l-2-羟基戊二酸 (l-2-HG) 是一种参与多种生理过程的功能性代谢物。然而，由于技术限制，其亚细胞分布和线粒体运输仍不清楚。在本研究中，开发了一种超灵敏的l-2-HG生物传感器sfLHGFRH，其由循环排列的黄色荧光蛋白和l-2-HG特异性转录调节因子组成。 sfLHGFRH 用于分析 l-2-HG 代谢的能力首先在人胚胎肾细胞 (HEK293FT) 和巨噬细胞中测定。然后，通过 sfLHGFRH 支持的时空 l-2-HG 监测来识别 HEK293FT 细胞中 l-2-HG 的亚细胞分布和线粒体 l-2-HG 的较低丰度。最后，使用 sfLHGFRH 阐明了 L-谷氨酸转运蛋白 SLC1A1 在线粒体 L-2-HG 摄取中的作用。基于sfLHGFRH的设计，开发了另一种低检测限的高灵敏度生物传感器sfLHGFRL，用于l-2-HG相关疾病的即时诊断。使用 sfLHGFRL 生物传感器测定肾癌患者尿液中 l-2-HG 的积累。© 2024 作者。 《Advanced Science》由 Wiley‐VCH GmbH 出版。

l-2-Hydroxyglutarate (l-2-HG) is a functionally compartmentalized metabolite involved in various physiological processes. However, its subcellular distribution and mitochondrial transport remain unclear owing to technical limitations. In the present study, an ultrasensitive l-2-HG biosensor, sfLHGFRH, composed of circularly permuted yellow fluorescent protein and l-2-HG-specific transcriptional regulator, is developed. The ability of sfLHGFRH to be used for analyzing l-2-HG metabolism is first determined in human embryonic kidney cells (HEK293FT) and macrophages. Then, the subcellular distribution of l-2-HG in HEK293FT cells and the lower abundance of mitochondrial l-2-HG are identified by the sfLHGFRH-supported spatiotemporal l-2-HG monitoring. Finally, the role of the l-glutamate transporter SLC1A1 in mitochondrial l-2-HG uptake is elucidated using sfLHGFRH. Based on the design of sfLHGFRH, another highly sensitive biosensor with a low limit of detection, sfLHGFRL, is developed for the point-of-care diagnosis of l-2-HG-related diseases. The accumulation of l-2-HG in the urine of patients with kidney cancer is determined using the sfLHGFRL biosensor.© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.