抗污染传感界面的构建及其在生物分子检测中的应用
发布时间：2019-07-09 10:46
【摘要】：制备能够用于实际生物样品中低丰度肿瘤标志物的高灵敏度,高选择性检测的电化学生物传感器存在很大的挑战。主要存在问题是实际生物样品中蛋白质,多糖,脂质等生物大分子对传感界面的非特异性吸附。因此构建具有优良抗污染性能的传感界面对于传感器的研制是十分必要的。本论文以电化学方法制备了几种纳米材料,以表面共价修饰的方法构建抗污染传感界面,系统地研究了传感器的抗污染传感性能。本论文的主要研究内容如下:(1)成功的制备了一种新颖简单的超灵敏且具有抗污染能力的电化学DNA传感器。首先通过电化学氧化玻碳电极的方式得到修饰有羧基官能团的界面,再通过共价链接的方法将抗污染材料PEG以及固定探针依次修饰到电极表面,从而制备了这种具有抗污染能力的电化学DNA传感器。传感器的检测线性范围为1.0 fM to 100.0 nM,最低检测限为0.3 fM。并且制备的生物传感器在实际生物样品检测中具有良好的表现,有望在实际临床检测中得到应用。(2)非特异性蛋白质吸附是免疫测定中面临的关键问题,构建可以在复杂生物介质中显著抵抗蛋白质非特异性吸附的界面是非常重要的。本研究,是基于掺杂有透明质酸(HA)的聚3,4-乙烯二氧噻吩(PEDOT)新型复合材料,制备了用于肿瘤标志物-癌胚抗原(CEA)检测的抗污染电化学免疫传感器。电沉积的PEDOT/HA复合材料显示出多孔微结构和很强的亲水性,并且在其表面上还具有许多羧基官能团,可以用于固定CEA抗体。基于anti-CEA/PEDOT/HA的免疫传感器在从1 pg/mL至0.1μg/m L的宽线性浓度范围内对CEA表现出高的检测灵敏度,检测限是0.3 pg/m L。此外,所开发的免疫传感器具有高度特异性和抗污染性能,可以用于在真实的人血清样品中测定CEA,避免由于非特异性蛋白吸附而导致的信号干扰。(3)通过共价链接的方法将MB-rep/probe(亚甲基蓝信号探针/固定探针)双链固定到AuNCs(金纳米簇)/HA(透明质酸)/Popd(聚邻苯二胺)/CoO(氧化钴)核/壳复合材料表面,得到一种新型的能够快速检测microRNA(miRNA)的电化学生物传感器。在检测目标物时,形成probe/target双链体(电极表面)和MB-rep发夹结构(溶液中),其迅速从生物传感器置换MB-rep,导致SWV(方波脉冲伏安法)信号的迅速变化。基于生物传感器的Au/HA/Popd/CoO复合材料提供更多的电活性表面积和优异的抗污染性能,其可以用于生物复杂介质中的miRNA的超灵敏检测。生物传感器显示出优异的特异性和检测灵敏性,具有从100 fM至1μM的宽线性范围,为临床生物应用提供了重要支撑。
[Abstract]:The preparation of an electrochemical biosensor capable of being used for high sensitivity and high-selectivity detection of low-abundance tumor markers in an actual biological sample has a great challenge. The main problems are the non-specific adsorption of the biological macromolecules such as proteins, polysaccharides and lipids in the actual biological sample to the sensing interface. Therefore, it is necessary to construct a sensing interface with excellent anti-pollution performance. In this paper, several nano-materials were prepared by electrochemical method, and the anti-pollution sensing interface was constructed by the method of surface covalent modification, and the anti-pollution sensing performance of the sensor was systematically studied. The main contents of this thesis are as follows: (1) A novel, simple, ultra-sensitive and anti-pollution electrochemical DNA sensor was successfully prepared. Firstly, the interface with the anti-pollution function is obtained through the method of electrochemical oxidation of the glassy carbon electrode, and the anti-pollution material PEG and the fixed probe are sequentially modified to the surface of the electrode through a covalent linkage method, so that the electrochemical DNA sensor with the anti-pollution capability is prepared. The sensor has a linear range of 1.0 fM to 100.0 nM and a minimum detection limit of 0.3 fM. And the prepared biosensor has good performance in actual biological sample detection and is expected to be applied in practical clinical detection. (2) Non-specific protein adsorption is a key problem in immunoassays, and it is very important to construct an interface that can significantly resist the non-specific adsorption of proteins in complex biological media. The present study was based on the novel composite of poly (3,4-ethylene) dioxolane (PEDOT) doped with hyaluronic acid (HA), and prepared the anti-pollution electrochemical immunosensor for tumor marker-carcinoembryonic antigen (CEA) detection. Electrodeposited PEDOT/ HA composites exhibit a porous microstructure and very strong hydrophilicity, and also have a number of base-based functional groups on their surface, which can be used to fix the CEA antibody. The immune sensor based on the anti-CEA/ PEDOT/ HA exhibited high detection sensitivity to CEA in a wide linear concentration range from 1 pg/ mL to 0.1. m u.g/ m L, with a detection limit of 0.3 pg/ m L. In addition, the developed immunosensor has a high specificity and an anti-contamination performance, CEA can be used in a real human serum sample to avoid signal interference due to non-specific protein adsorption. (3) the MB-rep/ probe (methylene blue signal probe/ fixed probe) double-chain is fixed to the surface of the AuNCs (gold nanocluster)/ HA (hyaluronic acid)/ Popd (poly (o-phenylenediamine)/ CoO (cobalt oxide) core/ shell composite material by a covalent link method, A novel electrochemical biosensor capable of rapidly detecting microRNA (miRNA) is obtained. When the target is detected, the probe/ target duplex (electrode surface) and the MB-rep hairpin structure (in solution) are formed, which quickly displaces the MB-rep from the biosensor, resulting in a rapid change in the SWV (square wave pulse voltammetry) signal. The Au/ HA/ Popd/ CoO composite based on the biosensor provides more electroactive surface area and excellent anti-pollution performance, which can be used for ultra-sensitive detection of miRNAs in a biological complex medium. Biosensors exhibit excellent specificity and detection sensitivity, with a wide linear range from 100 fM to 1. m u.M, providing important support for clinical biological applications.
【学位授予单位】：青岛科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.1
本文编号：2512090
[Abstract]:The preparation of an electrochemical biosensor capable of being used for high sensitivity and high-selectivity detection of low-abundance tumor markers in an actual biological sample has a great challenge. The main problems are the non-specific adsorption of the biological macromolecules such as proteins, polysaccharides and lipids in the actual biological sample to the sensing interface. Therefore, it is necessary to construct a sensing interface with excellent anti-pollution performance. In this paper, several nano-materials were prepared by electrochemical method, and the anti-pollution sensing interface was constructed by the method of surface covalent modification, and the anti-pollution sensing performance of the sensor was systematically studied. The main contents of this thesis are as follows: (1) A novel, simple, ultra-sensitive and anti-pollution electrochemical DNA sensor was successfully prepared. Firstly, the interface with the anti-pollution function is obtained through the method of electrochemical oxidation of the glassy carbon electrode, and the anti-pollution material PEG and the fixed probe are sequentially modified to the surface of the electrode through a covalent linkage method, so that the electrochemical DNA sensor with the anti-pollution capability is prepared. The sensor has a linear range of 1.0 fM to 100.0 nM and a minimum detection limit of 0.3 fM. And the prepared biosensor has good performance in actual biological sample detection and is expected to be applied in practical clinical detection. (2) Non-specific protein adsorption is a key problem in immunoassays, and it is very important to construct an interface that can significantly resist the non-specific adsorption of proteins in complex biological media. The present study was based on the novel composite of poly (3,4-ethylene) dioxolane (PEDOT) doped with hyaluronic acid (HA), and prepared the anti-pollution electrochemical immunosensor for tumor marker-carcinoembryonic antigen (CEA) detection. Electrodeposited PEDOT/ HA composites exhibit a porous microstructure and very strong hydrophilicity, and also have a number of base-based functional groups on their surface, which can be used to fix the CEA antibody. The immune sensor based on the anti-CEA/ PEDOT/ HA exhibited high detection sensitivity to CEA in a wide linear concentration range from 1 pg/ mL to 0.1. m u.g/ m L, with a detection limit of 0.3 pg/ m L. In addition, the developed immunosensor has a high specificity and an anti-contamination performance, CEA can be used in a real human serum sample to avoid signal interference due to non-specific protein adsorption. (3) the MB-rep/ probe (methylene blue signal probe/ fixed probe) double-chain is fixed to the surface of the AuNCs (gold nanocluster)/ HA (hyaluronic acid)/ Popd (poly (o-phenylenediamine)/ CoO (cobalt oxide) core/ shell composite material by a covalent link method, A novel electrochemical biosensor capable of rapidly detecting microRNA (miRNA) is obtained. When the target is detected, the probe/ target duplex (electrode surface) and the MB-rep hairpin structure (in solution) are formed, which quickly displaces the MB-rep from the biosensor, resulting in a rapid change in the SWV (square wave pulse voltammetry) signal. The Au/ HA/ Popd/ CoO composite based on the biosensor provides more electroactive surface area and excellent anti-pollution performance, which can be used for ultra-sensitive detection of miRNAs in a biological complex medium. Biosensors exhibit excellent specificity and detection sensitivity, with a wide linear range from 100 fM to 1. m u.M, providing important support for clinical biological applications.
【学位授予单位】：青岛科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.1
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