基于新型抗污染材料的电化学生物传感器的构建及其应用研究

发布时间：2018-01-16 05:34

本文关键词：基于新型抗污染材料的电化学生物传感器的构建及其应用研究　出处：《青岛科技大学》2017年博士论文　论文类型：学位论文

【摘要】：本课题主要以新型的抗污染材料为基础,以不同的导电聚合物修饰电极,通过不同的组装方式构建不同的电化学生物传感器,实现对多种疾病标志物的特异性、灵敏性检测。采用多种表征手段对不同生物传感界面的抗污染性能进行了探究,通过不同的电化学技术研究了各种生物传感器对目标物的传感响应能力,并分析了构建的生物传感器在真实血清样品中目标物检测方面的应用潜力。本论文的主要研究内容概括如下:(1)采用简单的电沉积方法在玻碳电极(GCE)表面成功制备了聚3,4-乙烯二氧噻吩/聚乙二醇衍生物(PEDOT/PEG)复合材料。采用扫描电子显微镜(SEM)和循环伏安(CV)技术对该复合材料修饰电极(PEDOT/PEG/GCE)的表面形貌和电化学稳定性进行了研究,结果表明该复合材料呈多层片状结构、比表面积大,而且电化学稳定性高。以牛血清白蛋白(BSA)溶液为测试对象,通过电化学交流阻抗(EIS)技术探究了该复合材料修饰界面在消除蛋白质非特异性吸附方面的性能表现,结果表明该复合材料修饰界面具有良好的消除蛋白质非特异性吸附的效能。通过自组装方式在聚3,4-乙烯二氧噻吩/聚乙二醇衍生物修饰电极的表面引入金纳米粒子(AuNPs),继而在金纳米粒子表面固定甲胎蛋白抗体(AFP-Ab),成功构建了甲胎蛋白(AFP)电化学生物传感器。该生物传感器的传感性能通过电化学交流阻抗技术进行了测试,得到的线性检测范围从0.001 fg/m L到10.0 fg/m L,最低检出限是0.0003 fg/m L,而且,构建的甲胎蛋白生物传感器表现出超高的选择性、灵敏度和实际应用潜力。该生物传感器优异的性能表现主要归因于制备的聚3,4-乙烯二氧噻吩/聚乙二醇衍生物复合材料具有较大的比表面积、较高的稳定性、抗污染性能和生物相容性。(2)具有超高的亲水性且呈现电中性的两性离子多肽(Peptide)是优良的抗污染材料。通过自组装方式将抗污染多肽(氨基酸序列为EKEKEKEPPPPC)固定在金电极(Au)表面,形成抗污染界面层,并采用电化学交流阻抗技术对该界面分别在单一蛋白质溶液(人血清白蛋白HSA和溶菌酶lysozyme)和复杂蛋白质溶液(人类血清human serum)中的抗污染性能进行了探索。采用水的接触角实验验证了该两性离子多肽界面的亲水性。基于该两性离子多肽薄膜优异的抗污染特性,利用多肽上自带的功能基团(-nh2),将羧基(-cooh)修饰的含有19个碱基的乳腺癌易感基因1(brca1)相关的dna序列连接到电极表面,结合双链dna杂交会引起电化学信号变化的原理,成功构建能够特异性、灵敏性识别乳腺癌易感基因1的电化学抗污染生物传感器。传感器的构建过程主要采用x射线光电子能谱(xps)和电化学交流阻抗技术进行表征。通过电化学交流阻抗技术得到的线性检测范围从1.0fm到10.0pm,最低检出限为0.3fm。诸多优异的传感性能,包括抗污染性、选择性、灵敏度、重现性等,表明该生物传感器有望用于乳腺癌易感基因1的临床分析。(3)抗污染材料与生物识别探针在电极表面的组装方式,是影响生物传感界面的抗污染性能和目标识别能力的关键因素之一。在本研究中,我们自主设计了具有亲水性和电中性的两性离子多肽(氨基酸序列为eesksesksggggc)。采用电化学差分脉冲伏安(dpv)法探究了短链的两性离子多肽(eesksesksggggc)和长链的生物识别探针(甲胎蛋白适配体afp-aptamer)在金电极表面的不同自组装方式对构建界面的抗污染性能和目标检测能力的影响。构建的生物传感界面的抗污染能力分别在不同浓度的蛋白质溶液,包括人血清白蛋白溶液和复杂的人血清溶液,通过灵敏的电化学差分脉冲伏安技术进行了测试。传感界面的构建过程、亲水特性以及目标响应能力分别通过电化学技术交流阻抗、差分脉冲伏安、x射线光电子能谱、水的接触角实验等不同技术进行了表征。该生物传感器对目标物甲胎蛋白的线性检测范围在10.0fg/ml到100.0pg/ml之间。将制备的生物传感器应用于实际人血清样本中甲胎蛋白含量的测定,得到的结果与医院标准检测结果的偏差在6.0%之内,显示出较高的实际应用潜力。(4)间氨基苯甲酸(m-aminobenzoicacid)是苯胺的一种衍生物。采用电化学技术在玻碳电极表面成功电聚合聚间氨基苯甲酸(p-maba),并通过扫描电子显微镜技术对其修饰电极的表面形貌进行了表征,结果显示聚间氨基苯甲酸修饰电极呈明显的褶皱形态,有效地增大了电极的比表面积。利用聚间氨基苯甲酸自身含有的羧基基团,先后固定氨基修饰的捕获探针(人免疫球蛋白e适配体ige-aptamer)和抗污染多肽(氨基酸序列为chhhddd)制备电化学生物传感器。基于目标蛋白(ige)与对应适配体(ige-aptamer)之间的特异性结合能力,实现对目标物人免疫球蛋白e的灵敏性检测。电化学交流阻抗、循环伏安、差分脉冲伏安等多种电化学技术被用于该生物传感器的构建、抗污染性能及传感性能的表征。聚间氨基苯甲酸修饰的电极界面在不同浓度(v/v)的胎牛血清(fbs)溶液中表现出较高的抗污染性能。构建的生物传感器对免疫球蛋白E的线性检测范围在0.01 ng/m L到50.0 ng/mL之间,表现出较高的选择特异性。
[Abstract]:This paper is mainly based on the anti pollution new material, with different conductive polymer electrodes, electrochemical biosensors based on different assembly through different ways, to achieve specific, a variety of markers of disease detection sensitivity. Using a variety of characterization methods of anti pollution performance of different biological sensing interface have been studied by electrochemical technology study of the variety of biological sensor on the target response ability, and analyzes the application potential of the biosensor was constructed on target detection in real serum samples. The main research contents of this thesis are summarized as follows: (1) by electrodeposition method simple on glassy carbon electrode (GCE) surface successfully. Preparation of poly ethylene 3,4- two oxygen thiophene / polyethylene glycol derivative (PEDOT/PEG) composite material. By scanning electron microscopy (SEM) and cyclic voltammetry (CV) technology of the complex The composite modified electrode (PEDOT/PEG/GCE) surface morphology and electrochemical stability were studied. The results show that the composite material is a multilayer structure, large surface area and high electrochemical stability. Using bovine serum albumin (BSA) solution as the test object, the electrochemical impedance (EIS) technique to explore the composite modification the interface in the elimination of protein non specific adsorption performance, results show that the interfacial modification of the composite material has a good elimination of nonspecific protein adsorption performance. Through self-assembly of poly 3,4- in two oxygen ethylene thiophene / polyethylene glycol derivatives modified electrode surface into gold nanoparticles (AuNPs), and then in a fixed on the surface of gold nanoparticles fetoprotein antibody (AFP-Ab), the successful construction of alpha fetoprotein (AFP) electrochemical biosensor. The biosensor sensitivity by electrochemistry AC impedance technique was tested. The linear detection range from 0.001 fg/m to 10 L fg/m L, the lowest detection limit is 0.0003 fg/m L, and the selectivity of AFP biosensor showed a super high sensitivity, and practical applications. The performance of the biosensor is mainly attributed to the excellent preparation of poly 3,4- two oxygen ethylene thiophene / polyethylene glycol derivatives of composite materials with high surface area, high stability, anti fouling properties and biocompatibility. (2) with high hydrophilicity and electrically neutral zwitterionic peptide (Peptide) is the excellent anti pollution materials. Through self assembling the anti pollution polypeptide (amino acids EKEKEKEPPPPC) immobilized on the gold electrode (Au) surface, the formation of anti pollution boundary layer, and the electrochemical impedance of the interface in a single protein solution (human blood. Protein HSA and lysozyme lysozyme) and complex protein solution (human serum human serum) in the anti pollution performance was explored. The water contact angle experiments verify the hydrophilicity of the zwitterionic peptide interface. Anti fouling characteristics of the zwitterionic peptide film with excellent functional groups based on the use of a polypeptide with (-nh2) the carboxyl (-cooh), modified to contain 19 nucleotides of breast cancer susceptibility gene 1 (BRCA1) DNA sequence is connected to the electrode surface, combined with the principle of double stranded DNA hybrid will cause the electrochemical signal changes, can successfully construct specific susceptibility gene 1, electrochemical sensitivity of breast cancer anti pollution biological recognition the construction process of the main sensor. Sensor using X ray photoelectron spectroscopy (XPS) and electrochemical impedance spectroscopy was investigated. The linear detection range obtained by electrochemical impedance technique from 1.0fm to 10 .0pm, the lowest detection limit was 0.3fm. many excellent sensing properties, including pollution resistance, selectivity, sensitivity, reproducibility, showed that the biosensor could be used in clinical analysis of breast cancer susceptibility gene 1. (3) anti pollution materials and biological recognition probe in the assembly of the electrode surface, is one of the effects of biological sensor the interface of the anti pollution performance and target recognition ability of the key factors. In this study, we designed the zwitterionic peptide with hydrophilic and neutral (amino acids eesksesksggggc). Differential pulse voltammetry using electrochemical (DPV) the zwitterionic peptide short chain of inquiry (eesksesksggggc) probe and biological identification long chain (AFP aptamer afp-aptamer) on the gold electrode surface of different self-assembly methods on anti pollution performance and target detection ability to construct interface. The influence of community construction of biosensors The anti pollution ability in protein solutions of different concentrations, including human serum albumin and human serum by complex solution, sensitive electrochemical differential pulse voltammetric techniques were tested. The construction process of sensing interface, hydrophilicity and target response ability respectively by electrochemical impedance technique, differential pulse voltammetry, X-ray photoelectron x energy spectrum, water contact angle experiments of different technology were investigated. The linear biosensor for target detection of alpha fetoprotein in the range of 10.0fg/ml to 100.0pg/ml. The determination of biosensor prepared in serum samples of the actual application of alpha fetoprotein detection results, the results obtained with the standard deviation of the hospital in 6%, showing high application potential. (4) m-aminobenzoic acid (m-aminobenzoicacid) is a derivative of aniline by electrochemical. Science and technology on the surface of glassy carbon electrode electro polymerization m-aminobenzoic acid (p-maba), and surface morphology were characterized by scanning electron microscopy on the modified electrode. The results showed that poly m-aminobenzoic acid modified electrode showed obvious fold shape, effectively increasing the specific surface area of the electrode. The carboxyl group poly m-aminobenzoate itself contains, has fixed amino modified capture probe (Human Immunoglobulin e aptamer ige-aptamer) and anti pollution peptide (amino acid sequence chhhddd). Preparation of electrochemical biosensor based on a target protein (IGE) and corresponding aptamer (ige-aptamer) specific binding capacity, to achieve the target Human Immunoglobulin e sensitivity detection. Electrochemical impedance spectroscopy, cyclic voltammetry, differential pulse voltammetry and other electrochemical techniques were used to construct the biosensor, anti pollution Characterization and sensing properties. Poly m-aminobenzoic acid modified electrode interface (v/v) in different concentrations of fetal bovine serum (FBS) showed the anti pollution performance of high solution. Linear biosensors for the detection of immunoglobulin E in the range of 0.01 ng/m L to 50 ng/mL, showing selection of high specificity.

【学位授予单位】：青岛科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TP212.3;TB34

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