Glucose biosensors based on the immobilization of copper oxide and glucose oxidase within a carbon paste matrix

The performance of amperometric glucose biosensors based on the dispersion of glucose oxidase (GOx) and copper oxide within a classical carbon (graphite) paste composite is reported in this work. Copper oxide promotes an excellent electrocatalytic activity towards the oxidation and reduction of hydrogen peroxide, allowing a large decrease in the oxidation and reduction overpotentials, as well as an important enhancement of the corresponding currents. Therefore, it is possible to perform the glucose biosensing at low potentials where there is no interference even in large excess of ascorbic acid, uric acid or acetaminophen. The influence of the copper oxide and glucose oxidase content in the paste on the analytical performance of the bioelectrode is discussed. The resulting biosensor shows a fast response, a linear relationship between current and glucose concentration up to 1.35 × 10⁻² M (2.43 g L⁻¹) and a detection limit of 2.0 × 10⁻⁵ M. The effect of the presence of the enzyme in the composite material on the dispersion of the copper oxide particles is also discussed.     

Biocatalytic anode for glucose oxidation utilizing carbon nanotubes for direct electron transfer with glucose oxidase

Covalently linked layers of glucose oxidase, single-wall carbon nanotubes and poly-l-lysine on pyrolytic graphite resulted in a stable biofuel cell anode featuring direct electron transfer from the enzyme. Catalytic response observed upon addition of glucose was due to electrochemical oxidation of FADH₂ under aerobic conditions. The electrode potential depended on glucose concentration. This system has essential attributes of an anode in a mediator-free biocatalytic fuel cell.     

Investigation of the origin of the glucose response in a glucose oxidase|polyaniline system

The origin of the signal seen in response to glucose in a polyaniline|glucose oxidase system is explored by immittance spectroscopy, by comparing data from an equivalent circuit model and the parameters obtained from a solution of the faradaic branch of the frequency dispersion for a coupled chemical—electrochemical reaction mechanism. It was shown that an RC subcircuit in the equivalent circuit model was sensitive to peroxide concentration, and the interaction of peroxide with polyaniline at potentials where it either oxidised or reduced the polyaniline was discussed. This information was used to compare the data obtained in a bulk and entrapped glucose oxidaselglucose system, and it was seen that the origin of the response could not be fully attributed to peroxide interaction in the latter case. Under anaerobic conditions with entrapped enzyme, it was proposed that a complex between the gluconolactone product of the enzyme reaction and the polymer leads to a more conducting polymer, with inherent charge compensation, and this results in the observed enhanced current signal.     

Fiber-optic glucose biosensor based on glucose oxidase immobilised in a silica gel matrix

A monolithic silica gel matrix with entrapped glucose oxidase was constructed as a bioactive element in an optical biosensor for glucose determination. Physicochemical and biochemical characterizations of the catalytic matrix were performed, and the intrinsic fluorescence of immobilised glucose oxidase (GOD) was investigated in the UV and visible range by performing steady state and time course measurements. In all cases, the silica gel matrix proved to be a suitable support for optical biosensing owing to its superior optical properties (e.g., high transmittance and reliable fluorescence and GOD absorption spectra after immobilisation). From steady state measurements, calibration curves were obtained as a function of glucose concentration. When time course measurements were performed, the silica gel support displayed a larger linear calibration range and higher sensitivity than other immobilisation systems. In addition, a glucose optical biosensor was developed and characterised using as catalytic element GOD immobilised on a gel disk bound to a bundle of optical fibres.     

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.     

Native and modified glucose oxidase in reversed micelles

The enzymatic activity of the native and modified glucose oxidase (GOx) from Aspergillus niger in the system of reversed micelles of Aerosol OT in octane was investigated. Two forms of the modified enzyme were studied: a hydrophobized form obtained by the attachment of palmitic chains to lysine amino groups by the reaction with palmitic acid ester of N-hydroxysuccinimide and a glycosylated (hydrophilized) form obtained by the attachment of the cellobiose moieties. The native glucose oxidase and its derivatives, while incorporated into micelles in a surfactant concentration range from 0.05 to 0.3 M, display an enzymatic activity, which is comparable with the activity in aqueous solution. The dependence of the enzymatic activity on hydration degree of surfactant (the molar ratio of water to surfactant, W₀) does not indicate the formation of qualitatively new associated forms of the enzyme subunits inside the micelles. The apparent size of Aerosol OT micelles obtained by dynamic light scattering gradually increases from 10±3 nm at low W₀ up to 25±5 nm at high W₀. Incorporation of the native and hydrophobized glucose oxidase into micelles does not affect their mean size. Kinetic analysis shows that the enzyme specificity is about an order of magnitude greater in the system of reversed micelles as compared with aqueous solution.     

Oxidation of glucose to gluconic acid by glucose oxidase in a membrane bioreactor

Glucose oxidase (GO) (EC 1.1.3.4) was used as catalyst for oxidizing glucose into gluconic acid utilizing a 10-mL Bioengineering Enzyme Membrane Reactor® or a 400-mL Millipore Stirred Ultrafiltration Cell (MSUC) coupled with a Millipore UF membrane (cutoff of 100 kDa) and operated for 12 h under an agitation of 100 rpm, pH 5.5, and 30°C. The effect of feeding rate (0.10, 0.15, or 0.20 min^?1), glucose (2.5 or 5.0 mM), and GO (1.0 or 2.0 mg/mL) concentrations on the catalysis were studied. A yield of about 75% was attained when the MSUC filled with 1.0 mg/mL of GO was fed with 2.5 mM glucose solution at a rate of 0.15 min^?1.     

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.     

Stereoselective synthesis of azasugars by electrochemical oxidation

A new method using electrochemical oxidation has been exploited for the stereoselective synthesis of 2,3,6-trihydroxylated 5S-piperidine derivatives. The electrochemical method was successively used for the conversion of N-protected piperidines to N-protected 1-methoxypiperidines and for the conversion of 1-methoxy-2,3-didehydropiperidine derivatives to 1,2,3-triacetoxypiperidine derivatives. The method provided a new synthetic route to 2S,3S,6-triacetoxy-5S-methylpiperidine and 2R,3R,6-triacetoxy-5S-methylpiperidine.     

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.     

Recent progress in electrochemical oxidation of saccharides at gold and copper electrodes in alkaline solutions

This article reviews the progress made in the past 10 years, on electrochemical oxidation of saccharides in alkaline media for gold and copper electrodes. The mechanism and processes associated with the electrochemical oxidation of saccharides at native and surface coated electrodes continues to be of great interest. Despite the effort and various mechanisms proposed, still the need for an electrochemically active material that understands the complexity associated with saccharides continues to increase as their detection poses a challenge for bioanalytical chemistry and liquid chromatography.     

Integration of a highly ordered gold nanowires array with glucose oxidase for ultra-sensitive glucose detection

A highly sensitive amperometric nanobiosensor has been developed by integration of glucose oxidase (GO_x) with a gold nanowires array (AuNWA) by cross-linking with a mixture of glutaraldehyde (GLA) and bovine serum albumin (BSA). An initial investigation of the morphology of the synthesized AuNWA by field emission scanning electron microscopy (FESEM) and field emission transmission electron microscopy (FETEM) revealed that the nanowires array was highly ordered with rough surface, and the electrochemical features of the AuNWA with/without modification were also investigated. The integrated AuNWA–BSA–GLA–GO_x nanobiosensor with Nafion membrane gave a very high sensitivity of 298.2 μA cm⁻² mM⁻¹ for amperometric detection of glucose, while also achieving a low detection limit of 0.1 μM, and a wide linear range of 5–6000 μM. Furthermore, the nanobiosensor exhibited excellent anti-interference ability towards uric acid (UA) and ascorbic acid (AA) with the aid of Nafion membrane, and the results obtained for the analysis of human blood serum indicated that the device is capable of glucose detection in real samples.     

Multicommutated flow system for the determination of glucose in honey with immobilized glucose oxidase reactor and spectrophotometric detection

A new automated method for the determination of glucose in honey is proposed. The method is based on multicommutated flow analysis (MCFA) and employs an immobilized glucose oxidase reactor and spectrophotometric detection at 505 nm of the red quinoneimine formed (Trinder's method).The calibration curve obeyed a second order equation in the range 0-0.14 g L⁻¹ (h = −2.2199 C² + 1.3741C + 0.0077, r² = 0.9991, where h is the peak height (absorbance) and C the concentration in g L⁻¹). The method was validated analyzing eight commercial samples, both by the AOAC 954.11 and 977.20 official methods. According to Student's t-test of mean values, at the confidence level of 95% the results obtained with the proposed method were in agreement with those obtained by the official methods. Precision (s_r(%), n = 10) was 3% and the sampling frequency of the system was 20 samples h⁻¹.     

脂质体为模板仿生硅化固定葡萄糖氧化酶

将脂质体囊泡与仿生硅化技术相结合,模拟细胞纳微环境,实现以脂质体为模板仿生制备氧化硅固定葡萄糖氧化酶(GOx),建立性能稳定的固定化酶.扫描电镜分析显示,固定化GOx为球形纳米粒子,粒径分布在200nm左右,在优化反应条件下GOx回收率达到71.8%.由于载体的空间限制作用及其提供的较稳定微环境,固定化GOx表现出良好的热稳定性和pH稳定性,其对变性剂耐受性和操作稳定性等也得到明显提高.     

Simultaneous detection of uric acid and glucose on a dual-enzyme chip using scanning electrochemical microscopy/scanning chemiluminescence microscopy

Scanning electrochemical microscopy (SECM) and scanning chemiluminescence microscopy (SCLM) were used for imaging an enzyme chip with spatially-addressed spots for glucose oxidase (GOD) and uricase microspots. For the SECM imaging, hydrogen peroxide generated from the GOD and/or uricase spots was directly oxidized at the tip microelectrode in a solution containing glucose and/or uric acid (electrochemical (EC) detection). For the SCLM imaging, a tapered glass capillary (i.d. of 1∼2 μm) filled with luminol and horseradish peroxidase (HRP) was used as the scanning probe for generating the chemiluminescence (CL). The inner solution was injected from the capillary tip at 78 pl s⁻¹ while scanning above the enzyme-immobilized chip. The CL generated when the capillary tip was scanned above the enzyme spots was detected using a photon-counter (CL detection). Two-dimensional mapping of the oxidation current and photon-counting intensity against the tip position affords images of which their contrast reflects the activity of the immobilized GOD and uricase. For both the EC and CL detections, the signal responses were plotted as a function of the glucose and uric acid concentrations in solution. The sensitivities for the EC and CL detection were found to be comparable.     

Direct electron transfer reaction of glucose oxidase at bare silver electrodes and its application in analysis

The direct electron transfer reaction of glucose oxidase (GOx) at a bare silver electrode is verified. The electron transfer number n = 2, electron transfer coefficient α = 0.45 and rate constant of the electrochemical reaction K_s = 0.1 s⁻¹ are obtained. This communication presents a multimolecular adsorption model to explain the properties of the direct electron reaction between GOx and bare silver electrodes. The residual valence force may be an important factor to ensure a direct electron transfer reaction on the bare electrode. On the basis of the experimental fact that only biologically active GOx exhibits electrochemical activity in solution, a facile analytical method for analyzing the active GOx concentration is developed. The results determined correspond very well to that of a spectrometric method.     

Modification of polypyrrole nanowires array with platinum nanoparticles and glucose oxidase for fabrication of a novel glucose biosensor

A novel glucose biosensor, based on the modification of well-aligned polypyrrole nanowires array (PPyNWA) with Pt nanoparticles (PtNPs) and subsequent surface adsorption of glucose oxidase (GOx), is described. The distinct differences in the electrochemical properties of PPyNWA–GOx, PPyNWA–PtNPs, and PPyNWA–PtNPs–GOx electrodes were revealed by cyclic voltammetry. In particular, the results obtained for PPyNWA–PtNPs–GOx biosensor showed evidence of direct electron transfer due mainly to modification with PtNPs. Optimum fabrication of the PPyNWA–PtNPs–GOx biosensor for both potentiometric and amperometric detection of glucose were achieved with 0.2 M pyrrole, applied current density of 0.1 mA cm⁻², polymerization time of 600 s, cyclic deposition of PtNPs from −200 mV to 200 mV, scan rate of 50 mV s⁻¹, and 20 cycles. A sensitivity of 40.5 mV/decade and a linear range of 10 μM to 1000 μM (R² = 0.9936) were achieved for potentiometric detection, while for amperometric detection a sensitivity of 34.7 μA cm⁻² mM⁻¹ at an applied potential of 700 mV and a linear range of 0.1–9 mM (R² = 0.9977) were achieved. In terms of achievable detection limit, potentiometric detection achieved 5.6 μM of glucose, while amperometric detection achieved 27.7 μM.     

苯甲酰肼在多壁碳纳米管修饰玻碳电极上的电催化氧化及其电化学动力学性质

研究了苯甲酰肼(BH)在MWCNT/GCE上的电化学行为。实验结果表明,BH在GCE上的直接电化学氧化十分迟缓,无氧化峰出现,但在MWCNT/GCE上BH在0.20 V处出现了一个不可逆氧化峰,且峰电流大幅度增大,表明MWCNT/GCE对BH电化学氧化具有良好的催化作用。同时用计时库仑法(Chro-nocoulometry,CC)和计时电流法(Chronoamperometry,CA)测定了电极过程动力学参数:扩散系数D=8.73×10-5cm2.s-1,电子转移系数α=0.85,电极反应速率常数kf=1.45×10-3s-1。稳态电流-时间实验结果表明,电流响应信号随其浓度成比例增长,响应时间小于6 s,最低响应浓度为1×10-6mol/L。该方法可用于BH电化学定量测定。     

Direct electrochemistry of glucose oxidase based on its direct immobilization on carbon ionic liquid electrode and glucose sensing

Direct electrochemistry of glucose oxidase (GOx) has been achieved by its direct immobilization on carbon ionic liquid electrode (CILE) with a conductive hydrophobic ionic liquid, 1-butyl pyridinium hexafluophosphate ([BuPy][PF₆]) as binder for the first time. A pair of reversible peaks is exhibited on GOx/CILE by cyclic voltammetry. The peak-to-peak potential separation (ΔE_P) of immobilized GOx is 0.056 V in 0.067 M phosphate buffer solution (pH 6.98) with scan rate of 0.1 V/s. The average surface coverage and the apparent Michaelis–Menten constant are 6.69 × 10⁻¹¹ mol·cm⁻² and 2.47 μM. GOx/CILE shows excellent electrocatalytic activity towards glucose determination in the range of 0.1–800 μM with detection limit of 0.03 μM (S/N = 3). The biosensor has been successfully applied to the determination of glucose in human plasma with the average recoveries between 95.0% and 102.5% for three times determination. The direct electrochemistry of GOx on CILE is achieved without the help of any supporting film or any electron mediator. GOx/CILE is inexpensive, stable, repeatable and easy to be fabricated.     

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).