Construction Of A Biosensor Based On Dihydroxyglutarate Dehydrogenase

2-Hydroxyglutaric acid（2HG）,also known asα-Hydroxyglutaric acid,which could be divided into D-2-hydroxyglutaric acid（D2HG）and L-2-hydroxyglutaric acid（L2HG）according to chirality.Mutations of genes related to 2HG metabolism increase its concentration in human blood,urine and cerebrospinal fluid,and lead to 2-hydroxyglutaric aciduria（2HGA）.At the same time,mutations of isocitrate dehydrogenase（IDH）are involved in a large number of human cancers such as glioma,glioblastoma,chondrosarcoma,bile duct epithelioma and acute myeloid leukemia.The mutations give cancer cells the ability to produce D2HG,which is also known as cancer metabolite.Therefore,the concentration of D2HG is an important indicator in the diagnosis and treatment of many diseases.At present,the detection methods of D2HG mainly focus on liquid chromatography-mass spectrometry,magnetic resonance spectroscopy,high resolution electrospray ionization mass spectrometry and so on.These methods rely on high precision equipment and instrument support,and cumbersome operation.In contrast,the electrochemical enzymatic biosensor have strong anti-interference ability to detect the substrate specifically.Such biosensors also have the advantages of fast detection speed,high sensitivity,good portability,low cost.Therefore,the detection of D2HG related diseases by electrochemical enzymatic biosensor is feasible and has outstanding advantages.A biosensor based on D-2-hydroxyglutaric acid dehydrogenase（D2HGDH）was developed to detect the concentration of D2HG acid in human blood and urine samples.The biosensor used biological enzymes to catalyze the dehydrogenation of D2HG in the sample toα-ketoglutarate,the generated electrons were transferred to the electrode through the electron mediators,and the current signal was generated as the detection basis.After comparative analysis,D2HGDH from Ralstonia solanacearum（RsD2HGDH）was selected as the sensing element of the biosensor.After constructing the recombinant plasmid,transforming the expression vector,fermentation and purification,the specific enzyme activity and enzymatic properties of the pure enzyme solution were determined.At pH 7.0 and 25℃,the specific enzyme activity of RsD2HGDH was 4.92 U/mg,the Km value for D2HG was 0.433 mM and Kcat was 4.86 s^-1.The prepared portable biosensor based on screen printed gold electrode（Au SPE）could detect D2HG with a linear range of 0.5-120μM.The sensitivity was 22.26μA mM^-1 cm^-2.When the signal-to-noise ratio is equal to 3,the detection limit of the biosensor for D2HG was 0.1μM.Meanwhile,the relative standard deviation（RSD）of D2HG analyzed by the biosensor was 2.65%.The biosensor showed excellent anti-interference ability when there were common electroactive interfering substances in the system.After 30 days of storage at 4℃,the biosensor still had 72.52%of initial performance,reflecting its good storage stability.In the spiked recovery experiment of real samples,the recoveries of the D2HG biosensor in serum and urine samples were 99.56%-106.83%and 97.3%-102.47%respectively,and the RSD were less than 7.21%and 5.25%respectively.This further verifies the accuracy and reliability of the D2HG biosensor in actual sample detection.Therefore,D2HG biosensor has important practical significance and broad application prospects in the diagnosis of D2HGA and IDH related cancers,and provides a new idea for the detection of related diseases.