Tetra-substituted Amino Aluminum Phthalocyanine as a New Red-region Fluorescent Substrate for Horseradish Peroxidase Based Enzyme-linked Immunosorbent Assay

ＩｎｔｒｏｄｕｃｔｉｏｎＥｎｚｙｍｅｉｍｍｕｎｏａｓｓａｙｂａｓｅｄｏｎａｓｅｌｅｃｔｉｖｅａｎｔｉｇｅｎ ａｎｔｉｂｏｄｙｂｉｎｄｉｎｇａｎｄａｌａｂｅｌｅｎｚｙｍｅｈａｓｇａｉｎｅｄｉｎｃｒｅａｓ ｉｎｇｉｍｐｏｒｔａｎｃｅｉｎｒｅｃｅｎｔｙｅａｒｓ .Ａｍｏｎｇｔｈｅｅｎｚｙｍｅｓｕｓｅｄ ,ｈｏｒｓｅｒａｄｉｓｈｐｅｒｏｘｉｄａｓｅ (ＨＲＰ)ｉｓｔｈｅｍｏｓｔｗｉｄｅｌｙｕｓｅｄｅｎｚｙｍｅｌａｂｅｌｂｅｃａｕｓｅｏｆｉｔｓｈｉｇｈｓｐｅｃｉｆｉｃｉｔｙａｎｄｓｅｎｓｉｔｉｖｉ ｔｙ .1 3 Ｆｌｕ…     

硝基磺酚S作为酶联免疫分析中辣根过氧化物酶测定底物的研究

提出一种新的ＨＲＰ底物－硝基磺酚Ｓ,初步探讨了在ＨＲＰ催化下被过氧化氢氧化的机制,以硝基磺酚Ｓ为底物测定了酶促反应动力学常数Ｋｍ和Ｋｓ分别为４．５１×１０＾－５ｍｏｌ／Ｌ和２８．１ｍｏｌ／Ｌ·ｓ＾－１。分别用于酶联免疫显色光度法和伏安酶联免疫分析法测定了ＩｇＧ－ＨＲＰ,其所能测定的最高稀释比分别为１：１．６×１０＾５和１：４．８×１０＾５。     

以1-萘胺为底物伏安法测定辣根过氧化物酶

提出了1-萘胺-过氧化氢-辣根过氧化物酶(HRP)伏安酶联免疫分析体系应用于HRP的伏安法测定.在pH 7.0的KH2>PO4-Na2>HPO4缓冲溶液中,HRP催化过氧化氢氧化1-萘胺生成电活性产物,在极谱仪上进行二阶导数伏安线性扫描,该产物在滴汞电极上发生还原反应,于-0.43 V(vs.SCE)左右产生一灵敏的还原峰.HRP浓度在3.0×10-7～5.0×10-5>g·L-1范围内与线性扫描峰电流的二阶导数值(△I"p)呈线性关系,检出限为2.5×10-7g·L-1.     

Horseradish Peroxidase Catalyzed Synthesis of Polycardanol Microcapsules

Polymers synthesized from naturally derived monomers are valuable since they decrease the reliance on petroleum based feed stocks. Cashew nut shell oil (CNSL) is a side-product from processing of edible Cashew nuts of Annacardium occidentale. One of the major components of CNSL is cardanol, which is a phenol derivative having an unsaturated pentadecyl substituent in the ‘meta’ position with varying amount of unsaturation (no double bonds to three double bonds). The substituent in the meta position can also be hydrogenated to yield completely saturated hydrogenated cardanol. Cardanol can be utilized to stabilize the dispersions of oil in water and vice versa since it displays amphiphilic behavior owing to the phenolic head and the C15 aliphatic tail. Here we report the horseradish peroxidase (HRP) catalyzed polymerization of cardanol at oil water interface to obtain polycardanol microcapsules. A synthetic analogue of hydrogenated cardanol, 3-pentadecylphenol (3PDP), was also oxidatively polymerized at the oil-water interface to obtain Poly(3-pentadecylphenol) microcapsules.     

隐性亮绿作为过氧化物酶催化测定过氧化氢底物的研究

研究了以隐性亮绿作为氢供体底物的辣根过氧化物酶－过氧化氢催化体系。该体系在ＰＨ＝６．４－７．５的条件下所形成的亮绿于６４０ｎｍ处有最大吸收。测定过氧化氢的摩尔吸光系数为９．３×１０＾４Ｌ．ｍｏｌ＾－１．ｃｍ＾－１；工作曲线的线性范围为０．０３６ｍｇ／Ｌ。方法可应用于雨水中过氧化氢含量的测定。     

3‐Indoxyl Phosphate as an Electrochemical Substrate for Horseradish Peroxidase

In this work 3‐indoxyl phosphate (3‐IP), an alkaline phosphatase substrate, is demonstrated to be a suitable substrate for horseradish peroxidase (HRP). HRP catalyzes the oxidation of 3‐IP in presence of hydrogen peroxide (H₂O₂) generating the product indigo blue, which is an aromatic heterocycle compound insoluble in aqueous solutions. This product was easily converted into its soluble parent compound indigo carmine (IC) (by addition of fuming sulfuric acid to the reaction media) which has a reversible voltammetric peak at the formal potential of ?0.15 V (vs. Ag pseudo‐reference electrode) when a screen‐printed carbon electrode (SPCE) is used. Parameters that influence the enzymatic reaction, such as pH, temperature, substrate concentration and reaction time have been optimized. Moreover, the enzyme apparent kinetic constants (V_max, K_M) for both substrates (3‐IP and H₂O₂) have been calculated. Indirect measurements of HRP activity in solution were carried out not only by cyclic voltammetry but also using amperometric detection in a flow system. The detection limits were 6.86×10^?12 and 5.68×10^?12 M, respectively. Thus, 3‐IP is the first substrate that could be used for alkaline phosphatase (AP) and HRP, the most common enzymatic labels in affinity assays.     

过氧化物酶新型荧光底物3,4-二氢-2(1H)-喹喔啉酮的反应机理及性能的研究

合成了过氧化物酶新型荧光底物3,4-二氢-2(1H)-喹喔啉酮(DHQXL). 在过氧化物酶催化下,该化合物可被H₂O₂氧化生成强荧光物质2(1H)－喹喔啉酮(QXL). 用高压液相色谱法分离得荧光产物纯品,经核磁共振谱和质谱证实其结构,确证了提出的酶催化反应机理. 对该化合物作为荧光底物的性能研究表明,它是一个很有应用前景的过氧化物酶的新型荧光底物.     

以变色酸2R为底物测定辣根过氧化物酶的研究

变色酸 2R(CT2R)是一种具有电化学活性的物质 ,在碱性条件下 ,该物质在滴汞电极上 - 6 6 0mV(vs.SCE)左右产生还原波 ,加入H2 O2 和辣根过氧化物酶 (HRP)后 ,该物质被氧化生成具有非电化学活性的物质 ,导致溶液中游离的CT2R浓度降低 ,相应还原波波高降低 ,HRP在一定含量范围内与伏安峰的降低值具有良好的线性关系。通过测定波高的降低值可测定HRP ,测定游离HRP的线性范围是 4 .0× 10 -5～ 4 .0× 10 -3 g/L。对酶促反应的动力学进行了研究 ,反应的米氏常数Km =1.38mmol/L ,最大反应速率Vmax=5 3.9nA/min。     

4-羟基苯乙烯基吡啶为辣根过氧化物酶底物的酶荧光免疫传感体系测定布氏杆菌抗体

合成了一种新型辣根过氧化物酶(HRP)荧光底物-4-羟基苯乙基吡啶(pHSP),并首次将它运用于酶联荧光免疫传感体系.对pHSP化学性质的研究证实,pHSP在空气中较稳定,对HRP,H2O2的荧光响应性能优于传统HRP荧光底物,如对羟苯乙酸、Amplex Red和佳昧醇等.在pH 5.8的Britton-Robinson缓冲溶液中,pHSP本身只有极弱的荧光,在HRP-IgG催化下可被H2O2氧化成二聚体产物,该二聚体在300 nm的激发光下能发射波长为437 nm的强荧光,并且反应体系的荧光增加与HRP量在一定浓度范围内成线性相关.根据此原理,建立了兔布氏杆菌抗体的酶联荧光传感分析新方法.运用制备的传感装置测定兔布氏杆菌抗体的线性范围为6.3×10-11～1×10-8mol·L-1,抗体检出限为6.3×10-11mol·L-1,相对标准偏差为4.1%(n=11).pHSP的二聚体产物水溶性很低,利用设计的装置较好地解决了传统测定溶液体系方法灵敏度不高的问题.     

Horseradish Peroxidase Enzyme Electrodes for Nadh and Nadph

Abstract

The aerobic oxidation of reduced pyridine phosphonucleotides catalyzed by horseradish peroxidase (E.C.1.11.1.7) (HRP) in combination with Mn²⁺-ions, phenolics and redox dyes was used to perform NADH and NADPH determinations with an amperometric HRP enzyme electrode. Optimal operational conditions have been elaborated for such a sensor and a linear relationship between derivative current (dI/dt) and NADH-concentration was obtained between 2.10^?5 and at least 2.10^?4 mol/l. The coefficient of variation was equal to or smaller than 4 %. The time for one measurement was less than 10 minutes including equilibration of the electrode.     

Horseradish Peroxidase as a Catalyst for Atom Transfer Radical Polymerization

The hemoprotein horseradish peroxidase (HRP) catalyzes the polymerization of N‐isopropylacrylamide with an alkyl bromide initiator under conditions of activators regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP) in the absence of any peroxide. This is a novel activity of HRP, which we propose to name ATRPase activity. Bromine‐terminated polymers with polydispersity indices (PDIs) as low as 1.44 are obtained. The polymerization follows first order kinetics, but the evolution of molecular weight and the PDI upon increasing conversion deviate from the results expected for an ATRP mechanism. Conversion, and PDI depend on the pH and on the concentration of the reducing agent, sodium ascorbate. HRP is stable during the polymerization and does not unfold or form conjugates.

     

氯化血红素作为过氧化物模拟酶与HRP的比较研究

用荧光光谱和紫外－可见吸收光谱研究了氯化血红素（hemin)催化过氧化氢与对羧基苯丙酸反应的动力学和反应产物,表明hemin同时具有模拟过氧化物酶、过氧化氢酶的活性,在反应过程中hemin自身被氧化分解。hemin催化反应的产物除与天然的辣根过氧化物酶（HRP）催化的产物相同（λex/λem=310nm/406nm）外,还有激发峰在258nm和300nm,发射峰在360nm的荧光物质。通过对产物光谱特性的比较,研究了hemin和HRP的特异性差异。该研究结果对进一步筛选和研究高特异性过氧化物模拟酶具有重要的指导意义。     

New Polymers from Natural Phenols Using Horseradish or Soybean Peroxidase

The flavonol quercetin, its glycoside rutin, the flavanol catechin, the isoflavone daidzein and 5,6,4′‐trihydroxyisoflavone can be oxidatively polymerized via a radical polyrecombination mechanism catalyzed by horseradish or soybean peroxidase in a mixture of phosphate buffer (pH 7) and 1,4‐dioxane. The average molecular weights of the polymers were found to be in the range 4 000–12 000 g/mol. Daidzein derivatives with a methoxy (formononetin) or nitro group or a hydrogen atom at C‐4′ could not be polymerized under these conditions. These results and molecular modeling studies suggest that polymerization preferentially occurs via the electron‐rich ring B of the isoflavonoids. The polymers were characterized by means of FT‐IR and UV spectroscopy, and their redox behavior was analyzed using cyclic voltammetry.     

纳米二氧化钛对辣根过氧化酶催化作用

将辣根过氧化物酶(HRP)固定在二氧化钛(TiO2)纳米颗粒或纳米管修饰玻碳电极(GC)上,形成纳米TiO2微粒/HRP修饰GC电极和TiO2纳米管/HRP修饰GC电极.比较了HRP在纳米TiO2微粒、TiO2纳米管电极上的直接电子转移反应.实验表明,HRP在TiO2纳米管电极表面更能有效地促进它的电活性中心发生电子交换反应.此外,还测定了HRP标记抗体的电化学性能,为抗原抗体免疫反应信号的选择提供了参考依据.     

新型荧光试剂N-2-噻唑基水杨醛亚胺Schiff碱的合成及荧光性质

合成了１０种新型荧光试剂Ｎ－２－噻唑基水杨醛亚胺Ｓｃｈｉｆ碱,通过元素分析,ＩＲ,１ＨＮＭＲ和ＵＶ－ｖｉｓ光谱进行了结构表征,并且测定了试剂的荧光光谱,微量的二价碱土金属离子对试剂的荧光有一定的熄灭作用     

无定形硒纳米粒子的交联酶模板法合成及其在生物传感器中的应用

本文以戊二醛交联的辣根过氧化物酶(HRP)为模板合成了均匀分散的无定形硒纳米粒子(粒径10~20 nm)，以所得合成产物为载体构建了HRP生物传感器。研究结果表明，无定形硒纳米粒子具有良好的生物相容性和吸附性，所得传感器灵敏度高，对测定底物的生物亲和性好。     

辣根过氧化物酶在海藻酸钠水凝胶中的电化学和电催化特性

辣根过氧化物酶在海藻酸钠水凝胶中的电化学和电催化特性;辣根过氧化物酶;海藻酸钠;直接电化学电;催化     

反相微乳液中辣根过氧化物酶催化反应的研究

研究了辣根过氧化物酶（ＨｏｒｓｅｒａｄｉｓｈＰｅｒｏｘｉｄａｓｅ，ＨＲＰ）在ＡＯＴ／水／异辛烷微乳液中的多底物酶促反应行为．实验结果表明，反相胶束中动力学反应机制与纯水中不同，并且利用紫外可见光度法研究了中间化合物Ⅲ在２种不同介质中的稳定性．     

碳纳米管电极上辣根过氧化物酶的直接电化学

制备了碳纳米管修饰玻碳电极(CNT/GC).将辣根过氧化物酶(HRP)固定在CNT/GC电极表面,形成HRP-CNT/GC电极.研究了HRP的直接电子转移.实验结果表明,HRP在CNT/GC电极表面能进行有效和稳定的直接电子转移反应,其循环伏安曲线上表现出一对良好的、几乎对称的氧化还原峰;式量电位E^0'几乎不随扫速(至少在20～100 mV/s的扫速范围内)而变化,其平均值为(-0.319±0.002) V (vs. SCE, pH 6.9); HRP在CNT/GC电极表面直接电子转移的速率常数为(2.07±0.56) s^-1;式量电位E^0'与溶液pH 的关系表明HRP的直接电化学是(1e+1H⁺)的电极过程.进一步的实验结果显示,固定在CNT/GC电极表面的HRP能保持其对H₂O₂还原的生物电催化活性,而且能快速地响应H₂O₂浓度的变化.本文制备碳纳米管修饰电极和固定酶的方法具有简单和易于操作等优点,可用于获得其它生物氧化还原蛋白质和酶的直接电子转移.