【作者】 张伟；

【导师】 陈林林；

【作者基本信息】 哈尔滨商业大学 ， 食品科学， 2018， 硕士

【摘要】 通过光谱法和电化学方法分析甲烷氧化菌素(Methanobactin,Mb)与铜的配合物模拟超氧化物歧化酶(superoxide dismutase,SOD)的活性,并进一步应用于电化学生物传感器。Mb是由甲烷氧化菌分泌到细胞外捕获和运输Cu2+的小分子荧光肽,它具有配合Cu2+等金属离子的能力,其铜配合物(Mb-Cu)可以清除超氧阴离子。实验第一部分从ethylosinus trichosporium 3011发酵液中提取Mb并与Cu2+配合成Mb-Cu,分别采用紫外光谱和荧光光谱考察了邻苯三酚自氧化的线性时间以及有Mb-Cu参与的荧光激发波长,确定邻苯三酚的自氧化线性时间是90s,荧光激发波长为446nm。进一步考察了不同条件(EDTA-Na2的浓度、缓冲溶液的pH值、Tris-HAc缓冲溶液浓度、邻苯三酚浓度、温度和Mb-Cu浓度)对邻苯三酚自氧化的影响,从而评价Mb-Cu模拟SOD的活性。讨论认为,当EDTA-Na2的浓度为2mmol/L,溶液的pH值为8.2,Tris-HAc缓冲溶液浓度为0.05mol/L,温度为20℃,邻苯三酚浓度为0.363mmol/L时,浓度为0.8mg/L的Mb-Cu表现出良好的SOD生物活性,能有效抑制超氧阴离子(O2-·)。在此光谱分析条件下Mb-Cu模拟SOD活性稳定。实验第二部分按照柠檬酸钠还原法制备了粒径大小在10～50(11.5、15、35、42、50)nm的纳米金(AuNps),并对其进行了 TEM扫描,可以观察到制得的纳米金颗粒表面光滑、大小均匀。将粒径为15nm的AuNps依次组装Mb和Cu2+并进行了红外光谱的表征,证明Mb与Cu(Ⅱ)是通过Mb结构中的-OH或酚-OH配合。又将15nm的AuNps与具有不同类型官能团的交联剂缔合,并利用紫外光谱、荧光光谱、红外光谱和TEM透射电镜表征,其中β-巯基乙胺可与AuNps与Mb-Cu之间建立更紧密的结合,形成树状纳米簇结构。分别考察了 AuNps粒径、AuNps:Mb-Cu摩尔比、交联剂种类和Mb-Cu:交联剂摩尔比对邻苯三酚自氧化的影响,并通过光谱分析认为当AuNps粒径为15nm、AuNps:Mb-Cu摩尔比为1:150、交联剂选择β-疏基乙胺、Mb-Cu:β-疏基乙胺摩尔比为1:3时,Mb-Cu-Au模拟酶活性最高。改变SOD活性测定体系发现,与黄嘌呤氧化酶法相比,模拟酶活性基本一致且方法稳定。实验最后一部分在0.1M的Fe(CN)63-/Fe(CN)64-的探针溶液中对空白、Au/β-疏基乙胺/AuNps、Au/β-巯基乙胺/AuNps/Mb和Au/β-巯基乙胺/AuNps/Mb/Cu电极进行了比较,通过循环伏安(CV)和交流阻抗(EIS)扫描得出Au/β-巯基乙胺/AuNps/Mb/Cu电极是以单层膜组装到金电极上,AuNps的存在可以加速异相电子传递,Cu2+反之。在研究SOD模拟酶自组装膜时,分别讨论了邻苯三酚自氧化基质作为探针溶液时,不同电解质、电解质浓度、邻苯三酚反应时间、邻苯三酚添加量和扫速对电极感应电流的影响,讨论认为当电解质KC1的浓度为0.3mol/L、超氧阴离子积累时间为3min、邻苯三酚添加量为20μL及扫速为300mV/s时,电极对电流的感应程度最高。并考察了不同模拟酶及组装方式、不同交联剂种类对模拟酶在电化学中对抑制邻苯三酚自氧化的影响,认为以β-巯基乙胺作为交联剂,以层层组装的形式组装的Au/β-巯基乙胺/AuNps/Mb/Cu电极的SOD模拟酶活性最强。该自组装膜检验测定电流的重现性较好,且有良好的稳定性和使用寿命。本文利用Mb-Cu模拟SOD,通过光谱法和电化学方法确定了模拟酶的活性。建立的SOD模拟酶自组装体系具有良好的重现性、稳定性,在检验O2-·浓度方面有较好的应用前景。更多还原

【Abstract】 The determination for mimic superoxide dismutase(SOD)activity of methanobactin(Mb)and copper complexes by spectroscopy and electrochemistry was established and further applied to electrochemical biosensors.Mb is a small molecule fluorescent peptide secreted by methanotrophs to capture and transport Cu2+ extracellularly.It has the ability of coordinating Cu2+ and other metal ions.The copper complex has the ability of scavenging superoxide anion.In the first part of the experiment,Mb was extracted from ethylosinus trichosporium 3011 and combined with Cu2+to form Mb-Cu.The linearization time of pyrogallol autoxidation and the fluorescence excitation wavelength with Mb-Cu added were investigated respectively.The autoxidative linear time of pyrogallol was 90s,and the fluorescence wavelength of pyrogallol system was 446nm.The effects of Tris-HAc concentration,pH,the concentration of EDTA-Na2 and pyrogallol,temperature and the concentration of Mb-Cu on the pyrogallol autoxidation were futher discussed by ultraviolet and fluorescence spectrum.So as to evaluate the SOD activity of Mb-Cu.The results showed that when the concentration of EDTA-Na2 was 2mmol/L,pH value was 8.2,Tris-HAc was 0.05mol/L,the temperature was 20 ℃,and the concentration of pyrogallol was 0.363mmol/L,Mb-Cu at a concentration of 0.8 mg/L showed better SOD activity and can inhibit superoxide anion(O2-·)effectively.Under this spectrum analysis conditions,Mb-Cu mimic SOD activity was stable.In the second part of the experiment,AuNps with particle sizes of 11.5,15,35,42 and 50 nm were prepared by sodium citrate reduction method and characterized by TEM.The results showed that the Au nanoparticles(AuNps)prepared have uniform size and smooth surface.Mb and Cu2+ were assembled by AuNps of a particle size of 15 nm successively and characterized by infrared spectrum.It was proved that Mb complexed Cu(Ⅱ)by the hydroxyl in Mb structure or phenolic hydroxyl group.In turn,15 nm AuNps were bound to crosslinkers of different types of functional groups and characterized by UV,fluorescence,IR and transmission electron microscope.β-mercaptoethylamine can bond closer between AuNps and Mb-Cu,which Forming a dendritic nanocluster structure.The effects of AuNps particle size,molar ratio of AuNps to Mb-Cu,different cross-linking agent and molar ratio of Mb-Cu to cross-linking agent on the pyrogallol autoxidation were investigated respectively.By spectrum analysis,it was considered that when the AuNps particle size was 15nm,molar ratio of AuNps:Mb-Cu was 1:150,the optimal crosslinking agent was P-mercaptoethylamine,molar ratio of Mb-Cu:β-mercaptoethylamine was 1:3,Mb-Cu-Au mimic has the optimum enzyme activity.When the SOD activity measurement system was changed to xanthine oxidase method,it was verified that,the activity of the mimic enzyme activity was basically consistent and the method was stable.The final part of the experiment was performed on blank,Au/β-mercaptoethylamine/AuNps,Au/β-mercaptoethylamine/AuNps/Mb and Au/β-mercaptoethylamine/AuNps/Mb/Cu electrodes to be compared in a probe solution of 0.1m Fe(CN)63-/Fe(CN)64-by cyclic voltammetry(CV)and electrochemical impedance spectroscopy(EIS)scanning.The Au/β-mercaptoethylamine/AuNps/Mb/Cu electrode was assembled as a monolayer on a gold electrode,AuNps can accelerate the transmission of heterogeneous electrons.Otherwise,Cu2+inhibited heterogeneous electron transfer.In the research of SOD mimic enzyme self-assembled membrane,the effects of different electrolytes,electrolyte concentrations,pyrogallol reaction time,pyrogallol dosage and sweeping rate on the electrode response of pyrogallol autotrophic matrix as probe solution.The effects of the current on the electrode current were investigated.When the the concentration of KCl was 0.3 mol/L,the superoxide anion accumulation time was 3 min,the amount of pyrogallol added was 20 μL and the sweep speed was 300 mV/s,the electrode has the highest degree of induction to the current.The effects of different mimic enzymes,assembly methods and crosslinker on the inhibition of pyrogallol autoxidation by mimic enzymes in the electrochemical method were investigated.It was believed that β-mercaptoethylamine can be used as a cross-linking agent and can be assembled in layers.The assembled Au/β-mercaptoethylamine/AuNps/Mb/Cu electrode exhibited the highest SOD mimic enzyme activity.The reproducibility of the current measured by self-assembled membrane test was better,and it had good stability and life.In this paper,Mb-Cu was chosen to mimic SOD,and the activity of mimics were determined by spectroscopic and electrochemical methods.The self-assembly system of SOD mimic enzyme has good reproducibility and stability,and has a better application prospect in detecting O2-·.更多还原