典型激素类污染物检测的双通道ICA与ECL-RET传感技术研究

发布时间：2018-08-30 18:20

【摘要】：兽药残留问题不仅严重威胁人们的身体健康,进入环境影响生态,而且还严重影响我国的进出口贸易。近几年我国在农副产品贸易中因残留物超标而导致的外贸经济损失每年高达数百亿美元。食品生产过程中兽药的非法、不合理使用是造成这种现状的重要因素。因而,面对我国食品检测量大,覆盖面广的问题,研发样品前处理简单、快速特异检测方法是形成食品安全监测能力的重要保障之一。本论文以兽药残留物己烯雌酚(DES)和雌二醇(E2)为靶标,抗体为识别分子,免疫纳米颗粒为信号元件,针对免疫层析色谱(ICA)快速检测技术的检测通量较低的不足,探索了双通道免疫层析试纸技术,实现了两种目标物在一个免疫层析色谱上的同时检测分析,且操作简单,响应迅速,为多元快速检测的ICA方法提供了技术支持;为了进一步提高检测灵敏度,引入“turn-on”模式的生物传感技术,制备了新型光谱可调的双功能磁性纳米颗粒,并在此基础上建立了灵敏检测雌二醇的电化学发光共振能量转移传感技术,实现了痕量残留的检测,为电化学发光共振能量转移技术在食品安全领域的应用提供技术支撑。1.双通道免疫层析技术研究首先合成了己烯雌酚半抗原,雌二醇半抗原,将其分别与OVA与BSA偶联制备完全抗原,优化后选取靶标抗原与BSA偶联物固定到硝酸纤维素膜上的两个检测区域,以形成T1和T2两条检测线;两种单克隆抗体与胶体金偶联物吸附于结合垫;在样品端口加入样品后,由于毛细作用力,样品流过结合垫和检测线,从而实现二种物质的同时检测,其不需借助仪器,可实现裸眼检测。优化了抗原抗体浓度,以及封闭液的选择,在最佳条件实现了对DES的快速检测,分别在实际样品如虾组织、液态奶中检测限达到25 ng/g,在奶粉中达到30ng/g,其结果可以在7-10分钟判读;E2在奶粉、液态奶及虾肉组织的检测限分别达到75ng/g、65ng/g和60ng/g,并且其结果可以3-4分钟内进行判断。采用HPLC-MS/MS进行方法比对,通过测试从当地市场购得的37份牛奶和虾组织样本,通过统计学分析,证明这两种方法之间结果无显著性差异,具有良好的一致性。新型二联免疫层析测试纸条的构建为多元快速检测的ICA方法提供了技术支持,如果进一步扩展,就可以在同一条的免疫层析试纸实现多种残留物的检测,从而使其在食品安全等领域发挥更加重要的作用。2.双功能复合纳米颗粒可控合成研究分别采用高温热分解法、溶剂热法、共沉淀法制备了四氧化三铁(Fe3O4),通过TEM结果可知,溶剂热法合成的Fe3O4纳米粒子粒径均一但是较大,不利于下一步的Au壳的包覆;高温热分解法得到了单分散的油酸、油胺包被的Fe3O4,但是不能分散在水溶液中,限制了其进一步应用,后续通过相转移手段,使其可分散在水溶液中,并在水相中完成了Au壳的包覆,通过调节巯基丙酸的量,成功实现Au壳的逐步生长,进而实现了复合纳米Fe3O4@Au光谱调控550-575nm;共沉淀法合成的Fe3O4,虽然形貌不如前两者,但其体积小、水溶性优,利于Au壳的包覆,成功制备了Fe3O4@Au,通过振动样品磁强计(VSM)表征,Fe3O4:49.83emu/g;Fe3O4@Au:39.22emu/g,表明了磁性较好的保留,可以用于样品前处理。3.多壁碳纳米管-量子点电极电化学发光行为研究本章以硫化镉(CdS)量子点为发光体,多壁碳纳米管为信号放大材料,构建了新型CdS量子点电化学发光体系。合成水溶性CdS量子点,并进行了荧光光谱(PL)、紫外可见(UV-vis)、透射电镜(TEM)表征。采用了CS-QD-MWCNTs-GCE的电极修饰方式,研究了量子点电化学发光机理,探讨了多壁碳纳米管(MWCNTs),壳聚糖(CS)对CdS量子点电化学发光的影响,考察了两种碳纳米管对ECL信号的影响,优化了发光条件,pH以及量子点的含量,最终实现了多壁碳纳米管对CdS的电化学发光(ECL)信号的放大,放大效果达10倍。这种简单、高效的修饰方法为量子点电化学发光免疫检测的研究奠定了基础。4.电化学发光共振能量转移免疫检测技术研究以一种新型双功能磁性纳米复合物Fe3O4@Au作为ECL能量受体,CdS量子点修饰电极为能量供体,建立了一种“turn-on”模式的ECL共振能量转移免疫分析方法。将Fe3O4@Au与雌二醇抗体偶联,通过间接竞争免疫的方法,构建了灵敏的雌二醇ECL-RET传感检测技术,其线性方程为A=472.2lgC+9523,相关系数(R2)为0.9936,线性范围为2.0×10-10~2.0×10-4mg/mL,最低检出限为3.2×10-12mg/mL,与二联试纸条相比提高了3个数量级。双功能纳米颗粒Fe3O4@Au与量子点电化学发光相结合,实现了新型共振能量转移的免疫检测策略,为高灵敏检测痕量污染物提供了一个富有应用前景的技术平台。
[Abstract]:The problem of veterinary drug residues not only seriously threatens people's health, enters the environment and ecology, but also seriously affects China's import and export trade. Therefore, in the face of the problem of large detection and wide coverage of food in China, the development of sample pretreatment is simple, rapid and specific detection method is one of the important guarantees to form food safety monitoring ability. In this paper, veterinary drug residues diethylstilbestrol (DES) and estradiol (E2) as the target, antibodies as recognition molecules, immunity. Nanoparticles are signal components. Aiming at the low detection flux of immunochromatographic (ICA) rapid detection technology, a dual-channel immunochromatographic test paper technology is explored, which realizes the simultaneous detection and analysis of two target substances on an immunochromatographic (ICT) system with simple operation and rapid response. In order to further improve the detection sensitivity, the "turn-on" mode biosensor technology was introduced to prepare a new type of dual-functional magnetic nanoparticles with adjustable spectra. On this basis, a sensitive electrochemiluminescence resonance energy transfer sensing technology for the detection of estradiol was established, which realized the detection of trace residues and electrochemiluminescence. Resonance energy transfer technology provides technical support for the application of food safety. 1. Two-channel immunochromatographic techniques were used to synthesize diethylstilbestrol hapten and estradiol hapten, and then coupled with OVA and BSA to prepare complete antigen respectively. After optimization, target antigen and BSA conjugate were selected and fixed on nitrocellulose membrane for two detection. Two detection lines, T1 and T2, were formed, two monoclonal antibodies and colloidal gold conjugates were adsorbed on the bonding pad, and after the sample was added at the sample port, the sample flowed through the bonding pad and the detection line due to capillary force, thus achieving the simultaneous detection of the two substances without the aid of an instrument. The concentration of antigen and antibody was optimized. The detection limits of DES in real samples such as shrimp tissues and liquid milk are 25 ng/g and 30 ng/g respectively, and the results can be interpreted in 7-10 minutes; the detection limits of E2 in milk powder, liquid milk and shrimp meat tissues are 75 ng/g, 65 ng/g and 60 ng/g respectively, and the results are satisfactory. The results can be judged in 3-4 minutes. The method of HPLC-MS/MS was used to compare 37 samples of milk and shrimp tissues purchased from the local market. Statistical analysis showed that there was no significant difference between the two methods and the results had good consistency. The ICA method provides technical support, if further expanded, it can be used in the same immunochromatographic test paper to detect a variety of residues, so that it can play a more important role in food safety and other fields. 2. The controllable synthesis of bifunctional composite nanoparticles were studied by high temperature thermal decomposition, solvothermal method and coprecipitation method, respectively. Fe3O4 was prepared. TEM results showed that the size of Fe 3O4 nanoparticles synthesized by solvothermal method was uniform but large, which was not conducive to the coating of Al shell. Monodispersed oleic acid and oleamine coated Fe 3O4 were obtained by high temperature pyrolysis method, but could not be dispersed in aqueous solution, which limited its further application. The Au shell can be dispersed in aqueous solution and coated in aqueous phase by transfer method. By adjusting the amount of Mercaptopropionic acid, the Au shell can be grown step by step, and then the composite nano-Fe3O4@Au spectrum can be controlled 550-575 nm. Fe3O4 @ Au was successfully prepared and characterized by vibrating sample magnetometer (VSM), Fe3O4:49.83emu/g; Fe3O4@Au:39.22emu/g, which indicated that the magnetic properties were well retained and could be used for sample pretreatment. A novel electrochemiluminescence system for CdS quantum dots was constructed. Water-soluble CdS quantum dots were synthesized and characterized by fluorescence spectroscopy (PL), ultraviolet-visible spectroscopy (UV-vis) and transmission electron microscopy (TEM). The effects of carbohydrate (CS) on the electrochemiluminescence (ECL) of CdS quantum dots were investigated. The luminescence conditions, pH and quantum dot content were optimized. Finally, the amplification effect of multi-walled carbon nanotubes (MWCNTs) on the electrochemiluminescence (ECL) of CdS quantum dots was 10 times. This simple and efficient modification method was quantum dot electrochemistry. 4. A novel bifunctional magnetic nanocomposite Fe3O4@Au was used as the ECL energy receptor and CdS quantum dot modified electrode as the energy donor to establish a "turn-on" mode of ECL resonance energy transfer immunoassay. 3O4@Au was coupled with estradiol antibody by indirect competitive immunization. A sensitive ECL-RET sensor for estradiol detection was constructed. The linear equation was A=472.2lgC+9523, the correlation coefficient (R2) was 0.9936, the linear range was 2.0 *10-10~2.0 *10-4mg/mL, and the minimum detection limit was 3.2 *10-12mg/mL, which was three orders of magnitude higher than that of the binary test strip. Bifunctional nanoparticles Fe3O4@Au combined with electrochemiluminescence of quantum dots (QDs) to achieve a novel resonance energy transfer immunoassay strategy, providing a promising platform for highly sensitive detection of trace pollutants.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：O657;R155.5

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