A series of genetically encoded sensors have been developed to detect the important signaling molecule H2O2 in living cells. However, more responsive and sensitive biosensors need to be developed. To address these demands, we used E. coli as a platform to develop a novel fluorescent H2O2 sensor, which we refer to as TScGP. This sensor employs a circularly permuted YFP (cpYFP) and is based on a redox relay between peroxiredoxin (Prx) and thioredoxin (Trx). Structurally, cpYFP is sandwiched between a fungal PrxA and a C-terminal cysteine mutated TrxA that can form a stabilized disulfide bond between PrxA and TrxA in response to H2O2. We confirmed that TScGP can be used for detecting exogenous H2O2 in the range of 0.5-5 mu M with high selectivity and rapidly detecting H2O2 within 30 s in E. coll. To demonstrate an application, cellular H2O2 production by menadione was detected directly by TScGP. Our results demonstrated that using Prx-Trx combination as a sensing moiety is another strategy in designing H(2)O(2 )sensor with high performance. (C) 2019 Elsevier Inc. All rights reserved.