Initially, we synthesized probe RhQ and a control probe, HC, from available starting materials (Scheme S1, S2 and S3). Herein, the naphthalimide based quencher group Q-1 (Scheme S1) was used for construction of the probe: i) Q-1 exhibited an excellent fluorescence quenching ability for rhodamine due to overlap with the absorption of Q-1 and fluorescence emission of the rhodamine group (Figure ); ii) the azo group is a great fluorescence quencher, but can be reduced under specific conditions such as hypoxic conditions, which results in the nonspecific imaging of protein vicinal dithiols. Hence, we used the naphthalimide based azo to monitor the stability of the azo group. The naphthalimide moiety can emit fluorescence at 540 nm when the naphthalimide azo is broken. As shown in Figure , no fluorescence signal changes for the quencher group (Q-1) were observed in the presence of reduced bovine serum albumin (rBSA, which contains 17 pair of vicinal dithiols as a model protein of VDPs) in HEPES buffer, indicating that Q-1 is a stable quencher. Then a pH titration was conducted to evaluate the pH effects on the fluorescence performance of RhQ. As shown in Figure , no significant changes in absorbance and fluorescence intensity were observed over a wide pH range between 2 and 10, suggesting that RhQ was stable over that pH range. Additionally, the effect of redox reagents on the fluorescence of RhQ were evaluated. Both DTT and H2O2 induced no significant fluorescence changes of RhQ even at concentrations as high as 5 mM (Figure ), indicating that DTT and H2O2 do not interfere with the interaction between RhQ and VDPs.