Design optimisation and characterisation of an amperometric glutamate oxidase-based composite biosensor for neurotransmitter l-glutamic acid.

A polymer/enzyme composite biosensor for monitoring neurochemical glutamate was performance optimised in vitro for sensitivity, selectivity and stability. This first generation Pt/glutamate oxidase-based sensor displayed appropriate sensitivity (90.4 ± 2.0 nA cm-2 µM-1). It also has ideal stability/biocompatibility with no significant decrease in response observed for repeated calibrations, exposure to electron beam sterilisation, or following storage at 4 °C either dry (28 days) or in ex-vivo rodent brain tissue (14 days). Potential non-glutamate contributing signals, generated by extracellular levels of the principal endogenous electroactive interferents, were typically <5% of the basal (10 µM) glutamate response. Changes in molecular oxygen (the natural enzyme mediator) over the normal brain tissue range of 40-80 µM had minimal effect on the glutamate signal for concentrations of 10 and 100 µM (Mean KMO2 = 1.86 ± 0.74 µM, [O2]90% = ca. 15 µM). Additionally, a low µM calculated limit of detection (0.44 ± 0.05) and rapid response time (ca. 1.67 ± 0.06 s), combined with no effect of pH and temperature changes over physiologically relevant ranges (7.2-7.6 and 34-40 °C respectively), collectively suggest that this composite biosensor should reliably detect l-glutamate when used for neurochemical monitoring. Preliminary experiments involving implantation in the striatum of freely moving rats demonstrated stable recording over several weeks, and reliable detection of physiological changes in glutamate in response to behavioural/neuronal activation (locomotor activity and restraint stress).

The impact of COVID-19 on obesity services across Europe: A physician survey.

Obesity is a risk factor for severe complications from coronavirus disease 2019 (COVID-19). During the COVID-19 pandemic in Spring 2020, many clinics and obesity centers across Europe were required to close. This study aimed to determine the impact of COVID-19 on the provision of obesity services across 10 European countries via a survey of physicians (n = 102) specializing in treating persons with obesity (PwO). In total, 62-95 out of 102 physicians reported that COVID-19 affected obesity-related services, with cancellations/suspensions ranging from 50% to 100% across the 10 countries. Approximately 75% of cancellations/suspensions were provider- rather than patient-initiated. A median increase of 20%-25% in waiting times was reported for most services across the countries. When services resume, 87 out of 100 physicians consider factors influencing down-stream patient outcomes as the most relevant factors for prioritizing interventional treatment. Responses showed that 65 out of 102 and 36 out of 102 physicians believed it (highly) likely that a change in treatment guidance will occur to prioritize earlier interventional treatment for the management of PwO, by either using bariatric surgery or pharmacotherapy, respectively. Results from this study provide important learnings, such as opportunities for, and discrepancies in, the provision of alternative care in light of services cancellations or delays, which may be important for the future management of obesity, especially during future waves of COVID-19 or other infectious pandemics.

Development and validation of a real-time microelectrochemical sensor for clinical monitoring of tissue oxygenation/perfusion.

Oxygen is of critical importance to tissue viability and there is increasing demand for its reliable real-time clinical monitoring in order to prevent, diagnose, and treat several pathological disorders, including hypoxia, stroke and reperfusion injury. Herein we report the development and characterisation of a prototype clinical O2 sensor, and its validation in vivo, including proof-of-concept monitoring in patients undergoing surgery for carpal tunnel release. An integrated platinum-based microelectrochemical device was custom designed and controlled using a miniaturised telemetry-operated single channel clinical potentiostat. The in vitro performance of different sensor configurations is presented, with the best sensor design (S2) displaying appropriate linearity (R2 = 0.994) and sensitivity (0.569 ± 0.022 nA µM-1). Pre-clinical validation of S2 was performed in the hind limb muscle of anaesthetised rats; tourniquet application resulted in a significant rapid decrease in signal (90 ± 27%, [ΔO2] ca. 140 ± 18 µM), with a return to baseline within a period of ca. 3 min following tourniquet release. Similar trends were observed in the clinical study; an immediate decrease in signal (39 ± 3%, [ΔO2] ca. 30 ± 20 µM), with basal levels re-established within 2 min of tourniquet release. These results confirm that continuous real-time monitoring of dynamic changes in tissue O2 can serve as an indicator of reperfusion status in patients undergoing carpal tunnel surgery, and suggests the potential usefulness of the developed microelectrochemical sensor for other medical conditions where clinical monitoring of O2 and perfusion is important.

A platinum oxide-based microvoltammetric pH electrode suitable for physiological investigations.

Attempts to develop miniaturised pH electrodes for in vivo monitoring have received much attention in recent years. Continuous real-time pH measurements may be predictive of potentially dangerous deviations in metabolic events that could improve patient prognosis. Herein, we report the in vitro investigation of a physiologically relevant, Pt oxide-based microvoltammetric pH electrode. Cycling through the potential window range -0.65 V to +0.8 V vs. SCE, gave rise to well-established monolayer oxide (MO) and hydrogen (H2) adsorption redox peaks in aqueous solution. The H2 desorption and MO reduction peaks demonstrated pH dependent, linear responses (49 ± 11 mV pH-1 and 76 ± 4 mV pH-1 respectively), following pre-activation of the electrode surface in HCl. Since in vivo monitoring is at the core of this design, the effect of incorporating a miniaturised pseudo reference electrode (PRE) was determined. The Ag/AgCl PRE demonstrated near Nernstian behaviour for the MO reduction peak (58 ± 5 mV pH-1) and sub-Nernstian behaviour (43 ± 6 mV pH-1) for its H2 desorption counterpart. Finally, a preliminary in vivo recording performed in the striatum of a freely moving mouse confirmed that the MO reduction peak was maintained under physiological conditions. These findings support the ability of the Pt oxide-based pH electrode to perform continuous, stable recordings in vivo and warrants further characterisation.

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 -0.80V. A non-significant (p=0.11) decrease in sensitivity was recorded when compared against an established Pt-based O2 sensor operating at -0.65V. Furthermore, a biocompatible clinical sensor (ClinOX) was designed, demonstrating excellent linearity (R2=0.99) and sensitivity (1.41±0.02nAµM-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<0.001) and ClinOX (p<0.01) and for ClinOX (p<0.001) in porcine limbs. Ex vivo sensocompatibility investigations identified no significant difference (p=0.08) in sensitivity values over 14days of exposure to tissue homogenate. The Pt-Pt based O2 design demonstrated high sensitivity for tissue ischaemia detection and thus warrants future clinical investigation.

A microelectrochemical biosensor for real-time in vivo monitoring of brain extracellular choline.

A first generation Pt-based polymer enzyme composite biosensor developed for real-time neurochemical monitoring was characterised in vivo for sensitive and selective detection of choline. Confirmation that the sensor responds to changes in extracellular choline was achieved using local perfusion of choline which resulted in an increase in current, and the acetylcholinesterase inhibitor neostigmine which produced a decrease. Interference by electroactive species was tested using systemic administration of sodium ascorbate which produced a rapid increase in extracellular levels before gradually returning towards baseline over several hours. There was no overall change in the response of the biosensor during the same period of monitoring. Oxygen interference was examined using pharmacological agents known to change tissue oxygenation. Chloral hydrate produced an immediate increase in O2 before gradually returning to baseline levels over 3 h. The biosensor signal displayed an initial brief decrease before increasing to a maximum after 1 h and returning to baseline within 2 h. L-NAME caused a decrease in O2 before returning to baseline levels after ca. 1.5 h. In contrast, the biosensor current increased over the same time period before slowly returning to baseline levels over several hours. Such differences in time course and direction suggest that changes in tissue O2 levels do not affect the ability of the sensor to monitor choline reliably. Although it was found to rapidly respond to behavioural activation, examination of baseline in vivo data suggests a stable viable signal for at least 14 days after implantation. Using in vitro calibration data the basal extracellular concentration of choline was estimated to be 6.3 µM.

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.

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

In vitro characterisation results for O(2) reduction 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(-2)µM(-1) vs. -0.048 ± 0.002 µAmm(-2)µM(-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 O(2) 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 O(2) 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 O(2) over extended periods in freely-moving animals.

Characterisation of carbon paste electrodes for real-time amperometric monitoring of brain tissue oxygen.

Tissue O2 can be monitored using a variety of electrochemical techniques and electrodes. In vitro and in vivo characterisation studies for O2 reduction at carbon paste electrodes (CPEs) using constant potential amperometry (CPA) are presented. Cyclic voltammetry indicated that an applied potential of -650 mV is required for O2 reduction at CPEs. High sensitivity (-1.49 ± 0.01 nA/µM), low detection limit (ca. 0.1 µM) and good linear response characteristics (R² > 0.99) were observed in calibration experiments performed at this potential. There was also no effect of pH, temperature, and ion changes, and no dependence upon flow/fluid convection (stirring). Several compounds (e.g. dopamine and its metabolites) present in brain extracellular fluid were tested at physiological concentrations and shown not to interfere with the CPA O2 signal. In vivo experiments confirmed a sub-second response time observed in vitro and demonstrated long-term stability extending over twelve weeks, with minimal O2 consumption (ca. 1 nmol/h). These results indicate that CPEs operating amperometrically at a constant potential of -650 mV (vs. SCE) can be used reliably to continuously monitor brain extracellular tissue O2.

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 50 microM 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.

Calibration of NO sensors for in-vivo voltammetry: laboratory synthesis of NO and the use of UV-visible spectroscopy for 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 NO(2) (-) 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 micromol(-1) L, while that for NO(2) (-) was 5.9 x 10(-3) nA micromol(-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.