Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, People's Republic of China.
Anal Chem. 2010 Aug 1;82(15):6440-6. doi: 10.1021/ac1006238.
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.
一种基于发夹 DNA 开关、纳米粒子和酶信号放大的新型 DNA 检测平台，用于超灵敏检测 DNA 杂交，已在本工作中开发。在这个 DNA 测定中，使用了一种双标记有巯基的“茎环”DNA 探针在其 5'端和生物素在其 3'端，分别。该探针固定在金纳米粒子（AuNPs）上，通过蛋白质，γ-球蛋白，在 96 孔微孔板上。在没有靶 DNA 的情况下，由于空间位阻，带有茎环结构的固定探针阻止生物素被连接辣根过氧化物酶的链霉亲和素（链霉亲和素-HRP）缀合物接近。然而，在存在靶 DNA 的情况下，发夹 DNA 探针与靶 DNA 之间的杂交导致探针的显著构象变化，这迫使生物素远离 AuNPs 的表面。结果，生物素可被链霉亲和素-HRP 接近，并且可以通过辣根过氧化物酶催化的底物 3,3',5,5'-四甲基联苯胺通过分光光度法敏感地检测靶杂交事件。已经优化了控制测定性能的一些实验参数。在最佳条件下，这种 DNA 测定可以通过基于酶和纳米粒子组合的信号放大策略检测纳摩尔级浓度的 DNA。由于发夹 DNA 探针的固有高选择性，这种方法还表现出优异的区分 DNA 靶标的单碱基错配的特异性。
