核酸适配体氯化血红素比色法检测牛奶中的氯霉素-《集美大学学报（自然科学版）》

文章信息/Info

Title:: Colorimetric Method for the Detection of Chloramphenicol in Milk Based on Aptamer and Hemin

Author(s):: SUN Huaixia; WANG Lingchen; LIN Zhengzhong; HONG Chengyi; HUANG Zhiyong; (College of Food and Biological Engineering，Jimei University，Xiamen 361021，China)

Keywords:: aptamer; hemin; peroxidase activity; colorimetric method; chloramphenicol

摘要:: 利用一种基于适配体氯化血红素(hemin)比色分析法快速检测牛奶中的氯霉素(chloramphenicol，CAP) 含量。设计两条DNA链，其中一条为CAP的核酸适配体，在其3′端修饰氯化血红素；另一条为CAP核酸适配体的互补链(cDNA)，在其5′端修饰hemin。当体系中无CAP时，两条DNA单链杂交形成DNA双链，两个hemin单体形成二聚体使其过氧化物酶活性降低，以致不能催化过氧化氢氧化3,3′,5,5′-四甲基联苯胺(3,3′,5,5′-tetramethylbenzidine，TMB)产生蓝色产物。而体系中有CAP时，CAP与CAP适配体特异性结合，DNA双链解离，hemin单体的过氧化物酶活性恢复，并催化过氧化氢氧化TMB产生蓝色产物，加入反应终止液后，检测452 nm处的吸光度值。在优化条件下，该比色法检测CAP的线性范围为1～30 nmol·L-1，以3倍标准偏差计算其检出限为0.315 nmol·L-1。将该方法用于牛奶样品的检测，加标回收率为955%～1160%，表明该方法能够快速、准确和灵敏地检测牛奶中CAP含量。

Abstract:: A colorimetric method for rapid detection of chloramphenicol(CAP) in milk was developed based on hemin-modified aptamer.Two DNA strands,the aptamer of CAP and its complementary strand(cDNA),were introduced.The hemin molecules were modified at the 3-end of the aptamer and the 5′ end of the cDNA,respectively.In the absence of CAP,two DNA single strands hybridized to form a double strand.Two hemin monomers formed dimers which reduced their peroxidase activities and was thus unable to catalyze the oxidization of 3,3′,5,5′-tetramethylbenzidine(TMB) by H2O2 to produce a blue colored product.In the presence of CAP,the CAP specifically hybridized with the aptamer.The DNA double strands were dissociated and the peroxidase activity of hemin monomers was restored which effectively catalyzed the oxidization of TMB by H2O2 to present blue colored product.The absorbance at 452 nm was recorded after adding of the stop solution.Under the optimal conditions,the colorimetric method showed a good linear response for CAP detection in the range of 130 nmol/L.The detection limit based on the 3σ/k criterion reached 0.315 nmol/L.The recovery tested by the spiked CAP in milk samples was 95.5%-116.0%,showing that the method can be used to sensitively and accurately detect CAP in milk.