Voltammetric detection of Neuropeptide Y using a modified sawhorse waveform.

The hormone Neuropeptide Y (NPY) plays critical roles in feeding, satiety, obesity, and weight control. However, its complex peptide structure has hindered the development of fast and biocompatible detection methods. Previous studies utilizing electrochemical techniques with carbon fiber microelectrodes (CFMEs) have targeted the oxidation of amino acid residues like tyrosine to measure peptides. Here, we employ the modified sawhorse waveform (MSW) to enable voltammetric identification of NPY through tyrosine oxidation. Use of MSW improves NPY detection sensitivity and selectivity by reducing interference from catecholamines like dopamine, serotonin, and others compared to the traditional triangle waveform. The technique utilizes a holding potential of -0.2 V and a switching potential of 1.2 V that effectively etches and renews the CFME surface to simultaneously detect NPY and other monoamines with a sensitivity of 5.8 ± 0.94 nA/µM (n = 5). Furthermore, we observed adsorption-controlled, subsecond NPY measurements with CFMEs and MSW. The effective identification of exogenously applied NPY in biological fluids demonstrates the feasibility of this methodology for in vivo and ex vivo studies. These results highlight the potential of MSW voltammetry to enable fast, biocompatible NPY quantification to further elucidate its physiological roles.

Perspective-Advances in Voltammetric Methods for the Measurement of Biomolecules.

Voltammetry is a powerful electroanalytical tool that makes fast, real-time measurements of neurotransmitters and other molecules. Electroanalytical methods like cyclic, pulse, and stripping voltammetry are useful for qualitative and quantitative examination. Neurochemical sensing has been enhanced using carbon-based electrodes and waveform modification methods that improve sensitivity and stability of electrode performance. Voltammetry has revolutionized neurochemical monitoring by providing real-time information on neurotransmitter dynamics for neurochemical studies. Selectivity and electrode fouling remain issues for biomolecule detection, but recent advances promise new methods of analysis for other applications to enhance spatiotemporal resolution, sensitivity, selectivity, and other important considerations.

Development of Carbon Nanotube Yarn Supercapacitors and Energy Storage for Integrated Structural Health Monitoring.

Developing efficient, sustainable, and high-performance energy storage systems is essential for advancing various industries, including integrated structural health monitoring. Carbon nanotube yarn (CNTY) supercapacitors have the potential to be an excellent solution for this purpose because they offer unique material properties such as high capacitance, electrical conductivity, and energy and power densities. The scope of the study included fabricating supercapacitors using various materials and characterizing them to determine the capacitive properties, energy, and power densities. Experimental studies were conducted to investigate the energy density and power density behavior of CNTYs embedded in various electrochemical-active matrices to monitor the matrices' power process and the CNTY supercapacitors' life-cyclic response. The results showed that the CNTY supercapacitors displayed excellent capacitive behavior, with nearly rectangular CV curves across a range of scan rates. The energy density and power density of the supercapacitors fluctuated between a minimum of 3.89 Wh/kg and 8 W/kg while the maximum was between 6.46 Wh/kg and 13.20 W/kg. These CNTY supercapacitors are being tailored to power CNTY sensors integrated into a variety of structures that could monitor damage, strain, temperature, and others.

Carbon Electrode Sensor for the Measurement of Cortisol with Fast-Scan Cyclic Voltammetry.

Cortisol is a vital steroid hormone that has been known as the "stress hormone", which is elevated during times of high stress and anxiety and has a significant impact on neurochemistry and brain health. The improved detection of cortisol is critically important as it will help further our understanding of stress during several physiological states. Several methods exist to detect cortisol; however, they suffer from low biocompatibility and spatiotemporal resolution, and they are relatively slow. In this study, we developed an assay to measure cortisol with carbon fiber microelectrodes (CFMEs) and fast-scan cyclic voltammetry (FSCV). FSCV is typically utilized to measure small molecule neurotransmitters by producing a readout cyclic voltammogram (CV) for the specific detection of biomolecules on a fast, subsecond timescale with biocompatible CFMEs. It has seen enhanced utility in measuring peptides and other larger compounds. We developed a waveform that scanned from -0.5 to -1.2 V at 400 V/s to electro-reduce cortisol at the surface of CFMEs. The sensitivity of cortisol was found to be 0.87 ± 0.055 nA/µM (n = 5) and was found to be adsorption controlled on the surface of CFMEs and stable over several hours. Cortisol was co-detected with several other biomolecules such as dopamine, and the waveform was fouling resistant to repeated injections of cortisol on the surface of the CFMEs. Furthermore, we also measured exogenously applied cortisol into simulated urine to demonstrate biocompatibility and potential use in vivo. The specific and biocompatible detection of cortisol with high spatiotemporal resolution will help further elucidate its biological significance and further understand its physiological importance and impact on brain health.

High resolution voltammetric and field-effect transistor readout of carbon fiber microelectrode biosensors.

Rapid and sensitive pH measurements with increased spatiotemporal resolution are imperative to probe neurochemical signals and illuminate brain function. We interfaced carbon fiber microelectrode (CFME) sensors with both fast scan cyclic voltammetry (FSCV) and field-effect transistor (FET) transducers for dynamic pH measurements. The electrochemical oxidation and reduction of functional groups on the surface of CFMEs affect their response over a physiologically relevant pH range. When measured with FET transducers, the sensitivity of the measurements over the measured pH range was found to be (101 ± 18) mV, which exceeded the Nernstian value of 59 mV by approximately 70%. Finally, we validated the functionality of CFMEs as pH sensors with FSCV ex vivo in rat brain coronal slices with exogenously applied solutions of varying pH values indicating that potential in vivo study is feasible.

Modified Sawhorse Waveform for the Voltammetric Detection of Oxytocin.

Carbon fiber microelectrodes (CFMEs) have been used to detect neurotransmitters and other biomolecules using fast-scan cyclic voltammetry (FSCV) for the past few decades. This technique measures neurotransmitters such as dopamine and, more recently, physiologically relevant neuropeptides. Oxytocin, a pleiotropic peptide hormone, is physiologically important for adaptation, development, reproduction, and social behavior. This neuropeptide functions as a stress-coping molecule, an anti-inflammatory agent, and serves as an antioxidant with protective effects especially during adversity or trauma. Here, we measure tyrosine using the Modified Sawhorse Waveform (MSW), enabling enhanced electrode sensitivity for the amino acid and oxytocin peptide. Applying the MSW, decreased surface fouling and enabled codetection with other monoamines. As oxytocin contains tyrosine, the MSW was also used to detect oxytocin. The sensitivity of oxytocin detection was found to be 3.99 ± 0.49 nA/µM, (n=5). Additionally, we demonstrate that applying the MSW on CFMEs allows for real time measurements of exogenously applied oxytocin on rat brain slices. These studies may serve as novel assays for oxytocin detection in a fast, sub-second timescale with possible implications for in vivo measurements and further understanding of the physiological role of oxytocin.

Multiplexing neurochemical detection with carbon fiber multielectrode arrays using fast-scan cyclic voltammetry.

Carbon fiber microelectrodes (CFMEs) have been extensively used to measure neurotransmitters with fast-scan cyclic voltammetry (FSCV) due to their ability to adsorb cationic monoamine neurotransmitters. Although FSCV, in tandem with CFMEs, provides high temporal and spatial resolution, only single-channel potentiostats and electrodes have been primarily utilized. More recently, the need and use of carbon fiber multielectrode arrays has risen to target multiple brain regions. Previous studies have shown the ability to detect dopamine using multielectrode arrays; however, they are not readily available to the scientific community. In this work, we interfaced a carbon fiber multielectrode array (MEA or multielectrode array), to a commercially available four-channel potentiostat for multiplexing neurochemical measurements. The MEA's relative performance was compared to single CFMEs where dopamine detection was found to be adsorption controlled to the electrode's surface. Multiple waveforms were applied to each fiber of the multielectrode array simultaneously to detect different analytes on each electrode of the array. A proof of concept ex vivo experiment showed that the multielectrode array could record redox activity in different areas within the mouse caudate putamen and detect dopamine in a 3-mm2 area. To our knowledge, this is the first use of the multielectrode array paired with a commercially available multichannel potentiostat for multi-waveform application and neurotransmitter co-detection. This novel array may aid in future studies to better understand complex brain heterogeneity, the dynamic neurochemical environment, and how disease states or drugs affect separate brain areas concurrently. Graphical abstract.

Polyvinyl alcohol-montmorillonite composites for water purification: Analysis of clay mineral cation exchange and composite particle synthesis.

Municipal and residential water purification rely heavily on activated carbon (AC), but regeneration of AC is costly and cannot be performed at the point-of-use. Clay minerals (CMs) comprise a class of naturally abundant materials with known capacities for analyte adsorbance. However, the gel-forming properties of CMs in aqueous suspension pose problems for these materials being used in water-purification. In this study, we have taken three main steps to optimize the use of CMs in these applications. First, we produced several variants of montmorillonite CMs to evaluate the effect of interstitial cation hydrophobicity on the ability of the CM to uptake chargecarrying organic pollutants. These variants include CMs with the following cations: sodium, hexyl(triphenyl) phosphonium, hexyadecyl(triphenyl)phosphonium, and hexyl(tributyl)phosphonium. Second, we synthesized polymer-clay mineral composite films composed of polyvinyl alcohol (PVA), crosslinked in the presence of a CM variant. These films were evaluated for their ability to uptake malachite green (MG). Finally, we developed a one-pot synthetic method for the generation of polymer-clay particles for use in a continuous column process. We synthesized polymer-clay mineral particles using the highest performing CM (based on the film experiments) and evaluated the equilibrium capacity and kinetics of MG uptake from solution.

Review-Recent Advances in FSCV Detection of Neurochemicals via Waveform and Carbon Microelectrode Modification.

Fast scan cyclic voltammetry (FSCV) is an analytical technique that was first developed over 30 years ago. Since then, it has been extensively used to detect dopamine using carbon fiber microelectrodes (CFMEs). More recently, electrode modifications and waveform refinement have enabled the detection of a wider variety of neurochemicals including nucleosides such as adenosine and guanosine, neurotransmitter metabolites of dopamine, and neuropeptides such as enkephalin. These alterations have facilitated the selectivity of certain biomolecules over others to enhance the measurement of the analyte of interest while excluding interferants. In this review, we detail these modifications and how specializing CFME sensors allows neuro-analytical researchers to develop tools to understand the neurochemistry of the brain in disease states and provide groundwork for translational work in clinical settings.

Direct Detection of DNA and RNA on Carbon Fiber Microelectrodes Using Fast-Scan Cyclic Voltammetry.

DNA and RNA have been measured with many techniques but often with relatively long analysis times. In this study, we utilize fast-scan cyclic voltammetry (FSCV) for the subsecond codetection of adenine, guanine, and cytosine, first as free nucleosides, and then within custom synthesized oligos, plasmid DNA, and RNA from the nematode Caenorhabditis elegans. Previous studies have shown the detection of adenosine and guanosine with FSCV with high spatiotemporal resolution, while we have extended the assay to include cytidine and adenine, guanine, and cytosine in RNA and single- and double-stranded DNA (ssDNA and dSDNA). We find that FSCV testing has a higher sensitivity and yields higher peak oxidative currents when detecting shorter oligonucleotides and ssDNA samples at equivalent nucleobase concentrations. This is consistent with an electrostatic repulsion from negatively charged oxide groups on the surface of the carbon fiber microelectrode (CFME), the negative holding potential, and the negatively charged phosphate backbone. Moreover, as opposed to dsDNA, ssDNA nucleobases are not hydrogen-bonded to one another and thus are free to adsorb onto the surface of the carbon electrode. We also demonstrate that the simultaneous determination of nucleobases is not masked even in biologically complex serum samples. This is the first report demonstrating that FSCV, when used with CFMEs, is able to codetect nucleobases when polymerized into DNA or RNA and could potentially pave the way for future uses in clinical, diagnostic, or research applications.

Carbon Nanotube Yarn Microelectrodes Promote High Temporal Measurements of Serotonin Using Fast Scan Cyclic Voltammetry.

Carbon fiber-microelectrodes (CFMEs) have been the standard for neurotransmitter detection for over forty years. However, in recent years, there have been many advances of utilizing alternative nanomaterials for neurotransmitter detection with fast scan cyclic voltammetry (FSCV). Recently, carbon nanotube (CNT) yarns have been developed as the working electrode materials for neurotransmitter sensing capabilities with fast scan cyclic voltammetry. Carbon nanotubes are ideal for neurotransmitter detection because they have higher aspect ratios enabling monoamine adsorption and lower limits of detection, faster electron transfer kinetics, and a resistance to surface fouling. Several methods to modify CFMEs with CNTs have resulted in increases in sensitivity, but have also increased noise and led to irreproducible results. In this study, we utilize commercially available CNT-yarns to make microelectrodes as enhanced neurotransmitter sensors for neurotransmitters such as serotonin. CNT-yarn microelectrodes have significantly higher sensitivities (peak oxidative currents of the cyclic voltammograms) than CFMEs and faster electron transfer kinetics as measured by peak separation (ΔEP) values. Moreover, both serotonin and dopamine are adsorption controlled to the surface of the electrode as measured by scan rate and concentration experiments. CNT yarn microelectrodes also resisted surface fouling of serotonin onto the surface of the electrode over thirty minutes and had a wave application frequency independent response to sensitivity at the surface of the electrode.

Timed Electrodeposition of PEDOT:Nafion onto Carbon Fiber-Microelectrodes Enhances Dopamine Detection in Zebrafish Retina.

Carbon fiber-microelectrodes (CFMEs) are one of the standards for the detection of neurotransmitters such as dopamine (DA). In this study, we demonstrate that CFMEs electrodeposited with poly (3,4-ethylenedioxythiophene) (PEDOT) in the presence of Nafion exhibit enhanced sensitivity for DA detection. Scanning electron microscopy (SEM) revealed the smooth outer surface morphologies of polymer coatings, which filled in the ridges and grooves of the bare unmodified carbon electrode and energy-dispersive X-ray spectroscopy (EDX) confirmed PEDOT:Nafion incorporation. PEDOT:Nafion coated CMFEs exhibited a statistically enhanced two-fold increase in DA sensitivity compared to unmodified microelectrodes, with stability and integrity of the coated microelectrodes maintained for at least 4 h. A scan rate test revealed a linear relationship with peak DA oxidative current (5 µM), indicating adsorption control of DA to the surface of the PEDOT:Nafion electrode. As proof of principle, PEDOT:Nafion coated electrodes were used to detect potassium chloride (KCl)-induced DA release in zebrafish (Danio rerio) retinal tissue ex vivo, thus illustrating their applicability as biosensors.

Polymer Modified Carbon Fiber Microelectrodes for Precision Neurotransmitter Metabolite Measurements.

Carbon fiber-microelectrodes (CFMEs) are considered to be one of the standard electrodes for neurotransmitter detection such as dopamine (DA). DA is physiologically important for many pharmacological and behavioral states, but is readily metabolized on a fast, subsecond timescale. Recently, DA metabolites such as 3-methoxytyramine (3-MT) and 3,4-dihydroxyphenylacetaldehyde (DOPAL) were found to be involved in physiological functions, such as movement control and progressive neuro degeneration. However, there is no current assay to detect and differentiate them from DA. In this study, we demonstrate the co-detection of similarly structured neurochemicals such as DA, 3-MT, and DOPAL. We accomplished this through electrodepositing CFMEs with polyethyleneimine (PEI) and poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) polymers. This endowed the bare unmodified CFMEs with surface charge, physical, and chemical differences, which resulted in the improved sensitivity and selectivity of neurotransmitter detection. The differentiation and detection of 3-MT, DOPAL, and DA will potentially help further understand the important physiological roles that these dopaminergic metabolites play in vivo.

Gold Nanoparticle Modified Carbon Fiber Microelectrodes for Enhanced Neurochemical Detection.

For over 30 years, carbon-fiber microelectrodes (CFMEs) have been the standard for neurotransmitter detection. Generally, carbon fibers are aspirated into glass capillaries, pulled to a fine taper, and then sealed using an epoxy to create electrode materials that are used for fast scan cyclic voltammetry testing. The use of bare CFMEs has several limitations, though. First and foremost, the carbon fiber contains mostly basal plane carbon, which has a relatively low surface area and yields lower sensitivities than other nanomaterials. Furthermore, the graphitic carbon is limited by its temporal resolution, and its relatively low conductivity. Lastly, neurochemicals and macromolecules have been known to foul at the surface of carbon electrodes where they form non-conductive polymers that block further neurotransmitter adsorption. For this study, we modify CFMEs with gold nanoparticles to enhance neurochemical testing with fast scan cyclic voltammetry. Au3+ was electrodeposited or dipcoated from a colloidal solution onto the surface of CFMEs. Since gold is a stable and relatively inert metal, it is an ideal electrode material for analytical measurements of neurochemicals. Gold nanoparticle modified (AuNP-CFMEs) had a stability to dopamine response for over 4 h. Moreover, AuNP-CFMEs exhibit an increased sensitivity (higher peak oxidative current of the cyclic voltammograms) and faster electron transfer kinetics (lower ΔEP or peak separation) than bare unmodified CFMEs. The development of AuNP-CFMEs provides the creation of novel electrochemical sensors for detecting fast changes in dopamine concentration and other neurochemicals at lower limits of detection. This work has vast applications for the enhancement of neurochemical measurements. The generation of gold nanoparticle modified CFMEs will be vitally important for the development of novel electrode sensors to detect neurotransmitters in vivo in rodent and other models to study neurochemical effects of drug abuse, depression, stroke, ischemia, and other behavioral and disease states.

Polymer Modified Carbon Fiber-Microelectrodes and Waveform Modifications Enhance Neurotransmitter Metabolite Detection.

Carbon-fiber microelectrodes (CFMEs) have been used for several years for the detection of neurotransmitters such as dopamine. Dopamine is a fundamentally important neurotransmitter and is also metabolized at a subsecond timescale. Recently, several metabolites of dopamine have been shown to be physiologically important such as 3-methoxytyramine (3-MT), 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA). Many of these neurotransmitter metabolites are currently only detected with microdialysis coupled with liquid chromatography with relatively low temporal and spatial resolution. Current electrochemical methods such as the dopamine waveform (scanning from -0.4 to 1.3 V at 400 V/sec) are utilized to electrostatically repel anions such as DOPAC and promote dopamine adsorption to the surface of the electrode. Moreover, polymer coatings such as Nafion have been shown to electrostatically repel anions such as 5-hydroxyindoleacetic acid (5-HIAA). In this study, we develop novel polymer and waveform modifications for enhanced DOPAC detection. Applying the DOPAC waveform (scanning from 0 to 1.3 V at 400 V/sec) enhances DOPAC detection significantly because it does not include the negative holding potential of the dopamine waveform. Moreover, positively charged cationic polymers such as polyethyleneimine (PEI) allow for the preconcentration of DOPAC to the surface of the carbon fiber through an electrostatic attraction. The limit of detection for DOPAC for PEI coated CFMEs with the DOPAC waveform applied is 58.2 ± 2 nM as opposed to 291 ± 10 nM for unmodified electrodes applying the dopamine waveform (n = 4). This work offers promise for the development of novel electrode materials and waveforms for the specific detection of several important biomolecules such as dopamine metabolite neurotransmitters.

Ruboxistaurin Reduces Cocaine-Stimulated Increases in Extracellular Dopamine by Modifying Dopamine-Autoreceptor Activity.

Cocaine is a highly abused drug, and cocaine addiction affects millions of individuals worldwide. Cocaine blocks normal uptake function at the dopamine transporter (DAT), thus increasing extracellular dopamine. Currently, no chemical therapies are available to treat cocaine abuse. Previous works showed that the selective inhibitors of protein kinase Cß (PKCß), enzastaurin and ruboxistaurin, attenuate dopamine overflow and locomotion stimulated by another psychostimulant drug, amphetamine. We now test if ruboxistaurin similarly affects cocaine action. Perfusion of 1 µM ruboxistaurin directly into the core of the nucleus accumbens via retrodialysis reduced cocaine-stimulated increases in dopamine overflow, measured using microdialysis sampling, with simultaneous reductions in locomotor behavior. Because cocaine activity is highly regulated by dopamine autoreceptors, we examined whether ruboxistaurin was acting at the level of the D2 autoreceptor. Perfusion of 5 µM raclopride, a selective D2-like receptor antagonist, before addition of ruboxistaurin, abrogated the effect of ruboxistaurin on cocaine-stimulated dopamine overflow and hyperlocomotion. Further, ruboxistaurin was inactive against cocaine-stimulated locomotor activity in mice with a genetic deletion in D2 receptors as compared to wild-type mice. In contrast, blockade or deletion of dopamine D2 receptors did not abolish the attenuating effect of ruboxistaurin on amphetamine-stimulated activities. Therefore, the inhibition of PKCß reduces dopamine overflow and locomotor activity stimulated by both cocaine and amphetamine, but the mechanism of action differs for each stimulant. These data suggest that inhibition of PKCß would serve as a target to reduce the abuse of either amphetamine or cocaine.

Use and Future Prospects of in Vivo Microdialysis for Epilepsy Studies.

Epilepsy is a common neurological disease characterized by recurrent unpredictable seizures. For the last 30 years, microdialysis sampling has been used to measure changes in excitatory and inhibitory neurotransmitter concentrations before, during, and after seizures. These advances have fostered breakthroughs in epilepsy research by identifying neurochemical changes associated with seizures and correlating them to electrophysiological data. Recent advances in methodology may be useful in further delineating the chemical underpinnings of seizures. A new model of ictogenesis has been developed that allows greater control over the timing of seizures that are similar to spontaneous seizures. This model will facilitate making chemical measurements before and during a seizure. Recent advancements in microdialysis sampling, including the use of segmented flow, "fast" liquid chromatography (LC), and capillary electrophoresis with laser-induced fluorescence (CE-LIF) have significantly improved temporal resolution to better than 1 min, which could be used to measure transient, spontaneous neurochemical changes associated with seizures. Microfabricated sampling probes that are markedly smaller than conventional probes and allow for a much greater spatial resolution have been developed. They may allow the targeting of specific brain regions important to epilepsy studies. Coupling microdialysis sampling to optogenetics and light-stimulated release of neurotransmitters may also prove useful for studying epileptic seizures.

Chemical biomarkers of epileptogenesis and ictogenesis in experimental epilepsy.

Epilepsy produces chronic chemical changes induced by altered cellular structures, and acute ones produced by conditions leading into individual seizures. Here, we aim to quantify 24 molecules simultaneously at baseline and during periods of lowered seizure threshold in rats. Using serial hippocampal microdialysis collections starting two weeks after the pilocarpine-induced status epilepticus, we evaluated how this chronic epilepsy model affects molecule levels and their interactions. Then, we quantified the changes occurring when the brain moves into a pro-seizure state using a novel model of physiological ictogenesis. Compared with controls, pilocarpine animals had significantly decreased baseline levels of adenosine, homovanillic acid, and serotonin, but significantly increased levels of choline, glutamate, phenylalanine, and tyrosine. Step-wise linear regression identified that choline, homovanillic acid, adenosine, and serotonin are the most important features to characterize the difference in the extracellular milieu between pilocarpine and control animals. When increasing the hippocampal seizure risk, the concentrations of normetanephrine, serine, aspartate, and 5-hydroxyindoleacetic acid were the most prominent; however, there were no specific, consistent changes prior to individual seizures.