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题名: 纳米增强单线态氧通道均相化学发光免疫分析研究及其在双酚A检测中的应用
作者: 侯长江
学位类别: 硕士
答辩日期: 2016-05
授予单位: 中国科学院研究生院
授予地点: 北京
导师: 赵利霞
关键词: 均相免疫,双酚A,化学发光,单线态氧,纳米金 ; Homogenous immunoassay, Bisphnol A, Chemiluminescence, Singlet oxygen, Gold nanoparticles
其他题名: Nanoparticles enhanced singlet oxygen channeling homogenous chemiluminescence immunoassay and its application for the detection of bisphenol A
学位专业: 环境工程
中文摘要:       双酚A(BPA)作为一种重要的化工原料,广泛应用于消费产品的生产制造如塑料瓶和食品容器,已对人类健康构成了严重威胁。因此,对其分析控制尤为重要。目前,对其分析测定常使用的方法有高效液相色谱法(HPLC)和酶联免疫吸附试验(ELISA)等,这些方法灵敏度高、特异性强,但因为它们复杂的预处理步骤或洗涤步骤使得这些方法既费时又费力。因此急需发展一种快速、灵敏度高、易于实现高通量检测的方法来定量分析环境样品中的BPA。
      在本研究工作中,首先发展了一种基于纳米粒子对的单线态氧通道均相化学发光免疫法,实现了双酚A的定量检测。分别采用两种纳米颗粒为给体珠和受体珠,给体珠上修饰酞菁染料,受体珠上修饰二甲基噻吩的衍生物及三价铕的掺杂物。这两种纳米微珠通过特异性的生物反应如抗原-抗体和生物素-链霉亲和素的特异性反应形成粒子对。当给体珠受光照射时,其上的光敏剂会将周围的常态氧分子转化为单线态氧,生成的单线态氧迁移到受体纳米颗粒上并与发光物质反应,产生激发光。基于此,以环境雌激素双酚A为污染物模型,建立了一种竞争性的单线态氧通道均相化学发光免疫分析体系。实验操作是在384微孔板上进行的,因此可以实现双酚A的高通量筛选测定,其线性检测范围为10~1000 ng/mL, 检测限为2.9 ng/mL,组内与组间精密度均小于5.1%。在实际环境样品自来水和河水中不同浓度的加标回收率均值为95.45~121%,并同高效液相色谱法做了比对,结果相关性很好。与已有的方法相比,该工作具有免洗涤、简单、快速、使用试剂量少、可以用于大量环境样品中双酚A的高通量测定等优点,但其灵敏度还有待进一步的提高。
      根据单线态氧通道均相化学发光免疫分析原理,单线态氧的产生和扩散是决定于化学发光信号的关键因素。而纳米粒子如纳米金的加入可能会提高单线态氧的产生,基于此,我们在该体系中加入金纳米粒子来考察其对体系化学发光信号的影响。首先研究了纳米金对亚甲基蓝-二甲基噻吩-Eu(TTA)3液相体系化学发光信号的影响,结果表明体系在多次光照之后,纳米金表现出了信号增强作用,并且不同粒径的纳米金的增强效果不同。此外,还用原位静态注射法研究了纳米金、Eu(TTA)3和过氧化氢对受体珠-碳酸氢盐缓冲溶液体系的信号增强作用,结果表明三价铕络合物和过氧化氢可以增强该体系的化学发光信号,并且论证了单线态氧的产生。最后,在酶标仪检测中,研究了纳米金和过氧化氢对亚甲基蓝-受体珠-碳酸盐溶液体系信号的影响,结果表明纳米金和过氧化氢对该体系的化学发光信号有明显的增强,并且浓度越高增强效果越强。
英文摘要:       Bisphenol A (BPA) is widely used in consumer products such as plastic bottles and food containers. It has become a ubiquitous environmental contaminant, and poses a serious risk to human health. However, traditional methods, such as high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA), allow high sensitivity and specificity, but are labor intensive, expensive, and time consuming because of their complex pretreatment steps. A rapid, sensitive and high-throughput method for detecting BPA is therefore desirable.
      Herein, a donor/acceptor nanoparticle pair-based singlet oxygen channeling chemiluminescence homogenous immunoassay is developed for the determination of BPA in our study work. The donor nanoparticles were modified with phthalocyanine as a photosensitizer, and were then coated with streptavidin. The acceptor nanoparticles were doped with thioxene derivatives and Eu (III) as a chemiluminescence emitter, and then coated with anti-BPA antibody. Under light irradiation, oxygen near the donor surface transforms to singlet oxygen (1O2), which migrates to the acceptor and reacts with it, generating luminescence. Because 1O2 has a very short lifetime, luminescence is generated only when the donor and acceptor are in close proximity. This occurs when they are brought together by the antigen/antibody and streptavidin/biotin reaction. Based on this singlet oxygen channeling mechanism, a competitive homogenous chemiluminescence immunoassay for BPA was developed on 384 microplates. The assay exhibited linear detection over the range 10~1000 ng/mL, and a limit of detection of 2.9 ng/mL. The intra- and inter-assay precisions were both below 5.1%. The average recoveries of three spiked samples in tap and river water samples were in the range 95.5~121.0%, in agreement with values obtained using high-performance liquid chromatography. The homogeneous assay is rapid, low cost, no wash steps, and allows high-throughput, so is well suited for screening large numbers of environmental samples.
      According to the principle of the singlet oxygen channeling chemiluminescence homogenous immunoassay, the key factor of generating chemiluminescence signal is the concentration of singlet oxygen diffusion. Hence, in this work gold nanoparticles (GNPs) had been added into the system to study its effect on the chemiluminescence signal. The influence of GNPs on methylene blue (MB)-thioxene-Eu(TTA)3 system had been studied, and after sometimes illumination the chemiluminescence signal enhancement had been observed, but different sizes of GNPs showed different enhancement effect. The influence of GNPS and H2O2 on acceptor — carbonate bicarbonate buffer (CBS) had been studied. The signal enhancement had been observed and the presence of 1O2 had been proved. Finally, the GNPs and H2O2 had been added into the MB-acceptor-CBS system and it showed a very strong chemiluminescence signal enhancement, and the higher concentration of GNPs and H2O2 will bring the stronger signal enhancement.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/36802
Appears in Collections:环境化学与生态毒理学国家重点实验室_学位论文

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Recommended Citation:
侯长江. 纳米增强单线态氧通道均相化学发光免疫分析研究及其在双酚A检测中的应用[D]. 北京. 中国科学院研究生院. 2016.
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