A Simple and Effective Strategy To Increase the Sensitivity of Fluorescence Probes in Living Cells
A Simple and Effective Strategy To Increase the Sensitivity of Fluorescence Probes in Living Cells
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DOI：
10.1021/ja902511p
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发表时间：
2009-07-29
影响因子：
15
通讯作者：
Nagano, Tetsuo
中科院分区：
文献类型：
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作者：
Izumi, Saki;Urano, Yasuteru;Nagano, Tetsuo
活细胞中蛋白质、生物分子和酶之间相互作用的非侵入性可视化和研究是生物学家的重要目标，荧光探针是实现这一目的的有力工具。由于许多靶分子仅以痕量存在，因此高灵敏度非常重要，通常通过关注高反应速度K-d来提高荧光探针的灵敏度。(Gee、K.的R.; Archer，E.一、拉帕姆湖一、伦纳德，M. E.中;周志; Bingham，J.; Diwu，Z. Bioorg.医学化学快报2000，10，1515 -1518.）基于上述概念，我们设计合成了多种高灵敏度的荧光探针。(Gabe，Y.; Urano，Y.;菊池; Kojima，H.; Nagano，T. J. Am. 2004，126，3357-3367中所述。Komatsu，K.; Urano，Y.; Kojima，H.; Nagano，T. J. Am. 2007，129，13447-13454）。然而，尽管在比色皿中具有高的化学灵敏度，但它们有时对检测活细胞中的生物分子不够灵敏。在这份报告中，我们提出了一种新的方法来增加荧光探针的灵敏度，专注于他们的细胞内保留。由于钙黄绿素是良好的保留，我们研究了它的结构，化学和光学特性，并发现亚氨基二乙酸基团（IAG）是细胞内保留的关键结构。我们接下来设计并合成了含有IAGs的新型荧光探针。它们显示出上级的细胞内保留，使得能够可视化用常规探针难以观察的低浓度靶分子，并允许在活细胞中进行长期观察。提高荧光探针的细胞内滞留作为开发用于研究各种生物现象的广泛的高灵敏度探针的策略具有很大的前景。
Noninvasive visualization and investigation of interactions among proteins, biomolecules, and enzymes in living cells is an important goal for biologists, and fluorescence probes are powerful tools for this purpose. Because many target molecules are present in only trace amounts, high sensitivity is very important, and it is common to improve the sensitivity of fluorescence probes by focusing on high reaction velocity, K-d. (Gee, K. R.; Archer, E. A.; Lapham, L. A.; Leonard, M. E.; Zhou, Z.; Bingham, J.; Diwu, Z. Bioorg. Med. Chem. Lett. 2000, 10,1515-1518.) So far, we have designed and synthesized various highly sensitive fluorescence probes based on the above concepts. (Gabe, Y.; Urano, Y.; Kikuchi, K.; Kojima, H.; Nagano, T. J. Am. Chem. Soc. 2004, 126, 3357-3367. Komatsu, K.; Urano, Y.; Kojima, H.; Nagano, T. J. Am. Chem. Soc. 2007, 129, 13447-13454.) Nevertheless, they were sometimes insufficiently sensitive to detect biomolecules in living cells, despite high chemical sensitivity in cuvette. In this report, we suggest a new approach to increase the sensitivity of fluorescence probes, focusing on their intracellular retention. Since calcein is well-retained, we investigated its structural, chemical, and optical characteristics and found that the iminodiacetic acid group (IAG) is a key structure for the intracellular retention. We next designed and synthesized novel fluorescence probes containing IAGs. They showed superior intracellular retention, making it possible to visualize low concentrations of target molecules that would be difficult to observe with conventional probes and permitting long-term observation in living cells. Improvement of intracellular retention of fluorescence probes holds great promise as a strategy for developing a wide range of highly sensitive probes for studies on various biological phenomena.