Abstract：
Trimethine cyanine dyes are widely used as probes for the detection, study and quantification of biomolecules. In particular, cationic trimethine cyanines noncovalently interact with DNA with growing fluorescence. However, their use is often limited by the tendency to self-association - to the formation of aggregates. Disubstituted trimethine cyanines with hydrophobic substituents are especially prone to aggregation. In this work, we studied the interaction of a number of substituted trimethine cyanines with DNA (in aqueous buffer solutions) and showed that their aggregation strongly interfered with their use as fluorescent probes for DNA. To eliminate this drawback, preliminary heating of dye solutions with DNA to 60-70 °C was used, followed by cooling to room temperature. Compared to the experiments without heating, an increase in the dye fluorescence intensity was observed due to the partial thermal decomposition of the aggregates and the interaction of the resulting monomers with DNA. To decompose aggregates, another method was also used - protonation of the dyes with amino substituents in buffer solutions with pH 5.0, which also led to growing the dye fluorescence intensity in the presence of DNA. Complexes of the dyes with DNA were modeled using molecular docking. Effective binding constants of the dyes to DNA and detection limits when using the dyes as probes for DNA (LOD and LOQ) were determined. It is shown that dye 3 with heating in neutral buffer and dye 1 in acidic buffer may be recommended as sensitive probes for DNA. It is concluded that the method of preliminary heating may be applied to dyes prone to aggregation, for improving their properties as biomolecular probes. Another possible means to reduce the interfering effects of dye aggregates is to use easily protonated dyes (with amino substituents) in slightly acidic media.
摘要：
三甲青花青染料被广泛用作检测的探针，生物分子的研究和定量。特别是,阳离子三甲基花青与DNA非共价相互作用，荧光增强。然而,它们的使用通常受到自缔合倾向的限制-形成聚集体。具有疏水取代基的二取代的三甲基花青特别易于聚集。在这项工作中,我们研究了许多取代的甲基花青与DNA（在水性缓冲液中）的相互作用，并表明它们的聚集强烈干扰了它们作为DNA荧光探针的使用。为了消除这个缺点，用DNA将染料溶液初步加热至60-70°C，然后冷却到室温。与没有加热的实验相比，由于聚集体的部分热分解和所得单体与DNA的相互作用，观察到染料荧光强度的增加。要分解聚集体，还使用了另一种方法-在pH5.0的缓冲溶液中用氨基取代基对染料进行质子化，这也导致在DNA存在下染料荧光强度的增加。使用分子对接对染料与DNA的复合物进行建模。确定了使用染料作为DNA探针时染料与DNA的有效结合常数和检测限（LOD和LOQ）。结果表明，在中性缓冲液中加热的染料3和在酸性缓冲液中加热的染料1可能被推荐作为DNA的敏感探针。结论初步加热的方法可以应用于容易聚集的染料，用于改善其作为生物分子探针的性能。降低染料聚集体的干扰作用的另一种可能的方法是在微酸性介质中使用容易质子化的染料(具有氨基取代基)。
