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.     

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 novel electrochemiluminescence glucose biosensor based on platinum nanoflowers/graphene oxide/glucose oxidase modified glassy carbon electrode

A novel electrochemiluminescence (ECL) biosensor based on platinum nanoflowers (PtNFs)/graphene oxide (GO)/glucose oxidase (GODx) was discovered for glucose detection. PtNFs/GO was synthesized using a nontoxic, rapid, one-pot and template-free method and characterized by transmission electron microscopy (TEM) and high-resolution TEM techniques. The as-prepared PtNFs/GO with clean surface and multiporous structure was used to assemble GODx to form a glucose biosensor. Based on ECL results, the PtNFs/GO/GODx film-modified electrode displayed a high electrocatalytic activity towards the oxidation of glucose, which generated hydrogen peroxide (H₂O₂) to react with the luminol radicals thus enhanced the luminol ECL. Under the optimized conditions, two linear regions of ECL intensity to glucose concentration were valid in the range from 5 to 80 μmol/L (r?=?0.9957) and 80 to 1,000 μmol/L (r?=?0.9909) with a detection limit (S/N?=?3) of 2.8 μmol/L. In order to verify the reliability, the thus-fabricated biosensor was applied to determine the glucose concentration in glucose injection, glucose functional drink, and blood serum. The results indicated that the proposed biosensor presented good characteristics in terms of high sensitivity and good reproducibility for glucose determination, promising the applicability of this sensor in practical analysis.     

A glucose biosensor using methyl viologen redox mediator on carbon film electrodes

A new methyl viologen-mediated amperometric enzyme electrode sensitive to glucose has been developed using carbon film electrode substrates. Carbon film electrodes from resistors fabricated by pyrolytic deposition of carbon were modified by immobilization of glucose oxidase through cross-linking with glutaraldehyde in the presence of bovine serum albumin. The mediator, methyl viologen, was directly immobilised with the enzyme together with Nafion cation-exchange polymer. The electrochemistry of the glucose oxidase/methyl viologen modified electrode was investigated by cyclic voltammetry and by electrochemical impedance spectroscopy. The biosensor response to glucose was evaluated amperometrically; the detection limit was 20 μM, the linear range extended to 1.2 mM and the reproducibility of around 3%. When stored in phosphate buffer at 4 °C and used every day, the sensor showed good stability over more several weeks.     

A stable glucose biosensor prepared by co-immobilizing glucose oxidase into poly(p-chlorophenol) at a platinum electrode

An amperometric glucose biosensor was successfully developed by electrochemical polymerization of p-chlorophenol (4-CP) at a Pt electrode in the presence of glucose oxidase. The amperometric response of this biosensor to hydrogen peroxide, formed as the product of enzymatic reaction, was measured at a potential of 0.6 V (vs. SCE) in phosphate buffer solution. The performances of sensors, prepared at different monomer concentrations and polymerization potentials, were investigated in detail. The biosensor prepared under optimal conditions had a linear response to glucose ranging from 2.5 x 10(-4) to 1.5 x 10(-2) mol L(-1) with a correlation coefficient of 0.997 and a response time of less than 2 s. Substrate selectivity of the polymer-based enzyme electrode was tested for coexisting interferents such as uric acid and ascorbic acid, and no discernible response was observed. After 90 days, the response of the biosensor remained almost unchanged, indicating very good stability.     

A stable glucose biosensor prepared by co-immobilizing glucose oxidase into poly(p-chlorophenol) at a platinum electrode

An amperometric glucose biosensor was successfully developed by electrochemical polymerization of p-chlorophenol (4-CP) at a Pt electrode in the presence of glucose oxidase. The amperometric response of this biosensor to hydrogen peroxide, formed as the product of enzymatic reaction, was measured at a potential of 0.6 V (vs. SCE) in phosphate buffer solution. The performances of sensors, prepared at different monomer concentrations and polymerization potentials, were investigated in detail. The biosensor prepared under optimal conditions had a linear response to glucose ranging from 2.5 × 10^–4 to 1.5 × 10^–2 mol L^–1 with a correlation coefficient of 0.997 and a response time of less than 2 s. Substrate selectivity of the polymer-based enzyme electrode was tested for coexisting interferents such as uric acid and ascorbic acid, and no discernible response was observed. After 90 days, the response of the biosensor remained almost unchanged, indicating very good stability.     

Biosensors for determination of glucose with glucose oxidase immobilized on an eggshell membrane

A glucose biosensor using an enzyme-immobilized eggshell membrane and oxygen electrode for glucose determination has been fabricated. Glucose oxidase was covalently immobilized on an eggshell membrane with glutaraldehyde as a cross-linking agent. The glucose biosensor was fabricated by positioning the enzyme-immobilized eggshell membrane on the surface of a dissolved oxygen sensor. The detection scheme was based on the depletion of dissolved oxygen content upon exposure to glucose solution and the decrease in the oxygen level was monitored and related to the glucose concentration. The effect of glutaraldehyde concentration, pH, phosphate buffer concentration and temperature on the response of the glucose biosensor has been studied in detail. Common matrix interferents such as ethanol, d-fructose, citric acid, sodium benzoate, sucrose and l-ascorbic acid did not give significant interference. The resulting sensor exhibited a fast response (100 s), high sensitivity (8.3409 mg L⁻¹ oxygen depletion/mmol L⁻¹ glucose) and good storage stability (85.2% of its initial sensitivity after 4 months). The linear response is 1.0×10⁻⁵ to 1.3×10⁻³ mol L⁻¹ glucose. The glucose content in real samples such as commercial glucose injection preparations and wines was determined, and the results were comparable to the values obtained from a commercial glucose assay kit based on a spectrophotometric method.     

Thin-film biosensor for the measurement of glucose concentration in human serum and urine

Solid-state technology and pulse electroplating were used to fabricate a glucose biosensor based on hydrogen peroxide detection. This glucose biosensor was composed of thin-film electrodes, and enzyme-immobilized and deactivated enzyme-immobilized membranes. The electrodes were fabricated by metallic film deposition. Cr and Ni adhesive layers were applied successively by vapour deposition on the thermally oxidized SiO₂ isolating layer on a silicon substrate, and then the two metallic layers were patterned by the photolithographic method. Subsequently, a 1 μm thick Au layer was applied by means of pulse electroplating, forming two anodes and one common cathode in each sensor chip. On one anode, glucose oxidase (GOD) was immobilized by cross-linking with bovin serum albumin and glutaraldehyde. A deactivated GOD-immobilized membrane was formed on the other anode, which worked as a reference working electrode. A novel differential measurement system was used to treat the output signals of the two anodes by adjusting the initial position of the response curves, compensating amplifications of the individual I—V converters and treating the output signals with a subtraction circuit in order to decrease measurement error. The test results showed that the signal of ascorbic acid up to 4.5 mmol 1⁻¹ or uric acid up to 1.2 mmol 1⁻¹ was successfully cancelled. Glucose concentrations in the range 0.02–4.0 mmol/1 could be detected and an excellent linear response was obtained in the low concentration range. The correlation coefficient between the result of the enzyme electrode and the clinically enzymatic method for glucose measurement in human serum was 0.9912. Correlated results between the biosensor method and the routine clinical method for the measurement of glucose concentration in urine were obtained. The lifetime of the enzyme electrode was over 2 months.     

Hydrogen peroxide and glucose biosensor based on silver nanowires synthesized by polyol process

Silver nanowires synthesized through a polyol process using polyvinylpyrrolidone as protection (PVP-AgNWs) were used as a new electrode material for constructing a sensor. Hydrogen peroxide (H(2)O(2)) and glucose were used as analytes to demonstrate the sensor performance of the PVP-AgNWs. It is found that the PVP-AgNWs-modified glassy carbon electrode (PVP-AgNWs/GCE) exhibits remarkable catalytic performance toward H(2)O(2) reduction. This sensor has a fast amperometric response time of less than 2 s and the catalytic current is linear over the concentration of H(2)O(2) ranging from 20 μM to 3.62 mM (R = 0.998) with a detection limit of 2.3 μM estimated on a signal-to-noise ratio of 3. A glucose biosensor was constructed by immobilizing glucose oxidase (GOD) onto the surface of the PVP-AgNWs/GCE. The resultant glucose biosensor can be used for glucose detection in human blood serum with a sensitivity of 15.86 μA mM(-1) cm(-2) and good selectivity and stability.     

Construction of bienzyme biosensors based on combination of the one-step electrodeposition and covalent-coupled sol-gel process

Horseradish peroxidase (HRP) and glucose oxidase (GOD) bienzyme biosensor was constructed by in-situ formation of the organic-inorganic biocomposite film based on the one-step electrodeposition and covalent-coupled sol-gel process. The electrodeposition was performed in the solution containing functional inorganic precursor possessing the epoxy groups, γ-glycidoxypropyltrimethoxysiloxane (GPTMS), a biopolymer chitosan (CS), HRP and GOD. The covalent-coupled sol-gel process was formed by self-hydrolysis and self-condensation of GPTMS, followed by in-situ covalent cross-linking of CS, HRP and GOD through covalent reaction between amino groups and epoxy groups. The developed bienzyme biosensor presented high stability in acidic solution owing to the covalent-coupled organic-inorganic hybridization. Compared with the non-hybrid HRP-GOD/CS/Au electrode, the bienzyme biosensor of HRP-GOD/GPTMS/CS/Au showed improved sensitivity and a wider linear range for the determination of glucose. The linear response of the developed HRP-GOD/GPTMS/CS/Au biosensor for the determination of glucose ranged from 1 to 351 μmol/L with a detection limit of 0.3 μmol/L.     

Enhanced direct electron transfer of glucose oxidase based on a protic ionic liquid modified electrode and its biosensing application

A novel glucose oxidase (GOD) biosensor was fabricated with a protic ionic liquid (PIL) N-ethylimidazolium trifluoromethanesulfonate ([EIm][TfO]) as the modifier of a carbon electrode. Due to the excellent conductivity and the conformational changes of the microenvironment around the GOD, the electrochemical and biocatalytic properties of GOD immobilized on the PIL-based electrode were dramatically enhanced. A couple of well-defined redox peaks could be observed, with a formal potential of −0.476 V. The GOD biosensor presented good catalytic activity to the oxidation of glucose in oxygen-saturated phosphate buffer solutions. The cathodic peak currents of GOD decreased along with glucose concentrations. A linear response in the range 0.005–2.8 mM was obtained with a detection limit of 2.5 μM. The sensitivity and the apparent Michaelis–Menten constant (K _m) were estimated to be 14.96 μA mM⁻¹ and 1.53 μM, respectively. In addition, the biosensor remained stable over 30 days, indicating its good chemical and mechanical stability. The glucose content of several serum samples was determined by using the newly developed biosensor, and the results were in good agreement with those obtained by hospital measurements. All results suggested that PILs were a good media for supporting biocatalytic processes on the bioelectrode.     

Mediatorless amperometric bienzyme glucose biosensor based on horseradish peroxidase and glucose oxidase cross-linked to multiwall carbon nanotubes

We report on a bienzyme-channeling sensor for sensing glucose without the aid of mediator. It was fabricated by cross-linking horseradish peroxidase (HRP) and glucose oxidase (GOx) on a glassy carbon electrode modified with multiwalled carbon nanotubes (MWNTs). The bienzyme was cross-linked with the MWNTs by glutaraldehyde and bovine serum albumin. The MWNTs were employed to accelerate the electron transfer between immobilized HRP and electrode. Glucose was sensed by amperometric reduction of enzymatically generated H₂O₂ at an applied voltage of ?50 mV (vs. Ag/AgCl). Factors influencing the preparation and performance of the bienzyme electrode were investigated in detail. The biosensor exhibited a fast and linear response to glucose in the concentration range from 0.4 to 15 mM, with a detection limit of 0.4 mM. The sensor exhibited good selectivity and durability, with a long-term relative standard deviation of <5 %. Analysis of glucose-spiked human serum samples yielded recoveries between 96 and 101 %.

Bienzymatic amperometric biosensor for glucose based on polypyrrole/ceramic carbon as electrode material

A novel amperometric biosensor utilizing two enzymes, glucose oxidase (GOD) and horseradish peroxidase (HRP), was developed for the cathodic detection of glucose. The glucose biosensor was constructed by electrochemical formation of a polypyrrole (PPy) membrane in the presence of GOD on the surface of a HRP-modified sol-gel derived-mediated ceramic carbon electrode. Ferrocenecarboxylic acid (FCA) was used as mediator to transfer electron between enzyme and electrode. In the hetero-bilayer configuration of electrode, all enzymes were well immobilized in electrode matrices and showed favorable enzymatic activities. The amperometric detection of glucose was carried out at +0.16 V (versus saturated calomel reference electrode (SCE)) in 0.1 M phosphate buffer solution (pH 6.9) with a linear response range between 8.0×10⁻⁵ and 1.3×10⁻³ M glucose. The biosensor showed a good suppression of interference in the amperometric detection.     

以天青Ⅰ为介体的纳米金颗粒增强的葡萄糖传感器

采用层层自组装的方法和异种电荷互相吸引的原理,将Nafion修饰在金电极上固载带正电荷的天青Ⅰ,并利用天青Ⅰ中的氨基固载纳米金,再通过纳米金将酶固定在金电极表面,制成了葡萄糖传感器.采用循环伏安法和交流阻抗法,研究了金电极表面组装各层之后的电化学特征,以及电极对葡萄糖的电化学催化作用. 结果表明,天青Ⅰ不仅可以固定酶和纳米金,而且还可以在酶和电极之间有效地传递电子.在优化的实验条件下,该传感器对葡萄糖响应的线性范围为5.1×10-6 ~4.0×10-3 mol/L,检出限(S/N=3)为1.0 μmol/L.该生物传感器显示出较好的稳定性和抗干扰能力,将其用于人体血清中葡萄糖的测定,结果令人满意.     

Amperometric glucose sensor using tetrathiafulvalene in Nafion gel as electron shuttle

An amperometric mediated sensor for glucose has been contrived by using bovine serum albumin and glutaraldehyde to immobilize glucose oxidase on a Nafion-tetrathiafulvalene (TTF) modified electrode. It is further coated by Nafion. The inner Nafion membrane can prevent leaking of tetrathiafulvalene; the outer Nafion film serves as a barrier to electroactive anionic interferents such as ascorbate and urate and protects the biosensor from fouling agents. The experiment shows that TTF⁺ and TTF²⁺ can oxidize the reduced flavin adenine dinucleotide (FADH₂) of glucose oxidase. The biosensor responds to glucose in less than 50 s and its calibration curve is linear from 3.0 × 10⁻⁴ to l.0 × 10⁻² M.     

表面可更新的石墨陶瓷葡萄糖生物传感器研究

A surface-renewable glucose biosensor is reported. The glucose biosensor is developed using glucose oxidase (GOD) encapsulated in organically modified solgel glass (ORMOSIL) network in the composite matetial. The organic group in the ORMOSIL network controls the hydrophobicity of the electrode surface and thus limits the wettability of the electrode surface. The graphite powder provides the conductivity for the electrode.Ferrocenecarboxylic acid in phosphate buffer solution (pH 7.0) transfers electron between enzyme and electrode. Cyclic volammetry and amperometric measurements have been used to exmine the electrochemical behavior of glucose biosensor as shown in Fig. 1 and Fig.2. The electrode gives a linear response range of 1 -20mM glucose with a sensitivity of 3.26 μA· mM-1. The electrode can be renewed easily in reproducible manner by a simple polishing step.     

Novel glucose biosensor based on a glassy carbon electrode modified with hollow gold nanoparticles and glucose oxidase

A novel glucose biosensor is presented as that based on a glassy carbon electrode modified with hollow gold nanoparticles (HGNs) and glucose oxidase. The sensor exhibits a better differential pulse voltammetric response towards glucose than the one based on conventional gold nanoparticles of the same size. This is attributed to the good biological conductivity and biocompatibility of HGNs. Under the optimal conditions, the sensor displays a linear range from 2.0?×?10^?6 to 4.6?×?10^?5?M of glucose, with a detection limit of 1.6?×?10^?6?M (S/N?=?3). Good reproducibility, stability and no interference make this biosensor applicable to the determination of glucose in samples such as sports drinks.

Development of an amperometric sulfite biosensor based on a gold nanoparticles/chitosan/multiwalled carbon nanotubes/polyaniline-modified gold electrode

A sulfite oxidase (SOx) purified from leaves of Syzygium cumini (Jamun) was immobilized covalently onto a gold nanoparticles (AuNPs)/chitosan (CHIT)/carboxylated multiwalled carbon nanotubes (cMWCNTs)/polyaniline (PANI) composite that was electrodeposited onto the surface of a gold (Au) electrode. A novel and highly sensitive sulfite biosensor was developed that used this enzyme electrode (SOx/AuNPs/CHIT/cMWCNT/PANI/Au) as the working electrode, Ag/AgCl as the standard electrode, and Pt wire as the auxiliary electrode. The modified electrode was characterized by Fourier transform infrared (FTIR) spectroscopy, cyclic voltammetry (CV), scanning electron microscopy (SEM), and electrochemical impedance spectroscopy (EIS) before and after the immobilization of the SOx. The sensor produced its optimum response within 3 s when operated at 50 mVs⁻¹ in 0.1 M phosphate buffer, pH 7.0, and at 35 °C. The linear range and detection limit of the sensor were 0.75–400 μM and 0.5 μM (S/N = 3), respectively. The biosensor was employed to determine sulfite levels in fruit juices and alcoholic beverages. The enzyme electrode was used 300 times over a period of three months when stored at 4 °C.     

Automated determination of glucose in soluble coffee using Prussian Blue-glucose oxidase-Nafion((R)) modified electrode

A highly selective, fast and stable biosensor for determination of glucose in soluble coffee has been developed. The biosensor electrode consist of a thin film of ferric hexacyanoferrate (Prussian Blue or PB) electrodeposited on the glassy carbon electrode (GCE) (to provide a catalytic surface for the detection of hydrogen peroxide) glucose oxidase immobilized on top of the electrode and a Nafion^® polymer layer. The stability of the PB film and the biosensor was evaluated by injecting standard-solution (50 μM H₂O₂ and 0.5 mM glucose) during 4 h in a flow-injection system with the electrodes polarized at −50 mV versus Ag/AgCl. The system is able to handle about 60 samples per hour and is very stable and suitable for industrial control. Determination of glucose in the range 2.5 and 15% (w/v) in phosphate buffer with precision (r.s.d. < 1.5%) has been achieved and is in agreement with the conventional procedures. Linear calibration in the range of 0.15 and 2.50 mM with detection limits of ca. 0.03 mM has been obtained. The morphology of the enzyme glucose oxidase on the modified electrode has been analyzed by scanning electron microscopy (SEM) measurements.     

Glucose biosensor based on a platinum electrode modified with rhodium nanoparticles and with glucose oxidase immobilized on gold nanoparticles

We have developed an enzymatic glucose biosensor that is based on a flat platinum electrode which was covered with electrophoretically deposited rhodium (Rh) nanoparticles and then sintered to form a large surface area. The biosensor was obtained by depositing glucose oxidase (GOx), Nafion, and gold nanoparticles (AuNPs) on the Rh electrode. The electrical potential and the fractions of Nafion and GOx were optimized. The resulting biosensor has a very high sensitivity (68.1 μA mM^?1 cm^?2) and good linearity in the range from 0.05 to 15 mM (r?=?0.989). The limit of detection is as low as 0.03 mM (at an SNR of 3). The glucose biosensor also is quite selective and is not interfered by electroactive substances including ascorbic acid, uric acid and acetaminophen. The lifespan is up to 90 days. It was applied to the determination of glucose in blood serum, and the results compare very well with those obtained with a clinical analyzer.