Characterisation of a Platinum-based Electrochemical Biosensor for Real-time Neurochemical Analysis of Choline

A choline biosensor was characterised in detail to determine the effects of physiologically relevant parameters on the ability of the sensor to reliably detect neurochemical changes in choline. This first generation Pt-based polymer enzyme composite sensor displayed excellent shelf-life and biocompatibility with no significant decrease in choline sensitivity observed following 14 days of storage dry, or in ex-vivo rodent brain tissue. However, subjecting the sensor to repeated calibrations and storage over the same period resulted in significant decreases (20–70 %) due to enzyme denaturation associated with the repeated calibration and storage cycles. Potential interference signals generated by the principal electroactive interferents present in the brain were minimal; typically <1 % of the choline current response at in vivo levels. Additionally, changing temperature over the physiologically relevant range of 34–40 °C had no effect on sensitivity, while increasing pH between 7.2 and 7.6 produced only a 5 % increase in signal. The limit of detection of the sensor was in the low μM range (0.11±0.02 μM), while the in vitro response time was determined to be less than the solution mixing time and within ca. 5 s, suggesting potential sub-second in vivo response characteristics. Finally, the sensor was implanted in the striatum of freely moving rats and demonstrated reliable detection of physiological changes in choline in response to movement, and pharmacological manipulation by injection of choline chloride

In vitro development and in vivo application of a platinum-based electrochemical device for continuous measurements of peripheral tissue oxygen

Acute limb ischaemia is caused by compromised tissue perfusion and requires immediate attention to reduce the occurrence of secondary complications that could lead to amputation or death. To address this, we have developed a novel platinum (Pt)-based electrochemical oxygen (O2) device for future applications in clinical monitoring of peripheral tissue ischaemia. The effect of integrating a Pt pseudo-reference electrode into the O2 device was investigated in vitro with an optimum reduction potential of −0.80 V. A non-significant (p = 0.11) decrease in sensitivity was recorded when compared against an established Pt-based O2 sensor operating at −0.65 V. Furthermore, a biocompatible clinical sensor (ClinOX) was designed, demonstrating excellent linearity (R2 = 0.99) and sensitivity (1.41 ± 0.02 nA μM−1 ) for O2 detection. Significant rapid decreases in the O2 current during in vivo ischaemic insults in rodent limbs were reported for Pt-Pt (p b 0.001) and ClinOX (p b 0.01) and for ClinOX (p b 0.001) in porcine limbs. Ex vivo sensocompatibility investigations identified no significant difference (p = 0.08) in sensitivity values over 14 days of exposure to tissue homogenate. The Pt-Pt based O2 design demonstrated high sensitivity for tissue ischaemia detection and thus warrants future clinical investigation

An in vitro characterisation comparing carbon paste and Pt microelectrodes for real-time detection of brain tissue oxygen

In vitro characterisation results for O2reduction at Pt-based microelectrodes are presented and compared with those for carbon-paste electrodes (CPEs). Cyclic voltammetry indicates a potential of −650 mV vs. SCE is required for cathodic reduction at both electrode types, and calibration experiments at this potential revealed a significantly higher sensitivity for Pt (−0.091 ± 0.006 μAmm−2μM−1vs. −0.048 ± 0.002 μAmm−2μM−1 for CPEs). Since Pt electrodes are readily poisoned through contact with biological samples selected surface coated polymers (polyphenylenediamine (PPD), polymethyl methacrylate (PMMA) and Rhoplex®) were examined in biocompatibility studies performed in protein, lipid and brain tissue solutions. While small and comparable decreases in sensitivity were observed for bare Pt, Pt-Rhoplex and PMMA there was minimal change at the Pt-PPD modified electrode for each 24h treatment, including an extended 3 day exposure to brain tissue. The polymers themselves had no effect on the O2 response characteristics. Further characterisation studies at the Pt-based microelectrodes confirmed interference free signals, no effect of pH and ion changes, and a comparable detection limit (0.08 ± 0.01 μM) and response time (<1 s) to CPEs. Although a significant temperature effect (ca. 3% change in signal for each 1 °C) was observed it is predicted that this will not be important for in vivo brain tissue O2 measurements due to brain temperature homeostasis. These results suggest that amperometric Pt electrodes have the potential to be used reliably as an alternative to CPEs to monitor brain tissue O2 over extended periods in freely-moving animals

Development of a microelectrochemical biosensor for the real-time detection of choline

Here we describe the development of a first generation biosensor for the detection of brain extracellular choline, investigating important considerations for in-vivo monitoring such as sensor sensitivity, O2 interference and selectivity. Extensive optimisation of choline biosensor designs resulted in a biosensor with excellent sensitivity towards choline (0.54 ± 0.03 nA/μM). Oxygen interference studies demonstrate a 1% reduction in current at 50 μM O2 when compared to atmospheric O2 levels (200 μM), indicating that the sensor can be used for reliable choline monitoring, free from changes in current associated with physiological O2 fluctuations. A negligible sensitivity of 0.0021 ± 0.0002 nA/μM n = 8 was achieved utilising poly-phenylenediamine (PPD) as a permselective membrane for interference rejection of ascorbic acid (AA), the most physiologically important endogenous electroactive species present in the brain. The optimised biosensor when implanted into the striatum of a freely moving rat successfully detected local perfusions of choline demonstrating the sensors ability to detect choline in-vivo

An integrative dynamic model of brain energy metabolism using in vivo neurochemical measurements

An integrative, systems approach to the modelling of brain energy metabolism is presented. Mechanisms such as glutamate cycling between neurons and astrocytes and glycogen storage in astrocytes have been implemented. A unique feature of the model is its calibration using in vivo data of brain glucose and lactate from freely moving rats under various stimuli. The model has been used to perform simulated perturbation experiments that show that glycogen breakdown in astrocytes is significantly activated during sensory (tail pinch) stimulation. This mechanism provides an additional input of energy substrate during high consumption phases. By way of validation, data from the perfusion of 50μM propranolol in the rat brain was compared with the model outputs. Propranolol affects the glucose dynamics during stimulation, and this was accurately reproduced in the model by a reduction in the glycogen breakdown in astrocytes. The model’s predictive capacity was verified by using data from a sensory stimulation (restraint) that was not used for model calibration. Finally, a sensitivity analysis was conducted on the model parameters, this showed that the control of energy metabolism and transport processes are critical in the metabolic behaviour of cerebral tissue

An integrative dynamic model of brain energy metabolism using in vivo neurochemical measurements

An integrative, systems approach to the modelling of brain energy metabolism is presented. Mechanisms such as glutamate cycling between neurons and astrocytes and glycogen storage in astrocytes have been implemented. A unique feature of the model is its calibration using in vivo data of brain glucose and lactate from freely moving rats under various stimuli. The model has been used to perform simulated perturbation experiments that show that glycogen breakdown in astrocytes is significantly activated during sensory (tail pinch) stimulation. This mechanism provides an additional input of energy substrate during high consumption phases. By way of validation, data from the perfusion of 50μM propranolol in the rat brain was compared with the model outputs. Propranolol affects the glucose dynamics during stimulation, and this was accurately reproduced in the model by a reduction in the glycogen breakdown in astrocytes. The model’s predictive capacity was verified by using data from a sensory stimulation (restraint) that was not used for model calibration. Finally, a sensitivity analysis was conducted on the model parameters, this showed that the control of energy metabolism and transport processes are critical in the metabolic behaviour of cerebral tissue

An integrative dynamic model of brain energy metabolism using in vivo neurochemical measurements

An integrative, systems approach to the modelling of brain energy metabolism is presented. Mechanisms such as glutamate cycling between neurons and astrocytes and glycogen storage in astrocytes have been implemented. A unique feature of the model is its calibration using in vivo data of brain glucose and lactate from freely moving rats under various stimuli. The model has been used to perform simulated perturbation experiments that show that glycogen breakdown in astrocytes is significantly activated during sensory (tail pinch) stimulation. This mechanism provides an additional input of energy substrate during high consumption phases. By way of validation, data from the perfusion of 50μM propranolol in the rat brain was compared with the model outputs. Propranolol affects the glucose dynamics during stimulation, and this was accurately reproduced in the model by a reduction in the glycogen breakdown in astrocytes. The model’s predictive capacity was verified by using data from a sensory stimulation (restraint) that was not used for model calibration. Finally, a sensitivity analysis was conducted on the model parameters, this showed that the control of energy metabolism and transport processes are critical in the metabolic behaviour of cerebral tissue

An Investigation of Hypofrontality in an Animal Model of Schizophrenia Using Real-Time Microelectrochemical Sensors for Glucose, Oxygen, and Nitric Oxide

Glucose, O2, and nitric oxide (NO) were monitored in real time in the prefrontal cortex of freely moving animals using microelectrochemical sensors following phencyclidine (PCP) administration. Injection of saline controls produced a decrease in glucose and increases in both O2 and NO. These changes were short-lived and typical of injection stress, lasting ca. 30 s for glucose and between 2 and 6 min for O2 and NO, respectively. Subchronic PCP (10 mg/kg) resulted in increased motor activity and increases in all three analytes lasting several hours: O2 and glucose were uncoupled with O2 increasing rapidly following injection reaching a maximum of 70% (ca. 62 μM) after ca. 15 min and then slowly returning to baseline over a period of ca. 3 h. The time course of changes in glucose and NO were similar; both signals increased gradually over the first hour post injection reaching maxima of 55% (ca. 982 μM) and 8% (ca. 31 nM), respectively, and remaining elevated to within 1 h of returning to baseline levels (after ca. 5 and 7 h, respectively). While supporting increased utilization of glucose and O2 and suggesting overcompensating supply mechanisms, this neurochemical data indicates a hyperfrontal effect following acute PCP administration which is potentially mediated by NO. It also confirms that long-term in vivo electrochemical sensors and data offer a real-time biochemical perspective of the underlying mechanisms

Calibration of NO sensors for In Vivo Voltammetry: Laboratory synthesis of NO and the use of UV-visible spectroscopy in determining stock concentrations

The increasing scientific interest in nitric oxide (NO) necessitates the development of novel and simple methods of synthesising NO on a laboratory scale. In this study we have refined and developed a method of NO synthesis, using the neutral Griess reagent, which is inexpensive, simple to perform, and provides a reliable method of generating NO gas for in-vivo sensor calibration. The concentration of the generated NO stock solution was determined using UV–visible spectroscopy to be 0.28±0.01 mmol L−1. The level of NO2− contaminant, also determined using spectroscopy, was found to be 0.67±0.21 mmol L−1. However, this is not sufficient to cause any considerable increase in oxidation current when the NO stock solution is used for electrochemical sensor calibration over physiologically relevant concentrations; the NO sensitivity of bare Pt-disk electrodes operating at +900 mV (vs. SCE) was 1.08 nA μmol−1 L, while that for NO2− was 5.9×10−3 nA μmol−1 L. The stability of the NO stock solution was also monitored for up to 2 h after synthesis and 30 min was found to be the time limit within which calibrations should be performed