酶联核酸适配体比色法检测奶粉中的乳铁蛋白  

Detection of Lactoferrin in Milk Powder by Enzyme-linked Nucleic Acid Aptamer Colorimetric Method

作　　者：冯子如 朱超 江利华 贺彦爽 颜朦朦 李腾飞 FENG Ziru;ZHU Chao;JIANG Lihua;HE Yanshuang;YAN Mengmeng;LI Tengfei(Laboratory of Natural Products and Functional Food Development,School of Life Sciences and Food Engineering,Hebei University of Engineering,Handan 056038,Hebei,China;Institute of Quality Standards and Testing Technology for Agro-products,Shandong Academy of Agricultural Sciences,Jinan 250100,Shandong,China)

机构地区：[1]河北工程大学生命科学与食品工程学院,天然产物与功能食品开发重点实验室,河北邯郸056038 [2]山东省农业科学院农业质量标准与检测技术研究所,山东济南250100

出　　处：《食品研究与开发》2024年第23期126-132,共7页Food Research and Development

基　　金：国家自然科学基金项目(22304106)。

摘　　要：建立一种酶联核酸适配体间接竞争型比色法检测奶粉中的乳铁蛋白(lactoferrin,Lf)。生物素化Lf适配体通过链霉亲和素-生物素作用与辣根过氧化物酶(horseradish peroxidase,HRP)缀合形成检测探针,与样品溶液混合孵育后加入96孔微孔板,样品中的Lf与提前包被在微孔板内的Lf共同竞争结合检测探针,洗涤后结合微孔板内Lf的检测探针中HRP高效催化底物3,3′,5,5′-四甲基联苯胺显色,进而间接实现样品中Lf的定性定量检测。在磷酸缓冲盐溶液及基质体系中,Lf浓度在50~300 nmol/L内均表现出良好的线性关系,相关系数(R^(2))分别为0.9933、0.9967,特异性较好,检出限为24.45 nmol/L,加标回收率91.3%~115.4%。采用该方法对3种市售奶粉开展Lf检测,并使用高效毛细管电泳法进行验证,一致率为94.39%~109.50%。该方法操作简单,成本较低,灵敏度较好,有望实现奶粉中Lf商业化检测。An indirect competitive enzyme-linked aptamer colorimetric method for the detection of lactoferrin(Lf)in milk powder was established.A biotinylated Lf aptamer was conjugated with horseradish peroxidase(HRP)via streptavidin-biotin interaction to form a detection probe.After incubation with the sample solution,the mixture was added to a 96-well microplate.The Lf in the sample competed with the Lf pre-coated on the microplate to bind the detection probe.After washing,the HRP in the detection probe bound to the Lf in the microplate efficiently catalyzed the substrate 3,3′,5,5′-tetramethylbenzidine(TMB)for color development,thereby indirectly achieving qualitative and quantitative detection of Lf in the sample.In phosphate-buffered saline and matrix systems,the method showed a good linear relationship in the Lf concentration range of 50-300 nmol/L,with correlation coefficients(R~2)of 0.993 3 and 0.996 7,respectively.The specificity was good,with a detection limit of 24.45 nmol/L,and the recovery rate of the added standard ranged from 91.3% to115.4%.The method was used to detect the Lf content in three commercially available milk powders,and validation using high-performance capillary electrophoresis(HPCE)showed a concordance rate of 94.39%-109.50%.This method was simple to operate,cost-effective,and highly sensitive,and was expected to enable the commercial detection of Lf in milk powder.

关 键 词：适配体 婴幼儿配方奶粉 乳铁蛋白 酶联适配体 比色法 

分 类 号：TS252.51[轻工技术与工程—农产品加工及贮藏工程] O657.3[轻工技术与工程—食品科学与工程]