Monitoring Real-time Metabolite Trafficking in the Brain using Microelectrochemical Biosensors

In a first series of experiments a glucose oxidase-based biosensor implanted in the striatum of freely moving rats was used to determine the concentration of brain extracellular (ECF) glucose in two distinct ways. With a modification of the zero-net-flux method, in which different concentrations of glucose are infused through a dialysis probe glued to the biosensor, the concentration at which there was no change in glucose current was calculated by regression analysis; this gave an ECF concentration of 351 ± 16 μM. The concentration calculated from the basal current and the in vitro calibration of the biosensor was not significantly different from this. The basal extracellular glucose concentration determined by either method remained constant over a period of several days. In a second series of experiments rats were implanted in the striatum with a Pt/Ir electrode for measurement of regional cerebral blood flow (rCBF, H 2 clearance technique), a carbon paste electrode for monitoring tissue oxygen, and a glucose biosensor for monitoring extracellular glucose. There was a parallel increase in rCBF and oxygen in response to neuronal activation (5 min tail pinch). During the neuronal activation there was a decrease in ECF glucose which was followed by a slow rise that took 30 min to return to basal levels. Finally, a group of rats implanted with a combined glucose biosensor and dialysis probe were given a 5 min tail pinch while the dialysis probe was perfused with either artificial cerebrospinal fluid (aCSF) alone or aCSF with the addition of the β -adrenoceptor antagonist propranolol. Perfusion with aCSF once again produced an initial reduction in extracellular glucose, which was co- extensive with the period of stimulation, followed by a delayed and long-lasting increase in glucose. Propranolol had no effect on basal levels of glucose but suppressed the delayed increase. These results suggests that extracellular glucose in the brain is not derived directly from the blood vascular system but from some other source which is most likely to be astrocyte

Microelectrochemical Sensors for In Vivo Brain Analysis: An Investigation of Procedures for Modifying Pt Electrodes Using Nafion®

Various Nafion® coating procedures were examined in order to design a simple and reproducible coating method to maximise permselective characteristics, and thus eliminate signals from electroactive interferents, in sensors designed for direct in vivo measurements in the brain. Interferents investigated included ascorbic acid (AA), the principal endogenous electroactive interferent present in the brain, and uric acid. Application of the Nafion® (5% commercial solution) using a thermally annealing procedure involving 5 pre-coats, and 2 subsequent dip-bake layers resulted in elimination of interferent signals. It also produced complete blocking of the signal for the neurotransmitter dopamine. The optimum time and temperature for annealing was found to be 5 min at 210 °C. An examination of shelf life over two weeks indicated negligible AA interference over this period. Preliminary investigations with respect to the potential use of these Nafion®-modified Pt electrodes in the design of implantable, first generation, peroxide detecting biosensors indicated that the modified electrode had no effect on O2 permeability but did produce a significant decrease in H2O2 sensitivity. While this may preclude their use in biosensor development they may be more suitable for detection of gaseous neurochemicals such as nitric oxide

Investigation of events in the EEG signal correlated with changes in both oxygen and glucose in the brain

Since the brain has no constant energy reserves, a continuous supply of energy substrates is central to all processes that maintain the functionality of the neuronal cells. EEG has been found to be tightly related to variations in the concentration of the energy substrates such as oxygen and glucose. Prediction of neural activation is particularly useful as it could contribute significantly in the prevention, stabilization, or treatment of diseases such as Alzheimer's disease, migraine headache, and ischemic stroke, in which signaling between neurons and brain vessels is threatened because of dysfunctions that affect the neuronal, astroglial, and/or vascular components of the neurovascular unit. This work deals with investigation of events in the EEG signal correlated with changes in both oxygen and glucose signals in the brain. The topic is to implement a model that through measures of oxygen and glucose in the brain of rats allow to achieve a good estimation of the neural signals, which reflecting the simultaneous metabolic changes, during spontaneous oscillation and electrical stimulation

Investigation of events in the EEG signal correlated with changes in both oxygen and glucose in the brain

Since the brain has no constant energy reserves, a continuous supply of energy substrates is central to all processes that maintain the functionality of the neuronal cells. EEG has been found to be tightly related to variations in the concentration of the energy substrates such as oxygen and glucose. Prediction of neural activation is particularly useful as it could contribute significantly in the prevention, stabilization, or treatment of diseases such as Alzheimer's disease, migraine headache, and ischemic stroke, in which signaling between neurons and brain vessels is threatened because of dysfunctions that affect the neuronal, astroglial, and/or vascular components of the neurovascular unit. This work deals with investigation of events in the EEG signal correlated with changes in both oxygen and glucose signals in the brain. The topic is to implement a model that through measures of oxygen and glucose in the brain of rats allow to achieve a good estimation of the neural signals, which reflecting the simultaneous metabolic changes, during spontaneous oscillation and electrical stimulation

Continuous monitoring of extracellular glucose concentrations in the striatum of freely moving rats with an implanted glucose biosensor

We have used a glucose oxidase-based sensor implanted in the striatum of freely moving rats to determine the concentration of extracellular glucose in two distinct ways. With a modification of the zero net flux method, in which different concentrations of glucose are infused through a dialysis probe glued to the biosensor, we calculated the concentration at which there was no change in glucose current by regression analysis; this gave a concentration of 0.351 ± 0.016 mM. Calculating the concentration from the basal current and the in vitro calibration of the biosensor was not significantly different from this. The basal extracellular glucose concentration determined by either method remained constant over a period of several days. Infusion of 50 µM veratridine through the adjacent dialysis probe caused a steep decrease in glucose current as soon as the drug reached the brain in contrast to the delayed fall (7.5 min) seen with microdialysis in previous experiments from this laboratory. These results demonstrate that this biosensor provides a direct, real-time measure of the extracellular concentration of glucose

Remediation of chromium(VI) at polypyrrole-coated titanium

The application of conducting polypyrrole (PPy) and polyaniline (PAni) coated substrates in remediation of chromium, Cr(VI), is an area of considerable interest. Here, we discuss the implementation of PPy-coated titanium as a new material for the reduction of Cr(VI) to the less toxic trivalent state, Cr(III). An alkaline-peroxide based etching process was used to ensure the adhesion of the PPy coatings to the underlying titanium. The PPy films showed excellent resistance to acidic Cr(VI) solutions and remained adherent after continuous exposure to the solutions. In order to optimise the remediation process a number of experimental parameters were investigated, including the thickness of the PPy coating, the reduction potential used in pre-treatment of the PPy and the degree of solution agitation. The durability of the materials on exposure to the Cr(VI) test solutions made them suitable for repeated remediation experiments. Following several test-runs, the cleanup efficiency of the material was found to decrease slightly, however, increasing the exposure/experiment time resulted in significantly improved cleanup ability

Control of the Oxygen Dependence of an Implantable Polymer/Enzyme Composite Biosensor for Glutamate

Biosensors for glutamate (Glu) were fabricated from Teflon-coated Pt wire (cylinders and disks), modified with the enzyme glutamate oxidase (GluOx) and electrosynthesized polymer PPD, poly(o-phenylenediamine). The polymer/enzyme layer was deposited in two configurations:  enzyme before polymer (GluOx/PPD) and enzyme after polymer (PPD/GluOx). These four biosensor designs were characterized in terms of response time, limit of detection, Michaelis−Menten parameters for Glu (Jmax and KM(Glu)), sensitivity to Glu in the linear response region, and dependence on oxygen concentration, KM(O2). Analysis showed that the two polymer/enzyme configurations behaved similarly on both cylinders and disks. Although the two geometries showed different behaviors, these differences could be explained in terms of higher enzyme loading density on the disks; in many analyses, the four designs behaved like a single population with a range of GluOx loading. Enzyme loading was the key to controlling the KM(O2) values of these first generation biosensors. The counterintuitive, and beneficial, behavior that biosensors with higher GluOx loading displayed a lower oxygen dependence was explained in terms of the effects of enzyme loading on the affinity of GluOx for its anionic substrate. Some differences between the properties of surface immobilized GluOx and glucose oxidase are highlighted

Electrochemistry at NUI Maynooth

Abstract included in text

The efficiency of immobilised glutamate oxidase decreases with surface enzyme loading: an electrostatic effect, and reversal by a polycation significantly enhances biosensor sensitivity

The apparent Michaelis constant, KM, for glutamate oxidase (GluOx) immobilised on Pt electrodes increased systematically with enzyme loading. The effect was due, at least in part, to electrostatic repulsion between neighbouring oxidase molecules and the anionic substrate, glutamate (Glu). This understanding has allowed us to increase the Glu sensitivity of GluOx-based amperometric biosensors in the linear response region (100 ± 11 nA cmâ2µMâ1 at pH 7.4; SD, n = 23) by incorporating a polycation (polyethyleneimine, PEI) to counterbalance the polyanionic protein. Differences in the behaviour of glucose biosensors of a similar configuration highlight a limitation of using glucose oxidase as a model enzyme in biosensor design

The efficiency of immobilised glutamate oxidase decreases with surface enzyme loading: an electrostatic effect, and reversal by a polycation significantly enhances biosensor sensitivity

The apparent Michaelis constant, KM, for glutamate oxidase (GluOx) immobilised on Pt electrodes increased systematically with enzyme loading. The effect was due, at least in part, to electrostatic repulsion between neighbouring oxidase molecules and the anionic substrate, glutamate (Glu). This understanding has allowed us to increase the Glu sensitivity of GluOx-based amperometric biosensors in the linear response region (100 ± 11 nA cmâ2µMâ1 at pH 7.4; SD, n = 23) by incorporating a polycation (polyethyleneimine, PEI) to counterbalance the polyanionic protein. Differences in the behaviour of glucose biosensors of a similar configuration highlight a limitation of using glucose oxidase as a model enzyme in biosensor design