基于金屬編碼探針的多元腫瘤標(biāo)志物早期篩查免疫傳感器研究
發(fā)布時間：2018-08-21 11:21
【摘要】：本論文是基于金屬編碼探針、生物夾心免疫及電化學(xué)溶出技術(shù)構(gòu)建了一系列簡單、快速、靈敏、低成本的多元電化學(xué)免疫分析方法,用于同時檢測多種腫瘤標(biāo)志物(甲胎蛋白AFP、癌胚抗原CEA、糖類抗原CA19-9)。多元電化學(xué)免疫分析方法是在單元檢測基礎(chǔ)上發(fā)展起來的一種新的分析方法,本文依據(jù)不同金屬的溶出峰電位不同,來區(qū)別定義腫瘤標(biāo)志物,對應(yīng)的溶出峰電流來定量腫瘤標(biāo)志物,達(dá)到單次運行同時測定多種腫瘤標(biāo)志物的目的。構(gòu)建的免疫分析方法使用免疫磁珠Dynabeads、磁性分子印跡MMIPs作為捕獲探針,不僅簡化了分離步驟,還增加了待測物的富集量;使用高聚物Envision、石墨烯(G)、聚賴氨酸(PLL)作為載體,進(jìn)一步負(fù)載金屬編碼標(biāo)簽(量子點QDs、納米金Au、重組脫鐵鐵蛋白r-Apo及脫鐵鐵蛋白Apo)分別制備可分辨的信號標(biāo)簽。當(dāng)抗原抗體之間發(fā)生特異性免疫反應(yīng)后,隨即形成夾心免疫復(fù)合物。磁性分離后,執(zhí)行高靈敏度的溶出伏安技術(shù),在同一體系同時定量分析金屬元素,以定量多種腫瘤標(biāo)志物的含量。本論文的研究工作從以下三個方面展開:1.基于聚合物-金屬信號放大標(biāo)簽和雙向溶出伏安法的多元電化學(xué)免疫分析方法本章構(gòu)建了一個雙向溶出伏安免疫分析方法,同時檢測多種腫瘤標(biāo)志物甲胎蛋白(AFP)、癌胚抗原(CEA)、糖類抗原CA19-9。我們采用Envision負(fù)載金屬納米粒子作為信號探針,免疫磁珠作為捕獲探針。Envision,一種長鏈聚合物,含有大量的辣根過氧化物酶和二抗。我們將其作為載體,標(biāo)記相應(yīng)的一抗,并負(fù)載納米Cd S、Pb S、Au制備可分辨的信號探針。我們在磁珠Dynabeads上同時固定三種一抗,制備捕獲探針。通過夾心型免疫反應(yīng),形成免疫復(fù)合物。進(jìn)一步使用雙向溶出伏安法,定量分析金屬成分Cd、Pb、Au以檢測三種腫瘤標(biāo)志物。AFP、CEA、CA19-9檢測范圍分別為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;檢測限分別為0.02 pg m L-1,0.05 pg m L-1,0.3 pg m L-1。實驗結(jié)果表明構(gòu)建的雙向多元免疫分析可同時檢測多種標(biāo)志物,具有靈敏度高、穩(wěn)定性良好等優(yōu)點,并在臨床免疫學(xué)方面擁有一定的應(yīng)用前景。2.基于重組脫鐵鐵蛋白編碼的金屬標(biāo)簽構(gòu)建多元電化學(xué)免疫方法本章構(gòu)建了一種多元電化學(xué)免疫分析方法,同時測定兩種腫瘤標(biāo)志物,采用AFP和CEA作為研究模型。我們使用重組脫鐵鐵蛋白編碼的金屬納米粒子(r Apo-M)作為信號標(biāo)簽,雙模板磁性分子印跡(MMIPs)作為捕獲探針。我們在石墨烯(G)上原位合成納米金(Au)制備G-Au納米復(fù)合物,以負(fù)載重組脫鐵鐵蛋白(r Apo-M)及標(biāo)記一抗(anti-AFP和anti-CEA)制備信號探針。弱堿性溶液中加入模板蛋白AFP和CEA,多巴胺(DA)在納米Fe3O4表面形成MMIPs以制備捕獲探針。夾心型免疫反應(yīng)后,信號探針固定到MMIPs表面。隨后使用電化學(xué)溶出技術(shù),分析檢測金屬成分Cd和Pb,量化腫瘤標(biāo)志物。實驗結(jié)果表明,同時測定AFP和CEA線性范圍為0.001-5ng m L-1。AFP和CEA的檢出限分別為0.3和0.35pg m L-1(S/N=3)。此外,結(jié)果表明我們構(gòu)建的多元電化學(xué)免疫分析可拓展為其他生物標(biāo)志物的臨床篩查。3.基于生物基聚合物納米探針構(gòu)建多元電化學(xué)免疫方法本章構(gòu)建了一種新穎的夾心型電化學(xué)多元免疫方式,同時檢測雙分析物AFP和CEA。將甲胎蛋白和癌胚抗原的一抗包被磁珠作為捕獲探針。金屬編碼的脫鐵鐵蛋白(Cd-Apo和Pb-Apo)及二抗分別通過納米金(Au NPs)交聯(lián)在聚賴氨酸(PLL)上,通過自組裝制備成信號探針。經(jīng)過夾心免疫反應(yīng),在磁珠上形成夾心免疫復(fù)合物。我們通過鉛(Cd)和鎘(Pb)的溶出電位不同來區(qū)別待測物。兩者的溶出峰電流分別與AFP和CEA的濃度相關(guān)。實驗結(jié)果表明構(gòu)建的免疫策略實現(xiàn)了對AFP和CEA的同時檢測,線性范圍都為0.01-50 ng m L-1,檢測限為4 pg m L-1。該方法簡單、靈敏且環(huán)保。此外該方法成功應(yīng)用于同時檢測血清樣品中的AFP和CEA,結(jié)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.
【學(xué)位授予單位】：寧波大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R730.4;TP212
本文編號：2195558
[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.
【學(xué)位授予單位】：寧波大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R730.4;TP212
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