基于末端脱氧核糖核酸转移酶的纳米界面上DNA的生长策略及用于恩诺沙星的适配体传感器构建研究  

Construction of an Aptasensor Based on DNA Growth at Nano-interface of Terminal Deoxynucleotidyl Transferase and Its Application in Enrofloxacin Detection

作　　者：杜玉梅 周洋洋[1] 卞晓军[1,2,3] 颜娟 DU Yu-mei;ZHOU Yang-yang;BIAN Xiao-jun;YAN Juan(College of Food Science and Technology,Shanghai Ocean University,Shanghai201306,China;Shanghai Engineering Research Center of Aquatic-Product Processing&Preservation,Shanghai201306,China;Laboratory of Quality&Safety Risk Assessment for Aquatic Products on Storage and Preservation(Shanghai),Shanghai201306,China)

机构地区：[1]上海海洋大学食品学院,上海201306 [2]上海水产品加工及贮藏工程技术研究中心,上海201306 [3]农业部水产品贮藏保鲜质量安全风险评估实验室(上海),上海201306

出　　处：《分析测试学报》2019年第7期784-791,共8页Journal of Instrumental Analysis

基　　金：国家自然科学基金资助项目(21775102,21405167);上海市教育发展基金会“晨光计划”项目(15CG54)

摘　　要：在纳米金(Au nanoparticles,AuNPs)表面通过组装巯基DNA制得AuNPs-DNA生物探针后,末端脱氧核糖核酸转移酶(Terminal deoxynucleotidyl transferase,TdTase)催化探针DNA的3′-OH 端,实现DNA的生长,并得到生长产物长单链DNA(Single-stranded DNA,ssDNA)。通过琼脂糖凝胶电泳和原子力显微镜成像(Atomic force microscope,AFM)对探针及ssDNA进行表征,结果表明TdTase能够实现纳米界面上DNA的高效生长,卷曲状态下链长可达850 nm。进一步将这种DNA的生长策略与适配体(Aptamer)技术相结合。首先在纳米金表面组装适配体片段1,在醛基化的磁珠颗粒表面组装适配体片段2,以此构建针对恩诺沙星的适配体生物传感器(Aptasensor)。该传感器对ENR在10 ~1.0×10 5 ng/mL具备良好的响应性能,检出限为1.0 ng/mL,能明显区分环丙沙星、氧氟沙星、诺氟沙星等对照组信号。研究表明,基于TdTase的适配体传感器操作便捷,具备良好的靶分子响应性能和特异性,可为食品安全检测等领域提供新的研究思路。A bio-probe was firstly prepared by assembling sulfhydryl DNA on the surface of Au nanoparticles(AuNPs).Then the 3′-OH end on the probe DNA was extended under the catalysis of terminal deoxynucleotidyl transferase(TdTase),and produced a long single chain DNA(single-stranded DNA,ssDNA).The probe and the long ssDNA were characterized by agarose gel electrophoresis and atomic force microscopy(AFM).Results showed that,under the action of TdTase,DNA containing 3′-OH achieved an efficient extension at the nano interface,and the chain length was up to 850 nm though it was in the irregular coiled state.Afterwards,this DNA growth strategy combined with aptamer technology was applied to construct an aptasensor for the detection of enrofloxacin(ENR).The aptamer fragment 1 was firstly assembled on the surface of the AuNPs,then the aptamer fragment 2 was assembled on the surface of the magnetic particle activated by glutaraldehyde.After experiencing the TdT-mediated DNA growth strategy,the aptasensor had a good response to ENR concentration in the range of 10-1.0×10 5 ng/mL with a detection limit of 1.0 ng/mL.At the same time,the detection signal for enrofloxacin was significantly different from those for ciprofloxacin,ofloxacin,norfloxacin and other control groups.The results showed that the TdTase-based aptasensor could provide a new research idea or method for food safety detection and other fields with the advantages of convenient operation,good sensitivity and excellent specificity.

关 键 词：纳米生物探针 末端脱氧核糖核酸转移酶(TdTase) DNA生长 适配体传感器 恩诺沙星 

分 类 号：O657.1[理学—分析化学] TQ460.72[理学—化学]