Development of amperometric glucose biosensor through immobilizing enzyme in a Pt nanoparticles/mesoporous carbon matrix

Pt nanoparticles were deposited on mesoporous carbon material CMK-3. Glucose oxidase (GOx) was immobilized in the resulting Pt nanoparticles/mesoporous carbon (Pt/CMK-3) matrix, and then the mixture was cast on a glassy carbon electrode (GCE) using gelatin as a binder. The glucose biosensor exhibited excellent current response to glucose after cross-linking with glutaraldehyde. At 0.6V (vs. SCE) the response current was linear to glucose concentration in the range of 0.04-12.2mM. The response time (time for achieving 95% of the maximum current) was 15s and the detection limit (S/N=3) was 1 microM. The Michaelis-Menten constant (K(m)(app)) and the maximum current density (i(max)) were 10.8 mM and 908 microAcm(-2), respectively. The activation energy of the enzymatic reaction was estimated to be 22.54 kJ mol(-1). The biosensor showed good stability. It achieved the maximum response current at about 52 degrees C and retained 95.1% of its initial response current after being stored for 30 days. In addition, some fabrication and operation parameters for the biosensor were optimized in this work. The biosensor was used to monitor the glucose levels of serum samples after being covered with an extra Nafion film to improve its anti-interferent ability and satisfied results were obtained.     

Direct electrochemistry of glucose oxidase in a colloid Au-dihexadecylphosphate composite film and its application to develop a glucose biosensor

Colloid Au (Au(nano)) with a diameter of about 10 nm was prepared and used in combination with dihexadecylphosphate (DHP) to immobilize glucose oxidase (GOD) onto the surface of a graphite electrode (GE). The direct electrochemistry of GOD confined in the composite film was investigated. The immobilized GOD displayed a pair of redox peaks with a formal potential of -0.475 mV in pH 7.0 O(2)-free phosphate buffers at scan rate of 150 mV s(-1). The GOD in the composite film retained its bioactivity and could catalyze the reduction of dissolved oxygen. Upon the addition of glucose, the reduction peak current of dissolved oxygen decreased, which could be developed for glucose determination. A calibration linear range of glucose was 0.5-9.3 mM with a detection limit of 0.1 mM and a sensitivity of 1.14 microA mM(-1). The glucose biosensor showed good reproducibility and stability. The general interferences that coexisted in human serum sample such as ascorbic acid and uric acid did not affect glucose determination.     

Direct electrochemistry of glucose oxidase immobilized on a hexagonal mesoporous silica-MCM-41 matrix

The direct electrochemistry of glucose oxidase (GOD) immobilized on a hexagonal mesoporous silica modified glassy carbon electrode was investigated. The adsorbed GOD displayed a pair of redox peaks with a formal potential of -417 mV in 0.1 M pH 6.1 phosphate buffer solution (PBS). The response showed a diffusion-controlled electrode process with a two-electron transfer coupled with a two-proton transfer reaction process. GOD immobilized on a hexagonal mesoporous silica retained its bioactivity and stability. In addition, the immobilized GOD could electrocatalyze the oxidation of glucose to gluconlactone by taking ferrocene monocarboxylic acid (FMCA) as a mediator in N(2) saturated solutions, indicating that the electrode may have the potential application in biosensors to analyze glucose. The sensor could exclude the interference of commonly coexisted uric acid, p-acetaminophenol and ascorbic acid and diagnose diabetes very fast and sensitively. This work demonstrated that the mesoporous silica provided a novel matrix for protein immobilization and the construction of biosensors.     

Mediator-free amperometric determination of glucose based on direct electron transfer between glucose oxidase and an oxidized boron-doped diamond electrode

A mediator-free glucose biosensor, termed a “third-generation biosensor,” was fabricated by immobilizing glucose oxidase (GOD) directly onto an oxidized boron-doped diamond (BDD) electrode. The surface of the oxidized BDD electrode possesses carboxyl groups (as shown by Raman spectra) which covalently cross-link with GOD through glutaraldehyde. Glucose was determined in the absence of a mediator used to transfer electrons between the electrode and enzyme. O₂ has no effect on the electron transfer. The effects of experimental variables (applied potential, pH and cross-link time) were investigated in order to optimize the analytical performance of the amperometric detection method. The resulting biosensor exhibited fast amperometric response (less than 5 s) to glucose. The biosensor provided a linear response to glucose over the range 6.67×10⁻⁵ to 2×10⁻³ mol/L, with a detection limit of 2.31×10⁻⁵ mol/L. The lifetime, reproducibility and measurement repeatability were evaluated and satisfactory results were obtained.     

A rapid and easy procedure of biosensor fabrication by micro-encapsulation of enzyme in hydrophilic synthetic latex films. Application to the amperometric determination of glucose

Novel enzyme electrodes based on synthetic hydrophilic latex matrices are described for the detection of glucose. Glucose oxidase was immobilised through micro-encapsulation, by the simple adsorption of enzyme–latex suspensions on the surface of a platinum electrode. Two latex films functionalised by a hydroxy or a gluconamide group were used. The response of these biosensors to glucose additions was measured by potentiostating the modified electrodes at 0.6 V/SCE in order to oxidise the hydrogen peroxide generated by the enzymatic oxidation of glucose in the presence of dioxygen. The response of such electrodes was evaluated as a function of film thickness and temperature. The sensitivity for a two-layer latex-based biosensor was found to be 38.78 mA M⁻¹ cm⁻² with a response time of 3–5 s. Moreover, a marked improvement of the thermal stability of the biosensor was observed. Only at temperatures higher than 65°C the enzyme started to be denatured and being inactive.     

Glucose biosensing based on the highly efficient immobilization of glucose oxidase on a Prussian blue modified nanostructured Au surface

We report on a novel glucose biosensor based on the immobilization of glucose oxidase (GOx) on a Prussian blue modified nanoporous gold surface. The amperometric glucose biosensor fabricated in this study exhibits a fast response and the very low detection limit of 2.5 μM glucose. The sensitivity of the biosensor was found to be very high, 177 μA/mM; the apparent Michaelis–Menten constant is calculated to be 2.1 mM. In addition, the biosensor has good reproducibility and remains stable over 60 days. The anti-interference ability of the biosensor was also assessed, showing little interference from possible interferents such as ascorbic acid (AA), acetaminophen (AP) and uric acid (UA).     

硫堇-牛血清白蛋白氧化还原复合膜修饰葡萄糖生物传感器的研究

通过交联法和自组装法制备了一种双酶型葡萄糖生物传感器.首先以牛血清白蛋白-戊二醛为交联剂以实现对辣根过氧化物酶(HRP)的固载,再利用凝集素-糖蛋白的识别作用将葡萄糖氧化酶(GOD)分子组装到电极表面,制得双酶型的葡萄糖生物传感器.采用原子力显微镜(AFM)考察了复合膜的性质,同时采用循环伏安法和计时电流法考察了该传感...     

An amperometric biosensor based on covalent immobilization of ascorbate oxidase on biocompatiable and low-toxic poly(thiophene-3-acetic acid) matrix

The biocompatiable and low-toxic poly（thiophene-3-acetic acid）（PTAA） matrix was successfully electrosynthesized in ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate（BmimPF₆） in comparison with the electrosynthesis of PTAA matrix in acetonitrile（ACN）.Ascorbate oxidase（AO） was used as a model for the development and application of biosensor.Vitamin C（VC） biosensors were facilely fabricated by the covalent immobilization of AO molecules on PTAA matrices electrosynthesized in ACN containing tetrabutylammonium tetrafluoroborate and BmimPF₆, respectively.Electrochemical impedance spectroscopy,scanning electron microscopy and FTIR spectroscopy indicated that AO molecules were covalently immobilized on PTAA matrices.Parameters of the as-obtained biosensors such as working potential,pH and temperature have been optimized.The amperometric biosensor based on PTAA matrix electrosynthesized in BmimPF₆ exhibited wider linear range,lower detection limit,higher sensitivity and bioaffinity,and better operational and storage stability than that electrosynthesized in ACN under optimal conditions.The as-obtained biosensor based on PTAA matrix electrosynthesized in BmimPF₆ was employed for the detection of VC content in commercial juices,and the result was close to the data given by manufacturers.Excellent results indicate that the PTAA matrix electrosynthesized in ionic liquid is a promising platform for the covalent immobilization of biologically-active species and the development of biosensors.     

Development of glucose amperometric biosensor based on a novel attractive enzyme immobilization matrix: amino derivative of thiacalix[4]arene

Calixarenes and their derivatives may be a promising material for enzyme immobilization owing to their particular configuration, unique molecule recognition function and aggregation properties. In this paper, p-tert-butylthiacalix[4]arene tetra-amine (TC4TA) was first used as enzyme immobilization material. This attractive material was exploited for the mild immobilization of glucose oxidase (GOD) to develop glucose amperometric biosensor. GOD was strongly adsorbed on the TC4TA modified electrode to form TC4TA/GOD composite membrane. The adsorption mechanism was driven from the covalent bond between amino-group of TC4TA and carboxyl group of GOD and molecule recognition function of TC4TA. Amperometric detection of glucose was evaluated by holding the modified electrode at 0.60 V (versus SCE) to oxidize the hydrogen peroxide generated by the enzymatic reaction. The sensor (TC4TA/GOD) showed a relative fast response (response time was about 5 s), low detection limit (20 μM, S/N = 3), and high sensitivity (ca. 10.2 mA M⁻¹ cm⁻²) with a linear range of 0.08–10 mM of glucose, as well as a good operational and storage stability. In addition, optimization of the biosensor construction, the effects of the applied potential as well as common interfering compounds on the amperometric response of the sensor were investigated and discussed herein.     

Immobilization of glucose oxidase on rod-like and vesicle-like mesoporous silica for enhancing current responses of glucose biosensors

The use of rod-like and vesicle-like mesoporous SiO₂ particles for fabricating high performance glucose biosensors is reported. The distinctively high surface areas of mesoporous structures of SiO₂ rendered the adsorption of glucose oxidase (GOx) feasible. Both morphologies of SiO₂ enhanced the sensitivities of glucose biosensors, but by a factor of 36 for vesicle-like SiO₂ and 18 for rod-like SiO₂, respectively. The greater enhancement of vesicle-like SiO₂ can be accounted for by its higher specific surface area (509 m² g⁻¹) and larger total pore volume (1.49 cm³ g⁻¹). Interestingly, the current responses of GOx immobilized in interior channels of the mesoporous SiO₂ were enhanced much more than those of simple mixtures of GOx and the mesoporous SiO₂. This suggests that the enhancement of current responses arise not only from the high surface area of SiO₂ for high enzyme loading, but also from the improved enzyme activity upon its adsorption on mesoporous SiO₂. Also compared were the performances of glucose biosensors with GOx immobilized on mesoporous SiO₂ by physical adsorption and by covalent binding to 3-aminopropyltrimethoxysilane (APTMS) modified SiO₂ using glutaraldehyde as the cross-linker. The covalent binding approach resulted in higher enzyme loading but lower current sensitivity than with the physical adsorption.     

Development of an amperometric glucose biosensor based on the immobilization of glucose oxidase in an ormosil-PVA matrix onto a Prussian Blue modified electrode

An amperometric glucose biosensor was developed based on the immobilization of glucose oxidase in the organically modified silicate (ormosil)-polyvinyl acetate (PVA) matrix onto a Prussian Blue (PB)-modified glassy carbon electrode. A higher stability PB-modified electrode was prepared by the electrochemical deposition of FeCl₃, K₃[Fe(CN)₆] and ethylenediamine tetraacetic acid (EDTA) under cyclic voltammetric (CV) conditions. The effects of the potential range of CV conditions, electrolyte cations, applied potential, pH, temperature and co-existing substances were investigated. The detection limit of the glucose biosensor was 8.1 μmol·L⁻¹ (S/N = 3) with a linear range from 20 μmol·L⁻¹ to 2 mmol·L⁻¹ (R = 0.9965). The biosensor presented a fast response and good selectivity. Additionally, excellent reproducibility and stability of the biosensor were observed. Supported by the National High Technical Development Project (863 project) Foundation (Grant No. 2006AA09Z160) and the National Natural Science Foundation of China (Grant No. 20775064)     

Immobilization of glucose oxidase on the films prepared by electrochemical copolymerization of pyrrole and 1-(2-carboxyethyl)pyrrole for glucose sensing

Glucose oxidase (GOx) was immobilized through amide linkages on the surfaces of the conducting polymer films prepared by electrochemical copolymerization of pyrrole (Py) and 1-(2-carboxyethyl)pyrrole (Py-COOH) for the purpose of fabricating GOx-immobilized electrodes for amperometric sensing of glucose. The conductivity of the copolymer film was in the range 10⁻⁸-10⁻³ S/cm and showed a tendency to decrease with increasing content of Py-COOH units in the copolymer. The amount of immobilized GOx increased significantly with increasing content of Py-COOH units in the copolymer film up to 30%, and showed a tendency to level off when the content of Py-COOH units became larger. The activity of immobilized GOx per area of the copolymer film decreased slightly with increasing content of Py-COOH units in the copolymer. Although the GOx-immobilized copolymer films gave the amperometric response to glucose depending on its concentration, the magnitude of the response to a given concentration was found to decrease with increasing content of Py-COOH units in the copolymer. The variation in the amperometric response was attributed to the difference in conductivity of the copolymer film. The appropriate content of Py-COOH units in the copolymer was considered to be 5% or less for the amperometric sensing of glucose with the GOx-immobilized copolymer film.     

Amperometric glucose-responding property of enzyme electrodes fabricated by covalent immobilization of glucose oxidase on conducting polymer films with macroporous structure

Macroporous conducting polymer films were prepared by the electrochemical copolymerization of 3-methylthiophene and thiophene-3-acetic acid on the ITO-coated glass plates bearing different sizes of polystyrene template particles, and enzyme electrodes were fabricated by covalent immobilization of glucose oxidase on the macroporous copolymer films. It was found that the doping level and conductivity of the copolymer films was significantly affected by the treatment with solvent to remove the polystyrene particles, which was considered to result in deterioration in amperometric glucose-responding property of the enzyme electrodes fabricated with the copolymer films. Three-dimensionally ordered macroporous structure on the copolymer films led to enhancement of amperometric response of the enzyme electrodes, and this effect was attributed to the geometry of the interconnected channel structure formed by the linkage of macropores. It was suggested that the amperometric response of the enzyme electrodes was determined by whether the interconnected channel structure on the copolymer films had long distance regularity and a proper size to allow the enzyme and electron-mediator molecules to penetrate into the interior pores of the copolymer film. In particular, the interconnected channel structure seemed to play an important role in the electron-transfer reaction between the mediator molecules and the surface of electrodes.     

Studies on optimization of a platinum catalyst and porphine modified, pyrolytic graphite, amperometric, glucose sensor by sequential level elimination experimental design

A new amperometric glucose sensor based on the glucose oxidase immobilized on pyrolytic graphite (PG) modified with tetraammineplatinum(II) chloride (TAPtCl) and 5,10,15,20-tetrakis (4-methoxy-phenyl)-21H,23H-porphine cobalt(II) (TMPPCo) as well as Nafion was studied. The performances amongst the glucose sensors with or without TAPtCl or/and TMPPCo measured with oxygen present in the solution were compared. The compositions of the membranes of the glucose sensors were optimized by a new orthogonal experimental design technique–sequential level elimination method according to chemometric approaches. Our studies show that the prepared sensor with optimal membrane composition in this study gives satisfactory performance in terms of long-term stability, fast amperometric response, good detection limits and satisfactory recovery. The study provides a useful basis for developing other sensors with corresponding optimal membranes.     

An electrochemiluminescent biosensor for glucose based on the electrochemiluminescence of luminol on the nafion/glucose oxidase/poly(nickel(II)tetrasulfophthalocyanine)/multi-walled carbon nanotubes modified electrode

A poly(nickel(II) tetrasulfophthalocyanine)/multi-walled carbon nanotubes composite modified electrode (polyNiTSPc/MWNTs) was fabricated by electropolymerization of NiTSPc on MWNTs-modified glassy carbon electrode (GCE). The modified electrode was found to be able to greatly improve the emission of luminol electrochemiluminescence (ECL) in a solution containing hydrogen peroxide. Glucose oxidase (GOD) was immobilized on the surface of polyNiTSPc/MWNTs modified GC electrode by Nafion to establish an ECL glucose sensor. Under the optimum conditions, the linear response range of glucose was 1.0 × 10⁻⁶ to 1.0 × 10⁻⁴ mol L⁻¹ with a detection limit of 8.0 × 10⁻⁸ mol L⁻¹ (defined as the concentration that could be detected at the signal-to-noise ratio of 3). The ECL sensor showed an outstanding well reproducibility and long-term stability. The established method has been applied to determine the glucose concentrations in real serum samples with satisfactory results.