Enzymatic activity of glucose oxidase covalently wired via viologen to electrically conductive polypyrrole films

The surface functionalization of an electrically conductive polypyrrole film (PPY) with a viologen, (N-(2-carboxyl-ethyl)-N'-(4-vinyl-benzyl)-4,4'-bipyridinium dichloride, or CVV) for the covalent immobilization of glucose oxidase (GOD) has been carried out. The viologen was first synthesized and graft polymerized on PPY film. It then served as an anchor via its carboxyl groups for the covalent immobilization of GOD. The surface composition of the as-functionalized substrates was characterized by X-ray photoelectron spectroscopy (XPS). The effects of the CVV monomer concentration on the CVV-graft polymer concentration and the amount of GOD immobilized on the surface were investigated. The activity of the immobilized GOD was compared with that of free GOD and the kinetic effects were also obtained. The cyclic voltammetric (CV) response of the GOD-functionalized PPY substrates was studied in a phosphate buffer solution under an argon atmosphere. The CV results support the mechanism in which CVV acts as a mediator to transfer electron between the electrode and enzyme, and hence regenerating the enzyme in the enzymatic reaction with glucose. High sensitivity and linear response of the enzyme electrode was observed with glucose concentration ranging from 0 to 20 mM.     

Direct electrochemistry of glucose oxidase and biosensing for glucose based on boron-doped carbon nanotubes modified electrode

Due to their unique physicochemical properties, doped carbon nanotubes are now extremely attractive and important nanomaterials in bioanalytical applications. In this work, selecting glucose oxidase (GOD) as a model enzyme, we investigated the direct electrochemistry of GOD based on the B-doped carbon nanotubes/glassy carbon (BCNTs/GC) electrode with cyclic voltammetry. A pair of well-defined, quasi-reversible redox peaks of the immobilized GOD was observed at the BCNTs based enzyme electrode in 0.1M phosphate buffer solution (pH 6.98) by direct electron transfer between the protein and the electrode. As a new platform in glucose analysis, the new glucose biosensor based on the BCNTs/GC electrode has a sensitivity of 111.57 microA mM(-1)cm(-2), a linear range from 0.05 to 0.3mM and a detection limit of 0.01mM (S/N=3). Furthermore, the BCNTs modified electrode exhibits good stability and excellent anti-interferent ability to the commonly co-existed uric acid and ascorbic acid. These indicate that boron-doped carbon nanotubes are the good candidate material for the direct electrochemistry of the redox-active enzyme and the construction of the related enzyme biosensors.     

Direct electrochemistry and electrocatalysis of glucose oxidase immobilized on glassy carbon electrode modified by Nafion and ordered mesoporous silica-SBA-15

In this paper, it was found that glucose oxidase (GOD) has been stably immobilized on glassy carbon electrode modified by ordered mesoporous silica-SBA-15 and Nafion. The sorption behavior of GOD immobilized on SBA-15 matrix was characterized by transmission electron microscopy (TEM), ultraviolet–visible (UV–vis), FTIR, respectively, which demonstrated that SBA-15 can facilitate the electron exchange between the electroactive center of GOD and electrode. The direct electrochemistry and electrocatalysis behavior of GOD on modified electrode were characterized by cyclic voltammogram (CV) which indicated that GOD immobilized on Nafion and SBA-15 matrices displays direct, nearly reversible and surface-controlled redox reaction with an enhanced electron transfer rate constant of 3.89 s⁻¹ in 0.1 M phosphate buffer solution (PBS) (pH 7.12). Furthermore, it was also discovered that, in the absence of O₂, GOD immobilized on Nafion and SBA-15 matrices can produce a wide linear response to glucose in the positive potential range. Thus, Nafion/GOD-SBA-15/GC electrode is hopeful to be used in the third non-mediator's glucose biosensor. In addition, GOD immobilized on SBA-15 and Nafion matrices possesses an excellent bioelectrocatalytic activity for the reduction of O₂. The Nafion/GOD-SBA-15/GC electrode can be utilized as the cathode in biofuel cell.     

A novel glucose biosensor based on the nanoscaled cobalt phthalocyanine-glucose oxidase biocomposite

We report on the utilization of a novel nanoscaled cobalt phthalocyanine (NanoCoPc)-glucose oxidase (GOD) biocomposite colloid to create a highly responsive glucose biosensor. The biocomposite colloid is constructed under enzyme-friendly conditions by adsorbing GOD molecules on CoPc nanoparticles via electrostatic interactions. The glucose biosensor can be easily achieved by casting the biocomposite colloid on a pyrolytic graphite electrode (PGE) without any auxiliary matter. It has been found that GOD can be firmly immobilized on PGE surface and maintain its bioactivity after conjugating with NanoCoPc. NanoCoPc displays intrinsic electrocatalytic ability to the oxidation of the product of enzymatic reaction H2O2 and shows a higher catalytic activity than that of bulk CoPc. Under optimal conditions, the biosensor shows a wide linear response to glucose in the range of 0.02-18 mM, with a fast response (5s), high sensitivity (7.71 microA cm(-2) mM(-1)), as well as good thermostability and long-term life. The detection limit was 5 microM at 3 sigma. The general interferences coexisted in blood except ascorbic acid and L-cysteine do not affect glucose determination, and further coating Nafion film on the surface of the biosensor can effectively eliminate the interference from ascorbic acid and L-cysteine. The biosensor with Nafion film has been used for the determination of glucose in serum with an acceptable accuracy.     

Miniaturized multi-enzyme biosensors integrated with pH sensors on flexible polymer carriers for in vivo applications

An electrochemical glucose sensor has been integrated, together with a pH sensor, on a flexible polyimide substrate for in vivo applications. The glucose sensor is based on the measurement of H₂O₂ produced by the membrane-entrapped enzyme glucose oxidase (GOD). To minimize electrochemical interference, an electrode configuration was designed to perform differential measurements. The solid-state pH sensor employs a PVC-based neutral carrier membrane. The enzymes GOD and catalase were immobilized into two layers of photolithographically patterned hydrogels. The intended use of this device is the short-term monitoring of glucose and pH in intensive care units and operating theatres, especially for neurosurgical applications. The developed immobilization technique can also be used to create integrated multi-sensor chips for clinical analysers. The glucose and pH sensor exhibited excellent performance during tests in buffer solutions, serum and whole blood.     

A glucose oxidase electrode based on polypyrrole with polyanion/PEG/enzyme conjugate dopant

This study investigated a new glucose sensor prepared by electrochemical polymerization of pyrrole with polyanion/poly(ethylene glycol) (PEG)/glucose oxidase (GOD) conjugate dopants. GOD was coupled to a strong polyanion, poly(2-acrylamido-2-methylpropane sulfonic acid) (AMPS) via PEG spacer to effectively and reproducibly immobilize GOD within a polypyrrole matrix onto a Pt electrode surface. PEGs with four different chain lengths (1000, 2000, 3000, and 4000) were used as spacers to study the spacer length effect on enzyme immobilization and electrode function. After conjugation, more than 90% of the GOD bioactivity was preserved and the bioactivity of the conjugated GOD increased with longer PEG spacers. The resulting polyanion/PEG/GOD conjugate was used as a dopant for electropolymerizing pyrrole. The activity of the immobilized enzyme on the electrode ranged from 119 to 209 mU cm(-2) and the bioactivity increased with the use of longer PEG spacers. The amperometric response of the enzyme electrode was linear up to 20 mM glucose concentration with a sensitivity ranging from 180 to 270 nA mM(-1) cm(-2). The kinetic parameters Michaelis-Menten constant (K(M)(app)) and maximum current density (j(max)) depended on the amount of active enzyme, level of substrate diffusion, and PEG spacer length. An increase in the electrical charge passed during polymerization (thus, increasing polypyrrole thickness) to 255 mC cm(-2) increased the sensitivity of the enzyme electrode because of the greater amount of incorporated enzyme. However, although the amount of incorporated GOD continued to increase when the charge increased above 255 mC cm(-2), the sensitivity began to decline gradually. The condition for preparing the enzyme electrode was optimized at 800 mV potential with a dopant concentration of 1 mg ml(-1).     

Glucose oxidase enzyme immobilized porous silica for improved performance of a glucose biosensor

High activity of glucose oxidase (GOD) enzyme (immobilized in porous silica particles) is desirable for a better glucose biosensor. In this work, effect of pore diameter of two porous hosts on enzyme immobilization, activity and glucose sensing was compared. The hosts were amine functionalized: (i) microporous silica (NH₂-MS) and (ii) mesoporous silica (NH₂-SBA-15). Based on whether the dimension of GOD is either larger or smaller than the pore diameter, GOD was immobilized on either external or internal surface of NH₂-MS and NH₂-SBA-15, with loadings of 512.5 and 634 mg/g, respectively. However, GOD in NH₂-SBA-15 gave a higher normalized absolute activity (NAA), which led to an amperometric sensor with a larger linear range of 0.4–13.0 mM glucose. In comparison, GOD in NH₂-MS had a lower NAA and a smaller linear range of 0.4–3.1 mM. In fact, the present GOD-NH₂-SBA-15 electrode based sensor was better than other MS and SBA-15 based electrodes reported in literature. Thus, achieving only a high GOD loading (as in NH₂-MS) does not necessarily give a good sensor performance. Instead, a host with a relatively larger pore than enzyme, together with optimized electrode composition ensures the sensor to be functional in both hyper- and hypoglycemic range.     

Enzyme immunosensors based on electropolymerized polytyramine modified electrodes

Highly sensitive amperometric enzyme immunosensors for human immunoglobulin G (IgG) were prepared on the basis of electrogenerated polytyramine (PTy, tyramine = p-(2-aminoethyl)-phenol) modified electrodes. Properties of PTy films changed depending on electrolysis conditions. On the basis of the found properties of the films, an effective IgG sensor was prepared: a PTy film was formed first from an acid solution on a Pt electrode, and the surface was further covered with a PTy film from an alkaline methanol solution to give a PTy doubly coated electrode on which anti-IgG was then immobilized. This electrode provided a large surface area with little non-specific adsorption of proteins. By means of the competitive enzyme immunoassay technique using glucose oxidase (GOD) labeled IgG conjugates, IgG was determined in the concentration range of c. 10 pg/ml-1 mg/ml from the oxidation current of H2O2 generated by the enzyme (GOD) reaction using the above IgG sensor. Also, an anti-IgG immobilized electrode, prepared by using a Pt electrode singly covered with a PTy film from an alkaline methanol solution, acted as an effective IgG sensor with a detection limit for IgG of c. 100 pg/ml.     

Amperometric glucose biosensor based on gold-deposited polyvinylferrocene film on Pt electrode

The preparations and performances of the novel amperometric biosensors for glucose based on immobilized glucose oxidase (GOD) on modified Pt electrodes are described. Two types of modified electrodes for the enzyme immobilization were used in this study, polyvinylferrocene (PVF) coated Pt electrode and gold deposited PVF coated Pt electrode. A simple method for the immobilization of GOD enzyme on the modified electrodes was described. The enzyme electrodes developed in this study were called as PVF-GOD enzyme electrode and PVF-Au-GOD enzyme electrode, respectively. The amperometric responses of the enzyme electrodes were measured at constant potential, which was due to the electrooxidation of enzymatically produced H2O2. The electrocatalytic effects of the polymer, PVF, and the gold particles towards the electrooxidation of the enzymatically generated H2O2 offers sensitive and selective monitoring of glucose. The biosensor based on PVF-Au-GOD electrode has 6.6 times larger maximum current, 3.8 times higher sensitivity and 1.6 times larger linear working portion than those of the biosensor based on PVF-GOD electrode. The effects of the applied potential, the thickness of the polymeric film, the amount of the immobilized enzyme, pH, the amount of the deposited Au, temperature and substrate concentration on the responses of the biosensors were investigated. The optimum pH was found to be pH 7.4 at 25 degrees C. Finally the effects of interferents, stability of the biosensors and applicability to serum analysis of the biosensor were also investigated.     

Characterization of thin layers of glucose oxidase

Glucose Oxidase (GOD) has been covalently bound to functionalized glass cover slips. The surface density of immobilized GOD molecules was measured by a method based on the amperometric determination of Flavin Adenine Dinucleotide (FAD). Atomic Force Microscopy (AFM) images, obtained in aqueous solution for the covalently bound enzyme, show a monomolecular layer of the enzyme on a functionalized glass surface. The catalytic constants were measured for the immobilized GOD and compared with those of the free enzyme.     

On-line determination of glucose concentration throughout animal cell cultures based on chemiluminescent detection of hydrogen peroxide coupled with flow-injection analysis.

A flow-injection analysis (FIA) system for the on-line determination of glucose in animal cell cultures is described. The system is based on immobilized glucose oxidase (GOD). The hydrogen peroxide generated in the enzyme reaction is determined via a highly sensitive chemiluminescent reaction with luminol. Based on the measurement of the maximum emitted light intensity, the system was able to analyse hydrogen peroxide over the concentration range of 10(-7) to 10(-2) M. For glucose determination, the system has a linear range of 10(-5) to 5 x 10(-2) M glucose, with an r.s.d. of 3% at the 1 mM level (5 measurements). The influence of luminol and buffer concentrations, pH and temperature on the chemiluminescent reaction were investigated. The enzyme reactor used was stable for more than 4 weeks in continuous operation, and it was possible to analyse up to 20 samples per h. The system has been successfully applied to on-line monitoring of glucose concentration during an animal cell culture, designed for the production of human antithrombin III factor. Results obtained with the FIA system were compared with off-line results, obtained with a Yellow Springs Instrument Company Model 27 (YSI).     

Amperometric glucose biosensor based on adsorption of glucose oxidase at platinum nanoparticle-modified carbon nanotube electrode

A new amperometric biosensor, based on adsorption of glucose oxidase (GOD) at the platinum nanoparticle-modified carbon nanotube (CNT) electrode, is presented in this article. CNTs were grown directly on the graphite substrate. The resulting GOD/Pt/CNT electrode was covered by a thin layer of Nafion to avoid the loss of GOD in determination and to improve the anti-interferent ability. The morphologies and electrochemical performance of the CNT, Pt/CNT, and Nafion/GOD/Pt/CNT electrodes have been investigated by scanning electron microscopy, cyclic voltammetry, and amperometric methods. The excellent electrocatalytic activity and special three-dimensional structure of the enzyme electrode result in good characteristics such as a large determination range (0.1-13.5mM), a short response time (within 5s), a large current density (1.176 mA cm(-2)), and high sensitivity (91mA M(-1)cm(-2)) and stability (73.5% remains after 22 days). In addition, effects of pH value, applied potential, electrode construction, and electroactive interferents on the amperometric response of the sensor were investigated and discussed. The reproducibility and applicability to whole blood analysis of the enzyme electrode were also evaluated.     

A chemiluminescence automatic analyser for the measurement of biological compounds

A compact automated analyser which could analyse constituents in biological fluids with a small sample volume and in a short time has been developed. The instrument was composed of a flow injection analysis system equipped with chemiluminometric detection and an immobilized enzyme column reactor used in combination. Chemiluminescence has high sensitivity, and its reaction proceeds very quickly. Furthermore, an immobilized enzyme column reactor can produce a sufficient amount of hydrogen peroxide from compounds in serum in a short time. When enzymes are used as reagents for the analysis of substances in blood or blood serum, the final signals emitted by different enzyme reactions are usually not only hydrogen peroxide but also ammonia, NAD(P)H and so on. However, the practical chemiluminescence method for ammonia and NAD(P)H has not been established. We have discovered a new practical method for ammonia and NAD(P)H using an enzyme column reactor consisting of both immobilized L -glutamate dehydrogenase and L -glutamate oxidase. The determinations of glucose and uric acid in serum by chemiluminometry after production of hydrogen peroxide by the respective oxidases are presented. A newly chemiluminometric determination of ammonia, NAD(P)H and its applications to other enzymatic analyses that give ammonia and NAD(P)H as a final signal are also described.     

Co-immobilization of glucose oxidase and hexokinase on silicate hybrid sol-gel membrane for glucose and ATP detections

The co-immobilization of glucose oxidase (GOD) and hexokinase/glucose-6-phosphate dehydrogenase (HEX) in the silica hybrid sol-gel film for development of amperometric biosensors was investigated. The silica hybrid film fabricated by hydrolysis of the mixture of tetraethyl orthosilicate and 3-(trimethoxysiyl)propyl methacrylate possessed a three-dimension vesicle structure and good uniformity and conformability, and was ready for enzyme immobilization. The electrochemical and spectroscopic measurements showed that the silica hybrid sol-gel provided excellent matrice for the enzyme immobilization and that the immobilized enzyme retained its bioactivity effectively. The immobilized GOD could catalyze the oxidation of glucose, which could be used to determine glucose at +1.0 V without help of any mediator. The competition between GOD and HEX for the substrate glucose involving ATP as a co-substrate led to a decrease of the glucose response, which allowed us to develop an ATP sensor with a good stability. The fabricated silica hybrid sol-gel matrice offered a stage for further study of immobilization and electrochemistry of proteins.     

蔗糖-葡萄糖双功能酶传感器的研究

结合蔗糖转化酶(INV)酶管与葡萄糖氧化酶(GOD)-葡萄糖变旋酶(MUT)双酶电极构成一种新的蔗糖传感器。该传感器可以分别用于蔗糖及葡萄糖的测定。蔗糖经酶管作用产生α-D-葡萄糖,再用COD-MUT双酶电极定糖。若是样品中蔗糖和葡萄糖共存,比较样品流经不同路径(Ws和Wg)时传感器的响应值,可以排除葡萄糖对蔗糖测定的干扰。传感器的最适pH和温度范围分别为:5.0—6.5和30—40℃。在稳态法实验中,传感器的线性范围为:2.5×10~(-4)—5×10~(-3)mol/L。传感器的重复性很好,CV<1％。该传感器在用于测定发酵培养基(含葡萄糖)的蔗糖含量,平均回收率为97.9％。传感器与糖度计法测定的相关系数为0.997。传感器至少可以稳定使用8天以上。     

Development of a high analytical performance amperometric glucose biosensor based on glucose oxidase immobilized in a composite matrix: layered double hydroxides/chitosan

This paper aimed at showing the interest of the composite material based on layered double hydroxides (LDHs) and chitosan (CHT) as suitable host matrix likely to immobilize enzyme onto electrode surface for amperometric biosensing application. This hybrid material combined the advantages of inorganic LDHs and organic biopolymer, CHT. Glucose oxidase (GOD) immobilized in the composite material maintained its activity well as the usage of glutaraldehyde was avoided. The process parameters for the fabrication of the enzyme electrode and various experimental variables such as pH, applied potential and temperature, were explored for optimum analytical performance of the enzyme electrode. The enzyme electrode provided a linear response to glucose over a concentration range of 1 x 10(-6) to 3 x 10(-3) M with a high sensitivity of 62.6 mA M(-1) cm(-2) and a detection limit of 0.1 muM based on the signal-to-noise ratio of 3.     

Nanoflake-like SnS₂ matrix for glucose biosensing based on direct electrochemistry of glucose oxidase

A novel biosensor is developed based on immobilization of proteins on nanoflake-like SnS? modified glass carbon electrode (GCE). With glucose oxidase (GOD) as a model, direct electrochemistry of the GOD/nanoflake-like SnS? is studied. The prepared SnS? has large surface area and can offer favorable microenvironment for facilitating the electron transfer between protein and electrode surface. The properties of GOD/SnS? are characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), UV-vis spectroscopy, Fourier transform infrared spectroscopy (FTIR) and cyclic voltammetry (CV), respectively. The immobilized enzyme on nanoflake-like SnS? retains its native structure and bioactivity and exhibits a surface-controlled, reversible two-proton and two-electron transfer reaction with the apparent electron transfer rate constant (k(s)) of 3.68 s?1. The proposed biosensor shows fast amperometric response (8s) to glucose with a wide linear range from 2.5 × 10?? M to 1.1 × 10?3 M, a low detection limit of 1.0 × 10?? M at signal-to-noise of 3 and good sensitivity (7.6 ± 0.5 mA M?1 cm?2). The resulting biosensor has acceptable operational stability, good reproducibility and excellent selectivity and can be successfully applied in the reagentless glucose sensing at -0.45 V. It should be worthwhile noting that it opens a new avenue for fabricating excellent electrochemical biosensor.     

Properties of a glucose oxidase covalently immobilized on amorphous AlPO4 support

Glucose oxidase (GOD) was covalently immobilized on amorphous AlPO₄ as well as on an AlPO₄/clay mineral Sepiolite system. Immobilization of the enzyme was carried out through the -amino group of lysine residues through an aromatic Schiff's-base. Activation of the support was obtained after reaction of appropriate molecules with support surface –OH groups. The enzymatic activities of native, and different immobilized GOD systems and filtrates, were followed by the amount of liberated -gluconic acid obtained in the enzymatic β- -glucose oxidation with the aid of an automatic titrator. The kinetic properties of native and immobilized GOD were obtained for glucose concentrations in the range of physiological conditions and at different working conditions such as reaction temperature, reaction pH, and enzyme concentration.

The binding percentage of enzymes was in the 50–80% range, with residual and specific activities in the 65–80% and 90–150% ranges, respectively. No change in the pH optimum and only slight changes in the V_max and K_M kinetic parameters with respect to native GOD were observed, so that not only was little deactivation of enzyme obtained throughout the immobilization process but also that the stability of the covalently bound enzyme in the two supports appeared to have increased with respect to the soluble enzyme. GOD immobilization also increased its efficiency and operational stability in repeated uses on increasing the amount of immobilized enzyme.     

一种免受乙醇干扰的GOD的酶电极

将酶电极应用于发酵糖的测定时。与糖共存的乙醇常常会影响测糖的准确性,通过对葡萄糖氧化酶(GOD)电极的研究,探讨了乙醇对测糖酶电极测定影响,进而研制出抗干扰的GOD酶电极,若是GOD电极的酶膜通过夹心法制备．在乙醇含量为0．1％(V／V)时·即产生显著的影响,使测定结果偏大4．3%,且乙醇的影响随浓度的升高而增大,若用尼龙网固定GOD膜,GOD电极在测定20mmol／L和5mm01／L左右的葡萄糖溶液时,含量高达9％的乙醇仍未对测定产生显著的影响．表现出良好的不受乙醇干扰的特性,并且,该尼龙网GOD电极具有良好的重复性、稳定的响应活性及较长的保存期．     

Flow system for fish freshness determination based on double multi-enzyme reactor electrodes

A double reactor system for the determination of fish and shellfish freshness using the freshness indicator, K-value (K=[(HxR+Hx)/(ATP+ADP+AMP+IMP+HxR+Hx)]x100), was developed, where ATP, ADP, AMP, IMP, HxR and Hx are adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, inosine monophosphate, inosine and hypoxanthine, respectively. The system consisted of a pair of enzyme reactors with an oxygen electrode positioned close to the respective reactor. The enzyme reactor (I) was packed with nucleoside phosphorylase and xanthine oxidase immobilized simultaneously on chitosan beads (immobilized enzyme A). Similarly, the enzyme reactor (II) was packed with immobilized enzyme A and immobilized enzyme B (co-immobilized alkaline phosphatase and adenosine deaminase). Moreover, this reactor consisted of two layers, the enzyme A and enzyme B (1:1). A good correlation was obtained between K values, which were determination by the proposed system and by the HPLC method. One assay could be completed within 5 min. The signal for the determination of K value of fish and shellfish was reproducible within 2.3%. The long-term stability of the enzyme reactors was evaluated at 30 degrees C for 28 days.