双重标记SiO2微球免疫比色法检测沙门氏菌抗体研究.pdf

浙江工商大学硕士学位论文双重标记SiO2微球免疫比色法检测沙门氏菌/抗体研究摘要沙门氏菌(Salmonella)是一种引起最为常见和高传染性人类疾 病的病原菌，该致病菌分布于世界各地，在人类和动物中具有高致病 率和死亡率。此外，国内外标准要求食品中不得检出沙门氏菌。因此,建立沙门氏菌及其抗体的更佳检测方法，对控制病原菌污染，保证畜 禽健康和食品安全尤为重要。目前，对沙门氏菌及其抗体的检测主要 包括标准检测法和快速检测法。标准检测法通常准确但繁琐耗时且不 经济。快速检测法近年来发展迅速且应用广泛，如荧光法、化学发光 法、电化学法和比色法等。其中，比色法由于具有简便、经济、信号 产生迅速和裸眼可观测结果等优点而成为最重要的分析方法。免疫比 色法是一种以抗原抗体反应为基础，根据颜色变化实现检测的免疫分 析技术o免疫比色法的反应结果可以通过肉眼观察或者借助于基础光 学仪器测得，已广泛用于现场检测。比色法的关键是设计一种新的信 号转导方式将肉眼不可见的检测反应转变为颜色改变反应。酶联免疫 吸附试验(Enzyme linked immuno so r bent assay,ELISA)因其诸多优 点而成为最常见检测致病菌及其血清抗体的免疫比色法,ELISA通常 通过酶标抗体及其底物实现免疫比色反应的信号转导。但其尚存在如 下缺点：酶标抗体制备过程繁杂，且标记和保存过程较易引起抗体和 酶分子生物活性降低；抗体包被、孵育抗原和洗涤过程均会消耗较长 时间；且易出现假阳性结果。I浙江工商大学硕士学位论文为改进和解决上述问题，本文以鸡白痢鸡伤寒沙门氏菌(.Salmonella pullorum and Salmonella gallinarum,S.pullorum and S.gallinarum)及其抗体为分析检测对象，利用活性翠兰KNG掺杂的蓝 色纳米二氧化硅微球(Blue silica nano par ticles,Blue-SiNP s)和酶标记 纳米二氧化硅微球(Enzyme-labeled silica nano par ticle)作为信号转导 探针结合磁性纳米微球(Magnetic nano par ticles,MNP s)的分离富集 作用，开发建立了以下几种分析检测方法：1基于蓝色SiO2纳米微球的竞争抑制免疫比色法检测抗沙门氏菌抗 体沙门氏菌感染与否可由血清中的抗鸡白痢鸡伤寒沙门氏菌抗体(Antibo dies against S.pullorum and S,gallinarum,anti-S.pullorum and S.gallinarum)水平表示。为建立一种新型竞争抑制免疫比色法用 于检测沙门氏菌感染，利于从根本上有效减少该菌对禽类食品的污 染。本方法合成了两种纳米颗粒并功能化：采用化学共沉淀法合成磁 性纳米核心并包覆二氧化硅外壳制成MNP s,经修饰制成抗鸡白痢鸡 伤寒沙门氏菌抗体偶联磁性纳米微球(Antibo dies against S.pullorum and S.gallinarum co njugated magnetic nano par ticles,IgG-MNP s)作为 捕获探针用于固定S.pullorum and S.gallinarum；采用反相微乳液法 合成并掺杂活性翠兰KNG制成Blue-SiNP s,经修饰后制成抗鸡白痢 鸡伤寒沙门氏菌抗体偶联二氧化硅纳米微球(Antibo dies against S.pullorum and S.gallinarum co njugated Blue silica nano par ticles,IgG-Blue-SiNP s)作为检测探针用于信号转导。IgG-MNP s首先与S.浙江工商大学硕士学位论文pullorum and S.gallinarum 孵育形成 IgG-MNP sS.pullorum and S.gallinarum免疫复合物，随后加入的阳性血清中的anti-5,pullorum and S.gallinarum与IgG-Blue-SiNP s共同竞争上述免疫复合物中有限的抗 原决定簇。通过磁铁分离 IgG-MNP s-S.pullorum and S.gallinarum-IgG-Blue-SiNP s夹心结构，用NaOH溶解蚀刻该夹心结构使活性翠兰 KNG染料释放出来。该染料在614 nm下的吸光度值与阳性血清中 anti-5,pullorum and S.gallinarum含量呈负相关。以酶标仪测量结果。在最佳检测条件下，可检测到稀释了 1：480的鸡白痢鸡伤寒沙门氏 菌阳性血清，该检测限明显低于国标法检测结果，且检测时间不超过 60 mino由此可见，该方法简单、快速、灵敏、特异性良好，应用潜 力较大。2基于HRP标记型SiO2纳米微球免疫夹心比色法检测S.pullorum and S.gallinarum对致病菌的快速、灵敏检测是食品安全和临床检验中的主要目 标。为构建一种优良的新型S.pullorum and S.gallinarum快速检测技 术，IgGMNP s被用作捕获探针；标记辣根过氧化物酶(Ho r ser adish per o xidase,HRP)和 anti-5.pullorum and S.gallinarum 的二氧化硅纳 米微球(HRP/anti-S.pullorum and S.gallinarum dual-labeled silica nano par ticles,HRP-IgG-SiNP s)作为检测探针。当存在目标菌S.pullorum and S.gallinarum时，被捕获探针特异性捕获后与检测探针 形成夹心结构，经磁性分离后催化氧化底物四甲基联苯胺(33,5,5，Tetr ameth ylbenzidine,TMB)，经硫酸终止反应后产生黄色产 in浙江工商大学硕士学位论文物，用酶标仪测得该有色产物的吸光度值与S.pullorum and S.gallinarum的浓度的对数成比例关系。在最优检测条件下，检测S.pullorum and S.gallinarum 的线性范围为 8.4x1(P CFU-mL 1 至 8.4x 107 CFUmL/，检测限为 1.7xl()3 CFU,mL/。该方法检测 S.pullorum and5.ga讥aw加具有简便、经济、灵敏、特异性良好的优点。3葡萄糖氧化酶触发的酶级联反应结合Fe3+-SCN络合物显色体系比 色检测 S.pullorum and S.gallinarum为建立一种基于葡萄糖氧化酶(Gluco se o xidase,GOx)触发的酶 级联反应的快速检测S.pullorum and S.gallinarum的免疫比色法，利 用标记葡萄糖氧化酶/抗鸡白痢鸡伤寒沙门氏菌抗体双标记纳米二氧 化硅微球(GOx/anti-S.pullorum and S.gallinarum dual-labeled silica nano par ticles,GOx-IgG-SiNP s)作为检测探针用于信号转导；利用 IgG-MNP s作为捕获探针分离和富集目的菌；以免疫夹心法检测目的 菌S.pullorum and S.gallinarum o GOx可催化氧化葡萄糖形成葡萄糖 酸和H2O2,Fe?+被H2O2氧化成Fe3+,Fe?+能迅速与SCN形成Fe3+-SCN-络合物。此络合物可使溶液从无色变为红色。目的菌S.pullorum and S.gallinarum被IgG-MNP s捕获和富集，随后与GOx-IgG-SiNP s形成夹 心结构。夹心结构中的GOx可以触发上述酶级联反应产生Fe3+-SCK 络合物。吸光度值由酶标仪测量。吸光度值强度与S%and S ga/wn/m浓度的对数值成比例关系。在最优条件下，该比色法检测 S.pullorum and S.gallinarum 的线性范围为 8.4xl03 CFU-mL-1 至 8.4X 107 CFU-mL1,检测限为2.36x103 CFUmL/。由此可见，这是一种优IV浙江工商大学硕士学位论文良的检测S.pullorum and S.gallinarum的比色分析法。关键词：磁性纳米微球；二氧化硅纳米微球；信号转导；比色法;Salmonella pullorum and Salmonella gallinarumv浙江工商大学硕士学位论文RESEARCH OF DUAL-LABELED SILICA NANOP ARTICLESFOR COLORIMETRIC DETECTION OFSALMONELLA/ANTLSALMONELLAABSTRACTSalmonella is th e causative agent o f th e mo st co mmo n and h igh ly infectio us h uman disease.Th is co smo po litan path o gen is r espo nsible fo r a significant mo r bidity and mo r tality in h umans as well as animals.In additio n,th e do mestic and inter natio nal standar ds r equir e th at th e Salmonella sh o uld no t be detected in fo o d.Th er efo r e,it is par ticular ly impo r tant to establish better meth o ds fo r detectio n o f Salmonella and its antibo dy to meet th e demo nds fo r co ntr o lling th e co ntaminatio n o f path o genic bacter ia,and secur ing th e fo o d safety and th e h ealth o f livesto ck and po ultr y.At pr esent,th e detectio n o f Salmonella and its antibo dy ar e mainly include standar d detectio n meth o ds and r apid detectio n meth o ds.Standar d detectio n meth o ds ar e usually accur ate but cumber so me,time co nsuming,and no t eco no mical.Rapid detectio n VII浙江工商大学硕士学位论文meth o ds h ave develo ped r apidly and widely used in r ecent year s,such as fluo r escence,ch emiluminescence,electr o ch emical and co lo r imetr ic meth o d.Amo ng all th e r apid detectio n str ategies,co lo r imetr ic immuo assay h as been well r eco gnized as vital analytical tech niques cur r ently used due to th eir advantages in simplicity,lo w-co st,r apid signal gener atio n and easily r eado ut with th e naked eyes.Th e co lo r imetr ic immuno assay is based o n th e r eactio n between th e antigen and antibo dy,and it can be detected by th e co lo r ch ange.Th e r esults o f th e co lo r imetr ic immuno assay can be detected by th e naked-eyes o r with th e aid o f th e basic o ptical instr uments,wh ich h as alr eady been widely used in o n-site test.A key str ategy fo r design a co lo r imetr ic immuno assay is to explo it a no vel signal tr ansductio n path way,wh ich can make th e detectio n event co nver ted into co lo r ch ange.Enzyme linked immuno so r bent assay(ELISA)is o ne o f th e mo st co mmo n co lo r imetr ic immuno assay fo r detectio n o f path o gens and th eir ser um antibo dies because o f its many advantages,and th e signal tr ansductio n o f ELISA-based co lo r imetr ic immuno assay is ach ieved by th e enzyme labeled antibo dy and enzymatic substr ate.But its sh o r tco mings ar e as fo llo ws:th e pr o cess fo r pr epar atio n o f enzyme labeled antibo dy is co mplicated and may cause th e bio lo gical activity decr ease o f th e antibo dy and enzyme;th e pr o cess fo r antibo dy co ating,incubatio n and wash ing will co nsume a lo t o f time,and pr o ne to false po sitive r esults.VIII浙江工商大学硕士学位论文In o r der to impr o ve and so lve th e abo ve pr o blems,Salmonella pullorum and Salmonella gallinarum(S.pullorum and S.gallinarum)and its antibo dy wer e used as detectio n o bjects,signal tr ansductio n pr o bes including r eactive tur quo ise blue KNG do ped silica nano par ticles(Blue-SiNP s)and enzyme labeled silica nano par ticles co mbine with th e separ atio n and enr ich ment o f magnetic nano par ticles(MNP s)wer e used to design co lo r imetr ic immuno assays.Th e study is divided into th r ee par ts:1 A competitive inhibition colorimetric immunoassay for detection of antibodies against Salmonella based on blue silica nanoparticlesSalmonella infectio n can be indicated by th e level o f antibo dies against Salmonella pullorum and Salmonella gallinarum(anti-5.pullorum and S.gallinarum)in ser um.In o r der to establish a no vel co mpetitive inh ibitio n immuno assay fo r detectio n o f Salmonella infectio n,and th us to r educe th e co ntaminatio n o f po ultr y fo o d.Two kinds o f nano par ticles wer e synth esized and functio nalized in th is study:MNP s wer e pr epar ed by co pr ecipitatio n meth o d and co ated with silica sh ell,and th en functio nalized with antibo dies to fo r m anti-5,pullorum and S.gallinarum co njugated magnetic nano par ticles(IgG-MNP s),wh ich wer e emplo yed as captur e pr o bes fbr immo bilizing S.pullorum and S.gallinarum,Blue-SiNP s wer e synth esized by do ping r eactive tur quo ise blue KNG into silica nano par ticles using r ever se micr o emulsio n meth o d,IX浙江工商大学硕士学位论文and th en functio nalized with antibo dies to fo r m anti-5,pullorum and S.gallinarum co njugated blue silica nano par ticles(IgG-Blue-SiNP s),wh ich wer e emplo yed as detectio n pr o bes fo r signal tr ansductio n.In a typical detectio n pr o cess,IgG-MNP s wer e fir stly incubated with S.pullorum and S.gallinarum to fo r m IgG-MNP s-5.pullorum and S.gallinarum co mplexes,and subsequent added anti-S.pullorum and S.gallinarum po sitive ser um to co mpete with IgG-Blue-SiNP s fbr th e limited antigenic deter minants o n th e abo ve co mplexes.Th e sandwich str uctur es o f IgG-MNP-S.pullorum and S.ga/ZZnar ww-IgG-Blue-SiNP s wer e separ ated with a magnet and etch ed with NaOH to r elease r eactive tur quo ise blue KNG.Th e abso r bance o f r eactive tur quo ise blue KNG at 614 nm was inver sely r elated to th e co ncentr atio n o f anti-S.pullorum and S.gallinarum in th e po sitive ser um.Th e co lo r imetr ic immuno assay can be easily car r ied o ut o n a micr o plate r eader.Under th e o ptimal co nditio ns,th e detectio n limit o f anti-S.pullorum and S.gallinarum po sitive ser um dilutio n titr e was 1:480,wh ich was lo wer th an th at o btained fr o m natio nal standar d meth o d,and th e detectio n time was less th an 60 min.Th us,th is meth o d is simple,r apid,sensitive,specific,and h as gr eat po tential fbr applicatio n.2 Colorimetric sandwich immunoassay fbr detection of pullorum and S.gallinarum based on horseradish peroxidase loaded silica nanoparticlesRapid and sensitive detectio n o f path o gens is a key r equir ement fo r x浙江工商大学硕士学位论文bo th fo o d safety and clinical settings.In o r der to co nstr uct a excellent no vel r apid detectio n str ategy,IgG-MNP s wer e used as captur e pr o bes;HRP/anti-5.pullorum and S.gallinarum dual-labeled silica nano par ticles(HRP-IgG-SiNP s)wer e used as detectio n pr o bes.In th e pr esence o f S.pullorum and S.gallinarum,th e tar get bacter ia wer e captur ed by captur e pr o bes and detectio n pr o bes to fo r m sandwich str uctur es.And th is sandwich co mplexes wer e th en magnetically iso lated and used to catalytically o xidize th e substr ate 3,3,5,5-Tetr ameth ylbenzidine(TMB),after ter minated by H2so4,th e abso r bance o f th e gener ated yello w pr o ducts wer e r eco r ded by micr o plate r eader,and th e abso r bance intensity is pr o po r tio nal to th e lo gar ith m o f th e co ncentr atio n o f S.pullorum and S.gallinarum.Under th e o ptimized co nditio ns,th e co lo r imetr ic sandwich assay h ad a quantitative detectio n r ange fr o m 8.4x103 to 8.4x1 O,CFU-mL1 with a limit o f detectio n o f 1.7xl03 CFU-mL1.Th is appr o ach was demo nstr ated as a simple,co st-effective,sensitive,specific,and r apid meth o d to detect S.pullorum and S.gallinarum.3 Glucose oxidase triggered enzymatic cascade reaction coupled with Fe3+SCN-clathrate chromogenic system for colorimetric detection of S,pullorum and S.gallinarumIn o r der to develo p a gluco se o xidase(GOx)tr igger ed enzymatic cascade r eactio n-based co lo r imetr ic immuno assay fo r detectio n o f S.XI浙江工商大学硕士学位论文pullorum and S.gallinarumy GOx/anti-5.pullorum and S.gallinarum dual labeled silica nano par ticles(GOx-IgG-SiNP s)wer e used as detectio n pr o bes fbr signal tr ansductio n,IgG-MNP s wer e used as captur e pr o bes fbr separ ate and enr ich th e tar get bacter ia,and sandwich pr o to co l was utilized fbr detectio n o f S.pullorum and S.gallinarum.GOx can catalytically o xidized gluco se leading to th e fo r matio n o f gluco nic acid and H2O2.Acco mpanying th e gener atio n with H2O2,th e fer r o us ir o n(II)was co nver ted into th e fer r ic ir o n(III).Fe3+can r apidly r eact with th e po tassium th io cyanat to fo r m Fe3+-SCN-clath r ate.Th e r esulting Fe3+-SCN-clath r ate can cause th e co lo r o f th e so lutio n to ch ange fr o m near ly co lo r less to r ed.In a gener al detectio n pr o cedur e,th e tar get bacter ia S.pullorum and S.gallinarum wer e fir stly captur ed and enr ich ed by IgG-MNP s and th en sandwich ed by GOx-IgG-SiNP s.Th e car r ied GOx in th e sandwich str uctur es can tr igger ed abo ve enzymatic cascade r eactio n to pr o duce Fe3+-SCN*clath r ate.Th e abso r bance was r eco r ded by a micr o plate r eader.Th e abso r bance intensity is pr o po r tio nal to th e lo gar ith m o f th e co ncentr atio n o f S.pullorum and S.gallinarum.Under th e o ptimal co nditio ns,th e develo ped co lo r imetr ic immuno assay exh ibited a wide dynamic r ange o f 8.4xl038.4xl07 CFU-mL1 to war d S.pullorum and S.gallinarum with a detectio n limit o f 2.36x10 CFU-mL.Th us,th is newly develo ped meth o d was pr o ved to be an excellent co lo r imetr ic immuno assay fo r detectio n o f S.pullorum and S.gallinarum.XII浙江工商大学硕士学位论文KEYWORDS:magnetic nano par ticles;silica nano par ticles;signal tr ansductio n;co lo r imetr ic assay;Salmonella pullorum and Salmonella gallinarumxni浙江工商大学硕士学位论文目录摘要.IABSTRACT.VII第1章绪论.11.1 免疫标记技术.11.1.1 免疫荧光技术.11.1.2 放射免疫技术.21.2 酶免疫技术.21.2.1 ELISA中常用的酶及底物.21.2.2 ELISA技术类型.31.3 微纳米材料作为指示物与标记载体在免疫分析中的应用.31.3.1 金属纳米颗粒作为标记载体.41.3.2 碳纳米材料作为标记载体.51.3.3 高分子纳米微球作为标记载体.61.3.4 脂质体作为标记载体.61.3.5 磁性纳米微球作为标记载体.7136量子点作为标记载体.813.7稀 土发光材料作为标记载体.81.3.8 纳米二氧化硅作为标记载体.91.4 二氧化硅微球的合成.101.4.1 Sto ber.101.4.2 反相微乳液法.101.5 鸡白痢鸡伤寒沙门氏菌的危害及研究现状.101.6 以二氧化硅作为信号指示物的免疫比色法的构建.111.6.1 彩色纳米二氧化硅微球作为信号指示物.111.6.2 酶标记纳米二氧化硅微球作为信号指示物.121.7 论文研究意义、目的和创新性.121.7.1 研究意义.121.7.2 研究目的.131.7.3 创新性.13第2章 基于蓝色Si。2纳米微球的竞争抑制免疫比色法检测抗沙门氏菌抗体.152.1 实验部分.162.1.1 试剂与设备.162.1.2 活性翠兰KNG掺杂二氧化硅纳米微球的合成.172.1.3 抗体共价结合至Blue-SiNP s表面.172.1.4 Fe3O4磁性纳米颗粒的合成.182.1.5 核壳式磁性纳米微球的合成.182.1.6 抗体改性磁性纳米微球的制备.182.1.7 细菌菌株的制备.182.1.8 纳米微球的表征.192.1.9 竞争抑制免疫比色法检测步骤.192.2 结果和讨论.212.2.1 MNP s的制备和表征.21XV浙江工商大学硕士学位论文2.2.2 Blue-SiNP s的合成和表征.242.2.3 检测条件优化.252.2.4 竞争抑制免疫比色法对不同稀释滴度鸡白痢鸡伤寒阳性血清的检测灵敏度282.2.5 特异性.302.3 结论.31第3章 基于HRP标记型Si。?纳米微球免疫夹心比色法检测*5*.pullorum and S.gallinarum.333.1 实验部分.343.1.1 试剂与设备.343.1.2 SiNP s 的合成.343.1.3 Anti-S.pullorum and S.gallinarum 和 HRP 共价结合至 SiNP s 表面(HRP-IgG-SiNP s).353.1.4 Fe3O4磁性纳米颗粒的合成.353.1.5 MNP s 的合成.363.1.6 IgG-MNP s 的制备.363.1.7 细菌菌株的制备.363.1.8 纳米微球的表征.373.1.9 检测过程.373.2 结果与讨论.393.2.1 比色法测定 S.pullorum and S.gallinarum 基本原理.39322 MNP s和SiNP s合成与表征.393.2.3 条件优化.423.2.4 灵敏度.443.2.5 特异性.463.2.6 稳定性.473.2.7 实际样品的准确性试验.483.3 结论.49第4章葡萄糖氧化酶触发的酶级联反应结合Fe3+-SCN络合物显色体系比色检测S.pullorumand S.gallinarum.514.1 实验部分.524.1.1 试剂与设备.524.1.2 SiNP s 的合成.524.1.3 Anti-5,pullorum and S.gallinarum 和 GOx 共价结合到 SiNP s 表面(GOx-IgG-SiNP s).534.1.4 Fe3()4磁性纳米颗粒的合成.534.1.5 MNP s 的合成.534.1.6 IgG-MNP s 的制J备.544.1.7 细菌菌株的制备.544.1.8 纳米微球的表征.544.1.9 GOx触发酶促级联反应结合Fe-SCN-显色系统对S.pullorum and S.gallinarum 检测步骤.554.2 结果与讨论.564.2.1 检测的基本原理.564.2.2 MNP s和SiNP s的合成和表征.57XVI浙江工商大学硕士学位论文4.2.3 对照实验.57424检测条件优化.594.2.5 灵敏度.614.2.6 特异性.624.2.7 稳定性.634.2.8 实际样品检测适用性.644.3 结论.65第5章总结与展望.675.1 课题总结.675.1.1 基于蓝色SiO2纳米微球的竞争抑制免疫比色法检测抗沙门氏菌抗体.675.1.2 基于HRP标记型SiO2纳米微球免疫夹心比色法检测S.pullorum and S.gallinarum.675.1.3 葡萄糖氧化酶触发的的酶级联反应结合Fe3+=SCNT络合物显色体系比色检测Spullo rum and S.gallinarum.685.2 课题展望.685.2.1 寻找新的标记物载体.685.2.2 建立新的显色信号转导方式.685.2.3 扩大该新型比色免疫法的应用范围.695.2.4 快速检测试剂盒的开发.69参考文献.71附录.77感谢基金支持.79致谢.81独创性声明.83XVII浙江工商大学硕士学位论文第1章绪论目前，食品安全问题已成为全球关注的焦点。致病菌污染是最主要的危害因 素，其中，沙门氏菌常成为首要食源性致病菌。国内外标准规定，食品中不得检 出沙门氏菌。鸡白痢鸡伤寒沙门氏菌是危害养禽业和禽类食品安全的重要致病 菌。集新知识、新材料