胰蛋白酶催化罗丹明110双酰胺衍生物荧光酶联免疫吸附方法检测桔青霉素  

Fluorescence ELISA based on trypsin-catalyzed rhodamine 110 bis-amide derivative for the detection of citrinin

作　　者：邹回 聂丽娟 熊勇华[1,2] 李响敏[1,2] ZOU Hui;NIE Lijuan;XIONG Yonghua;LI Xiangmin(State key Laboratory of Food Science and Technology,Nanchang University,Nanchang 330047,China;Jiangxi-OAI Joint Research Institute,Nanchang University,Nanchang 330047,China)

机构地区：[1]南昌大学食品科学与技术国家重点实验室,江西南昌330047 [2]南昌大学中德联合研究院,江西南昌330047

出　　处：《食品与发酵工业》2022年第11期246-252,共7页Food and Fermentation Industries

基　　金：“十三五”国家重点研发计划项目(2018YFC1602203)。

摘　　要：桔青霉素(citrinin,CIT)是一种广泛污染玉米、大米等农作物,且对人和动物具有肾毒性、致畸性等毒害的真菌毒素。目前的国家标准检测方法中,需要先用免疫亲和柱或固相萃取柱净化,再用配荧光检测器的高效液相色谱仪测定,需要专业的大型仪器和检测场所。因此,有必要开发一种更简便、灵敏的方法用于谷物中CIT的检测。该实验构建了一种胰蛋白酶催化的荧光酶联免疫吸附方法用于玉米中CIT的检测。该方法利用胰蛋白酶催化罗丹明110双酰胺荧光衍生物裂解产生荧光,以及CIT和胰蛋白酶-CIT与CIT抗体的竞争结合关系,通过记录竞争抑制率B_(0)与CIT浓度的变化关系,从而实现对玉米中CIT的定量检测,可用方程y=18.606 lnx-18.713(R^(2)=0.992),定量范围为12.5~400 ng/mL,通过检测4个不同CIT含量(0.22~3.5 mg/kg)的玉米样本,结果显示该方法的加标回收率为90.10%~110.58%,批内、批间的变异系数为3.65%~14.08%,该研究所构建的荧光酶联免疫吸附方法适用于玉米中不同浓度的CIT快速、灵敏定量检测。Citrinin(CIT)is a fungal toxin that is widely found in crops such as maize and rice and is toxic to animals in terms of nephrotoxicity,genotoxicity,embryo toxicity and teratogenicity.In addition,CIT contamination in crops such as maize and fruit has caused large losses to the production economies of industrialized countries.In view of its harmful effects on human health and economic development,it is important to develop a sensitive and rapid method for the detection of CIT.Currently,Japan and the European Union have issued limits for CIT,while China's Mainland has not yet issued limits for CIT and has only issued methods for its detection.In the national standard detection method,it is necessary to clean up with an immunoaffinity column or a solid phase extraction column,and then use HPLC with fluorescence detector to determine it.Based on the limitations of large instrumental methods and the current situation of CIT contamination,it is necessary to develop a simpler and more sensitive method for the determination of CIT in cereals.As a sensitive,high-throughput,simple and low-cost method for sample screening and quantification,enzyme-linked immunosorbent assay(ELISA)has been rapidly developed and widely used in recent years.Traditional ELISA methods are based on horseradish peroxidase-catalyzed chromogenic substrates and thus quantitative detection of the analytes by colorimetric signals,which are susceptible to large interferences during the detection of complex substrates,thus affecting the accuracy of the results.The ELISA method,which uses fluorescence as the signal output,is considered to be one of the most sensitive methods for the screening of hazardous foodborne substances due to its ability to resist substrate interference and its high sensitivity.In order to achieve high sensitivity,a fluorescent ELISA(FELISA)method was constructed for the detection of CIT in maize by introducing trypsin,which has high specificity for the arginine site in the peptide,and a rhodamine 110 bis-amide derivative,which is a

关 键 词：荧光酶联免疫吸附方法 胰蛋白酶 罗丹明110双酰胺衍生物 桔青霉素 

分 类 号：O657[理学—分析化学] TS210.7[理学—化学]