Multiplex Detection of Endonucleases by Using a Multicolor Gold Nanobeacon
Multiplex Detection of Endonucleases by Using a Multicolor Gold Nanobeacon
复制标题
使用多色金纳米信标多重检测核酸内切酶。
DOI：
10.1002/chem.201003765
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发表时间：
2011-06-01
影响因子：
4.3
通讯作者：
Liu, Yi-Ming
中科院分区：
文献类型：
--
作者：
Huang, Yong;Zhao, Shulin;Liu, Yi-Ming
基于一种新型的酶响应型多色金纳米粒子，建立了一种高灵敏度和选择性的多重检测核酸内切酶的方法。该纳米粒子利用了DNA裂解反应的高度特异性和金纳米粒子(AuNPs)独特的荧光猝灭特性。为了制备纳米核酸，在AuNPs(15 nm)的表面共组装了三个发夹状DNA记者，每个发夹DNA记者在5‘端标记了荧光染料(即荧光素酰胺、羧基-X-罗丹明、菁染料)，它们对三种不同的内切酶中的一种有反应。这种组装使染料与AuNP非常接近，这导致了由于纳米表面能量转移(NSET)效应而导致的荧光显著猝灭。当纳米粒子暴露于目标内切酶时，特定的DNA裂解发生，DNA片段与荧光染料一起从AuNP表面释放出来，这导致了荧光恢复，为定量测量内切酶活性提供了基础。研究了三种内切酶，即HaeIII、EcoRI和EcoRV，作为概念验证分析物。该方法灵敏度高、特异度高，可同时定量均一混合溶液中的核酸内切酶。该方法的检测限为5.0×10(-4) U m L(-1)~1.0×10(-3) U m L(-1)，灵敏度至少为文献报道的10 0倍。核酸内切酶抑制剂会损害DNA的切割，因此本方法在筛选核酸内切酶抑制剂方面具有很大的潜力。为了证明这一应用，研究了某些抗癌药物对HaeIII、EcoRI和EcoRV活性的抑制作用。实践证明，该方案具有灵敏、可靠、易实现等特点。
A highly sensitive and selective assay based on a novel enzyme-responsive multicolor gold nanobeacon has been developed for the multiplex detection of endonucleases, a group of very important nucleases. The nanobeacon takes advantage of the high specificity of DNA cleavage reactions combined with the unique fluorescence-quenching property of gold nanoparticles (AuNPs). To prepare the nanobeacon, three hairpin DNA reporters, each labeled at the 5' terminus with a fluorescent dye (i.e., fluorescein amidite (FAM), carboxy-X-rhodamine (ROX), cyanine dye (Cy5)), that respond to one of three different endonucleases are co-assembled at the surface of AuNPs (15 nm). This assembly brings the dyes into very close proximity with the AuNP, which leads to significant quenching of the fluorescence due to the nanosurface energy-transfer (NSET) effect. When the nanobeacon is exposed to the targeted endonucleases, specific DNA cleavage occurs and pieces of DNA fragments are released from the AuNP surface along with the fluorescent dye, which results in the fluorescence recovery that provides the basis for a quantitative measurement of endonuclease activity. Three endonucleases, namely HaeIII, EcoRI, and EcoRV, were studied as the proof-of-concept analytes. These endonucleases in homogeneous mixture solutions were simultaneously quantified by the proposed assay with high sensitivity and specificity. The limits of detection obtained were in the range of 5.0×10(-4) U mL(-1) to 1.0×10(-3) U mL(-1) of endonuclease; these limits are at least 100 times more sensitive than the previously reported endonuclease assays. Endonuclease inhibitors impair the DNA cleavage, so it is anticipated that the present method has great potential for screening inhibitors of endonucleases. To demonstrate this application, the inhibitory effects of certain anticancer drugs on HaeIII, EcoRI, and EcoRV activities were studied. The present protocol proved to be sensitive, reliable, and easy to carry out.