基于复合纳米材料组装的信号增强的电化学免疫传感器的研究
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摘要
电化学免疫传感器是一种将电化学分析方法与免疫学技术相结合而发展起来的具有快速、灵敏、选择性高、操作简便等特点的生物传感器。免疫传感器主要由生物识别系统(感受器)和换能器组成。将抗原或者抗体分子固定到换能器表面称为传感器敏感界面的构建,这是免疫传感器的研究和开发中最为重要和关键的步骤之一。本文从功能化复合纳米材料的制备,传感器敏感界面的构建以及新型信号增强的免疫传感器的研制等方面进行了探索和研究。本文主要分为以下几个部分:
第一章综述简要概述了现代免疫分析方法,详细介绍了免疫生物传感器的基本原理和敏感界面的固定化技术,着重评述了多种纳米材料在生物传感器方面的应用;详细介绍了电化学信号增强的免疫传感器的基本策略和信号增强的新型电化学免疫传感器的研究进展。
第二章由于抗原抗体蛋白均为非电活性物质,因此电化学免疫传感器一般需要对抗体或抗原进行生物酶或者电活性物质的标记,从而实现把抗体和抗原结合信息转变为可测的电化学信息。然而标记步骤复杂、耗时,且标记过程可能会影响免疫蛋白的特异性位点,致使生物活性丧失。基于自组装技术和纳米技术,制各了染料硫堇包覆的SiO_2复合纳米粒子,再结合具有比表面积大、吸附力强、生物亲和性好等优点的金纳米粒子,研制了纳米金/硫堇-SiO_2/纳米金复合膜修饰的电流型癌胚抗原(CEA)免疫传感器。值得注意的是,基于硫堇固有的电化学活性,该硫堇-SiO_2复合纳米粒子修饰电极呈现出良好的氧化还原活性,其可以起到氧化还原探针的作用,用以指示免疫反应发生的进程。从而无需在测试溶液中加入其他氧化还原探针,进一步简化操作步骤,构建了无试剂型免疫传感器。与传统的免疫物质测试方法相比,该方法具有无需对生物分子进行标记,不需竞争或者夹心反应,测试液中亦不需加入电活性物质,只需在传感器表面进行常规免疫反应后,将其转移至测量池中测量响应电流信号即可,具有操作简单、响应快、特异性强等特点。
第三章以具有独特的电学和光学特性的有机半导体材料苝四甲酸二酐(PTCDA,3,4,9,10-perylenetetracarboxylic dianhydride)为原料,通过两步合成法制备了二茂铁基修饰的新型成膜材料(PTC-Fc)。PTC-Fc具有导电性好,氧化还原活性强,易于成膜且比表面积大、表面活性位点多等优点,是一种理想的电极修饰材料。结合有机相合成和相转移技术制备的带正电荷的金纳米粒子构建了电流型CEA免疫传感器。正电荷金纳米粒子的引入不仅牢固的固定了抗体蛋白,很好的保持了抗体蛋白的生物活性,同时与纳米多孔材料PTC-Fc形成镶嵌结构,起到了纳米导线的作用,加快了PTC-Fc的氧化还原电子转移速率。该传感器具有制备简单、灵敏度高、稳定性好、响应时间短等特点。
第四章酶免疫传感器将酶的化学放大作用与免疫物质的专一性识别相结合,兼有电化学分析的灵敏性和免疫反应的特异性,为进一步提高免疫传感器的灵敏度,设计了免去传统酶标操作的新型多层酶修饰的信号增强的电流型免疫传感器。以牛血清白蛋白(BSA)为固酶基质,通过交联作用固载HRP以提高酶膜的稳定性,基于联吡啶钴(Co(bpy)_3~(3+))与BSA之间的静电作用和疏水作用,Co(bpy)_3~(3+)能够进入BSA-HRP复合膜中形成的具有电化学活性和生物相容性的复合基质。固载抗体后又提出了使用HRP封闭免疫电极上的非特异性吸附位点,并同时利用HRP的生物催化放大作用放大响应电流信号,进而提高免疫传感器的灵敏度的新方法。经实验研究证明,该方法操作简单,切实可行,大大提高了免疫生物传感器的灵敏度。
第五章磁性纳米粒子具有易于磁性分离的特点,常用于标记免疫蛋白分子,构建可多次再生的免疫传感器。本研究中合成了以磁性纳米颗粒Fe_3O_4为核,普鲁士蓝(PB)为中间层并作为电活性物质,Au为外壳的多层结构的功能化磁性纳米颗粒(Au-PB-Fe_3O_4),该多层结构纳米颗粒兼有在外磁场中可分离性以及生物分子快速固定化等特点,且由于中间层PB的引入,其还具有良好的氧化还原电活性。同时,将辣根过氧化物酶(HRP)、葡萄糖氧化酶(GOD)和蛋白抗体同时标记到该纳米粒子表面。该多功能生物分子标记材料具有生物亲和性、催化活性、电化学活性和易分离等优点。基于夹心免疫反应模式,借助生物标记的三层纳米粒子的多重电化学催化作用可显著放大响应电流信号,构建了超灵敏易再生的电化学免疫传感器。
第六章在敏感界面的构建方面:将碳纳米管用惰性蛋白BSA分散,制得BSA包覆的功能化CNT复合材料修饰到金电极表面,利用BSA的-NH_2残基吸附小粒径的纳米金颗粒。通过恒电位沉积法,以小粒径的纳米金颗粒为晶种,在HAuCl_4溶液中电沉积纳米金,制得具有高比表面积、强吸附作用和良好的生物亲和性的DpAu-CNT纳米复合膜用以固载抗体蛋白。在信号检测模式方面,设计了进一步增强电流响应信号,提高电极灵敏度的信号放大新策略:首先制备了具有稳定性好、阻抗高等特点的聚合物Nafion(Nf)修饰的纳米SiO_2粒子,以此标记二抗蛋白。当采用夹心免疫反应模式时,由于Nf-SiO_2复合纳米粒子在电极敏感界面的引入,能显著阻碍电子的传输,使电极的有效截面积大大减少,从而起到放大响应信号的作用,进而提高传感器灵敏度。与直接免疫反应的方法相比,以二抗标记的Nf-SiO_2纳米复合物为信号增强手段的夹心免疫反应模式具有更宽的线性范围,高的灵敏度和强的特异性。
Electrochemically based immunosensors are valuable analytical tools for monitoring of the antibody or antigen as the advantages of short response time,high sensitivity, high specificity and easy manipulation,which combined the merits of electrochemical technology and immunoassay.An immunosensor is an analytical device that responds to a suitable combination of a biological recognition system and an electrochenmical transducer,thus the platforms construction of the antigen or antibody biomolecules immobilization have been the vital step in successful development of.an immunosensor. Therefore,this research focuses on the preparation of multi-functionalized nanomaterials,the construction of the immunoreaction interface and the development of sensitivity enhancement electrochemical immunosensors.The detail contents are as follows:
1.In the review section,after general introduction of immunoassay including its principle and the construction of immunoreaction interface,the application of nanotechnology into immunosensors was highlighted.Moreover,the research development of immunosensors based on novel signal amplificatory strategy was presented.Finally,the work and significance of this thesis was briefly introduced.
2.Since most immune protein are not intrinsically able to act as redox partners in an electrochemical reaction,most amperometric immunoassay techniques are relied on the label of either antigen or antibody,which requires highly qualified personnel, tedious assay time,or Sophisticated instrumentation.In this experiment,we described a new strategy for the development of a novel reagentless amperometric immunosensor based on the carcinoembryonic antibody(anti-CEA) molecule that was coupled at high density to sandwich(nano-Au/SiO_2@Thionine/nano-Au) nanocomposite layers for efficient carcinoembryonic antigen(CEA) recognition. Herein,the immobilized SiO_2@Thionine may act as electrochemical redox probe to monitor the variety of the kinetic-barrier of the electrode interface.Thus,the detection mode is based on the change of the current response before and after the specific binding of anti-CEA to CEA,due to the immunocomplex inhibiting the access of redox probe to electrode.The assay format avoids the label of antibody or antigen,the competitive or sandwich formats,the addition of an electron transfer mediator to the solution and the separation step,This significantly simplifies the immunoassay,procedure and shortens assay times.
3.This work describes a two-step conjugate synthesis of a porous organometallic nanostructured materials composed of ferrocenemonocarboxylic(Fc-COOH)and 3,4,9,10-perytenetetracarboxilic diarthydride.(PTCDA) and then a reagentless amperometric immunosensor prepared with positively charged gold nanoparticles (PGN) immobilized in this nanostructure Conductive film is developed.This nanostructured material comtaining ferrocenyt(PTC-Fc) coutd easily form stable film on the electrode surface with efficient redox-activity and excellent conductivity. Furthermore,with the negatively charged surface,this film can be used as an interface to adsorb the PGN,which were prepared in-organic solvents at relatively high concentrations with improved monodispersity compared to those prepared in aqueous solution.The presence of PGN provided a congenial microenvironment for adsorbed biomolecules and decreased the electron transfer impedance.Thus,with anti-CEA as a model antibody,the proposed immunosensor showed rapid and highly sensitive amperometric response to CEA with acceptable preparation reproducibility and stability.
4.Although the label-free immunosensors are able to detect the physical changes during the immune complex formation,whereas the enzyme-labeled imrnunosensors use signal-generating labels which allow more sensitive and versatile detection modes when incorporated into the complex.Thus,a concept based on a novel redox-biocompatible composite protein membrane fabrication,double enzyme membrane modification technique and antibody immobilization,was exploited to develop a highly sensitive amperometric enzyme immunosensor for detection of CEA. In this concept,a solution of bovine serum albumin(BSA)Containing horseradish peroxidase(HRP) is coated on the gold electrode in such a way that the first enzyme membrane is achieved.Then Co(bpy)_3~(3+),as a redox probes,was embedded in BSA-HRP composite membrane vis the electrostatic force and hydrophobe functions. Moreover,the new strategy of the employment of HRP to block the possible remaining active sites and amplify the response of the antigen-antibody reaction was proposed.The immunosensor constructed with the double layer biocatalytic HRP membranes and the desirable Co(bpy)_3~(3+)/BSA redox-biocompatible composite membrane performed high sensitivity and a wide linear response to CEA,as well as good Stability and long-term life.
5.With their unique property-superparamagnetism,magnetic nanoparticles have become attractive for exploitation mainly in biology and medicine because they can simplify the process of proteins immobilization and separation.In this work,the preparation,characterization and application of a three-layer magnetic nanoparticle composed of a Fe_3O_4 magnetic core,a Prussian Blue(PB) interlayer and a gold shell (it can be abbreviated as Au-PB-Fe_3O_4) for an ultrasensitive and reproducible electrochemical immunosensing fabrication was described for the first time.With the employment of the Au-PB-Fe_3O_4 nanoparticle,a new signal amplification strategy was developed based on bienzyme(horseradish peroxidase and glucose oxidase) functionalized Au-PB-Fe_3O_4 nanoparticles for an electrochemical immunosensing fabrication by using CEA andα-fetoprotein(AFP) as model systems,respectively. The experiment results show that the multilabeled Au-PB-Fe_3O_4 nanoparticles exhibit satisfying redox electrochemical activity and highly enzyme catalysis activity,which predetermines their utility in high sensitivity antibody detection schemes. Furthermore,this immunosensor could be regenerated,by simply using an external magnetic field which ensured a reproducible immunosensor with high sensitivity.
6.In this work,the multi-walledcarbon nanotubes(CNTs) were individually dispersed in an aqueous solution by a kind of natural proteins,bovine serum albumin(BSA),to obtain BSA molecules coated CNTs(BSA-CNTs).Then the gold colloids(nano-Au) were absorbed on the BSA-CNTs surface by the amido and disulfide groups of BSA. Later,a functionalized gold/carbon nanotube composite nanohybrid (DpAu/nano-Au/BSA-CNTs) modified electrode was developed based on electrochemical deposition of Au~(3+) onto nano-Au/BSA-MWNTs surface.Thus,a sensitive immunosensor for carbohydrate antigen 19-9(CA19-9) based on antibody immobilization on a functionalized gold/carbon nanotube composite nanohybrid has been constructed with the amplification response of antigen-antibody by the employment of Nation-coated SiO_2 nanoparticles labeled secondary antibody based on a sandwich immunoassay.More importantly,the special biomolecule-binding can not only cause the construction of the dielectric immunocomptex layer but also introduce the insulated Nation coated SiO_2 nanoparticles which demonstrate the relatively high resistance,resulting in a strong detection signal.Moreover,the extremely high stability of the functionalized gold/carbon nanotube composite nanohybrid monolayer allows the designed biosensing interface to obtain a good stability and long-term life.
第一章综述简要概述了现代免疫分析方法,详细介绍了免疫生物传感器的基本原理和敏感界面的固定化技术,着重评述了多种纳米材料在生物传感器方面的应用;详细介绍了电化学信号增强的免疫传感器的基本策略和信号增强的新型电化学免疫传感器的研究进展。
第二章由于抗原抗体蛋白均为非电活性物质,因此电化学免疫传感器一般需要对抗体或抗原进行生物酶或者电活性物质的标记,从而实现把抗体和抗原结合信息转变为可测的电化学信息。然而标记步骤复杂、耗时,且标记过程可能会影响免疫蛋白的特异性位点,致使生物活性丧失。基于自组装技术和纳米技术,制各了染料硫堇包覆的SiO_2复合纳米粒子,再结合具有比表面积大、吸附力强、生物亲和性好等优点的金纳米粒子,研制了纳米金/硫堇-SiO_2/纳米金复合膜修饰的电流型癌胚抗原(CEA)免疫传感器。值得注意的是,基于硫堇固有的电化学活性,该硫堇-SiO_2复合纳米粒子修饰电极呈现出良好的氧化还原活性,其可以起到氧化还原探针的作用,用以指示免疫反应发生的进程。从而无需在测试溶液中加入其他氧化还原探针,进一步简化操作步骤,构建了无试剂型免疫传感器。与传统的免疫物质测试方法相比,该方法具有无需对生物分子进行标记,不需竞争或者夹心反应,测试液中亦不需加入电活性物质,只需在传感器表面进行常规免疫反应后,将其转移至测量池中测量响应电流信号即可,具有操作简单、响应快、特异性强等特点。
第三章以具有独特的电学和光学特性的有机半导体材料苝四甲酸二酐(PTCDA,3,4,9,10-perylenetetracarboxylic dianhydride)为原料,通过两步合成法制备了二茂铁基修饰的新型成膜材料(PTC-Fc)。PTC-Fc具有导电性好,氧化还原活性强,易于成膜且比表面积大、表面活性位点多等优点,是一种理想的电极修饰材料。结合有机相合成和相转移技术制备的带正电荷的金纳米粒子构建了电流型CEA免疫传感器。正电荷金纳米粒子的引入不仅牢固的固定了抗体蛋白,很好的保持了抗体蛋白的生物活性,同时与纳米多孔材料PTC-Fc形成镶嵌结构,起到了纳米导线的作用,加快了PTC-Fc的氧化还原电子转移速率。该传感器具有制备简单、灵敏度高、稳定性好、响应时间短等特点。
第四章酶免疫传感器将酶的化学放大作用与免疫物质的专一性识别相结合,兼有电化学分析的灵敏性和免疫反应的特异性,为进一步提高免疫传感器的灵敏度,设计了免去传统酶标操作的新型多层酶修饰的信号增强的电流型免疫传感器。以牛血清白蛋白(BSA)为固酶基质,通过交联作用固载HRP以提高酶膜的稳定性,基于联吡啶钴(Co(bpy)_3~(3+))与BSA之间的静电作用和疏水作用,Co(bpy)_3~(3+)能够进入BSA-HRP复合膜中形成的具有电化学活性和生物相容性的复合基质。固载抗体后又提出了使用HRP封闭免疫电极上的非特异性吸附位点,并同时利用HRP的生物催化放大作用放大响应电流信号,进而提高免疫传感器的灵敏度的新方法。经实验研究证明,该方法操作简单,切实可行,大大提高了免疫生物传感器的灵敏度。
第五章磁性纳米粒子具有易于磁性分离的特点,常用于标记免疫蛋白分子,构建可多次再生的免疫传感器。本研究中合成了以磁性纳米颗粒Fe_3O_4为核,普鲁士蓝(PB)为中间层并作为电活性物质,Au为外壳的多层结构的功能化磁性纳米颗粒(Au-PB-Fe_3O_4),该多层结构纳米颗粒兼有在外磁场中可分离性以及生物分子快速固定化等特点,且由于中间层PB的引入,其还具有良好的氧化还原电活性。同时,将辣根过氧化物酶(HRP)、葡萄糖氧化酶(GOD)和蛋白抗体同时标记到该纳米粒子表面。该多功能生物分子标记材料具有生物亲和性、催化活性、电化学活性和易分离等优点。基于夹心免疫反应模式,借助生物标记的三层纳米粒子的多重电化学催化作用可显著放大响应电流信号,构建了超灵敏易再生的电化学免疫传感器。
第六章在敏感界面的构建方面:将碳纳米管用惰性蛋白BSA分散,制得BSA包覆的功能化CNT复合材料修饰到金电极表面,利用BSA的-NH_2残基吸附小粒径的纳米金颗粒。通过恒电位沉积法,以小粒径的纳米金颗粒为晶种,在HAuCl_4溶液中电沉积纳米金,制得具有高比表面积、强吸附作用和良好的生物亲和性的DpAu-CNT纳米复合膜用以固载抗体蛋白。在信号检测模式方面,设计了进一步增强电流响应信号,提高电极灵敏度的信号放大新策略:首先制备了具有稳定性好、阻抗高等特点的聚合物Nafion(Nf)修饰的纳米SiO_2粒子,以此标记二抗蛋白。当采用夹心免疫反应模式时,由于Nf-SiO_2复合纳米粒子在电极敏感界面的引入,能显著阻碍电子的传输,使电极的有效截面积大大减少,从而起到放大响应信号的作用,进而提高传感器灵敏度。与直接免疫反应的方法相比,以二抗标记的Nf-SiO_2纳米复合物为信号增强手段的夹心免疫反应模式具有更宽的线性范围,高的灵敏度和强的特异性。
Electrochemically based immunosensors are valuable analytical tools for monitoring of the antibody or antigen as the advantages of short response time,high sensitivity, high specificity and easy manipulation,which combined the merits of electrochemical technology and immunoassay.An immunosensor is an analytical device that responds to a suitable combination of a biological recognition system and an electrochenmical transducer,thus the platforms construction of the antigen or antibody biomolecules immobilization have been the vital step in successful development of.an immunosensor. Therefore,this research focuses on the preparation of multi-functionalized nanomaterials,the construction of the immunoreaction interface and the development of sensitivity enhancement electrochemical immunosensors.The detail contents are as follows:
1.In the review section,after general introduction of immunoassay including its principle and the construction of immunoreaction interface,the application of nanotechnology into immunosensors was highlighted.Moreover,the research development of immunosensors based on novel signal amplificatory strategy was presented.Finally,the work and significance of this thesis was briefly introduced.
2.Since most immune protein are not intrinsically able to act as redox partners in an electrochemical reaction,most amperometric immunoassay techniques are relied on the label of either antigen or antibody,which requires highly qualified personnel, tedious assay time,or Sophisticated instrumentation.In this experiment,we described a new strategy for the development of a novel reagentless amperometric immunosensor based on the carcinoembryonic antibody(anti-CEA) molecule that was coupled at high density to sandwich(nano-Au/SiO_2@Thionine/nano-Au) nanocomposite layers for efficient carcinoembryonic antigen(CEA) recognition. Herein,the immobilized SiO_2@Thionine may act as electrochemical redox probe to monitor the variety of the kinetic-barrier of the electrode interface.Thus,the detection mode is based on the change of the current response before and after the specific binding of anti-CEA to CEA,due to the immunocomplex inhibiting the access of redox probe to electrode.The assay format avoids the label of antibody or antigen,the competitive or sandwich formats,the addition of an electron transfer mediator to the solution and the separation step,This significantly simplifies the immunoassay,procedure and shortens assay times.
3.This work describes a two-step conjugate synthesis of a porous organometallic nanostructured materials composed of ferrocenemonocarboxylic(Fc-COOH)and 3,4,9,10-perytenetetracarboxilic diarthydride.(PTCDA) and then a reagentless amperometric immunosensor prepared with positively charged gold nanoparticles (PGN) immobilized in this nanostructure Conductive film is developed.This nanostructured material comtaining ferrocenyt(PTC-Fc) coutd easily form stable film on the electrode surface with efficient redox-activity and excellent conductivity. Furthermore,with the negatively charged surface,this film can be used as an interface to adsorb the PGN,which were prepared in-organic solvents at relatively high concentrations with improved monodispersity compared to those prepared in aqueous solution.The presence of PGN provided a congenial microenvironment for adsorbed biomolecules and decreased the electron transfer impedance.Thus,with anti-CEA as a model antibody,the proposed immunosensor showed rapid and highly sensitive amperometric response to CEA with acceptable preparation reproducibility and stability.
4.Although the label-free immunosensors are able to detect the physical changes during the immune complex formation,whereas the enzyme-labeled imrnunosensors use signal-generating labels which allow more sensitive and versatile detection modes when incorporated into the complex.Thus,a concept based on a novel redox-biocompatible composite protein membrane fabrication,double enzyme membrane modification technique and antibody immobilization,was exploited to develop a highly sensitive amperometric enzyme immunosensor for detection of CEA. In this concept,a solution of bovine serum albumin(BSA)Containing horseradish peroxidase(HRP) is coated on the gold electrode in such a way that the first enzyme membrane is achieved.Then Co(bpy)_3~(3+),as a redox probes,was embedded in BSA-HRP composite membrane vis the electrostatic force and hydrophobe functions. Moreover,the new strategy of the employment of HRP to block the possible remaining active sites and amplify the response of the antigen-antibody reaction was proposed.The immunosensor constructed with the double layer biocatalytic HRP membranes and the desirable Co(bpy)_3~(3+)/BSA redox-biocompatible composite membrane performed high sensitivity and a wide linear response to CEA,as well as good Stability and long-term life.
5.With their unique property-superparamagnetism,magnetic nanoparticles have become attractive for exploitation mainly in biology and medicine because they can simplify the process of proteins immobilization and separation.In this work,the preparation,characterization and application of a three-layer magnetic nanoparticle composed of a Fe_3O_4 magnetic core,a Prussian Blue(PB) interlayer and a gold shell (it can be abbreviated as Au-PB-Fe_3O_4) for an ultrasensitive and reproducible electrochemical immunosensing fabrication was described for the first time.With the employment of the Au-PB-Fe_3O_4 nanoparticle,a new signal amplification strategy was developed based on bienzyme(horseradish peroxidase and glucose oxidase) functionalized Au-PB-Fe_3O_4 nanoparticles for an electrochemical immunosensing fabrication by using CEA andα-fetoprotein(AFP) as model systems,respectively. The experiment results show that the multilabeled Au-PB-Fe_3O_4 nanoparticles exhibit satisfying redox electrochemical activity and highly enzyme catalysis activity,which predetermines their utility in high sensitivity antibody detection schemes. Furthermore,this immunosensor could be regenerated,by simply using an external magnetic field which ensured a reproducible immunosensor with high sensitivity.
6.In this work,the multi-walledcarbon nanotubes(CNTs) were individually dispersed in an aqueous solution by a kind of natural proteins,bovine serum albumin(BSA),to obtain BSA molecules coated CNTs(BSA-CNTs).Then the gold colloids(nano-Au) were absorbed on the BSA-CNTs surface by the amido and disulfide groups of BSA. Later,a functionalized gold/carbon nanotube composite nanohybrid (DpAu/nano-Au/BSA-CNTs) modified electrode was developed based on electrochemical deposition of Au~(3+) onto nano-Au/BSA-MWNTs surface.Thus,a sensitive immunosensor for carbohydrate antigen 19-9(CA19-9) based on antibody immobilization on a functionalized gold/carbon nanotube composite nanohybrid has been constructed with the amplification response of antigen-antibody by the employment of Nation-coated SiO_2 nanoparticles labeled secondary antibody based on a sandwich immunoassay.More importantly,the special biomolecule-binding can not only cause the construction of the dielectric immunocomptex layer but also introduce the insulated Nation coated SiO_2 nanoparticles which demonstrate the relatively high resistance,resulting in a strong detection signal.Moreover,the extremely high stability of the functionalized gold/carbon nanotube composite nanohybrid monolayer allows the designed biosensing interface to obtain a good stability and long-term life.
引文
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