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D-2-hydroxyglutaric acid (D2HG) is overproduced as a result of the D-2-hydroxyglutaric aciduria and relevant cancers, caused by gene mutation. Accurate analysis of D2HG could help rapid diagnosis of these diseases and allow for timely treatment. In this work, a D-2-hydroxyglutarate dehydrogenase from <i>Ralstonia solanacearum</i> (<i>Rs</i>D2HGDH) is cloned and recombinantly expressed. This enzyme features the direct electron transfer to chemical electron mediators (such as methylene blue (MB)) in the absence of additional coenzymes. Therefore, NAD⁺, a natural electron acceptor for the commercial D2HGDH and usually known for being unstable and difficult for immobilization can be avoided in the preparation of biosensors. The <i>Rs</i>D2HGDH and MB are co-immobilized on a two-dimensional material, Ti₃C₂ MXene, followed by drop-coating on the gold screen-printed electrode (AuSPE) to construct a compact and portable biosensor. The D2HG in samples can be catalyzed by <i>Rs</i>D2HGDH, where the current change is measured by chronoamperometry at −0.23 V. The biosensor shows a D2HG detection range of 0.5 to 120 µM (R² = 0.9974) with a sensitivity of 22.26 μA mM⁻¹ cm⁻² and a detection limit of 0.1 µM (S/N = 3). The biosensor retains 72.52% performance of its incipient state after 30 days of storage. The samples of D2HG-containing fetal bovine serum and artificial urine were analyzed with the recovery of 99.56% to 106.83% and 97.30% to 102.47% further indicating the great application potential of our portable D2HG biosensor.