硫化物微生物传感器的研制与应用

研制了硫化物微生物传感器并用于测定硫化物。该传感器对 S2 -的测定线性范围为 0 .0 3～ 3.0 0 mg/L,测定一个样品需 2～ 3min;实验选出了最佳测试条件和用于实际样品分析     

Amperometric Choline Sensor with Enzyme Immobilized by Gamma-Irradiation in a Biocompatible Membrane

Abstract

An amperometric choline biosensor was constructed using choline oxidase immobilized on poly(2-hydroxyethylmethacrylate) membranes obtained by gamma radiation-induced polymerization at low temperature. The measurements were carried out by Clark-type oxygen or hydrogen peroxide electrodes. Calibration curves were linear in the 10-200 umol · 1^?1 range for the oxygen probe and 5-250 umol · 1^?1 for the H₂O₂-based probe. Temperature and pH effects on the activity of immobilized enzyme are described and the response characteristics of the sensor are summarized. The immobilized enzyme membranes stored in glycine buffer or in a dry state were very stable and no significant decrease in the electrode response was observed after three months. The biosensor was employed also to analyse a choline-containing pharmaceutical product and the results were compared to those obtained by enzymatic-spectrophotometric detection.     

光致电化学鸟嘌呤传感器的制备及其应用

将硫堇电聚合在光透电极的表面,再利用壳聚糖将黄嘌呤氧化酶固定在具有光电活性的聚硫堇光透电极的表面上制备了光致电化学鸟嘌呤传感器.基于同时具有光敏和电子受体功能的聚硫堇光电界面,该传感器能与黄嘌呤氧化酶催化鸟嘌呤氧化而产生的电子供体(过氧化氢)产生光致电化学响应,通过测量光致电化学反应产生的光电流实现了对鸟嘌呤的检测.文中探讨了传感器的光致电化学响应机理,讨论了偏压、酶量、电解质溶液pH对传感器测定鸟嘌呤的影响.在优化的实验条件下,该传感器对鸟嘌呤的测定范围为1.00～200μmol/L,检出限为0.55μmol/L,9次测定的相对标准偏差小于3.92%.应用该传感器对酸解DNA脱出的鸟嘌呤基和药品阿昔洛韦进行的检测实验显示,相对标准偏差小于5.37%,加标回收率为96.8%～106%.该传感器的制备和对鸟嘌呤的检测不需要过氧化物酶,不需要除氧,有经济、简便等优点.     

The Application of an Enzyme Electrode for Blood Glucose Determination in the Automated Flow System

Abstract

An enzyme electrode containing immobilized GOD was utilized in a continuous flow system for the automatical determination of glucose. The advantages of kinetic measurement of H₂ O ₂ are used. Characteristic parameters of device and results in routine are shown. Particular advantages are: low requirement on material (20 μl blood, < l μg GOD/measurement) and time (60 samples/h at 15 sec response time) as well as sufficient precision and accuracy (S % - serial: < 2 %).     

利用壳聚糖的絮凝性质包埋酶的葡萄糖氧化酶电极

本文利用壳聚糖的絮凝性质，将葡萄糖氧化酶包埋在壳聚糖与多聚磷酸盐的絮絮沉淀中，操作简单，固定化效率高。研究了适合了酶电极使用的最佳固定化条件。     

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.     

A Stable Enzyme Biosensor Using Concanavalin a Complex of Glucose Oxidase for Glucose Determination

Abstract

The thermal stability of an insoluble concanavalin A (ConA) complex of glucose oxidase (GOD) was researched. The thermal deactivation rate constants of the complexes were obtained. It was found that the GOD-ConA complexes were less sensitive to thermal inactivation than the native enzyme GOD. By using the complexes, ferrocene-mediated enzyme electrodes were constructed. The results suggested that the GOD-Con A complex electrodes had good thermal stability at room temperature.     

压电晶体传感器阵列用于含乙醇的蒸气检测

将多个表面涂有不同选择性吸附材料的压电晶体传感器组成阵列，用于混合有机溶剂蒸气中乙醇的检测。对阵列输出的数据，采用逐步判别法进行二类判别，可以检测混合蒸气中少量的乙醇，采用主成分回归法，可由阵列输出数据预测未知样品中乙醇的浓度。     

Preparation of a Xanthine Sensor Based on the Immobilization of Xanthine Oxidase on a Chitosan Modified Electrode by Cross‐linking

Here in this paper, xanthine oxidase (XOD) was immobilized onto the chitosan (CHT) modified electrode by a simple way of cross‐linking with glutaraldehyde (GTD) and 3‐aminopropyltriethoxysilane (KH). The electrode displayed a sharp peak to the oxidation of xanthine at a potential about 0.67 V and the optimum of pH for determination was investigated. Under the optimum conditions, the biosensor fabricated on the KH/GTD/XOD/CHT modified electrode showed excellent response to the oxidation of xanthine within the range of 0.5 to 18 μmol/L with a low detection limit of 0.0215 µmol/L, a good stability and a high selectivity. The sensor can also be used for the determination of hypoxanthine. The electrochemical results indicated that the immobilized enzyme still retained its biological activity and this provided a new way for the construction of biosensors and determination of xanthine.     

Enzyme Electrode Sensor for Hydrogen Peroxide

Abstract

An enzymatic sensor was constructed from an oxygen amperometric sensor on whose surface a dialysis membrane containing covalently bonded catalase was set. Immobilization of the enzyme on the dialysis membrane surface was achieved with 2,4-dichloro-6-methoxy-s-triazine, a derivative of cyanuric chloride, which unlike others of its monosubstituted derivatives ensures some advantages. The time of measurement is less than 12 sec, for the kinetic method of the initial slope and one minute for the steady-state method. The sensor responds linearly to hydrogen peroxide in the concentration range 10^?3 - 10^?5 moles/1. The utilization of this sensor in intermittent and continuous operation in a laboratory enzymatic minireactor is discussed.     

Functionalized Gold Nanoparticles – Octadecylamine Hybrid Langmuir‐Blodgett Film for Enzyme Sensor

Mediator free enzyme sensor has been fabricated by covalently immobilizing cholesterol oxidase (ChOx) onto 11‐mercaptoundecanoic acid functionalized gold nanoparticles (MUDA‐AuNPs) – octadecylamine (ODA) hybrid Langmuir–Blodgett film. The cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) studies reveal that MUDA‐AuNP/ODA LB film has good affinity for ChOx and provides favorable microenvironment for direct electron transfer between enzyme and electrode. Interference free estimation of cholesterol has been realized at 0.3 V with linear range from 25 to 500 mg/dL, detection limit of 23.38 mg/dL, sensitivity of 1.085 μA mM^?1 and response time of 20 s at pH 7.0.     

盐酸阿霉素分子印迹传感器的制备及识别特性

采用自组装以及电聚合的方法,在磷酸盐缓冲液(PBS)中以3,4-乙烯二氧噻吩(EDOT)为功能单体,盐酸阿霉素(DOX)为模板,在金电极表面电聚合制备DOX印迹敏感膜(MIPs),构建了一种选择性检测DOX的分子印迹电化学传感器.采用循环伏安法(CV)及交流阻抗法(EIS)对其性能进行了表征.优化实验条件后,在含0.005 mol/L K3[Fe(CN)_6]及0.1 mol/L KCl的PBS中,应用差分脉冲伏安法(DPV)测试了该传感器的响应性能.实验结果表明,该传感器检测DOX的线性范围为4.0×10~(-7)~1.0×10~(-6)mol/L,相关系数为0.9967,检出限(S/N=3)达6.5×10~(-8)mol/L;采用电化学洗脱法可使传感器再生,对DOX的测定具有良好重现性及稳定性;该传感器对于干扰物长春碱、放线菌素D及5-氟尿嘧啶有微弱的电流响应,显示出良好的选择性.将该传感器用于人体血样中盐酸阿霉素的分析,回收率为96.0%~106.7%,表明其具有潜在的实用价值.     

Preparation and characterization of sensing membranes for the detection of glucose, lactate and tyramine in microtiter plates

In this work, sensing membranes for the detection of glucose, lactate and tyramine were successfully prepared by immobilizing enzymes and fluorophore on sol-gels. The membranes were fabricated on the bottom of the wells in a microtiter plate. Glucose oxidase (GOD), lactate oxidase (LOD) and tyramine oxidase (TOD) were immobilized on individual sol-gels or a mixture of different sol-gels (3-glycidoxypropyl-trimethoxysilane (GPTMS), methyl-triethoxysilane (MTES), aminopropyl-trimethoxysilane (APTMS)). The oxidation of the analytes specifically catalyzed by the enzymes resulted in the reduction of the oxygen concentration, which changed the fluorescence intensity (FI) of the oxygen sensitive ruthenium complex acting as the transducer. The linear calibration graphs were in the ranges of 0.0-5.0 g/l for glucose, 0.0-9.0 mg/l for lactate and 0.0-100 mg/l for tyramine. The values of the detection limit were found to be 0.10-0.52 g/l for glucose, 7.77 mg/l for lactate and 6.30-8.73 mg/l for tyramine. The covalent binding between the epoxy and amine groups of the sol-gels and enzymes, respectively, prevented the enzymes from being washed out and preserved the high stability of the sensing membranes. The different ratios of silanes in the sol-gels, which were used as the supporting matrix for the immobilization of the enzymes led to different responses of the sensing membranes to various concentrations of glucose, lactate and tyramine. The kinetic parameters of the enzymatic reactions, and the stability and other parameters for the sensing membranes were also investigated.     

Development and Application of an Immobilized Enzyme Electrode for the Determination of Sulfite in Foods and Feeds

Abstract

An enzyme electrode was developed using the enzyme sulfite oxidase (EC 1.8.3.1) immobilized onto pig intestine. All experimental parameters such as pH, temperature, buffer constituent concentration, etc., were thoroughly investigated and optimized when appropriate. Hydrogen peroxide was monitored amperometrically. The response to sulfite concentration was linear in the range of 5.2 × 10^?6 to 1.0×10^?3M. The proposed method was found to have a correlation coefficient of 0.952 when evaluated versus the standard titration method.     

利用60%季铵化聚(4-乙烯)吡啶固定化酶的电位型尿素酶电极的研制

在电位型尿素酶电极的组装过程中，要求尽量不改变尿素酶的构型和构象，使其在酶膜中保持其自然状态，从而可获得较高的酶活力．用ｐＨ玻璃电极作原电极，将尿素酶固定在其表面，常用的有戊二醛交联法［１］和各种聚合物膜法［２～４］．本文利用６０％季铵化的聚（４－乙...     

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.     

分子印迹荧光传感器的研制及其对呋喃妥因的特异性识别

采用柠檬酸作为碳源合成了荧光碳量子点(CDs),经硅烷化试剂改性后,以呋喃妥因为模板分子,在其表面合成了分子印迹聚合物(MIP-CDs)。以傅立叶变换红外光谱仪(FTIR)和扫描电镜(SEM)对合成产物进行结构和形貌表征;通过一系列吸附实验研究了MIP-CDs对呋喃妥因的识别性能。实验结果表明:在最佳吸附条件下,MIP-CDs的荧光猝灭程度与呋喃妥因的浓度符合Stern-Volmer方程,呋喃妥因在0~10mg/L质量浓度范围内具有良好的线性关系(r2=0.991 4),方法检出限(S/N=3)为2μg/L。在水样和饲料样品中添加不同浓度的呋喃妥因,加标回收率为78.0%~95.2%,相对标准偏差(RSD)不大于6.4%,符合分析方法学要求。该方法简单、快速、选择性好,可直接用于水体和饲料中痕量呋喃妥因的监测。     

聚乙烯醇/聚吡咯复合导电水凝胶应变传感器的制备及性能

以聚乙烯醇(PVA)为原料, 植酸(PA)和氨基-聚倍半硅氧烷(NH₂-POSS, NP)为交联剂, 通过冻融循环法制得PVA/PA/NP复合水凝胶, 再以其为模板, 通过吡咯的原位聚合制得PVA/PA/NP-PPy复合导电水凝胶, 克服了聚吡咯材料易脆、 疏水的特性, 进一步改善了水凝胶的导电性和灵敏性. 循环拉伸实验结果表明该水凝胶具有良好的自回复能力, 电导率高达7.53 S/m, 从I-V曲线可知其作为柔性可穿戴应变传感器的最高检测电流可达 9.029 mA, 灵敏度因子可达6.796, I-T曲线表明该传感器可以准确地通过电流信号变化来监测人体的各种微小运动.     

The Preparation and Enzyme Immobilization of Hydrophobic Polysiloxane Supports

Enzymes are versatile biocatalysts and find increasing applications in many areas. The major advantages of using enzymes in biocatalytic transformations are their chemo‐, regio‐, and stereospecificity, as well as the mild reaction conditions that can be used. However, even when an enzyme is identified as being useful for a given reaction, its application is often hampered by its lack of long‐term stability under process conditions, and also by difficulties in recovery and recycling. For ease of application and stabilization purposes, enzymes are often immobilized on solid supports. Among support matrices, hydrophobic biomaterials have been extensively used as supports for enzyme immobilization because the hydrophobic interactions not only can effectively increase the amount of enzyme immobilization, but also exhibit higher activity and retention of activity compared with hydrophilic supports. On the other hand, polysiloxane can evidently increase the amount of enzyme immobilization because of its hydrophobicity and strong affinity with enzyme. Therefore, this research details the first preparation and use of a hydrophobic polysiloxane support for enzyme immobilization in which the structural and functional characteristics of new supports have been investigated by using glucose oxidase (GOD) and a simple Fenton's assay method, and extremely interesting features were revealed. The results showed that the amount of GOD immobilization and the stability of GOD loaded, which are fundamental properties for enzyme separation and purification, can be significantly improved by adsorption. Moreover, the results indicated that hydrophobic polysiloxane supports can effectively increase the enzymatic affinity and durability of GOD, and decrease the rate of GOD desorbed.

     

菊酯类农药代谢物分子印迹荧光传感器的制备及应用

制备了一种分子印迹荧光传感器用于菊酯类农药代谢物间苯氧基苯甲醛(3-PBD)的选择性测定。以3-PBD为模板分子,3-氨丙基三乙氧基硅烷为功能单体,四乙氧基硅烷为交联剂,采用溶胶-凝胶法在Mn掺杂ZnS量子点表面成功合成了荧光分子印迹聚合物。采用红外光谱、X射线衍射及X射线光电子能谱对聚合物进行表征,并对检测条件进行了优化。在最佳条件下,该荧光传感器对3-PBD的响应线性范围为0.3～5μmol/L,检出限为0.267μmol/L。此分子印迹荧光传感器可与农药的酶解过程相结合,用于菊酯类农药的快速检测。对实际样品的测定结果显示,3-PBD的回收率为84.0%～119%,相对标准偏差不超过5.0%。结果表明,该分子印迹荧光传感器可以用于食品中3-PBD的测定。