## 1 Introduction  
Hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)) is a strong oxidant, which is widely used in bleaching agents [1; 2] and disinfect [3; 4]. In vivo, it is a main component of reactive oxygen species (ROS), which has long been considered to be harmful to cells [5] and involved in the development of many diseases [6; 7]. Nonetheless, it is also found to participate in many signaling pathways [8; 9] and to help defend against microbe infection [10; 11] and abiotic stress [12]. Moreover, it is a by-product of many enzymatic reactions [13; 14], with glucose oxidase (GOX) as a typical example [15; 16]. The unique and significant role of H\({}_{2}\)O\({}_{2}\) attracts great research interest in biosensor development because one can know the amount of specific enzyme substrate indirectly by measuring its quantity. Therefore, the detection of H\({}_{2}\)O\({}_{2}\) is of practical significance. At this moment, quite a lot of methods exist to detect H\({}_{2}\)O\({}_{2}\), and they can be classified into four categories: colorimetric [17], spectrophotometry [18], electrochemistry [19], and fluorescence [20]. Among these, fluorescence-based methods have shown advantages like high sensitivity, fast response, and ability to fulfill in situ measurement in organelles within the cell. Besides fluorescent small molecules [21] and various fluorescent nano materials [22], fluorescent protein (FP) is another frequently utilized constituent in H\({}_{2}\)O\({}_{2}\) biosensors. Many proteins in cell recognize H\({}_{2}\)O\({}_{2}\) with high selectivity and sensitivity by the advantage of specific activity of biological enzymes towards their substrate. One can easily fuse these recognition elements to FPs through regular genetic manipulations to construct H\({}_{2}\)O\({}_{2}\) probes. HyPer [23; 24] and roGFP2-Orp1 [25; 26] are two prototypes of this kind, which involve a single FP to measure the excitation ratio, and both are frequently exploited in H\({}_{2}\)O\({}_{2}\)-related biochemical research. [PERSON] et al. [27] developed two fluorescence resonance energy transfer (FRET)-based H\({}_{2}\)O\({}_{2}\) probes, which were different from Hyper and roGFP2-Orp1, involving two FPs and measuring emission ratio other than excitation. Compared to single FP, FRET-based protein sensors are easier to construct, and it is unnecessary to optimize the insertion site of response element into FP, and their ratiometric readouts can eliminate background interference. To the best of our knowledge, these are the only two pioneers in this type, therefore we would like to explore more. Peroxiredoxin (Prx) is a ubiquitous antioxidant protein [28]. As a member of the Prx family, Prx2 reacts with H\({}_{2}\)O\({}_{2}\) to form two intermolecular disulfide bonds in a homodimer, which can be reduced by thioredoxin/thioredoxin reductase (Trx/TrxR) system [29]. Both Prx and Trx were fused to a single FP to construct two efficient genetic redox probes, roGFP2-Prx [30] and TrxRFP [31], respectively. It was reported that transient mixed disulfide bond existed between Prx and Trx during the PRx reduction process [32], as verified by the observation of stable intermediate of mixed disulfide dimer between Trx active-site cysteine mutant (mTrx) and Prx [33; 34]. This stable intermediate inspired us to construct a Prx/mTrx and FRET-based H\({}_{2}\)O\({}_{2}\) probe. In fact, at the same time as conducting this study, we also employed this Prx/mTrx combination to develop a cpYFP-based H\({}_{2}\)O\({}_{2}\) sensor and have recently proved its feasibility [35]. In the present study, we chose cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) as the energy donor and acceptor, respectively, to construct two fusion proteins of CFP-Prx and mTrx-YFP. After the addition of H\({}_{2}\)O\({}_{2}\) 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 H\({}_{2}\)O\({}_{2}\) probe.