新型水溶性不对称五甲川菁染料的合成、光稳定性及蛋白质的荧光标记

合成并表征了系列水溶性五甲川菁染料, 研究了其在不同溶剂中的光谱性能. 结果表明, 染料在水中的最大吸收和荧光光谱在647~665 nm波长范围内, 荧光量子产率达到0.1左右. 考察了N位取代基对染料水溶液光稳定性的影响, 结果表明, 在N原子上引入带有苯环结构和大体积的磺酸基, 可以提高染料的光稳定性. 高效液相色谱(HPLC)分析结果表明, 染料4a的N-羟基琥珀酰亚胺(NHS)活性酯标记牛血清白蛋白(BSA)的检测限为1.2×10^-8 mol/L, 与紫外检测相比, 检测灵敏度提高了近2个数量级.

Novel Water-soluble Asymmetric Cy5 Dyes: Synthesis,Photo-stabilities and Fluorescent Labeling Protein

In the post-genome sequencing era, there has been more and more attention paid to protein analysis. Because of their large molar extinction coefficients, moderate-to-high fluorescence quantum yields and their spectra in the near-infrared(NIR) region, pentamethine cyanine(Cy5) dyes have received considerable attention and is considered as the main fluorescent labeling compounds for proteins. In order to improve detection sensitivity for proteins, a series of water-soluble asymmetric Cy5 dyes were synthesized and their spectral properties were tested in different solvents. The maximum absorption and emission wavelengths of these dyes in water are in the range from 647 nm to 665 nm. The fluorescence quantum yields(Φ) of these dyes are about 0.1 in water. The photo-stabilities of these dyes in water were investigated as well. It is demonstrated that benzyl group and sulfo-group on N-position of cyanine dyes improved the photo-stability owing to their steric hindrance. The limit of detection(LOD) of succinimidyl ester of dye 4a(4a-NHS) for BSA is 1.2×10-8 mol/L by HPLC with fluorescent director. Compared with UV detection, the sensitivity of detection for BSA is increased about 100 times by fluorescent labeling of dye 4a. Therefore, dye 4a could be used to improve potostability and detection sensitivity in protein analysis.