铜离子生物传感器电极的构建及铜识别膜的筛选

发布时间：2018-04-18 08:24

本文选题：铜离子 + 丝网印刷电极　；参考：《暨南大学》2016年硕士论文

【摘要】：目的:铜离子存在于多种氧化还原酶的活性位点,对酶的功能发挥有重要作用;然而铜过量易导致阿尔茨海默病,对人体产生危害;目前铜盐在水产养殖业中大量使用,易导致水体污染、鱼类含铜量超标等危害;构建快速检测铜含量的方法非常必要。本研究利用多壁碳纳米管(MWNT)和半胱氨酸(Cys)的结合产物(MWNT-Cys)组装在丝网印刷金电极表面,利用差分脉冲阳极溶出伏安法(DPSVO)作为表征方法,构建一个快速检测鱼肉样品中铜离子浓度的传感器。此外,蛋白质的活性位点具有许多铜离子结合基序,本研究从铜离子结合基序中选择常见氨基酸残基,设计Lys-Cys-Met-Cys(KCMC)、Lys-Cys-Met-Met-Cys(KCMMC)、Lys-Cys-His-Cys(KCHC)、Lys-Cys-His-His-Cys(KCHHC)、Lys-Gly-His-Gly-His(KGHGH)五条寡肽序列,利用电化学方法研究寡肽在构建检测铜离子传感器中的可行性。方法:利用傅里叶变换红外光谱(FT-IR)对多壁碳纳米管(MWNT)的羧基化程度进行表征;利用纳米粒度及Zeta电位分析仪对碳纳米管的粒径进行测量;利用差分脉冲阳极溶出伏安法(DPSVO)对铜浓度进行表征;利用电感耦合等离子体质谱法(ICP-MS)检测标准铜离子溶液浓度;利用循环伏安法(CV)、差分脉冲伏安法(DPV)、交流阻抗法(EIS)对寡肽的构象变化进行表征。结果:Cu~(2+)浓度在10nmol/l~1μmol/l、1μmol/l~9μmol/l、9μmol/l~0.9mmol/l范围内具有良好的线性关系;利用红外湿法消解法消解鱼肉样品,ICP结果测得铜离子加标回收率为101.00%,相对标准偏差(RSD)为3.5%;利用传感器对样品进行三次加标检测,相对标准偏差(RSD)5%;对锑(Sb)和铅(Pb)实现半定量检测,最低检出浓度可达到nmol/l级别;不含巯基(-SH)的寡肽组装稳定,寡肽传感器对铜的结合需要单一的环境,多离子的共存对寡肽与铜的结合影响较大;KGHGH与金属离子的结合具有p H值依赖性。结论:所构建的丝网印刷电极传感器能较准确的定量Cu~(2+)的浓度,并且具有制作简便、检测快速、可弃式使用等特点;寡肽在结合金属离子的过程中,需要单一环境才能与金属离子特异性结合,具有p H值依赖性。
[Abstract]:Objective: copper ions exist in the active sites of many redox enzymes and play an important role in the function of the enzymes. However, excessive copper can lead to Alzheimer's disease and cause harm to human body. At present, copper salt is widely used in aquaculture.It is easy to lead to water pollution and fish copper content exceeds the standard, so it is necessary to establish a method for rapid detection of copper content.In this study, multiwalled carbon nanotubes (MWNTs) and cysteine (CysCys) binding products, MWNT-Cys), were assembled on the surface of silk-screen printed gold electrodes. Differential pulse anodic stripping voltammetry (DPSVOO) was used as a characterization method.A sensor for rapid detection of copper ion concentration in fish samples was constructed.In addition, the active sites of proteins have many copper ion binding motifs. In this study, we selected common amino acid residues from copper ion binding motifs and designed Lys-Cys-Met-Cys-Cys-Met-Cys-Cys-Cys-Met-Cys-Cys-Cys-Cys-His-Cys-Cys-Cis-Cis-Cys-CKCHH KCHHC-Lys-Gly-His-Gly-Hisy-HisKGHGHH5 oligoseptide sequences from copper ion binding motifs.The feasibility of oligoseptide in the construction of copper ion sensor was studied by electrochemical method.Methods: the carboxylation degree of MWNTs was characterized by Fourier transform infrared spectroscopy (FTIR), and the diameter of MWNTs was measured by nano-particle size and Zeta potential analyzer.The copper concentration was characterized by differential pulse anodic stripping voltammetry (DPSVOO), and the concentration of standard copper ion solution was detected by inductively coupled plasma mass spectrometry (ICP-MS).The conformation of oligopeptides was characterized by cyclic voltammetry, differential pulse voltammetry (DPVN) and AC impedance spectroscopy (EIS).Results there was a good linear relationship in the range of 10nmol/l~1 渭 mol / L ~ (1 渭 mol / L ~ (-1) mol/l~9 / L ~ (-1) 渭 mol / L ~ (9) 渭 mol/l~0.9mmol/l), the recovery rate of Cu ~ (2 +) was 101.00% and the relative standard deviation (RSD) was 3.5% by using infrared wet digestion method.Relative standard deviation (RSD5); semi-quantitative detection of antimony, SB) and lead, the lowest detectable concentration can reach nmol/l level; oligoseptides without sulfhydryl group are assembled stably, and oligoseptide sensors require a single environment for the binding of copper.The coexistence of multi-ions has a significant effect on the binding of oligopeptide to copper. The binding of KGHGH to metal ions is pH dependent.Conclusion: the proposed screen-printing electrode sensor can accurately quantify the concentration of Cu~(2, and has the advantages of simple fabrication, rapid detection and disposable use.It needs a single environment to specifically bind to metal ions, which is dependent on pH value.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：TP212.3;O657.1

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