Multifunctional Biocompatible Membrane and Its Application to Fabricate A Miniaturized Glucose Sensor with Potential for Use In Vivo

A multifunctional membrane with biocompatibility, diffusion-limiting effect, and the ability to curtail the responses of an H₂O₂ electrode to ascorbate and urate was prepared. It was composed of MB, AB, and CTA, where MB is the copolymer of 2-methacryloyloxyethyl phosphorylcholine (MPC) and n-butylmethacrylate (BMA), AB is the copolymer of acrylamide-2-methylpropane sulfonic acid (AMPS) and BMA, CTA is cellulose triacetate. Investigation of the biocompatibility of this membrane showed that, compared with CTA, relatively few platelets bound to it. The membrane was coated onto the working electrode of a needle-type glucose sensor on which immobilized glucose oxidase membrane has been coated. The sensor did not respond to ascorbate and urate at their concentration normally encountered in blood. Its response was not inhibited by metal ions in blood at usual concentration. The sensor exhibited superior thermostability in addition to a rapid response (<90 seconds in batch operation), good reproducibility (RE<5%), good stability (more than 36 hours continuously in heparinized whole blood), and a wide dynamic range (5–650 mg/dl glucose). The sensor was used to determine glucose in serum. The data obtained from the sensor showed good agreement with that from a clinical autoanalyzer (R=0.973). This revised version was published online in July 2006 with corrections to the Cover Date.     

Glucose biosensors based on oxygen electrode with sandwich-type membranes

A glucose biosensor based on an amperometric oxygen electrode has been developed. Polycarbonate and Silastic membranes were assembled (glued together) to form a multilayer sandwich glucose diffusion barrier. The effects of the glue layer composition and thickness of the Silastic membrane on sensor response parameters have been investigated in order to optimize the sensor. The parameters measured were the sensitivity, the concentration range of the linear dependence of the sensor response to glucose, and the long-term operation time. The sensors with the sandwich-type glucose diffusion membrane (Silastic membrane prepared from 20% Silastic suspension, glue layer prepared from polyurethane, 0.5 w/v % in THF solution and standard polycarbonate membrane) demonstrated linearity of response up to 520 mg/dl glucose at 25°C and up to 400 mg/dl at 37°C. These sensor showed good reproducibility of response without significant interference effects (from 1 to 5% of the background current value). The long-term continuous operational time of the sensors was over 40 days at 37°C, and over 60 days at 25°C.     

Polymeric membranes for silicon based (bio)sensors.

During the last decade, chemical and biochemical sensor research has benefited from the availability of new technologies and materials. New embodiments of classical devices have resulted from the use of e.g., solid state technology for the realization of the transducers. In this paper we describe several examples of membrane deposition techniques used in connection with planar, silicon based electrochemical transducers. Casting and electrochemical deposition of glucose oxidase containing membranes are described for the fabrication of glucose enzyme electrodes. Photolithographic patterning of polyacrylamide hydrogel and of siloxane based gas permeable membrane is used for the realization of an amperometric oxygen sensor and an ISFET-based pCO2 device. The last example is that of a free-chlorine sensor for which the photolithographic patterning of the polyHEMA hydrogel layer is described.     

Use of charged membranes to control interference by body chemicals in a glucose biosensor

The prerequisite for the continuous in vivo monitoring of glucose concentration is the development of an implantable glucose sensor with long-term stability. A new enzyme electrode concept featuring fluid-state glucose oxidase modified carbon powder along with a cross-linked glucose oxidase enzyme layer has been developed. The glucose sensor incorporating this enzyme electrode has been tested in vitro at 37°C. It has a lifetime of three months after which it can be recharged with fresh enzyme. The next step in the characterization of this sensor is its in vitro behaviour in the presence of interfering substances commonly encountered in human blood. Here we report such a study of the sensor. The glucose diffusion membranes used were polycarbonate membranes. We used standard polycarbonate membranes (membranes treated with polyvinylpyrrolidone or PVP), PVP-free polycarbonate membranes, and standard polycarbonate membranes coated with positively and negatively charged hydrogel layers. The sensors showed a response to glucose concentrations <300 mg dL⁻¹, both in pure phosphate buffer and in the presence of interferences. The influence of ascorbic acid, bilirubin, creatinine, L-cystine, glycine, uric acid and urea on the amperometric signal of the sensor was investigated. The polycarbonate membrane coated with the negatively charged hydrogel layer provided good protection for the enzyme electrode, especially in the presence of ascorbic acid and uric acid.     

Enhancement of implantable glucose sensor function in vivo using gene transfer-induced neovascularization

The in vivo failure of implantable glucose sensors is thought to be largely the result of inflammation and fibrosis-induced vessel regression at sites of sensor implantation. To determine whether increased vessel density at sites of sensor implantation would enhance sensor function, cells genetically engineered to over-express the angiogenic factor (AF) vascular endothelial cell growth factor (VEGF) were incorporated into an ex ova chicken embryo chorioallantoic membrane (CAM)-glucose sensor model. The VEGF-producing cells were delivered to sites of glucose sensor implantation on the CAM using a tissue-interactive fibrin bio-hydrogel as a cell support and activation matrix. This VEGF-cell-fibrin system induced significant neovascularization surrounding the implanted sensor, and significantly enhanced the glucose sensor function in vivo. This model system, for the first time, provides the "proof of principle" that increasing vessel density at the sites of implantation can enhance glucose sensor function in vivo, and demonstrates the potential of gene transfer and tissue interactive fibrin bio-hydrogels in the development of successful implants.     

Mediation of in vivo glucose sensor inflammatory response via nitric oxide release

In vivo glucose sensor nitric oxide (NO) release is a means of mediating the inflammatory response that may cause sensor/tissue interactions and degraded sensor performance. The NO release (NOr) sensors were prepared by doping the outer polymeric membrane coating of previously reported needle-type electrochemical sensors with suitable lipophilic diazeniumdiolate species. The Clarke error grid correlation of sensor glycemia estimates versus blood glucose measured in Sprague-Dawley rats yielded 99.7% of the points for NOr sensors and 96.3% of points for the control within zones A and B (clinically acceptable) on Day 1, with a similar correlation for Day 3. Histological examination of the implant site demonstrated that the inflammatory response was significantly decreased for 100% of the NOr sensors at 24 h. The NOr sensors also showed a reduced run-in time of minutes versus hours for control sensors. NO evolution does increase protein nitration in tissue surrounding the sensor, which may be linked to the suppression of inflammation. This study further emphasizes the importance of NO as an electroactive species that can potentially interfere with glucose (peroxide) detection. The NOr sensor offers a viable option for in vivo glucose sensor development.     

Development of a transcutaneous blood-constituent monitoring method using a suction effusion fluid collection technique and an ion-sensitive field-effect transistor glucose sensor

The paper describes a method for the transcutaneous monitoring of blood constituents. It combines the use of a suction effusion fluid (SEF) collecting technique with a silicon on sapphire/ion-sensitive field-effect transistor (SOS/ISFET) biosensor. SEF is directly collected by a weak evacuation through skin from which the stratum corneum has been removed. An SEF collecting cell with a stainless-steel mesh at the bottom is kept in a weak vacuum condition, and SEF is sucked up through the mesh and deposited in a reservoir above. An ISFET glucose sensor is able to detect glucose concentrations in very small SEF samples through the use of two small ISFETs and an immobilised enzyme membrane. The reliability of transcutaneously obtained SEF was first confirmed in an experiment using rabbits. A clinical analyser was used to determine levels of glucose, urea nitrogen and creatinine in SEF obtained transcutaneously; these results are compared with results obtained by the same analyser directly from sera. The ISFET glucose sensor was successfully tested on human subjects for the monitoring of blood glucose levels. During these tests, glucose level changes in the SEF followed acutal blood glucose level changes with a slight time delay. Results suggest the feasibility of non-invasive, transcutaneous monitoring of low molecular weight substances in the blood without the use of ordinary blood sampling.     

A flexible and wearable biosensor for tear glucose measurement

A flexible and wearable amperometoric glucose sensor was fabricated and tested. Also, the sensor was utilized to tear glucose monitoring. The sensor was constructed by immobilizing GOD onto a flexible oxygen electrode (Pt working electrode and Ag/AgCl counter/reference electrode), which was fabricated using “Soft-MEMS” techniques onto a functional polymer membrane. In purpose of bioinstrumentation, adhesive agents were not used for constructing the flexible biosensor. Linear relationship between glucose concentration and output current was obtained in a range of 0.025–1.475 mmol/l, with a correlation coefficient of 0.998. Current dependences on pH and temperature were also evaluated. The current was largest at pH 7.0 and the current increased when temperature increased. This indicates that the output current depends on enzyme activity. Based on the basic characteristics investigation, the glucose sensor was applied to measurement of glucose in tear fluids on an eye site of a Japan white rabbit. The change of tear glucose level induced by oral-administration of glucose was monitored as a current change of the sensor attached on the eye site. In this investigation, the tear glucose level varied from 0.16 to 0.46 mmol/l. Although there was a delay of several tens of minutes towards blood sugar level, it is considered to be possible that non-invasive continuous glucose monitoring can be realized using the flexible biosensor.     

改良的YSI膜在葡萄糖传感器中的作用

为提高葡萄糖传感器的稳定性、检测精度、抗干扰能力及改善电极输出电流与葡萄糖浓度间的线性相关。在ＹＳＩ膜上固植过氧化氢酶，并在不同浓度的葡萄糖液、抗环血酸液和醋氨酚液中进行体外实验。结果显示：此传感器的背景电流为０．３７±０．０６ｎＡ；漂移值在０ｍＭＧ．Ｓ．中为（０．２０±０．１４）％／３ｈｒ，在１０ｍＭＧ．Ｓ．中为（３．２２±０．４０）％／３０ｈｒ，单位浓度单位时间的均值为０．１０３％；电极输出电流与葡萄糖浓度间的线性相关系数均值为０．９８６，线性范围为０～２０ｍＭＧ．Ｓ．；低浓度的抗坏血酸不能干扰传感器，高浓度的抗坏血酸和醋氨酚仅产生很小的干扰电流。因此，改良的ＹＳＩ酶膜使整个葡萄糖传感器系统性能、检测精度、抗干扰能力得到较明显的改善。     

A soft and flexible biosensor using a phospholipid polymer for continuous glucose monitoring

A flexible biosensor using a phospholipid polymer to immobilization of glucose oxidase (GOD) was fabricated and tested. At first, an enzyme membrane formed by immobilizing GOD onto a porous polytetrafluoroethylene (PTFE) membrane using the phospholipid polymer (2-methacryloyloxyethyl phosphorylcholine (MPC) copolymerized with 2-ethylhexylmethacrylate (EHMA) : PMEH) was evaluated. According to the result of amperometric measurement, average density of GOD to be immobilized was optimized to 38.9 units cm⁻². Temperature and pH dependences were also investigated. Then, a flexible glucose sensor was fabricated by immobilizing GOD onto a flexible hydrogen peroxide electrode using PMEH. The flexible glucose sensor showed a linear relationship between output currents and glucose concentration in 0.05–1.00 mmol L⁻¹, with a correlation coefficient of 0.999. The calibration range covered the normal tear glucose level of 0.14–0.23 mmol L⁻¹. This indicates that the flexible biosensor is considered to be useful for monitoring of glucose in tear fluids.     

Protein interactions with subcutaneously implanted biosensors

Biofouling of in vivo glucose sensors has been indicated as the primary reason for sensitivity losses observed during the first 24 h after implant [Wisniewski N, Moussy F, Reichert WM. Characterization of implantable biosensor membrane biofouling. Fresen J Anal Chem 2000; 366(6-7): 611-621]. Identification of the biomolecules that contribute to these sensitivity perturbations is the primary objective of the research presented. Active needle-type glucose sensors were implanted in Sprague-Dawley rats for 24h, and then a proteomics approach was used to identify the substances absorbed to the sensors. MALDI-TOF mass spectrometry was the primary tool utilized to identify the biomolecules in sensor leachate samples and species absorbed directly on sensor membranes excised from explanted in vivo sensors. Not surprisingly serum albumin was identified as the primary biomolecule present, however, predominantly as endogenous fragments of the protein. In addition, several other biomolecule fragments, mainly less than 15 kD, were identified. Based on these findings, it is concluded that fragments of larger biomolecules infiltrate the sensor membranes causing diminished glucose diffusivity, thus decreasing in vivo sensitivity.     

植入式葡萄糖传感器膜材料壳聚糖与Nafion的组织相容性比较

背景：壳聚糖是天然高分子多糖,可单独或者与其他材料复合制作敷料、药物、基因载体、生物涂层、组织工程支架、传感器膜材料等。 目的：了解壳聚糖作为植入式葡萄糖传感器膜材料的组织相容性,并与Nafion膜进行对比。 方法：制备壳聚糖膜并对其理化性质进行表征,比较壳聚糖膜皮下植入与肌肉植入、Nafion膜肌肉植入的生物相容性。 结果与结论：壳聚糖膜的厚度、溶胀率、表观密度等理化参数可以通过浓度、铸膜液体积来控制;壳聚糖膜能生物降解,63 d皮下植入的降解率为(17.0±9.9)%,说明壳聚糖的体内降解速度较慢。壳聚糖膜皮下植入引起的炎症反应较肌肉植入重,63 d后形成的纤维包膜比肌肉植入要厚(P < 0.05);肌肉植入Nafion与壳聚糖膜引起材料周围纤维包膜厚度差异无显著性意义(P > 0.05),两者均在15 d以后趋于稳定。证明壳聚糖膜能生物降解,与Nafion膜均有较好的组织相容性。     

Monitoring the kinetics of glucose transfer across a bioreactor membrane with a glucose sensor

Bioreactors for cell culture, in which hollow fibers are sealed into a protective jacket, cells are seeded in the fibers' outer surface and a culture medium circulates through the fibers, have been proposed as a bioartificial pancreas. We used a needle-type glucose sensor to study the kinetics of glucose transfer across the membrane of one such device. The glucose transfer was found to be dependent on the flow rate of the circulating medium, which suggests the involvement of an ultrafiltration flux across the membrane. The glucose concentration was heterogeneous within the cell compartment. This heterogeneity, and the delay in transmission of changes in glucose concentration from the circulating medium to the cell compartment, can be ascribed to the large volume of the compartment. The design of these bioreactors should therefore be modified, in order to meet the requirements of glucose transfer kinetics of a bioartificial pancreas.     

Mathematical simulation of an amperometric enzyme-substrate electrode with apO2 basic sensor

In the first part of the paper a mathematical model of the enzyme-substrate electrode with a pO₂ basic sensor was outlined. This model is used to simulate the dependencies of the measuring characteristics (calibration curve, measuring range, sensitivity, response time) on the design parameters (geometric arrangement, membrane properties, enzyme kinetic quantities) of an enzyme-based glucose sensor. The simulated and measured calibration curves are in good agreement with each other. With decreasing pO₂ a stoichiometric limitation occurs, and the linear range of measurement is reduced. If catalase is co-immobilised together with glucose oxidase the oxygen consumption is halved and the measuring range is doubled. The infuences of the diffusion coefficients and of the specific enzyme activity on sensitivity and response time are simulated. The results are in good correspondence with the theoretical statements and the experimental results. The limits of the model are determined by its convergence properties.     

Development of a highly responsive needle-type glucose sensor using polyimide for a wearable artificial endocrine pancreas

To produce a long-life, stable, miniature glucose sensor for a wearable artificial endocrine pancreas (WAEP), we developed a novel microneedle-type glucose sensor using polyimide, designated the PI sensor (outer diameter, 0.3 mm; length, 16 mm), and investigated its characteristics in vitro and in vivo. In the in vitro study, we tested the sensor in 0.9% NaCl solution with varying glucose concentrations and observed an excellent linear relationship between the sensor output and glucose concentration (range: 0–500 mg/100 ml). In in vivo experiments, the PI sensor was inserted into the abdominal subcutaneous tissue of beagle dogs (n = 5), and interstitial fluid glucose concentrations were monitored after sensor calibration. Simultaneously, blood glucose concentrations were also monitored continuously with another PI sensor placed intravenously. The correlation and time delay between subcutaneous tissue glucose (Y) and blood glucose concentrations (X: 30–350 mg/100 ml) were Y = 1.03X + 7.98 (r = 0.969) and 6.6 ± 1.2 min, respectively. We applied the new WAEP system/PI sensor and an intravenous insulin infusion algorithm developed previously for glycemic control in diabetic dogs. The use of the WAEP system resulted in excellent glycemic control after an oral glucose challenge of 1.5 g/kg (post-challenge blood glucose levels: 176 ± 18 mg/100 ml at 65 min and 93 ± 23 mg/100 ml at 240 min), without any hypoglycemia. Thus, we confirmed that our new PI sensor has excellent sensor characteristics in vitro and in vivo. The new WAEP using this sensor is potentially suitable for clinical application.     

Microwave sensor for the investigation of glucose-dependent reflection properties in aqueous samples

Abstract

The reported paper presents the development of a microwave sensor with a resonant frequency 2.4?GHz. The sensor is also demonstrated in vitro to investigate the variation in its response as a function of glucose concentration. The sensor could be used for the monitoring of blood glucose level in diabetics through non-invasive technology. The approach followed is based on the notion that, change in glucose concentration in the blood affects dielectric properties of blood which in turn produce an impact on reflection properties of the sensor. This effect on response of sensor will be ultimately used to estimate blood glucose concentration. The design specifications considered for the development of sensor are defined in the paper. The experimental setup for in vitro experiment and procedure employed for the investigation of the reflection properties of the sensor as a function of glucose concentration are also discussed in detail. The shift in resonance frequency and the change in the magnitude of the reflection coefficient of proposed sensor have been observed. The reported measurement results are the preliminary results in exploring the implementation of proposed sensor for non-invasive blood glucose monitoring.     

Electrocatalytic glucose sensor

An electrocatalytic glucose sensor for in vivo application has been developed. The sensor is a flow-through cell with three electrodes and can be integrated into a blood vessel. The principle of measurement is based on the direct electrochemical oxidation of glucose at a membrane-covered noble-metal electrode. To test the potential long-term in vivo function of the sensor, it was implanted in the carotid artery of a sheep. Thus, the sensor performance was verified over a period of 71 days. During this time, a nearly constant blood flow through the cell was achieved, which indicates good blood compatibility of the materials used. It was possible to set up a calibration that was valid over 24 days (mean error 2.3 mmol l⁻¹). The tested cross-sensitivity of the sensor towards cysteine, acetyl salicylic acid and other small molecules shows tolerable effects on this type of glucose measurement. Only high concentrations of lactate and ethanol require a special adaptation of the calibration to suppress their influence. Minor crossensitivity and promising long-term stability recommend this type of sensor for in vivo monitoring of blood sugar level. However, for intravasal application, it is necessary to modify the present sensor design to a catheter-type construction.     

Flat-Chip Microanalytical Enzyme Sensor for Salivary Amylase Activity

It is considered that measurement of salivary α-amylase activity is a useful tool for evaluating the sympathetic nervous system. The purpose of this research is to demonstrate a new design of a flat-chip microanalytical enzyme sensor (flat-chip sensor) for salivary amylase activity as a Micro-Electro-Mechanical Systems (MEMS), which may be used for wearable analytical systems. To meet this purpose, the biosensor needs to be miniaturized and to possess high-sensitivity. A pre-column and a flat-enzyme electrode were incorporated in a flow cell of volume 25.7 ml. In order to miniaturize the flow cell, two enzymatic membranes containing maltose phosphorylase obtained from Enterococcus hirae (MP membrane) and glucose oxidase and peroxidase (GOD-POD membrane) were immobilised on the same planar surface. As a result, a flat-chip sensor incorporating a flow cell as small as a C battery was produced. The optimum conditions of three parameters of the fabricated flat-chip sensor, the immobilising method of the enzymatic membrane, dropping volume of the mixed enzymatic solution and flow rate of the sample solution, were examined. An analytical system for 0–190 kU/l amylase activity with R² of 0.97 was fabricated with a sample volume of 50 μl. This research indicates the possibility of a wearable biosensor for continuous monitoring of salivary amylase activity.     

Sensors of extracellular nutrients in Saccharomyces cerevisiae

It has been known for a long time that yeast are capable of making rapid metabolic adjustments in response to changing extracellular nutrient conditions. Until recently it was thought that yeast, in contrast to mammalian cells, primarily monitored nutrient availability through the activity of intracellular sensors. Recent advances in our understanding of nutrient sensing indicate that yeast cells possess several nutrient-sensing systems localized in the plasma membrane that transduce information regarding the presence of extracellular amino acids, ammonium. and glucose. Strikingly, the transmembrane components of several of these sensors, Ssylp, Mep2p, Snf3p. and Rgt2p, are unique members of nutrient-transport protein families. Perhaps with the exception of Mep2p, the ability of these transporter homologues to transduce nutrient-(ligand)-induced signals across the plasma membrane appears to be independent of nutrient uptake; and thus these sensor components may function analogously to traditional ligand-dependent receptors. Additionally, the G protein-coupled receptor Gpr1p has been shown to exhibit properties consistent with it being a sensor. These recent advances indicate that yeast cells obtain information regarding their growth environments using sensing systems that are more similar to those present in mammalian cells than previously thought. The fact that yeast plasma membrane nutrient sensors have only recently been discovered reveals how little is understood regarding the molecular signals that enable eukaryotic cells to adapt to changing environments.     

Development of an enzyme-free glucose sensor using the gate effect of a molecularly imprinted polymer

The instability of enzymatic glucose sensors has prevented the development of a practical artificial pancreas for diabetic patients. We therefore developed an enzyme-free glucose sensor using the gate effect of a molecularly imprinted polymer (MIP). This sensor has the advantages of improved stability and a simplified manufacturing procedure. An adduct of glucose and 4-vinylphenylboronic acid (VPBA) was synthesized by esterification and was then purified. The copolymer of the glucose/VPBA adduct and methylene bisacrylamide was grafted onto an indium tin oxide electrode surface. Glucose was washed out from the copolymer to obtain an MIP layer. Cyclic voltammetry of ferrocyanide in aqueous solution was performed using an MIP-grafted electrode, and the effect of glucose on the anodic current intensity was evaluated. The anodic current intensity was sensitive to the glucose concentration, and the dynamic range (0–900 mg/dl) covered the typical range of diabetic blood glucose levels. The response time of the MIP-grafted electrode to a stepwise change in the glucose concentration was approximately 3–5 min. Thus, we can conclude that, by taking advantage of its gate effect, it is feasible to use an MIP-grafted electrode as a glucose sensor for monitoring blood sugar in diabetic patients.