Metal-enhanced PicoGreen® fluorescence: Application to fast and ultra-sensitive pg/ml DNA quantitation
Metal-enhanced PicoGreen® fluorescence: Application to fast and ultra-sensitive pg/ml DNA quantitation
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DOI：
10.1016/j.jim.2010.09.011
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发表时间：
2010-10-31
影响因子：
2.2
通讯作者：
Geddes, C. D.
中科院分区：
文献类型：
--
作者：
Dragan, A. I.;Bishop, E. S.;Geddes, C. D.
在本文中，我们提供了两个理论和实验分析的灵敏度的DNA定量分析使用荧光发色团非共价结合dsDNA。众所周知，可用于荧光定量的DNA浓度范围取决于发色团的浓度、其对核酸的亲和力、DNA上的结合位点大小以及与DNA结合时的发色团与溶液中自由发色团的荧光强度之比。我们提出了PicoGreen(R) (PG)/DNA定量分析的实验数据，这与我们的理论分析结果完全一致。实验测量的PG荧光强度与DNA浓度函数通过导出的解析表达式拟合，其中PG与DNA的结合参数和发色团荧光性质包括在内。我们发现，由于金属增强荧光效应（MEF），银纳米粒子显著提高了与DNA结合的PG和游离PG的荧光之比，从而将DNA浓度的检测下限提高了几个数量级。另外一个数量级增加的PG/DNA测定灵敏度（类似于1 PG/ ml）可以通过降低PG浓度来实现。我们在这里表明，在表面分析中使用MEF底物对分析灵敏度有深远的影响。(C) 2010 Elsevier B.V.版权所有
In this paper we provide both a theoretical and experimental analysis of the sensitivity of a DNA quantitation assay using a fluorescent chromophore which non-covalently binds dsDNA. It is well-known that the range of DNA concentrations available for fluorescence quantitation depends on the concentration of the chromophore, its affinity for nucleic acids, the binding site size on DNA and the ratio between the fluorescence intensity of the chromophore when bound to DNA compared to free chromophore in solution. We present experimental data obtained for a PicoGreen(R) (PG)/DNA quantitation assay, which is in complete agreement with the results of our theoretical analysis. Experimentally measured PG-fluorescence intensity vs DNA concentration functions were fitted by a derived analytical expression, in which parameters of PG binding to DNA and chromophore fluorescence properties were included. We show that silver nanoparticles significantly increase the ratio between the fluorescence of PG bound to DNA and free PG, due to the metal-enhanced fluorescence effect (MEF), which enhances the lower limit of detectability of DNA concentrations by several orders of magnitude. An additional order of magnitude increase of PG/DNA assay sensitivity (similar to 1 pg/ml) can be achieved by decreasing the PG concentration. We show herein that the use of MEF substrates in surface assays has a profound effect on assay sensitivity. (C) 2010 Elsevier B.V. All rights reserved.