基于适配体-杂交链式反应比色检测生鲜牛乳中四环素类抗生素

Colorimetric Detection of Tetracyclines in Fresh Milk Based on Aptamer-Hybridization Chain Reaction

)以特异性识别四环素类抗生素(TCs)的广谱型适配体为识别元件,结合杂交链式反应(HCR)信号放大策略,提出了一种TCs多残留比色检测方法,并优化了检测条件和进行了方法学考察。取40 nmol·L^(-1)生物素化检测探针(bio-DP)溶液加入到包被有亲和素的酶标板中,室温孵育后加入牛血清白蛋白(BSA)溶液,封闭反应1 h。加入生鲜牛乳样品稀释液,室温孵育20 min,如果样品中含有TCs,TCs与bio-DP的适配体序列结合,其发夹结构被打开。加入200 nmol·L^(-1)生物素化发夹DNA1(bio-H1)溶液和200 nmol·L^(-1)生物素化发夹DNA2(bio-H2)溶液,室温下进行HCR 40 min,从而形成具有多个重复单元的双链DNA(dsDNA)纳米线。加入辣根过氧化物酶(HRP)标记链霉亲和素(SA-HRP),室温孵育标记dsDNA。加入显色剂[含3,3′,5,5′-四甲基联苯胺(TMB)],HRP催化TMB生成蓝色物质,显色5~8 min后终止反应,在酶标仪中于450 nm测量上述体系的吸光度。结果显示,bio-DP对四环素、土霉素、金霉素和多西环素具有高特异性,和卡那霉素、庆大霉素、氨苄青霉素、泰乐菌素、磺胺嘧啶和恩诺沙星等抗生素无交叉反应。四环素、土霉素、金霉素和多西环素的质量浓度总和在0.8~100μg·L^(-1)内与对应的吸光度总和呈线性关系,检出限(3s/k)为0.18μg·L^(-1)。对生鲜牛乳样品进行加标回收试验,TCs回收率为86.4%~106%,测定值的相对标准偏差(n=3)为2.5%~7.1%。方法应用于生鲜牛乳样品的分析,检测结果与国家标准方法GB 31658.6-2021差异不显著(P>0.05),检出的TCs总量也均未超标(GB 31650-2019)。与文献报道的其他方法相比,上述方法兼具简单、快速、检出限低、准确度高等优点,适用于食品中四环素类抗生素多残留的快速检测。

A colorimetric method for the detection of multi-residues of tetracyclines(TCs)was proposed by using broad-spectrum aptamer against TCs as the recognition element,and hybridization chain reaction(HCR)as signal amplification strategy.The detection conditions were optimized and methodological studies were carried out.The 40 nmol·L^(-1)biotinylated detection probe(bio-DP)solution was added to enzyme plate coated with avidin,and the mixture was incubated at room temperature.The enzyme plate was blocked with bovine serum albumin(BSA)solution for 1 h.The flesh milk sample diluent was added,and the mixture was incubated at room temperature for 20 min.When the TCs were present in the sample,TCs were captured by the aptamer sequence of bio-DP,and the hairpin structure would be opened.Then 200 nmol·L^(-1)biotinylated hairpin DNA1(bio-H1)solution and 200 nmol·L^(-1)biotinylated hairpin DNA2(bio-H2)solution were added,and the HCR was performed at room temperature for 40 min to form double stranded DNA(dsDNA)nanowires with multiple repeating units.Horseradish peroxidase(HRP)labeled streptavidin(SA-HRP)was added,and the mixture was incubated to label dsDNA at room temperature.The chromogenic agent containing 3,3′,5,5′-tetramethylbenzidine(TMB)was added,and catalyzed to format blue reactive organisms by HRP.The coloration reaction was terminated after 5-8 min,and the absorbance of the above system was measured at 450 nm by an enzyme-labelling measuring instrument.As found by the results,the bio-DP had high specificity for tetracycline,oxytetracycline,aureomycin and doxycycline,and had no cross reactivity with antibiotics such as kanamycin,gentamicin,ampicillin,tylosin,sulfadiazine and enrofloxacin.Linear relationships between values of the mass concentration sum and the absorbance sum of tetracycline,oxytetracycline,aureomycin,and doxycycline were kept in the range of 0.8-100μg·L^(-1),with detection limits(3s/k)of 0.18μg·L^(-1).Test for recovery was made by the standard addition method on actual fresh milk samples,giving recoveries in the range of 86.4%-106%,and RSDs(n=3)of the determined values ranged from 2.5%to 7.1%.The proposed method was applied to the analysis of fresh milk samples,and there was no significant difference(P>0.05)between the detection results of the proposed method and the national standard method of GB 31658.6-2021,and the detected amount of TCs detected did not exceed the standard(GB 31650-2019).Compared with other methods reported in the literatures,the above method had advantages of simplicity,speed,low detection limit,and high accuracy,and was suitable for the rapid detection of multi-residues of TCs in food.