【作者】 张婷婷；

【导师】 赵慧敏；

【作者基本信息】 大连理工大学 ， 环境工程， 2015， 博士

【摘要】 近年来,由环境污染引起的人类健康问题逐年增多,在这种情况下污染物的生物毒性效应受到了人们的广泛关注。传统的毒性检测方法存在操作繁琐、灵敏性低、耗时长等问题,难以满足实际需求。因此,开发快速、高灵敏和高选择性的毒性检测方法成为环境领域重要的研究课题。与常规分析方法相比,电化学发光传感方法具有灵敏度高、线性范围宽、反应可控、无电子干扰、操作简单、响应速度快等优点,为污染物毒性的检测和评价提供了新的思路。本论文以污染物基因毒性检测为目标,围绕构建高灵敏的电极反应界面和增强传感信号等问题开展相关研究,建立了几种基于微纳米材料的电化学发光传感方法用于基因毒性检测。主要研究内容和结果如下：(1)结合MIL-100(Fe)和氧化石墨烯(GO)对鲁米诺-H202发光反应的催化作用,建立了MIL-100(Fe)-GO基的鲁米诺电化学发光传感方法,实现了对H202的灵敏检测。采用水热法合成了MIL-100(Fe),将MIL-100(Fe)和GO修饰在Pt电极表面,制备了Pt/MIL-100(Fe)-GO电极。在MIL-100(Fe)和GO的共同催化作用下,鲁米诺-H202反应产生的电化学发光信号强度较MIL-100(Fe)和GO单独催化产生的电化学发光信号强度分别提高了1.43倍和10.86倍。H202在0.1 μmol/L-1000 μmol/L的浓度范围内与电化学发光信号强度呈线性关系,定量限为0.1 μmol/L。该电化学发光传感方法对肿瘤细胞所释放出的H202响应信号明显,能够为肿瘤疾病的研究提供技术支持。该方法检测实际水样中的H202回收率在96.0%-102.8%之间。(2)利用碳量子点(CQDs)优良的电化学发光特性,结合二氧化硅包裹纳米金(Au/SiO2)核壳结构的信号放大作用,建立了一种新型电化学发光传感方法用于污染物造成的DNA氧化损伤标志物八羟基脱氧鸟苷(8-OHdG)的检测。以Au/SiO2作为CQDs的修饰平台制备了CQDs包裹的Au/SiO2核壳结构纳米颗粒(Au/SiO2/CQDs)。在Au/SiO2核壳结构的优良信号放大作用下,Au/SiO2/CQDs修饰的铂电极产生的电化学发光信号强度较单纯CQDs修饰的铂电极产生的电化学发光信号强度提高了8倍。该方法定量检测8-OHdG的线性范围为0.2 μg/L-200 μg/L,最低检出限为0.085 μg/L (3σ/k),该电化学发光传感方法检测牛奶样品和实际水样中的8-OHdG回收率分别在102.0%-110.0%和90.0%-115.0%之间。(3)利用环糊精的主-客体识别作用,结合Au/SiO2核壳结构优良的信号放大作用,建立了一种新型电化学发光传感方法实现了对DNA氧化损伤的修复标志物人源DNA糖基化酶(hOGGl)的灵敏检测。分别合成了α环糊精(a-CD)功能化的Au/SiO2核壳结构纳米颗粒(Au/SiO2/α-CD),以及由双链DNA、钌化合物[Ru(bpy)23+]以及单壁碳纳米管(SWNTs)构成电化学发光探针(RuSD)。以Au/SiO2为修饰平台增加了α-CD的主体识别位点,提高主-客体识别的灵敏度,使Pt/Au/SiO2/α-CD/RuSD工作电极产生的电化学发光信号强度较未引入Au/SiO2的电极产生电化学发光信号强度提高了4倍。用此电化学发光传感方法对hOGG1的活性进行分析,线性范围为2×10。U/L-2×105 U/L,定量限为2×103 U/E,比目前仅有的DNA糖基化酶电化学发光检测方法的定量限(5×105U/L)低两个数量级。该电化学发光传感方法检测实际水样中的hOGG1回收率在104.1%-105.0%之间。(4)利用硼掺杂量子点(BGQDs)优良的发光稳定性和生物相容性,建立了新型电化学发光传感方法,用于检测污染物基因毒性所致肿瘤疾病的生物标志物miRNAs。采用电化学剥离法制备了BGQDs。通过考察不同硼掺杂量对BGQDs性能的影响,得出掺杂硼元素的原子百分比为1.29%时,BGQDs的电阻最低、荧光稳定性最高,其电阻与GQDs相比下降了11%,60天-荧光稳定性比GQDs高51%。采用交联法将BGQDs固定在发夹状探针DNA的一端,并将其修饰于铂电极表面制备成工作电极。目标miRNAs与发夹状探针DNA杂交后,发夹状探针DNA的空间结构改变,其末端连接BGQDs远离工作电极表面,导致电化学发光信号降低。基于该原理实现了对miRNA-20a的定量检测,线性范围为0.1 pmol/L-1×104 pmol/L,定量限为0.1 pmol/L 。该方法用于检测胞外分泌物和实际水样中miRNA-20a的回收率分别在103.1%-110%和85.3%-102.3%之间,为检测其它种类的miRNAs提供了新的思路。综上所述,本论文以污染物毒性检测趋于简单快速化的需求和发展趋势为导向,利用电化学发光传感技术和微纳米材料的优异特性,结合污染物基因毒性生物标志物的不同特点建立了几种新型电化学发光传感方法,实现了污染物基因毒性的简单快速检测,为电化学发光传感方法应用于污染物毒性的检测和评价领域的提供了新思路。更多还原

【Abstract】 In recent years, the biological toxicity effect of pollutants has received the widespread attention because of the increasing human health problems caused by environmental pollution. The traditional methods for toxicity detection were difficult to meet the practical detection requirements due to its disadvantages of complicated operation, low sensitivity and time-consuming. Therefore, it is necessary to develop a rapid, high sensitive and high selective method for toxicity detection. Electrochemiluminescence (ECL) sensing as a detection technology was applicable to detect and evaluate the toxicity of pollutants due to its advantages of high sensitivity, wide linear range, controllable reaction, no electronic interference, simple operation and fast response speed. In this thesis, several novel ECL sensing methods were developed for detection of the genetic toxicity of pollutants based on micro/nanomaterials. Relevant work was done on the key technologies for construct of the high sensitive response interface and enhance the sensing signals. The main contents and results are as follows:(1) A MIL-100 (Fe)-GO-based ECL sensing method was structured for the detection of H2O2, which combination with the catalytic property of MIL-100 (Fe) and GO. MIL-100 (Fe) was firstly prepared by hydrothermal reduction method. And then the Pt/MIL-100(Fe)-GO electrode was fabricated by immobilizing MIL-100 (Fe) and GO on Pt electrode. With the synergistic catalysis of MIL-100 (Fe) and GO, the ECL signal of Pt/MIL-100(Fe)-GO was 1.43 and 10.86 times as much as that of Pt/MIL-100(Fe) and Pt/GO, respectively. In the linear range of 0.1 μmol/L-1000 μmol/L, the ECL signal was proportional to the H2O2 concentration with a limit of quantitation (LOQ) of 0.1 μmol/L. This ECL sensing method has significant signal response on H2O2 released by tumor cells, and could provide technical support for the research of tumor diseases. The spiked recoveries for real water sample analysis were 96.0%-102.8%.(2) A novel ECL sensing method was developed for the highly sensitive detection of 8-hydroxy-2’-deoxyguanosine (8-OHdG) in combination with the advantages of the excellent ECL efficiency of carbon quantum dots (CQDs) and the signal amplification of Au/SiO2. CQDs coated Au/SiO2 core-shell nanoparticles (Au/SiO2/CQDs) was synthesized by using Au/SiO2 as the modified platform of CQDs. The ECL signal of Au/SiO2/CQDs modified Pt electrode was 8 times as much as that of CQDs modified Pt electrode due to the excellent signal amplification of Au/SiO2. This ECL sensing method displayed a linear range of 0.2 μg/L-200 μg/L for the quantitative detection of 8-OHdG with a limit of detection (LOD) of 0.085 μg/L (3σ/k). The spiked recoveries for milk sample and real water sample analysis were 102.0%-110.0% and 90.0%-115.0%.(3) A novel ECL sensing method was constructed by taking advantage of the host-guest recognition mechanism and the signal amplification of Au/SiO2, which realized the highly sensitive assay of hOGG1 The a-cyclodextrin functionalized Au/SiO2 (Au/SiO2/a-CD) and ECL probe (RuSD) which composed of double-stranded DNA, [Ru(bpy)23+] and single-walled carbon nanotubes (SWNTs) were synthesized, respectively. The sensitivity of host-guest recognition was enhanced due to the increased recognition site of a-CD by using Au/SiO2 as the modified platform of a-CD, and the ECL signal of Pt/Au/SiO2/α-CD/RuSD was 4 times as much as that of Pt/a-CD/RuSD. This ECL sensing method provided a linear range of 2×103 U/L-2×105 U/L with a LOQ of 2×103 U/L for assay hOGGl. The spiked recoveries for real water sample analysis were 104.1%-105.0%.(4) A boron doped graphene quantum dots (BGQDs)-based sensing method was designed by taking advantage of excellent luminescence stability and biocompatibility of BGQDs for the highly sensitive detection of miRNAs. BGQDs was prepared by electrolytical exfoliation method. The PL stability and the electrochemical impedance text indicated that the 60 days-PL stability of BGQDs can be improved 51% and the resistance of BGQDs can be decreased 11% by doping B with atomic percentage 1.29%. After hybridization with the target miRNAs, the folded structure of the hairpin DNA was transformed into a linear double helix, leading to the BGQDs tag was removed away from the electrode surface, causing the decrease of the ECL intensity. On the basis of this principle, this ECL sensing method displayed a linear range of 0.1 pmol/L-1×104 pmol/L for the quantitative detection of miRNA-20a with a LOQ of 0.1 pmol/L. The spiked recoveries for extracellular secretion sample and real water sample analysis were 103.1%-110.0% and 85.3%-102.3%. Furthermore, this convenient approach offers a new direction for detection other kinds of miRNAs.Above all, with the requirements of simple and rapid detection of pollutant toxicity, we designed several novel ECL sensing methods for the biomarkers of genetic toxicity by combining the ECL technology and the micro/nanometer materials, and realized the simple and rapid detection of the genetic toxicity. It provided new developing orientation on the application and development of ECL sensing in the field of pollutant toxicity detection.更多还原