All nuclear magnetic resonance (NMR) spectra of BPN-NBD were collected by a Bruker Avance 400 MHz spectrometer. A Zeiss LSM710 Wetzlar (German) laser scanning confocal microscope was used for fluorescence imaging. High-resolution mass spectrometry (HRMS) data of new compounds were tested on AB Sciex TripleTOF 4600. UV–vis absorption and fluorescence data were collected through a Shimadzu UV-2450 spectrophotometer and a HITACHI F-4600 fluorescence spectrophotometer, respectively. Ultra-high performance liquid chromatography (UHPLC) analyses were conducted on a Shimadzu NexeraX2 UHPLC LC-30A. Unless otherwise specified, all raw materials used for synthesis were purchased from the chemical suppliers in China and used directly without further refining.

BPN-OH (195 mg, 1.0 mmol) and NBD-Cl (238.8 mg, 1.2 mmol) were dissolved in 30 ml dichloromethane, and then, the Et₃N (121.3 mg, 1.2 mmol) was added to the above-mentioned solution under stirring. The resulting mixture reacted at room temperature for 4 h. The color of the solution gradually deepened as time progressed. After the reaction was completed, the yellow precipitated was extracted three times with dichloromethane (10 ml/3). Next, the solvent was removed under reduced pressure, and a crude product was produced. Then, the crude product was purified by column chromatography (CH₂Cl₂:PE = 1:1) to obtain a light-yellow solid BPN-NBD (272.1 mg, 76% yield). ¹H NMR (400 MHz, DMSO-d ₆ ) δ 8.67 (d, J = 8.4 Hz, 1H), 8.00 (d, J = 2.0 Hz, 1H), 7.98 (d, J = 2.0 Hz, 1H), 7.97 (s, 4H), 7.62 – 7.52 (m, 2H), 6.84 (d, J = 8.4 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d ₆ ) δ 153.45, 152.87, 145.43, 144.40, 143.33, 136.75, 135.43, 132.93, 130.51, 129.52, 127.65, 121.48, 118.76, 110.26. HRMS (ESI) m/z: calcd for [C₁₉H₁₁N₄O₄]⁺ 359.0739, found 359.0780.

The 10.0 mM source of analytes (amino acids and ions) was freshly prepared by being dissolved in twice-distilled water. Fluorescence spectra were measured in 7.4 20 mM PBS (containing 1.0 mM CTAB), in which a certain amount of biothiols (Cys, Hcy, or GSH) standard solution (3.0 mM) and other competitive analytes reacted with BPN-NBD in a 3.0 ml quartz cuvette. The testing solution was completely incubated at ambient conditions within a short time (about 150 s) to record. The fluorescence spectra were obtained with the excitation wavelength at 365 nm and 5.0/5.0 nm for slit width.

The MCF-7 cells were used to perform the fluorescent imaging of BPN-NBD. The cytotoxicity of BPN-NBD was evaluated by MCF-7 cells cultured in 96-well plates and placed in a medium with different BPN-NBD concentrations (0.0–100.0 µM) for another 24 h. The MTT reagent was added to each well of the plate to obtain data of absorbance measurement at 490 nm for testing cell viability. As considerable previous literature described, the MCF-7 cells were grown on glass-bottom dishes in Dulbecco’s Modified Eagle’s Medium with a humidified atmosphere of 5% CO₂ overnight for cell attachment. For imaging experiments, the MCF-7 cells were first seeded in probe BPN-NBD (10.0 μM) for 30 min and then washed with PBS prior to imaging. The adhered MCF-7 cells, as control experiments, were pre-treated with thiol scavenger N-ethylmaleimide (NEM, 1.0 mM) for 30 min, and fluorescence image was acquired by incubated in probe BPN-NBD (10.0 μM) for another 30 min. In addition, NEM-stained MCF-7 cells were treated with biothiol (150.0 μM Cys, Hcy, or GSH) for 30 min and probe BPN-NBD (10.0 μM) for another 30 min. Before the imaging, all the abovementioned dyed MCF-7 cells were washed three times with PBS and recorded with a confocal fluorescence microscope.

To validate the biological potential of BPN-NBD as a fluorescent scaffold, fluorescence imagings toward biothiols in living zebrafish were further explored. Three-day-old zebrafish were obtained from Eze-Rinka Company (Nanjing, China). Five groups were operated in different ways. For the first group, the zebrafish were stained with BPN-NBD alone for 30 min. For visualizing biothiol-induced fluorescence, the other test groups were all pre-treated with NEM. The second test group was then given an incubation of BPN-NBD for 30 min, while the remaining three experimental groups were incubated with biothiol (Cys, Hcy, or GSH, respectively) for 30 min followed by treatment with BPN-NBD for another 30 min. After the excess BPN-NBD adopted with PBS flushing solution was removed, the zebrafish fluorescence imagings were captured by a confocal microscope at green and blue channels.

The Balb/c mice (female) were purchased from Liaoning Changsheng biotechnology Co., Ltd. The animals were then evaluated using the protocols approved by the Animal Ethics Committee of Qiqihar Medical University. Nine female Balb/c mice at 4 weeks of age were divided into three groups (groups A, B, and C). Next, 0.1 ml of a 4T1 cell suspension containing 5 × 106 cells was injected into the left armpit of each mouse. When the tumors reached approximately 0.8 cm in diameter, the experiments were performed (induced by tail vein injection of 200.0 μM Cys(A)/GSH(B) for 1.5 h and 50.0 μM BPN-NBD for another 1.5 h). The Balb/c mice of the control experiment (C) were individually injected with BPN-NBD (50.0 μM) for 1.5 h. After the mice were anesthetized with sodium pentobarbital anesthetic administered, the tumor tissue was harvested from these mice for confocal fluorescence imaging. Fresh tumor tissues were fixed in 4% paraformaldehyde solution for over 48 h. The tissue was cut into a suitable tissue block (nearly 5 mm), placed in a tissue-embedded box, and dehydrated in a Leica ASP200S automatic vacuum dehydrator. The preparation and seal of tissue slices with a section thickness of 4 μm were operated in the conventional processing (Wan et al., 2021).

The molecular structure of BPN-NBD was designed based on the following considerations. 4′-hydroxy-[1,1′-biphenyl]-4-carbonitrile, BPN-OH, was selected as the chromophore because of its good biocompatibility, intense luminescence, large Stokes shift, and brilliant photostability (Chen et al., 2018c). A literature survey indicated that the NBD group could function not only as the potential green fluorophore but also as an outstanding response site for Cys/Hcy and GSH (Jiang et al., 2021; Yang et al., 2021). On the basis of the above-mentioned concerns, two fragments (BPN-OH and NBD) were fused into one molecule to develop a new 4′-hydroxy-[1,1′-biphenyl]-4- carbonitrile-based fluorescent probe BPN-NBD. The design rationale was illustrated in Scheme 2. BPN-NBD was nearly nonfluorescent in the blue and green region because of the PET process ascribed to the NBD group. However, the NBD moiety in probe BPN-NBD was attacked by Cys/Hcy via nucleophilic substitution reaction when BPN-NBD reacted with Cys/Hcy. Subsequently, a strong blue fluorescent product BPN-OH and the Smile rearrangement product (green fluorescence) NBD-Cys/Hcy were formed. When BPN-NBD encountered GSH, BPN-NBD only output the brilliant fluorescence in the blue region derived from chromophore BPN-OH. These properties enabled BPN-NBD to be used for the detection of Cys/Hcy from GSH through a dual-emission signal manner. Probe BPN-NBD was synthesized by simple one-step (Scheme 1), and the corresponding characterization data (HRMS, ¹³C NMR, and ¹H NMR) of BPN-NBD were depicted in the Supporting Information (Supplementary Figures S15–S17).

With BPN-NBD in hand, the fluorescence spectral properties of BPN-NBD (10.0 µM) were measured in pH 7.4 20 mM PBS (containing 1.0 mM CTAB) with varying concentrations (0.0–50.0 µM) of three biothiols (Cys, Hcy, and GSH) under a single-wavelength excitation at 365 nm. As seen in Figure 1, free BPN-NBD (10.0 µM) initially exhibited no fluorescence. However, after Cys/Hcy (Figures 1A1,B1) was added to probe BPN-NBD (10.0 µM), a concentration-dependent fluorescence emission spectra increment appeared at two-emission fluorescence of 475 and 545 nm (approximately 80-fold and 120-fold increase for 5 equiv. of Cys and Hcy, respectively). Compared with Cys/Hcy, GSH (50.0 µM) directly reacting with BPN-NBD (10.0 µM) only triggered a remarkable fluorescence enhancement at 475 nm (up to about 120-fold) (Figure 1C1). The results indicated that there were good linearities between the emission intensities and Cys/Hcy (Figures 1A2,B2) or GSH (Figure 1C2) content in the range of 0.0–6.0 µM (R ² = 0.9982, 0.9981, 0.9961). Based on the equation of intensities as the amount, the limit of detection concentration (LOD) of BPN-NBD was calculated (0.011 µM for Cys, 0.015 µM for Hcy, 0.003 µM for GSH, respectively), which are comparable with previously reported results (Supplementary Table S1). The abovementioned data demonstrated that BPN-NBD had excellent sensitivity for quantitative analysis toward Cys/Hcy and GSH under the experimental conditions.

To inspect the availability of BPN-NBD toward biothiols with high specificity, fluorescent probe BPN-NBD (10.0 µM) related to other potentially competitive species was investigated in pH 7.4 20 mM PBS (containing 1.0 mM CTAB). As depicted in Figure 2A1, only the three biothiols (Cys, Hcy, and GSH) displayed significant fluorescence increases in BPN-NBD (10.0 µM) detection system, whereas the fluorescence responses barely varied with other amino acids (including Leu, Ser, Ile, Asp, Ala, Val, Pro, Gly, Met, Phe, His, Trp, Arg, Tyr, Glu, and Thr) at 475 nm. The great selectivity of BPN-NBD (10.0 µM) toward biothiols (Figure 2A2) was also found compared with potential biologically relevant ions (including F⁻, Cl⁻, SO₄ ²⁻, SO₃ ²⁻, NO₃ ⁻, AcO⁻, K⁺, Na⁺, Al³⁺, Mg²⁺, Zn²⁺, Cu²⁺, Mn²⁺, Fe²⁺, Fe³⁺, and Ca²⁺), and a nearly imperceptible change was observed for even ions employed at much higher concentration (500.0 µM) at 475 nm. Noticeably, when Cys/Hcy (100.0 µM) was added to BPN-NBD (10.0 µM) system, the fluorescence at 545 nm was also lit up at the same time differently from that of GSH.1 Furthermore, the coexistence experiments in the presence of biothiols with the competitive analytes were performed (Supplementary Figures S2–S11). As expected, exposing BPN-NBD to the mixture of abovementioned analytes and biothiols rendered similar results to the case of biothiols only. The coexistence of the competitive analytes did not cause interference in the analysis of biothiols. These observations clearly proved that BPN-NBD not only responded to Cys/Hcy or GSH selectively against other related substances but also discriminated Cys/Hcy from GSH in aqueous media.

Response time is an important index for reaction-based fluorescence probes. Thus, the dynamic reaction of BPN-NBD incubated with different biothiols (Cys, Hcy, and GSH) was acquired to evaluate the time-dependent fluorescence response. As shown in Figures 2B1,B2, BPN-NBD (10.0 µM) held stable emission signal output (at 475/545 nm) with the extended response time. When BPN-NBD mixed with the biothiols (50.0 µM), we found the dramatic spectrum increase reached a plateau state within 150 s for three states (BPN-NBD upon addition of Cys/Hcy or GSH) at 475 nm. Similar results of the fluorescence spectrum of BPN-NBD with Cys/Hcy emerged at the emission wavelength of 545 nm, which indicated that BPN-NBD could be used as an effective candidate for real-time sensing biothiols. The pH effect on probe BPN-NBD (10.0 µM) with and without biothiols (50.0 µM) was evaluated (ranging from 2 to 11) (Figures 2C1,C2) to verify the sensing property of BPN-NBD for biothiols under physiological environment. In the absence of biothiols, no remarkable intensity change was observed. BPN-NBD exhibited excellent stability under a wide range of pH conditions. In the case of Cys/Hcy and GSH, the strong fluorescence augmentation of BPN-NBD appeared with altering pH (5.0–9.0), whereas a weak fluorescence behavior occurred when the pH range was 2.0–4.0 or over 10. The optimal range of pH values was 6.0–8.0 at 475 nm or 545 nm, which meant BPN-NBD could appreciably monitor biothiols with a pH value around 7.4. These results implied that BPN-NBD had a latent capability for biological applications in the typical physiological conditions.

The HRMS spectra of BPN-NBD + Cys and BPN-NBD + GSH were checked first to verify the sensing mechanism of BPN-NBD toward Cys/Hcy and GSH. As displayed in Supplementary Figures S12, S13, a mass spectral peak at m/z 285.0263 corresponding to [M + H⁺]⁺ for NBD-Cys and a peak at m/z 196.0744 corresponding to [M + H⁺]⁺ for BPN-OH were represented in the mixture of Cys with BPN-NBD. The desired products (m/z 471.0886 for NBD-GSH and m/z 196.0750 for BPN-OH) were also obtained in the mixed solution of BPN-NBD and GSH. UHPLC was used to obtain insights into the corresponding sensing mechanism. As displayed in Figure 3, BPN-NBD exhibited a main peak with a retention time of 7.41 min. However, two new peaks with retention times of 3.22 and 11.12 min emerged when BPN-NBD encountered Cys, and the two new peaks were further confirmed to be BPN-OH and NBD-Cys, respectively. The data powerfully support the proposed response mechanism in Scheme 2.

The biological applicability of BPN-NBD imaging of biothiols in a living system was conducted as encouraged by the excellent spectroscopic performance of BPN-NBD in aqueous media. Prior to fluorescence imaging, cytotoxicity of the BPN-NBD to living MCF-7 cells was checked by MTT staining method (Supplementary Figure S14). The related results revealed that different concentrations (5.0, 10.0, 20.0, 50.0, 100.0 μM) of BPN-NBD all displayed stable high levels of cell survival rate (over 80%) after 24 h in MCF-7 cells, which suggested that BPN-NBD had negligible toxicity and good biocompatibility to the live-cell imaging. Then, the imaging results in living MCF-7 cells were obtained. As shown in Figure 4, living MCF-7 cells were incubated in BPN-NBD (10.0 μM) medium for 30 min at room temperature. Owing to the merits of MCF-7 cell membrane permeability for BPN-NBD, endogenous biothiols were detected with obvious fluorescence signals observed in blue and green channels (Figures 4A1,A2). As a sharp contrast, the treatment of MCF-7 cells pre-incubated with thiol-blocking reagent (1.0 mM for N-ethylmaleimide, NEM) induced a result of no fluorescence response under the same conditions (Figures 4B1,B2). The subsequent experiments as envisaged, after adding Cys or Hcy for 30 min and BPN-NBD for an additional 30 min, the NEM-treated MCF-7 cells send out remarkable fluorescence signals in blue (Figures 4C1,C2) and green channels (Figures 4D1,D2). When the NEM-treated MCF-7 cells were co-incubated with GSH and then BPN-NBD successively (Figures 4E1,E2), an obvious fluorescence signal was researched only in the blue channel rather than in two channels.

We sought to evaluate the ability for Cys/Hcy and GSH detection of BPN-NBD in living zebrafish because of the promising performance of BPN-NBD in MCF-7 cells. As shown in Figure 5, zebrafish pre-incubated with BPN-NBD (10.0 μM) for 30 min displayed intense fluorescence in green (Figure 5A2) and blue channels (Figure 5A3). However, NEM-loaded zebrafish treated with BPN-NBD (10.0 μM) did not exhibit fluorescence emission in green (Figure 5B2) and blue channels (Figure 5B3). Next, when zebrafish were pretreated with NEM for 30 min, treated with Cys/Hcy (150.0 μM) for 30 min, and treated with BPN-NBD (10.0 μM) for 30 min, distinct strong fluorescence signals were collected from green (Figures 5C2,D2) and blue channels (Figures 5C2,D2). With regard to GSH (150.0 μM), strong fluorescence was observed in the blue channel (Figure 5E2) and no fluorescence was found in the green channel (Figure 5E3). These phenomena indicated that BPN-NBD could detect Cys/Hcy and GSH in zebrafish through a dual-emission signal manner.

Probe BPN-NBD for imaging endogenous and exogenous Cys/GSH in 4T1 breast cancer cell inoculated 4-week-old female BALB/c mice was evaluated. As displayed in Figure 6, we found green (Figure 6A1) and blue (Figure 6A2) fluorescences when tumor tissues in female BALB/c mice were injected and stained with 50.0 μM BPN-NBD for 1.5 h. As expected, the apparent fluorescence enhancement in green (Figure 6B1) and blue (Figure 6B2) channels were detected by treating the tumor tissues in female BALB/c mice with 200.0 μM Cys and 50.0 μM BPN-NBD for 1.5 h. Then, tumor tissues in female BALB/c mice treated with 200.0 μM GSH and 50.0 μM BPN-NBD for 1.5 h showed a pronounced fluorescence enhancement only in the blue channel (Figure 6C2). The outstanding performance of BPN-NBD towards Cys/GSH detection in tumor tissues indicated that BPN-NBD had great potential for clinical application in cancer diagnosis.