Gold nanoparticle based photometric determination of tobramycin by using new specific DNA aptamers
Gold nanoparticle based photometric determination of tobramycin by using new specific DNA aptamers
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DOI：
10.1007/s00604-017-2568-6
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发表时间：
2018-01-01
影响因子：
5.7
通讯作者：
Zhou, Nandi
中科院分区：
文献类型：
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作者：
Han, Xuyan;Zhang, Yuhong;Zhou, Nandi
应用基于磁珠的SELEX，经过10轮筛选，鉴定出37个针对妥布霉素的单链DNA适体。根据序列分析将这些适体分为9个家族。其中，选择了几个具有典型序列的适体，并通过荧光法测定了它们的解离常数（K（d）s）。称为“Ap 32”的适体具有最高的亲和力和令人满意的特异性，其Kd值为56.8 +/- 4.6 nM。理论模拟表明，核苷酸14-18和26-29在适体与妥布霉素的相互作用中起着最重要的作用。随后，通过合理设计的截短，对Ap 32的序列进行了优化.截短的适体Ap 32-2由34个核苷酸组成，并且具有与原始适体相似的Kd。选择其作为用于测定的最佳适体，并将其固定在金纳米颗粒上。添加妥布霉素后，颜色从红色变成紫色。这些发现被用来设计一个光度测定（最好在520 nm处进行），具有线性响应在100 nM至1.4 μ M的浓度范围内，与37.9 nM的检测限。该方法已成功地应用于蜂蜜样品中妥布霉素的测定。
A magnetic bead-based SELEX was applied to identify 37 single-stranded DNA aptamers specific for tobramycin after ten rounds of selection. The aptamers were classified into nine families according to sequence analysis. Among them, several aptamers with typical sequences were selected and their dissociation constants (K(d)s) were determined by a fluorescent method. An aptamer termed "Ap 32", with a K-d value of 56.8 +/- 4.6 nM, possesses the highest affinity and satisfactory specificity. Theoretical modeling showed that nucleotides 14-18 and 26-29 play a most significant role in the interaction between aptamer and tobramycin. Subsequently, the sequence of Ap 32 was optimized through rationally designed truncation. The truncated aptamer Ap 32-2 consists of 34 nucleotides and has a K-d that is similar to the original one. It was chosen as the optimal aptamer for use in the assay and was immobilized on gold nanoparticles. On addition of tobramycin, the color turns from red to purple. The findings were used to design a photometric assay (best performed at 520 nm) that has a linear response in the 100 nM to 1.4 mu M concentration range, with a 37.9 nM detection limit. The method was successfully applied to the determination of tobramycin in (spiked) honey samples.