Abstract
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.
Highlights
Hydrogen peroxide (H2 O2 ) is a strong oxidant, which is widely used in bleaching agents [1,2] and disinfectant [3,4]
Developed two fluorescence resonance energy transfer (FRET)-based H2 O2 probes, which were different from Hyper and roGFP2-Orp1, involving two fluorescent protein (FP) and measuring emission ratio other than excitation
We developed a mild, sensitive, and fast detection method for H2 O2 by the distinctive
Summary
Hydrogen peroxide (H2 O2 ) is a strong oxidant, which is widely used in bleaching agents [1,2] and disinfectant [3,4]. Fluorescence-based methods have shown advantages like high sensitivity, fast response, and ability to fulfill in situ measurement in organelles within the cell. Developed two fluorescence resonance energy transfer (FRET)-based H2 O2 probes, which were different from Hyper and roGFP2-Orp, involving two FPs and measuring emission ratio other than excitation. Both Prx and Trx were fused to a single FP to construct two efficient genetic redox probes, roGFP2-Prx [30] and TrxRFP [31], respectively. After the addition of H2 O2 to the mixture of these two fusion proteins (CPmTY), the FRET ratio increased immediately as a result of the CFP-Prx/mTrx-YFP heterodimer formation through a mixed disulfide bond, showing the feasibility of CPmTY as a H2 O2 probe. Further research indicates good performance of CPmTY in glucose detection by the transformation of glucose into H2 O2 with GOX
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