期刊
SENSORS AND ACTUATORS B-CHEMICAL
卷 385, 期 -, 页码 -出版社
ELSEVIER SCIENCE SA
DOI: 10.1016/j.snb.2023.133681
关键词
d-2-Hydroxyglutarate; Biosensor; Fo ?rster resonance energy transfer; Allosteric transcription factor
A FRET-based biosensor for detecting D-2-Hydroxyglutarate (D-2-HG) was developed, which showed excellent sensitivity and specificity. It can accurately measure the concentration of D-2-HG in body fluids and cell-related samples in vitro.
D-2-Hydroxyglutarate (D-2-HG) is an oncometabolite aberrantly accumulated in patients with D-2-hydrox-yglutaric aciduria or various isocitrate dehydrogenases mutation-associated cancers. The detection of D-2-HG is of great significance for the diagnostic and therapeutic aspects of these diseases. Herein, a Fo center dot rster resonance energy transfer (FRET)-based D-2-HG biosensor was developed. The D-2-HG-specific allosteric transcription factor DhdR from Achromobacter denitrificans NBRC 15125 was used as its biorecognition element and inserted between two fluorescent proteins Clover and mRuby2. The response magnitude of the biosensor was improved by trun-cation of the terminal amino acids of DhdR and addition of artificial linkers. The optimized variant DHGFR1.0 exhibited excellent sensitivity and specificity with a maximum emission ratio change (Delta Rmax) of 30.77%, a half -maximal effective concentration (EC50) of 2.78 mu M, and a dynamic detection range of 0.17-36.22 mu M. It was able to detect the concentration of D-2-HG in body fluids and cell-related samples with high accuracy and precision in vitro. The ligand-binding site of DhdR was predicted and several biosensors with low affinity for D-2-HG were also generated by site-directed mutagenesis of DhdR.
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