Amperometric Enzyme Sensor-Based Enzyme Immunoassay for Factor VIII-Related Antigen

Abstract

The coupling of an enzyme immunoassay for factor VIII-related antigen with a commercial glucose oxidase based amperometric sensor permits the determination of 1.6 to 16 ng of factor XIII-related antigen in human plasma. Further pure amperometric sensors or amperometric enzyme sensors for determination of the main marker-enzymes of enzyme immunoassays are described.     

交联酶聚体技术研究进展*

交联酶聚体（CLEAs）是一类新型的固定化酶技术,具有制备简单、酶活回收率高、操作和保存稳定性强等优点。近年来,CLEAs技术与材料学、印迹工程、介质工程、反应工程学等相结合取得了一系列新进展,包括载体固定化CLEAs、包埋CLEAs、印迹法CLEAs、多酶CLEAs、CLEAs膜浆反应器等,在手性分子拆分与合成。抗生素生产等领域取得了一些成果。本文对CLEAs酶活影响因素及CLEAs技术的最新研究进展进行了分析与总结,并展望了需进一步深入开展的内容,有助于生物工程、酶工程、化学工程和材料科学等领域相关研究工作的开展。     

Glutathione Amperometric Enzyme Microsensor

Abstract

Glutathione micro-enzyme sensors were developed based on the immobilization of glutathione oxidase at the tip of a 25 μm Pt wire sealed in glass. An inner membrane constructed from cellulose acetate (CA) was deposited onto the tip of the Pt microelectrode prior to enzyme immobilization with glutaraldehyde. The final outer diameter of the microelectrodes is approximately 30 μm. The analytical characteristics of the microelectrodes, including calibration curves, apparent K_M′, pH response curves, stability and selectivity over enzymatic interferences were determined. The response was linear in the concentration range 1.0 × 10^?5 M - 1.9 × 10^?4 M glutathione, reaching 95% steady-state current in 15–20 seconds. The microelectrodes were useful for more than two months.     

大环化合物(macrocycles)酶模型

对大环化合物的结构特点及催化作用进行了综述。     

A Split-Flow Enzyme Thermistor

Abstract

The split-flow system is comprised of two identical micro-columns, one of which contains an immobilized enzyme preparation, the other an inert support material.

The heat produced in each column on introduction of a sample is measured with thermistors placed in these columns. The use of a reference column virtually eliminates the influence on the measurements of artifactual signals as unspecific heat, i.e., heat not produced by the enzymic reaction. The performance of the split-flow enzyme thermistor at a variety of pH's, ionic strengths or viscosities associated with the sample has been investigated and compared with previously described alternative enzyme thermistor arrangements. In this comparative study glucose at a concentration of 5 · 10^?4 M was used throughout. On passage through the imnobilized glucose oxidase preparation this solution gave rise to a heat change At of about 0.01°C. The insensitivity of the system described herein towards such variations makes it particularly suitable for the analysis of metabolities present in crude solutions such as urine and skim-milk.     

基于固定化酶的乙酰胆碱酯酶抑制剂体外筛选模型的建立

以可重复使用的固定化酶代替游离态酶, 建立一种基于比色分析的乙酰胆碱酯酶(AChE)抑制剂体外筛选新模型. 采用以氨基化硅胶为载体固定的AChE优化了实验条件, 用AChE抑制剂阳性对照物他克林和毒扁豆碱对该模型进行验证, 还对模型技术参数进行评价, 并将新模型用于单体化合物及天然产物粗提物AChE抑制活性评价. 结果表明, 最佳实验条件为: 固定化酶用量55 μL, 底物浓度5 mmol/L, 甲醇、 乙醇及体积分数不高于6%的二甲基亚砜水溶液均可作为样品溶剂; 模型验证及模型技术参数评价结果良好, 该模型对AChE抑制剂筛选有较好的特异性和灵敏度, 可用于筛选AChE抑制剂. 该模型具有适用性强、 固定化酶可重复使用及结果可靠等优点, 是单体化合物及天然产物粗提物AChE抑制剂活性评价的有效方法.     

溶解性可调节的酶载体制备和固定化酶的研究

本文利用自由基沉淀聚合反应,合成了甲基丙烯酸-丙烯酰胺-顺丁烯二酸酐三元共聚物,测定了这些共聚物形成水不溶性的大分子氢键复合物的临界pH值.利用共聚物上的酸酐基团,直接进行了木瓜蛋白酶的固定化,得到了具有液相酶与固相酶两者优点的新型修饰酶。     

交联血管紧张素转化酶聚集体的制备及性质

制备了交联血管紧张素转化酶聚集体（ACE-CLEAs）,比较了ACE-CLEAs及游离ACE的酶学性质,包括最适酶促反应温度、最适pH值、Km、vmax、温度稳定性及pH稳定性等。 以酶活力回收率为参考,确定了制备ACE-CLEAs的最佳条件为：饱和度为80%的(NH4)2SO4溶液作为沉淀剂,沉淀时间0.5 h,质量分数为0.02%的戊二醛作为交联剂,交联时间1 h。 通过比较酶学性质发现,ACE-CLEAs比游离ACE具有更好的温度稳定性及pH稳定性,且与游离ACE接近的Km值表明,ACE-CLEAs对底物的亲和力与游离酶几乎相当。     

Comparison of Oxalate Oxidase Enzyme Electrodes for Urinary Oxalate Determinations

Abstract

Enzyme electrodes for oxalate were constructed by immobilizing oxalate oxidase enzyme (E, C, 1,2,3,4) on both potentiometric carbon dioxide and amperometric hydrogen peroxide sensors. These systems were optimized, and the response characteristics of the two biosensors examined in a comparison study. Areas investigated include linear range, calibration curve slope, response time, limit of detection, and lifetime. Selectivity studies were carried out with the system and showed no measurable response to millimolar levels of various L-amino acids, metal ions or ascorbic acid.

This comparison was extended into the choice of an optimal sensor for urinary oxalate determinations with minimal sample pretreatment. The peroxide sensor performed best in urine samples, allowing for the 1:40 dilution necessary to minimize the effect of any interferents present. At this or greater dilution, the recovery of standard additions of oxalate averaged 95.5%, with an average relative standard deviation of 4.9%. The system retained its activity throughout two weeks of continued use.     

An Enzyme Immunoassay of Catalytically Active Proteases

Abstract

A method is proposed to assay catalytically active protease. The method is based on the subsequent inter action of the enzyme with a protein inhibitor immobi lized on polysterene and with antibodies labelled with a high-activity enzyme. Optimal conditions have been chosen for assaying alkaline protease from Bacillus subtilis (subtilisin) by ELISA with peroxidase as label, employing two protein serine protease inhibitors isolated from duck egg white and maize seeds. The proposed method enables the detection of the catalyti cally active enzyme at concentrations as low as 1 ng/ml for 1.5 hr. The c. v. of the assay is less than 5%. The method can be used to determine the equilibrium con stant of the enzyme-inhibitor reaction in solution.     

丝素膜固定化丝网印刷酶电极

采用丝网印刷技术制作的一次性薄层碳电极具有良好的分析性能。经化学修饰二茂铁后,以丝素为固定化载体固定含有多酚氧化酶的蘑菇萃取蛋白,所得酸电极对其底物邻苯二酚和多巴胺有良好响应,线性范围为２．０×１０－８～２．０×１０－２,ｍｏｌ／Ｌ,在３０ｓ内即可达到９５％稳态电流。该电极的ＲＳＤ为２．７％,且可保持１０天以上的工作寿命。该酶电极在低温湿润环境中可保存较长时间,因此有较好的商品化前景。     

Enzyme based assays in a sequential injection format: a review

Sequential injection analysis systems have been extensively exploited in the last decades for the implementation of enzyme based assays aiming the evaluation of enzyme activity or the determination of specific analytes. The most prominent aspects of the automation of enzymatic assays in these systems are discussed in this review. Special attention is devoted to the mode of enzyme manipulation in homogeneous or heterogeneous media and to the comparison with batch and flow injection enzyme methodologies. The possibility of implementing strategies for the enhancement of selectivity in specific determinations is also addressed. The more recent trends in this field are discussed focusing mainly on the miniaturization resorting to the lab-on valve platform as well as on the bead injection concept.     

胰蛋白酶交联聚体的制备及性质研究

交联酶聚体(CLEA)是一种新型的无载体固定化技术. 以胰蛋白酶为模型体系, 系统地研究了CLEA技术的制备工艺、应用条件、稳定性及结构形貌. (1)在制备工艺中考察了各步骤对CLEA酶活保留的影响, 重点分析了沉淀剂浓度、类型的影响, 结果表明100%乙醇是较为理想的沉淀剂; (2)在应用条件确定中测定CLEA的最适催化温度为70 ℃, 最适催化pH为9.0, 并解释了温度-活性、pH-活性曲线的漂移现象; (3)稳定性研究结果表明CLEA技术大幅提高了胰蛋白酶的热稳定性、溶剂稳定性, 且无酶泄漏; (4)在形貌表征中分别利用扫描电子显微镜、光学显微镜和激光粒度分析对CLEA的微观结构进行了多尺度研究, 着重讨论了其独特结构与优良特性间的关系. 本文所得结论为胰蛋白酶CLEA的应用提供基础数据, 并为CLEA技术应用于其它酶种提供参考.     

Improved Blood Compatibility at a Glucose Enzyme Electrode Used for Ejctraoorpokeal Monitoring

Abstract

A glucose enzyme electrode based on glucose oxidase has been used in an extracorporeal flow system which allows standardization during monitoring. The detection limit for glucose was 0.02 mmol/l, and the linear range, which extended to 5 mmol/l glucose, allowed measurement of whole blood after dilution. Restandardization following whole blood exposure led to a loss of sensitivity which was attributed to coating of electrode membranes by blood constituents. The problem was largely overcome through use of silane-treated membranes and the operation of electrodes in a narrow channel flow cell.     

毛细管电泳法在酶抑制剂筛选中的应用

毛细管电泳法以其分离效率高、耗时短、样品用量少、灵敏度高以及易于实现自动化等诸多特点,在筛选酶抑制剂研究中有着不可替代的优势.本文综述了毛细管电泳法在酶抑制剂筛选中的应用.主要介绍了应用最广泛的离线分析(Off-line assay)和在线分析(In-line assay),即柱前酶反应(Pre-capillary enzyme reaction)和在柱酶分析(In-capillary enzyme reaction).并对该领域的未来发展进行了展望.     

电化学酶传感器在环境污染监测中的应用

电化学酶传感器是一种应用广泛的生物传感器,它将酶及其底物相互作用的特异性与电化学的强大分析功能相结合,已经被广泛应用于药理学、临床、食品、农业以及环境监测中。制备电化学酶传感器的关键步骤是酶的固定化,选择用于制备电化学酶传感器的合适的酶固定化方法,在传感器电子转移动力学、稳定性和重现性等方面起着主要作用。本文在阐述电化学酶传感器工作原理的基础上,简要介绍了用于电化学酶传感器制备过程中的酶固定化方法,重点讨论了电化学酶传感器在监测环境中广泛存在的有机污染物、无机污染物和重金属等方面的应用,并对电化学酶传感器的发展方向进行了展望。     

Detection of Sulfatase Enzyme Activity with a CatalyCEST MRI Contrast Agent

A chemical exchange saturation transfer (CEST) MRI contrast agent has been developed that detects sulfatase enzyme activity. The agent produces a CEST signal at δ=5.0 ppm before enzyme activity, and a second CEST signal appears at δ=9.0 ppm after the enzyme cleaves a sulfate group from the agent. The comparison of the two signals improved detection of sulfatase activity.     

生物素标识酶在生物传感器中的应用研究

利用生物素与亲合素之间的特异性结合,在恒电位下,首先将亲合素吸附在电极上,然后使标识在生物素上的葡萄糖氧化酶固定于电极表面,制成具有高活性的多层固定化酶的葡萄糖传感器。电极线性范围:1.0×10^-5～1.0×10^-2mol/L,检测限:6.0×10^-6mol/L,响应时间:6s.本传感器具有线性范围宽、灵敏度高、响应速度快、可反复活化使用等优点。     

Valence Bond and Enzyme Catalysis: A Time To Break Down and a Time To Build Up

Understanding enzyme catalysis and developing ability to control of it are two great challenges in biochemistry. A few successful examples of computational‐based enzyme design have proved the fantastic potential of computational approaches in this field, however, relatively modest rate enhancements have been reported and the further development of complementary methods is still required. Herein we propose a conceptually simple scheme to identify the specific role that each residue plays in catalysis. The scheme is based on a breakdown of the total catalytic effect into contributions of individual protein residues, which are further decomposed into chemically interpretable components by using valence bond theory. The scheme is shown to shed light on the origin of catalysis in wild‐type haloalkane dehalogenase (wt‐DhlA) and its mutants. Furthermore, the understanding gained through our scheme is shown to have great potential in facilitating the selection of non‐optimal sites for catalysis and suggesting effective mutations to enhance the enzymatic rate.