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类型 基础研究 预答辩日期 2018-04-20
开始(开题)日期 2016-11-28 论文结束日期 2018-01-22
地点 化工老楼大会议室307 论文选题来源 国家自然科学基金项目     论文字数 7.4 (万字)
题目 基于功能型纳米材料的电化学发光免疫传感器的制备与应用
主题词 电化学发光,免疫传感器,纳米材料,双极电极,癌胚抗原
摘要 近年来,磁性纳米材料、量子点发光材料、催化纳米材料由于其独特的结构和性质受到广大科研工作者的关注。磁性纳米颗粒除了具备纳米材料普遍的性质—量子尺寸效应、表面效应及宏观量子隧道效应之外,还具备良好的磁导向性、生物相容性等特性,可用作磁性分离,因而常被应用于生物分析领域;半导体量子点因其具有较高的电化学发光(Electrochemiluminescence, ECL)效率,光学稳定性等优势,成为ECL分析检测方法中的首选标记物;而具有催化性能的纳米材料在生物分析领域占有持久性的优势,这主要因为它可以和生物体分泌的电活性物质相关联。本文旨在基于这些功能型纳米材料,制备高灵敏度、专一性好、准确度高的ECL免疫传感器。然而,将这些功能性纳米材料应用于免疫传感分析时,还具有一定的挑战性,主要体现在以下几方面:(1) 尽管磁性纳米材料可以通过磁性分离的方式避免孵育期间的非特异性免疫结合,但是由于其导电性并不是太好,应用在传感器中时,可能会影响到最终的灵敏度;(2) 尽管半导体材料的ECL发光性质很好,但是重金属Cd的存在会使生物蛋白变性,因而影响检测效果;(3) 现有的ECL免疫传感器多构建在玻碳电极上,需要技术人员仔细操作,设施成本较贵,不方便临床检测。因此,如何利用好这些材料的优点,弥补它们的不足,证实它们与蛋白之间的偶联效果,最终提高传感器的灵敏度都是需要我们深入研究的。本论文的内容主要包括以下四个部分,概括如下: (1) 以核-壳型CdTe@ZnS量子点作为发光标记物、Fe3O4@SiO2纳米粒子作为磁性分离基底,制备ECL夹心型免疫传感器。主要通过水热法,合成了一系列低毒的、不同核径的CdTe@ZnS量子点,比较了它们的ECL发光行为,由于量子点的ECL与其带隙、表面陷阱有关,故其ECL效率存在差异。将具有最高ECL发光量子产率的量子点选作二抗的标记物,通过琼脂糖凝胶电泳证明其成功偶联与否;同时将具有磁分离能力的、大的比表面积的、易生物官能化的Fe3O4@SiO2纳米粒子作为磁性载体固载一抗。在最优实验条件下,该ECL传感器针对癌胚抗原CEA具有较宽的线性检测范围0.01-125 ng mL-1,检测限为3.0 pg mL-1。而且,该传感器显示了极好的选择性、稳定性和重现性。 (2) 基于双稳定剂保护的CdTe量子点作为信号探针、Fe3O4-Au纳米复合物作为磁性分离载体的夹心免疫型ECL传感器。以3-巯基丙酸和六偏磷酸钠同时作为保护剂,制备CdTe量子点,并标记二抗。这是由于双稳定剂保护的量子点表面陷阱较少,表面钝化作用更加完全,相对于单保护剂的量子点ECL效率也就更高。磁性Fe3O4-Au纳米复合物作为基底固载一抗。对照实验表明,相对上个体系中使用的Fe3O4@SiO2,磁性Fe3O4-Au纳米复合物的导电性更好,可以显著的提高该ECL传感器的灵敏度。传感器的构建过程利用循环伏安、交流阻抗、透射电镜、X-射线能谱表征,最终针对CEA检测的线性范围是0.005-80 ng mL-1,检测限为1 pg mL-1。同时,该传感器也成功地应用于实样检测。 (3) 石墨碳纸基的双极电极传感平台。为了能更好的构建无线、低价、灵敏的生物检测平台,我们首次用石墨碳纸构建了一个闭合式的双极电极ECL传感平台。由于石墨纸具有极好的导电性,材质均一,可以通过剪裁得到理想的图案,所以可作合格的双极电极。该传感平台被用于检测氧化物H2O2和肿瘤标记物CEA。在分析H2O2时,Pt纳米粒子被电镀在双极电极的阴极端,用来催化H2O2还原,该传感器对H2O2的检测线性范围为0.001-15 mM,检测限为0.5 μM。在检测CEA时,壳聚糖-多壁碳管(CS-MWCNTs)被用来为固载一抗,为其提供一个亲水界面,Au@Pt纳米材料用来偶联二抗,作为还原H2O2的催化剂。在最优条件下,该传感器对CEA的检测范围为0.01-60 ng mL-1,检测限为5.0 pg mL-1。 (4) 具有高电催化活性的补丁金-Fe3O4纳米球应用于纸基双极电极适配体传感器。我们通过简单的吸附-还原方法,在水相中成功地合成了补丁金包覆的Fe3O4纳米球(PG-Fe3O4 NPs),并且进一步讨论了它的合成机理。该材料具有很好的磁性和催化H2O2电还原的性质。计时安培和i-t实验表明它具有高的催化速率常数3.13×10-5 M-1 s-1,较高的灵敏度578.87 μA mM-1 cm-2以及较低的米氏常数462 μM。同时,由于补丁金的引入,方便了其生物官能化。在这里,用巯基修饰的适配体固载在补丁金部分作为信号探针来检测CEA。并且构建了一个相关的纸基双极电极适配体传感器。为了提高该传感器的性能,Au纳米枝晶也首次电镀在双极电极阴极上方的多孔纸中,用来偶联另一个适配体。最终该传感器对CEA的检测线性范围为0.1 pg mL-1-15 ng mL-1,检测限为0.03 pg mL-1,证实了PG-Fe3O4具有很好的催化性能,可用作生物分析领域。 综上所述,本论文基于不同功能型的纳米材料,成功构建了四种ECL免疫传感器。同时,还制备出一种新型纳米材料,并将其应用于生物分子的定量分析中。
英文题目 Preparation and application of electrochemiluminescence immunosensor based on functional nanomaterials
英文主题词 Electrochemiluminescence,immunosensor,nanomaterials,bipolar electrode, carcinoembryonic antigen
英文摘要 Recently, magnetic nanomaterials, quantum dots, catalytic nanomaterials have gained increasing attention because of their unique structure and properties. Besides the universal nature of nanomaterial (like quantum size effect、surface effect and macroscopic quantum tunneling effect), magnetic nanoparticles possess other properties such as magnetic guidance and good biocompatibility, and thus are often employed in bioassay; semiconductor quantum dots are viewed as candidates of Ab2 label owing to their high ECL efficiency; The nanomaterials with catalytic activity have overwhelming superiority, mainly for their relationship with electroactive molecules released from organism. The target of this thesis is to construct sensitive and selective ECL immunosensors with these functional nanomaterials. However, there are still some challenges: (1) Although the magnetic property could facilitate the wash process during immune recognition and avoid nonspecific adsorption, the conductivity of these materials is not good enough, which will affect the final sensitivity of the immunosensor. (2) The ECL behavior of semiconductor quantum dots is excellent, but the existence of Cd will make the protein denatured easily and thereby influence the results; (3) Now, most of the ECL immunosensors are fabricated on the glassy carbon electrode, the incubation process needs specialists, and the instrument is expensive. Thus how to utilize the advantages of these materials, make up for the deficiency, confirm the interaction between these nanoparticles and protein, and improve the sensitivity are our research content. In brief, this thesis includes four parts as followed. (1) General strategy to fabricated Electrochemiluminescence sandwich-type nanoimmunosensors using CdTe@ZnS as luminescent labels and Fe3O4@SiO2 nanoparticles as magnetic separable scaffolds. A series of low-toxic CdTe@ZnS QDs with different core diameters are prepared via hydrothermal method. And their ECL behaviors are compared. The difference of the ECL efficiency mainly comes from their bandgaps and surface trap. The QDs with best ECL performance are used as label to conjugate with detection antibody (Ab2), and the successful conjugation is verified by running agarose gel electrophoresis. Meanwhile, Fe3O4@SiO2 nanoparticles with large surface area and good magnetic performance are employed to immobilize the capture antibody (Ab1). Under the optimum condition, this ECL immunosensor achieves a wide linear range of 0.01-125 ng mL-1 with a detection limit of 3.0 pg mL-1. Also, this immunosensor displays good selectivity, stability and reproducibility. (2) Sandwich-structured electrogenerated chemiluminescence immunosensor based on dual-stabilizers-capped CdTe quantum dots as signal probes and Fe3O4-Au nanocomposites as magnetic separable carriers. Dual-stabilizers-capped CdTe quantum dots are synthesized with 3-mercaptopropionic acid (MPA) and sodium hexametaphosphate (HMP) as cappers, and used to conjugate with Ab2. The ECL quantum efficiency of these QDs is high due to the less surface traps and high-passivated surface. Fe3O4-Au nanocomposites are used to immobilize the Ab1 for the better electro-conductivity than previous Fe3O4@SiO2. The successful fabrication of the immunosensor is confirmed by CV, EIS, TEM and EDS. For CEA detection, the linear range is 0.005-80 ng mL-1, and the detection limit is 1 pg mL-1. Meanwhile, this sensor is also applied in real sample. (3) Graphite paper-based bipolar electrode (BPE) electrochemiluminescence sensing platform. Graphite paper is firstly used as BPE for its good conductivity, uniform composition, and ease of operation. This platform is used for H2O2 and CEA detection. For H2O2 detection, Pt NPs are electrodeposited at BPE cathode to catalyze the reduction of H2O2. The linear range is 0.001-15 mM and the detection limit is 0.5 μM. For CEA detection, CS-MWCNTs are utilized to supply a hydrophilic platform for the immobilization of Ab1. Au@Pt nanostructures are employed to conjugate with Ab2 as the catalyst of H2O2 reduction in the cathodic cell. Under the optimum condition, this immunosensor achieves wide linear range of 0.01-60 ng mL-1 with a low detection limit of 5.0 pg mL-1. (4) Patchy gold coated Fe3O4 nanospheres with enhanced catalytic activity applied for paper-based bipolar electrode-Electrochemiluminescence aptasensors. In this part, patchy gold coated Fe3O4 nanospheres (PG-Fe3O4) are synthesized for the first time in aqueous solution via a simple adsorption-reduction process. The formation mechanism is discussed detail. This nanomaterial possesses good magnetic property and catalytic activity toward H2O2 electro-reduction. Chronoamperometric and amperometric experiments indicate a relatively high catalytic rate constant of 3.13×105 M-1s-1, a high sensitivity of 578.87 μA·mM-1·cm-2 and a low Michaelis-Menten constant of 462 μM. The introduction of patchy gold facilitates the biofunctionalization. Thiol-terminated aptamers are immobilized onto the patchy gold part as signal probe to detect CEA. A related paper-based BPE aptasensor is fabricated. To enhance the sensitivity of the sensor, Au nanodendrites are electrodeposited at BPE cathode in the porous paper. The aptasensor achieves wide linear range of 0.1 pg mL-1-15 ng mL-1 with detection limit of 0.03 pg mL-1, suggesting the good catalytic activity of PG-Fe3O4 and their potential application in bioassay. In summary, this thesis introduces four ECL immunosensor based on different functional nanomaterials. One novel nanomaterial is applied in biosensors.
学术讨论
主办单位时间地点报告人报告主题
丁收年课题组 2016年12月2日 化工院319会议室 张鑫及课题组同学 组会工作汇报(一)
丁收年课题组 2016年12月15日 化工院319会议室 张鑫及课题组同学 组会工作汇报(二)
丁收年课题组 2016年12月30日 化工院319会议室 张鑫及课题组同学 组会工作汇报(三)
丁收年课题组 2017年4月22日 化工院319会议室 张鑫及课题组同学 组会工作汇报(四)
丁收年课题组 2017年6月3日 化工院319会议室 课题组同学 组会工作汇报(五)
丁收年课题组 2017年6月18日 化工院319会议室 课题组同学 组会工作汇报(六)
化学化工学院 2017年12月12日 化工院319会议室 鲍宁教授 一次性纸基电化学检测装置及其应用
化学化工学院 2017年12月12日 化工院319会议室 张庆研究员 消费品中有害物质检测技术及标准化
     
学术会议
会议名称时间地点本人报告本人报告题目
国家自然科学基金委员会化学科学部:2016全国生命分析化学学术大会 2016年12月16-19日 南京国展中心 基于核壳结构CdSe@ZnSe量子点和Fe3O4@SiO2磁性载体的电化学发光免疫传感器
瑞典化学会分析化学部:2017年第19届欧洲分析化学会议 2016年12月16-19日 瑞典斯德哥尔摩大学 Sandwich-structured Electrochemiluminescence immunosensor based on CdSe@ZnSe quantum dots as signal probes and Fe3O4@SiO2 nanoparticles as magnetic separable scaffolds
     
代表作
论文名称
General Strategy to Fabricate Electrochemiluminescence Sandwich-Type nanoimmunosensors
Sandwich-structured electrogenerated chemiluminescenceimmunosensor based on dual-stabilizers-capped
Graphite paper-based bipolar electrode electrochemiluminescence sensing
Facile and large-scale synthesis of green-emitting carbon nanodots from aspartame and the applicatio
 
答辩委员会组成信息
姓名职称导师类别工作单位是否主席备注
徐静娟 正高 教授 博导 南京大学
张庆 正高 研究员 博导 中国检验检疫科学研究院
孙岳明 正高 教授 博导 东南大学
刘松琴 正高 教授 博导 东南大学
周建成 正高 教授 博导 东南大学
      
答辩秘书信息
姓名职称工作单位备注
卫伟 正高 教授 东南大学