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Quantum Dot-Fluorescent Protein FRET Probes for Sensing Intracellular pH

机译:量子点荧光蛋白FRET探针用于感测细胞内pH值

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摘要

Intracellular pH (pHi) plays a critical role in the physiological and pathophysiological processes of cells, and fluorescence imaging using pH-sensitive indicators provides a powerful tool to assess the pHi of intact cells and sub-cellular compartments. Here we describe a nanoparticle-based ratiometric pH sensor, comprising a bright and photostable semiconductor quantum dot (QD) and pH-sensitive fluorescent proteins (FPs), exhibiting dramatically improved sensitivity and photostability compared to BCECF, the most widely used fluorescent dye for pH imaging. We found that Förster resonance energy transfer (FRET) between the QD and multiple FPs modulates the FP/QD emission ratio, exhibiting a >12-fold change between pH 6 and 8. The modularity of the probe enables customization to specific biological applications through genetic engineering of the FPs, as illustrated by the altered pH range of the probe through mutagenesis of the fluorescent protein. The QD-FP probes facilitate visualization of the acidification of endosomes in living cells following polyarginine-mediated uptake. These probes have the potential to enjoy a wide range of intracellular pH imaging applications that may not be feasible with fluorescent proteins or organic fluorophores alone.
机译:细胞内pH(PHI)在细胞的生理和病理生理过程中起着关键作用,并且使用pH敏感指标的荧光成像提供了评估完整细胞和亚细胞室的PHI的强大工具。在这里,我们描述了一种基于纳米粒子的比例pH传感器,其包含明亮和光稳定的半导体量子点(QD)和pH敏感的荧光蛋白(FPS),与BCECF相比,显着提高了敏感性和光稳定性,最广泛使用的pH值最广泛的荧光染料成像。我们发现QD和多个FPS之间的Förster共振能量转移(FRET)调节FP / QD发射比,在pH 6和8之间表现出> 12倍变化。探头的模块化使定制通过遗传算法对特定的生物学应用来定制通过通过荧光蛋白诱变的探针的改变的pH范围来说明FPS的工程。 QD-FP探针促进了Polyarnine介导的摄取后活细胞中底体酸化的可视化。这些探针有可能享受各种细胞内pH成像应用,这些应用可能不可能与荧光蛋白或有机荧光团可行。

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