## 3 Results and Discussion
### Construction of the Proposed H2O2 Probe
In this condition, the FRET ratio was very small, even after adding an excess amount of H\({}_{2}\)O\({}_{2}\) (much like Figure 3b). We then removed NaCl from the buffer and found the FRET ratio increased significantly in response to H\({}_{2}\)O\({}_{2}\) (much like Figure 2b). Salt ions may bind on protein interfaces to destroy hydrophobic interactions between protein dimer and prevent proteins from getting close to each other by electrostatic repulsion. Thus, NaCl may decrease the FRET ratio by impeding CFP-Prx dimerization and separating CFP and YFP in the CFP-Prx/mTrx-YFP conjugate. Moreover, YFP used in this study is pH sensitive, and shows stronger fluorescence intensity in pH 6-8. Therefore, we finally chose to examine the detection performance of CpnTY in 1 mM phosphate buffer (pH 7.0). Based on the working principle, the detection range of this method depends on the concentration of the two chimeric proteins. Here, we set the molar ratio as 1:1. With 200 nM CFP-Prx and 200 nM mTrx-YFP, the FRET ratio of CpnTY increased linearly in the range from 0 to 80 nM of H\({}_{2}\)O\({}_{2}\). At a concentration four times of the former (i.e., 800 nM CFP-Prx and 800 nM mTrx-YFP) the linear range expanded to 120-320 nM. Based on these results, it could be concluded that H\({}_{2}\)O\({}_{2}\) detection range by CpnTY varies with CFP-Prx and mTrx-YFP protein concentrations. However, the protein concentrations should neither be too high nor too low. As the concentration increased, spontaneous FRET occurred between separate CFP-Prx and mTRX-YFP, diminishing detection sensitivity (i.e., the difference between largest and smallest FRET ratio). As shown in Figure 4, the slope of the left fitting curve (i.e., 0.01) is greater than four times of the right (i.e., 0.0015). If the protein concentrations continue to increase, slope value of the fitting curve would tend to be zero, meaning that spontaneous FRET between separate CFP-Prx and mTrx-YFP equals  
Figure 3: Confirmation of disulfide bonds formation as the mechanism of H\({}_{2}\)O\({}_{2}\) induced FRET in CFP-Prx/mTrx-YFP system (CPmTY). (**a**) Fluorescence spectrum variation in 6 min when excess amount of DTT was added after CPmTY reaction with and H\({}_{2}\)O\({}_{2}\). (**b**) Fluorescence spectra of CFP-Prx/wtTrx-YFP (wild-type Trx that retained the resolving cysteine residue, C39) mixture in the absence (black line) and presence (red line) of abundant H\({}_{2}\)O\({}_{2}\). to that within the CFP-Prx/mTrx-YFP conjugate. On the other hand, too low protein concentrations also damage detection performance, as the fluorescence intensity is similar to the background interference, and at the same time both reaction possibilities between reduced CFP-Prx and H\({}_{2}\)O\({}_{2}\) and between oxidized CFP-Prx dimer and mTrx-YFP decrease drastically. The optimal detection limit of CPmTY was determined to be 4 nM (calculated as 3S\({}_{\text{B}}\)/m, \(S_{\text{B}}\) is the standard deviation of 20 blank responses and m the slope of calibration curve), which was much lower than most of the present H\({}_{2}\)O\({}_{2}\) detection methods (as shown in Table 1). In the general detection test, we preferred to set the protein concentrations to a relatively low level to increase detection sensitivity. Hence, when applying a low detection range, the dilution of samples with high H\({}_{2}\)O\({}_{2}\) concentration can eliminate the interference of other compositions such as salts in the sample.