Improved biosensor for glucose based on glucose oxidase-immobilized silk fibroin membrane

Based on glucose oxidase-immobilized silk fibroin membrane and oxygen electrode, the authors have developed an amperometric glucose sensor in flow-injection analysis. After the sensor was improved by the configuration of oxygen electrode and a temperature control system was added to the electrode body, its sensitivity, analytical precision, and stability were enhanced greatly. The authors first introduced a tailing inhibitor-ion pair reagent into a buffer system in the biosensor so as to eliminate all interference from hemacyte, macromolecules, and small mol wt charged species besides electroactive specie ascorbate in complex matrices. A considerably serious tailing of the biosamples, such as whole blood, plasma, serum, or urine on the sensor, based on enzyme electrode, entirely disappeared, their response times were shortened, and base lines became more smooth and stable. The glucose sensor has a broad range of linear response for glucose (up to 25.0 mmol/L) and a good correlation (γ = 0.999) under conditions of control temperature 32.0°C and 1.6 mL/min 0.02 mol/L phosphate buffer containing 0.5% tailing inhibitor (v/v). Recoveries of glucose in these biosamples are within the range of 93.71–105.88%, and its repeatabilities for determining glucose, repeated 100 times, human blood dilution 125 times, and serum 128 times, are 1.81,2.48, and 2.91% (RSD), respectively. The correlation analysis for 200 serum samples showed that the correlation (γ) is 0.9934 between the glucose sensor and Worthington method for determining serum glucose used conventionally in a hospital laboratory. Moreover, the enzyme membrane used in the biosensor can be stored for a long time (over 2 yr) and measured repeatedly over 1000 times for biosamples. The glucose sensor is capable of detecting over 60 biosamples/hr.     

Glucose Biosensor Based on Oxygen Electrode. Part II: Long-Term Operational Stability of the Rechargeable Glucose Biosensor

Abstract

A potentially implantable glucose biosensor for measuring blood or tissue glucose levels in diabetic patients has been developed. The glucose biosensor is based on an amperometric oxygen electrode and immobilized glucose oxidase enzyme, in which the immobilized enzyme can be replaced (the sensor recharged) without surgical removal of the sensor from the patient. Recharging of the sensor is achieved by injecting fresh immobilized enzyme into the sensor using a septum. A special technique for immobilization of the enzyme on Ultra-Low Temperature Isotropic (ULTI) carbon powder held in a liquid suspension has been developed.

In vitro studies of the sensors show stable performance during several recharge cycles over a period of 3 months of continuous operation.

Diffusion membranes which ensure linear dependence of the sensor response on glucose concentration have been developed. These membranes comprise silastic latex-rubber coatings over a microporous polycarbonate membrane. Calibration curves of the amperometric signal show linearity over a wide range of glucose concentrations (up to 16 mM), covering hypoglycemic, normoglycemic and hyperglycemic conditions.

The experimental results confirm the suitability of the sensors for in vitro measurements in undiluted human sera.     

A potentiometric polypyrrole-based glucose biosensor

A new concept is described for monitoring a biomolecule with a sensor having an enzyme entrapped in a conducting polymer. This is based on the sensitivity of the electroactive polymer itself to changes of pH in solution. The concept has been investigated for a glucose sensor with glucose oxidase (GOD) immobilized in a polypyrrole (PPy) layer on an inert platinum electrode. Measurements with a Pt/PPy/GOD electrode for glucose concentrations in the physiological range gave a linear correlation with logarithm of concentration over one decade with a satisfactory dynamic response. There was practically no change of slope or range of linear response to glucose after several days of use; this was in contrast to the amperometric response of the detector when there was about a 50% loss of sensitivity.     

A biocompatible needle-type glucose sensor based on platinum-electroplated carbon electrode

A biocompatible needle-type glucose sensor with a 3-electrode configuration was constructed. A platinum-electroplated carbon stick was used as the working electrode, Ag/AgCl as the reference electrode, and a disposable hypodermic needle made of stainless steel as the counter electrode. A Nafion membrane, an immobilized glucose oxidase (GOD) membrane, and a biocompatible membrane with diffusion-limiting effect were coated successively onto the working electrode. The sensor showed a rapid response (< 120 s in batch operation), good reproducibility (RE < 3%), good stability (over 36 h in control serum), a wide dynamic range (5-600 mg/dL glucose), and superior biocompatibility. It was used to determine glucose in serum. The data obtained from the sensor showed good agreement with that from a clinical autoanalyzer (R > 0.95).     

Immobilized Fullerene C60‐Enzyme‐Based Electrochemical Glucose Sensor

A mixed‐valence cluster of cobalt(II) hexacyanoferrate and fullerene C60‐enzyme‐based electrochemical glucose sensor was developed. A water insoluble fullerene C60‐glucose oxidase (C60‐GOD) was prepared and applied as an immobilized enzyme on a glassy carbon electrode with cobalt(II) hexacyanoferrate for analysis of glucose. The glucose in 0.1 M KCl/phosphate buffer solution at pH = 6 was measured with an applied electrode potential at 0.0 mV (vs Ag/AgCl reference electrode). The C60‐GOD‐based electrochemical glucose sensor exhibited efficient electro‐catalytic activity toward the liberated hydrogen peroxide and allowed cathodic detection of glucose. The C60‐GOD electrochemical glucose sensor also showed quite good selectivity to glucose with no interference from easily oxidizable biospecies, e.g. uric acid, ascorbic acid, cysteine, tyrosine, acetaminophen and galactose. The current of H₂O₂ reduced by cobalt(II) hexacyanoferrate was found to be proportional to the concentration of glucose in aqueous solutions. The immobilized C60‐GOD enzyme‐based glucose sensor exhibited a good linear response up to 8 mM glucose with a sensitivity of 5.60 × 10² nA/mM and a quite short response time of 5 sec. The C60‐GOD‐based glucose sensor also showed a good sensitivity with a detection limit of 1.6 × 10^‐6 M and a high reproducibility with a relative standard deviation (RSD) of 4.26%. Effects of pH and temperature on the responses of the immobilized C60‐GOD/cobalt(II) hexacyanoferrate‐based electrochemical glucose sensor were also studied and discussed.     

Covalent attachment of glucose oxidase to an Au electrode modified with gold nanoparticles for use as glucose biosensor

A feasible method to fabricate glucose biosensor was developed by covalent attachment of glucose oxidase (GOx) to a gold nanoparticle monolayer modified Au electrode. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) of ferrocyanide followed and confirmed the assemble process of biosensor, and indicated that the gold nanoparticles in the biosensing interface efficiently improved the electron transfer between analyte and electrode surface. CV performed in the presence of excess glucose and artificial redox mediator, ferrocenemethanol, allowed to quantify the surface concentration of electrically wired enzyme (Gamma(E)(0)) on the basis of kinetic models reported in literature. The Gamma(E)(0) on proposed electrode was high to 4.1 x 10(-12) mol.cm(-2), which was more than four times of that on electrode direct immobilization of enzyme by cystamine without intermediate layer of gold nanoparticles and 2.4 times of a saturated monolayer of GOx on electrode surface. The analytical performance of this biosensor was investigated by amperometry. The sensor provided a linear response to glucose over the concentration range of 2.0 x 10(-5)-5.7 x 10(-3) M with a sensitivity of 8.8 microA.mM(-1).cm(-2) and a detection limit of 8.2 microM. The apparent Michaelis-Menten constant (K(m)(app)) for the sensor was found to be 4.3 mM. In addition, the sensor has good reproducibility, and can remain stable over 30 days.     

Oxygen electrode-based enzyme immunoassay for the amperometric determination of hepatitis B surface antigen

Specific antibodies labelled with glucose oxidase are immobilized onto a gelatin membrane, which is fixed over an oxygen electrode. The sensor is immersed in a standard glucose solution and a signal is obtained by measuring the consumption of oxygen by the enzyme catalyzed reaction. The response increases linearly with increasing antigen concentration over the range 0.1–100 μg 1^?1. A microcomputer is used for data acquisition and processing.     

Biosensor system for continuous glucose monitoring in fish

A biosensor system was developed for continuous estimation of blood glucose in fish. Because it is difficult to measure blood components in real-time due to decreased sensor output resulting from blood coagulation and coalescing blood proteins at the sensor placement site, we used the eyeball scleral interstitial fluid (EISF) as the site of sensor implantation. Evaluation of the relationship between EISF and blood glucose concentrations revealed that the blood glucose concentration correlated closely with the EISF glucose concentration (y = 2.2996 + 0.9438x, R = 0.960, n = 112). To take advantage of the close correlation between blood and EISF glucose, we prepared a needle-type enzyme sensor for implantation in the fish sclera using a flexible wire electrode. The sensor provided a rapid response, good linearity, and reproducibility. Continuous glucose monitoring could be carried out by implanting this needle-type glucose sensor onto the eye. The findings indicated that the glucose concentration increased with sensor output current over time, and that changes in the blood glucose were continuously reflected in the EISF. The glucose concentration was estimated based on the one-point or two-point calibration methods. The two-point calibration method yielded the most accurate glucose monitoring (blood glucose range of 70-420 mg dL⁻¹) over 160 min. Sensor-estimated glucose and whole blood glucose values were highly correlated (y = 0.4401 + 0.8656x, R = 0.958).     

纳米增强型毛细管酶柱用于葡萄糖液滴生物传感器的研究

葡萄糖的检测在临床医学以及食品工业等领域中十分重要.以往的检测方法主要包括化学发光法_{[1]、吸光光度法[2]、电化学法[3]和荧光法[4]等.固定化酶柱的制作是发展葡萄糖传感器的关键技术之一.传统的固定化方法主要是将具有生物活性的酶通过物理吸附、共价键合和交联的方法固定于载体基质上或包埋于有机聚合物的基质中.近期研究[5,6]表明,采用溶胶凝胶(Sol-gel)法将蛋白质和酶等生物活性物质包埋于无机陶瓷或玻璃材料内,保持生物组分的活性,且SiO2作为基质材料具有较好的坚固性、抗磨性、化学惰性以及高的光稳定性和透过性,但目前该法多用于电化学型生物传感器[7,8].本文利用纳米颗粒的比表面积大和吸附能力强等特点,将酶吸附在SiO2纳米颗粒表面,用易成膜的聚乙烯醇缩丁醛(PVB)作辅助基质在毛细管上固定酶,并采用分立式酶柱,克服了以往混合型酶柱普遍存在的酶促效率不高和使用寿命较短的局限性.所制得的酶柱具有表面反应活性高、表面活性中心多和催化效率高等特点.结合自行设计的液滴光化学传感装置[9,10],建立了一种高效、快速、微量的葡萄糖实时检测方法.}     

Glucose sensor based on a field-effect transistor with a photolithographically patterned glucose oxidase membrane

A photopolymer solution consisting of polyvinylpyrrolidone and 2,5-bis(4′-azido-2′-sulfobenzal)cyclopentanone is used to make a patterned glucose oxidase membrane for a FET-glucose sensor by photolithography. A small patterned glucose oxidase membrane, 0.2 mm wide and 1 mm long, is made on the gate surface of an ISFET by developing a photocross-linked glucose oxidase membrane with aqueous 1–3% glutaraldehyde solution. The optimum composition of the enzyme/photopolymer solution is described. The sensor with the patterned membrane showed linear response to glucose concentration from 0.3 to 2.2 mM and useful response up to 5 mM.     

A Tubulin‐Based Quantitative Assay for Taxol (Paclitaxel) with Enzyme Channeling Sensing

We report the development and characterization of a biosensor for sensitive and rapid determination of the anticancer agent Taxol (paclitaxel). The sensor is based on the interaction of Taxol with its receptor protein tubulin in conjunction with the separation‐free immunosensor technique of enzyme channeling. The sensor system consisted of a three electrode electrochemical cell containing a graphite carbon electrode modified with glucose oxidase and tubulin as working electrode poised at +40 mV (vs. Ag/AgCl). Addition of Taxol, horseradish peroxidase labeled Taxol, glucose and potassium iodide to the cell generated a cathodic current response that was proportional to the concentration of Taxol in the range of 10 to 1 000 pM.     

Frequency response of the glucose sensor based on immobilized glucose oxidase membrane

Frequency response of the glucose sensor based on the immobilized glucose oxidase membrane was investigated experimentally by giving the sinusoidal change of glucose concentration to the glucose sensor and observing its output signal. Observed values of gains and phase lags of the frequency response of the glucose sensor followed the frequency response model of the first-order with dead time; The time constant and also the dead time were estimated and found to decrease as the amount of enzyme immobilized in the membrane increased and the thickness of the membrane decreased.

     

Response characteristics of the glucose-sensitive field-effect transistor. Computer simulation of the effect of gluconolactonase coimmobilization in a glucose oxidase membrane

The bienzyme system consisting of glucose oxidase and gluconolactonase was investigated using a conventional diffusion-kinetics model for an enzyme-modified field-effect transistor (FET) to clarify the effect of gluconolactonase coimmobilization in a glucose oxidase membrane on the steady-state response amplitude of a glucose sensor based on a FET. The model includes spontaneous and enzymatic hydrolysis reactions of d-glucono-δ-lactone and it elucidated the following experimental results: a glucose sensor with a membrane (about 1 μm in thickness) coimmobilizing these enzymes showed a sufficient response amplitude, whereas without coimmobilization of gluconolactonase no detectable response was observed up to 3 mM glucose; and the response amplitude depended strongly on the amount of lactonase in the membrane. The model also predicted an optimum enzyme ratio for coimmobilization in a membrane.     

生物功能电极(Ⅴ)─葡萄糖氧化酶在环糊精聚合物中的固定化

将葡萄糖氧化酶(GOD)固定在α-环糊精聚合物中,而电子传递体分子被包含在环糊精腔穴中。固定化酶膜的FTIR测定表明,GOD与环糊精聚合物发生共价连接。制备了含电子传递体的不同GOD酶电极并比较了它们的性能。含四硫代富瓦烯的酶电极具有良好的电流响应特性,可望成为第二代葡萄糖酶电极的新构型。     

α‐Fe2O3 Film with Highly Photoactivity for Non‐enzymatic Photoelectrochemical Detection of Glucose

A non‐enzyme photoelectrochemical (PEC) glucose sensor based on α‐Fe₂O₃ film is investigated. The α‐Fe₂O₃ film was fabricated via a simple spin coating method. The proposed glucose sensor exhibits good selectivity, a fast response time of <5 s, a linear range of 0.05 to 6.0 mM, sensitivity of 17.23 μA mM^?1 cm^?2 and a detection limit of 0.05 μM. Meanwhile, the excellent performances of the α‐Fe₂O₃ sensor were obtained in reproducibility and the long‐term stability under ambient condition. The linear amperometric response of the sensor covers the glucose levels in physiological and clinical for diabetic patients. Therefore, this non‐enzyme PEC sensor based on α‐Fe₂O₃ film has a great potential application in the development of glucose sensors.     

Glucose sensor using a phospholipid polymer-based enzyme immobilization method

An electroenzymatic glucose sensor based on a simple enzyme immobilization technique was constructed and tested. The glucose sensor measures glucose concentrations as changes of oxygen concentrations induced by enzymatic reactions. The immobilizing procedure was developed with the purpose of producing wearable biosensors for clinical use. Two types of biocompatible polymers, 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymerized with dodecyl methacrylate (PMD) and MPC copolymerized with 2-ethylhexyl methacrylate, were compared as a sensitive membrane of biosensors. The PMD enzyme membrane had a better response time. Linearity, reproducibility, effect of the concentrations of immobilized enzyme and drifts of sensor characteristics in long-term tests were also investigated. The linear characteristics were confirmed with glucose concentration from 0.01 to 2.00 mmol/l, with a coefficient of determination of 0.9999. The average output current for 1 mmol/l and the standard deviation were 0.992 and 0.0283 muA. Significant changes in the sensor's characteristics were not observed for 2 weeks when it was kept in a refrigerator at 4 degrees C. Because of the simple procedure, the enzyme immobilization method is not only useful for wearable devices but also other devices such as micro total analysis systems.     

Immobilization of lactate oxidase in a poly(vinyl alcohol) matrix on platinized graphite electrodes by chemical cross-linking with isocyanate

A new method for development of an electrochemical sensor based on lactate oxidase is dedbed. Platinized spectroscopic-grade graphite electrodes were modified by chemically cross-linking l-lactate oxidase from Pediococcus species into a poly(vinyl alcohol) network through reaction with a tri-isocyanate. The immobilized enzyme exhibits high activity and long-term stability. The sensor provides a linear response to l-lactate over a concentration range of 2 x 10(-5)-4 x 10(-3)M and a sensitivity of 1.71 muA.1. mmole(-1). The response time of the sensor is 10-45 sec and the detection limit is 10muM. Stable response to the substrate was obtained over a period of 3 months. The new sensor was also used for the analysis of some dairy products without any special pretreatment.     

Glucose oxidase-incorporated hydrogel thin film for fast optical glucose detecting under physiological conditions

Hydrogel biosensors usually suffer from a slow response, which severely hinders their practical applications. Here a new optical glucose biosensor was designed, using glucose-sensitive hydrogel films as both glucose-sensing material and Fabry-Perot cavity. The film was fabricated by layer-by-layer assembly from partially oxidized dextran (PO-Dex), chitosan, and glucose oxidase (GOD). The film responds to glucose because the incorporated GOD converts glucose to gluconic acid, and thus lowers the local pH in the film, and, in turn, triggers the pH-sensitive film to swell. The glucose-induced swelling causes a shift of Fabry?Perot fringes on the reflection spectra of the film, from which the glucose concentration can be reported. The new sensor works well under physiological conditions. Potential interferents, such as diols for phenylboronic acid-based sensors and electroactive compounds for electrochemical sensors, do not influence the new sensor. The sensor can respond reversibly over a wide range of glucose concentration. Particularly, it responds linearly within the clinically relevant glucose range (0–20 mM). More importantly, because the film is very thin, the new sensor can respond quickly, making it potential for real-time, continuous glucose monitoring.     

Microfluidic device to investigate factors affecting performance in biosensors designed for transdermal applications

In this work we demonstrate a novel microfluidic based platform to investigate the performance of 3D out-of-plane microspike array based glucose and lactate biosensors. The microspike array was bonded with a glass slide and modified with glucose oxidase or lactate oxidase using covalent coupling chemistry. An epoxy-polyurethane based membrane was used to extend the linear working range (from 0 to 25 mM of substrate) of these biosensors. Both lactate and glucose sensors performed well in the clinically relevant substrate concentration range. Glucose microspikes were further investigated with respect to the effects of substrate transfer by incorporation into a microfluidic system. Data from the microfluidic system revealed that the sensor response is mainly dependent on enzyme kinetics rather than membrane permeability to glucose. The robustness of the sensors was demonstrated by its consistency in performance extending over 48 h.     

Layer-layer assembly for extremely stable glucose sensors

A novel fabrication of an amperometric glucose sensor by layer after layer approach is described. The sensor electrode is fabricated by arranging a layer of Pt black, a layer of glucose oxidase (GOD) and a layer of stabilizer gelatin on a shapable electro-conductive (SEC) film surface. Finally, the dried layered-assembly is cross-linked by exposing to a diluted glutaraldehyde solution. The performance of the developed sensor is evaluated by a FIA system at 37°C and under a continuous polarization at 0.4 V (vs. Ag/AgCl). The sensitivity of the sensor was dependent on the amount of GOD loaded. The highest sensitivity (3.6 μA/mM cm⁻²) of the sensor was obtained at a GOD loading of 160 μg/cm², and the linear dynamic range was extended to 80 mM level when the sensor was covered with a polycarbonate membrane. The sensor shows an extremely stable response for several weeks and a storage stability of over 2 years.