【作者】 李萌； 黄想容； 李芯蕊； 尚亚卓；

【Author】 Meng Li;Xiang-Rong Huang;Xin-Rui Li;Ya-Zhuo Shang;Key Laboratory for Advanced Materials,School of Chemistry & Molecular Engineering,East China University of Science and Technology;

【机构】 华东理工大学结构可控先进功能材料及其制备教育部重点实验室；

【摘要】 辣根过氧化物酶（HRP）作为生化实验中最常用的血红素酶之一,具有性质稳定,催化活性高,易与抗体或聚合物偶联的特性;又因其可催化氧化芳香胺或酚类化合物发生颜色反应,该酶在目前商品化酶联免疫吸附试验（ELISA）试剂盒中应用最为广泛。在HRP的众多色原底物中,3,3’,5,5’-四甲基联苯二胺（TMB）是较为理想的一种,具有灵敏度高,毒性低,无生物致畸性等优点;但TMB水溶性差,反应过程需酸终止,色原产物（黄色的二亚胺终产物）易分解的缺点却限制了其应用[1]。Kireyko等[2]的研究表明一定浓度的阴离子表面活性剂十二烷基硫酸钠（SDS）可提高TMB的氧化中间体（蓝色的二胺/二亚胺电子转移复合物）的稳定性,基于此,我们系统地研究了SDS对HRP-H2O2-TMB反应的作用机理,结果表明SDS溶液中该反应的酶动力学机制符合乒乓机制;当SDS浓度低于2.5m M时,胶束的存在不但有利于提高酶与底物的亲和性、促进酶和底物之间的直接电子转移,而且可以稳定蓝色色原产物,提高酶催化反应的效率;当SDS浓度高于2.5m M时,SDS与HRP之间强烈的静电相互作用和疏水相互作用使酶结构改变,致使酶活性降低,进而抑制酶促反应。在上述研究的基础上,我们创新性地引入"绿色溶剂"咪唑类离子液体[Cnmim][BF4]（n=2, 4, 6, 8, 10）,把SDS/[Cnmim][BF4]复配体系作为酶促反应介质,研究结果显示,该反应介质可显著提高蓝色色原体的生成速率和稳定性,且影响程度随离子液体烷基链长的改变而略有差异（见Fig.1（a））。介质的胶体化学性质是影响酶催化反应过程的重要因素,研究结果显示离子液体链长越长,界面活性越强,越易于参与胶束的形成,提高体系中SDS胶束密度,这在一定程度上利于易分解的蓝色产物的生成和稳定;然而,离子液体参与胶束的形成能力越强,体系中形成的混合胶束所带负电荷越少,不利于蓝色产物的生成和稳定。这可能是导致SDS/[C4mim][BF4]体系中HRP-H2O2-TMB反应效率最高的直接原因（见Fig.1（b））。实验结果证实,HRP与不同链长的离子液体相互作用不同,影响HRP与底物的亲和性（与量化计算结果（Fig.1（c））一致）,从而影响酶促反应行为。以SDS/[C₄mim][BF4]混合体系为HRP-H₂O₂-TMB反应介质,进一步优化介质中离子液体及表面活性剂浓度、配比及p H。我们发现p H=7.4时,TMB-HRP-H₂O₂反应在SDS/[C₄mim][BF4]（2.5 mM/2.5 m M）混合介质中反应效率最高,且色原体的OD值能在较长的时间保持稳定（24h内?OD≤1.67%）,显色过程无需酸终止,操作便捷。实验结果也显示,与其它色原底物（OPD, ABTS, ODA, 5-As）相比SDS/[C4mim][BF4]混合反应介质对底物TMB的HRP催化反应具有特殊的增效性。我们已将SDS/[C₄mim][BF4]反应介质成功用于HRP活性的测定和H₂O₂的检测[3],拟将其用于商品化ELISA试剂盒。更多还原

【Abstract】 Horseradish peroxidase（HRP）,a sort of heme-containing enzyme,is used extensively in biochemical detection and other fields because of relative stable property,effective catalytic activity and combining with antibody and polymer easily.HRP can catalyze various of aromatic amine and phenolic compounds oxidation in the presence of H₂ O₂ to produce chromogenic reaction,which endows it with potential applications in enzyme linked immunosorbent assay（ELISA）.Among the various substrates of HRP,3,3’,5,5’-tetramethylbenzidine is an optimum substrate due to its excellent sensitivity,low toxicity,and non-teratogenicity.However,the intrinsic disadvantages of TMB,such as poor water solubility,request for the acid to stop the reaction,short color developing time derived from the highly unstable chromogens（yellow diimine end-product）,limit the scope of its practical application [1].Kireyko et al.found the existence of sodium dodecyl sulfate（SDS） with certain concentration can stabilize intermediates（blue diamine/diimine charge transfer complex） formed in the peroxidase oxidation of TMB[2].Based on the above research,we study the action mechanism of SDS on the entire process of the catalysis of HRP-H₂ O₂-TMB comprehensively.The results indicate that the catalysis of HRP-H₂ O₂-TMB takes place in a ping-pong kinetic mechanism in SDS solutions; when the concentrations of SDS are under 2.5 mM,the existence of SDS micelles not only improve the affinity between enzymes and substrates,facilitate the electron transfer between enzymes and substrates,but also stabilize the blue chromogen so that increase the efficiency of the catalysis; when the concentrations of SDS exceed 2.5 mM,SDS takes work as the inhibitor of HRP that is mainly attributed to strong electrostatic interaction and hydrophobic interaction between SDS and HRP,which change the structure of enzymes and decrease their activity.Based on the above study,creatively,the "Green solvent"-midazolium-based ionic liquids（ILs） [Cnmim][BF4]（n=2,4,6,8,10） are introduced to create SDS/[Cnmim][BF4] combination as a novel efficient medium for the chromogenic catalysis.The results indicate that SDS/[Cnmim][BF4] combinations can improve the rate of formation of the blue chromogen and its stability,additionally,the effects of ILs on the catalysis vary with alkyl chain length of ILs slightly（Fig.1（a））.The colloidal properties of mediums are the key factors of affecting the process of enzymatic catalytic reactions,the research findings show that ILs with longer chain length are liable to form the micelles with SDS further increase the density of micelles in the mediums because of increased interfacial activity,which facilitate to form and stabilize the blue chromogens to a certain extent; however,more ILs with positive charges participate in forming micelles would cause the decrease of the negatively charges of the mixed micelles in the combinations,so that hinder the formation and stabilization of the blue chromogens.The SDS/[C₄ mim][BF₄] combination performs best in improving the catalytic efficiency comparing with others,which maybe the results of the above synthetic action（Fig.1（b））.The results of experiment combing with the results of quantum chemical calculation verify that the interactions between HRP and ILs with different chain length are distinct(（Fig.1（c））,which affect the affinities between HRP and substrates further effect the behaviors of enzymatic reaction.Fourthly,the SDS/[C₄ mim][BF₄] combination is selected to be the reaction medium for the catalysis of HRP-H₂ O₂-TMB,in which the concentrations of ILs and surfactant,the ratios between the two,and the pH of the combinations have been optimized.The results show that the catalysis performs highest catalyticefficiency in SDS/[C₄ mim][BF4]（2.5 mM/2.5 mM） combination at pH=7.4,besides,the OD values of the blue chromogens are constant for a long time（?OD≤1.67% in 24 h）.Compared with its traditional application in ELISA,the chromogenic system without acid termination is more convenient.Compared with the other chromogenic substrates of HRP（OPD,ABTS,ODA,5-As）,the amplification effects of SDS/[C₄ mim][BF₄]（2.5 mM/2.5 mM） combination on the chromogenic catalysis for TMB with HRP is unique.We have applied SDS/[C₄ mim][BF₄] combination as reaction medium to detecting HRP activity and H₂ O₂ concentration successfully[3],the following schedule is using it for refining the commercial ELISA kits.更多还原