Ultra-sensitive hydrogen peroxide sensor based on peroxiredoxin and fluorescence resonance energy transfer

In this paper, a fluorescence resonance energy transfer (FRET)-based sensor for ultra-sensitive detection of H₂O₂ was developed by utilizing the unique enzymatic properties of peroxiredoxin (Prx) to H₂O₂. Cyan and yellow fluorescent protein (CFP and YFP) were fused to Prx and mutant thioredoxin (mTrx), respectively. In the presence of H₂O₂, Prx was oxidized into covalent homodimer through disulfide bonds, which were further reduced by mTrx to form a stable mixed disulfide bond intermediate between CFP-Prx and mTrx-YFP, inducing FRET. A linear quantification range of 10-320 nM was obtained according to the applied protein concentrations and the detection limit (LOD) was determined to be as low as 4 nM. By the assistance of glucose oxidase to transform glucose into H₂O₂, the CFP-Prx/mTrx-YFP system (CPmTY) was further exploited for the detection of glucose in real sample with good performance, suggesting this CPmTY protein sensor is highly practical.