【作者】 关桦楠；

【导师】 迟德富；

【作者基本信息】 东北林业大学 ， 森林生物工程， 2011， 博士

【摘要】 长期使用农药引起农产品中农药残留超标,对生态环境和人类健康造成极大危害。有机磷和氨基甲酸酯类农药是最常见的两类农药,也是目前为止仍然最广泛使用的农药,它们是一种神经毒物,会抑制人及动物体内胆碱酯酶活性,引起中枢神经系统功能紊乱,出现中毒症状,以至危及生命。近年来,农药残留问题已经受到了世界范围的关注。随着人们食品安全意识的不断增强,改善农药残留检测技术已迫在眉睫。农药残留常用的检测方法为气相、液相色谱法、质谱法以及波谱法等,通常耗时长、成本高,难以满足现场快速检测的需要。因此,急需研究一些快速、灵敏、便捷的检测方法,将高农药残留的农产品阻挡在市场之外,以杜绝食品安全事故的发生。生物传感分析技术与传统方法相比具有选择性好、灵敏度高、分析速度快、成本低、能在线检测等优点,在环境监测、食品检验等方面得到高度重视和广泛应用。乙酰胆碱酯酶(AChE)生物传感器广泛应用于有机磷和氨基甲酸酯类农药的测定。由于乙酰胆碱酯酶能够选择性的催化底物水解,且其催化活性能被有机磷农药所抑制,利用这一特性可制成用于测定有机磷农药含量生物传感器。为了提高传感器的灵敏度,通常在制备传感器时,在基础电极上修饰某种电化学催化剂,以增强传感器对底物的响应信号。纳米材料具有独特的物理和化学特性,能促进生物分子的活性中心与电极间的直接电子交换,同时最大限度地保持生物分子的活性。因此,将纳米技术应用于生物分子电化学分析研究,有利于创新性地建立一些新理论、新技术和新方法,将是一个很有前景的领域。本文旨在建立快速检测果蔬中有机磷和氨基甲酸酯农药残留的电化学酶生物传感器。试验以玻碳电极作为信号转换器,分别利用纳米氧化锌和纳米氧化硅以及多壁碳纳米管作为电极修饰材料,构建新型生物传感器,并对其电化学性质进行研究。主要研究结果如下：(1)实验中对粗酶液的提取条件进行了研究。采用了Sephadex G-200层析,对鞘翅目昆虫洋虫的头胸部组织粗酶提取液中的乙酰胆碱酯酶进行了分离纯化,并对纯化后的酶液的性质进行了研究。结果表明,洋虫头胸部乙酰胆碱酯酶粗酶的最适提取条件为：pH7.5,Triton X-100浓度为2.0%,按料液比为1：5,4℃下浸提10个小时。采用Sephadex G-200层析纯化粗酶液,AChE的纯化倍数为52.76倍,总蛋白含量2.37 mg,酶比活力达到13.19 OD/(min/mg),酶活回收率为85.67%。纯化后的样品经SDS-PAGE电泳测定其分子量为78.6 KDa。通过纯化后的酶性质研究,确定检测条件为：最佳反应温度为36℃时,抑制时间为35 min。通过敌敌畏抑制试验检测酶的特性,结果表明,敌敌畏在0.2-2μM范围内,AChE抑制率与农药浓度呈良好的线性关系,抑制率等式为I%=(18.38c+11.25)%。当敌敌畏在2-10μM范围内,AChE抑制率与农药浓度呈另一线性关系,抑制率等式为I%=(1.53c+51.66)%。检出限为1.58±0.37μg/L。(2)采用薄膜蒸发-冻融法制备AChE脂质体,通过正交设计优化制备工艺,以鱼精蛋白沉淀法分离脂质体与游离酶。通过计算活性包封率,确定AChE脂质体最佳制备工艺为：根据正交试验结果,确定最佳处方工艺,即卵磷脂与胆固醇的质量比为2：1,(卵磷脂+胆固醇)的质量与AChE的质量之比1：2,冻融时的循环次数为30次,旋转速度为150 rpm,最佳处方工艺制得的5组脂质体的活性包封率的平均值为89.5%。AChE脂质体生物反应器有效平均粒径为7.3±0.85μm,其中85%(体系分数)的生物反应器微粒集中在7μm。AChE脂质体生物反应器表面电势-78.6 mV。AChE脂质体生物反应器相比于同等酶含量的游离酶,具有较高的稳定性。Porin蛋白嵌入磷脂层,构成底物进入通道的同时控制酶的逸出,因为酶的大小无法通过孔道。脂质体内酶的活性根据荧光指示剂的信号强度来测定。通过敌敌畏和西维因抑制试验结果表明,AChE抑制率与农药浓度呈良好的线性关系,检出限分别为1.134±0.29μg/L和1.364±0.35μg/L。(3)采用三电极系统对玻碳电极进行了预处理及活化,实验结果表明,GCE-(CS/ALB)n对底物ATChCl的催化电流值随着组装层数的增多而明显增大,(CS/ALB)多层膜最适层数为5层,(CS/ALB)5同时体现出了较好的稳定性。抑制率与敌敌畏的浓度在一定范围内存在线性关系。在0.25-1.5μM的范围内抑制率回归方程为I%=(24.45c+12.35)%,相关系数R2为0.9981。在1.75-10μM的范围内抑制率回归方程为I%=(1.86c+58.76)%,相关系数R2为0.9914。检出限为0.86±0.098μg/L。(4)采用硅酸钠为硅源,氯化铵为沉淀剂制备纳米二氧化硅。研究了硅酸钠的浓度、乙醇与水的体积比以及pH值对纳米二氧化硅粉末比表面积的影响。利用XRD衍射和扫描电子显微镜对产物进行表征。选用最适体系制备出的纳米氧化硅比表面积平均值为297.3 m2/g,表面电势为-42.5 mV,平均粒径为115.5±1.86 nm。(ALB/SiO2)n最适层数为6。GCE-(ALB/SiO2)6对农药的响应实验结果表明,该电极灵敏度较高且抑制率与农药的浓度在一定范围内存在线性关系。GCE-(ALB/SiO2)6具有较好的重复性和稳定性。(5)将多壁碳纳米管(MWNTs)置于混酸(硝酸：硫酸=1：3)中,利用超声波振荡截短碳纳米管、并使其与羧基链接,而后基于阳离子聚合电解质壳聚糖(CS)和带有阴离子的碳纳米管之间的静电作用,通过层层自组装的模式均匀稳定地形成复合壳聚糖多层膜。GCE-(ALB/MWNTs)n最适层数为6层。在GCE-(ALB/MWNTs)6对农药的响应实验结果表明,农药抑制率与农药的浓度在一定范围内存在线性关系,检出限为0.68±0.076μg/L。(6)采用溶胶-凝胶法制备Al掺杂的ZnO纳米粉体。结合正交设计对各因素进行优化,得到最适制备条件为：醋酸锌浓度为1.5 mol/L,((NH)3C6H5O7浓度为2 mol/L,Al掺杂质量分数1.5%,V(乙醇)：V(H2O)为1.5：1和煅烧温度1200℃。XRD分析和EDS能谱分析表明Al元素已经融合进了ZnO的晶格当中。GCE-(ALB/ZnO)n最适层数为5层。GCE-(ALB/ZnO)5对展现了良好的光电性能,当紫外光与可见光同时存在时,经Al-ZnO修饰的电极具有较高的响应电流。农药抑制率与农药的浓度在一定范围内存在线性关系,检出限为0.76±0.087μg/L(7)将纳米ZnO、MWNTs和纳米氧化硅这三种纳米材料,经过有序的组合构建酶最终获得了以(ALB/MWNTs/SiO2/ZnO)4膜为基础的性能优越的电化学生物传感器。根据新型传感器的性能优化工作条件,即反应体系总体积10 mL,底物反应时间15 min,体系温度36℃,同时伴随光源辐射,反应体系pH值为7.4,农药样品抑制12 min,加入1.25 mM底物,反应15 min。在敌敌畏响应实验中,农药抑制率与农药的浓度在一定范围内存在线性关系,检出限为0.53±0.096μg/L。(8)实验中考察了新型传感器的抗干扰能力和可再生能力,这两方面的特性直接影响着传感器的实际应用。在研究重金属离子、农药以及其他化合物对AChE生物传感器测定时的影响过程中,发现酶纳米复合结构生物传感器在含有重金属离子和不同农药的溶液中基本不受影响,含有抗坏血酸的体系对传感器的影响较大。筛选出三种物质来活化酶传感器可再生的活化物。通过比较选择碘解磷定(2-PAM)作为传感器可再生的活化物。结果表明,所制备的传感器可以重复使用6次,活性仍在80%以上。(9)采用优化好的体系制备酶纳米生物传感器,测定对两类中的8种农药的电化学响应,结果表明8种农药抑制率分别在一定范围内与农药浓度呈良好的线性关系,且具有较低的检出限。利用乙酰胆碱酯酶生物传感器技术,以苹果、小白菜和黄瓜为样品,采用标准加入法进行分析,测定蔬菜水果中有机磷和氨基甲酸酯类农药的残留。酶纳米传感器检测三种果蔬中两类农药残留时,均表现出良好的精确度、重现性和准确性,可以用于实际的检测,且不需要繁琐的样品预处理过程,能够满足快速检测的需要。更多还原

【Abstract】 With the long-term use of pesticides in agricultural products, excessive pesticide residues has caused considerable hazard on ecological environment and health of human. Organophosphorus and carbamate pesticides are two common pesticides which are still widely used in agriculture production. They can inhibit cholinesterase activity in body and cause poisoning symptom, even endanger the life of human being. So pesticide residues in food have been paid more attention over the world. Now, people would pay growing awareness of food safety, since it’s extremely urgent to strengthen the detection of pesticide residues.Traditionally, the widely used methods for determination of organophosphorus pesticides are liquid/gas chromatography, mass chromatography and spectroscopy, these methods are sensitive and allow discrimination among different organophosphorus compounds but they are expensive and require a long time not to satisfy the need of fast detection. So research a rapid, sensitive and convenient method for detection pesticide residues is our imperative needs, it will play an important role in prevention high pesticide residues farm products from the marketplace and to reduce the incidents of food safety. Enzymatic biosensors have emerged as an ultra sensitive, selective cost-effective and rapid technique for pesticides residue analysis in environmental monitoring, food and quality control.In recent years, it has been given serious attention and extensive research, becoming important trends for detection pesticide residues.The acetylcholinesterase biosensors are applied to detect organophosphorus pesticides widely. The acetylcholinesterase can catalyze the substance to hydrolyze selectively. And the activity of acetylcholinesterase can be restrained by the organophosphorus pesticides. We make use of the characteristic to design the biosensors to detect the content of organophosphorus pesticides. In order to improve the sensitivity of the biosensor, the electrochemical catalyst usually be used in the preparation of the biosensor.Due to their special physical and chemical properties, nanosturctured materials can activate while active electrode surface, and promote the direct electron transfer between the active center in biomolecule and the electrode surface. Therefore, the application of nano-technology on electrochemical analysis of biological molecules is a promising area and conducive to the establishment of some new theories, new technologies and new methods.In this paper, we will propose a simple and efficient method for detection trace pesticide residues based on immobilization of AChE on nanosturctured materials modified electrode, which called electrochemical biosensor for screening of organophosphorus and carbamate pesticides. In this thesis, the novel biosensors were constructed based on nano-ZnO, nano-SiO2 and multi-walled carbon nanotubes. The electrochemical properties and catalytic effects were studied. The main contents and results are summarized as follows:(1) This experiment is mainly for the extraction of the crude enzyme solution. Acetylcholinesterase (AChE) from cephalothorax of Martianus dermestoides Chevrolat., was separated from the crude extract and purified to electrophoretic homogeneity by Sephadex G-200 chromatography. Then the properties of crude extract and purified enzyme were studied. The results show that the acetylcholinesterase from cephalothorax of Martianus dermestoides Chevrolat. optimum conditions for the extraction is,2.0%Trition X-100, pH 8.0, the ratio of material and extract 1:5, the extraction for 10 h at 4℃. The crude enzyme was purified 52.76-fold. And the content of total protein was 2.37 mg. The specific activity of enzyme was 13.19 OD/(min/mg). The 85.67% of activity recovery was obtained. The Molecular weight of the purified enzyme was 78.6 KDa, measured by SDS-PAGE. Studies on the properties of the AChE showed that the optimum temperature of the enzyme was 36℃and the exhibited optimum inhibition time about 35 min. The inhibition efficiency of dichlorvos was a linear function of its concentration from 0.2 to 2 and 2 to 10.00μM. The linearization equation were I%=(18.38c+11.25)% and I%=(1.53c+51.66)%. The detection limit was calculated to be 1.58±0.37μg/L(2) The formulation was optimized on the basis of orthogonal design and its entrapment efficiency was performed by the protamine sedimentation method. The film-evaporation combining with freeze-thawing method was used to prepare AChE liposome. The optimal conditions were found to be that the ratio of (cholesterol+egg phospholipid) and AChE was 1:2, the times of freeze-thawing was 30 times, the rotate speed was 150 rpm, the ratio of cholesterol and egg phospholipid was 2:1. The average activity entrapment efficiency of the optimized AChE liposomes was 89.5%. AChE liposomes had a mean diameter of 7.3±0.85μm and 85% (by volume) of the microspheres were below 7μm and a zeta potential of-34.7 mV. AChE liposomes bioreactor compared to the same content of the free enzyme has high stability. Porins are embedded into the lipid membrane, allowing for the free substrate transport, but not that of the enzyme due to size limitations. The enzyme activity within the liposome is monitored using pyranine, a fluorescent pH indicator. The inhibition efficiencies of dichlorvos and sevin were a linear function of their concentrations. The detection limit was calculated to be 1.13±0.29μg/L and 1.36±0.35μg/L, respectively.(3) A three-electrode analysis system linked to LK98 electrochemical analyzer was used as a detection system. The results showed that the GCE-(CS/ALB)5 Could improve the catalytic current response to ATChCl significantly with the increased number of the multilayer films. The optimum assembly number was 5. The inhibition efficiency of dimethoate was a linear function of its concentration from 0.25 to 1.5 and 1.75 to 10.00μM. The linearization equation were I%= (24.45c+12.35)% and I%= (1.86c+58.76)%, with the correlation coefficients squre of 0.9981 and 0.9914, respectively. The detection limit was calculated to be 0.86±0.098μg/L.(4) Sodium silicane and anmonium chbride were used m prepare nanometer SiO2. The effects of concentration of sodium silicane, volume ratio of ethanol to water and pH value on specific surface area of SiO2 powder were investigated. The nanometer SiO2 was characterized by SEM and XRD. The SiO2 had a specific surface area of 297.3 m2/g. The results showed that SiO2 which were obtained by association had a mean length of 115.5±1.86 nm and a zeta potential of-42.5 mV. The optimum assembly number was 6. The GCE-(ALB/SiO2)6 inhibition efficiency of dimethoate was a linear function of its concentration. The developed GCE-(ALB/SiO2)6 exhibited good reproducibility and acceptable stability, thus providing a new promising tool for analysis of enzyme inhibitors.(5) Multiwalled carbon nanotubes (MWNTs) were treated in 1:3 concentrated nitric-sulfuric acid to cut them into short tubes and to create carboxyl groups at their ends. Homogeneous multilayer films of the shortened MWNTs were assembled by a layer-by-layer method, based on electrostatic interaction of positively charged cationic polyelectrolyte chitosan (CS) and the negatively charged and MWNTs. The optimum assembly number was 6. The GCE-(ALB/MWNTs)6 inhibition efficiency of dimethoate was a linear function of its concentration. The detection limit was calculated to be 0.68±0.076μg/L.(6) The main content of this chapter was to build the chemically modified electrode for the detection of pesticide residues based on (ALB/ZNO)n multilayer films.ZnO nanoparticles intermingled with Al(Al-ZnO) were gained by the sol-gel method The slid solution structure of Al-ZnO was confirmed by X-ray diffraction (XRD)and energy dispersive spectroscopy (EDS). The optimization experimental conditions were calciming temperature of 1200℃, the ratio of water and alcohol of 1.5, Zn(CH3COO)2 concentration of 1.5 mol/L, concentration of 2 mol/L and the mass fraction of Al3+adulteration of 1.5%. The optimum assembly number of (ALB/ZNO)n was 5. GCE-(ALB/ZnO)5 showed the good optical properties. The Al-ZNO-modified electrode had a higher response current with UV and visible light. The GCE-(ALB/ZNO)5 inhibition efficiency of dimethoate was a linear function of its concentration. The detection limit was calculated to be 0.76±0.087μg/L.(7) In this experiment, the nano-ZnO, MWNTs and nano-SiO2 which were through a combination of ordered nano-composite structure constructed enzyme biosensor. We got a superior performance electrochemical biosensor based on (ALB/MWNTs/SiO2/ZnO)4 film by re-optimize their working conditions. The optimal conditions were found to be that the total reaction volume of 10 mL, the reaction time of substrate 15 min, system temperature of 36℃, accompanied by UV and visible light radiation, the reaction system pH value 7.4, inhibition of pesticide samples 12 min, added 1.25 mM substrate, the reaction time 15 min. The GCE-(ALB/MWNTs/SiO2/ZnO)4 inhibition efficiency of dimethoate was a linear function of its concentration. The detection limit was calculated to be 0.53±0.096μg/L.(8) The anti-interference ability and reactivation of AChE biosensors were examinated, the characteristics of both were a direct impact on the practical application of the biosensor. The AChE biosensors were tested to study the influence of heavy metal ions, pesticides and other compounds on the corresponding enzyme. It has finally appeared that heavy metal ions and pesticides gave no significant inhibition. For ascorbic acid, the experiments showed that biosensors are quite sensitive to it. In the present study, enzyme reactivations by three oximes were explored for reactivation of the enzyme for repeated use.2-PAM was found to be a more efficient reactivator under repeated use, retaining more than 80% of initial activity after 6 reuses.(9) The electrochemical responses of 8 pesticides were detected with enzyme nano-biosensors. The results showed that the biosensor inhibition efficiencies of 8 kinds of pesticides were a linear function of their concentrations and with the low detection limit. Using acetylcholinesterase biosensor technology, taking apples, cucumbers and cabbages examples, the organophosphorus pesticide residues were determined with standard addition method. AChE had shown good accuracy, reproducibility and accuracy. It could be used for wild testing, and did not require tedious sample preparation process. It met the rapidly testing needs.更多还原