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

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 attached to both ends of the fluorescent molecule through the arginine-containing peptide,as the enzyme and substrate in the ELISA method as follows:non-fluorescent rhodamine 110,the bis-amide fluorescent derivative was hydrolyzed by trypsin,the arginine present between the rhodamine 110 molecule and the two terminal peptide chains,and the resulting rhodamine 110 molecule was highly fluorescent.Trypsin-CIT was synthesized by formaldehyde addition,retaining 47.09%of the enzymatic activity,and after immobilization of the CIT antibody on the enzyme plate by protein G,using the ELISA was established using the competitive binding relationship between trypsin-CIT and CIT and CIT antibodies.When both were present in the assay system and the concentration of trypsin-CIT was certain,the number of trypsin-CIT bound to CIT antibodies was stronger because of the stronger binding ability of CIT and CIT antibodies,when the concentration of CIT gradually increased,the number of trypsin-CIT bound to CIT antibodies Therefore,the concentration of rhodamine bis-amide derivatives was firstly optimized in this experiment,and the concentration of the derivative that was catalytically hydrolyzed to produce the strongest fluorescence signal at a certain enzyme concentration was selected as the optimal substrate concentration,and in order to obtain the best performance of the assay,the concentration of the derivative that was catalytically hydrolyzed to produce the strongest fluorescence signal was determined by B_(0)=(1-F/F_(0))×100.In order to obtain the best performance of the assay,B_(0)=(1-F/F_(0))×100(F is the fluorescence value under negative conditions,F_(0) is the fluorescence value under 50 ng/mL spiking conditions)was used to reflect the performance of the established method.The optimal conditions were:20μg/mL of encapsulated ascites,5.5μg/mL of trypsin-CIT,pH 6.5,10%of methanol,80 mmol/L of NaCl,30 min of immunoincubation time and 30 min of enzyme catalytic time.The standard curve can be expressed by the equation y=18.606 ln x-18.713(R2=0.992).The quantitative range of the method is 12.5-400 ng/mL,with a minimum sensitivity of 8.01 ng/mL and an IC_(50) of 40 ng/mL.The IC_(50) was up to 40 ng/mL,which is 1.34 times the sensitivity and 2.741 times the IC_(50) of the horseradish peroxidase-catalyzed colorimetric ELISA.Subsequently,six common mycotoxins were assayed at high concentrations and the results showed that the method had no significant cross-reactivity with the common mycotoxins and had good specificity.The results showed that the spiked recoveries of the method ranged from 90.10%-110.58%and the intra-and inter-batch coefficients of variation ranged from 3.65%-14.08%,indicating that the method has good reproducibility and precision.The results showed that the method is suitable for the rapid and sensitive quantification of different concentrations of CIT in maize.In conclusion,this experiment has established a FELISA method for the determination of CIT in maize by introducing fluorescence signals,which has good performance and application prospects and provides a reference for subsequent use in the determination of other targets.