An Ultrasensitive Biosensor for Probing Subcellular Distribution and Mitochondrial Transport of l‐2‐Hydroxyglutarate

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, sfLHGFR_H, composed of circularly permuted yellow fluorescent protein and l-2-HG-specific transcriptional regulator, is developed. The ability of sfLHGFR_H 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 sfLHGFR_H-supported spatiotemporal l-2-HG monitoring. Finally, the role of the l-glutamate transporter SLC1A1 in mitochondrial l-2-HG uptake is elucidated using sfLHGFR_H. Based on the design of sfLHGFR_H, another highly sensitive biosensor with a low limit of detection, sfLHGFR_L, 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 sfLHGFR_L biosensor.