基于金属编码探针的多元肿瘤标志物早期筛查免疫传感器研究

发布时间：2018-08-21 11:21

【摘要】：本论文是基于金属编码探针、生物夹心免疫及电化学溶出技术构建了一系列简单、快速、灵敏、低成本的多元电化学免疫分析方法,用于同时检测多种肿瘤标志物(甲胎蛋白AFP、癌胚抗原CEA、糖类抗原CA19-9)。多元电化学免疫分析方法是在单元检测基础上发展起来的一种新的分析方法,本文依据不同金属的溶出峰电位不同,来区别定义肿瘤标志物,对应的溶出峰电流来定量肿瘤标志物,达到单次运行同时测定多种肿瘤标志物的目的。构建的免疫分析方法使用免疫磁珠Dynabeads、磁性分子印迹MMIPs作为捕获探针,不仅简化了分离步骤,还增加了待测物的富集量;使用高聚物Envision、石墨烯(G)、聚赖氨酸(PLL)作为载体,进一步负载金属编码标签(量子点QDs、纳米金Au、重组脱铁铁蛋白r-Apo及脱铁铁蛋白Apo)分别制备可分辨的信号标签。当抗原抗体之间发生特异性免疫反应后,随即形成夹心免疫复合物。磁性分离后,执行高灵敏度的溶出伏安技术,在同一体系同时定量分析金属元素,以定量多种肿瘤标志物的含量。本论文的研究工作从以下三个方面展开:1.基于聚合物-金属信号放大标签和双向溶出伏安法的多元电化学免疫分析方法本章构建了一个双向溶出伏安免疫分析方法,同时检测多种肿瘤标志物甲胎蛋白(AFP)、癌胚抗原(CEA)、糖类抗原CA19-9。我们采用Envision负载金属纳米粒子作为信号探针,免疫磁珠作为捕获探针。Envision,一种长链聚合物,含有大量的辣根过氧化物酶和二抗。我们将其作为载体,标记相应的一抗,并负载纳米Cd S、Pb S、Au制备可分辨的信号探针。我们在磁珠Dynabeads上同时固定三种一抗,制备捕获探针。通过夹心型免疫反应,形成免疫复合物。进一步使用双向溶出伏安法,定量分析金属成分Cd、Pb、Au以检测三种肿瘤标志物。AFP、CEA、CA19-9检测范围分别为1 pg m L-1-50 ng m L-1,1 pg m L-1-50 ng m L-1,5 pgm L-1-100 ng m L-1;检测限分别为0.02 pg m L-1,0.05 pg m L-1,0.3 pg m L-1。实验结果表明构建的双向多元免疫分析可同时检测多种标志物,具有灵敏度高、稳定性良好等优点,并在临床免疫学方面拥有一定的应用前景。2.基于重组脱铁铁蛋白编码的金属标签构建多元电化学免疫方法本章构建了一种多元电化学免疫分析方法,同时测定两种肿瘤标志物,采用AFP和CEA作为研究模型。我们使用重组脱铁铁蛋白编码的金属纳米粒子(r Apo-M)作为信号标签,双模板磁性分子印迹(MMIPs)作为捕获探针。我们在石墨烯(G)上原位合成纳米金(Au)制备G-Au纳米复合物,以负载重组脱铁铁蛋白(r Apo-M)及标记一抗(anti-AFP和anti-CEA)制备信号探针。弱碱性溶液中加入模板蛋白AFP和CEA,多巴胺(DA)在纳米Fe3O4表面形成MMIPs以制备捕获探针。夹心型免疫反应后,信号探针固定到MMIPs表面。随后使用电化学溶出技术,分析检测金属成分Cd和Pb,量化肿瘤标志物。实验结果表明,同时测定AFP和CEA线性范围为0.001-5ng m L-1。AFP和CEA的检出限分别为0.3和0.35pg m L-1(S/N=3)。此外,结果表明我们构建的多元电化学免疫分析可拓展为其他生物标志物的临床筛查。3.基于生物基聚合物纳米探针构建多元电化学免疫方法本章构建了一种新颖的夹心型电化学多元免疫方式,同时检测双分析物AFP和CEA。将甲胎蛋白和癌胚抗原的一抗包被磁珠作为捕获探针。金属编码的脱铁铁蛋白(Cd-Apo和Pb-Apo)及二抗分别通过纳米金(Au NPs)交联在聚赖氨酸(PLL)上,通过自组装制备成信号探针。经过夹心免疫反应,在磁珠上形成夹心免疫复合物。我们通过铅(Cd)和镉(Pb)的溶出电位不同来区别待测物。两者的溶出峰电流分别与AFP和CEA的浓度相关。实验结果表明构建的免疫策略实现了对AFP和CEA的同时检测,线性范围都为0.01-50 ng m L-1,检测限为4 pg m L-1。该方法简单、灵敏且环保。此外该方法成功应用于同时检测血清样品中的AFP和CEA,结果良好。
[Abstract]:A series of simple, rapid, sensitive and low-cost multivariate electrochemical immunoassays based on metal-coded probes, bio-sandwich immunoassay and electrochemical stripping techniques were developed for simultaneous detection of multiple tumor markers (AFP, CEA, CA19-9). A new analytical method developed on the basis of meta-assay is presented in this paper. According to the different dissolution peak potentials of different metals, tumor markers are defined differently, and the corresponding dissolution peak currents are used to quantify tumor markers to achieve the purpose of simultaneous determination of multiple tumor markers in a single run. Ads, magnetic molecular imprinting MMIPs as capture probes, not only simplified the separation process, but also increased the amount of enrichment to be measured; using polymer Envision, graphene (G), polylysine (PLL) as the carrier, further loaded with metal coding tags (QDs, nano-Au, recombinant de-ferritin r-Apo and de-ferritin Apo) to prepare cocoa, respectively. After magnetic separation, a highly sensitive stripping voltammetry technique is performed to simultaneously quantify metal elements in the same system to quantify the contents of various tumor markers. The research work in this paper is progressed from the following three aspects Polymer-metal signal amplification tag and bidirectional stripping voltammetry were used to construct a bidirectional Stripping Voltammetric Immunoassay Method for the simultaneous detection of a variety of tumor markers, including alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), and carbohydrate antigen CA19-9. Envision-loaded metal nanoparticles were used as the samples. Envision, a long-chain polymer containing a large number of horseradish peroxidase and antibodies, is used as a signal probe. We labeled the corresponding primary antibody as a carrier and loaded nano-Cd S, Pb S, Au to prepare the resolvable signal probe. We immobilized three primary antibodies on the magnetic beads Dynabeads to prepare the capture probe. Immune complexes were formed by sandwich immunoreaction. The metal components Cd, Pb and Au were quantitatively analyzed by two-way stripping voltammetry to detect three tumor markers. AFP, CEA and CA19-9 ranged from 1 pg m L-1 to 50 ng m L-1, 1 pg m L-1 to 50 ng m L-1, 5 pg m L-1 to 100 ng m L-1, and the detection limits were 0.02 PG m L-1, 0.05 PG m L-1, respectively. 1,0.3 PG m L-1. The experimental results show that the constructed BIMA can simultaneously detect multiple markers with high sensitivity and good stability, and has certain application prospects in clinical immunology. 2. The construction of multivariate electrochemical immunoassay method based on the metal tags encoded by recombinant ferritin. A multivariate electrochemical immunoassay was developed for the simultaneous determination of two tumor markers. AFP and CEA were used as the study models. The metal nanoparticles encoded by recombinant deferrin (r Apo-M) were used as signal labels and the double template magnetic molecular imprinting (MMIPs) as capture probes. We synthesized gold nanoparticles (Au) on graphene (G) in situ. G-Au nanocomposites were prepared by loading recombinant ferritin (r Apo-M) and labeling antibody (anti-AFP and anti-CEA). In weak alkaline solution, template protein AFP and CEA were added and dopamine (DA) was added to form MMIPs on the surface of nano-Fe3O4 to prepare capture probes. The results showed that the linear ranges of AFP and CEA were 0.001-5 ng m L-1. The detection limits of AFP and CEA were 0.3 and 0.35 PG m L-1 (S/N=3), respectively. In addition, the results showed that the multiple electrochemical immunoassay constructed by us could be extended to other biomarkers. Clinical screening. 3. Multivariate electrochemical immunoassay based on biopolymer nanoprobes. In this chapter, a novel sandwich electrochemical immunoassay was constructed for the simultaneous detection of dual analytes AFP and CEA. B-Apo and anti-cadmium were crosslinked on polylysine (PLL) by Au NPs, and signal probes were prepared by self-assembly. Sandwich immune complexes were formed on the magnetic beads by sandwich immune reaction. The experimental results show that the proposed immune strategy can simultaneously detect AFP and CEA with a linear range of 0.01-50 ng m L-1 and a detection limit of 4 PG m L-1. The method is simple, sensitive and environmentally friendly. In addition, the method has been successfully applied to the simultaneous detection of AFP and CEA in serum samples with good results.
【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R730.4;TP212

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