A ratiometric optical imaging probe for intracellular pH based on modulation of europium emission
A ratiometric optical imaging probe for intracellular pH based on modulation of europium emission
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DOI：
10.1039/b718993a
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发表时间：
2008-01-01
影响因子：
3.2
通讯作者：
Parker, David
中科院分区：
文献类型：
--
作者：
Pal, Robert;Parker, David
已经合成了一组三个pH响应的比率Eu（III）配合物，将协调的氮杂硫杂蒽酮敏化剂和pH依赖的烷基磺酰胺部分。发射特性，阴离子结合亲和力，pH响应曲线和蛋白质结合常数进行了详细研究，在水介质中，和含有各种浓度的干扰阴离子和蛋白质的溶液也进行了检查。配合物[EuL 3]对蛋白质和内源性阴离子有一定的干扰作用。G.乳酸盐和碳酸氢盐，但在人血清溶液中质子化常数为7.2。获得了合适的校准曲线，并使用680/589 nm强度比与pH值的关系图确定局部pH值。共聚焦荧光显微镜图像显示快速摄取的复合物和细胞内分布良好的定位，快速出口也发生。核糖体定位，与高浓度的蛋白质密集的核仁内观察到，以类似的方式结构相关的复合物具有相同的协调敏化部分。使用MTT测定法估计IC 50值为67（+/- 20）μ M。选定的发射带比与pH值的曲线允许pH值测量范围为6至8，使细胞内的pH值进行测量，通过显微镜。NIH 3 T3细胞在核仁和核糖体的蛋白质丰富区域中的值估计为7.4。
A set of three pH-responsive ratiometric Eu(III) complexes has been synthesised incorporating a coordinated azathioxanthone sensitiser and a pH dependent alkylsulfonamide moiety. Emission properties, anion binding affinities, pH response curves and protein binding constants were studied in detail in aqueous media, and solutions containing various concentrations of interfering anions and protein were also examined. The complex, [EuL3] exhibited some interference from protein and endogenous anions, e. g. lactate and hydrogen carbonate, but possessed a protonation constant of 7.2 in human serum solution. A suitable calibration curve was obtained and was used to determine the local pH using a 680/589 nm intensity ratio vs. pH plot. Confocal fluorescence microscopy images revealed fast uptake of the complex and a well distributed localisation within the cell; fast egress also occurred. Ribosomal localisation, with a high concentration within the protein-dense nucleoli was observed, in a similar manner to structurally related complexes bearing the same coordinated sensitising moiety. An IC50 value of 67 (+/- 20) mu M was estimated using an MTT assay. Selected emission band ratio versus pH plots allow pH measurement in the range 6 to 8, enabling intracellular pH to be measured by microscopy. A value of 7.4 was estimated for NIH 3T3 cells in the protein rich regions of the nucleolus and ribosomes.