Yanan Zhang

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.

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.

Electrochemical oxidation of H2O2 on Pt and Pt + Ir electrodes in physiological buffer and its applicability to H2O2-based biosensors

Abstract The electrochemical oxidation of hydrogen peroxide on Pt disk and Pt + Ir wire electrodes was studied in physiological buffer at pH 7.40 in order to establish a practical protocol for H 2 O 2 -based biosensors. The electrode reaction is a two-electron irreversible process. The oxidation of H 2 O 2 on Pt and Pt+Ir electrode surfaces is a catalytic reaction which is H 2 O 2 concentration dependent but O 2 pressure independent at low H 2 O 2 concentration. Amperometric detection of H 2 O 2 is strongly affected by pH, temperature and electrode surface conditioning. An oxidized surface is required to obtain a stable H 2 O 2 response, and reductive pretreatment of the electrode always yields prolonged stabilization times. The application of a cellulose acetate membrane to the surface can protect the oxidized surface and therefore substantially reduce the stabilization time for H 2 O 2 measurements.

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.

In vitro and in vivo evaluation of oxygen effects on a glucose oxidase based implantable glucose sensor

Abstract The oxygen effect on a glucose oxidase based implantable glucose sensor was investigated based on studies of tissue oxygen availability and demand for oxygen by the glucose sensor. In vitro measurements showed that the oxygen effect is directly related to the glucose sensor sensitivity and linearity. Results of in vivo measurements in rat subcutaneous tissue with simultaneously implanted oxygen sensor and glucose sensor also suggested similar influence. A practically useful glucose sensor that is essentially free of oxygen fluctuation can be constructed by controlling its sensitivity and linearity.

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.

Application of cell culture toxicity tests to the development of implantable biosensors.

Cell culture toxicity testing methods were modified and applied to the development of implantable glucose microsensors, and positive and negative control materials suitable for the microsensor assessment were established. The location, source and degree of the toxic effect in a multi-component biosensor was spatially visualized with cell monolayers. A freshly prepared sensor showed moderate toxicity, mainly as a result of the presence of glutaraldehyde and the residual solvents in the polymer layers. However, it was possible to reduce the toxicity by removing the leachable toxic substances through extraction in phosphate buffer, and a non-toxic sensor was readily obtained.

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.