摘要 核酸适体作为一种新型分子识别元件,具有可体外合成、易修饰、稳定性好等优势,被认为是抗体的良好替代物。自玉米赤霉烯酮(ZEA)核酸适体被成功筛选以来,基于核酸适体与靶标的高特异性结合,构建了酶联适体间接竞争法检测玉米赤霉烯酮的新方法。ZEA与ZEA适体互补链DNA1竞争性结合酶标板上固定的ZEA适体,未结合的DNA1链被DNA2链捕获并标记辣根过氧化物酶,通过酶联显色反应实现对ZEA的可视化检测。优化了包被亲和素的质量浓度、封闭液、适体及互补链浓度等关键参数,并考察了酶联适体竞争法检测ZEA的灵敏度、特异性和实用性。结果表明:在0.5~100 ng/m L范围内,ZEA的质量浓度与反应体系的吸光度呈良好的线性关系(R2=0.995 9),检出限为0.44 ng/m L。该方法的特异性良好,对玉米和玉米油的检测与酶联免疫试剂盒测定的结果一致,具有简便、经济和高灵敏度的特点。 As a new type of molecular recognition element,aptamer has the advantages of being able to be synthesized in vitro,easy to modify,and good stability. It is also considered as a good substitute for antibodies.Since the zearalenone aptamer was successfully screened,based on the high affinity and specific binding of the aptamer to the target,an enzyme-linked aptamer( ELAA) indirect competition method was developed for rapid detection of zearalenone( ZEA). Through the combination of biotin and streptavidin,aptamer was immobilized on the microplate 1;DNA1 was a partially complementary strand of the aptamer labeled with horseradish peroxidase( HRP);DNA2 was completely complementary to DNA1 single-stranded and fixed to the microplate 2 by biotin and avidin. When the sample has no ZEA,DNA1 and aptamer are partially complementary to each other and then fixed on the microplate 1. After adding TMB to the microplate 2,the color will not develop;when the sample contains ZEA,the target will specifically bind to the aptamer,making DNA1 melt in a free state. DNA1 solution was pipetted to the microplate 2,the DNA1 labeled with HRP is captured by the DNA2 in the microplate 2 based on the principle of double-strand complementary pairing,catalyzing the TMB substrate turn to blue,and H2SO4 was added to stop the reactionand the color will turn to yellow. The absorbance was detected at the wavelength of 450 nm. Therefore,the higher the ZEA content,the more it binds to the ZEA aptamer,and the more free DNA1,that is,the more DNA1 captured on the microplate 2,and the deeper the color of the catalytic TMB and the greater the absorbance value. The result of the enzyme-catalyzed color reaction was used for ZEA detection. Key parameters,such as streptavidin,blocking solution,aptamer and complementary chain concentration were optimized.The optimized conditions were as follows: dry coating method was used;SA was 4 μg/mL;mass fraction of BSA was 2%;aptamer was 270 nmol/L;Bio-DNA1 was 10 nmol/L;Bio-DNA2 was 90 nmol/L. Moreover,the sensitivity,sp
机构地区 河南工业大学粮油食品学院
出处 《河南工业大学学报:自然科学版》 CAS 北大核心 2021年第1期56-62,共7页 Journal of Henan University of Technology:Natural Science Edition
基金 国家自然科学基金资助项目(32001808) 河南省科技攻关项目(202102310469)。
