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.
Bibliographical noteFunding Information:
This research was funded by National Science Foundation of China (21672065 and 21636003), the Fundamental Research Funds for the Central Universities (22221818014), the 111 Project (B18022), the Project Funded by the International S&T Innovation Cooperation Key Project (2017YFE0129600), and National Major Scientific and Technological Special Project for "Significant New Drugs Development" (2019ZX09739-001).
- Hydrogen peroxide