【作者】 董静；

【导师】 艾仕云；

【作者基本信息】 山东农业大学 ， 土壤学， 2013， 博士

【摘要】 农药是目前世界农业生产活动中最重要的生产资料之一。作为农业增产的重要因素，农药在避免病虫害、减轻劳动强度等方面起到了重要作用。农药的使用给人们创造了巨大的经济效益和社会效益。但是，随着农药的品种和使用量的不断增加，农药残留给生态环境和人类健康也带来了严重威胁。农药进入人体后可抑制血液和组织中的乙酰胆碱酯酶的活性，造成神经递质乙酰胆碱大量蓄积，导致神经麻痹。有些农药虽然急性毒性不高，但在人畜体内有慢性积累性毒性，长期暴露于微量农药下有可能引起慢性中毒。不仅如此，据报道有些农药还有致畸、致癌、致突变的作用。随着健康意识和环保意识的增强，农药残留分析技术的研究也受到了高度重视。电化学方法以其成本低廉、操作简单、灵敏度高、快速方便、仪器小巧便携等特点引起了广大研究者的关注。近年来，电化学检测方法已被广泛应用于药物分析、疾病诊断和环境监测等领域。本文构建了四种不同类型的电化学传感器，研究了以甲基对硫磷为模型药物的有机磷农药在传感器上的电化学行为，并对河水和土壤实际样品中的农药残留进行了检测。主要分为以下四个方面：（1）以石墨烯-壳聚糖纳米复合物（GR-Chi）为修饰材料，以硼酸功能化的ZnSe量子点（F-ZnSe QDs）为酶固定材料，构建了一种响应快、灵敏度高的安培型生物传感器用于定量检测有机磷农药。传感器构建过程包括：（1）借助于壳聚糖良好的成膜性将GR-Chi固定在玻碳电极的表面；（2）通过静电吸引、氢键等作用将F-ZnSe QDs固定于GR-Chi膜表面；（3）通过硼酸羟基与多糖的特异性结合将乙酰胆碱酯酶（AChE）固定于F-ZnSe QDs表面。GR-Chi良好的导电性可以提高电子传递速率，降低硫代胆碱的氧化电位，生物相容性的ZnSe量子点的高比表面积不仅可以为酶的固定提供更多有效位点，而且可以保持酶的生物活性；二者协同作用不仅能加速电子传导，而且可以同时促进酶催化反应，从而增大电极的检测灵敏度。以甲基对硫磷为模型药物，利用农药对AChE催化活性的抑制作用原理来检测有机磷农药，在0.5nM-0.5μM范围内，酶抑制率与甲基对硫磷浓度呈线性关系，检出限可达0.2nM （S/N=3）。该传感器响应速度快，检测范围宽，稳定性较好。对河水和土壤样品中甲基对硫磷检测的平均回收率在94.5%到108.6%之间（2）结合Ellman试剂的电化学还原和酶抑制原理，提出了一种检测农药残留的新方法。首先以多壁碳纳米管-壳聚糖纳米复合物为修饰材料构建了一种乙酰胆碱酯酶生物传感器。多壁碳纳米管良好的导电性可以提高电极界面的电子传递速率，壳聚糖的生物相容性可以保持酶的生物活性，二者的协同作用保证了酶传感器具有良好的电化学响应。同时，Ellman试剂的电化学还原相对容易，受干扰较少，因此可以进一步提高检测的灵敏度。实验优化了应用电位、缓冲溶液pH、酶催化时间和抑制时间等检测条件，在最优条件下，利用Ellman试剂的电化学还原信号，以甲基对硫磷为模型药物对有机磷农药进行了检测。在甲基对硫磷浓度范围5.0×10^-7-1.0×10^-12M内，Ellman试剂的电化学还原电流变化值与农药浓度呈线性关系。根据信噪比S/N=3得出检出限为7.5×10^-13M。该方法具有良好的重现性和稳定性，并且成功实现了土壤和水实际样品中农药的检测。（3）以二步沉淀法制备了多壁碳纳米管-二氧化铈-金（MWCNTs-CeO₂-Au）纳米复合物，用红外吸收光谱、扫描电镜和X-射线衍射等手段对其形貌和结构进行了表征。多壁碳纳米管对芳香类化合物具有很强的吸附作用，CeO₂和Au纳米颗粒不仅具有较大的比表面积，而且对电活性物质的氧化还原具有较强的催化活性，结合多壁碳纳米管和纳米粒子的优点于一体，以MWCNTs-CeO₂-Au纳米复合物为修饰材料构建了一种新型的电化学传感器，以甲基对硫磷为模型药物对有机磷农药进行了检测。实验结果表明，MWCNT-CeO₂-Au提高了电极有效表面积，增加了待测溶液中甲基对硫磷的吸附量；同时，该纳米复合物具有良好的导电性，促进了电极表面电子传递和传感器的响应速度，提高了甲基对硫磷在修饰电极上的电流响应，从而提高了检测的灵敏度。在最优条件下利用吸附-溶出伏安法对甲基对硫磷进行了检测，在浓度范围1.0×10^-101.0×10^-7M内，甲基对硫磷的氧化峰电流与浓度对数呈线性关系，检出限达到7.5×10^-11M （S/N=3）。该传感器响应速度快，检测范围宽，灵敏度较高，稳定性较好，有望成为一种新型的农药残留检测工具。（4）利用超声辅助下水热合成法合成了石墨烯量子点（GQDs），用透射电镜、红外吸收光谱进行了表征。以GQDs为修饰材料制备了GQDs/GCE的玻碳电极，然后将此电极在酸性条件下电聚合了一层聚三聚氰胺（PAM）导电膜，制备了PAM/GODs/GCE用于甲基对硫磷的检测。GQDs大的表面积不仅为三聚氰胺提供了更多的聚合位点，而且还为三聚氰胺的电化学聚合提供了必要的含氧表面；PAM膜含有大量的氨基，可以吸附大量的甲基对硫磷，而且其较好的导电性也为甲基对硫磷的溶出提供了条件；同时，GQDs和PAM对甲基对硫磷的催化还原进一步提高了检测灵敏度。在优化条件下，用示差脉冲伏安法对不同浓度的MP进行了电化学富集-溶出检测。甲基对硫磷还原电流与其浓度在0.1nM到50.0nM范围内呈线性关系，检出限为0.06nM。该传感器响应速度快，检测范围宽，灵敏度较高，稳定性较好。水和土壤样品中的平均加标回收率在94.6%到109.2%之间，表明该传感器可以用于实际样品的检测。更多还原

【Abstract】 Pesticides play an important role in the high productivity achieved in agriculture throughthe control of pests. However, pesticides are intentionally toxic, and the presence of pesticideresidues in food, water, and soil has become a major issue in environmental chemistry.Worldwide, organophosphorus pesticides （OPs） compounds account for over38%of thetotal pesticides used. Their toxicity is based on the inhibition of acetylcholinesterase （AChE,EC3.1.1.7）, which is essential for the functioning of the centralnervous system of humansand insects. This results in the accumulation of the acetylcholine neurotransmitter, whichinterferes with muscular responses and causes respiratory and myocardial malfunctions andeven death. As a result, effective monitoring of OPs in the environment is very desirable.Among many analysis methods, electrochemical analysis has been confirmed as one of themost promising method for pesticide measurement by virtue of its simplicity, rapidity, highsensitivity and versatility. In this paper, four different electrochemical sensors were fabricatedbased on new nanomaterials and their applications in OPs determination were investigated.The main contents could be divided into four aspects as follow:（1） A sensitive acetylcholinesterase biosensor was fabricated based on dual-signalamplification. A large amount of enzyme was immobilized on a glassy carbon electrode viaspecific binding between functionalized ZnSe quantum dots and acetylcholinesterase, and agraphene-chitosan nanocomposite was introduced as electrode modifier that improvesresponse. These two factors render the biosensor highly sensitive to acetylthiocholine chloride.Organophosphate pesticides were detected with this biosensor using methyl parathion as amodel enzyme inhibitor. Under optimal conditions, there is a linear relationship between thepercentage of inhibition（I%）and the log of the concentration of methyl parathion in the0.5nM to0.5μM range, with a0.2nM detection limit （at an S/N of3）. The biosensor displaysacceptable reproducibility and relatively good storage stability. It was successfully employedto the determination of methyl parathion in spiked water and soil samples.（2） A novel method was proposed for ultra-trace detection of pesticides combining electrochemical reduction of Ellman′s reagent （DTNB） with AChE inhibition. Theamperometric biosensor, fabricated by immobilizing AChE on multi-walled carbonnanotubes-chitosan （MWCNTs-Chi） nanocomposites modified glassy carbon electrode,enjoyed high sensitivity owing to the excellent conductivity and favorable biocompatibility ofMWCNTs-Chi nanocomposites. Meanwhile, the sensitivity of the biosensor was furtherenhanced using the electrochemical reduction signal of DTNB for determination. Underoptimum conditions, methyl parathion was detected based on its inhibition effect on AChEactivity and the subsequent change in electrochemical reduction response of DTNB. Goodrelationship was obtained between the reduction current and pesticide concentration in theranges of5.0×10^-7-1.0×10^-12M with a detection limit of7.5×10^-13M （S/N=3）. Moreover,the proposed protocol was successfully employed for the determination of methyl parathion inspiked water and soil samples.（3） A novel multi-walled carbon nanotubes-CeO₂-Au nanocomposite（MWCNTs-CeO₂-Au） was synthesized by a facile two-step precipitation method and usedfor solid-phase extraction of methyl parathion （MP）. The MWCNTs-CeO₂-Aunanocomposite combined the individual properties of MWCNTs （high conductivity andexceptional adsorption affinity） and nanoparticles （high surface area and special catalyticactivity）, and realized the efficient enrichment and electrochemical stripping voltammetricdetection of MP. An ultra-low detection limit of7.5×10^-11M （S/N=3） for MP wasobtained at MWCNTs-CeO₂-Au nanocomposite modified electrode, suggesting the highsensitivity of the nanocomposite based electrochemical sensor. Moreover, the proposedelectrochemical sensor was successfully employed for the determination of MP in water andsoil samples.（4） With the assistance of ultrasound, graphene quantum dots （GQDs） weresuccessfully prepared via cleaving graphene oxide under acid conditions and werecharacterized by TEM and FT-IR. Then, a novel OPs electrochemistry sensor was fabricatedby electro-polymerization melamine film onto GODs modified glassy carbon electrode（PAM/GODs/GCE）. A large amount of MP was absorbed on the electrode surface via thestatic electricity and hydrogen bonds between PAM film and MP molecules. GQDs wereintroduced as electrode modifier that improves the electrode response. In addition, sensitivityof the sensor was further enhanced by the catalytic action of PAM and GODs towards theelectrochemistry reduction of MP. Under optimum conditions, good relationship was obtained between the reduction current and pesticide concentration in the ranges of0.1nM to50nm,with a detection limit of0.06nM （S/N=3）. Moreover, the proposed protocol wassuccessfully employed for the determination of MP in spiked water and soil samples.更多还原