Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, College of Chemistry and Chemical Engineering, Guangxi Normal University, Guilin 541004, PR China.
Chemistry. 2011 Jun 20;17(26):7313-9. doi: 10.1002/chem.201003765. Epub 2011 May 9.
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.
一种基于新型酶响应多色金纳米荧光探针的高灵敏和高选择性检测方法已被开发出来，用于检测内切酶，这是一组非常重要的核酸内切酶。该纳米荧光探针利用 DNA 切割反应的高特异性和金纳米粒子（AuNPs）独特的荧光猝灭特性。为了制备纳米荧光探针，将三条发夹 DNA 报告分子，每条报告分子的 5' 端标记有一个荧光染料（即荧光素 amidite（FAM）、羧基-X-罗丹明（ROX）、Cy 染料（Cy5）），共同组装在 AuNPs（15nm）表面，这些报告分子对应于三种不同的内切酶之一。这种组装使染料非常接近 AuNP，这导致由于纳米表面能量转移（NSET）效应，荧光显著猝灭。当纳米荧光探针暴露于靶向内切酶时，会发生特异性的 DNA 切割，DNA 片段从 AuNP 表面释放出来，并与荧光染料一起释放，这导致荧光恢复，为内切酶活性的定量测量提供了依据。以 HaeIII、EcoRI 和 EcoRV 三种内切酶作为概念验证分析物进行研究。通过该方法，可在均相混合溶液中同时对这些内切酶进行高灵敏度和高特异性的定量检测。获得的检测限范围为 5.0×10(-4) U mL(-1) 至 1.0×10(-3) U mL(-1) 的内切酶；这些检测限比以前报道的内切酶检测方法至少灵敏 100 倍。内切酶抑制剂会损害 DNA 切割，因此预计该方法在筛选内切酶抑制剂方面具有很大的应用潜力。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。该方案被证明具有灵敏、可靠和易于操作的特点。
