3.1 Fluorescent properties of probe to small molecular thiols
To verify the thiol-specific reactivity of FL-DT , the probe was first mixed with 10 equivalents of various amino acids in HEPES buffer, respectively. As shown in Figure 1A , only the thiol-containing Cys triggers the fluorescence enhancement (𝜆max = 526 nm), which is ~ 28-fold higher than the cases of other analytes. The results demonstrate that FL-DT specifically responds to the thiol group (Figure S1A ). We next performed the kinetics studies for the fluorescent responses of FL-DT to Cys at 526 nm. As shown in Figure 1B , upon the addition of Cys, the reaction between α , β -unsaturated ketones, and thiols complete within 30 sec as the fluorescence intensity reaches a plateau. Additionally, the rate constants of the consecutive thiol addition were measured by the resulting time-dependent fluorescence intensity with a large excess Cys (Figure S4 and Table S1 ). The first thiol addition is 25 times faster than the second addition. The probeFL-DT (10 μM) was then titrated by Cys (0-100 μM) to lead the fluorescence intensity at 526 nm emitted from the activatedFL-DT enhanced gradually and determine its limit of detection (LOD) as 0.32 μM for thiol group (Figure 1C and S1B ). The mechanism of the fluorescent response of FL-DT was further investigated to support our assumption (Scheme 1 ). Mass spectrometry analysis of the product obtained from FL-DTreacted with Cys in HEPES buffer exhibits a peak at 757.25, which corresponds to [FL-DT -2Cys−H](Figure S1C ). The stoichiometry between FL-DT and Cys was also determined to be 1:2 from a Job’s plot (Figure 1D ). The obtained binding stoichiometry is consistent with the mass spectrometry analysis, demonstrating that one FL-DT molecule eventually reacts with two thiols. This event is required to trigger the activation of fluorescence enhancement of fluorescein since the presence of etherified phenol group by 2-cyclopentenone quenches the emission.19
In order to label a protein with FL-DT , fusing a diCys-containing peptide sequence to POI is necessary. As reported previously, the properties of the peptide sequence, including the conformation and the distance between Cys residues, affect the performance of the labeling agent significantly.13, 24In this work, we chose a hairpin sequence (-CPGC-) as the template to design the diCys-containing peptides because hairpin is reported to be a preferred conformation for multiple-thiol binding.13 A number of peptides with varying lengths and distances between two Cys residues (Figure 1E ) were prepared and tested for inducing the fluorescence response of FL-DT . According to the titration experiments, the fluorescence enhancement from the thiol reactedFL-DT generally reached a maximum when 1 equivalent of each peptide was added (Figure S2 ), while the reaction betweenFL-DT and each peptide mostly completed within 60 s (Figure S3 ). Moreover, the shortest peptide 4C2C induces the strongest fluorescence enhancement, which is ~ 80% of that induced by Cys (Figure 1G ). Increasing the length of the peptide tag to 10 and 15 amino acids (10C2C and 15C2C) generally reduces the fluorescence intensity to ~ 60% of that induced by Cys, and the role of the distance between two Cys residues (15C2C, 15C4C, 15C6C, and 15C8C) is not critical; however, the direct connection of Cys residues (10CC and 15CC) causes a low fluorescence efficiency (Figure 1G ), demonstrating the necessity of a spacer between the two Cys. Mass spectrometry was also carried out on the product from the reaction between FL-DT and the tetrapeptide 4C2C in HEPES buffer. The peak at 893.2348 represents for [FL-DT -4C2C−H] (Figure 1F ), confirming that FL-DT preferably reacts with two thiols on one peptide chain, thus, reliable for quantifying the diCys-containing peptide.