G. Reach

Implantable glucose sensor

Two new approaches for construction and testing of a miniature in vivo implantable glucose sensor are described. The first approach is to immobilize glucose oxidase onto a platinum wire, coated with special glass (Corning), and to cover the immobilized enzyme with a thin layer of polymer to prevent large molecules from poisoning the electrode. The second, and more successful, approach is to use the principle of coated wire electrodes by incorporating a quaternary ammonium salt with an insoluble glucose salt and poly(vinyl chloride). Potentiometric measurements were used to test the two electrodes, as well as records of voltage and current responses. Responses of the electrodes to varying glucose concentrations are described. Current work is concerned with the responses of these electrodes to plasma and blood, and in examining interference effects from blood chemicals and electrolytes. Alternative methods for preparation of the coated wire electrodes are under investigation, as is their mechanism of action.

A glucose monitoring system for on line estimation in man of blood glucose concentration using a miniaturized glucose sensor implanted in the subcutaneous tissue and a wearable control unit

SummaryWe have developed a miniaturized glucose sensor which has been shown previously to function adequately when implanted in the subcutaneous tissue of rats and dogs. Following a glucose load, the sensor output increases, making it possible to calculate a sensitivity coefficient to glucose in vivo, and an extrapolated background current in the absence of glucose. These parameters are used for estimating at any time the apparent subcutaneous glucose concentration from the current. In the previous studies, this calibration was performed a posteriori, on the basis of the retrospective analysis of the changes in blood glucose and in the current generated by the sensor. However, for clinical application of the system, an on line estimation of glucose concentration would be necessary. Thus, this study was undertaken in order to assess the possibility of calibrating the sensor in real time, using a novel calibration procedure and a monitoring unit which was specifically designed for this purpose. This electronic device is able to measure, to filter and to store the current. During an oral glucose challenge, when a stable current is reached, it is possible to feed the unit with two different values of blood glucose and their corresponding times. The unit calculates the in vivo parameters, transforms every single value of current into an estimation of the glucose concentration, and then displays this estimation. In this study, 11 sensors were investigated of which two did not respond to glucose. In the other nine trials, the volunteers were asked to record every 30 s what appeared on the display during the secondary decrease in blood glucose. The results were analysed by comparing these readings (approximately 220 measurements per trial) to the changes in plasma glucose, measured every 15 min. The Error Grid Analysis indicated that 84.1±3.6% of the measurements were in zone A (accurate) and 15±3.6% were in zone B (acceptable). Considering each individual trial, the differences between the displayed value and the concomitant plasma glucose concentration ranged between −1.7 and 0.69 mmol/l. These excellent results were due to the absence of any significant lag between the changes in plasma glucose concentration and the changes in the result on the display. We conclude that this glucose monitoring system, based on subcutaneous sensing of glucose, is able to provide a direct on line estimation of blood glucose concentration.

Study and development of multilayer needle-type enzyme-based glucose microsensors

Glucose oxidase (GOD) was covalently coupled to a cellulose acetate (CA) layer, using bovine serum albumin (BSA) and parabenzoquinone (PBQ) linkages. Prior to GOD coupling this CA layer was deposited on the platinum tip of a needle-type sensor and covered with an outer layer of polyurethane (PU). Such microsensors were found to be active, their GOD load reaching 1.6 to 3.0 micrograms mm-2 and their glucose response reaching 1 to 3 microA M-1 mm-2, even when the upper limit of their linear range reached 10-30 mM. Due to the multilayer structure and composition of these microsensors, small anions such as ascorbate were partially discriminated from neutral molecules such as hydrogen peroxide. When implanted subcutaneously in anaesthetized rats, sensor responses correlated correctly with blood glucose concentration but presented sensitivity coefficients significantly different to those determined in vitro: a 2 point calibration procedure was found necessary for in vivo experiments.

Towards continuous glucose monitoring: in vivo evaluation of a miniaturized glucose sensor implanted for several days in rat subcutaneous tissue

SummaryA miniaturized amperometric, enzymatic, glucose sensor (outer diameter 0.45 mm) was evaluated after implantation in the subcutaneous tissue of normal rats. A simple experimental procedure was designed for the long-term assessment of the sensors function which was performed by recording the current during an intraperitoneal glucose load. The sensor was calibrated by accounting for the increase in the current during the concomitant increase in plasma glucose concentration, determined in blood sampled at the tail vein. This made it possible to estimate the glucose concentration in subcutaneous tissue. During the glucose load, the change in subcutaneous glucose concentration followed that in blood with a lag time consistently shorter than 5 min. The estimations of subcutaneous glucose concentration during these tests were compared to the concomitant plasma glucose concentrations by using a grid analysis. Three days after implantation (n=6 experiments), 79 estimations were considered accurate, except for five which were in the acceptable zone. Ten days after implantation (n=5 experiments), 101 estimations were accurate, except for one value, which was still acceptable. The sensitivity was around 0.5 nA mmol−1·l−1 on day 3 and day 10. A longitudinal study on seven sensors tested on different days demonstrated a relative stability of the sensors sensitivity. Finally, histological examination of the zone around the implantation site revealed a fibrotic reaction containing neocapillaries, which could explain the fast response of the sensor to glucose observed in vivo, even on day 10. We conclude that this miniaturized glucose sensor, whose size makes it easily implanted, works for at least ten days after implantation into rat subcutaneous tissue.

A method for evaluating in vivo the functional characteristics of glucose sensors

Abstract A simple system for evaluating ex vivo the functional characteristics of glucose sensors was set up. Normal rats implanted with carotid and jugular catheters were connected under free-moving conditions to an extracorporeal circuit. Blood was allowed to circulate in contact with an enzyme glucose electrode. Glucose or insulin was infused intravenously at different rates to produce glycaemic alterations appropriate for sensor checking. Comparison of the changes in signal output with the corresponding variations in plasma glucose enabled in vivo evaluation of the performances of the sensor, i.e. of the linearity and of the speed of its response to glucose. This method, suitable for small laboratory animals, could therefore be used for the preliminary evaluation of glucose sensors, under in vivo conditions.

Evaluating in vitro and in vivo the interference of ascorbate and acetaminophen on glucose detection by a needle-type glucose sensor

The aim of this work was to assess, in vitro and in vivo, the interference of ascorbate and acetaminophen on glucose measurements by a needle-type glucose sensor detecting hydrogen peroxide generated during the enzymatic oxidation of glucose, and to ascertain whether the protection against interference by the membranes used in the construction of the electrode is feasible. The oxidation of ascorbate and acetaminophen on a platinum electrode set at a 650 mV potential yielded a current representing 75 +/- 5% and 25 +/- 6% of that generated by the oxidation of an equimolar concentration of hydrogen peroxide, respectively. The bias introduced by the presence of 100 mumol l-1 ascorbate on the reading of 5 mmol l-1 glucose by the complete sensor (electrode + membranes) would be minimal (approximately 0.4 mmol l-1). By contrast, the bias introduced by 200 mumol l-1 of acetaminophen (a plasma concentration easily reached in clinical practice) was about 7 mmol l-1. The sensor was implanted subcutaneously in anaesthetized rats (n = 3). Using the current generated in the presence of a plasma acetaminophen concentration of about 200 mumol l-1 for glucose monitoring would lead to a major underestimation (approx. 6 mmol l-1) of subcutaneous glucose concentrations.

In vitro kinetics of insulin release by microencapsulated rat islets: effect of the size of the microcapsules

SummaryMicroencapsulation has been proposed to protect islets of Langerhans against immune rejection in xenogenic transplantation. However, to achieve glucose homeostasis in human diabetic patients, insulin release by microencapsulated islets must increase in response to a glucose load. We microencapsulated isolated rat islets using the alginate-polylysine procedure. Capsule size was found to range from 300 to 800 μm, and microencapsulated islets were separated according to their size. Groups of 10 microencapsulated islets, either small (350 μm) or large (650 μm) were placed in plastic microwells, in minimal Eagles culture medium containing either 5.5 mol/l glucose (basal) or 16.5 mol/l glucose and 5.5 mol/l theophylline (stimulatory medium). The increase in insulin concentration in the surrounding medium was then serially determined over 30 min: (1) With the small capsules, insulin concentration rose from 199 ±20 to 297 ±58 μU/ml in basal medium, and from 236 ±23 to 510 ±121 μU/ml in stimulatory medium (n = 10 preparations), the difference between the data obtained with the basal or the stimulatory medium being significant (p<0.01) from the 5th min onwards. (2) With large capsules, insulin concentration increased from 182±9 to 266±44 μU/ml, and from 216 ±19 to 297 ±34 μU/ml in basal and stimulatory medium, respectively, with no apparent significant difference. The magnitude of insulin secretion in response to glucose by unencapsulated islets was, under similar conditions, seven-fold greater. We conclude therefore that the size of the microcapsules is an essential parameter which has to be considered for the optimisation of the microencapsulation procedure.

Diabetes mellitus following pentamidine-induced hypoglycemia in humans.

Four patients, treated with pentamidine because of Pneumocystis carinii pneumonitis, displayed severe fasting hypoglycemia during this treatment. Diabetes mellitus appeared later, requiring insulin therapy in the three of them who survived more than a few weeks. The metabolic study, performed in two cases during the hypoglycemic period, demonstrated inappropriately high insulin levels in the postaborptive state. 28 ± 1 μU/ml (blood glucose 41 ± 4 mg/dl) and 86 ± 5 μU/ml (blood glucose 15 ± 5 mg/dl) vs. 15 ± 3 μU/ml in 10 control subjects and 55 ± 3 μU/ml in 6 patients with a verified B-cell tumor, respectively. Poor B-cell secretory responses followed the stimulations by oral glucose (maximal increment over basal: +5 μU/ml vs. +40 μU/ml in control group and +77 μU/ml in the insulinoma group), by i.v. arginine (maximal increment +10 and +28 μU/ml, respectively, vs. +55 in the controls and +90 μU/ml in the insulinoma group) and by i.v. glucagon (+10 and +23 μU/ml, respectively) vs. +40 μU/ml in both the control and the insulinoma groups). Plasma cortisol and glucagon, and the A-cell response to arginine were higher than normal. These high, nonsuppressible, nonstimulable insulin levels and the sequence of hypoglycemia followed by insulin-dependent diabetes mellitus is consistent with the hypothesis of a selective toxicity turned towards the B-cells. In vitro incubation of islets with pentamidine 10−10 M produced a passive release of insulin, followed by a significant decrease in B-cell response to glucose + theophylline. It is suggested that pentamidine can induce hypoglycemia because of an early cytolytic release of insulin, and then diabetes mellitus because of B-cell destruction and insulin deficiency.

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.

Calibration of a subcutaneous amperometric glucose sensor implanted for 7 days in diabetic patients: Part 2. Superiority of the one-point calibration method

UNLABELLED Calibration, i.e. the transformation in real time of the signal I(t) generated by the glucose sensor at time t into an estimation of glucose concentration G(t), represents a key issue for the development of a continuous glucose monitoring system. OBJECTIVE To compare two calibration procedures. In the one-point calibration, which assumes that I(o) is negligible, S is simply determined as the ratio I/G, and G(t) = I(t)/S. The two-point calibration consists in the determination of a sensor sensitivity S and of a background current I(o) by plotting two values of the sensor signal versus the concomitant blood glucose concentrations. The subsequent estimation of G(t) is given by G(t) = (I(t)-I(o))/S. RESEARCH DESIGN AND METHODS A glucose sensor was implanted in the abdominal subcutaneous tissue of nine type 1 diabetic patients during 3 (n = 2) and 7 days (n = 7). The one-point calibration was performed a posteriori either once per day before breakfast, or twice per day before breakfast and dinner, or three times per day before each meal. The two-point calibration was performed each morning during breakfast. RESULTS The percentages of points present in zones A and B of the Clarke Error Grid were significantly higher when the system was calibrated using the one-point calibration. Use of two one-point calibrations per day before meals was virtually as accurate as three one-point calibrations. CONCLUSION This study demonstrates the feasibility of a simple method for calibrating a continuous glucose monitoring system.