固定化对动植物酯酶性质的影响

比较了离子交换法得到的固定化小麦酯酶和固定化鸡肝酯酶的主要性质与相应的游离酶的性质。结果表明 ,固定化小麦酯酶的最适pH在 5 5附近 ,比游离小麦酶的最适 pH增加了0 8个 pH单位 ;固定化鸡肝酯酶的最适pH在 8 0附近 ,比游离鸡肝酯酶的最适 pH增加了 0 5个pH单位 ;固定化小麦酯酶的最适温度与游离酶的相当 ,但酶活力 温度曲线的峰形变小 ;固定化鸡肝酯酶的最适温度比游离鸡肝酯酶的下降了 5℃ ,酶活力 温度曲线的峰形也略变陡峭。这 2种固定化酯酶对有机磷类农药 (敌敌畏 )抑制的响应明显高于游离酶对农药抑制的响应     

农药残留快速检测固定化酶片的研究

采用硝酸纤维素膜(Nc)和植物酯酶研究了农药残留快速检测固定化酶片.通过对酶用量、戊二醛浓度、牛血清白蛋白浓度、固定化pH值和温度的参数优化,建立了NC膜固定化植物酯酶的方法,即:20μl游离酶,0.05%戊二醛2μl,0.5%牛血清白蛋白15μl,pH7.0缓冲液体系,4℃下固定8h.对游离酶和固定化酶的特性进行了比较,发现固定化植物酯酶对底物的最适反应温度为35℃,比游离酶提高了5℃;最适pH为8.0,比游离酶提高了0.5个pH单位.游离酶和固定化酶对四种农药(敌敌畏、灭多威、氧乐果、久效磷)的敏感性进行了比较,发现固定化酶对四种农药敏感性都较游离酶有所提高,固定化酶更适合于农药的快速检测.     

电沉积法构建AChE/COD@AuNPs共固定化酶生物传感器差分脉冲伏安法快速检测有机磷农药

有机磷农药残留危害环境和人类健康。本文采用电沉积法将乙酰胆碱酯酶(AChE)与胆碱氧化酶(COD)固定化,共修饰于裸金电极表面,构建新型固定化酶生物传感器。通过电子显微镜对固定化酶形貌进行表征,结果表明：当固定化酶生物传感器上,乙酰胆碱酯酶与乙酰胆碱氧化酶的共修饰层数为2层,修饰圈数为35圈,在氯化乙酰胆碱浓度为2 mmol/L,pH 7.8的检测体系中,该生物传感器能够识别有机磷农药信号,且检测性能尚佳。传感器在有机磷农药质量浓度为10^-9～10-7mg/L时电流响应良好,工作曲线方程为y=0.0468x+1.2124(R²=0.9985),最低检出限1.15×10^-11 mg/L(S/N=3)。本研究为现场快速检测有机磷农药提供了新的研究方法。     

小麦酯酶提取及其对有机磷农药检测初探

研究从小麦面粉提取小麦酯酶提取条件及其粗酶液对4种有机磷农药最低检测限和灵敏度。运用单因素实验和正交实验对小麦酯酶提取条件进行优化,确定适宜提取条件,提取液为0.15 mol/L NaCl溶液、料液比1∶4、40℃振荡提取60 min,提取粗酶液酶活为1.675 U/mL;并确定粗酶液对敌敌畏、敌百虫、乐果和毒死蜱等4种有机磷农药最低检测限分别为0.05 mg/kg、0.04 mg/kg、0.05 mg/kg和0.07 mg/kg,均低于国家规定农药最大残留量,且粗酶液对敌敌畏灵敏度最佳。     

流动注射式酶生物传感器检测蔬菜中农药残留的定量标准的研究

本文基于酶抑制原理,采用固定化鸡肝酯酶和自行研制的流动注射式酶生物传感器,研究了不同农药对酶的抑制程度,确定了甲胺磷作为农药检测的定量标准。同时,针对不同的抑制前的初始酶活,绘制了相应的抑制率--甲胺磷含量的标准曲线。并在此基础上,选用了甲胺磷、氧化乐果、乙酰甲胺磷、毒死蜱、西维因和灭多威作为强化农药,对添加了一定强化农药量的黄瓜、青菜进行了定量测定,发现通过标准的建立可以较真实地反映样品中的农药残留量。     

小麦酯酶的纯化、冻干及对有机磷农药的检测

该文研究从小麦面粉提取的小麦酯酶粗酶液的纯化、冻干和其有机磷农药的最低检测限。确定了用饱和度为30%和60%的硫酸铵对小麦酯酶粗酶液进行分段盐析,纯化后酶液的比酶活是纯化前的2.4倍;最佳保护剂为5%的聚乙二醇,它不仅可以有效保护纯化后的小麦酯酶在冷冻干燥过程中不失活,而且还具有良好的稳定性;最后确定了冻干小麦酯酶酶粉中敌敌畏、敌百虫、毒死蜱、乐果和辛硫磷等5种有机磷农药最低检测限分别为0.01 mg/kg、0.02 mg/kg、0.05 mg/kg、0.07 mg/kg和0.03 mg/kg,均低于国家规定农药最大残留量,冻干酶粉对敌敌畏灵敏度最佳。     

驴血清胆碱酯酶抑制法快速检测蔬菜中农药残留

采用驴血清丁酰胆碱酯酶作为酶源,对不同的有机磷农药的检测条件和方法进行了研究,同时通过酶抑制法对锦州地区的部分蔬菜进行了农药残留检测.结果表明:乐果、敌百虫、敌敌畏、马拉硫磷对驴血清丁酰胆碱酯酶(BChE)的IC50分别为0.094、0.179、0.124、0.0078mg/mL,驴血清BChE对这4种有机磷农药灵敏度的大小顺序为:马拉硫磷＞乐果＞敌敌畏＞敌百虫 乐果、敌敌畏对丁酰胆碱酯酶的最佳抑制时间为15 min;敌百虫、马拉硫磷对丁酰胆碱酯酶的最佳抑制时间为20min 用酶抑制法检测3个农贸市场的蔬菜样品,发现韭菜有2例检测结果超标,油菜有1例检测结果超标,尖椒有2例检测结果超标;蔬菜样品合格率为86.1％.     

采用酶偶联体系量热法检测蔬菜中农药残留的实验研究

通过筛选合适的酚氧化酶,与能被农药抑制的鸡肝酯酶组成了可用于农残检测的酶偶联体系。同时采用Micro DSCⅢ量热法,分别针对鸡肝酯酶单酶体系和该酶偶联体系,比较研究了1mg/L的敌百虫溶液和几种蔬菜中的农药残留引起这两种酶体系催化反应放热的变化。结果发现,1mg/L的敌百虫导致酶偶联体系催化反应放热的变化是其导致单酶体系催化反应热量变化的48倍。同时通过抑制率计算分析,发现针对相同样品,采用酶偶联体系,可以获得较高的抑制率响应。     

用于有机磷农药残留快速检测的固定化小麦酯酶研究

以大孔径强碱性阴离子交换树脂作为固定化载体，采用离子结合法，对植物粗酶液进行固定化。在室温及酶液对载体比例为4(ml)：1(g)的条件下，对酶固定化的环境pH值、缓冲液浓度及固定化特性进行了研究。得到的固定化酶对l0^-7mol/L水平下敌敌畏的抑制仍有明显的响应，这一效果明显优于游离酶的抑制响应。     

豌豆酯酶的纯化和酯酶活性表征及其对农药的敏感性

由于食品和环境中的农药残留对人体健康构成威胁,使得发展农残快速检测方法显得十分重要,从植物中发现新的酶源有助于降低酶抑制法快速检测农药的成本。实验从豌豆中分离纯化豌豆酯酶,并进行了酶类鉴别、特性表征和农药敏感性研究。纯化后的豌豆酯酶分子质量约为26. 0 k Da,具有较高的酯酶活性。通过底物和抑制剂特异性试验,将豌豆酯酶分类为羧酸酯酶。豌豆酯酶在质量浓度为0. 45μg/m L,缓冲液p H 6. 5和反应温度40℃时,表现出最好的催化活性。豌豆酯酶可以被有机磷和氨基甲酸酯类农药抑制。豌豆酯酶属于B型酯酶,可用于酶抑制法检测有机磷和氨基甲酸酯类农药残留,且经过纯化的豌豆酯酶满足大多数农药最大残留限值(MRL)检测的要求。     

Performance comparison of free and immobilised chicken liver esterase inhibited by four different pesticides

BACKGROUND: In enzyme inhibition‐based biosensors for the detection of organophosphate and carbamate pesticides, the biological element is the immobilised esterase which is inhibited selectively by the pesticides. The free chicken liver esterase, which possesses comparable inhibition response to the pesticides as AChE, has been reported. However, the responses to the pesticides are different between the free and immobilised enzyme due to the diffusion limitation. Therefore, in this study four typical pesticides were selected to compare the ability of free and (ion exchange) immobilised chicken enzyme for pesticide detection and reasons for the difference were investigated. RESULTS: For dichlorvos and malathion at a concentration of 0.1 mg L^?1, the inhibition of the immobilised enzyme was 26.98% and 48.72%, respectively, higher than that for the free enzyme, while the percentage inhibition of free and immobilised enzyme differed very little for trichlorfon at 0.1 mg L^?1. In the meantime, carbaryl at a concentration of 2.5 mg L^?1 showed a 17.72% inhibition for immobilised enzyme which was 13.64% higher than that for the free enzyme. The Michaelis constant of immobilised enzyme was lower than that of the free one and suitable pH values for the free and immobilised enzyme were 7.5 and 8.0, respectively. CONCLUSIONS: The sensitivity of chicken liver esterase to the pesticide inhibition could be improved by immobilisation. Smaller K_m and a lower pH for the micro‐environment of immobilised enzyme should result in higher percentage inhibition compared with that of the free enzyme. Copyright © 2008 Society of Chemical Industry     

影响农残快检仪抑制率测定结果的因素

考察不同因素对农残快检仪抑制率的影响。以GDYN-1096SC型农药残留快检仪为例,考察快检仪的重复性、稳定性、吸光度线性、通道一致性4个技术参数,并使用乙酰胆碱酯酶和小麦酯酶两种来源不同的酶,分别对甲胺磷、对硫磷、敌敌畏、氧化乐果、毒死蜱、甲萘威、灭多威、克百威8种农药进行检测。结果显示：农残快检仪各技术参数在满足测试要求的情况下,不影响抑制率;来源不同的酶试剂是影响快检仪抑制率测定结果的主要因素。     

Simultaneous determination of residues of trichlorfon and dichlorvos in animal tissues by LC-MS/MS

Trichlorfon is a thermally unstable insecticide that can be easily decomposed to dichlorvos at high temperatures. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for simultaneous determination of the residues of trichlorfon and dichlorvos in animal tissues. Trichlorfon and dichlorvos in animal tissues were extracted with dichloromethane, homogenized/shaken, concentrated, and determined by LC-MS/MS. The two insecticides can be effectively separated and individually determined. The limits of detection (LODs) of trichlorfon and dichlorvos were 0.04 and 0.07 µg kg^?1, respectively; the limits of quantification (LOQs) of the two insecticides were both 5 µg kg^?1. The average recoveries for three spiked blank samples at 10, 20 and 40 µg kg^?1 were in the range 85–106%, with relative standard deviations (RSDs) below 10.6%, which falls in a reasonable range for the analysis of the two insecticides in animal-derived foods. The method is fit-for-purpose for the simultaneous determination of residues of trichlorfon and dichlorvos in animal tissues.     

基于多克隆抗体包被磁性纳米粒子的ELISA法检测西维因

建立一种基于抗体包被氨基化Fe3O4磁性纳米粒子,建立免疫磁珠酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)法用于检测氨基甲酸酯农药西维因。结果表明:在最佳条件下,西维因质量浓度在1×10-3~10 mg/L范围内,建立的免疫磁珠ELISA法具有较好的线性关系(y=8.87lnx+72.77,R2=0.994),抑制率最高可达90.6%;测得的半抑制浓度(half maximal inhibitory concentration,IC50)为0.077 mg/L,检出限(IC15)为1.48×10-3 mg/L。以质量浓度1 mg/L的西维因在相同条件下重复检测3次,相对标准偏差为1.67%。以大米和卷心菜为实际样品进行西维因加标回收率实验,通过免疫磁珠ELISA法测得的回收率为70.5%~123.1%,同时采用高效液相色谱法进行相关性验证,结果表明,2种方法检测结果的相关性较好(R2=0.91)。通过一系列分析表明,所建立的抗体包被磁性纳米粒子ELISA法可用于快速检测西维因。     

一种商品化酶抑制-比色法快速检测农药残留试剂盒对10种有机磷农药的敏感性测定

目的了解酶抑制-比色法快速检测试剂盒对不同农药的敏感性。方法通过使用一种商品化酶抑制-比色法试剂盒对敌敌畏等10种农药残留分别迚行5个浓度的测定,用抑制率和农药浓度的常用对数拟合回归曲线,同时计算对应的IC50和IC10。结果商品化的酶抑制-比色法试剂盒不是对所有的有机磷农药都敏感,即不是蔬菜中的所有的有机磷农药用酶抑制法都可以测定出来;该试剂盒对10种农药残留的敏感性依照敌敌畏、敌百虫、辛硫磷、喹硫磷、巴胺磷的顺序依次降低;试剂盒对地虫磷、杀螟硫磷、乐果、甲基嘧啶磷、倍硫磷不敏感,该试剂盒对含有浓度为3mg/kg以下的这些农药的样品没有响应。结论建议在日常检测中只要抑制率高于10%的样品建议复检,同时使用色谱、质谱定性定量。     

Cutinase inhibition by means of insecticidal organophosphates and carbamates Part 2: screening of representative insecticides on cutinase activity

Representative organophosphorus and carbamate insecticides were subjected to a recently developed, efficient spectrophotometric cutinase assay. The inactivation ability of ten organophosphate oxons [chlorpyrifos oxon, chlorpyrifos-methyl oxon, paraoxon, paraoxon-methyl, malaoxon, dichlorvos, monocrotophos, chlorfenvinphos, demeton-S-methyl, acephate], five organophosphate thions [chlorpyrifos, chlorpyrifos-methyl, parathion, parathion-methyl, malathion] and six carbamates [methomyl, carbaryl, propoxur, carbofuran, ethiofencarb, pirimicarb] was examined and characterized in terms of inhibitory rate constants. Regarding their strengths as cutinase inhibitors, organophosphate oxons were found to exceed by far the corresponding thions, whereas ethyl esters proved to be superior to their methyl analogues. Chlorpyrifos oxon with an inhibition constant k _i of 9.4 × 10⁵ L/(mol min) was identified as strongest cutinase inhibitor, resulting in a detection limit of 2 μg/L (standard solution/sample extract). As novel result it is established that also carbamates are cutinase inhibitors, though of minor strength as compared to organophosphate oxons. Most efficient carbamates are methomyl and carbaryl with inhibition constants of 7.5 × 10² and 2.6 × 10² L/(mol min), respectively, i.e., well in the range of organophosphorous insecticides.