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。     

开管柱固定化葡萄糖氧化酶反应器的制备及其在流动注射分析中的应用

本文将葡萄糖氧化酶化学键合到粗糙化的玻璃毛细管内壁上,用开管柱固定化酶反应器-安培检测器进行葡萄糖的流动注射分析。讨论了流速对流动注射分析体系峰电流和分散度的影响,提出了测量开管柱固定化酶表观活力的方法。     

Electrochemical Measurements of Glucose Using a Micro Flow‐Through Immobilized Enzyme Reactor

A new fast, cheap and efficient epoxy‐amine immobilized enzyme reactor (IMER) is demonstrated. Polyethyleneimine (PEI) was used as the curing agent of an epoxy resin (bisphenol‐A diglycidyl ether (BADGE)) in order to introduce a high number of reactive –NH₂ groups at the substrate. A ratio mixture of 3:2 PEI:BADGE (w/w) was shown to be the most suitable for curing, taking into account the number of immobilization sites and the resistance of the material towards channeling. Electrochemical measurements made by a three electrode system, adapted at the IMER, showed that the K_m value for immobilized glucose oxidase (GOx) was half the value commonly reported for GOx immobilized at PEI derived substrates. The IMER response decreased by around 41 % after one week of intense usage. Even so, the remaining activity was sufficient for quantifications of approximately 0.1 mmol L⁻¹, merely requiring a new calibration of the reactor before its utilization. The developed system was applied to D‐glucose quantification of beverage samples, requiring an injection volume of only 10 μL and a flow rate of 150 μL min⁻¹ (almost 60 samples per hour). Low detection and quantification limits (1.94 and 5.89 μmol L⁻¹, respectively) and a wide linear range (from 8 μmol L⁻¹ to 2 mmol L⁻¹) were also found, which are useful characteristics for quality control analysis.     

固定化葡萄糖氧化酶活性的X射线微区分析

利用X射线微区分析方法,对固定化活性葡萄糖氧化酶进行了定位分析;葡萄糖作为底物,FeSO4和KI作为捕捉剂,底物经固定化葡萄糖氧化酶催化产生H2O2,后者和捕捉剂反应生成沉淀,可以确定固定化葡萄糖氧化酶的催化活性部位。结果表明：颗粒越小,酶活越高,活性葡萄糖氧化酶在凝胶内分布均匀,且绝大多数葡萄糖氧化酶固定在凝胶的内部。作者还研究了固定化活性葡萄糖氧化酶定位的最佳条件。     

Amperometric Biosensor Coupled to Capillary Electrophoresis for Glucose Determination

A novel glucose biosensor has been fabricated and employed as the amperometric detector of a capillary electrophoresis (CE) system. (±)-1-Ferrocenylethylamine and chitosan were successively modified on a 500-µm diameter disc platinum electrode by dip-coating. The modified electrode was subsequently immersed in glucose oxidase (GOx) solution to entrap the enzyme in the chitosan membrane. The primary amino groups of 1-ferrocenylethylamine, GOx, and chitosan were cross-linked by glutaraldehyde to obtain a biosensing membrane so as to reduce leaching of 1-ferrocenylethylamine and GOx. The electrochemical behavior of the target biosensor was investigated. It was demonstrated that the investigated biosensor features fast response, high stability, long lifetime, and ideal compatibility with the CE system. When CE was employed to introduce a glucose plug into the surface of the biosensor, the current response was linear to the glucose concentration in the range of 0.0025 to 2.5 mM with a detection limit of 1.2 µM (S/N = 3) at a working potential of +0.6 V (vs. SCE). The CE-biosensor system was applied to the determination of the glucose level in human serum. The results were satisfactory and in good agreement with the hospital assay results.     

Amperometric Determination of Catalase in Brazilian Commercial Honeys

A sensor for catalase (CAT) amperometric detection in association with flow injection analysis and a tubular reactor containing Amberlite IRA-743 resin was developed. Catalase was quantified in 14 samples of Brazilian commercial honeys. At flow rate of 1.5 mL min^?1 and injecting 200 µL of sample volumes, a sampling frequency of 90 determinations per hour was afforded. The linear dynamic range in CAT extends from 50 to 5000 UI mL^?1, at pH 7.4. The levels of CAT vary from 9.97 to 99.07 UI mg^?1. Taking into account these results, an inverse correlation was obtained with CAT and H₂O₂ levels.     

Determination of Urinary Glucose by a Flow Injection Analysis Amperometric Biosensor and Ion-Exchange Chromatography

A practical biosensor system has been developed for the determination of urinary glucose using a flow-injection analysis (FIA) amperometric detector and ion-exchange chromatography. Glucose oxidase was immobilized onto porous aminopropyl glass beads via glutaraldehyde activation to form an immobilized enzyme column. On the basis of its negative charge at pH 5.5, endogenous urate in urine samples was effectively retained by an upstream anion-exchange resin column. The biosensor system possessed a sensitivity of 160 ±2.4 RU μM^-1 (RU or relative unit is defined as 2.86 μV at the detection output) for glucose with a minimum detection level of 10 μM. When applied for the determination of urinary glucose, the result obtained compared very well with that of the widely accepted hexokinase assay. The immobilized glucose oxidase could be reused for more than 1000 repeated analyses without losing its original activity. The reuse of the acetate anion-exchange column before replacement would be about 25–30 analyses. Acetaminophen and ascorbic acid were also effectively adsorbed by the acetate anion exchanger. The introduction of this type of anion exchanger thus greatly improved the selectivity of the FIA biosensor system and fostered its applicability for the determination of glucose in urine samples.     

固定化酶法测定血清中葡萄糖和胆固醇

本文讨论了一种灵敏、高选择性的方法分别测定葡萄糖和胆固醇,并介绍了成二醛固定化酶(葡萄糖氧化酶或胆固醇氧化酶)后产生的过氧化氢借助于鲁米诺-铁氰化钾发光反应由流动注射法测定。对血清中样品的前处理和反应试剂浓度的影响也分别作了研究。本法有良好的线性工作范围,连续进样100次仍能保持工作的稳定性,相对标准误差为3.8～5.2%。     

纳米金-还原氧化石墨烯修饰葡萄糖氧化酶传感器的制备及其电流法检测饮料中的葡萄糖

利用纳米金(Au NPs)与还原氧化石墨烯(rGO)复合纳米材料制备了葡萄糖氧化酶生物传感器并用于饮料中葡萄糖含量的检测。将壳聚糖作为还原剂及稳定剂,通过一步法合成了Au NPs-rGO复合材料,并通过物理吸附固定葡萄糖氧化酶(GOx)来制作GOx生物传感器。该传感器在磷酸盐缓冲溶液(0.1 mol/L,p H6.0)中,-0.45 V(vs.Ag/Ag Cl)电位下电流法检测葡萄糖含量,线性检测范围为0.01~0.88 mmol/L,灵敏度为22.54μA·mmol-1·L·cm-2,检出限为1.01μmol/L,且表观米氏常数为0.497 mmol/L。该传感器用于多种饮料中葡萄糖含量的直接检测,结果满意。     

Biosensing Membrane Base on Ferulic Acid and Glucose Oxidase for an Amperometric Glucose Biosensor

A biosensing membrane base on ferulic acid and glucose oxidase is synthesized onto a carbon paste electrode by electropolymerization via cyclic voltammetry in aqueous media at neutral pH at a single step. The developed biosensors exhibit a linear response from 0.082 to 34 mM glucose concentration, with a coefficient of determination R² equal to 0.997. The biosensors display a sensitivity of 1.1 μAmM⁻¹ cm⁻², a detection limit of 0.025 mM, and 0.082 mM as glucose quantification limit. The studies reveal stable, repeatable, and reproducible biosensors response. The results indicate that the novel poly-ferulic acid membrane synthesized by electropolymerization is a promising method for glucose oxidase immobilization towards the development of glucose biosensors. The developed glucose biosensors exhibit a broader linear glucose response than other polymer-based glucose biosensors.     

The Amperometric Glucose Enzyme Electrode by Immobilized Glucose Oxidase in Glucose Oxidase/N-ethyl-Poly (4-vinyl)pyridine Multilayer

Enzylnebasedampcr()metricbiosensorsha\'eattractedincreasinginterestinthe1astt`xodecades.Inordertofabricate[heenzyn1elayer,considerahlceff()rtsha\,eheendcv()tcdt(1thedeveloprnent()fvari()ustechniquesf()rin1n1obilizingthcen/yn1c"2.Rcccntly.thcp()lyelectrolyte-proteincomplcx111ultilayerassen1blybyaIternateelectr()staticadsorptionhasbcenrep()rted',amullilaycrc()nsistingofalterl1atepolyethylenimineandglucoseoxidasclaycrswasassemb1edhyLv()veltl/>.ThesebioIllo1eculararthitecturesopenawaytoconstruct…     

Amperometric Glucose Determination by Means of Glucose Oxidase Immobilized on a Cellulose Acetate Film: Dependence on the Immobilization Procedures

Glucose microelectrodes were prepared by immobilizing glucose oxidase onto a cellulose acetate film coating a platinum wire. Hexamethylenediamine (HMDA) and Glutaraldehyde (GA) were employed as spacer and coupling agent, respectively. Sensitivities and linear response ranges were studied as a function of the relative amounts of HMDA and GA. The best sensitivity was found when HMDA and GA were 5% and 2.5% in aqueous solutions, respectively. Taking as a reference the functioning of this biosensor, the roles of HMDA and GA percentages appear to be opposed when the extension of the linear response range is considered. Indeed, an increase of one unit in HMDA percentage (from 5 to 6 %) induces an increase in the extension of the linear response range equal to that obtained with a decrease of one unit of GA percentage (from 2.5 to 1.5%).     

Amperometric Detection of Glucose with Glucose Oxidase Immobilized in Layered Double Hydroxides

A novel cheap and simple amperometric biosensor, based on the immobilization of glucose oxidase (GOD) into anionic clay; layered double hydroxides (LDHs) [Zn₃‐Al‐Cl] is presented. GOD can be entrapped in the LDHs gel via electrostatic interaction. Amperometric detection of glucose with an unmediated sensor at 0.6 V (vs. SCE) results in a rapid response (5 s), a wide linear range of 0.001–12 mM, as well as good operational stability. The low detection limit was 0.1 μM at 3σ. The apparent Michaelis‐Menten constant (K is 4.4 mM. The general interferences that coexisted in blood serum do not affect glucose determination, except for uric acid. In addition, optimization of the biosensor construction and the effects of the applied potential on the amperometric response of the sensor were investigated and discussed herein.     

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.     

Prussian Blue Modified Amperometric Hg2+ Ion Biosensor Based on Glucose Oxidase Inhibition

In this research a Hg²⁺ ion biosensor was developed by combining Prussian blue (PB) with glucose oxidase (GOx) – an enzyme that can be inhibited by Hg²⁺ ions. An application of PB in the design of Hg²⁺ ion biosensor enabled detecting changes in hydrogen peroxide reduction current at low operational potential of 0.2 vs Ag|AgCl,KCl_sat. The described Hg²⁺ ion biosensor exhibited wide linear range from 27 μM to 247 μM of Hg²⁺ and higher maximal detectable concentration of Hg²⁺ than other GOx inhibition-based biosensors, making it convenient for the analysis of samples with high concentration of Hg²⁺ ions.     

Amperometric Sensor and Immobilized Enzyme Electrode for the Determination of Enzymatic Activities

Abstract

A sensitive method for the rapid determination of activities of soluble or immobilized enzymes, based on the electrochemical detection of hydrogen peroxide is described. Kinetic studies (Vmax and K_M determinations) can be performed for all H₂O₂ generating enzymes (i.e. most of the oxidases) using an amperometric probe with a platinum anode at a fixed potential.

When associated with an immobilized glucose oxidase membrane, this sensor constitutes a glucose electrode and the activity of any hydrolase which releases glucose can be measured. There is no need for other auxiliary enzymes and no preincubation step is required. The possibility to carry out continuous analysis constitutes the main advantage of the described method.     

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.     

Continuous Amperometric Determination of Glucose Using an Immobilized Enzyme Reactor in Combination with an Immersible Dialysis Probe

Abstract

Glucose was continuously determined by reaction in a packed-bed enzyme reactor containing glucose oxidase and catalase. Oxygen consumption was measured amperometrically with a polarographic Clark electrode. Glucose was sampled through a dialysis probe immersed in the solution to be measured. An extension of the normal range for the enzyme was achieved by modulating the flow rate through the dialysis probe and a linear response was obtained in the range of 1.0-60 mM glucose. The correlation between the glucose transfer and the membrane area of the dialysis probe was also studied. Six different membranes were used, all showing variations in the adhesion of yeast cells.     

Amperometric biosensors based on the immobilization of oxidases in a Prussian blue film by electrochemical codeposition

Prussian blue has been formed by cyclic voltammetry onto the basal pyrolytic graphite surface to prepare a chemically modified electrode which provides excellent electrocatalysis for both oxidation and reduction of hydrogen peroxide. It is found for the first time that glucose oxidase or -amino oxidase can be incorporated into a Prussian blue film during its electrochemical growth process. Two amperometric biosensors were fabricated by electrochemical codeposition, and the resulting sensors were protected by coverage with a thin film of Nafion. The influence of various experimental conditions was examined for optimum analytical performance. The glucose sensor responds rapidly to substrates with a detection limit of 2 × 10⁻⁶ M and a linear concentration range of 0.01–3 mM. There was no interference from 2 mM ascorbic acid or uric acid. Another ( -amino acid) sensor gave a detection limit of 3 × 10⁻⁵ M -alanine, injected with a linear concentration range of 7.0 × 10⁻⁵-1.4 × 10⁻² M. Glucose and -amino acid sensors remain relatively stable for 20 and 15 days, respectively. There is no obvious interference from anion electroactive species due to a low operating potential and excellent permselectivity of Nafion.     

Amperometric Biosensor Based on Enzymatic Reactor for Choline Determination in Flow Systems

A simple, selective and stable biosensor with the enzymatic reactor based on choline oxidase (ChOx) was developed and applied for the determination of choline (Ch) in flow injection analysis with amperometric detection. The enzyme ChOx was covalently immobilized with glutaraldehyde to mesoporous silica powder (SBA‐15) previously covered by NH₂‐groups. This powder was found as an optimal filling of the reactor. The detection of Ch is based on amperometric monitoring of consumed oxygen during the enzymatic reaction, which is directly proportional to Ch concentration. Two arrangements of an electrolytic cell in FIA, namely wall‐jet cell with working silver solid amalgam electrode covered by mercury film and flow‐through cell with tubular detector of polished silver solid amalgam were compared. The experimental parameters affecting the sensitivity and stability of the biosensor (i. e. pH of the carrier solution, volume of reactor, amount of the immobilized enzyme, the detection potential, flow rate, etc.) were optimized. Under the optimized conditions, the limit of detection was found to be 9.0×10^?6 mol L^?1. The Michaelis‐Menten constant for covalently immobilized ChOx on SBA‐15 was calculated. The proposed amperometric biosensor with the developed ChOx‐based reactor exhibits good repeatability, reproducibility, long‐term stability, and reusability. Its efficiency has been confirmed by the successful application for the determination of Ch in two commercial pharmaceuticals.