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.     

Amperometric determination of glucose,based on the direct electron transfer between glucose oxidase and tin oxide

An amperometric glucose biosensor was designed for the detection of glucose in blood, urine, beverages, and fermentation systems. In typical glucose biosensors that employ enzymes, mediators are used for efficient electron transfer between the enzymes and the electrode. However, some of these mediators are known to be toxic to the enzymes and also must be immobilized on the surface of the electrode. We propose a mediator-free glucose biosensor that uses a glucose oxidase immobilized on a tin oxide electrode. Direct electron transfer is possible in this system because the tin oxide has redox properties similar to those of mediators. The method for immobilization of the glucose oxidase onto the tin oxide is also very simple. Tin oxide was prepared by the anodization and annealing of pure tin, and this provides a large surface area for the immobilization step because of its porosity. Glucose oxidase was immobilized onto the tin oxide using the membrane entrapment method. The proposed method provides a simple process for fabricating the enzyme electrode. Glucose oxidase immobilized onto the tin oxide, prepared in accordance with this method, has a relatively large current response when comparedto those of other glucose biosensors. The sensitivity of the biosensor was 19.55 μA/mM, and a linear response was observed between 0∼3 mM glucose. This biosensor demonstrated good reproducibility and stability.     

Amperometric glucose biosensors based on layer-by-layer assembly of chitosan and glucose oxidase on the Prussian blue-modified gold electrode

A glucose biosensor based on layer-by-layer (LBL) self-assembling of chitosan and glucose oxidase (GOD) on a Prussian blue film was developed. First, Prussian blue was deposited on a cleaned gold electrode then chitosan and GOD were assembled alternately to construct a multilayer film. The resulting amperometric glucose biosensor exhibited a fast response time (within 10 s) and a linear calibration range from 6 μM to 1.6 mM with a detection limit of 3.1 μM glucose (s/n = 3). With the low operating potential, the biosensor showed little interference to the possible interferents, including ascorbic acid, acetaminophen and uric acid, indicating an excellent selectivity.     

An amperometric glucose biosensor based on poly(o-aminophenol) and Prussian blue films at platinum electrode

Prussian blue (PB), as a good catalyst for the reduction of hydrogen peroxide, has been combined with nonconducting poly(o-aminophenol) (POAP) film to assemble glucose biosensor. Compared with PB-modified enzymatic biosensor, the biosensor based on glucose oxidase immobilized in POAP film at PB-modified electrode shows much improved stability (78% remains after 30 days) in neutral medium. Additionally, the biosensor, at an applied potential of 0.0 V, exhibits other good characteristics, such as relative low detection limit (0.01 mM), short response time (within 5s), large current density (0.28 mA/cm2), high sensitivity (24 mAM(-1)cm(-2)), and good antiinterferent ability. The apparent activation energy of enzyme-catalyzed reaction and apparent Michaelis-Menten constant are 34.2 KJmol(-1) and 10.5 mM, respectively. In addition, effects of temperature, applied potential used in the determination, pH value of the detection solution, and electroactive interferents on the amperometric response of the sensor were investigated and are discussed.     

Glucose biosensor based on immobilization of glucose oxidase in poly(o-aminophenol) film on polypyrrole-Pt nanocomposite modified glassy carbon electrode

Novel Pt nanoclusters embedded polypyrrole nanowires (PPy-Pt) composite was electrosynthesized on a glassy carbon electrode, denoted as PPy-Pt/GCE. A glucose biosensor was further fabricated based on immobilization of glucose oxidase (GOD) in an electropolymerized non-conducting poly(o-aminophenol) (POAP) film that was deposited on the PPy-Pt/GCE. The morphologies of the PPy nanowires and PPy-Pt nanocomposite were characterized by field emission scanning electron microscope (FE-SEM). Effect of experimental conditions involving the cycle numbers for POAP deposition and Pt nanoclusters deposition, applied potential used in glucose determination, temperature and pH value of the detection solution were investigated for optimization. The biosensor exhibited an excellent current response to glucose over a wide linear range from 1.5 × 10⁻⁶ to 1.3 × 10⁻² M (r = 0.9982) with a detection limit of 4.5 × 10⁻⁷ M (s/n = 3). Based on the combination of permselectivity of the POAP and the PPy films, the sensor had good anti-interference ability to ascorbic acid (AA), uric acid (UA) and acetaminophen. The apparent Michaelis–Menten constant (K_m) and the maximum current density (I_m) were estimated to be 23.9 mM and 378 μA/cm², respectively. In addition, the biosensor had also good sensitivity, stability and reproducibility.     

葡萄糖氧化酶在枯草芽孢杆菌芽孢表面的展示及其酶电极制备

葡萄糖生物传感器是目前最为常见的电化学生物传感器,绝大多数葡萄糖生物传感器采用在电极表面修饰葡萄糖氧化酶的方法来制备,但是,在电极的固定化过程中需要酶的纯化,使得成本增加,已成为固定化酶电极开发领域的瓶颈。文中主要以芽孢衣壳蛋白CotX为锚定蛋白将葡萄糖氧化酶(Glucose oxidase,GOD)展示到枯草芽孢杆菌芽孢表面,通过Western blotting分析、免疫荧光分析以及酶活检测均证明GOD在芽孢表面有效表达,发酵获得重组芽孢(Spore-GOD)。再采用滴涂法和电沉积法制备了氧化石墨烯/普鲁士蓝沉积膜修饰玻碳电极,将Spore-GOD固定在修饰电极表面,最后滴加一层Nafion溶液,制成了电化学生物传感器,用于葡萄糖的灵敏测定。葡萄糖在该酶电极传感器上的循环伏安图表明,该反应在0.42V处出现明显的氧化峰,并且氧化还原峰电流与葡萄糖浓度在0.1–7.0mmol/L之间具有良好的线性关系,校正曲线方程为:I=1.3047C_glucose+3.639(R²=0.9929),其检测限为7.5μmol/L(S/N=3)。此修饰电极具有...     

An amperometric pyruvate biosensor based on pyruvate oxidase nanoparticles immobilized onto pencil graphite electrode

This present study was aimed to fabricate a sensitive and improved amperometric biosensor by the nanoparticles of pyruvate oxidase, which were prepared and immobilized covalently onto pencil graphite electrode. The biosensor showed ideal working within 5 s under defined conditions of pH 6.0 and incubation temperature of 30 °C at an applied voltage of -0.1 V. Under standard assay conditions, a linear response was obtained between pyruvate concentration ranging from 0.001 to 6000 μM and current (μA). A lower detection limit (0.58 μM) and an excellent correlation coefficient (R² = 0.999) with standard spectrophotometric assay was obtained for the present biosensor. Within and between batches of coefficients of variation were calculated and found to be 3.61 % and 3.33 %, respectively. The biosensor was put to continual use for over 210 days. The biosensor was employed for the measurement of pyruvate level in sera of normal healthy individuals and persons suffering from heart disease.     

Poly(3-hydroxybutyrate) synthesis in fed-batch culture of Ralstonia eutropha with phosphate limitation under different glucose concentrations

Poly(3-hydroxybutyrate) (PHB) was produced by fed-batch cultures of Ralstonia eutropha with phosphate limitation under different glucose concentrations. When glucose was kept at 2.5 g l^–1, cell growth and PHB synthesis were limited due to the shortage of carbon source but a higher PHB content occurred in the cell-growth stage. This shows that a low glucose concentration is favorable for PHB accumulation in R. eutropha. PHB obtained with glucose at 9 g l^–1 is 1.6 times that obtained with 40 g l^–1. When glucose was in the range of 9 to 40 g l^–1, PHB concentration and productivity decreased significantly with the increase of glucose concentration. The highest PHB productivity was obtained with glucose at 9 g l^–1.     

The chemical mechanism of action of glucose oxidase from Aspergillus niger

Glucose oxidase from Aspergillus niger (EC 1.1.3.4) is able to catalyze the oxidation of -D-glucose with p-benzoquinone, methyl-1,4-benzoquinone, 1,2-naphthoquinone, 1,2-naphthoquinone-4-sulfonic acid, potassium ferricyanide, phenazine methosulfate, and 2,6-dichloroindophenol. In this work, the steady-state kinetic parameters, V ₁/K _B , for reactions of these substrates were collected from pH 2.5–8. Further, the molecular models of the enzyme's active site were constructed for the free enzyme in the oxidized state, the complex of -D-glucose with the oxidized enzyme, the complex of reduced enzyme with methyl-1,4-benzoquinone, the reduced enzyme plus 1,2-naphthoquinone-4-sulfonic acid, oxidized enzyme plus reduced 1,2-naphthoquinone-4-sulfonic acid (hydroquinone anion), and oxidized enzyme plus fully reduced 1,2-naphthoquinone-4-sulfonic acid.Combining the steady-state kinetic and structural data, it was concluded that Glu412 bound to His559, in the active site of enzyme, modulates powerfully its catalytic activity by affecting all the rate constants in the reductive and the oxidative half-reaction of the catalytic cycle. His516 is the catalytic base in the oxidative and the reductive part of the catalytic cycle. It was estimated that the pK _a of Glu412 (bound to His559) in the free reduced enzyme is 3.4, and the pK _a of His516 in the free reduced enzyme is 6.9.     

Interfacial electron transfer of glucose oxidase on poly(glutamic acid)-modified glassy carbon electrode and glucose sensing

The interfacial electron transfer of glucose oxidase (GOx) on a poly(glutamic acid)-modified glassy carbon electrode (PGA/GCE) was investigated. The redox peaks measured for GOx and flavin adenine dinucleotide (FAD) are similar, and the anodic peak of GOx does not increase in the presence of glucose in a mediator-free solution. These indicate that the electroactivity of GOx is not the direct electron transfer (DET) between GOx and PGA/GCE and that the observed electroactivity of GOx is ascribed to free FAD that is released from GOx. However, efficient electron transfer occurred if an appropriate mediator was placed in solution, suggesting that GOx is active. The PGA/GCE-based biosensor showed wide linear response in the range of 0.5–5.5 mM with a low detection limit of 0.12 mM and high sensitivity and selectivity for measuring glucose.     

Neoglycoconjugates Based on Dendrimer Poly(aminoamides)

Neoglycoconjugates containing 4, 8, 32, and 64 terminal residues of B-disaccharide (B_DI) or N-acetylneuraminic acid (Neu5Ac) attached to poly(aminoamide)-type dendrimers (PAMAMs) were synthesized. The ability of B_DI conjugates to bind natural xenoantibodies (anti-B_DI antibodies) and the ability of Neu5Ac conjugates to inhibit the hemagglutinin-mediated adhesion of influenza virus were studied. The biological activity of PAMAM conjugates turned out to be higher than that of free carbohydrate ligands, but less than that of multivalent glycoconjugates based on other types of synthetic polymeric carriers. A conformational analysis of PAMAM matrices and resulting conjugates was performed to determine the statistical distances between carbohydrate ligands. The computations revealed the tendency of the PAMAM chains toward compaction and formation of dense globules. The process results in a decrease in the distances between the carbohydrate ligands in the conjugates and, hence, could affect the ability of glycoconjugates to efficiently bind the polyvalent carbohydrate-recognizing proteins.     

Co-immobilization of manganese peroxidase from Phlebia radiata and glucose oxidase from Aspergillus niger on porous silica beads

Manganese peroxidase (MnP) from Phlebia radiata and glucose oxidase from Aspergillus niger were co-immobilized on porous silica beads. Immobilization of both enzymes on the same carrier provided an integrated system in which H₂O₂ required by MnP was produced by glucose oxidase. The immobilization process resulted in a decrease of both enzymatic activities and substrate affinities. However, immobilization improved the stability of MnP against H₂O₂ or high pH, as well as the storage stability of this enzyme.     

Poly(l-lactide) degradation by Kibdelosporangium aridum

A new poly(L-lactide) (PLA)-degrading actinomycete, Kibdelosporangium aridum, degraded more than 97 mg out of 100 mg added high molecular weight PLA film (Mn: 3.4 x 10(5)) within 14 d in liquid culture. L-Lactic acid, the monomeric degradation product of PLA, was totally assimilated by the strain. In solid culture, many distinct grooves formed by the morphology of filamentous microorganisms on the surface of a PLA film were observed by scanning electron microscopy.     

Isolation and characterization ofPenicillium funiculosum mutants with enhanced glucose oxidase production

After the mutagenesis ofPenicillium funiculosum with UV light andN-nitroso-N-methylurea, 83 of 2237 grown colonies were surrounded with increased zones of glucose oxidase diffusion. Analysis of the glucose oxidase activity of selected mutant strains grown in submerged cultures allowed 18 mutant strains to be obtained whose glucose oxidase activity was 5–153% higher (in a medium with glucose) and 4–83% higher (in a medium with sucrose) than that of the parent strain. Two of these mutant strains, UV6.31 and NMU95-132, possessed high glucose oxidase activity when grown in media with glucose or sucrose and produced large amounts of mycelia. The active and morphologically stable mutantP. funiculosum NMU95-132 was chosen for further selection work.     

Amperometric glucose biosensor based on multilayer films via layer-by-layer self-assembly of multi-wall carbon nanotubes, gold nanoparticles and glucose oxidase on the Pt electrode

A novel amperometric glucose biosensor based on the nine layers of multilayer films composed of multi-wall carbon nanotubes (MWCNTs), gold nanoparticles (GNp) and glucose oxidase (GOD) was developed for the specific detection of glucose. MWCNTs were chemically modified with the H₂SO₄–HNO₃ pretreatment to introduce carboxyl groups which were used to interact with the amino groups of poly(allylamine) (PAA) and cysteamine via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide cross-linking reaction, respectively. A cleaned Pt electrode was immersed in PAA, MWCNTs, cysteamine and GNp, respectively, followed by the adsorption of GOD, assembling the one layer of multilayer films on the surface of Pt electrode (GOD/GNp/MWCNTs/Pt electrode). Repeating the above process could assemble different layers of multilayer films on the Pt electrode. PBS washing was applied at the end of each assembly deposition for dissociating the weak adsorption. Film assembling and characterization were studied by transmission electron microscopy and quartz crystal microbalance, and properties of the resulting glucose biosensors were measured by electrochemical measurements. The marked electrocatalytic activity of Pt electrode based on multilayer films toward H₂O₂ produced during GOD enzymatic reactions with glucose permitted effective low-potential amperometric measurement of glucose. Taking the sensitivity and selectivity into consideration, the applied potential of 0.35 V versus Ag/AgCl was chosen for the oxidation detection of H₂O₂ in this work. Among the resulting glucose biosensors, the biosensor based on nine layers of multilayer films was best. It showed a wide linear range of 0.1–10 mM glucose, with a remarkable sensitivity of 2.527 μA/mM, a detection limit of 6.7 μM estimated at a signal-to-noise ratio of 3 and fast response time (within 7 s). Moreover, it exhibited good reproducibility, long-term stability and the negligible interferences of ascorbic acid, uric acid and acetaminophen. The study can provide a feasible approach on developing new kinds of oxidase-based amperometric biosensors, and can be used as an illustration for constructing various hybrid structures.     

拟南芥多聚ADP核糖聚合酶Ⅰ的原核表达与活性检测

采用RT-PCR技术获得了拟南芥多聚ADP核糖聚合酶[poly(ADP-ribose)polymerase,PARP]PARP1基因的全长cDNA,转入原核表达载体pET32a并转化宿主菌Origami(DE3),加入终浓度为0.3mmol/L IPTG,在16℃下诱导可获得较多的可溶重组蛋白。纯化TRX-PARP1,在反应液中加入NAD+和断裂DNA,通过SDS-PAGE凝胶电泳和Western blotting分析,TRX-PARP1分子量可随着时间的延长逐渐增大,产生向上的弥散,表明蛋白质连上了ADP核糖分子;与此对比,作为参照的标签蛋白TRX无此现象。实验结果显示原核表达拟南芥PARP1能够催化自身多聚ADP核糖化修饰,为深入研究植物多聚ADP核糖聚合酶的功能奠定了基础。     

Glucose biosensor based on immobilization of glucose oxidase in poly(o-aminophenol) film on polypyrrole-Pt nanocomposite modified glassy carbon electrode

Novel Pt nanoclusters embedded polypyrrole nanowires (PPy-Pt) composite was electrosynthesized on a glassy carbon electrode, denoted as PPy-Pt/GCE. A glucose biosensor was further fabricated based on immobilization of glucose oxidase (GOD) in an electropolymerized non-conducting poly(o-aminophenol) (POAP) film that was deposited on the PPy-Pt/GCE. The morphologies of the PPy nanowires and PPy-Pt nanocomposite were characterized by field emission scanning electron microscope (FE-SEM). Effect of experimental conditions involving the cycle numbers for POAP deposition and Pt nanoclusters deposition, applied potential used in glucose determination, temperature and pH value of the detection solution were investigated for optimization. The biosensor exhibited an excellent current response to glucose over a wide linear range from 1.5 × 10⁻⁶ to 1.3 × 10⁻² M (r = 0.9982) with a detection limit of 4.5 × 10⁻⁷ M (s/n = 3). Based on the combination of permselectivity of the POAP and the PPy films, the sensor had good anti-interference ability to ascorbic acid (AA), uric acid (UA) and acetaminophen. The apparent Michaelis–Menten constant (K_m) and the maximum current density (I_m) were estimated to be 23.9 mM and 378 μA/cm², respectively. In addition, the biosensor had also good sensitivity, stability and reproducibility.