An effective colorimetric and ratiometric fluorescent probe based FRET with a large Stokes shift for bisulfite

Bisulfite plays crucial roles in diverse physiological processes. Therefore, the efficient detection of bisulfite is very important. In this study, we report a colorimetric and ratiometric fluorescent probe (CPT) with a large Stokes shift (162 nm) for bisulfite (HSO3−) based FRET mechanism. The probe can quantitatively detect HSO3− with low detection limit (45 nM) and high specificity over other common anions and biothiols. A nucleophilic addition reaction was proposed for the sensing mechanism, which was confirmed by HRMS spectra. The test strips of the probe were made and used easily. Moreover, probe CPT was used to ratiometric fluorescent imaging of exogenous and endogenous HSO3− in living cells.


Fluorescence quantum yield
Fluorescence quantum yield was determined by the relative comparison with quinine sulfate (Φ s = 0.56 in 0.1 N H 2 SO 4 aqueous solution) and rhodamine B (Φ s = 0. 69 in ethyl alcohol solution) as standard, and it was calculated by equation following. Φ = Φ s (IA s /I s A)(η 2 /η s 2 ) (1) in which, A is the absorbance, I is the integrated fluorescence intensity, and η is the refractive index of the solvent.

Calculation of energy transfer efficiency
Energy transfer efficiency (E) was calculated using the following equation: Where, F DA and F D denote the donor fluorescence intensity with and without an acceptor, respectively.

Calculation of the detection limit (LOD)
Where, σ is the standard deviation of the blank solution and k is the slope of the linear calibration plot between the fluorescence emission intensity and the concentration of HSO 3 -.

Preparation for UV -vis and fluorescence spectral measurements
Phosphate buffered saline (PBS, 10 mM) was used throughout the absorption and fluorescence determination. Probe CPT was dissolved in ethanol (EtOH) to get the stock solution (1 × 10 -3 M).

Cytotoxicity Assay
HeLa Cells were cultured in Dulbecco's modified Eagle's medium(DMEM) supplemented with 10% followed by addition of probe CPT with final concentrations of 1, 5, 10 µM, respectively. The cells were then incubated for 3 h, followed by SRB assays.

Preparation of CPT Test Strips
Probe CPT was dissolved in EtOH to afford the test solution (0.1 mg/mL, 10 mL). Filter paper was soaked in the test solution for 30 seconds, and then dried in a vacuum drying oven. 4 test strips were soaked in the 4 bisulfite solution (0, 10 -4 , 10 -3 , 10 -2 M) for 2 min, respectively, then dried. The photographs were taken in visible light after 5 min.

Test strips application
Encouraged by the results, we made bisulfite test strips to detect different concentrations of bisulfite in water. With the concentration of HSO 3 increasing (from 0, 10 -4 , 10 -3 , 10 -2 M), the color of the test strips gradually faded from red in visual light (Fig. S2), which was consistent with that in solution. The results showed that probe CPT could sensitively and simply detect bisulfite in practical water sample with the naked eye.

Figure S23
High resolution mass spectrum of the donor.