Electrochemistry at the Synapse.

Microphysiological systems have attracted attention because of their use in drug screening. However, it is challenging to measure cell functions in real time using a device. In this study, we developed a cell culture device using a porous membrane electrode for in situ electrochemical glucose measurements for cell analysis. First, a porous membrane electrode was fabricated and electrochemically evaluated for enzyme-free glucose measurement. Subsequently, the glucose uptake of MCF-7 spheroids was evaluated using living spheroids, fixed spheroids, supernatants, and glucose transporter inhibitor-treated spheroids. Conventionally, the direct optical measurement of glucose uptake requires fluorescence-labeled glucose derivatives. In addition, the glucose uptake can be evaluated by measuring the glucose concentration in the medium by optical or electrochemical measurements. However, glucose needs to be consumed in the entire cell culture medium, which needs a long culture time. In contrast, our system can measure glucose in approximately 5 min without any labels because of in situ electrochemical measurements. This system can be used for in situ measurements in in vitro cell culture systems, including organ-on-a-chip for drug screening.

Fast-scan cyclic voltammetry (FSCV) is an electrochemical technique for measuring rapid changes in the extracellular concentration of neurotransmitters within the brain. Due to its fast scan rate and large output-data size, the current analysis of the FSCV data is often conducted on a computer external to the FSCV device. Moreover, the analysis is semi-automated and requires a good understanding of the characteristics of the underlying chemistry to interpret, making it unsuitable for real-time implementation on low-resource FSCV devices. This paper presents a hardware-software co-design approach for the analysis of FSCV data. Firstly, a deep neural network (DNN) is developed to predict the concentration of a dopamine solution and identify the data recording electrode. Secondly, the DNN is pruned to decrease its computation complexity, and a custom overlay is developed to implement the pruned DNN on a low-resource FPGA-based platform. The pruned DNN attains a recognition accuracy of 97.2% with a compression ratio of 3.18. When the DNN overlay is implemented on a PYNQ-Z2 platform, it achieves the execution time of 13 ms and power consumption of 1.479 W on the entire PYNQ-Z2 board. This study demonstrates the possibility of operating the DNN for FSCV data analysis on portable FPGA-based platforms.

Detection of neurochemicals voltammetrically can be challenging in complex matrices like tissue. Norepinephrine (NE) is a neurotransmitter in the brain and is directly released by the sympathetic nervous system in the periphery. Fast-scan cyclic voltammetry (FSCV) is an electrochemical technique previously used to detect NE from sympathetic neurons in lymphoid tissues. Mesenteric lymph nodes present a unique challenge to FSCV due to their complex tissue matrix, which includes immune cells and neurons. These cells release various neuropeptides, cytokines, and other chemical signaling molecules, which can interfere with FSCV. Notably, Neuropeptide Y (NPY) from sympathetic neurons, and Substance P (SubP) and Calcitonin Gene-Related Peptide (CGRP) from sensory neurons are common peptides that are released locally near NE sites. These peptides are regulated by and interact with NE through complex neuronal circuits, potentially impacting FSCV NE detection. We demonstrate that increasing levels of each peptide alter voltammetric NE detection. We observed that the NE oxidation potential shifts with each peptide in vitro, and NE cyclic voltammograms exhibit unique peak broadening specific to NPY compared to SubP and CGRP, indicating that each peptide affects the carbon fiber microelectrode (CFME) differently. Overall, we show significant convolution of voltammetric NE peaks in the presence of peptides, providing evidence that future developments in materials to reduce protein fouling could significantly improve the robustness and accuracy of NE FSCV detection.

Direct electrochemical measurements of biological events are often challenging because of the low signal relative to the magnitude of the background and noise. When choosing a data processing approach, the frequency and phase content of the data must be considered. Here, we employ a zero-phase (infinite impulse response (IIR)) filter to remove the noise from the analytical signal, while preserving the phase content. In fast-scan cyclic voltammetry, the frequency content of the signal is a function of the scan rate of the applied waveform. Fourier analysis was used to develop a relationship between scan rate and the filter cutoff frequency to maximize the reduction in noise, while not altering the true nature of the analytical signal. The zero-phase filter has the same effect as traditional filters with regards to increasing the signal-to-noise ratio. Because the zero-phase filter does not introduce a change to ΔEpeak, the heterogeneous electron rate transfer constant (0.10 cm/s) for ferrocene is calculated accurately. The zero-phase filter also improves electrochemical analysis of signaling molecules that have their oxidation potential close to the switching potential. Lastly, a quantitative approach to filtering amperometric traces of exocytosis based on the rise time was developed.

Neuroplasticity, the ability of the brain to form new neural pathways and synaptic connections, is gaining renewed interest for therapeutic purposes in stroke or spinal cord injury patients. Earlier work in this area in animal models has shown that behavior modulation was associated with the release of serotonin that subsequently leads to changes in gene expression and new neural growth [1]. A separate work has also demonstrated that using serotonin 1A agonists in rats with respiratory dysfunction due spinal cord injury results in better respiratory function [2]. Taken together, these studies indicate that neurotransmitters such as serotonin are closely linked to neuroplasticity, suggesting that changes in levels of these neurotransmitters can be correlated to synaptic changes. Currently, there is growing interest in the use of electrical stimulation as a means for inducing neuroplasticity in stroke and spinal cord injury patients [3]. However, while the data suggests that plasticity could be induced by electrical stimulation, neither the mechanism nor the protocols of stimulation are understood. We submit that a possible path to understanding these mechanisms involves simultaneous real-time monitoring of neurotransmitters such as serotonin electrochemically and electrical signals during stimulation and finding a correlation between these electrical signals and neurotransmitters. In cases where detection of several key electroactive neurotransmitters like serotonin and dopamine is sought, fast scan cyclic voltammetry (FSCV) with its high temporal resolution is the preferred electrochemical method of obtaining real time data [4]. Now, to be able to simultaneously monitor these electrochemical signals along with electrical signals, new approaches in neural probe material and architecture selection are required, as almost all neural probes currently in use are capable of recording only electrical or chemical signals, but not both. Thus, implementation of electrochemical monitoring techniques in animal models requires an integrated electrode array with a size suitable for an animal containing a counter electrode, working electrodes and a reference electrode. Furthermore, electrode materials capable of electrochemically detecting the presence of neurotransmitters without undergoing non-reversible reactions are sought. To meet this need, we investigate and report on the use of glassy carbon, a form of carbon that has been demonstrated to possess ideal characteristics, such as a wide electrochemical window with no irreversible reactions as a coupled electrical and electrochemical sensing platform. In this particular study, we introduce a 12-channel glassy carbon (GC) microelectrode array fabricated on a flexible substrate. The specific design introduced here consists of electrodes supported on a polyimide substrate with penetrating probes at their edges that can penetrate up to 1.25 mm into an animal's cortex or spinal cord. Each probe contains a glassy carbon electrode that can be used to detect serotonin, stimulate a neuron, or read an electrophysiological signal. Our preliminary results show that serotonin can be detected using a polyimide-glassy carbon device down to at least 1 uM in vitro by stimulating through one portion of the device and reading through another (Figure 1). Furthermore, we have shown that our electrodes can read electrophysiological data in-vivo. Figure 2b shows the Fourier Transform of a few seconds of electrical brain signals from a song bird without stimulus. In this graph low frequency alpha waves are most prominent. Figure 2c, the stimulus graph, on the other hand, shows that the beta and gamma waves became more prominent under external, audio stimulation. We believe that the ability to couple this kind of electrical signal detection with electrochemical sensing through FSCV (fast-scan CV) in a single device for in-vivostudies give promise for the disambiguation behind the mechanisms involved in neuroplasticity. Figure 1: Background subtracted cyclic voltagramms for various concentrations of serotonin Figure2: In vivo ECoG signals recorded with carbon electrodes. (a) Time lapse of the audio stimulus (b) Amplitude spectra of the animal during quiet time (c) Amplitude spectra of the signal during the stimulus phase. [1] Kandel, Eric R. Science 294, no. 5544 (2001): 1030-1038. [2] Choi, Howard, Wei-Lee Liao, Kimberly M. Newton, Renna C. Onario, Allyson M. King, Federico C. Desilets, Eric J. Woodard et al. The Journal of neuroscience 25, no. 18 (2005): 4550-4559. [3] Dimyan, Michael A., and Leonardo G. Cohen. Nature Reviews Neurology 7, no. 2 (2011): 76-85. [4] Lama, Rinchen D., Karl Charlson, Arun Anantharam, and Parastoo Hashemi. Analytical chemistry 84, no. 19 (2012): 8096-8101. Figure 1

In a companion study, the authors describe the development of a new instrument named the Wireless Instantaneous Neurotransmitter Concentration System (WINCS), which couples digital telemetry with fast-scan cyclic voltammetry (FSCV) to measure extracellular concentrations of dopamine. In the present study, the authors describe the extended capability of the WINCS to use fixed potential amperometry (FPA) to measure extracellular concentrations of dopamine, as well as glutamate and adenosine. Compared with other electrochemical techniques such as FSCV or high-speed chronoamperometry, FPA offers superior temporal resolution and, in combination with enzyme-linked biosensors, the potential to monitor nonelectroactive analytes in real time. The WINCS design incorporated a transimpedance amplifier with associated analog circuitry for FPA; a microprocessor; a Bluetooth transceiver; and a single, battery-powered, multilayer, printed circuit board. The WINCS was tested with 3 distinct recording electrodes: 1) a carbon-fiber microelectrode (CFM) to measure dopamine; 2) a glutamate oxidase enzyme-linked electrode to measure glutamate; and 3) a multiple enzyme-linked electrode (adenosine deaminase, nucleoside phosphorylase, and xanthine oxidase) to measure adenosine. Proof-of-principle analyses included noise assessments and in vitro and in vivo measurements that were compared with similar analyses by using a commercial hardwired electrochemical system (EA161 Picostat, eDAQ; Pty Ltd). In urethane-anesthetized rats, dopamine release was monitored in the striatum following deep brain stimulation (DBS) of ascending dopaminergic fibers in the medial forebrain bundle (MFB). In separate rat experiments, DBS-evoked adenosine release was monitored in the ventrolateral thalamus. To test the WINCS in an operating room setting resembling human neurosurgery, cortical glutamate release in response to motor cortex stimulation (MCS) was monitored using a large-mammal animal model, the pig. The WINCS, which is designed in compliance with FDA-recognized consensus standards for medical electrical device safety, successfully measured dopamine, glutamate, and adenosine, both in vitro and in vivo. The WINCS detected striatal dopamine release at the implanted CFM during DBS of the MFB. The DBS-evoked adenosine release in the rat thalamus and MCS-evoked glutamate release in the pig cortex were also successfully measured. Overall, in vitro and in vivo testing demonstrated signals comparable to a commercial hardwired electrochemical system for FPA. By incorporating FPA, the chemical repertoire of WINCS-measurable neurotransmitters is expanded to include glutamate and other nonelectroactive species for which the evolving field of enzyme-linked biosensors exists. Because many neurotransmitters are not electrochemically active, FPA in combination with enzyme-linked microelectrodes represents a powerful intraoperative tool for rapid and selective neurochemical sampling in important anatomical targets during functional neurosurgery.

Electron Beam Induced Deposition (EBID) allows deposition of three-dimensional micro- and nano-structures of conductive and insulating materials on a wide range of substrates. The process is based on the decomposition of molecules of a pre-selected precursor by a focused electron beam. In recent decades EBID of several metals, namely Au, W, Cu and Pt, from different families of precursors, has been achieved and the technique has found some application for small-scale production of laboratory devices and for repair of masks and micro-optoelectronic devices. The weak point of the technique is at present the low purity of the deposited material, caused by metal-organic precursors and by the lack of selectivity of the electron-induced decomposition process. This work is dedicated to EBID of Rh nanostructures from the precursor [RhCl(PF3)2]2. High metal content deposits are expected because the precursor does not contain C atoms and because Rh is one of the less reactive metals. [RhCl(PF3)2]2 as EBID precursor has been characterized by vapor pressure, mass spectrometry and surface residence time measurements. The vapor pressure of 7.5 Pa at room temperature reveals that the precursor is sufficiently volatile for room temperature EBID. The knowledge of the vapor pressure allows also to estimate the number of precursor molecules delivered to the reaction area per unit time. Mass spectrometry measurements allow to know the decomposition path of the precursor under electron impact in the gas phase. The measured spectrum indicates that the molecule decomposes by successive loss of PF3 groups, as confirmed by density functional theory calculations. This is compatible with a high metal content deposit. Residence time measurements show that [RhCl(PF3)2]2 does not decompose on stainless steel surfaces. The measured residence time of 2 ms allows to estimate that the activation energy for desorption of [RhCl(PF3)2]2 on stainless steel is about 0.6 eV and that precursor molecules can travel distances in the micrometer range before being desorbed. EBID structures obtained from [RhCl(PF3)2]2 have been characterized with a wide range of techniques for a better knowledge of the material properties and the deposition process. The deposit morphology has been studied by Transmisssion Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) to characterize the different steps of the deposition process. Crystallographic analysis is carried out by TEM in diffraction mode. Chemical analysis is carried out by Auger Electron Spectroscopy (AES) and Electron Energy Loss Spectroscopy (EELS). Morphological analysis of deposits carried out at different exposure times reveals that the first phase of the growth process, in close proximity to the substrate, is characterized by an increase of the deposit height and the deposit diameter. On the other hand the second phase of the growth process is characterized by increasing height and constant diameter. TEM contrast profiles of dots and Atomic Force Microscopy (AFM) sections of lines have clearly shown that the EBID rate is highest in the center of the beam and decreases in the peripheral regions. Deposition at variable distances from the precursor source allowed to obtain hollow structures, whose morphology reveals that the precursor reaches the reaction area mainly by direct gas phase transport. Structural analysis and TEM revealed that, independently of the deposition conditions, the deposited material is made up of Rh nanocrystals immersed in a lighter amorphous matrix. Chemical analysis by Auger Electron Spectroscopy revealed that, after removal of the C rich contamination layer by Ar ion sputtering, the average composition of the deposits is about: 60 at.% Rh, 20 at.% P, 5 at.% Cl, 7 at.% N, 8 at.% O. The absence of C and the presence of N and O in the deposit bulk have been confirmed by Electron Energy Loss Spectroscopy. This technique allowed also to prove that Rh is dominant also in deposits of sub-micrometer size (not analyzable with AES) and to determine the elemental distribution in the deposit with nanometer resolution. Comparison of the deposit composition and the positive ions detected by mass spectrometry revealed that EBID, compared to low pressure gas phase ionization, involves a higher number of events, i.e. multi-electron decomposition and rearrangements of partially decomposed species.

Pipettes are a versatile laboratory tool able to manipulate precise volumes of liquid and are prevalent in biology and chemistry. Exploiting nanopipettes as probes for scanning probe microscopy (SPM) has led to a straightforward route to high resolution, spatially resolved electrochemical measurements. In this chapter, the electrochemical nanopipette-based techniques including ITIES, scanning ion conductance microscopy (SICM), and electrochemical scanning droplet-contact methods are discussed. Characterization of micro/nanopipettes is critical to obtain quantitative electrochemical measurements. Pipette dimensions can be characterized electrochemically by placing the pipette filled with electrolyte solution and a back-inserted electrode into a bath of the same electrolyte solution, which houses a second electrode. Electrodes can also be characterized with a scanning electrochemical microscopy (SECM) approach curve. Micro/nanoscale electrochemistry requires electrodes suitable for the confined environment of a liquid filled pipette. Quasi-reference counter electrodes (QRCE) are back inserted into the barrel(s) of a filled pipette to control the potential between the pipette and external electrodes.

Impedance-based dopamine detection neural probe utilizing dopamine antibody: Real-time quantification of dopamine.

Parkinson’s disease (PD) is a chronic neurodegenerative disorder characterized by the preferential loss of dopaminergic neurons stemming from the substantia nigra pars compacta and innervating the dorsal striatum. The substantial decreases in striatal dopamine (DA) result in devastating hypokinetic movements and motor disturbances. Increased generation of reactive oxygen species, such as hydrogen peroxide (H2O2), is also thought to contribute to Parkinsonian symptoms. However, the precise role of H2O2 in the initiation, progression, and maintenance of the disease remains unclear, as reactive oxygen species are difficult to monitor in brain tissue. Further, several lines of evidence suggest that the standard treatment strategy of dopaminergic replacement therapy via administration of Levodopa (L-DOPA; L-3,4 dihydroxyphenylalanine) may serve to increase oxidative stress and potentiate cell death. We are investigating how striatal H2O2 and DA dynamics underlie behavioral changes that result from chronic L-DOPA administration in a rodent model of PD (unilateral 6-OHDA lesion) using fast-scan cyclic voltammetry, an electrochemical technique that affords precise spatial and temporal resolution, as well as selective detection of these neurochemicals. Specifically, carbon-fiber microelectrodes are used to simultaneously quantify rapid H2O2 and DA fluctuations at single recording sites in the dorsal striatum over several weeks of L-DOPA administration. The chemical fluctuations are correlated with behavioral abnormalities that develop over the course of treatment. These studies are advancing our understanding of how oxidative stress modulates nigrostriatal DA signaling, and are demonstrating how these signals correspond with dyskinetic movements in the treatment of PD.

Free, ionic zinc (Zn2+) modulates neurotransmitter dynamics in the brain. However, the sub-s effects of transient concentration changes of Zn2+ on neurotransmitter release and uptake are not well understood. To address this lack of knowledge, we have combined the photolysis of the novel caged Zn2+ compound [Zn(DPAdeCageOMe)]+ with fast scan cyclic voltammetry (FSCV) at carbon fiber microelectrodes in live, whole brain preparations from zebrafish (Danio rerio). After treating the brain with [Zn(DPAdeCageOMe)]+, Zn2+ was released by application of light that was gated through a computer-controlled shutter synchronized with the FSCV measurements and delivered through a 1 mm fiber optic cable. We systematically optimized the photocage concentration and light application parameters, including the total duration and light-to-electrical stimulation delay time. While sub-s Zn2+ application with this method inhibited DA reuptake, assessed by the first-order rate constant (k) and half-life (t1/2), it had no effect on the electrically stimulated DA overflow ([DA]STIM). Increasing the photocage concentration and light duration progressively inhibited uptake, with maximal effects occurring at 100 μM and 800 ms, respectively. Furthermore, uptake was inhibited 200 ms after Zn2+ photorelease, but no measurable effect occurred after 800 ms. We expect that application of this method to the zebrafish whole brain and other preparations will help expand the current knowledge of how Zn2+ affects neurotransmitter release/uptake in select neurological disease states.

The aim was to develop an electrochemical/optical set-up and correlate it (as validation) with other chemical and physical methods to obtain a simple and cost-effective system to study biofilm formation. A simple microfluidic cell and methods allowed continuous monitoring of the first, critical steps of microbial attachment. We monitored sulfate-reducing bacteria (SRB) at the early stages of biofilm formation. Herein, we studied the formation and adherence of SRB consortium biofilms over an indium tin oxide (ITO) conducting surface using microbiological and chemical methods, microscopic observations [scanning electron microscopy (SEM) and optical], and electrochemical impedance spectroscopy (EIS) measurements. The SRB biofilm formation was evaluated for 30 d by SEM and EIS. Charge transfer resistance decreased when the microbial population colonized the electrode. The monitoring of early-stage biofilm formation was performed using EIS at a single frequency of 1Hz during the first 36 h. The simultaneous use of optical, analytical, and microbiological methods allowed us to connect the kinetics of the growth of the microbial consortium to the values obtained via the electrochemical technique. The simple setup we present here can help laboratories with limited resources to study biofilm attachment and facilitates the development of various strategies to control biofilm development in order to avoid damage to metallic structures (microbiologically influenced corrosion, MIC) or the colonization of other industrial structures and medical devices.

Effects of social deprivation in prepubescent rhesus monkeys: immunohistochemical analysis of the neurofilament protein triplet in the hippocampal formation

Post-chemotherapy cognitive impairment, also known as 'chemobrain,' is a neurological condition in which cognitive function is impaired as a result of cancer chemotherapy treatment. In this work, we used fast-scan cyclic voltammetry (FSCV) to measure electrically evoked dopamine release and uptake in whole brain preparations from zebrafish that have been treated with carboplatin, an agent associated with chemobrain. We administered carboplatin by addition to the fish's tank water or their food. One week of treatment with 100 μM carboplatin in the water was needed to significantly impair dopamine release (∼40 % of control); however, only one day of treatment through the zebrafish's food was needed to cause a similar impairment. Atomic absorption spectroscopy measurements suggested that administration through food resulted in higher initial levels of carboplatin compared to water administration, but water administration resulted in an increase over time. Uptake, determined by modeling stimulated release plots, was unaffected. These results are consistent with our previous findings of diminished neurotransmitter release in rats and support a role for zebrafish in chemobrain-related studies.

Thermal aging embrittlement of newly developed W alloyed 9%Cr ferritic steel KA-STBA29/KA-STPA29 (ASME T92/P92) has been investigated. In order to clarify the controlling factor of the embrittlement, Charpy impact tests and determination of precipitates have been carried out using materials aged under various conditions. The decrease in the upper and lower shelf energies and the increase in the fracture appearance transition temperature (FATT) due to thermal aging were caused by the precipitation and coarsening of Laves phase. The decrease in the impact toughness was found to be uniquely correlated with the increase in the area fraction of Laves phase. In order to develop a non-destructive technique for detection of the thermal aging embrittlement, changes in electrochemical properties of KA-STBA29/KA STPA29 due to thermal aging have been investigated. The electrochemical polarization technique has been applied to thermally aged materials. Experimental results on electrochemical polarization measurements revealed that the peak current density “Ip” which appeared at a specific potential during potentiodynamic polarization measurements in 1N KOH solution linearly increased with the degree of embrittlement evaluated by the impact energy at 0°C. The increase in Ip corresponded to the increase in selective dissolution of Laves phase. As a consequence, the degree of thermal aging embrittlement can be estimated by the electrochemical technique.