## 3 Results and Discussion  
### Construction of the Proposed H2O2 Probe  
We employed CFP and YFP to construct the proposed H2O2 probe, as a FRET donor and acceptor, respectively. As shown in Figure 1, CFP was linked to Prx (to form Prx-CFP) and YFP to mutant Trx (to form mTrx-YFP). Two intermolecular disulfide bonds formed in Prx homodimer in the presence of H2O2, and then mTrx reacted with the homodimer to generate mixed disulfide dimers, bringing the tethered CFP and YFP close enough to generate FRET signals, which in reverse could indicate H2O2 concentration in the medium. We named this new probe CPmTY, the abbreviation of the essential mixed disulfide dimer CFP-Prx/mTrx-YFP. Figure 1: Schematic illustration of the working principle of proposed H2O2 probe CFP-Prx/mTrx-YFP. CFP—cyan fluorescent protein, Prx—peroxiredoxin, mTrx—mutant thioredoxin, YFP—yellow fluorescent protein. FRET Signals Respond to H\({}_{2}\)O\({}_{2}\) Concentration as a Result of CFP-Prx/mTrx-YFP Conjugation through Disulfide Bond  
CFP-Prx and mTrx-YFP were successfully expressed and purified in this work. The fluorescence properties of both FPs were not interfered in each chimeric protein. At the same time, CFP-Prx retained the ability to react with H\({}_{2}\)O\({}_{2}\) to form dimer as free Prx did. When mixing and adding H\({}_{2}\)O\({}_{2}\) into the mixture, CFP-Prx/mTrx-YFP heterodimer formed as verified by SDS-PAGE (Figure 2a). If one disulfide bond in Prx dimer remained, an mTRX and a PRX-CFP dimer formed a covalent heterotrimer. Otherwise, this heterotrimer further resolved by another mTrx-YFP and decomposed into two CFP-Prx/mTrx-YFP heterodimers. Therefore, there were five kinds of entities in the mixture with the presence of H\({}_{2}\)O\({}_{2}\) corresponding to the five bands in each lane of SDS-PAGE image from top to bottom: (1) CFP-Prx/CFP-Prx/mTrx-YFP heterotrimer, (2) CFP-Prx/CFP-Prx homodimer, (3) CFP-Prx/mTrx-YFP heterodimer, (4) monomer CFP-Prx, and (5) monomer mTrx-YFP, where (2) and (3) were very close to each other. Moreover, as the H\({}_{2}\)O\({}_{2}\) amount increased, the monomers decreased in concentration while the trimer and dimer increased. FRET happened between CFP-Prx and mTrx-YFP in hetero dimer and trimer, as revealed in the fluorescence spectra (Figure 2b). Each spectrum corresponds to one lane in the SDS-PAGE image and more hetero dimer and trimer lead to higher FRET signals (decrease of CFP fluorescence and increase of YFP's). In this way, the H\({}_{2}\)O\({}_{2}\) concentration can be reflected by the FRET signal intensity. Figure 2: Demonstrations of H\({}_{2}\)O\({}_{2}\) induced homo/hetero dimerization between CFP-Prx and mTrx-YFP and corresponding fluorescence resonance energy transfer (FRET) signal changes. (**a**) SDS-PAGE of CFP-Prx and mTrx-YFP mixtures after reacting with H\({}_{2}\)O\({}_{2}\), in 0# sample no H\({}_{2}\)O\({}_{2}\) existed, from 1# to 7# sample H\({}_{2}\)O\({}_{2}\) concentration increased gradually. (**b**) Fluorescence spectra of the same sample as indicated in panel (**a**). To further prove that intermediate disulfide bonds was the cause for the FRET signal, we add DTT, which can destroy the bond, after H\({}_{2}\)O\({}_{2}\) and FRET signal diminished gradually in six minutes (Figure 3a). The reaction between DTT and protein disulfide bond was rather slow compared with that between Prx and H\({}_{2}\)O\({}_{2}\), which completed in seconds. Thus we can measure the FRET signal immediately after H\({}_{2}\)O\({}_{2}\) addition, which is a benefit of this detection method. We also constructed wild-type Trx fused with YFP (wtTrx-YFP), which cannot induce FRET with CFP-Prx in the presence of H\({}_{2}\)O\({}_{2}\) (Figure 3b), as the resolving cysteine in wtTrx destroys the intermediate disulfide bond. Ability of CpnTY to Detect H\({}_{2}\)O\({}_{2}\) with Low Limit and Wide Range by Optimal Protein Concentration  
First, we conducted the detection experiment in 20 mM Tris-HCl (pH 8.0) with 150 mM NaCl, the same buffer as used for protein purification.