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

Abstract 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.