Yibai Hu

Direct measurement of glutamate release in the brain using a dual enzyme-based electrochemical sensor

The in vivo measurement of the rapid changes in the extracellular concentrations of L-glutamic acid in the mammalian brain during normal neuronal activity or following excessive release due to episodes of anoxia or ischemia has not been possible to this date. Current techniques for the measurement of the release of endogenous glutamate into the extracellular space of the central nervous system are relatively slow and do not measure the actual concentration of free glutamate in the extracellular space. An enzyme-based electrode with rapid response times (about 1 s) and high degree of sensitivity (less than 2 microM) and selectivity for L-glutamic acid is described in this paper. This electrode has both L-glutamate and ascorbate oxidase immobilized on its surface. The latter enzyme removes almost completely any interferences produced by the high levels of extracellular ascorbate present in brain tissue. The response of the electrode to glutamate and other potentially interfering substances was fully characterized in vitro and its selectivity, sensitivity and rapidity in responding to a rise in extracellular glutamate concentrations was also demonstrated in vivo. Placement of the electrode in the dentate gyrus of the hippocampus led to the detection of both KCl-induced release of L-glutamic acid and the release induced by stimulation of the axons in the perforant pathway. The development of this selective, sensitive and rapidly responding glutamate sensor should make it now possible to measure the dynamic events associated with glutamate neurotransmission in the central nervous system.

Reduction of acetaminophen interference in glucose sensors by a composite Nafion membrane: demonstration in rats and man

SummaryAmperometric glucose sensors typically monitor the production of hydrogen peroxide generated in the course of the enzymatic oxidation of glucose. At the applied potential necessary to oxidize the peroxide produced, other species are also electroactive and contribute to the signal. Interference of ascorbate or urate has been effectively eliminated, but that resulting from the widely used analgesic acetaminophen is not. The aim of this work was to reduce this interference, which was found to be possible by introducing a membrane constructed of Nafion. We compared the in vitro sensitivity to acetaminophen of five Nafion sensors with that of five non-Nafion sensors with identical glucose sensitivity (2.0±0.4 vs 1.9±0.1 nA·mmol−1· 1−1, NS): sensitivity to acetaminophen was 12.2±2.7 vs 30.8±6.3 nA·mmol−1·1−1, respectively (p<0.05). These sensors were tested in rats by implanting in each animal one Nafion and one non-Nafion sensors. The in vivo sensitivity to glucose was similar (0.33±0.09 vs 0.30±0.05 nA·mmol−1·1−1, NS). The current generated by an acetaminophen infusion (plasma acetaminophen plateau=140±10 μmol/l) was much decreased in the case of the Nafion sensor: 0.5±0.3 vs 2.0±0.7 nA, p<0.05). Five Nafion sensors were implanted in the subcutaneous tissue of normal human volunteers who were given on oral dose of 500 mg acetaminophen. No change in the sensor current was observed, although plasma acetaminophen reached a peak (35±6 μmol/l) at 60 min and decreased by 50% at 180 min. In contrast, the current increased from 2.1±0.7 to 3.9±1.2 nA (p<0.05) at 60 min of a subsequent oral glucose tolerance test when plasma glucose concentration increased from 4.8±0.3 to 8.0±0.9 mmol/l. This paper provides the evidence that the presence of a Nafion membrane is a solution to acetaminophen interference in glucose sensing.

Glucose microbiosensor based on alumina sol–gel matrix/electropolymerized composite membrane

A procedure is described that provides co-immobilization of enzyme and bovine serum albumin (BSA) within an alumina sol-gel matrix and a polyphenol layer permselective for endogenous electroactive species. BSA has first been employed for the immobilization of glucose oxidase (GOx) on a Pt electrode in a sol-gel to produce a uniform, thin and compact film with enhanced enzyme activity. Electropolymerization of phenol was then employed to form an anti-interference and protective polyphenol film within the enzyme layer. In addition, a stability-reinforcing membrane derived from (3-aminopropyl)-trimethoxysilane was constructed by electrochemically-assisted crosslinking. This hybrid film outside the enzyme layer contributed both to the improved stability and to permselectivity. The resulting glucose sensor was characterized by a short response time (<10 s), high sensitivity (10.4 nA/mM mm(2)), low interference from endogenous electroactive species, and a working lifetime of at least 60 days.

A user-friendly method for calibrating a subcutaneous glucose sensor-based hypoglycaemic alarm

A crucial step in developing a glucose monitoring system using a subcutaneous implanted glucose sensor is the transformation of the sensor signal (a current) into an estimation of a blood glucose concentration. We have developed an Electronic Control Unit (ECU) able to recognize, before and after a glucose load, that the sensor current presents a plateau, thus triggering an alarm asking for blood glucose determination. The system, fed with these results, subsequently transforms the current into an estimation of glucose concentration by linear extrapolation based on the sensor sensitivity and the background current computed from the two sets of current and glycaemia values (two-point calibration). In addition, the system is able to trigger an alarm when this estimation decreases below a threshold that can be set by the user. This system was evaluated in experiments performed in 12 normal rats. The quality of the calibration was assessed by comparing, by error grid analysis, the data displayed on the liquid-crystal display of the ECU to concomitant plasma glucose concentration determined at frequent intervals, 65 +/- 6 and 26 +/- 5% of the values were in zones A (good) and B (acceptable estimation) of the grid, respectively. The system was set to trigger an alarm when the estimation of glucose concentration decreased below 70 mg/dl. Following an insulin administration, the alarm was triggered when the system displayed a 64 +/- 2 mg/dl glucose concentration. The concomitant plasma glucose concentration was 59 +/- 5 mg/dl (NS). In conclusion, this work validates experimentally the new, user-friendly method for calibrating the glucose sensor integrated into the ECU, based on an automatic detection of plateaus. The quality of the sensor calibration performed with this procedure is compatible with the appropriate functioning of this continuous glucose monitoring system, which was demonstrated by its ability to detect mild hypoglycaemia following insulin injection.

A needle-type enzyme-based lactate sensor for in vivo monitoring

Abstract A miniature needle-type lactate sensor prepared by coupling the enzyme lactate oxidase (LOD) with an hydrogen peroxide probe has been developed which makes possible real-time in vivo lactate measurements. The sensor has a size of 0.35mm o.d. with a cylindrical sensing cavity in which a multilayer detection element was placed. The linearity of sensor response was extended beyond the clinically relevant concentration range and the response time was about 30 seconds. Measurement of incubated plasma samples with lactate oxidase and catalase showed that the overall endogenous interferences caused a negligible error. In vitro characterization including effects of temperature, pH and P O 2 and stability was carried out as well. In vivo tests in rat subcutaneous tissue showed that the sensors functioned reliably. Good correlation was observed between the sensor output and plasma lactate measured simultaneously in situ with a flow injection system. Potential oxygen effects were also measured in vivo and the results showed that the sensor can tolerate reasonably low tissue oxygen levels even for hypoxic conditions in which P O 2 was as low as 10 mmHg.

Continuous glucose monitoring in the free-moving rat☆

The aim of this work was to set up an experimental model of glycemic fluctuations for assessing in the conscious freely moving rat, the performance of a continuous glucose-monitoring system, using a pocket-calculator-size electronic control unit and a miniaturized subcutaneous glucose sensor. The well-known triphasic glycemic pattern following streptozotocin injection (initial peak and secondary hypoglycemia preceding the establishment of permanent hyperglycemia) was used as a way to obtain spontaneous changes in blood glucose level over a wide concentration range. This report demonstrates that streptozotocin injection produced highly reproducible changes in the current generated by the sensor: an initial peak and a secondary nadir, during which blood sampling provided the evidence of hyperglycemia associated with immunoreactive hypoinsulinemia, and of hypoglycemia associated with hyperinsulinemia, respectively. This reproducible experimental model should be valuable for the assessment of a continuous glucose-monitoring system.