基于DNA核酸适配体序列的超灵敏比色法检测牛乳中的雌二醇  

A Supersensitive Colorimetric Method Based on DNA Aptamers for Estradiol Detection in Milk

作　　者：付田 曹锦轩 潘道东[1,2] 程克文 曾小群 吴振 孙杨赢 FU Tian;CAO Jinxuan;PAN Daodong;CHENG Kewen;ZENG Xiaoqun;WU Zhen;SUN Yangying(Key Laboratory of Animal Protein Food Deep Processing Technology of Zhejiang Province, Ningbo 315211, China;Branch of National Dairy Processing Technology Developing Center, Nanjing Normal University, Nanjing 210097, China)

机构地区：[1]浙江省动物蛋白精深加工技术重点实验室,浙江宁波315211 [2]南京师范大学国家乳品加工技术研发分中心,江苏南京210097

出　　处：《食品科学》2017年第8期264-270,共7页Food Science

基　　金："十二五"国家科技支撑计划项目(2015BAD17B02-3);浙江省攻关项目(2014C02020)

摘　　要：分别利用最适长度的雌二醇DNA核酸适配体的特异性识别和胶体金聚集前后颜色及吸光度的变化实现雌二醇的定性和定量检测。本研究制备了胶体金,并设计了75-mer、35-mer和22-mer的雌二醇核酸适配体,依据没有核酸适配体保护的胶体金在最适氯化钠浓度条件下聚集,而有核酸适配体保护的胶体金在此浓度条件下不聚集以及雌二醇的核酸适配体与雌二醇特异性结合的特性,实现对雌二醇的超灵敏检测。结果表明,在Na Cl浓度30 mmol/L、35-mer核酸适配体质量浓度30 nmol/L条件下,雌二醇质量浓度与胶体金在625 nm和523 nm波长条件下,吸光度的比值(A_(625) nm/A_(523) nm)在13.6~54.4 pg/m L的范围内呈良好的线性关系,检测限为2.7 pg/m L。所构建的比色法的特异性、稳定性和重复性均良好。应用该方法对牛乳样品进行检测,雌二醇的最低检测限为13.6 pg/m L,因此可用于奶制品中雌二醇的快速检测。A novel method was developed for the qualitative and quantitative determination of estradiol based on its pecific recognition by the DNA aptamer of optimum length as well as the changes in the color and absorbance of colloidal old before and after aggregation.In this work,colloidal gold particles were prepared and the75-mer,35-mer and22-mer stradiol-specific nucleic acid aptamers were designed for the ultrasensitive colorimetric detection of estradiol.The olorimetric method was based on the fact that the optimal concentration of sodium chloride leads to the aggregation of olloidal gold without the protection of the aptamer,while the colloidal gold does not aggregate under the protection of the ptamer.The results showed that the concentration of estradiol in the range of13.6?54.4pg/mL exhibited a good linear elationship with the absorbance ratio(A625nm/A523nm)of colloidal gold at625nm and523nm with a limit of detection(LOD)f2.7pg/mL,under the condition of30mmol/L sodium chloride and30nmol/L35-mer nucleic acid aptamer.The developed olorimetric sensor had the advantages of high specificity,stability and reproducibility.The practicality of this proposed ethod was further confirmed through the detection of estradiol in milk samples with LOD of13.6pg/mL.Therefore,this olorimetric method will be useful for the rapid and sensitive detection of estradiol in milk products.

关 键 词：雌二醇 胶体金 DNA核酸适配体 比色法 

分 类 号：TS252.7[轻工技术与工程—农产品加工及贮藏工程]