A Selective Fluorescent Probe for the Determination and Imaging of Fluoride in Living Cells

Fluoride anions are indispensable trace elements required for sustaining life. To investigate the homeostasis and action of fluoride in the body, a new highly sensitive and selective fluorescence detection method was designed for fluoride in aqueous solutions. A fluorescent probe for fluoride (FP-F) was synthesized for imaging F^- in living cells. The design strategy for the probe was based on the specific reaction between fluoride and silica to mediate deprotection of this probe to fluorescein. Upon treatment with F^-, FP-F, a closed and weakly fluorescent lactone, was transformed into an open and strongly fluorescent product. Under the optimum conditions, the detection limit for fluoride was 0.526 nM. FP-F could detect micromolar changes in F^- concentrations in living cells by confocal microscopy.

Keywords:
Fluoride determination; Indispensable trace element; Fluorescent probe; Fluorescence image; Macrophages

Authorship

Xuejun Cui

School of Chemistry and Materials Science, Ludong University, Yantai, Shandong, People’s Republic of ChinaLudong UniversityPeople’s Republic of ChinaYantai, Shandong, People’s Republic of ChinaSchool of Chemistry and Materials Science, Ludong University, Yantai, Shandong, People’s Republic of China

Xingong Wang

Department of Traditional Chinese Medicine, Shandong College of Traditional Chinese Medicine, Jinan, Shandong, People’s Republic of ChinaShandong College of Traditional Chinese MedicinePeople’s Republic of ChinaJinan, Shandong, People’s Republic of ChinaDepartment of Traditional Chinese Medicine, Shandong College of Traditional Chinese Medicine, Jinan, Shandong, People’s Republic of China

Xiaoyan Lin

Department of Nursing, Shandong College Of Traditional Chinese Medicine, Yantai, Shandong, People’s Republic of ChinaShandong College Of Traditional Chinese MedicinePeople’s Republic of ChinaYantai, Shandong, People’s Republic of ChinaDepartment of Nursing, Shandong College Of Traditional Chinese Medicine, Yantai, Shandong, People’s Republic of China

Yanchao Liang ^**Correspondence: Y. Liang. Institute of Shandong Xinhua Pharmaceutical Co., Ltd., 1, Zibo, Shandong, People’s Republic of China. Phone: +86-535-66-72429. Fax: +86-535-66-72429. E-Mail: liangyanchao2012@126.com

Institute of Shandong Xinhua Pharmaceutical Co., Ltd., 1, Zibo, Shandong, People’s Republic of ChinaInstitute of Shandong Xinhua Pharmaceutical Co., Ltd.People’s Republic of ChinaZibo, Shandong, People’s Republic of ChinaInstitute of Shandong Xinhua Pharmaceutical Co., Ltd., 1, Zibo, Shandong, People’s Republic of China

http://orcid.org/0000-0001-8162-8446

*Correspondence: Y. Liang. Institute of Shandong Xinhua Pharmaceutical Co., Ltd., 1, Zibo, Shandong, People’s Republic of China. Phone: +86-535-66-72429. Fax: +86-535-66-72429. E-Mail: liangyanchao2012@126.com

AUTHOR CONTRIBUTIONS

Xuejun Cui contributed to the whole process of the preparation of the fluorescent probe, characterization, confocal fluorescence imaging tests, interpretation of the results and preparation of the manuscript. Yanchao Liang supervised the whole procedure and also contributed to the data interpretation and preparation of the manuscript. Xingong Wang and Xiaoyan Lin contributed significantly to data analysis and probe characterization.

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

SCIMAGO INSTITUTIONS RANKINGS

Figures

Figures (7)

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Scheme 1
Synthesis of FP-F and the reaction of FP-F with F^-

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FIGURE 1
(a) The relative fluorescence intensity of 10 μM FP-F to F^-. Spectra were acquired in 0.02 M PBS buffer at pH 7.40 (λex = 490 nm, λem =513 nm, slit width 1.0 nm). Spectra shown are for buffered [F^-] of 4, 8, 10 20 nM, and 0.1, 0.2, 2 and 4 μM. (b) The linear correlation between the emission intensity and the concentration of F^- (ranged from 1.75×10^-9~ 2.0×10^-8 M). (c) The linear calibration curve between the emission intensity and the concentration of F^- (ranged from 1.0×10^-7~4×10^-6 M).

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FIGURE 2
(a). Effect of pH. (b) Effect of buffer concentration. FP-F (10 µM), NaF (4 µM), PBS (0.02 M).

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FIGURE 3
Effect of the fluorescent probe concentration. NaF (4.0 µM), PBS (pH 7.40, 0.02 M).

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FIGURE 4
Effects of reaction time. FP-F (10 μM), NaF (4 μM), PBS (pH 7.40, 0.02 M).

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FIGURE 5
Selectivity of FP-F for F^- at pH 7.40 (20 mM PBS). Fluorescence response of FP-F to F^-, other anions and oxidative species; bars represent the relative fluorescence intensity (RFI). Initial spectra were acquired in a 10 μΜ solution of FP-F. White bars represent the addition of an excess of the appropriate anion or oxidative species (1.0 mM for Cl^-, Br^-, I^-, SO₄ ^2-, and NO₃ ^-, 20 μΜ for HCO₃ ^- and HPO₄ ^2-, 4.0 μΜ for H₂O₂, HO , O₂ ^- , and F^- ) to a 10 μΜ solution of FP-

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FIGURE 6
Confocal fluorescence and brightfield images of live RAW264.7 cells. (a) Cells incubated with 10 μΜ FP-F for 30 min at 37 °C. (b) Cells supplemented with 4 μΜ F^- in the growth media for 1 h at 37 °C and stained with 10 μΜ FP-F for 30 min at 37 °C. (c) F^- -supplemented cells pretreated with 0.4 mM of a F^- scavenger, CaCl₂ , for 0.5 h at 37 °C before staining with 10 μΜ FP-F. Scale bar=20 μm.

Scheme 1 Synthesis of FP-F and the reaction of FP-F with F^-

FIGURE 1 (a) The relative fluorescence intensity of 10 μM FP-F to F^-. Spectra were acquired in 0.02 M PBS buffer at pH 7.40 (λex = 490 nm, λem =513 nm, slit width 1.0 nm). Spectra shown are for buffered [F^-] of 4, 8, 10 20 nM, and 0.1, 0.2, 2 and 4 μM. (b) The linear correlation between the emission intensity and the concentration of F^- (ranged from 1.75×10^-9~ 2.0×10^-8 M). (c) The linear calibration curve between the emission intensity and the concentration of F^- (ranged from 1.0×10^-7~4×10^-6 M).

FIGURE 2 (a). Effect of pH. (b) Effect of buffer concentration. FP-F (10 µM), NaF (4 µM), PBS (0.02 M).

FIGURE 3 Effect of the fluorescent probe concentration. NaF (4.0 µM), PBS (pH 7.40, 0.02 M).

FIGURE 4 Effects of reaction time. FP-F (10 μM), NaF (4 μM), PBS (pH 7.40, 0.02 M).

FIGURE 5 Selectivity of FP-F for F^- at pH 7.40 (20 mM PBS). Fluorescence response of FP-F to F^-, other anions and oxidative species; bars represent the relative fluorescence intensity (RFI). Initial spectra were acquired in a 10 μΜ solution of FP-F. White bars represent the addition of an excess of the appropriate anion or oxidative species (1.0 mM for Cl^-, Br^-, I^-, SO₄ ^2-, and NO₃ ^-, 20 μΜ for HCO₃ ^- and HPO₄ ^2-, 4.0 μΜ for H₂O₂, HO , O₂ ^- , and F^- ) to a 10 μΜ solution of FP-

FIGURE 6 Confocal fluorescence and brightfield images of live RAW264.7 cells. (a) Cells incubated with 10 μΜ FP-F for 30 min at 37 °C. (b) Cells supplemented with 4 μΜ F^- in the growth media for 1 h at 37 °C and stained with 10 μΜ FP-F for 30 min at 37 °C. (c) F^- -supplemented cells pretreated with 0.4 mM of a F^- scavenger, CaCl₂ , for 0.5 h at 37 °C before staining with 10 μΜ FP-F. Scale bar=20 μm.

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A Selective Fluorescent Probe for the Determination and Imaging of Fluoride in Living Cells

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[1] *Correspondence: Y. Liang. Institute of Shandong Xinhua Pharmaceutical Co., Ltd., 1, Zibo, Shandong, People’s Republic of China. Phone: +86-535-66-72429. Fax: +86-535-66-72429. E-Mail: liangyanchao2012@126.com