【作者】 杨丽娟；

【导师】 于海业；

【作者基本信息】 吉林大学 ， 农业生物环境与能源工程， 2009， 硕士

【摘要】 长期使用农药引起农产品农药残留超标,对生态环境和人类健康造成极大危害,加强农药残留检测迫在眉睫。生物传感分析技术与传统方法相比具有灵敏度高、分析速度快、成本低等优点,在环境监测、食品检验等方面得到高度重视和广泛研究。本文自行设计加工一次性丝网印刷电极,并通过超声清洗、电化学氧化等处理,使普通油墨批量生产的电极取得了良好电化学响应特性,为后续试验提供基础;同时以玻碳电极为信号转换器,将乙酰胆碱酯酶通过多壁碳纳米管-戊二醛交联固定在电极表面,构建了用于农药残留检测的电流型生物传感器。传感器对底物响应时间小于1分钟,对敌百虫和灭多威检测响应灵敏,线性关系良好,检测限分别为0.026μg/mL,1.5×10-3μg/mL;检测时间少于30分钟。通过组内偏差计算传感器的精确性、稳定性和重复性,变异系数均小于5%;在此基础上重点对一次性丝网印刷集成电极构建的电流型酶生物传感器进行研究。丝网印刷生物传感器制作时间短,测定电位比前者降低了100mV(vs.Ag/AgCl)。对马拉硫磷和呋喃丹的检测响应快,检测限分别可达0.01μg/mL和1.5×10-3μg/mL。通过组内偏差计算传感器的精确性、重复性和稳定性,除电极重复性外变异系数均小于5%;实际样品以黄瓜和苹果为例,将集成电极生物传感器用于农药残留检测。结果表明,实际样品基体效应对测定结果基本没有影响,有机磷和氨基甲酸酯回收率在90%~117%之间,传感器满足实际快速预警筛选需要。创新点:首先,自行设计了SPE电极、商业化批量加工后结合本文的预处理方法,使电极具有良好的化学响应,为SPE电极商业化生产提供了可能;其次,新型纳米材料MWNTs经过本文方法处理后能够均匀分散于乙醇中修饰SPE电极后,构建的这种无介体电流型酶传感器能够有效降低巯基胆碱的氧化电位(至+0.10V),不仅克服了DMF分散MWNTs有毒、挥发慢的缺点,而且氧化电压大大低于国内同期的报道;最后,研究了实际样品溶液基体效应对测定结果的影响,结果表明,在pH7.4的缓冲溶液做提取液的条件下,基体效应几乎没有影响,这于国内文献首次提出。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 human health. Now,People would pay growing awareness of food safety, since it’s extremely urgent to strengthen the detection of pesticide residues. Enzymatic biosensors (EBS) 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.In this paper, we will propose a simple and efficient method for detection trace pesticide residues based on immobilization of AChE on the multiwall carbon nanotube modified electrode, which called amperometric biosensor for screening of organophosphorus and carbamate pesticides. Using the traditional glass carbon electrode and self-design screen-printing electrode as signal converter, we investigated the influence factors and results of the biosensor that functionalized with acetylcholinesterase and modified with acid purified multi-wall carbon nanotubes. On that basis, exploit the screen-printed electrode enzyme biosensor (SPE) for measurement of real-sample that containing trace organophosphorus and carbamate pesticide residues. Work and conclusion as following:We design the thick film circle electrode, and subcontract by mass producing. With a pre-oxidation at +1.7V for 3min, the potential difference of the electrode has brought down from 0.52V to 0.15V and the current response doubled, which improved the electrode reversibility apparently. Under the mentioned pretreatment, the electrode has a good linear relationship between the reduction current and scan rate square, the related coefficient is 0.9973. Except for reduction peak potentials, the relative standard deviation of repeated measurements is less than 3%, which indicate the electrodes have good consistency and stability, provided a basis for following experiments.Using the traditional glassy carbon electrode as signal converter, an electrochemical method has been devised for investigation of pesticide. The multi-wall carbon nanotube (MWNTs) dispersed by N, N-Dimethylformamide (DMF) can promote the electron transfer effectively, and bring down the voltage for acetylcholine oxidation from +0.7V to +0.25V, reducing the interference of impurities solution. Meanwhile, optimize the experimental conditions: +0.25 V for working potential, pH7.4, 0.5% for the amount of cross-linking agent glutaraldehyde, and 1.5mmol/L for the substrate thio-acetylcholine chloride concentration. Under the optimal conditions, Pesticides of trichlorfon and methomyl are selected to discuss their inhibition efficiencies to acetylcholinesterase. Inhibition time of both pesticides is 10min. The GC/EBS for trichlorphon determination has a good linear relationship among the concentration range 0.005~100.0μg/mL, related coefficient 0.9973, and for the methomyl linear range is 0.01~100.0μg/mL, related coefficient 0.9934. The biosensor detection limit for trichlorfon and methomyl is 0.026μg/mL and 1.5×10-3μg/mL, respectively, with the whole detection time no longer than 60min. The investigation of group categories deviation is no more than 5%, demonstrated that biosensor has good precision, electrode to electrode reproducibility and stability.Based on above research efforts, we develop screen-printed electrode enzymatic biosensors (SPE/EBS) as a novel device for trace organophosphorus and carbamate pesticides measurement. The MWNTs dispersed by ethanol is used to modify the working electrode surface, which promotes the electron transfer high efficiently. Compared with GC/EBS, the SPE/EBS has a shorter response time and low detection potential (150mVvs.Ag/AgCl), 100mV lower than the GC/EBS, without any use of mediating redox species, further enhancing electrode selectivity and sensitivity. we also optimize the experimental conditions: +0.15V for working potential, pH7.4, 0.5% for the amount of cross-linking agent glutaraldehyde, and 1.4mmol/L for the substrate thio-acetylcholine chloride concentration. Under the optimal conditions, Pesticides of malathion and carbofuran are selected to discuss their inhibition efficiencies to acetylcholinesterase. Inhibition time of both pesticides is 8min. The SPE/EBS for malathion determination has a good linear relationship among the concentration range 0.005~10.0μg/mL, related coefficient 0.9926, and for the methomyl linear range is 0.001~0.1μg/mL, related coefficient 0.9874. The biosensor detection limit for malathion and carbofuran is as low as 0.01μg/mL and 1.5×10-3μg/mL, respectively. Investigation of group categories deviation to determinate the biosensor accuracy, and stability, both the characters are no more than 5%. To measure the repeatability, we use AChE/MWNTs/SPE to hydrolyze the substrate acetylcholine chloride for indirect determination. Prepare 6 identical SPE/EBS under the same conditions, and record their current responses in 1.4mmol/L acetylcholine chloride solution. The relative standard deviation (R.S.D) of 6 electrodes is 5.9%, which not satisfied and need to increase. As a result of the printing materials and technique influence, SPE electrode repeatability is always poorer than GC. However, it is disposal, inexpensive and simple operation, which allows the R.S.D less than 7% for practical application. Using the SPE/EBS to analyze the real cucumber and apple samples, SPE/EBS avoids the interference from matrix effects effectively, by phosphate buffer solution (pH 7.4) as the extracting solution. Take AChE inhibited by malathion in apple and cucumber samples for example, the accuracy, repeatability and stability of biosensors are evaluated. Conclusion is that all the relative standard deviations are less than 5%. In addition investigated the coefficient of recovery by spike recovery, which among 90%~117%, demonstrating the feasibility of the application of the sensor for rapid, sensitive, extremely inexpensive, and yet highly reproducible monitoring of pesticide residue. As a result, it could be achieved through further improvement in miniaturization of electrochemical system for toxic substances control in food and environment.Innovations: firstly, we designed the screen-printed electrode and subcontracted by mass producing, combining with the mentioned pretreatment in 2nd chapter, we got good consistency and stability for the electrodes, which would provide a basis for following experiments, also a potential possibility for future commercial application. Then the newly materials, MWNTs, could disperse in ethanol homogeneously after treated by the referred method in the paper. The SPE modified with that material has lower detection potential than GC electrodes, to be +0.1V, without any use of mediating redox species, enhancing electrode selectivity and sensitivity in further, not only overcome the shortcomings that the toxicity caused by MWNTs dispersed in DMF and time-consumed to volatilize, but also the oxidation voltage is much lower than the same period in the domestic. Finally, measurement of real sample solution was carried out to evaluate the matrix effect on the impact of the results. Conclusions is that using pH7.4 buffer solution to extract trace pesticides in samples, there is almost no effect for the final determination, which is introduced in the domestic literature for the first time.更多还原