Glucose-sensitive field-effect transistor with a membrane containing co-immobilized gluconolactonase and glucose oxidase

A glucose-sensitive field-effect transistor (FET) with a two-enzyme membrane containing gluconolactonase and glucose oxidase is investigated. The two-enzyme membrane (ca. 1 μm thick) is formed on the ion-sensitive gate of the FET by photopolymerization. The gluconolactonase used was a partially purified product prepared from crude glucose oxidase by gel filtration. A glucose sensor with only purified glucose oxidase has little response for glucose, but the co-immobilization of gluconolactonase and glucose oxidase considerably enhanced the response amplitude of the glucose sensor. The composition of the two-enzyme/photopolymer solution is optimized; gluconolactonase with an activity at least twice that of glucose oxidase is necessary. The linear calibration graph extends from 0.2 to 2 mM glucose.     

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.     

Glucose-sensitive field effect transistor using totally synthetic compounds

A field effect transistor (FET)-based glucose sensor was fabricated. As a totally synthetic and thus stable glucose-sensing moiety, 3-acrylamidophenylboronic acid was chemically introduced onto the FET gate surface in the form of a thin copolymer gel layer. Excellent transistor characteristics were confirmed even after the surface modification, ensuring validity of the modification procedure herein developed. Glucose-induced changes in the FET’s electric characteristics were obtained in quantitative as well as reversible manners. It was also demonstrated that the prepared FET is able to continuously perceive the change in the glucose concentration in the milieu. The detected signals were attributed to the faction change of the gate-introduced phenyborate anions, also presumably involving other parameter changes such as permittivity and conductivity. The use of the fabricated FET could further be extended to the construction of stable, readily minutualizable, and label-free carbohydrate molecule-sensing systems.     

Glucose Sensor Based On an Amorphous Silicon Isfet

Abstract

An amorphous silicon ion sensitive field effect transistor (a-ISFET) was first applied to glucose sensors. When glucose oxidase was immobilized on the membrane, the sensor gave a linear relationship between the initial rate of the gate output voltage change and the logarithmic value of glucose concentration between 0.1 and 1 mg/ml at pH 7.0, 37°C. Determination of glucose was possible within 1 min. the system can be used for three weeks with only slight loss of enzymatic activity.     

Disposable Sandwich‐type Electrochemical Sensor for Selective Detection of Glucose Based on Boronate Affinity

A disposable sandwich‐type electrochemical sensor for selective detection of glucose was established. The primary receptor, 3‐aminophenylboronic acid was grafted covalently onto the surface of screen‐printed carbon electrodes through an in situ‐generated diazo‐reaction. Glucose was first captured by boronic acid group on the electrode, followed by captureing an electroactive ferroceneboronic acid (FcBA) as the secondary receptor to form bidentate glucose‐boronic complex. Electrochemical impedance spectroscopy was applied to characterize the construction of sandwich‐type disposable sensor. In the sandwich assay, current response of captured FcBA on the electrode was dependent on the concentration of glucose. The sandwich assay showed higher selectivity for glucose than that for fructose, mannose, galactose and other electroactive interferences including uric acid, ascorbic acid and dopamine, and exhibited a dynamic concentration range of glucose from 0.5 to 20.0 mmol L⁻¹. The disposable sensor demonstrated a good reproducibility with 2.2 % relative standard deviation (RSD). In addition, the disposable glucose sensor was used in detection of the trace glucose in the clinical urine samples.     

Needle-type glucose microbiosensor based on glucose oxidase immobilised in an overoxidised polypyrrole film (an in-vitro study)

A fast response, needle-type glucose microbiosensor has been fabricated by a one-step electrochemical immobilisation of glucose oxidase in a polypyrrole film. The sensor shows a remarkable rejection of electroactive interferences, especially paracetamol. The maximum bias observed in the worst situation never exceeded the value of 6%. The fabrication procedure delivered very reproducible devices and the sensitivity of a newly prepared biosensor was typically 650 nA mM(-1) cm(-2). The kinetic parameters, obtained from an existing model, permitted to understand the sensor behaviour.     

Needle-type glucose microbiosensor based on glucose oxidase immobilised in an overoxidised polypyrrole film (an in-vitro study)

A fast response, needle-type glucose microbiosensor has been fabricated by a one-step electrochemical immobilisation of glucose oxidase in a polypyrrole film. The sensor shows a remarkable rejection of electroactive interferences, especially paracetamol. The maximum bias observed in the worst situation never exceeded the value of 6%. The fabrication procedure delivered very reproducible devices and the sensitivity of a newly prepared biosensor was typically 650 nAmM^–1cm^–2. The kinetic parameters, obtained from an existing model, permitted to understand the sensor behaviour.     

Fluorometric sensors based on chemically modified enzymes Glucose determination in drinks

In this paper an enzymatic fluorometric sensor for glucose determination in drinks is presented. The sensor film was obtained by immobilisation of glucose oxidase chemically modified with a fluorescein derivative (GOx-FS) in a polyacrylamide polymer. During the enzymatic reaction the changes in the fluorescence intensity of the GOx-FS are related to the glucose concentration. Working in FIA mode, the optimum conditions found were: 0.7 ml min(-1) flow rate, 300 mul sample injection and pH 6.5. The sensor responds to glucose concentrations ranging from 400 to 2000 mg l(-1), the reproducibility is around 3% and the life-time is at least 3 months (more than 350 measurements). The sensor was applied to direct glucose determination in drinks with good accuracy; interference caused by the filter effect was avoided by the kinetics of the reaction.     

Field-effect-transistor sensor based on enzyme-functionalized polypyrrole nanotubes for glucose detection

We describe the detection of glucose based on a liquid-ion gated field-effect transistor configuration in which enzyme-functionalized polypyrrole nanotubes are employed as the conductive channel. First of all, carboxylated polypyrrole nanotubes (CPNTs) were successfully fabricated by the chemical polymerization of an intrinsically functionalized monomer (pyrrole-3-carboxylic acid, P3CA) without degradation in major physical properties. The CPNTs possessed not only well-defined functional groups but also electrical properties comparable to nonsubstituted polypyrrole. Importantly, the carboxylic acid functional group can be utilized for various chemical and biological functionalizations. A liquid-ion gated FET sensor was readily constructed on the basis of the chemical functionality of CPNTs. In the first stage, the CPNTs were immobilized onto a microelectrode substrate via covalent linkages. It was noteworthy that the covalent immobilization allowed high-quality contact between the nanotubes and the microelectrodes in the liquid phase. The second stage involved the covalent binding of glucose oxidase (GOx) enzyme to the nanotubes. The covalent functionalization generally provides excellent enzymatic activity and thermal stability. The fabricated FET sensor provided real-time response (an increase in source-drain current) and high sensitivity toward the various concentrations (0.5-20 mM) of glucose. The enzymatic reaction product, hydrogen peroxide, played pivotal roles in modulating the charge transport property of CPNTs.     

Bio-field-effect transistors as detectors in flow-injection analysis

The development of enzyme-modified bio-field-effect transistors (BioFETs) for the determination of glucose, urea, penicillin G, penicillin V and cephalosporin C is reported. BioFETs are produced by covering the pH-sensitive gate areas of ion-selective field-effect transistors with enzyme membranes. The characteristics of the resulting BioFETs and the influence of several parameters, e.g., pH and buffer capacity, are described. The measuring range covers 1–2 orders of magnitude of substrate concentration, and the BioFETs are applicable for 3–12 weeks, depending on the enzyme. They show a short response time and are well suited for detection in flow systems. The frequency of determination with BioFETs in flow systems is high (15–20 measurements per hour). The application of a BioFET in on-line bioprocess control is described. A glucose oxidase FET monitors the glucose concentration during cultivation of Escherichia coli. The results correspond well with off-line liquid chromatographic determinations.     

Entrapment of both glucose oxidase and peroxidase in regenerated silk fibroin membrane

Two enzmyes, glucose oxidase and peroxidase, were for the first time simultaneously immobilized in regenerated silk fibroin membrane. The structure and morphology of the regenerated silk fibroin membrane containing both glucose oxidase and peroxidase were investigated with IR spectra and SEM. The bienzymes do not change the structures of the regenerated silk fibroin in the membrane, which has an islands-sea structure. For the first time, an amperometric methylene green mediating sensor for glucose based on co-immobilization of both glucose oxidase and peroxidase in regenerated silk fibroin was constructed. Cyclic voltammetry and amperometry were used to test the suitability of methylene green shuttling electrons between peroxidase and the glassy carbon electrode. The bienzyme-based system offers fast response and high sensitivity of the sensor to glucose. The effects of pH, temperature, and the concentration of the mediator on the response current were evaluated, and the dependence of the Michaelis-Menten constant K(m)(app) on the concentration of the mediator was investigated.     

Carbon nanotube enhanced mediator-type biosensor for real-time monitoring of glucose concentrations in fish

We have developed a mediator-type biosensor to rapidly monitor blood glucose concentrations in fish, which are an indicator of stress. Glucose oxidase was used to detect glucose concentrations and ferrocene was used to limit the effect of oxygen. We also improved the sensitivity and durability of the sensor for better performance. Single-walled carbon nanotubes were used to enhance sensor sensitivity. Affixing the carbon nanotubes (30 mg ml^-1) to the working electrode increased the sensor sensitivity to 61.9 mM nA^-1 mm^-2, twice the value for the sensor without single-walled carbon nanotubes. A fabricated mediator-type biosensor sensor was used to perform real-time in vivo measurements. The sensor was implanted into the interstitial fluid of a fish eyeball, and detection was transmitted to a personal computer by a wireless potentiostat. Continuous measurement of the glucose concentration was possible for 78 hours. Stress was artificially applied to the fish during the measurement, and the change of blood glucose concentrations were observed. Our proposed sensor is applicable for effectively monitoring stress in free-swimming fish.     

超微电极上半微分电分析法

介绍了超微半球电极上具有简单反应时的半微分循环伏安理论。理论表明,超微电极上半微分电流与电活性物质的浓度成正比,并据此提出了一种利用超微半球电极上半微分曲线进行电化学分析测量的新方法。该法具有灵敏度高、分辨率好等特点。     

Study of carbon nanotubes-HRP modified electrode and its application for novel on-line biosensors

In this paper, multi-walled carbon nanotubes (MWCNTs) were successfully immobilized on the surface of a glassy carbon electrode by mixing with horse-radish peroxidase (HRP). The electrochemical behavior of H2O2 was also studied with the MWCNTs-HRP modified electrode as a working electrode. The MWCNTs-HRP modified electrode showed excellent response of reduction current for the determination of H2O2 at the potential of -300 mV (vs. Ag/AgCl). We assembled the MWCNTs-HRP modified electrode in a thin-layer flow cell and the H2O2 solution was continuously introduced into the cell with a syringe pump. We optimized the sensitivity of the H2O2 sensor by adjusting the working potential and the pH of the buffer solution. The peak current increased linearly with the concentration of H2O2 in the range 3.0 x 10(-7) to approximately 2.0 x 10(-4) mol L(-1). The detection limit is 1.0 x 10(-7) mol L(-1) (S/N = 3). The interferences from ascorbic acid, uric acid and other electroactive substances can be greatly excluded since the sensor can be operated at -300 mV. Stability and reproducibility of the MWCNTs-HRP chemically modified electrode were also studied in this paper. Fabricated with glucose and lactate oxidase, the MWCNTs-HRP electrode was also applied to prepare the on-line glucose and lactate biosensors because of the high sensitivity for the determination of H2O2.     

Entrapment of both glucose oxidase and peroxidase in regenerated silk fibroin membrane

Two enzmyes, glucose oxidase and peroxidase, were for the first time simultaneously immobilized in regenerated silk fibroin membrane. The structure and morphology of the regenerated silk fibroin membrane containing both glucose oxidase and peroxidase were investigated with IR spectra and SEM. The bienzymes do not change the structures of the regenerated silk fibroin in the membrane, which has an islands-sea structure. For the first time, an amperometric methylene green mediating sensor for glucose based on co-immobilization of both glucose oxidase and peroxidase in regenerated silk fibroin was constructed. Cyclic voltammetry and amperometry were used to test the suitability of methylene green shuttling electrons between peroxidase and the glassy carbon electrode. The bienzyme-based system offers fast response and high sensitivity of the sensor to glucose. The effects of pH, temperature, and the concentration of the mediator on the response current were evaluated, and the dependence of the Michaelis-Menten constant K_m^app on the concentration of the mediator was investigated.     

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.     

Tailoring Zinc Oxide Nanowires for High Performance Amperometric Glucose Sensor

ZnO nanowire was tailored both physically and chemically to immobilize the enzyme glucose oxidase (GOD) for construction of a glucose sensor with high performance, which was ascribed to its high specific surface area and high isoelectric point value for efficient immobilization of high concentration of acidic enzymes and the mediating effect by the redox reaction of ZnO nanowires. The apparent Michaelis constants J_max, and K_M were adjusted in a large scope by tailoring the thickness of the GOD/ZnO nanowire layer and the enzyme load in the nanowired network. Thus, a variety of linear region, sensitivities and reaction rates of the sensor could be easily achieved. Moreover, the glucose sensor showed long term stability with the incorporation of the inorganic zinc oxide nanowire.     

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.     

In vitro and short-term in vivo characteristics of a Kel-F thin film modified glucose sensor.

A new outer layer composition, consisting of polytetrafluoroethylene (PTFE), Kel-F oil, and Nafion, is suggested to minimize the detrimental effect of dissolved oxygen and to extend the linear response range of a glucose oxidase(GOx)-based sensor using nonconducting polymer. The morphology of Kel-F/PTFE/Kel-F/Nafion polymeric laminate was followed during fabrication by SEM. When Kel-F film was formed on the PTFE outer layer, the linear response was extended to 21 mM, at a sensitivity of 2.8 +/- 0.8 nA/mM mm2. We demonstrate that a sensor without Kel-F/PTFE/Kel-F/Nafion outer layer is relatively oxygen dependent, whereas by comparison a sensor with Kel-F/PTFE/Kel-F/Nafion outer layer is oxygen independent. The current of such a glucose sensor implanted in the subcutaneous tissue stabilized within 60 min, and the lag between blood glucose changes and sensor output was within 1 min. The in vivo characteristics of the glucose sensor described show great promise for one-point in vivo calibration.     

Activity enhancement of a screen-printed carbon electrode by modification with gold nanoparticles for glucose determination

We have successfully developed a highly sensitive electrochemical sensor strip for a home blood-sugar monitoring device by a single-step straightforward procedure. The strip consists of a pair of screen-printed carbon electrodes, which work as counter and working electrodes in the chronoamperometric mode. To remedy the poor electrochemical activity of the printed carbon electrode, a small amount of gold nanoparticles was immobilized on the electrode. In the presence of glucose oxidase, the electrode modified with 2-nm particles showed about a five times higher sensitivity for glucose oxidation than the bare printed carbon electrode, and there was a significant dependence of the current on the particle diameter. Based on these observations, we have elucidated the glucose oxidation mechanism, which is comprised of two key factors, i.e. (1) electron transfer between the gold particles, and (2) electronic coupling between the gold particles and glucose oxidase.