逐层交替自组装法制备葡萄糖氧化酶电极

采用了分子沉积法制备葡萄糖氧化酶电极.将带有正电荷的聚二甲基二丙烯铵盐(PDDA)和带有负电荷的葡萄糖氧化酶(GOD)交替沉积在修饰有3-巯基-1-丙基磺酸盐(MPS)的金电极表面.该电极以甲酸二茂铁衍生物(Fc-COOH)为电子媒介体,促进电子在葡萄糖氧化酶和金电极表面的传递.通过循环伏安法(CV)检测酶电极的活性,在0.332 V出现一对典型的可逆二茂铁氧化还原峰(见图1),其峰电流的大小和扫描速率的平方根成正比,即由扩散控制电流大小.该电极在溶液中对葡萄糖的线性响应范围上限为6.63 mmol/L(见图2),检测限为0.547mmol/L,响应时间为9.44s.在不同的pH值测试其对葡萄糖的响应,结果表明pH为7.0时为最佳反应条件.在同一浓度下重复测试电流响应,其标准偏差为0.152,其重复使用性能稳定.     

铁氰酸镍膜修饰金电极的研制及应用

通过层层组装的方法,将Ni^2＋和[Fe（CN）6]^3-交替沉积在巯基乙酸功能化的金电极表面．首次成功制备了铁氰酸镍多层膜修饰电极,用循环伏安法研究了该多层膜的电化学行为,实验表明峰电流随膜层数的增加而增加,膜均匀增长．该修饰电极对一价金属离子Na^＋,K^＋,NH4^＋具有选择性响应,尤其对K^＋存在准能斯特响应,响应范围0．01～1．0mol／L;而且该电极对抗坏血酸（AA）和S2O3^2-体系的氧化具有良好的电催化作用,线性范围分别为：1．14×10^-4～1．14×10^-3mol／L和5．0×10^-4～3．1×10^-3mol／L．     

镀金和碳纳米管修饰金电极上吸附态葡萄糖氧化酶比活性的EQCM研究

采用石英晶体微天平(EQCM)技术监测了裸金电极、镀金和碳纳米管修饰金电极上葡萄糖氧化酶(GOD)的吸附过程. 通过EQCM测量吸附固定的GOD质量, 并实时检测酶反应产物H2O2的氧化电量, 求算了各表面上吸附态GOD的比活性(ESAi). 结果表明, 各表面上均可吸附一定的GOD, 且吸附态GOD均有一定的酶活性; 修饰CNTs可增大酶吸附量和酶电极对葡萄糖的响应电流, 但ESAi随CNTs修饰量的增大而降低; Au电极上电镀金后, 酶吸附量和酶电极对葡萄糖的响应电流亦增大, 但ESAi与裸金电极上的基本一致.     

一种噻吩基双偶极半菁与普鲁士蓝的静电自组装薄膜

通过交替沉积普鲁士蓝和一种含噻吩的半菁, 制备了一种新的无机-有机杂化静电自组装膜. 用紫外-可见吸收光谱、循环伏安技术和光电化学实验对薄膜进行了表征或光电性质研究. 376和698 nm处薄膜的吸光度随薄膜层数增加线性增加, 表明薄膜的沉积是均匀和可重复的. 薄膜中的普鲁士蓝具有良好的表面控制而非扩散控制的电化学活性, 膜的层数从1增加至5时, 阳极峰电流随膜层数增加而线性增加. 100 mW·cm^-2的白光照射下, 薄膜产生稳定的阴极光电流, 随层数增加线性增长, 层数增加到4层时, 光电流达到最大值. 饱和甘汞电极为参比电极, -0.4 V 偏压下, 4层薄膜产生的光电流密度高达0.28 μA·cm^-2.     

基于多壁碳纳米管/二茂铁接枝壳聚糖的核/壳结构组合物多层膜电极的组装及其电催化

以聚二烯丙基二甲基氯化铵(PDDA)修饰的玻碳电极(GCE)为基础电极, 利用静电层-层自组装的方法将多壁碳纳米管/二茂铁接枝壳聚糖的核/壳结构组合物(MWCNTs@CHIT-Fc)和聚苯乙烯磺酸钠(PSS)在该电极表面进行交替多层组装. 用循环伏安(CV)、紫外-可见光谱(UV-Vis)和扫描电子显微镜(SEM)等方法对组装过程进行了跟踪表征. 用CV方法研究了抗坏血酸(AA)在该多层膜电极上的电催化氧化. 研究结果表明, 当MWCNTs@CHIT-Fc为6个双层时, AA在该修饰电极上的氧化峰电位与裸GCE相比降低了约0.4 V. 用控制电位电流法研究了不同MWCNTs@CHIT-Fc双层时AA的电流响应. 用该电极测定了AA的灵敏度, 检测限及线性范围等性能参数可通过控制组装的MWCNTs@CHIT-Fc双层数进行调节.     

层层自组装制备基于多壁碳纳米管的胆碱生物传感器

以经混酸处理的多壁碳纳米管(MWCNTs)修饰铂(Pt)电极,在此基础上固定(PAA/PVS)3复合膜,采用层层自组装技术将高分子聚电解质PDDA与胆碱氧化酶交替组装在已修饰的电极上,构建了电流型胆碱生物传感器。实验结果表明,MWCNTs的引入使电极对H2O2的催化电流明显增大,制成的酶电极可以有效控制酶量的使用,酶膜组装层数为8时最优,对胆碱的线性响应范围为5×10-7～1×10-4mol/L;灵敏度为12.53μA/mmol;响应时间为7.60s;检出限为2×10-7mol/L(S/N=3)。传感器的抗干扰能力强,稳定性好,30d时的响应电流值仍保持最初的89.5%。3次平行实验的RSD为3.64%。     

基于室温离子液体的有序多孔金膜葡萄糖传感器

将1-丁基-3-甲基咪唑四氟硼酸盐（[BMIm][BF4]）、N,N-二甲基甲酰胺（DMF）与葡萄糖氧化酶（GOD）的混合物修饰于三维有序大孔（3DOM）金膜电极上,构建了一种新型的葡萄糖传感器．固定的GOD在pH7．0的磷酸缓冲液（PBS）中展现出一对可逆性好的氧化还原峰,这归因于GOD的活性中心黄素腺嘌呤二核苷酸（FAD）的直接电化学行为．研究表明,离子液体（IL）、DMF以及3DOM金膜对GOD的直接电化学都起到了重要的作用．3DOM金膜修饰电极作为基底提高了酶的负载量,加速了GOD与电极表面的电子传递;IL的应用增加了固定GOD的电化学活性;DMF与IL、GOD的协同作用更好地保持了GOD的生物活性．固定在电极表面的GOD对葡萄糖显示出良好的催化性能,其检测线性范围为10～125nmol／L,检测限为3．3nmol／L（S／N=3）,酶催化反应的表观米氏常数Km为0．018mmol／L．     

基于层-层自反应的葡萄糖氧化酶有序多层膜电极

以胱胺修饰的金电极为基础电极, 利用席夫碱反应使经高碘酸根氧化的葡萄糖氧化酶在该电极表面进行自身的层-层有序组装. 用电化学交流阻抗法对多层酶膜形成过程的跟踪结果表明, 该多层酶膜的生长是一个逐步形成的均匀过程. 用循环伏安法和I-t曲线法研究了该酶电极对葡萄糖的电催化氧化. 实验结果表明, 当采用羟基二茂铁作为人工电子转移媒介体时, 该酶电极对葡萄糖具有很好的电催化氧化功能. 该传感器制作简便, 响应迅速, 性能稳定, 催化电流与葡萄糖浓度在一定范围内成正比, 并且可以通过控制葡萄糖氧化酶的组装层数来调节该生物传感器的灵敏度与检测限.     

基于多壁碳纳米管/壳聚糖多层膜修饰玻碳电极邻苯二酚的测定

改进了碳纳米管在壳聚糖溶液中的分散方法,制备了多壁碳纳米管/壳聚糖多层膜修饰玻碳电极,对比了不同修饰层数膜电极的循环伏安和电化学阻抗行为,5层多壁碳纳米管/壳聚糖膜修饰玻碳电极的电化学性能优良.在最优实验条件下,该修饰玻碳电极对邻苯二酚(CAT)有灵敏的响应,CAT浓度在3.99×10-6～9.09×10-4mol/L范围内与氧化峰电流呈良好的线性关系,检出限为2.39×10-6mol/L(S/N=3).该修饰玻碳电极性能稳定,测定4×10-5mol/LCAT溶液,RSD(n=10)为2.1%;15周后,该电极的响应值仅降低1.9%.     

以对乙酰氨基酚为媒介体的葡萄糖传感器研究

电化学葡萄糖传感器检测血浆中的葡萄糖时,血浆中的电活性物质-对乙酰氨基酚会严重干扰检测结果。本研究以对乙酰氨基酚(ACP)为电子转移媒介体,研究了葡萄糖氧化酶(GOD)修饰电极的电化学行为,并探讨了不同pH条件下该修饰电极催化葡萄糖氧化的能力。研究发现ACP能有效地促进酶活性中心与电极间的电子转移,且Nafion涂覆的GOD修饰电极能有效排除尿酸(UA)和抗坏血酸(AA)的干扰。实验结果表明Nafion/GOD电极的氧化电流与葡萄糖浓度在0.6～15mmol/L范围内呈良好的线性关系,且对葡萄糖的检测限为59μmol/L。     

1-芘丁酸/石墨烯复合物的电化学性质及其在葡萄糖传感器上的应用

本文采用一步法制备了1-芘丁酸/石墨烯复合物(PBA/G),研究了其电化学性质. 采用铁氰化钾和亚铁氰化钾电化学探针测定了电化学阻抗滴定曲线,确定了PBA/G的表观pK_a为6.2. 此外,将葡萄糖氧化酶(GOD)共价键合在PBA/G表面构建了葡萄糖电化学传感器,其电化学响应与葡萄糖浓度（5 mmol L^-1浓度范围内）呈线性,检测限为0.085 mmol L^-1. 实验还测定了固定在PBA/G表面的GOD的表观米氏常数为5.40 mmol L^-1,表明固定化的GOD对葡萄糖有较高的催化活性。     

Application of polymaleimidostyrene as a convenient immobilization reagent of enzyme in biosensor

Sulfhydryl groups of glucose oxidase (GOD) were reacted with maleimide groups of polymaleimidostyrene (PMS) which was coated onto the porous carbon sheet, and the carbon sheet immobilized by GOD was combined with an oxygen electrode to fabricate a glucose sensor. The activity of thiolated GOD immobilized to PMS is much larger than that of native GOD immobilized to PMS. The good linear relationship of glucose and oxygen current response was obtained in a concentration range from 0.1 to 2 mM and upper limit of linear range was found to be 3.0 mM. The immobilized GOD activity is highly dependent on pH at immobilization and the maximum activity was obtained at pH 5.5, probably because the SH groups of GOD that are indispensable for generation of enzyme activity is not exposed at this pH. It was found that PMS is very effective reagent to immobilize enzyme strongly via covalent bond, because high density of maleimide groups of PMS can catch not only exposed SH groups but also buried SH groups.     

Immobilized Fullerene C60‐Enzyme‐Based Electrochemical Glucose Sensor

A mixed‐valence cluster of cobalt(II) hexacyanoferrate and fullerene C60‐enzyme‐based electrochemical glucose sensor was developed. A water insoluble fullerene C60‐glucose oxidase (C60‐GOD) was prepared and applied as an immobilized enzyme on a glassy carbon electrode with cobalt(II) hexacyanoferrate for analysis of glucose. The glucose in 0.1 M KCl/phosphate buffer solution at pH = 6 was measured with an applied electrode potential at 0.0 mV (vs Ag/AgCl reference electrode). The C60‐GOD‐based electrochemical glucose sensor exhibited efficient electro‐catalytic activity toward the liberated hydrogen peroxide and allowed cathodic detection of glucose. The C60‐GOD electrochemical glucose sensor also showed quite good selectivity to glucose with no interference from easily oxidizable biospecies, e.g. uric acid, ascorbic acid, cysteine, tyrosine, acetaminophen and galactose. The current of H₂O₂ reduced by cobalt(II) hexacyanoferrate was found to be proportional to the concentration of glucose in aqueous solutions. The immobilized C60‐GOD enzyme‐based glucose sensor exhibited a good linear response up to 8 mM glucose with a sensitivity of 5.60 × 10² nA/mM and a quite short response time of 5 sec. The C60‐GOD‐based glucose sensor also showed a good sensitivity with a detection limit of 1.6 × 10^‐6 M and a high reproducibility with a relative standard deviation (RSD) of 4.26%. Effects of pH and temperature on the responses of the immobilized C60‐GOD/cobalt(II) hexacyanoferrate‐based electrochemical glucose sensor were also studied and discussed.     

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.     

Hydrogen peroxide-induced deacetylation of acetyl resorufin as a novel indicator reaction for fluorometric detection of glucose using only glucose oxidase

Hydrogen peroxide (H2O2)-induced deacetylation of non-fluorescent acetyl resorufin (1) to fluorescent resorufin (2) as a novel indicator reaction for fluorometric detection of glucose using only glucose oxidase (GOD) is described. When a 1:1:1 mixture of 1 (in CH3CN), glucose, and GOD (each in pH 7.4 phosphate buffer) was incubated at 25 degrees C under aerobic conditions, the resulting solution turned yellow to fluorescent pink due to 2. The formation of 2 was markedly retarded on incubation under anaerobic conditions. When a mixture of 1 and H2O2 was incubated under aerobic conditions, the formation of 2 was noted as in the case of the enzymatic reaction of 1. These results demonstrated that the observed color change is brought about through deacetylation of 1 to 2 induced by H2O2 generated in GOD-catalyzed oxidation of glucose. With regard to the fluorometric traces of the enzymatic reaction with 1 (0.2 mM), GOD (0.5 mg/ml), and glucose at 25 degrees C, fluorescence intensity exhibited a linear relationship against glucose concentration between 0.2 and 2.0 mm, with a correlation coefficient of 0.997. Neither ascorbic acid, uric acid, nor bilirubin significantly interfered with the transformation of 1 to 2 through GOD-catalyzed oxidation of glucose.     

Low‐Potential Detection of Glucose with a Biosensor Based on the Immobilization of Glucose Oxidase on Polymer/Manganese Oxide Layered Nanocomposite

A nanocomposite with poly(diallyldimethylammonium), PDDA, intercalated between manganese oxide layers is constructed on a graphite electrode surface through one‐step electrodeposition and used to adsorb glucose oxidase (GOD). The immobilized GOD displays a pair of stable and quasireversible redox peaks with a formal potential of ?468 mV in pH 7.0 buffer solutions and exhibits excellent electrocatalysis to the reduction of oxygen. In the presence of dissolved oxygen, the reduction peak current decreased gradually with the addition of glucose, indicating that the immobilized GOD kept its bioactivity. Thus a reagentless biosensor for glucose at a low detection potential was established. The linear concentration range is from 0.02 to 2.78 mM with a detection limit of 9.8 μM. The proposed glucose biosensor was insensitive to common interferences such as ascorbic and uric acids etc.     

Nafion coating the ferrocenylalkanethiol and encapsulated glucose oxidase electrode for amperometric glucose detection

This paper describes the fabrication and application of a complex electrode--Nafion film coating ferrocenylalkanethiol (FcC(11)SH) and encapsulated glucose oxidase (GOD) on a gold electrode. FcC(11)SH is employed as a mediator enabling the electron transfer between GOD and the electrode, GOD is encapsulated in polyacrylamide gel to improve the stability of the enzyme, and the Nafion film is coated on the modified electrode to eliminate interferents such as ascorbic acid, uric acid and acetaminophen in amperometric glucose detection. It is noticed that such a complex electrode exhibits excellent catalytic activity for glucose oxidation, and preserves the native structure of GOD and therefore its enzymatic activity. The encapsulated GOD retains more than 80% of its original biocatalytic activity even after 24 days, much longer than that of naked GOD molecules attached directly to the electrode. The oxidation peak current at the modified electrode shows a linear relationship with the glucose concentration in the range from 0.05 to 20 mM with a detection limit of 2.4 μM. In addition, the electrode displays a rapid response and good reproducibility for glucose detection, and has been successfully employed for glucose detection in blood plasma samples.     

Direct Electron Transfer of Glucose Oxidase and Glucose Biosensor Based on Nano-structural Attapulgite Clay Matrix

The direct electrochemistry of glucose oxidase (GOD) was achieved based on the immobilization of GOD on a natural nano‐structural attapulgite (ATP) clay film modified glassy carbon (GC) electrode. The immobilized GOD displayed a pair of well‐defined quasi‐reversible redox peaks with a formal potential (E^0′) of ?457.5 mV (vs. SCE) in 0.1 mol·L^?1 pH 7.0 phosphate buffer solution. The peak current was linearly dependent on the scan rate, indicating that the direct electrochemistry of GOD in that case was a surface‐controlled process. The immobilized glucose oxidase could retain bioactivity and catalyze the oxidation of glucose in the presence of ferrocene monocarboxylic acid (FMCA) as a mediator with the apparent Michaelis‐Menten constant K^app_m of 1.16 mmol·L^?1. The electrocatalytic response showed a linear dependence on the glucose concentration ranging widely from 5.0×10^?6 to 6.0×10^?4 mol·L^?1 (with correlation coefficient of 0.9960). This work demonstrated that the nano‐structural attapulgite clay was a good candidate material for the direct electrochemistry of the redox‐active enzyme and the construction of the related enzyme biosensors. The proposed biosensors were applied to determine the glucose in blood and urine samples with satisfactory results.     

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.     

An Amperometric Glucose Electrode Based on Adsorbed Glucose Oxidase on Palladium/Gold Modified Graphite

Abstract

A glucose electrode was constructed by adsorbing glucose oxidase (GOD) on a modified electrode for H₂ O ₂ oxidation, consisting of Pd/Au sputtered on graphite. Maximally, 0.8 U cm^?2 of GOD could be adsorbed. The electrode was used in a f.i.a. manifold for determination of glucose. Linear calibration curves were obtained in the concentration range 3. 10^?6 4. 10^?3 mol L^?1 glucose. The applied potentials for glucose determination were + 300 mV vs. Ag/AgCl at pH 8.0, + 350 mV at pH 7.0, + 400 mV at pH 6.0 and + 500 mV at pH 5.0. The activity vs. pH profile of adsorbed GOD was broad having an optimum between pH 5 and 6. The apparent kinetic parameters for adsorbed GOD, K_M ^app and i_max, were found to be 50 mM and 160 uA at optimal pH.