of tissue content in nanoliter samples, leading to a better understanding of how diseases or drugs affect dopamine, serotonin, and adenosine content.

Analysis of biogenic amines in a single Drosophila larva brain by capillary electrophoresis with fast-scan cyclic voltammetry detection.

Drosophila, the fruit fly, is a common model organism in biology; however, quantifying neurotransmitters in Drosophila is challenging because of the small size of the central nervous system (CNS). Here, we develop neurotransmitter quantification by capillary electrophoresis with fast-scan cyclic voltammetry (CE-FSCV) detection, which allows peak identification by both migration time and the cyclic voltammogram, in contrast to traditional amperometric detection which provides no chemical identification. Tissue content of biogenic amine neurotransmitters was determined in a single CNS dissected from a Drosophila larva. Low detection limits, 1 nM for dopamine and serotonin, 2.5 nM for tyramine, and 4 nM for octopamine, were achieved using field-amplified sample stacking by diluting the homogenized tissue with percholoric acid and acetonitrile. Two different strains of wild-type flies, Oregon R and Canton S, have similar dopamine and serotonin levels but different octopamine content. When flies are fed NSD-1015, which inhibits dopamine decarboxylase (Ddc) a synthesis enzyme in the dopamine and serotonin pathways, dopamine significantly decreases by 52%. A genetically altered driver line, Ddc-GAL4, had lower serotonin and dopamine content as did w(118) flies. When the Ddc-GAL4 line was used to produce flies overexpressing the serotonin synthesis enzyme tryptophan hydroxylase (Ddc-GAL4;UAS-Trh), the serotonin tissue content was greater than for Ddc-GAL4 but not significantly different than the wild-type. These results show that CE-FSCV is useful for monitoring the impact of genetic and pharmacological manipulations on the content of multiple neurotransmitters in a CNS from a Drosophila larva.