Concatemeric dsDNA-templated copper nanoparticles strategy with improved sensitivity and stability based on rolling circle replication and its application in microRNA detection.
Concatemeric dsDNA-templated copper nanoparticles strategy with improved sensitivity and stability based on rolling circle replication and its application in microRNA detection.
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DOI：
10.1021/ac500955r
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发表时间：
2014-07
影响因子：
7.4
通讯作者：
Fengzhou Xu;Hui Shi;Xiaoxiao He;Kemin Wang;Dinggeng He;Qiuping Guo;Zhihe Qing;Lv’an Yan;
中科院分区：
文献类型：
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作者：
Fengzhou Xu;Hui Shi;Xiaoxiao He;Kemin Wang;Dinggeng He;Qiuping Guo;Zhihe Qing;Lv’an Yan;
以DNA为模板的铜纳米粒子（CuNPs）已成为生物化学检测中有前途的荧光探针，但报道的单体CuNPs由于荧光弱和稳定性差而仍然存在问题。为了解决这一问题，将滚环复制（RCR）技术引入到CuNPs的合成中，提出了一种新的多联体双DNA模板CuNPs（dsDNA-CuNPs）合成策略。在这种策略中，短的寡核苷酸引物可以触发RCR，并通过杂交进一步转化为长的多联体dsDNA支架。在加入铜离子和抗坏血酸后，多联体dsDNA-CuNPs可以有效地形成并在340 nm激发下在500-650 nm范围内发射强烈的荧光。与单体dsDNA-CuNP相比，串联dsDNA-CuNP的灵敏度大大提高，扩增约10，000倍。并且检测到它们的荧光信号在形成后2.5h保留约60%，显示约2倍的增强稳定性。基于这些优点，选择microRNA let-7 d作为模型靶标，以证明该策略作为通用测定平台。通过直接使用let-7 d作为RCR中的引物，成功地实现了简单、低成本和选择性的microRNA检测，在10 - 400 pM之间具有良好的线性，检测限为10 pM。多联体dsDNA-CuNP策略可能广泛适用于可以直接或间接诱导RCR的各种分析物。
DNA-templated copper nanoparticles (CuNPs) have emerged as promising fluorescent probes for biochemical assays, but the reported monomeric CuNPs remain problematic because of weak fluorescence and poor stability. To solve this problem, a novel concatemeric dsDNA-templated CuNPs (dsDNA-CuNPs) strategy was proposed by introducing the rolling circle replication (RCR) technique into CuNPs synthesis. In this strategy, a short oligonucleotide primer could trigger RCR and be further converted to a long concatemeric dsDNA scaffold through hybridization. After the addition of copper ions and ascorbate, concatemeric dsDNA-CuNPs could effectively form and emit intense fluorescence in the range of 500-650 nm under a 340 nm excitation. In comparison with monomeric dsDNA-CuNPs, the sensitivity of concatemeric dsDNA-CuNPs was greatly improved with ~10,000 folds amplification. And their fluorescence signal was detected to reserve ~60% at 2.5 h after formation, revealing ~2 times enhanced stability. On the basis of these advantages, microRNA let-7d was selected as the model target to testify this strategy as a versatile assay platform. By directly using let-7d as the primer in RCR, the simple, low-cost, and selective microRNA detection was successfully achieved with a good linearity between 10 and 400 pM and a detection limit of 10 pM. The concatemeric dsDNA-CuNPs strategy might be widely adapted to various analytes that can directly or indirectly induce RCR.