All reagents were purchased from reagent companies and used directly, if not otherwise specified. The water used in the experiment was double distilled water. UV-Vis spectra were measured with Shimadzu UV 2600. Fluorescence spectra were measured by F-7000 fluorescence spectrophotometer. High-resolution mass spectra of compounds were measured by Agilent Q-TOF6510 spectrograph. The NMR spectra of compounds were recorded by Bruker Advance 500 spectrometer. The pH was measured by PHS-3C. Absorbance for MTT assay was determined by TECAN Austria Gmbh A-5082. Confocal imagings were carried out by Nikon A1R MP.

As shown in Scheme 1, compound 1 (2 g, 10.72 mmol) and p-hydroxy benzaldehyde (2 g, 16.3 mmol) were dissolved in 50 ml CH₃CN before 2 ml acetic acid and 2 ml piperidine were added. The reaction mixture was heated to 120°C and kept in reflux for 12 h under the protection of argon. The reaction mixture was cooled and the solvent was removed under reduced pressure to give the crude product. Finally, the crude product was purified by silica gel column chromatography to give BC-OH as a red solid.

Under the protection of argon, the fluorophore BC-OH (261 mg, 0.9 mmol), 4-bromomethyl phenylboronic acid pinacol ester (88 mg, 0.296 mmol), anhydrous potassium carbonate (369 mg, 0.89 mmol) and NaI (440 mg, 2.93 mmol) were added into 15 ml anhydrous CH₃CN, and reacted at room temperature for 24 h. After completion of the reaction, the solvent was removed by evaporation under reduced pressure. DCM and saturated sodium chloride solution were used to extract repeatedly. The organic phase was collected and dried by anhydrous sodium sulfate. The filtrate was collected after filtration, and the organic solvent was removed under reduced pressure to give crude product, which was further purified by silica gel column chromatography (DCM: PE = 1: 1, v/v), to obtain orange solid (BC-B) (73 mg, yield 48.74%). ¹H NMR (500 MHz, DMSO) δ 7.70 (dd, J = 16.2, 8.3 Hz, 4H), 7.47 (d, J = 7.9 Hz, 2H), 7.29 (d, J = 4.6 Hz, 2H), 7.07 (d, J = 8.6 Hz, 2H), 6.85 (s, 1H), 5.22 (s, 2H), 1.31 (s, 14H), 1.03 (s, 6H), 0.89 (s, 2H). ¹³C NMR (126 MHz, DMSO) δ 160.01, 156.90, 140.66, 138.06, 135.05, 130.09, 129.42, 127.90, 127.33, 122.36, 115.80, 113.70, 84.16, 75.83, 69.58, 42.79, 38.66, 32.16, 27.93, 25.15.

Cytotoxicity was evaluated in HeLa cells using the MTT assay. HeLa cells were inoculated in culture plate. As adhered to the walls, HeLa cells were incubated with different concentration of BC-B (0, 3, 10, 30 μM) for 24 h. Then, MTT (10 μL) was added and HeLa cells were further cultured for 4 h. Finally, the plate was shaken for about 30 min, and each well was analyzed by the microplate reader (TECAN Austria GmbH A-5082) and detected at the absorbance of 492 nm.

Confocal imaging experiments were divided into three groups, each of which is parallel for three times (Ex = 561 nm). In the first group, the HeLa cells were incubated with 2 ml BC-B (10 μmol/L) for 110 min. In the second group, HeLa cells were incubated with 2 ml H₂O₂ (50 μmol/L) for 30 min and washed with PBS for three times, then incubated with 2 ml BC-B (10 μmol/L) for 110 min. The third group of HeLa cells was incubated with 2 ml H₂O₂ (50 μmol/L) for 30 min and washed with PBS for three times, then incubated with 2 ml of NAC (1 mmol/L) (NAC is N-Acetyl-l-cysteine, a remover of endogenous H₂O₂) for 1 h, washed with PBS three times, and then incubated with 2 ml of BC-B (10 μmol/L) for 110 min. In endogenous H₂O₂ imaging, PMA(1 g/mL)(PMA is Phorbol 12-myristate 13-acetate, a mature H₂O₂ inducer) is used to replace H₂O₂ added in the second and third groups of exogenous experiments, stimulate cells to produce endogenous hydrogen peroxide, and study its imaging by BC-B. All cells were washed three times with PBS buffer, and the fluorescence images of cells were observed with confocal fluorescence microscope by Nikon A1R MP.

At first, the fluorescence response of BC-B to H₂O₂ was measured in PBS solution at pH7.4 with excitation of 550 nm. As shown in Figure 1A, the BC-B did not emit fluorescence at 672 nm, suggesting that the phenylboronic acid pinacol ester group of the probe BC-B quenched the fluorescence of BC-OH fluorophore. While H₂O₂ (50 μmol/L) was added into the BC-B (10 μmol/L) system, the system emitted strong fluorescence at 672 nm with excitation of 550 nm. The emission wavelength of BC-B with H₂O₂ is the same to that of the fluorophore BC-OH. The results suggested that the probe BC-B can react with H₂O₂ to release BC-OH fluorophore, showing strong fluorescence. The H₂O₂ (50 μmol/L) solution pretreated with reactive oxygen scavenger NAC (1 mmol/L) for 5 min was added into BC-B solution (10 μmol/L) and incubated for 110 min, the fluorescence intensity of the system at 672 nm is significantly weaker than that of probe BC-B with H₂O₂ group, but stronger than that of the free probe BC-B group. When the probe BC-B was incubated with NAC (1 mmol/L) for 110 min, the fluorescence intensity was the same to that of BC-B. As the probe BC-B reacted with H₂O₂, the UV absorption peak appeared at 550 nm. The UV absorption spectral is similar to that of BC-OH (Supplementary Figure S1). These data further illustrated that H₂O₂ made BC-B probe release BC-OH fluorophore and showing strong fluorescence, we used HPLC to examine the mechanism of the probe BC-B (Scheme 2 and Supplementary Figure S2).

The fluorescence intensity of BC-B system at 672 nm increased with the concentration of H₂O₂, and a good linearity was built between the fluorescence intensity of BC-B probe and the concentration of H₂O₂ (y = 38.35 + 9.29196x, R ² = 0.98892) (Supplementary Figure S3A). The limit of detection was calculated to be 3 nmol/L according to the equation LOD = 3σ/k. These results show BC-B has high sensitivity to H₂O₂ and can quantitatively detect H₂O₂ in vitro.

First, the time-dependence of BC-B for detecting H₂O₂ was evaluated through measuring the fluorescence of BC-B after incubated with or without H₂O₂ for different time. As shown in Figure 2A and Supplementary Figure S3B, the free probe BC-B shows a little fluorescence in 120 min, indicating that BC-B probe is very stable in PBS solution. As H₂O₂ was added into the solution of BC-B, the fluorescence intensity (672 nm) of the system increased gradually during the incubation time of 0–110 min, and reached the maximum value at 110 min. Then, we measured the fluorescence changes of BC-B in presence or absence of H₂O₂ at different pH ranging from 3.4 to 10.5. As shown in Figure 2B and Supplementary Figure S3C, the fluorescence intensity of BC-B (10 μmol/L) at 672 nm was relatively stable with excitation at 550 nm. After incubation of BC-B with H₂O₂ (20 μmol/L) for 110 min, there was no fluorescence response under acidic conditions, while a good fluorescence response under alkaline conditions (pH = 7.4), this indicated that the BC-B has an ability to detect H₂O₂ under alkaline conditions.

The selectivity of analytes is an important parameter to evaluate property of fluorescent probes. It can be seen from Figure 2C that H₂O₂ induces a significant increase in the fluorescence intensity of BC-B, while the fluorescence intensity of other active species changes slightly, which indicates that the BC-B can specifically respond to H₂O₂. Then, add other active species to the solution of BC-B and H₂O₂ for interference test. Compared with the BC-B probe using H₂O₂, the fluorescence of these test samples has almost no change, showing good anti-interference ability. These results revealed that BC-B has excellent selectivity for H₂O₂ and has potential application in visualization of H₂O₂ in complex cell environment (Figure 2D ).

Cell survival percentage was calculated by MTT assay. As shown in Figure 3, the survival percentage of the 0 μmol/L BC-B was considered equal to 1 and the survival percentage of other samples was calculated according to the 0 μmol/L BC-B. High viability values of HeLa cells were obtained when treated with 30 μmol/L BC-B. Cell viability values measured by the MTT assay demonstrate that BC-B has a little cytotoxicity, and has the potential to be used to visualize H₂O₂ in HeLa.

Based on the excellent sensitivity, selectivity and biocompatibility of BC-B for H₂O₂, the ability of BC-B to visualize intracellular H₂O₂ was tested. As shown in Figure 4, compared with the BC-B group, the fluorescence intensity of the red channel in the BC-B + H₂O₂ group was significantly enhanced, this result indicates that BC-B can be used to visualize exogenous H₂O₂ in HeLa cells. After treating H₂O₂ incubated HeLa cells with active oxygen scavenger NAC, the fluorescence intensity of HeLa cells (NAC + BC-B group) incubated by BC-B was significantly weak comparing to that of BC-B group, which indicates that BC-B can be used to visualize endogenous H₂O₂ in HeLa cells.

In order to detect endogenous H₂O₂, PMA is used to stimulate the excessive production of H₂O₂ in cells. As expected, PMA-treated cells showed brighter fluorescence than untreated cells (Figure 5A). In addition, as shown in the third column of Figure 5A, the bright fluorescence is inhibited after adding NAC. The normalized fluorescence intensity shows the fluorescence change of the three groups (Figure 5B). Therefore, BC-B can image the endogenous H₂O₂ produced by living cells.