Degenerative diseases are closely related to the changes of protein conformation beyond the steady state. The development of feasible tools for quantitative detection of changes in the cellular environment is crucial for investigating the process of protein conformational variations. Here, we have developed a near-infrared AIE probe based on the rhodamine fluorophore, which exhibits dual responses of fluorescence intensity and lifetime to local viscosity changes. Notably, computational analysis reveals that NRhFluors fluorescence activation is due to inhibition of the RACI mechanism in viscous environment. In the chemical regulation of rhodamine fluorophores, we found that variations of electron density distribution can effectively regulate CI states and achieve fluorescence sensitivity of NRhFluors. In addition, combined with the AggTag method, the lifetime of probe A9-Halo exhibits a positive correlation with viscosity changes. This analytical capacity allows us to quantitatively monitor protein conformational changes using fluorescence lifetime imaging (FLIM) and demonstrate that mitochondrial dysfunction leads to reduced protein expression in HEK293 cells. In summary, this work developed a set of near-infrared AIE probes activated by the RACI mechanism, which can quantitatively detect cell viscosity and protein aggregation formation, providing a versatile tool for exploring disease-related biological processes and therapeutic approaches.

中文翻译：

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NRhFluors：通过荧光寿命成像定量揭示蛋白质稳态与线粒体功能障碍之间的相互作用

退行性疾病与蛋白质构象超出稳态的变化密切相关。开发定量检测细胞环境变化的可行工具对于研究蛋白质构象变化的过程至关重要。在这里，我们开发了一种基于罗丹明荧光团的近红外 AIE 探针，它对局部粘度变化表现出荧光强度和寿命的双重响应。值得注意的是，计算分析表明 NRhFluors 荧光激活是由于粘性环境中 RACI 机制的抑制所致。在罗丹明荧光团的化学调控中，我们发现电子密度分布的变化可以有效地调控CI态并实现NRhFluors的荧光灵敏度。此外，结合AggTag方法，探针A9-Halo的寿命与粘度变化呈现正相关。这种分析能力使我们能够使用荧光寿命成像 (FLIM) 定量监测蛋白质构象变化，并证明线粒体功能障碍导致 HEK293 细胞中蛋白质表达减少。综上所述，本工作开发了一套由RACI机制激活的近红外AIE探针，可以定量检测细胞粘度和蛋白质聚集形成，为探索疾病相关的生物过程和治疗方法提供了通用工具。