Bi-Enzyme Alcohol Biosensors Based on Genetically Engineered Alcohol Oxidase and Different Peroxidases

We report on the development of a bi-layer bi-enzyme biosensor architecture using different peroxidases and alcohol oxidase from Hansenula polymorpha C-105 as biological recognition elements. The sensor architecture comprises a first layer containing either horseradish peroxidase or royal palm tree peroxidase crosslinked with an Osmium complex-modified redox hydrogel. On top, a second layer was formed by electrochemically induced precipitation of a cathodic electrodeposition paint simultaneously entrapping alcohol oxidase isolated from a genetically modified strain of Hansenula polymorpha C-105. The sensor architecture was optimized with respect to effective electron transfer and stability of the enzyme. The main characteristics of the biosensors are an apparent maximal current I_max^app of 572–940 nA, an apparent Michaelis constant K_M^app of 9.5 mM, a sensitivity of 60–98 nA mM⁻¹ and an improved operational stability represented by a deactivation constant of 1.5–2.0 × 10⁻⁴min⁻¹.     

Sensitive Biosensors Based on (Dendrimer Encapsulated Pt Nanoparticles)/Enzyme Multilayers

A sensitive enzymed‐based biosensor for glucose has been obtained by introducing dendrimer encapsulated Pt nanoparticles via a layer‐by‐layer assembling method. The free amine groups located on each poly(amidoamine) dendrimer molecule were exploited to covalently attach enzyme to the dendrimer chains using carbodiimide coupling. The resultant enzyme electrodes are shown to have excellent sensitivity (as high as 30.33 μA mM^?1 cm^?2) and a limit of detection (about 0.1 μmol L^?1), depending on metal nanoparticles within dendrimers and the biocompatibility of dendrimers, the linear response range to glucose (from 5 μM to 1.0 mM), a fast response time (within 5 s), and good reproducibility (<8% relative standard deviation between electrodes at low substrate concentration). The sensitivities, and stabilities determined experimentally have demonstrated the potential of dendrimer encapsulated Pt nanoparticles as a novel candidate for enzymatic glucose biosensors.     

Electrochemical Biosensors Based on Enzyme Inhibition Effect

Several electrochemical biosensors based on various enzyme inhibition effects have been designed; their laboratory prototypes have been manufactured and thoroughly investigated. It should be noted that such biosensors are adapted to large-scale production technologies. A number of advantages and disadvantages of developed biosensors based on enzyme inhibition has been discussed. It is important that all developed biosensors are not opposite to traditional analytical methods, but complement them. This is an additional system of quick and early warning about the presence of toxic substances in the environment. Such systems can save time and money in emergencies due to the possibility of quick decision-making on local environmental problems. If necessary, more accurate, but time-consuming and expensive traditional methods could be used for further validation and additional research of samples previously tested by biosensors.     

Glucose Biosensor Based on Carbon/PVC-COOH/Ferrocene Composite with Covalently Immobilized Enzyme

A carbon/PVC-COOH/ferrocene composite electrode used for the determination of glucose has been prepared. The ferrocene acted as mediator was incorporated into the PVC-COOH polymer and the leakage could be prevented. The presence of carboxyl groups on the electrode surface allowed immobilizing enzyme via EDC and NHS. The ratio of PVC-COOH to graphite powder （w/w） has been studied. Amperometric determination of glucose has been performed at potential of 0.30 V vs SCE. The response time was 〈 15 s. The linear response range was of 0.1-20 mmol/L with a detection limit of 48μmol/L.     

基于对氨基苯甲酸/硫堇/纳米金共价修饰玻碳电极的葡萄糖生物传感器

实验制备了以对氨基苯甲酸(4-ABA)、硫堇(TH)、纳米金(Au NPs)共价键合葡萄糖氧化酶的新型葡萄糖生物传感器. 主要采用循环伏安法, 以羟基二茂铁作为电子媒介体, 对含葡萄糖和未含葡萄糖的电解液进行了研究. 结果表明: 传感器的响应电流值随葡萄糖氧化酶膜层数的不同而变化. 考虑到酶电极的长期稳定性与构造简单性, 我们制作了两层葡萄糖氧化酶膜的酶电极. 该传感器对1×10^－2 mol/L葡萄糖的响应电流达2.47 μA, 响应时间仅4.7 s. 该生物传感器检测的线性范围为3×10^－5～1×10^－3 mol/L, 最低检测浓度可达5.8×10^－6 mol/L. 该传感器制备简单, 稳定好, 具有一定的使用价值.     

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。     

Efficient and Facile Fabrication of Glucose Biosensor Based on Electrochemically Etched Porous HOPG Platform

Herein, a uniform porous highly oriented pyrolytic graphite (HOPG) electrode was prepared via diazonium salt assisted electrochemical etching method and firstly utilized to immobilize enzymes for the construction of a high‐performance glucose biosensor. The formation mechanism and morphology structure of the porous HOPG electrode were investigated using atomic force microscopy (AFM), X‐ray photoelectron spectroscopy (XPS) and X‐ray diffraction (XRD) characterizations. The glucose oxidase (GOx) was functionalized with pyrene groups and then immobilized on the porous HOPG substrate through π‐π stacking interactions and hydrogen bonding. As a result, eight times higher oxidation current density can be obtained for a given glucose concentration for the porous HOPG electrode than the pristine one. Detection limit of 5 μM for glucose was achieved for the as‐fabricated biosensor. It was obtained that 78 % biocatalytical activity of GOx can be retained after the pyrene functionalization and 65.7 % one can even be maintained after four weeks, which confirmed the high efficiency and good stability of the as‐prepared biosensor. What's more, it can be anticipated that various other enzymes can be loaded into this porous HOPG platform using the same enzyme modification methodology for the construction of efficient biosensors.     

Nafion-二茂铁-双酶修饰的葡萄糖传感器

用二茂铁作为过氧化物酶与玻碳电极的电子传递体,通过牛血清白蛋白-戊二醛交联剂把葡萄糖氧化酶和过氧化物酶固定在Nafion-二茂铁修饰玻碳电极上,制备成葡萄糖传感器。由于工作电位低,电活性物质如抗坏血酸、尿酸等对测定无干扰。该传感器的线性范围为5.0×10～(-4)～2.5×10～(-2)mol/L,响应时间小于30s.     

An Amperometric Glucose Biosensor Based on Poly (Pyrrole‐2‐Carboxylic Acid)/Glucose Oxidase Biocomposite

A newly developed amperometric glucose biosensor based on graphite rod (GR) working electrode modified with biocomposite consisting of poly (pyrrole‐2‐carboxylic acid) (PCPy) particles and enzyme glucose oxidase (GOx) was investigated. The PCPy particles were synthesized by chemical oxidative polymerization technique using H₂O₂ as initiator of polymerization reaction and modified covalently with the GOx (PCPy‐GOx) after activation of carboxyl groups located on the particles surface with a mixture of N‐(3‐dimethylaminopropyl)‐N′‐ethylcarbodiimide hydrochloride (EDC) and N‐hydroxysuccinimide (NHS). Then the PCPy‐GOx biocomposite was dispersed in a buffer solution containing a certain amount of bovine serum albumin (BSA). The resulting biocomposite suspension was adsorbed the on GR electrode surface with subsequent solvent airing and chemical cross‐linking of the proteins with glutaraldehyde vapour (GR/PCPy‐GOx). It was determined that the current response of the GR/PCPy‐GOx electrodes to glucose measured at +300 mV vs Cl reference electrode was influenced by the duration of the PCPy particles synthesis, pH of the GOx solution used for the PCPy particles modification and the amount of immobilized PCPy‐GOx biocomposite. An optimal pH of buffer solution for operation of the biosensor was found to be 8.0. Detection limit was determined as 0.039 mmol L⁻¹ according signal to noise ratio (S/N: 3). The proposed glucose biosensor was tested in human serum samples.     

Amperometric Glucose Biosensor Based on Ultrafine Platinum Nanoparticles

Abstract

In the present paper the ultrafine and highly dispersed platinum nanoparticles (average size 3 nm) were used for the construction of a glucose biosensor in a simple method. An excellent response to glucose has been obtained with a high sensitivity (137.7 µA mM^?1 cm^?2) and fast response time (5 s). The biosensor showed a detection limit of 5 µM (at the ratio of signal to noise, S/N=3) and a linear range form 0.2 to 3.2 mM with a correlation coefficient r=0.999. The apparent Michaelis–Menten constant (k _m) and the maximum current were estimated to be 9.36 and 1.507 mA mM^?1 cm^?2, respectively. In addition, effects of pH value, applied potential and the interferents on the amperometric response of the sensor were investigated and discussed.     

基于多壁碳纳米管和氧化锌纳米棒复合物的葡萄糖生物传感器(英文)

利用多壁碳纳米管(MWCNTs)和氧化锌(ZnO)纳米棒复合物膜构建了一种新的电流型葡萄糖生物传感器。MWCNTs-ZnO复合物在超声协助下通过静电配位的方式产生。其中,ZnO纳米棒的存在加强了该复合物催化氧化H2O2的能力,增加了响应电流。与单一的MWCNTs和ZnO相比,这种纳米复合物显示了更为有效地电催化活性。在此基础上,我们以MWCNTs-ZnO复合物膜为基底,用戊二醛交联法固定葡萄糖氧化酶,电聚合邻苯二胺(PoPD)膜为抗干扰层,构建了抗干扰能力强,稳定性好,灵敏度高,响应快的葡萄糖传感器。在+0.8V的检测电位下,该传感器对葡萄糖响应的线性范围为5.0×10-6~5.0×10-3mol·L-1(R=0.997),检测限为3.5×10-6mol·L-1(S/N=3),响应时间小于10s的葡萄糖生物传感器,常见干扰物质如抗坏血酸和尿酸不影响测定。     

基于葡萄糖/葡萄糖氧化酶/H2O2/L-酪氨酸/辣根过氧化物酶反应体系的荧光毛细分析法测定血糖

建立了葡萄糖(G)/葡萄糖氧化酶(GOD)/H2O2/L-酪氨酸/辣根过氧化物酶(HRP)新荧光反应新体系,并将此反应体系与荧光毛细分析法(FCA)结合,开发一种能够真正实现血糖测定的新葡萄糖酶荧光毛细分析法(GE-FCA).经优选得到的新反应体系最佳条件为:GOD和HRP浓度均为2000 U/L,L-酪氨酸浓度为1....     

酶荧光毛细分析法测定葡萄糖

基于酶催化和荧光毛细分析法(FCA)开发了一种微量、快速测定葡萄糖的新方法(GE-FCA)。优选的实验条件为:反应时间15min;反应温度30℃;磷酸盐缓冲液(pH6.0),HRP浓度和GOD浓度分别是200和150U/L;线性范围0.1～4mg/L;检出限0.070mg/L。GE-FCA对血液中葡萄糖进行测定,其回收率在98.9%～103.8%之间。与其它方法相比,GE-FCA法操作简单,而且试样用量少(仅为18μL),节省了酶试剂用量,实验成本低,易于普及推广。     

基于固定化酶的流动荧光法测定血清中的葡萄糖

本文将固定化酶柱，光纤以及停流技术结合起来，研究了以廉价而易得的硫胺素为荧光底物的葡萄糖的荧光分析，提出了一种快速、简便、精确地测定葡萄糖的新方法。其线性范围为１．０￣６０．０ｍｇ／Ｌ，线性相关系数为０．９９９，检测限为０．１５ｍｇ／Ｌ。以５．０ｍｇ／Ｌ的葡萄糖作精度试验，ＲＳＤ％＝２．１％（ｎ＝１１）。该方法已成功地用于血清中葡萄糖的测定，结果与光度法相符。     

Tannic Acid Modified Electrochemical Biosensor for Glucose Sensing Based on Direct Electrochemistry

A novel glucose biosensor was constructed through the immobilization of glucose oxidase (GOx) on gold nanoparticles (Au NPs) deposited, and chemically reduced graphene oxide (rGO) nanocomposite. In the synthesis, tannic acid (TA) was used for the reduction of both graphene oxide, and Au³⁺ to rGO, and Au NPs, respectively. Also, by harnessing the π‐π interaction between graphene oxide and TA, and protein‐TA interaction, a novel nanocomposite for the fabrication of a third generation biosensor was successfully constructed. Upon the oxidation of TA to quinone, which is easily reducible at the negative potential range, enhanced electron transfer was obtained. The cyclic voltammetry (CV) results demonstrated a pair of well‐defined and quasi‐reversible redox peaks of active site molecule of GOx. The biosensor exhibited a linear response to glucose concentrations varying from 2 to 10 mM with a sensitivity of 18.73 mA mM⁻¹ cm⁻². The fabricated biosensor was used for the determination of glucose in beverages.     

共反应试剂增强电致化学发光信号生物传感器

在电致化学发光（ECL）生物传感器的构建中,利用共反应试剂促进发光基团的发光效率是一种常见、方便且非常有效的方法. 然而,如何更好地利用共反应试剂使其更加有效地与发光基团作用是提高该类生物传感器灵敏度的重要因素. 本文结合作者课题组部分工作综述了三种共反应试剂放大ECL信号的构建：共反应试剂内置于检测底液;共反应试剂共存于电极表面;酶促生成共反应试剂,并提出了今后ECL信号放大构建的展望.     

基于纳米材料电化学生物传感器的研究进展

近年来,纳米材料在电化学生物传感器领域的研究已成为前沿性的内容.纳米材料具备优异的物理、化学、电催化等性能,加之其量子尺寸效应和表面效应,可将传感器的性能提高到一个新的水平.基于纳米材料的电化学生物传感器呈现出体积更小、速度更快、检测灵敏度更高和可靠性更好等优异性能.该文按照纳米结构的分类,综述了近几年基于以下纳米材料...     

葡萄糖浓度的酶联荧光毛细分析法测定

该文以葡萄糖氧化酶和辣根过氧化物酶为催化剂,使含有对甲基酚的葡萄糖溶液体系通过酶偶联催化反应生成荧光物质,从而实现对葡萄糖浓度的测定。优化的实验条件为:反应时间20 min;NH3-NH4Cl缓冲溶液(pH 10.4);对甲基酚浓度30.0μmol/L。分别采用荧光毛细分析法和荧光光谱分析法测定了相同浓度的系列葡萄糖溶液的荧光强度。在5.0~500.0μmol/L范围内,两种方法测得的荧光强度均与葡萄糖浓度的对数成正比。通过对比测试结果分析了两种方法的优缺点。     

碳纳米管负载铂颗粒酶电极葡萄糖传感器

以碳纳米管负载纳米铂颗粒修饰玻碳电极 (CNT Pt/GCE)为基底 ,用明胶固定葡萄糖氧化酶(GOD) ,构建了电流型葡萄糖生物传感器 (GOD/CNT Pt/GCE)。在实验中 ,GOD/CNT Pt/GCE显示了良好的分析性能 ,与常规铂电极葡萄糖传感器 (GOD/Pt)相比较 ,测定葡萄糖的检出限从 6 .7× 10 -3 mol/L下降到8.3× 10 -4mol/L ;工作电位从 0 .6 5V下降至 0 .4 5V ;响应时间从 30s下降至 5s左右。实验结果表明 ,具有高度电催化活性的CNT Pt/GCE可作为酶传感器的一种新型基体电极。