CN105181685A - 一种Et试剂及其应用 - Google Patents
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Abstract
本发明公开了一种Et试剂及其应用，由胆碱酯酶和5,5ˊ-二硫代双(2-硝基苯甲酸)混合而成。在农药残留检测时直接将其与样品待测液进行酶抑制反应，使得在进行酶抑制反应整个过程仅需三种试剂即可完成，检测方法更加简便，并减少实验器材的使用和环境的污染。
Description
技术领域
本发明属于分析化学领域，具体涉及一种Et试剂及其应用。
背景技术
DTNB是5,5'-二硫代双(2-硝基苯甲酸)(5,5'-Dithiobis-(2-nitrobenzoicacid))的简称。一方面它和碳酸氢钠或磷酸盐缓冲溶液以一定的比例混合后用作产物乙酰胆碱的显色剂，显黄色，通常用于农残检测，以吸光度的大小来反映酶活性的大小并计算胆碱酯酶的抑制率值，最终用来初步判断农药是否超标。另一方面常用于检测游离的巯基（-SH），此时也称作ELLman试剂。
在现有的农药残留快速检测方法中，DTNB是作为一种检测胆碱酯酶活性的单独使用显色剂，样品中的农药与胆碱酯酶作用抑制其酶活性后，再加入DTNB显色剂和底物后放到农药残留检测仪上检测胆碱酯酶的活性并计算胆碱酯酶的抑制率值。无论在农药残留检测还是在酶活性检测过程中还未见有将DTNB与胆碱酯酶复合成一种应用于农药残留检测的专用试剂的报道。
发明内容
本发明的目的在于提供一种Et试剂及其应用，由胆碱酯酶和5,5'-二硫代双(2-硝基苯甲酸)混合而成一种应用于食品安全检测和生化检测的Et试剂。在运用胆碱酯酶抑制法检测农药残留时只需吸取一次量的Et试剂，既可完成两套器皿对两种试剂的加样操作。为了解决胆碱酯酶与DTNB复合所引起的胆碱酯活性的降低失活，采用牛血清白蛋白和海藻糖作为保护剂，以及牛磺胆酸钠作为稳定剂使Et试剂的稳定性很好，在（0~5℃）的温度下可以保存一年以上，胆碱酯酶的活性没有明显降低，且可很好地应用于不同样品中的农药残留的快速检测。
为实现上述目的，本发明采用如下技术方案：
一种Et试剂，由胆碱酯酶和5,5'-二硫代双(2-硝基苯甲酸)混合而成，两者的重量比为1︰40～100。
所述的Et试剂用于动植物材料及食品、饮品、土壤中的有机磷和氨基甲酸酯类农药残留检测。
本发明的显著优点在于：在动植物材料及食品、饮品、土壤中的农药残留检测时直接将Et试剂与样品待测液进行酶抑制反应，在运用胆碱酯酶抑制法检测农药残留时只需吸取一次量的Et试剂，既可完成两套器皿对两种试剂的加样操作，使得操作过程更加简便，检测方法更加实用易于推广普及，并减少实验器材的使用和试剂的营运成本。
具体实施方式
实施例1
一种茶叶或茶青中农药残留的检测方法：茶叶或茶青中有机磷和氨基甲酸酯类农药残留的检测步骤：
（1）试剂的配制：
Et试剂：2mg（或1ku）胆碱酯酶溶解于500ml磷酸盐缓冲溶液（称取磷酸氢二钠5~15g和磷酸二氢钾0.2~0.9g于试剂瓶中加入500mL蒸馏水溶解，混匀；）中，加入2～10g牛血清白蛋白和3～15g海藻糖搅拌溶解；另称取80～200mg的DTNB和0.5～5g的牛磺胆酸钠加入，并搅拌至完全溶解后即成为Et试剂，将其封装在5ml的安瓿中并置于冰箱（0～5℃）保存。使用时取出开封即可。
Sub试剂：称取分析纯的碘化硫代乙酰胆碱10~60mg加入10mL蒸馏水，摇匀使之溶解；
农药提取液：吸取100~300mL的丙三醇加入50mL的蒸馏水混匀后再加入160mL95wt.%乙醇溶液，混匀；
缓冲液：称取磷酸氢二钠5~15g和磷酸二氢钾0.2~0.9g于试剂瓶中加入500mL蒸馏水溶解，混匀；
（2）检测
样品提取：
称取0.5克茶叶样品，加入1.0mL农药提取液，使样品充分浸润并静置10分钟，底部溶液即为待测液，作为检测之用；
对照测定：
吸取Et试剂100μL，加入1.5mL缓冲液，摇匀，加入200μL农药提取液，摇匀，静置10分钟，加入100μLSub试剂，摇匀后移入0.5cm比色皿，及时放入农药测试仪进行对照测试；
样品测定：
吸取Et试剂100μL，加入1.5mL缓冲液，摇匀，加入200μL待测液，摇匀，静置反应10分钟，加入100μLSub试剂，摇匀后移入0.5cm比色皿，及时放入农药测试仪进行样品测试；
测定结果的判定：
当检测结果的酶抑制率读数超过50%时，表明样品农药残留毒性可能超过安全标准，应当确认操作步骤无误、仪器工作正常、试剂没有污染失效，重新进行对照测试并对此样品进行2次以上的重复检测，如检测结果重复并仍然超过50%，则需用气相色谱作进一步的确认。
在铁观音茶叶和实验室栽种的无农药的茶青进行农药加样提取测定，得到的不同农药的检测灵敏度如下：
实施例2
果蔬中农药残留的快速检测方法：
（1）试剂的配制：
Et试剂：2mg（或1ku）胆碱酯酶溶解于500ml磷酸盐缓冲溶液（称取磷酸氢二钠5~15g和磷酸二氢钾0.2~0.9g于试剂瓶中加入500mL蒸馏水溶解，混匀；）中，加入2～10g牛血清白蛋白和3～15g海藻糖搅拌溶解；另称取80～200mg的DTNB和0.5～5g的牛磺胆酸钠加入，并搅拌至完全溶解后即成为Et试剂，将其封装在5ml的安瓿中并置于冰箱（0～5℃）保存。使用时取出开封即可。
Sub试剂：称取分析纯的碘化硫代乙酰胆碱10~60mg加入10mL蒸馏水，摇匀使之溶解；
缓冲液：称取磷酸氢二钠5~15g和磷酸二氢钾0.2~0.9g于试剂瓶中加入500mL蒸馏水溶解，混匀；
（2）检测
样品提取：取2g果蔬样品（叶菜剪成宽度为1厘米左右的菜样，块根菜取横截面样品）放入三角瓶中，加入10mL缓冲液，振荡提取２分钟后过滤，取出清液即为待测试液。
对照测试：取Et试剂100μL，缓冲液2.5mL，加入专用反应瓶中，混匀，再加入Sub试剂100μL。摇匀后立即转移到比色皿中，及时放入仪器，按对照测试键进行对照测试。
样品测试：取Et试剂100μL，待测试液2.5mL，加入专用反应瓶中，混匀静置抑制10分钟（秒表计时）后，再加入Sub试剂100μL。摇匀后立即转移到比色皿中，及时放入仪器，按样品测试键进行测试。
测试结果的判读：若被测样品的酶抑制率读数超过50（即抑制率达到50％以上），则表明被测样品的农药残留毒性可能超过安全的界定标准，需用气相色谱等仪器分析法作进一步的确认。
应用上述的检测方法和配套检测试剂进行果蔬中的农药残留快速检测，所得到的样品检测灵敏度如下：
以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围。
Claims (4)
1.一种Et试剂，其特征在于：由胆碱酯酶和5,5'-二硫代双(2-硝基苯甲酸)混合而成。
2.根据权利要求1所述的Et试剂，其特征在于：胆碱酯酶和5,5'-二硫代双(2-硝基苯甲酸)的重量比为1︰40~100。
3.一种如权利要求1所述的Et试剂的应用，其特征在于：所述的Et试剂用于动植物材料及食品、饮品、土壤中的农药残留检测。
4.根据权利要求3所述的应用，其特征在于：所述的农药为有机磷和氨基甲酸酯类农药。
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