Robert D. O'Neill

Characterization in vitro and in vivo of the oxygen dependence of an enzyme/polymer biosensor for monitoring brain glucose

The oxygen dependence of a first generation amperometric biosensor was investigated in vitro and in vivo by monitoring its glucose response as a function of solution pO(2). The biosensor was a glucose oxidase (GOx) modified poly(o-phenylenediamine) coated Pt cylinder electrode (Pt/PPD/GOx) that has been designed for neurochemical analysis in vivo. Two types of oxygen probes were used: a self-calibrating commercial macroelectrode in vitro; and a carbon paste microelectrode in vivo. Calibrations in vitro showed that oxygen interference in the operation of Pt/PPD/GOx electrodes was minimal for concentrations of glucose (approximately 0.5 mM) and oxygen (approximately 50 microM) found in brain ECF. This observation was confirmed by simultaneous monitoring in vivo of brain glucose and oxygen in the awake rat. However, at levels of glucose normally found in peripheral tissues (approximately 5 mM), the oxygen dependence was severe. We conclude that the oxygen sensitivity of Pt/PPD/GOx biosensors does not preclude their reliable use in media containing low glucose levels, such as brain ECF.

Continuous Monitoring of Extracellular Glucose Concentrations in the Striatum of Freely Moving Rats with an Implanted Glucose Biosensor

Abstract: 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.

An amperometric glucose-oxidase/poly(o-phenylenediamine) biosensor for monitoring brain extracellular glucose: in vivo characterisation in the striatum of freely-moving rats.

Amperometric glucose biosensors based on the immobilization of glucose oxidase (GOx) on Pt electrodes with electropolymerized o-phenylenediamine (PPD) were implanted in the right striatum of freely-moving rats. Carbon paste electrodes for the simultaneous monitoring of ascorbic acid (AA) and/or tissue O2 were implanted in the left striatum. A detailed in vivo characterization of the Pt/PPD/GOx signal was carried out using various pharmacological manipulations. Confirmation that the biosensor responded to changing glucose levels in brain extracellular fluid (ECF) was obtained by intraperitoneal (i.p.) injection of insulin that caused a decrease in the Pt/PPD/GOx current, and local administration of glucose (1 mM) via an adjacent microdialysis probe that resulted in an increase in the biosensor current. An insulin induced increase in tissue O2 in the brain was also observed. Interference studies involved administering AA and subanaesthetic doses of ketamine i.p. Both resulted in increased extracellular AA levels with ketamine also causing an increase in O2. No significant change in the Pt/PPD/GOx current was observed in either case indicating that changes in O2 and AA, the principal endogenous interferents, have minimal effect on the response of these first generation biosensors. Stability tests over a successive 5-day period revealed no significant change in sensitivity. These in vivo results suggest reliable glucose monitoring in brain ECF.

The oxidation of ascorbic acid at carbon paste electrodes: Modified response following contact with surfactant, lipid and brain tissue

Abstract The electrochemical characteristics of ascorbic acid and ferrocyanide were investigated at untreated, surfactant (Triton-X) treated and lipid (phosphatidylethanolamine) treated carbon paste electrodes (CPEs), at carbon powder electrodes, and at CPEs following contact with brain tissue. The results indicate that, following contact with brain tissue, pasting oil is removed (apparently by lipids present in the tissue) from the active surface of the electrode. Resistance and capacitance studies support this interpretation. Electron transfer at the resulting powder-type surface is faster than at the original CPE, leading to a shift in the oxidation wave for ascorbic acid to lower potentials. Adsorption of substrate on the electrode is evident following, but not before, the treatments. Implications for the development of chemically modified CPEs for use in vivo are discussed.

Real-Time Monitoring of Brain Tissue Oxygen Using a Miniaturized Biotelemetric Device Implanted in Freely Moving Rats

A miniaturized biotelemetric device for the amperometric detection of brain tissue oxygen is presented. The new system, derived from a previous design, has been coupled with a carbon microsensor for the real-time detection of dissolved O(2) in the striatum of freely moving rats. The implantable device consists of a single-supply sensor driver, a current-to-voltage converter, a microcontroller, and a miniaturized data transmitter. The oxygen current is converted to a digital value by means of an analog-to-digital converter integrated in a peripheral interface controller (PIC). The digital data is sent to a personal computer using a six-byte packet protocol by means of a miniaturized 434 MHz amplitude modulation (AM) transmitter. The receiver unit is connected to a personal computer (PC) via a universal serial bus. Custom developed software allows the PC to store and plot received data. The electronics were calibrated and tested in vitro under different experimental conditions and exhibited high stability, low power consumption, and good linear response in the nanoampere current range. The in vivo results confirmed previously published observations on oxygen dynamics in the striatum of freely moving rats. The system serves as a rapid and reliable model for studying the effects of different drugs on brain oxygen and brain blood flow and it is suited to work with direct-reduction sensors or O(2)-consuming biosensors.

Characterization of carbon paste electrodes in vitro for simultaneous amperometric measurement of changes in oxygen and ascorbic acid concentrations in vivo

Differential-pulse amperometry, an established technique for the in vivo monitoring of dopamine in brain extracellular fluid (ECF), was extended to the simultaneous electrochemical detection of molecular oxygen (O2) and ascorbic acid (AA). Lipid-treated carbon paste electrodes (LCPEs) were characterized in vitro using this technique and found to be ideally suited for the detection of both compounds. For O2 reduction, two equally sized cathodic pulses were applied, the first from a resting potential at -150 to -350 mV, which corresponds to the foot of the reduction wave for O2 at LCPEs, and the second from -350 to -550 mV, which corresponds to the peak of the reduction wave. Following the same criterion, equally sized anodic pulses were then applied from -150 to +50 mV and from +50 to +250 mV for AA oxidation. The complete sequence of pulses for O2 and AA detection lasts 1 s. Interference by O2 with AA currents and vice versa was not a problem. Also, several compounds present in brain ECF were tested and shown not to interfere appreciably with the amperometric signal for either compound. The technique was tested in vivo, and results from behavioural stimulation, achieved by the application of tail pinch, support the conclusion of simultaneous independent detection of changes in O2 and AA at LCPEs.

Fixed Versus Removable Microdialysis Probes for In Vivo Neurochemical Analysis: Implications for Behavioral Studies

Abstract: The levels of several neurochemicals, i.e., uric acid (UA), dopamine (DA), dihydroxyphenylacetic acid, and 5‐hydroxyindoleacetic acid, collected daily from the rat striatum with either fixed or removable microdialysis probes for 7 days after surgery were compared. The implantation of the fixed cannula was followed by a 10‐fold increase in the UA content in the dialysates collected from the first day after surgery onward and by a steady decrease in dihydroxyphenylacetic acid levels, whereas those of DA remained fairly stable. With the removable cannula system, only a smaller, transient increase in UA during the first 3 days after surgery was observed, with no change in DA or monoamine metabolites. The glial reaction around the cannula tracks was assessed by both quantitative histological techniques and measuring the glutamine levels in the dialysates collected at the time of surgery and 7 days later. Both the glial cell number and nuclear size, as well as the glutamine outflow, were considerably larger in the animals implanted with the fixed probes. It is, therefore, likely that the UA levels in the dialysate reflect the glial reaction to the probe. The suitability of the removable probe system for behavioral experiments involving repeated microdialysis sampling was illustrated in an experiment showing that the DA release in the nucleus accumbens of male rats assessed daily at postsurgery days 5–10 was virtually identical in three alternating sessions of sexual behavior as was the smaller release of this neurotransmitter detected during intervening nonsexual social interactions.

Anomalously High Concentrations of Brain Extracellular Uric Acid Detected with Chronically Implanted Probes: Implications for In Vivo Sampling Techniques

Abstract: The height of peak 2, h2, recorded using linear sweep voltammetry with 350‐μm‐diameter carbon paste electrodes in rat striatum was measured from the day of implantation (day 0) to 4 months after surgery. The value of h2was at a minimum on day 0 (0.6 ± 0.2 nA; n = 20), rose sharply to a maximum on day 2 (6.3 ± 0.9 nA; n = 12), and decreased to a stable level by day 7 (3.3 ± 0.7 nA; n = 16), which lasted for 4 months (3.2 ± 0.6 nA; n = 9). These changes were shown by microinfusion of uricase to be due to variations in the concentration of extracellular uric acid, although h2 appears to have a small baseline contribution of ∼0.3 nA from 5‐hydroxyindoleacetic acid. The stable value of h2 recorded under chronic conditions was estimated to correspond to a minimal uric acid concentration of 50 μmol/ L, which represents a 10‐fold increase in the extracellular level of this purine metabolite compared with the initial (acute) value. Very similar results were obtained using a mi‐crodialysis technique that detected uric acid directly. These estimates of striatal uric acid concentration are in marked contrast to those obtained using 40‐μm diameter carbon fiber electrodes, which showed a decrease from the acute preparation to < 1 μmol/L under chronic conditions. Large values of h2 were also recorded with chronically implanted paste electrodes in the hippocampus and frontal cortex. The results suggest that large in vivo probes, such as carbon paste electrodes and dialysis tubes, markedly disturb the neurochemical balance in the extracellular fluid even 1 day following implantation and emphasize the need to develop further small sensors for in vivo neurochemical analysis with minimal perturbation.

Strategies for decreasing ascorbate interference at glucose oxidase-modified poly(o-phenylenediamine)-coated electrodes

Monitoring glucose using biosensors in biological systems is complicated by the presence of reducing agents such as ascorbic acid (AA). This is particularly so in brain extracellular fluid (ECF), where glucose concentrations may be as low as 1 mmol l–1 and AA levels are approximately 500 µmol l–1. Since glucose oxidase-modified poly(o-phenylenediamine)-coated Pt (Pt/PPD/GOx) electrodes show good stability in vivo, glucose sensitivity and AA-blocking properties, attempts were made to improve the latter characteristic further by two distinct strategies: incorporating non-enzyme protein into the polymer film and underlaying the polymer with a lipid coat. Both tactics significantly decreased interference by AA without changing the sensitivity to glucose, the lipid modification being the more effective. The current ratio IGluc/IAA for 1 mmol l–1 glucose and 500 µmol l–1 AA for the best 50% of the lipid-modified Pt/PPD/GOx electrodes was approximately 30:1, indicating that these sensors are well suited for monitoring brain glucose in vivo.

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, K(M), 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 cm(-2)microM(-1) 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.