Electrochemical Monitoring of Propagative Fluctuation of Ascorbate in the Live Rat Brain during Spreading Depolarization.

Spreading depolarization (SD) occurs frequently in the injured brain, and its neurochemical effects are detrimental to brain function. We report the first observation that the release of ascorbate, an important neurochemical in the brain, is closely accompanied with SD. Ascorbate was monitored with carbon nanotube (CNT)-sheathed carbon fiber microelectrodes (CFEs). This system features high selectivity and temporal/spatial resolution. With our sensor, we observed a significant increase in the concentration of ascorbate in response to SD induction. Mechanistic studies show a contrasting behavior; with a SD specific inhibitor, release was completely suppressed, whereas with inhibition of commonly employed glutamate transporters, ascorbate release was increased, demonstrating a powerful means of discriminating ascorbate release between disputed pathways. Most importantly, we observed the propagative nature of ascorbate release following SD.

Controllable and Reproducible Sheath of Carbon Fibers with Single-Walled Carbon Nanotubes through Electrophoretic Deposition for In Vivo Electrochemical Measurements.

The unique electronic and chemical structures of carbon nanotubes (CNTs) have well enabled their applications in electrochemistry and electroanalytical chemistry; however, the difficulty in reproducibly confining CNTs onto substrate electrodes, particularly onto microelectrodes, still remains to be addressed. In this study, we develop a method to reproducibly confine single-walled carbon nanotubes (SWNTs) onto carbon fiber microelectrodes (CFEs) with electrophoretic deposition (EPD) for in vivo measurement of ascorbate. Under 2.5 V, acid-treated SWNTs are uniformly deposited on CFEs. After thermal treatment at 300 °C followed by electrochemical treatment in 0.5 M H2SO4, the SWNT-sheathed CFEs exhibit good activity to accelerate the electrochemical oxidation of ascorbic acid (i.e., ascorbate, in a neutral solution) at an onset potential of -0.15 V vs Ag/AgCl and could in vivo selectively detect ascorbate. The controllable procedures employed for EPD and pretreatment avoid the deviation in the conventional manual modification methods such as drop casting and hand rolling previously used for confining SWNTs onto an electrode surface. With the electrodes prepared here, we find that level of extracellular ascorbate in the rat cortex increases by 20.4 ± 4.8% ( n = 4), relative to its basal level, within 9 min after infusion of kainic acid into the hippocampus to evoke epilepsy. This study offers a reproducible method to prepare SWNT-sheathed CFEs for in vivo monitoring ascorbate that would largely facilitate future studies on neurochemical processes of ascorbate in various physiological and pathological events.

In Vivo Monitoring of Oxygen Fluctuation Simultaneously at Multiple Sites of Rat Cortex during Spreading Depression.

Spreading depression (SD) is a common pathological process in the brain shown as propagating neuronal depolarization followed by activity depression over the brain, and it is closely related to migraines and epilepsy. Although O2 is known to fluctuate during SD, the difference of O2 responses at different sites in the same brain region remains unknown. In this study, we develop an in vivo electrochemical method with microelectrode arrays (MEAs) to monitor, in real time, O2 fluctuation at multiple sites of rat cortex during SD with high spatial/temporal resolution. Platinum nanoparticles are electrochemically deposited on the multiplexed electrodes of the MEAs to monitor O2 fluctuation simultaneously and selectively via a four-electron reduction process. Configuration of electrode arrays is designed rationally to exclude the probable crosstalk between neighbor recording electrodes during simultaneous measurements. With the MEAs, we find both the basal O2 levels and O2 fluctuations at different sites of the cortex during SD exhibit significant differences, indicating the intensity of energy metabolism and oxidative stress vary at different sites even in the same brain region. Further studies prove that O2 fluctuation is mostly caused by the increase of brain blood flow and the consumption of neuronal O2 during SD. Our study reveals that energy metabolism varies at different sites in brain cortex during SD propagation, which may provide new understanding for SD-related pathological processes.

Selective Amperometric Recording of Endogenous Ascorbate Secretion from a Single Rat Adrenal Chromaffin Cell with Pretreated Carbon Fiber Microelectrodes.

Quantitative description of ascorbate secretion at a single-cell level is of great importance in physiological studies; however, most studies on the ascorbate secretion have so far been performed through analyzing cell extracts with high performance liquid chromatography, which lacks time resolution and analytical performance on a single-cell level. This study demonstrates a single-cell amperometry with carbon fiber microelectrodes (CFEs) to selectively monitor amperometric vesicular secretion of endogenous ascorbate from a single rat adrenal chromaffin cell. The CFEs are electrochemically pretreated in a weakly basic solution (pH 9.5), and such pretreatment essentially enables the oxidation of ascorbate to occur at a relatively low potential (i.e., 0.0 V vs Ag/AgCl), and further a high selectivity for ascorbate measurement over endogenously existing electroactive species such as epinephrine, norepinephrine, and dopamine. The selectivity is ensured by much larger amperometric response at the pretreated CFEs toward ascorbate over those toward other endogenously existing electroactive species added into the solution or ejected to the electrode with a micropuffer pipet, and by the totally suppressed current response by adding ascorbate oxidase into the cell lysate. With the pretreated CFE-based single-cell amperometry developed here, exocytosis of endogenous ascorbate of rat adrenal chromaffin cells is directly observed and ensured with the calcium ion-dependent high K+-induced secretion of endogenous ascorbate from the cells. Moreover, the quantitative information on the exocytosis of endogenous ascorbate is provided.

Ultrathin Cell-Membrane-Mimic Phosphorylcholine Polymer Film Coating Enables Large Improvements for In†Vivo Electrochemical Detection.

Resisting biomolecule adsorption onto the surface of brain-implanted microelectrodes is a key issue for in vivo monitoring of neurochemicals. Herein, we demonstrate that an ultrathin cell-membrane-mimic film of ethylenedioxythiophene tailored with zwitterionic phosphorylcholine (EDOT-PC) electropolymerized onto the surface of a carbon fiber microelectrode (CFE) not only resists protein adsorption but also maintains the sensitivity and time response for in vivo monitoring of dopamine (DA). As a consequence, the as-prepared PEDOT-PC/CFEs could be used as a new reliable platform for tracking DA in vivo and would help understand the physiological and pathological functions of DA.

High Antifouling Property of Ion-Selective Membrane: toward In Vivo Monitoring of pH Change in Live Brain of Rats with Membrane-Coated Carbon Fiber Electrodes.

In vivo monitoring of pH in live brain remains very essential to understanding acid-base chemistry in various physiological processes. This study demonstrates a potentiometric method for in vivo monitoring of pH in the central nervous system with carbon fiber-based proton-selective electrodes (CF-H+ISEs) with high antifouling property. The CF-H+ISEs are prepared by formation of a H+-selective membrane (H+ISM) with polyvinyl chloride polymeric matrixes containing plasticizer bis(2-ethylhexyl)sebacate, H+ ionophore tridodecylamine, and ion exchanger potassium tetrakis(4-chlorophenyl)borate onto carbon fiber electrodes (CFEs). Both in vitro and in vivo studies demonstrate that the H+ISM exhibits strong antifouling property against proteins, which enables the CF-H+ISEs to well maintain the sensitivity and reversibility for pH sensing after in vivo measurements. Moreover, the CF-H+ISEs exhibit a good response to pH changes within a narrow physiological pH range from 6.0 to 8.0 in quick response time with high reversibility and selectivity against species endogenously existing in the central nervous system. The applicability of the CF-H+ISEs is illustrated by real-time monitoring of pH changes during acid-base disturbances, in which the brain acidosis is induced by CO2 inhalation and brain alkalosis is induced by bicarbonate injections. The results demonstrate that brain pH value rapidly decreases in the amygdaloid nucleus by ca. 0.14 ± 0.01 (n = 5) when the rats breath in pure CO2 gas, while increases in the cortex by about 0.77 ± 0.12 (n = 3) following intraperitoneal injection of 5 mmol/kg NaHCO3. This study demonstrates a new potentiometric method for in vivo measurement of pH change in the live brain of rats with high reliability.

Protein Pretreatment of Microelectrodes Enables in Vivo Electrochemical Measurements with Easy Precalibration and Interference-Free from Proteins.

In vivo electrochemistry is one powerful strategy for probing brain chemistry. However, the decreases in sensitivity mainly caused by the adsorption of proteins onto electrode surface in short-term in vivo measurements unfortunately render great challenges in both electrode calibration and selectivity against the alternation of proteins. In this study, we observe that the pretreatment of carbon fiber microelectrodes (CFEs) with bovine serum albumin (BSA) would offer a simple but effective strategy to the challenges mentioned above. We verify our strategy for dopamine (DA) with conventionally used CFEs and for ascorbate with our previously developed carbon nanotube-modified CFEs. We find that, in artificial cerebral spinal fluid (aCSF) solution containing BSA, the current responses of the microelectrodes equilibrate shortly and the results for precalibration carried out in this solution are found to be almost the same as those for the postcalibration in pure aCSF. This observation offers a new solution to electrode calibration for in vivo measurements with a technical simplicity. Furthermore, we find that the use of BSA pretreated CFEs to replace bare CFEs would minimize the interference from the alternation of proteins in the brain. This study offers a new general and effective approach to in vivo electrochemistry with a high reliability and a simplified procedure.

Rational Design of Bioelectrochemically Multifunctional Film with Oxidase, Ferrocene, and Graphene Oxide for Development of in Vivo Electrochemical Biosensors.

This study demonstrates a new strategy to develop in vivo electrochemical biosensors through rational design and simple formation of bioelectrochemically multifunctional film (BMF). The BMF is rationally designed by first efficiently incorporating oxidase, ferrocene mediator, and graphene oxide into polymaleimidostyrene/polystyrene (PMS/PS) matrix to form a homogeneous mixture and then simply formed by drop-coating the mixture onto solid conducting substrate. By using the as-formed BMF, electrochemical biosensors could be constructed with a technical simplicity and high reproducibility. To illustrate the BMF-based biosensors for in vivo applications, we directly couple the biosensors to in vivo microdialysis to establish an online electrochemical system (OECS) for in vivo monitoring of glucose in rat auditory cortex during salicylate-induced tinnitus model. The OECS with the BMF-based biosensor as the detector shows a linear response toward glucose within a concentration range from 50 to 500 µM with a detection limit of 10 µM (S/N = 3). Additionally, the OECS is stable and does not suffer from the interference from the electroactive species endogenously coexisting in the brain microdialysate. With the BMF-based OECS, the basal level of glucose in the microdialysate continuously sampled from rat auditory cortex is determined to be 120 ± 10 µM (n = 5). After the rats were administrated with salicylate to induce transient tinnitus, the microdialysate glucose concentration in the rat auditory cortex remarkably increased to 433 ± 190 µM (n = 5) at the time point of 1.5 h. This study essentially offers a new, technically simple and reproducible approach to development of in vivo electrochemical biosensors, which is envisaged to be relatively useful for understanding of the molecular basis of brain functions.

Online electrochemical systems for continuous neurochemical measurements with low-potential mediator-based electrochemical biosensors as selective detectors.

This study demonstrates a new strategy to develop online electrochemical systems (OECSs) for continuously monitoring neurochemicals by efficiently integrating in vivo microdialysis with an oxidase-based electrochemical biosensor with low-potential electron mediators to shuttle the electron transfer of the oxidases. By using thionine and xanthine oxidase (XOD) as examples of low-potential mediators and oxidases, respectively, we demonstrate that the use of low-potential mediators to shuttle the electron transfer of oxidases would offer a new approach to the development of oxidase-based biosensors with theoretical and technical simplicity. To construct the XOD-based biosensor, thionine was adsorbed onto carbon nanotubes and used to shuttle the electron transfer of XOD. The XOD-based biosensor was positioned into an electrochemical cell that was directly coupled with in vivo microdialysis to form an online electrochemical system (OECS) for continuous and selective measurements of the substrate of XOD (with hypoxanthine as an example). The OECS based on the low-potential mediators is highly selective against the species endogenously existing in the brain system, which is attributed to the low operation potential benefited from the low redox potentials of the mediators. Moreover, the OECS demonstrated here is stable and reproducible and could thus be envisaged to find some interesting applications in physiological and pathological investigations. This study essentially offers a new strategy to develop online electrochemical systems, which is of great importance in understanding the molecular basis of physiological and pathological events.