基于功能化碳纳米材料构建的信号放大型电化学免疫传感器的研究

发布时间：2018-04-23 02:03

本文选题：电化学免疫传感器 + 肿瘤标志物　；参考：《西南大学》2015年硕士论文

【摘要】：在医学免疫分析中,发展一些快速、灵敏和准确的检测方法是科研工作者研究的重点、热点问题,这在临床分析中对于疾病的诊断具有非常重要的意义。电化学免疫传感器是一种将电化学技术、传感器技术和免疫分析技术三者相结合而发展来的,集灵敏度高、选择性好、操作简便等优点于一身的分析技术。近几十年来,发展新型功能化的纳米材料、探索生物大分子的固定化技术和构建信号增强技术等用于电化学免疫传感器检测生物分子的研究受到了广泛的关注。本文主要从功能化纳米材料的制备,传感器仿生界面的构建,信号放大技术的创新等方面来进行研究,并制备了一系列高灵敏的电化学免疫传感器。主要研究内容如下：1.基于纳米金功能化的多壁碳纳米管复合材料标记抗体的电化学免疫传感器的研究本研究工作构建了一种基于纳米金/(3-巯丙基)三甲氧基硅烷(Au/MPTS)修饰传感器界面和纳米金功能化的多壁碳纳米管复合材料标记生物分子用于信号探针的夹心型电化学免疫传感器,用于肝癌肿瘤标志物甲胎蛋白(AFP)的检测。首先,纳米金通过PDDA共价交联在多壁碳纳米管表面制备得到了纳米金功能化的碳纳米材料,接着进一步把电活性物质硫堇(Thi)标记的抗体生物分子和辣根过氧化物酶(HRP)固定在其表面,制备得到了纳米生物探针。纳米金功能化的多壁碳纳米管固载了大量的生物分子和电活性物质,从而可以有效的提高免疫传感器的灵敏度。该纳米生物探针不仅能识别目标分析物,而且在HRP对底物H2O2的催化作用下,可以进一步放大电化学反应信号。另外,Au/MPTS修饰传感器界面不仅可以用来固载生物分子、保持其生物活性,还能进一步促进电子的传递。采用夹心免疫反应模式,实现对AFP的定量检测,其线性范围为0.01-50.0 ng·mL-1,检测下限为3 pg·mL-1该传感器具有良好的选择性、稳定性和重现性。2.基于双酶催化沉淀技术和碳纳米角构建的信号增强型电化学免疫传感器的研究在本实验中,结合碳纳米角标记和双酶催化产物沉淀法构建了一种新颖的电化学免疫传感器,实现了对AFP的高灵敏检测。首先,制备金-石墨稀复合纳米材料(Au-Gra)用于修饰玻碳电极,不仅能促进氧化还原探针与电极表面的电子传递,也能增加电极的有效面积提高AFP抗体的固载量。其次,将功能化处理后的碳纳米角用于标记抗体和固载葡萄糖氧化酶(GOD)和HRP得到生物纳米探针,碳纳米角较大的比表面积增加了抗体和双酶的固载量。实验采用夹心免疫反应模式,利用4-萘酚作为沉淀底物,该探针不仅能识别目标分析物,还能通过双酶催化沉淀实现信号放大,提高了免疫传感器的灵敏度,从而实现对肿瘤标志物的高灵敏检测。免疫分析过程采用电化学交流阻抗和循环伏安法进行表征。免疫传感器对目标分析物AFP的线性响应范围为0.001-60 ng·mL-1,检测下限为0.33pg-mL-13.基于核-壳结构Au@Pd双金属纳米粒子功能化石墨稀构建的双重信号放大型电化学免疫传感器的研究该工作基于纳米金功能化的多孔石墨稀复合材料(Au-PGO)作为抗体的固定化基质,利用Au@Pd双金属纳米粒子修饰的石墨烯(Au@Pd-Gra)标记生物分子制备信号探针,构建了一种用于检测胰腺癌肿瘤标志物(CA19-9)的夹心型电化学免疫传感器。一方面,Au-PGO不仅可以固载大量的抗体生物分子,还能有效增加电极的活性面积,提高电极和氧化还原探针之间的电子传输能力；另一方面,Au@Pd-Gra不仅本身具有过氧化物模拟酶性质,其较大的比表面积和优良的生物相容性等特点,还可以用来固载更多的生物分子并保持生物分子的活性。同时,在Au@Pd-Gra和HRP的协同催化作用下,电化学信号实现了双重信号放大,免疫传感器的灵敏度得到了极大的提高。在优化的实验条件下,免疫传感器对目标分析物的检测范围为0.015-150 U·mL-1,最低检测限为0.006 U·mL-1。该方法操作简便、灵敏度高,在定量检测的临床分析中具有很大的应用潜力。
[Abstract]:In the analysis of medical immunoassay, the development of rapid, sensitive and accurate detection methods is the focus of research workers and hot issues. This is of great significance for the diagnosis of disease in clinical analysis. Electrochemical immunosensor is a combination of electrochemical technology, sensor technology and immunoassay technology three. In recent decades, the development of new functionalized nanomaterials, the exploration of biomolecular immobilization technology and the construction of signal enhancement techniques have attracted wide attention in recent decades. Mainly from the preparation of functional nanomaterials, the construction of biomimetic interfaces and the innovation of signal amplification technology, a series of highly sensitive electrochemical immunosensor are prepared. The main contents are as follows: 1. electrochemistry immunity of multi walled carbon nanometers based on gold nanoparticles A sandwich type electrochemical immunosensor based on nano gold / (3- mercapto propyl) trimethoxy silane (Au/MPTS) modified sensor interface and nano gold functionalized multi walled carbon nanotube composite biomolecules used for signal probe was constructed for the study of tumor marker alpha fetoprotein (AFP), a tumor marker of liver cancer. First, nano gold was prepared by PDDA covalent crosslinking on the surface of multi walled carbon nanotubes to obtain nano gold functionalized carbon nanomaterials. Then, the antibody biologically and horseradish peroxidase (HRP) labeled by the electroactive active substance (Thi) was immobilized on the surface, and nanoscale probes were prepared. The multi walled carbon nanotubes hold a large number of biomolecules and electroactive substances, which can effectively improve the sensitivity of the immunosensor. The nanoscale probe can not only identify the target analyte, but also further amplify the electrochemical reaction signal under the catalysis of HRP to the substrate H2O2. In addition, the interface of the Au/MPTS modified sensor is not It can only be used to immobilizing biomolecules, maintaining their biological activity, and further promoting electron transfer. The quantitative detection of AFP by sandwich immunoreaction model is realized. The linear range is 0.01-50.0 ng. ML-1, the detection limit is 3 pg. ML-1, the sensor has good selectivity, stability and reproducibility.2. based on double enzyme catalytic precipitation. In this experiment, a novel electrochemical immunosensor was constructed with carbon nanometers and double enzyme catalyzed product precipitation in this experiment. The high sensitivity detection of AFP was realized. First, the preparation of gold graphite thin composite nanomaterial (Au-Gra) was used in modified glassy. Carbon electrode can not only promote the electron transfer of the redox probe and the surface of the electrode, but also increase the effective area of the electrode to increase the solid load of the AFP antibody. Secondly, the carbon nano angle after the functionalized treatment is used to mark the antibody and the immobilized glucose oxidase (GOD) and HRP to get the nano probe, and the specific surface area of the carbon nano angle is increased. Using the sandwich immunoreaction model and using 4- naphthol as the precipitation substrate, the probe can not only identify the target analyte, but also enlarge the sensitivity of the immune sensor by double enzyme catalyzed precipitation, so as to realize the high sensitivity detection of the tumor marker. Characterization with electrochemical impedance and cyclic voltammetry. The linear response range of the immune sensor to the target analyte AFP is 0.001-60 ng. ML-1, and the detection limit is 0.33pg-mL-13. based on the dual signal amplified electrochemical immunosensor based on the nuclear shell structure Au@Pd bimetallic nanoparticles. Porous graphite thin composite (Au-PGO) is used as a immobilized matrix for antibody, and Au@Pd bimetal nanoparticles modified graphene (Au@Pd-Gra) biomolecules are used to prepare signal probes. A sandwich electrochemical immunosensor for detecting tumor markers (CA19-9) of pancreatic cancer (CA19-9) is constructed. On one hand, Au-PG O can not only immobilizing a large number of antibody biologic molecules, but also effectively increasing the active area of the electrode and improving the electronic transmission ability between the electrode and the redox probe. On the other hand, Au@Pd-Gra not only has the properties of the peroxidase mimic enzyme, but also the larger surface area and the excellent biocompatibility and so on. At the same time, under the synergistic catalysis of Au@Pd-Gra and HRP, the electrochemical signal has double signal amplification and the sensitivity of the immune sensor has been greatly improved. Under the optimized experimental conditions, the detection range of the immune sensor to the target analysis is 0.015-150 U. ML-1. The minimum detection limit is 0.006 U mL-1.. This method is simple and sensitive, and has great potential in clinical analysis of quantitative detection.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：TP212;TB383.1

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