Hairpin DNA switch for ultrasensitive spectrophotometric detection of DNA hybridization based on gold nanoparticles and enzyme signal amplification.
Hairpin DNA switch for ultrasensitive spectrophotometric detection of DNA hybridization based on gold nanoparticles and enzyme signal amplification.
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DOI：
10.1021/ac1006238
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发表时间：
2010-07
影响因子：
7.4
通讯作者：
Youyu Zhang;Zhiwen Tang;Jun Wang;Hong Wu;A. Maham;Yuehe Lin
中科院分区：
文献类型：
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作者：
Youyu Zhang;Zhiwen Tang;Jun Wang;Hong Wu;A. Maham;Yuehe Lin
本研究开发了一种基于发夹DNA开关、纳米颗粒和酶信号放大的DNA杂交超灵敏检测平台。在该DNA测定中，使用分别在其5'端用硫醇和在其3'端用生物素双重标记的“茎-环”DNA探针。将该探针固定在96孔微孔板上由蛋白质γ-球蛋白锚定的金纳米颗粒（AuNPs）上。在不存在靶DNA的情况下，具有茎环结构的固定化探针由于空间位阻而保护生物素不被庞大的辣根过氧化物酶连接的链霉亲和素（streptavidin-HRP）缀合物接近。然而，在靶DNA的存在下，发夹DNA探针和靶DNA之间的杂交引起探针的显著构象变化，这迫使生物素远离AuNP的表面。结果，生物素变得可被链霉亲和素-HRP接近，并且可以使用分光光度法经由HRP催化的底物3，3 '，5，5'-四甲基联苯胺灵敏地检测靶杂交事件。一些实验参数的测定性能进行了优化。在最佳条件下，该DNA测定可以通过基于酶和纳米颗粒的组合的信号放大策略检测飞摩尔水平的浓度的DNA。由于发夹DNA探针固有的高选择性，这种方法还显示出区分DNA靶的单碱基错配的优异特异性。
A novel DNA detection platform based on a hairpin DNA switch, nanoparticles, and enzyme signal amplification for ultrasensitive detection of DNA hybridization has been developed in this work. In this DNA assay, a "stem-loop" DNA probe dually labeled with a thiol at its 5' end and a biotin at its 3' end, respectively, was used. This probe was immobilized on the gold nanoparticles (AuNPs) anchored by a protein, gamma-globulin, on a 96-well microplate. In the absence of target DNA, the immobilized probe with the stem-loop structure shields the biotin from being approached by a bulky horseradish peroxidase linked streptavidin (streptavidin-HRP) conjugate due to the steric hindrance. However, in the presence of target DNA, the hybridization between the hairpin DNA probe and the target DNA causes significant conformational change of the probe, which forces biotin away from the surface of AuNPs. As a result, the biotin becomes accessible by the streptavidin-HRP, and the target hybridization event can be sensitively detected via the HRP catalyzed substrate 3,3',5,5'-tetramethylbenzidine using a spectrophometric method. Some experimental parameters governing the performance of the assay have been optimized. At optimal conditions, this DNA assay can detect DNA at the concentration of femtomolar level by means of a signal amplification strategy based on the combination of enzymes and nanoparticles. This approach also has shown excellent specificity to distinguish single-base mismatches of DNA targets because of the intrinsic high selectivity of the hairpin DNA probe.