Introduction

Fluoride ions, the smallest and most electronegative anions, are of paramount importance to organisms [1,2,3,4]. Adequate intake of F− plays a crucial role in health, such as maintaining skeletal structure and physiological functions [5,6,7,8]. However, excessive fluoride can have harmful effects on health, including acute and chronic fluoride poisoning, fluorosis, neurodegenerative diseases, gastric and renal issues, and even death [9,10,11,12,13,14,15,16]. Therefore, developing highly selective and sensitive detection and quantification methods for fluoride ions to visualize their distribution in organisms is crucial [17,18,19,20,21,22,23,24].

Fluorescence sensing is considered an ideal technique among commonly used methods like colorimetry [25, 26], atomic absorption spectroscopy, and ion chromatography for F− detection [27, 28], especially in bioimaging applications due to its non-invasive and real-time nature [29,30,31,32,33]. Currently, despite significant progress in the development of F− fluorescent probes, there are still some drawbacks in their application in live cells and in vivo, such as high background interference, insufficient sample penetration, and long response times. Therefore, designing fluoride ion probes that can rapidly respond and have a large Stokes shift remains a challenge that needs to be addressed [34,35,36,37].

Therefore, we have designed a new fluorescence probe, PTC, for detecting fluoride ions in cells. It utilizes a fluorescein moiety and relies on fluoride-induced Si–O bond cleavage as its recognition mechanism (Scheme 1). We aim to develop a fluorescence probe with fast response time, large Stokes shift, and excellent specificity for fluorescence imaging of fluoride ions in live cells.

Scheme 1
scheme 1

Recognition mechanism of PTC for F−

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Experimental

Materials and Instruments

All chemicals were sourced as analytical-grade from Energy Chemical Ltd. (Shanghai) and Sigma-Aldrich (Shanghai) Co., Ltd. NMR spectra (1H and 13C) were recorded on a Bruker (Avance) 400 MHz NMR instrument at Guizhou University’s School of Pharmacy. Absorption spectra were captured on a UV-5500PC UV–Vis spectrophotometer, and fluorescence spectra were measured using a Hitachi F4700 fluorescence spectrometer, both provided by Guizhou University’s School of Pharmacy. Mass spectra were obtained using a TSQ 8000 high-resolution mass spectrometer (Thermo Fisher Scientific Co., Ltd.). Melting points were determined with an X-4X digital melting point apparatus (uncorrected, Shanghai Microelectronics Technology Co., Ltd.). For biological imaging, an inverted fluorescence microscope (NIB600, Ningbo Novel Co., Ltd.) was utilized.

Preparation and Measurement of Probe Solutions

To prepare a 1 mM PTC stock solution, the probe PTC was dissolved in DMSO. A stock solution of tetrabutylammonium fluoride (TBAF) at a concentration of 100 mM was produced using THF. Analytes were made as 100 mM stock solutions in deionized water for storage, including common cations (Na+, Ca2+, K+, Fe2+, Fe3+, Cu2+, Ag+, Zn2+) and anions (Cl−, Br−, I−, SO4 2−, HSO3−, H2PO42−, HCO3−, SCN−, NO3−, HPO4−). The fluorescence spectra of PTC for various analytes were investigated using 4 mL of DMSO solution at room temperature. Unless otherwise noted, fluorescence spectra were typically collected after 1 h of analyte addition. All aqueous solutions were made using ultrapure water from a Milli-Q purifier.

Cell Cytotoxicity Assay and Cell Image

Human hepatocellular carcinoma cells (HepG2 cells) were procured from the Kunming Cell Bank of Chinese Academy of Sciences and maintained in our laboratory. The cytotoxicity of the probe PTC against HepG2 cells was evaluated using the MTT assay. HepG2 cells were sown in 96-well plates and cultivated for 24 h. PTC solutions at different concentrations (0, 1, 5, 10, and 20 µM) were added, incubated with cells for 24 h. The MTT assay was performed using the literature methods [38]. In cellular imaging experiments, HepG2 cells were initially incubated with PTC (3 µM, in PBS) for 30 min, then TBAF (200 µM) was added, further incubated for 30 min. After three times washes with PBS, imaging was performed using laser confocal scanning microscopy.

Synthesis and Characterization

Synthesis of Compound 2

Thiophene boronic acid ester (367 mg, 1 mmol) was added to a round-bottom flask containing Dioxane/H2O (1:1, 5 mL), followed by the addition of 4-bromo-2-methoxybenzaldehyde (215 mg, 1.1 mmol) and anhydrous Na2CO4 (212 mg, 2 mmol). Subsequently, Pd(PPh3)4 (11.55 mg, 0.01 mmol) was introduced under argon protection, and the reaction was carried out at 110 ℃ for 3 h. After cooling to room temperature, the reaction was quenched with water (5 mL), and the mixture was extracted with DCM (10 mL × 3). The combined organic phases were washed with saturated brine, dried over anhydrous Na2SO4, and concentrated under vacuum. The resulting crude product was purified by column chromatography (eluent: PE:EA = 5:1; stationary phase: 200–300 mesh silica gel) to afford compound 2 as pale yellow oil (220 mg, 58.7% yield). 1H NMR (400 MHz, CDCl3) δ 10.45 (d, J = 0.8 Hz, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.43 – 7.35 (m, 2H), 7.19 – 7.13 (m, 3H), 7.08 (d, J = 1.6 Hz, 1H), 6.95 – 6.86 (m, 3H), 3.98 (s, 3H), 3.87 – 3.83 (m, 2H), 1.89 – 1.83 (m, 2H), 1.03 (t, J = 7.6 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 189.4, 162.2, 147.8, 145.8, 144.7, 134.0, 129.1, 127.5, 127.4, 126.2, 125.9, 125.5, 124.1, 123.4, 122.7, 118.9, 115.6, 109.4, 55.7, 49.3, 20.1, 11.3. ESI-HRMS C23H21NO2S ([M + H]+): calcd 376.1366, found 376.1360.

Synthesis of Compound 3

AlCl3 (1.16 g, 9 mmol) was introduced into a pear-shaped flask, followed by the addition of anhydrous CH2Cl2 (10 ml). Compound 2 (1.08 g, 3 mmol) was dissolved in anhydrous CH2Cl2 (5 mL) and carefully added dropwise. The reaction proceeded at ambient temperature for 12 h. Upon completion, HCl solution (2 mol/L, 8 mL) was added dropwise and stirring continued for an additional 0.5 h. Following stirring, the organic solvent was evaporated under reduced pressure. The residual liquid was subsequently subjected to extraction with EA (20 mL × 3), and the combined organic phases were then washed with saturated brine, dried over anhydrous Na2SO4, and concentrated under vacuum. The resulting crude product underwent purification via column chromatography (eluent: PE:EA = 5:1; stationary phase: 200–300 mesh silica gel) to afford compound 3 as yellow-green solid (0.85 g, 81.2% yield). M.P: 98.3–99.4℃.1H NMR (400 MHz, DMSO) δ 11.06 – 10.60 (m, 1H), 10.22 (d, J = 1.6 Hz, 1H), 7.71 (dd, J = 8.0, 1.6 Hz, 1H), 7.53 (dt, J = 8.4, 2.0 Hz, 1H), 7.47 (d, J = 2.0 Hz, 1H), 7.26 (d, J = 8.0 Hz, 1H), 7.24 – 7.19 (m, 2H), 7.17 (d, J = 7.6 Hz, 1H), 7.10 (dd, J = 8.4, 1.6 Hz, 1H), 7.04 (d, J = 8.0 Hz, 1H), 6.96 (t, J = 7.6 Hz, 1H), 3.88 (d, J = 14.0 Hz, 2H), 1.73 (p, J = 7.2 Hz, 2H), 0.96 (td, J = 7.6, 1.6 Hz, 3H).13C NMR (100 MHz, CDCl3) δ 195.8, 162.0, 148.6, 146.1, 144.6, 134.1, 133.2, 127.5, 127.4, 126.3, 126.0, 125.5, 124.1, 122.8, 119.3, 118.1, 115.6, 115.5, 114.8, 49.3, 20.1, 11.3. ESI-HRMS C22H19NO2S ([M + H]+): calcd 362.120926, found 362.11946.

Synthesis of Compound 4

Compound 3 (100 mg, 1 mmol) was added to a two-neck flask along with DCM (5 mL), followed by the addition of DMAP (44 mg, 0.36 mmol) and TEA (91 mg, 0.9 mmol) under argon protection, and cooled to 0 ℃. Finally, tert-butyldimethylchlorosilane (181 mg, 1.2 mmol) was dissolved in DCM (5 mL) and added dropwise. The reaction mixture was stirred at room temperature for 12 h. Upon completion, deionized water (10 mL) was added to quench the reaction, and the mixture was extracted with DCM (10 mL × 3). The combined organic phases were washed with saturated brine, dried over anhydrous Na2SO4, and concentrated under vacuum. The resulting crude product was purified by column chromatography (eluent: PE:EA = 5:1; stationary phase: 200–300 mesh silica gel) to yield compound 4 as yellow-green oil (120 mg, 84% yield). 1H NMR (400 MHz, CDCl3) δ 10.44 (d, J = 0.8 Hz, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.37 – 7.32 (m, 2H), 7.21 – 7.13 (m, 3H), 7.00 (d, J = 1.6 Hz, 1H), 6.95 – 6.87 (m, 3H), 3.87 – 3.83 (m, 2H), 1.89 – 1.83 (m, 2H), 1.04 (d, J = 5.6 Hz, 12H), 0.31 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 189.6, 159.2, 147.5, 145.7, 133.8, 128.8, 127.5, 127.4, 126.1, 125.7, 124.2, 122.8, 119.8, 117.8, 115.6, 115.6, 49.3, 27.0, 25.7, 18.4, 11.3. ESI-HRMS C28H33NO2SSi ([M + Na]+): calcd 498.189348, found 498.18874.

Synthesis of Compound PTC

Compound 4 (950.4 mg, 2 mmol) was added to a round-bottom flask containing anhydrous THF (15 mL), followed by the addition of methyl cyanoformate (99 mg, 4 mmol), and tetrahydrofuran (0.53 mg, 0.0075 mmol), under argon protection, and the reaction mixture was cooled to 0 ℃ and stired for 4 h. Upon completion, the solvent was removed under vacuum, and the crude product was purified by column chromatography (eluent: PE:EA = 5:1; stationary phase: 200–300 mesh silica gel) to yield compound PTC as orange solid (420 mg, 37.7% yield). M.P:120.1–121.2℃.IR (KBr, ν, cm−1): 2948.7, 1709.6, 1579.1, 1467.0, 1245.2, 1135.65, 966.1. 1H NMR (400 MHz, DMSO) δ 8.69 (d, J = 3.6 Hz, 1H), 8.29 (dd, J = 22.4, 8.4 Hz, 1H), 7.56 (d, J = 8.0 Hz, 2H), 7.23 (dd, J = 13.6, 8.6 Hz, 3H), 7.16 (d, J = 8.6 Hz, 1H), 7.09 (d, J = 8.0 Hz, 1H), 7.01 (t, J = 7.6 Hz, 1H), 3.91 (d, J = 10.4Hz, 5H), 1.77 (q, J = 7.2 Hz, 2H), 1.07 – 0.88 (m, 12H), 0.34 (s, 5H). 13C NMR (100 MHz, CDCl3) δ 163.6, 157.2, 149.6, 146.8, 145.8, 144.6, 133.4, 129.6, 127.5, 127.4, 126.1, 125.7, 125.6, 124.1, 122.8, 121.7, 120.1, 117.1, 116.2, 115.7, 115.6, 100.0, 53.1, 49.4, 25.7, 20.1, 18.3, 11.3. ESI-HRMS C32H36N2O3SSi ([M + Na]+): calcd 579.210811, found 597.20996.

Results and Discussion

Synthesis

The fluorescent probe PTC, with a benzothiazole moiety as the fluorophore and utilizing Si–O bonds as recognition groups, was synthesized. Starting from benzothiazole boronate, it underwent a series of reactions including Suzuki coupling (yield: 58.7%), demethylation (yield: 81.2%), nucleophilic substitution (yield: 84%), and nucleophilic addition (yield: 37.7%), as depicted in Scheme 2, resulting in an overall yield of 15.1%. The structural characterization data for all intermediates and the probe can be found in the supporting information (Figures S1-S13). Mass spectrometric analysis of the fluorescent substance generated by the reaction of probe PTC with fluoride ions (Figure S14) showed consistency with the predicted reaction product, validating the fluorescence detection mechanism of this probe.

Scheme 2
scheme 2

Synthetic route of PTC

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Photophysical Properties

Absorption and Fluorescence Spectrum of PTC

The detection capability of the probe PTC for fluoride ions was investigated using absorption spectroscopy and fluorescence spectroscopy. As shown in Fig. 1a, the first absorption band of PTC (30 μM DMSO) appears at 425 nm. Upon response to fluoride ions in DMSO solution, the absorption band shifts to around 520 nm, with an isosbestic point emerging at 475 nm. Figure 1b illustrates that under an excitation wavelength of 520 nm, the probe PTC exhibits weak fluorescence at 580 nm. Upon addition of F−, the fluorescence intensity at 580 nm significantly increases under the same excitation wavelength, with the solution color turning pink. Furthermore, spectra of the probe PTC response to F− were recorded separately at an excitation wavelength of 580 nm and 520 nm, forming a mirror relationship with the emission spectrum. The result shows that, in DMSO solution, the response of the probe PTC to fluoride ions is evident, accompanied by a change in solution color from light to pink, had the ability to detect F−.

Fig. 1
figure 1

(a) Absorption spectra of probe PTC (30 μM) in DMSO solvent with (3 mM) and without F− at room temperature; (b) Fluorescence spectra of probe PTC (5 μM) in DMSO solution with and without F− (0.5 mM) at room temperature; Insert: color change of solution with and without F− under 365 nm light

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The excitation and emission peaks intersect, termed the fluorescence resonance energy transfer (FRET) mechanism. Four common strategies construct fluorescence probes using FRET [39]. This study transforms non-fluorescent receptors into fluorescent structures. In the PTC molecule, the phenothiazine ring serves as the fluorescent donor, with the receptor as a substituent on this ring. Fluorescence at 580nm is minimal without F−. Upon reacting with F−, a new ring forms, initiating FRET and yielding strong emission fluorescence at 580 nm.

Fluorescence Spectra of in Different Solvents

Subsequently, the fluorescence response of probe PTC to F− in various solvents was investigated (Fig. 2). The results reveal that its fluorescence response is most pronounced in THF solution. In acetone (DMK), acetonitrile (CAN), and DMSO solvents, probe PTC also demonstrates a noticeable fluorescence response to F−, with significant changes in fluorescence intensity observed pre- and post-fluoride ion addition. Considering the favorable biocompatibility of DMSO, it was selected as the foundational solvent for subsequent performance tests.

Fig. 2
figure 2

Fluorescence spectra of PTC (5 μM) in various solvents with and without F− (0.5 mM)

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Response Time of PTC

Building on the favorable fluorescence characteristics of probe PTC in DMSO solution, we conducted a detailed examination of the fluorescence intensity variations of probe PTC in response to F− at various time intervals in DMSO solution to elucidate its response kinetics to F-. As illustrated in Fig. 3, the fluorescence intensity of the solution peaked at 3200 a.u. within 5 min post addition of fluoride ions and remained relatively constant within 45 min. This observation suggests that PTC exhibits a rapid and substantial response to fluoride ions, manifested by notable changes in fluorescence intensity.

Fig. 3
figure 3

Time-dependent fluorescence intensity of PTC (5 μM) to F− (0.5 mM), λex = 520 nm, λem = 580 nm

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Job’s Curve and Detection Limit of PTC

To investigate the quantitative relationship between probe PTC and F− concentrations, fluorescence titration spectroscopy was employed. Figure 4a illustrates that at an excitation wavelength of 520 nm, the solution exhibited its maximum emission fluorescence at 580 nm. As the equivalent ratio of F− to probe PTC increased, the fluorescence intensity of the solution gradually intensified. Within the range of 0 to 12.5 μM, the fluorescence intensity exhibited a linear correlation with F− concentration (y = 124.49143C + 1084.7619, R2 = 0.98773) (Fig. 4b). The detection limit (LOD) was calculated using the formula LOD = 3 σ/κ and was found to be 0.51 μM.

Fig. 4
figure 4

(a) Fluorescence titration of PTC (5 μM) upon of F− (0–47.5 μM); Insert: the fluorescence changes with F− concentration (0–47.5 μM) under 365 nm light; (b) Linear correlation between the intensity of fluorescence and F− concentration (0–12.5 μM), λex = 520 nm, λem = 580 nm; Inset: the correlation between the fluorescence intensity and F− concentration (0–47.5 μM), λex = 520 nm

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Selectivity of PTC

To ensure the precise recognition of the target by probe PTC, a comprehensive array of common cations (Na+, Ca2+, K+, Fe2+, Fe3+, Cu2+, Ag+, Zn2+) and anions (Cl−, Br−, I−, SO4 2−, HSO3−, H2PO42−, HCO3−, SCN−, NO3−, HPO4−) were utilized as interferents to assess the selectivity of probe PTC towards F−. The outcomes, as illustrated in Fig. 5, reveal minimal interference from common cations and anions, thereby affirming the probe's efficacy in detecting F− within complex biological environments.

Fig. 5
figure 5

Fluorescence intensity of PTC (5 μM) with and without F− (0.5 mM) in the presence of other cations and anions. (a) various cations (sodium salt, 0.5 mM); (b) various anions (sulfate, 0.5 mM). λex = 520 nm, λem = 580 nm

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Cell Imaging of PTC

Initially, the cytotoxicity of probe PTC towards HepG2 cells was assessed using the MTT colorimetric method. HepG2 cells were treated with probe PTC at concentrations ranging from 0 to 20 μM. The results (Fig. 6) indicate minimal toxicity of PTC, with the cell viability remaining at 90% even at a compound concentration of 20 μM. Subsequently, selecting a concentration of 5 μM for probe PTC in cellular imaging experiments is feasible.

Fig. 6
figure 6

Cell viability of HepG2 cells treated with PTC (0–20 μM) after 24 h

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PBS was selected as the solvent for incubating the probe with cells. Initially, HepG2 cells were treated with PTC for 30 min, followed by the addition of F− for another 30 min, and the fluorescence changes were observed. The probe itself exhibited weak fluorescence in the green channel, but upon the addition of F−, fluorescence intensity notably increased in this channel (Fig. 7), indicating the applicability of probe PTC for fluorescent imaging of F− within living cells.

Fig. 7
figure 7

Cell imaging of HepG2 cells co-incubated with PTC (3 μM in PBS) and F− (100 μM), λex = 488 nm

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Conclusion

In summary, utilizing thiophene boronic acid ester as a starting material, the probe PTC was designed and synthesized, which exhibits specific recognition towards F−. This probe demonstrates good photophysical properties in DMSO solution, showing a good linear fluorescence response in the F− concentration range of 0 to 12.5 μM (R2 = 0.98773). It possesses a low detection limit (0.51 μM), rapid response rate (reaching peak within 5 min), and significant color change of the solution before and after response (colorless to pink), which is discernible to the naked eye. Furthermore, it exhibits strong interference resistance, low cytotoxicity, and is suitable for live cell imaging, holding promise for further development as a rapid detection reagent for F− in biological systems.