- Email: [email protected]
A two-photon fluorescent probe for imaging aqueous fluoride ions in living cells and tissues
Accepted Manuscript A two-photon fluorescent probe for imaging aqueous fluoride ions in living cells and tissues Xiaomin Shi, Wenlong Fan, Chunhua Fan, Zhengliang Lu, Qibing Bo, Zhuo Wang, Cory A. Black, Fangfang Wang, Yanqing Wang PII:
S0143-7208(16)31265-7
DOI:
10.1016/j.dyepig.2017.01.038
Reference:
DYPI 5737
To appear in:
Dyes and Pigments
Received Date: 25 November 2016 Revised Date:
10 January 2017
Accepted Date: 16 January 2017
Please cite this article as: Shi X, Fan W, Fan C, Lu Z, Bo Q, Wang Z, Black CA, Wang F, Wang Y, A two-photon fluorescent probe for imaging aqueous fluoride ions in living cells and tissues, Dyes and Pigments (2017), doi: 10.1016/j.dyepig.2017.01.038. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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A Two-Photon Fluorescent Probe for Imaging Aqueous Fluoride Ions in Living Cells and Tissues
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Xiaomin Shi, a Wenlong Fan, a Chunhua Fan, a Zhengliang Lu, *,a and Qibing Bo, *,a Zhuo Wang,b,* Cory A. Black, c Fangfang Wang a, and Yanqing Wang a a
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Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong (University of Jinan), Shandong Provincial Key Laboratory of Fluorine Chemistry and Chemical Materials, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China. E-Mail:[email protected] b College of Science, Beijing University of Chemical Technology, Beijing, 100029, China. BDG Synthesis, PO Box 38627, Wellington Mail Centre, Wellington 5045, New Zealand.
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Corresponding Author: [email protected], [email protected],
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[email protected]
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Abstract
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Fluoride ions are extensively found in aqueous solution, body fluids and tissues, and play an important function in life, so the detection and stable imaging of fluoride ions
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in living organs is of considerable importance. Herein, we designed and synthesized a new two-photon probe TXS to detect fluoride ions in aqueous solution with turn-on fluorescence. The chemical probe was constructed from tetrahydroxanthenone with a Si-O bond to provide excellent selectivity for fluoride ions. Based on the emission
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properties of tetrahydroxanthenone, two-photon microscopy (TPM) was applied to image fluoride ions in the organs, and the results showed good images in liver tissue at a depth of 60 µm. TPM images of TXS supported a feasible application in
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biological analyses.
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1.Introduction
Fluoride, the smallest and most electronegative anion, has attracted considerable attention due to its essential role in chemical and biological processes of living things, from bacteria to animals [1]. Appropriate levels of fluoride are conducive to
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preventing teeth caries and osteoporosis [2, 3]. High doses of fluoride anion, however, may cause dental and skeletal fluorosis, nephrotoxic changes and urolithiasis in humans [4-7]. Even higher exposure to fluoride ions might cause mitochondrial
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dysfunction due to damage of mitochondria by elevated oxidative stress and decrease of mitochondrial respiratory chain efficiency, and finally lead to serious late-onset
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neurodegenerative diseases [8, 9]. Therefore, sensitive and selective recognition of fluoride is an urgent need for the medical community. In this regard, molecular probe-based fluorescent methodology is a more
convenient technique towards imaging in cells and in vivo than other traditional techniques such as ion-selective electrode and ion chromatography. Furthermore, molecular probe fluorescence has simple sample preparation, high selectivity and sensitivity, noninvasive visibility and a limited requirement for expensive equipment
ACCEPTED MANUSCRIPT [10-17]. Therefore, in recent years, numerous turn-on fluorescent probes for fluoride containing common fluorophores including BODIPY, 6-hydroxybenzothiazole, and luciferase, have been developed according to various mechanisms, especially excitedstate intramolecular proton (ESIPT), photoinduced electron transfer (PET), and
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intramolecular charge transfer (ICT) [18-35]. Unfortunately, some fluoride probes have long reaction times, high detection limits, or non-aqueous working conditions possibly because of the recognition mechanism such as reaction-based fluoride-
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hydrogen bonding, stibonium-fluoride complex formation and boron-fluoride bond formation, which limit their applications for biomedical science [36-38]. Moreover,
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these probes were based on one-photon microscopy (OPM), which showed only very weak penetration ability in tissues with potential damage to cells and tissues from the short one-photon (OP) excitation wavelength. These disadvantages restrict applications of OP probes for fluoride ions on living samples.
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By contrast, two-photon microscopy (TPM) can more effectively bioimage cells and tissues than OPM due to the low energy near-infrared excitation wavelength, with deep penetration ability and weak autofluorescence, lowered phototoxicity and
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photobleaching, and reduced photo damage [39]. Furthermore, TPM not only
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provides the possibility of three-dimensional temporal/spatial localization, but it also can extend the observation window. Therefore, a straightforward direction of effort would be to combine the advantages of fluorescent probes with two-photon microscopy imaging for precise recognition and tracking of target fluoride in complex biological systems. To the best of our knowledge, two-photon (TP) probes for visualization of fluoride ions in tissue have not been demonstrated previously, although a few two-photon fluorescent probes for fluoride have been reported and applied in buffers or cells [30, 40-43], in which only two of them were developed and
ACCEPTED MANUSCRIPT applied for bioimaging fluoride in tumor tissue or zebrafish. Zhang and coworkers developed a probe (Z2) by anchoring tert-butyldiphenylsilane to 1,8-naphthalimide, which successfully detected fluoride in 30% DMSO solution. The high amount of DMSO limited the application in biological analysis because of the obvious toxicity.
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Very recently, another probe (HQB) based on quinoline-2-benzothiazole was reported to sense fluoride in zebrafish with low cytotoxicity. Currently there is no probe capable of detecting fluoride ions in mammalian animals as OP probes cannot
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effectively penetrate tissues to image the target. We have designed a TP probe to image fluoride ions in opaque tissues with greater penetration than has been observed
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previously with OP probes. Generally, the OP probes have a lower two-photon absorption cross section, which is one fundamental parameter of TPM. For example, the fluorophores of both fluorescent probes (Z2 and HQB) with donor-π-acceptor (Dπ-A) structure have a low two-photon absorption cross section (50 GM for Z2 and
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121 GM for HQB) [30, 44]. In this work we have applied new TP fluorophores in order to construct the TP probe.
The tetrahydroxanthenone fluorophore has been shown to display excellent two-
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photon properties with a very big two-photon absorption cross section up to 810 GM,
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good solubility and low cytotoxicity and biocompatibility [45, 46]. We envisioned that the introduction of a Si-O bond into tetrahydroxanthenone would conceivably produce a new two-photon fluorescent F‒ probe. Herein, we report a turn-on F‒specific two-photon fluorescent probe (TXS) based on a tetrahydroxanthenone fluorophore with high selectivity and sensitivity. For the first time, TXS is applied for imaging fluoride ions in liver tissues. The Si-O bond between the tertbutyldimethylsilyl (TBDMS) group and TXS could specifically be cleaved by fluoride ions, which granted the probe TXS with the ability to penetrate live cell
ACCEPTED MANUSCRIPT membranes and rapidly and selectively detect and image the fluoride anions. TXS not only is readily prepared, but also shows excellent F‒-sensing properties in living cells and deep tissues.
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2.Experimental section Materials and instruments. All reagents were purchased from commercial suppliers and used without further purification. Solvents were purified following standard
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methods prior to use. TX was synthesized using a revised procedure [46]. NMR spectra were recorded on a Bruker AVANCE III 400 MHz Digital NMR
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Spectrometer, using TMS as an internal standard. MS spectra were collected from a Bruker apex Ultra 7.0 T FTMS mass spectrometer in electrospray ionization mode (ESI). UV/Vis absorption spectra were obtained on a Shimadzu UV-2600 UV-vis spectropotometer. Fluorescent spectra were recorded at room temperature with an F4600 fluorescence spectrophotometer with the excitation and emission slit widths at
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10.0 and 10.0 nm, respectively. OP and TP images were obtained with an Olympus FV1000-IX81 confocal-laser scanning microscope and Olympus FV1000 two-photon
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fluorescence microscope, respectively. TLC analysis was performed on silica gel plates and column chromatography was conducted over silica gel from the Qingdao
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Ocean Chemicals.
Synthesis of TXS. A mixture of TX (0.1 g, 2 mmol), tert-butyldimethylsilyl chloride (0.33 g, 2.2 mmol), and imidazole (0.16 g, 2.4 mmol) in 10 mL of anhydrous CH2Cl2 was stirred overnight at room temperature. At the end of the reaction, the solvent was removed in vacuo. After purification by silica gel flash chromatography using petroleum ether/EtOAc, the pure title compound as a light yellow solid was obtained in moderate yield (0.232 g, 69%).1H NMR (400 MHz, DMSO-d6) δ (ppm): 0.21 (d, J = 1 Hz, 6H), 0.94 (s, 9H), 1.61-1.73 (m, 1H), 1.86-1.98 (m, 2H), 2.33-2.40 (m, 3H),
ACCEPTED MANUSCRIPT 4.98-5.03 (m, 1H), 6.37-6.38 (d, J = 2.2 Hz, 1H), 6.38-6.46 (dd, J1 = 8.2 Hz, J2 = 2.4 Hz, 1H), 7.09 (d, J = 8.2 Hz, 1H), 7.42 (s, 1H);
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C NMR (400 MHz, DMSO-d6): ‒
4.11, ‒4.07, 17.79, 18.44, 25.93, 29.47, 38.72, 74.94, 107.51, 114.62, 116.50, 128.56,
m/z calcd. 331.17295; Found 331.17245.
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130.58, 131.75, 157.27, 159.10, 196.75; HR-MS: calculated for C19H26O3Si, [M+H]+
Cell culture and imaging. The HeLa cells were washed with PBS buffer three times and first loaded with F- at 37oC for 30 min. After removal of excess F‒ by washing the
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cells with PBS buffer, TXS was added to stain the cells for 30 min. The fluorescence
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images were collected with an Olympus FV1000-IX81 confocal-laser scanning microscope.
Liver slices and TP fluorescence imaging. Slices were prepared from the liver of 4week-old mice, cut to 400 µm thickness and washed with PBS buffer. One set of the
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liver tissue slices was treated with 30 µM TXS for 40 min, and then the culture medium was removed and washed three times with PBS. The other set of the liver slices in the culture medium was treated with 30 µM TXS for 40 min, and then
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incubated with F‒ for 1 h. The residual probe was removed and washed three times using PBS before imaging with an Olympus FV1000 two-photon fluorescence
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microscope equipped with 40× objective lens. The two-photon fluorescence emission was collected between 500 and 550 nm upon excitation at 800 nm with a femtosecond pulse.
3.Results and discussion TXS was obtained in moderate yield by an elimination reaction between tertbutyldimethylsilyl chloride and hydroxyl tetrahydroxanthenone, the latter of which was synthesized according to a previously reported procedure. All compounds were
ACCEPTED MANUSCRIPT characterized by 1H NMR,
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C NMR, and MS (ESI, Fig. S1-S3). Detailed synthetic
procedures are described in the ESI. With TXS in hand, the optical properties of TXS were determined in 1% EtOH aqueous solution. In the absence of fluoride ions, the solution of probe TXS (10 µM)
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in H2O (1% EtOH) is colorless and exhibits a major absorption band centered at 368 nm with slight negligible fluorescence (Fig. S4). The absorption band of probe TXS bathochromically shifted to 377 nm upon addition of 10 equiv. of fluoride ions. A
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strong fluorescence emission at 512 nm was also observed upon exposure to UV light after adding fluoride ions to the solution, with a maximum intensity observed after the
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addition of 30 equiv. of fluoride (Fig. 1). A plot of fluorescence intensity at 512 nm vs. the concentrations of fluoride ions ranging from 0 to 100 µM showed a good linear relationship (R2 = 0.995) in 1% EtOH aqueous solution (HEPES 20 mM, pH 7.4) (Inset of Fig. 1). The detection limit was calculated to be 0.74 µM based on a signal-
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to-noise (S/N = 3) ratio, which is much lower than those previously reported and favorable for direct imaging of intracellular fluoride in a submicromolar range [18, 24, 38, 47-49]. The absorption and fluorescence results indicated that the probe TXS
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possessed high sensitivity for fluoride ions over a large dynamic range concentration.
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The proposed reaction mechanism is shown in Scheme 1. With the anchored TBDMS moiety, intra-molecular charge transfer (ICT) cannot take place in TXS due to lack of an electron-donor group. The presence of fluoride ions selectively cleaves the Si-O bond of TXS, which releases the phenolic hydroxylate group ultimately fulfilling the requirement of a donor-π-acceptor structure. Therefore, ICT from the phenolic hydroxylate group to the xanthenone moiety is activated, leading to enhanced fluorescence. This hypothesis was further confirmed by HRMS (Fig. S5), which displayed a m/z 215.07124 peak for the deprotonated TX (agreeing well with
ACCEPTED MANUSCRIPT the calcd. 215.07082 value) existing in 10 µM TXS solution in HEPES buffer containing 1% EtOH upon addition of fluoride ions (2 mM). Another direct evidence is from the 1 H NMR titration experiments of TXS with F‒. As shown in Fig. 2, when F- in D2O was added to the DMSO-d6 solution of TXS, Ha-f chemical shifts from
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TXS, especially tert-butyldimethylsilyl group all shifted upfield, which evidenced cutting down of Si-O bond (Fig. 2A, B). The phenolic hydroxyl proton signal from the reaction product TX disappeared probably because of the formation of O-H…F
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hydrogen bond with the hydroxyl group and deprotonate the phenolic proton [50-52]. The downfield shift of proton signal of D2O further indicated this phenomena (Fig.
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2B) which was confirmed by the broad OH proton signal at 10.18 ppm from isolated TX from the mixture of TXS + F‒ (Fig. 2C).
In order to investigate the utility of TXS in biology, the pH influence on fluorescence response of TXS to F‒ was examined at different pH values in 1% EtOH
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aqueous solution. As shown in Fig. S6, no fluorescence emission of probe TXS was observed in the absence of fluoride in the pH range 2-7.8. In the presence of fluoride, fluorescence emissions were detected in weakly acidic or near neutral environment
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(pH 5.5-7.8). These experimental results indicated that both probe TXS and TX was
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not sensitive and could be applied for the detection of fluoride ions in a physiological pH range.
As one of the most important fundamental parameters for reaction-based probes, response time of TXS when reacted with various concentrations of fluoride was further examined (Fig. S7). A range of fluoride concentrations (1 equiv., 5 equiv. and 30 equiv.) in 1% EtOH aqueous solution (HEPES 10 mM, pH 7.4) resulted in remarkable enhancement of fluorescence intensity. An apparent plateau was reached upon addition of 30 equiv. of F‒ after 50 min with a pseudo-first-order rate constant
ACCEPTED MANUSCRIPT (k) for F‒ calculated to be 0.044 min‒1 (Fig. S8). The above results therefore indicated that the probe TXS could be used for detecting F‒ at biologically relevant pH. To clarify the selectivity of fluoride, competition experiments to study the selectivity of TXS with various metal cations and anions were performed and the
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respective fluorescence intensities are displayed in Fig. 3. When the titration was conducted in 1% EtOH aqueous solution (HEPES 20 mM, pH 7.4), no competitive metal ions including Na+, K+, Ag+, Hg2+, Cd2+, Cr3+, Mn2+, Cu2+, Ca2+, Ni2+, Li+, Ni2+,
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Ba2+, Al3+, Zn2+, Fe2+, Fe3+, Pb+ induced any obvious fluorescence emission change, even at a concentration of 50 equiv. under physiological conditions. By contrast, only
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fluoride led to a significant fluorescence enhancement at 512 nm compared to other representative anions (Br‒, CH3COO‒, Cl‒, CO32‒, I‒, N3‒, NO3‒, PO43‒, SO32‒, SO42‒ and CN‒) as well as important biothiols (Cys, Hcy and GSH). This data demonstrate
analytes.
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that the probe TXS has exceptional selectivity in vitro towards fluoride against other
To ascertain the biocompatibility of probe TXS, first cytotoxicity experiments were
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carried out using standard cell viability protocols for bioimaging in living HeLa cells by MTT assay. At least 95% of HeLa cells remained in good condition over 12 h,
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after treatment with TXS at concentrations up to 50 µM (Fig. S9), indicating that probe TXS has good cytotoxicity and biocompatibility. To further corroborate the potential biological applications of this newly developed probe with these excellent fluorescence properties, we performed a series of cellbased experiments using confocal microscopy to study whether the probe TXS would selectively bioimage fluoride in living cells in both one-photon (444 nm) and twophoton (800 nm) modes. As shown in Fig. 4a-d, as expected, the untreated living
ACCEPTED MANUSCRIPT HeLa cells did not show any fluorescence with either excitation mode. In fact, regardless of the type of excitation (OP or TP), the incubation of the HeLa cells with either fluoride (40 µM) or TXS (10 µM) did not result in any observable fluorescence emission over 30 min at 37oC in PBS (Fig. 4e-l). Another control experiment was
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carried out to determine whether fluoride selectively triggers the cleavage of the Si-O bond from TXS to emit fluorescence in the living biological samples. First, the Hela cells were incubated with 20 µM TXS for 30 min, then 1 mL of the culture medium
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containing sodium fluoride (100 µM) for another 30 min. Finally, the residual fluoride was removed by washing three times using PBS before imaging with a two-photon
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fluorescence microscope equipped with a 40×objective lens. The incubation of Hela cells with TXS and then fluoride afforded a remarkable OP excited fluorescence enhancement under one-photon excitation (Fig. 4m-o). The fluorescence image collected from the green channel for the same samples under two-photon excitation
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demonstrated a strong fluorescence, which is in excellent agreement with those obtained in the one-photon mode (Fig. 4p). It should be noted that bright-field images of the cells treated with fluoride or TXS, and TXS+F‒ showed that the cells
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maintained good morphology, implying that TXS did not induce cytotoxicity and cell
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death. These results established that TXS is a promising turn-on fluorescent probe for fluoride in living HeLa cells in both the one- and two-photon modes. The favorable performance of the living cell studies inspired us to further explore the probe’s ability to tracking fluoride inside living tissues. Thick tissues slices (1.0 mm) were obtained and treated with 100 µM fluoride and the probe TXS for 30 min, respectively. After washing with PBS, imaging was accomplished using a two-photon fluorescence microscope with a femtosecond laser of 800 nm. As shown in Fig. 5A-C no fluorescence emission was observed in either the untreated liver slices, or liver
ACCEPTED MANUSCRIPT slices incubated with fluoride or the probe, respectively. However, liver tissue slices first treated with fluoride for 30 min and then with the probe for another 30 min exhibited strong green fluorescence, which was enhanced and then decreased gradually with increasing imaging depth (Fig. 5D). The penetration depth of the
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signals from the green channel was up to 60 µm. These studies strongly verified that TXS is able to image basal levels of fluoride deep inside liver tissues under twophoton excitation.
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4.Conclusions
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In summary, we have demonstrated the use of the first two-photon excited fluorescent probe based on the tetrahydroxanthenone fluorophore for sensing fluoride in aqueous buffer. This probe, TXS, displayed highly favorable two-photon fluorescence properties as well as low cytotoxicity and low detection limit. TXS has been successfully applied to image and track fluoride in HeLa cells. Moreover, the
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probe is also applicable for fluoride visualization in liver tissues. Thus, we anticipate that the probe will help to disclose both the physiological and pathological roles of
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fluoride and understand the therapeutic mechanism pertinent to fluoride in biomedical science. Moreover, the rational strategy of TXS for fluoride will promote the
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development of more fluorescent fluoride probes for better imaging performance with other two-photon fluorophores. Acknowledgements
We thank the national Natural Science Foundation of China (grant No. 21101074, 21575032 and 21403088), Shandong Provincial Natural Science Foundation of China (grant No. ZR2013BQ009 and ZR2016BL17), the Doctor’s Foundation of University of Jinan (Grant No. XBS1320), and the startup fund of University of Jinan, Research
ACCEPTED MANUSCRIPT Foundation for Advanced Talents of Beijing University of Chemical Technology. Appendix A. Supplementary information Supporting Information (ESI) including detailed procedures, characterization data,
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and additional plots associated with this article can be found in the online version at.
References:
[1] Newbrun E. What we know and do not know about fluoride. J Public Health Dent 2010;70:227-33.
SC
[2] Farley JR, Wergedal JE, Baylink DJ. Fluoride directly stimulates proliferation and alkaline phosphatase activity of bone-forming cells. Science 1983;222(4621“:330-2.
[3] Yang Z, Zhang K, Gong F, Li S, Chen J, Ma JS, et al. A new fluorescent chemosensor for
M AN U
fluoride anion based on a pyrrole-isoxazole derivative. Beilstein J Org Chem 2011;7:46-52. [4] Kleerekoper M. The role of fluoride in the prevention of osteoporosis. Endocrinol Metab Clin North Am 1998;27(2“:441-52.
[5] Upadhyay KK, Mishra RK, Kumar V, Chowdhury PKR. A coumarin based ICT probe for fluoride in aqueous medium with its real application. Talanta 2010;82(1“:312-8. [6] Singh PP, Barjatiya MK, Dhing S, Bhatnagar R, Kothari S, Dhar V. Evidence suggesting 2001;29(4“:238-44.
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that high intake of fluoride provokes nephrolithiasis in tribal populations. Urol Res [7] Kirk KL. Biochemistry of the elemental halogens and inorganic halides. Plenum Press: New York1991.
[8] Mahaboob Basha P, Saumya SM. Suppression of Mitochondrial Oxidative Phosphorylation
EP
and TCA Enzymes in Discrete Brain Regions of Mice Exposed to High Fluoride: Amelioration by Panax ginseng (Ginseng“ and Lagerstroemia speciosa (Banaba“ Extracts. Cell Mol Neurobiol 2013;33(3“:453-64.
AC C
[9] Li P, Xue Y, Zhang W, Teng F, Sun Y, Qu T, et al. Sodium fluoride induces apoptosis in odontoblasts via a JNK-dependent mechanism. Toxicology 2013;308:138-45. [10] Breadmore MC, Palmer AS, Curran M, Macka M, Avdalovic N, Haddad PR. On-column ion-exchange
preconcentration
electrochromatography
with
of
elution
inorganic using
anions
in
open
tubular
transient-isotachophoretic
capillary
gradients.
3.
implementation and method development. Anal Chem 2002;74:2112-8. [11] De Marco R, Clarke G, Pejcic B. Ion-selective electrode potentiometry in environmental analysis. Electroanalysis 2007;19(19-20“:1987-2001. [12] Gong Q, Shi W, Li L, Wu X, Ma H. Ultrasensitive fluorescent probes reveal an adverse action of dipeptide peptidase IV and fibroblast activation protein during proliferation of cancer cells. Anal Chem 2016;88(16“:8309-14.
ACCEPTED MANUSCRIPT [13] Li X, Gao X, Shi W, Ma H. Design strategies for water-soluble small molecular chromogenic and fluorogenic probes. Chem Rev 2014;114(1“:590-659. [14] Wan Q, Chen S, Shi W, Li L, Ma H. Lysosomal pH rise during heat shock monitored by a lysosome-targeting near-infrared ratiometric fluorescent probe. Angew Chem, Int Ed 2014;53(41“:10916-20. [15] Hu F, Huang Y, Zhang G, Zhao R, Yang H, Zhang D. Targeted bioimaging and
RI PT
photodynamic therapy of cancer cells with an activatable red fluorescent bioprobe. Anal Chem 2014;86(15“:7987-95.
[16] Huang Y, Zhang G, Hu F, Jin Y, Zhao R, Zhang D. Emissive nanoparticles from pyridinium-substituted tetraphenylethylene salts: imaging and selective cytotoxicity towards cancer cells in vitro and in vivo by varying counter anions. Chem Sci 2016;7:7013-9.
SC
[17] Jiang T, Guo D, Wang Q, Wu X, Li Z, Zheng Z, et al. Developing a genetically encoded green fluorescent protein mutant for sensitive light-up fluorescent sensing and cellular imaging of Hg(II“. Anal Chim Acta 2015;876:77-82.
M AN U
[18] Ke B, Wu W, Wei L, Wu F, Chen G, He G, et al. Cell and in Vivo Imaging of Fluoride Ion with Highly Selective Bioluminescent Probes. Anal Chem 2015;87(18“:9110-3. [19] Hu R, Feng J, Hu D, Wang S, Li S, Li Y, et al. A Rapid Aqueous Fluoride Ion Sensor with Dual Output Modes. Angew Chem, Int Ed 2010;49(29“:4915-8.
[20] Xu S-Y, Sun X, Ge H, Arrowsmith RL, Fossey JS, Pascu SI, et al. Synthesis and evaluation of a boronate-tagged 1,8-naphthalimide probe for fluoride recognition. Org Biomol Chem 2015;13(14“:4143-8.
TE D
[21] Zhou Y, Zhang JF, Yoon J. Fluorescence and Colorimetric Chemosensors for FluorideIon Detection. Chem Rev 2014;114(10“:5511-71. [22] Li Y, Cao L, Tian H. Fluoride ion-triggered dual fluorescence switch based on naphthalimides winged zinc porphyrin. J Org Chem 2006;71(21“:8279-82.
EP
[23] Vazquez M, Fabbrizzi L, Taglietti A, Pedrido RM, Gonzalez-Noya AM, Bermejo MR. A colorimetric approach to anion sensing: A selective chemosensor of fluoride ions, in which color
is
generated
by
anion-enhanced
delocalization.
Angew
Chem,
Int
Ed
AC C
2004;43(15“:1962-5.
[24] Wei G, Yin J, Ma X, Yu S, Wei D, Du Y. A carbohydrate modified fluoride ion sensor and its applications. Anal Chim Acta 2011;703(2“:219-25. [25] Liu XY, Bai DR, Wang S. Charge-transfer emission in nonplanar three-coordinate organoboron compounds for fluorescent sensing of fluoride. Angew Chem, Int Ed 2006;45(33“:5475-8. [26] Ke B, Chen W, Ni N, Cheng Y, Dai C, Dinh H, et al. A fluorescent probe for rapid aqueous fluoride detection and cell imaging. Chem Commun 2013;49(25“:2494-6. [27] Zhou X, Lai R, Li H, Stains CI. The 8-silyloxyquinoline scaffold as a versatile platform for the sensitive detection of aqueous fluoride. Anal Chem 2015;87(8“:4081-6. [28] Kim SY, Park J, Koh M, Park SB, Hong J-I. Fluorescent probe for detection of fluoride in water and bioimaging in A549 human lung carcinoma cells. Chem Commun 2009(31“:4735-7.
ACCEPTED MANUSCRIPT [29] Qiu B, Zeng Y, Cao L, Hu R, Zhang X, Yu T, et al. A colorimetric and ratiometric fluorescence sensor for sensitive detection of fluoride ions in water and toothpaste. RSC Adv 2016;6(54“:49158-63. [30] Wu Z, Tang X. Visualizing fluoride ion in mitochondria and lysosome of living cells and in living mice with positively charged ratiometric probes. Anal Chem 2015;87(17“:8613-7. [31] Fan G, Lin Y-X, Yang L, Gao F-P, Zhao Y-X, Qiao Z-Y, et al. Co-self-assembled
RI PT
nanoaggregates of BODIPY amphiphiles for dual colour imaging of live cells. Chem Commun 2015;51(62“:12447-50.
[32] Wu X, Chen X-X, Song B-N, Huang Y-J, Li Z, Chen Z, et al. Induced helical chirality of perylenebisimide aggregates allows for enantiopurity determination and differentiation of
-
hydroxy carboxylates by using circular dichroism. Chem - Eur J 2014;20(37“:11793-9.
SC
[33] Wu X, Chen X-X, Song B-N, Huang Y-J, Ouyang W-J, Li Z, et al. Direct sensing of fluoride in aqueous solutions using a boronic acid based sensor. Chem Commun 2014;50(90“:13987-9.
M AN U
[34] Zhang S, Fan J, Zhang S, Wang J, Wang X, Du J, et al. Lighting up fluoride ions in cellular mitochondria using a highly selective and sensitive fluorescent probe. Chem Commun 2014;50(90“:14021-4.
[35] Yoo M, Park S, Kim H-J. Activatable colorimetric and fluorogenic probe for fluoride detection
by
oxazoloindole-to-hydroxyethylindolium
2016;6(24“:19910-5.
transformation.
RSC
Adv
[36] Wade CR, Ke I-S, Gabbai FP. Sensing of aqueous fluoride anions by cationic stibine-
TE D
palladium complexes. Angew Chem, Int Ed 2012;51(2“:478-81. [37] Zhang G, Wang L, Cai X, Zhang L, Yu J, Wang A. A new diketopyrrolopyrrole (DPP“ derivative bearing boronate group as fluorescent probe for fluoride ion. Dyes Pigm 2013;98(2“:232-7.
EP
[38] Gupta AS, Paul K, Luxami V. Ratiometric fluorescent chemosensor for fluoride ion based on inhibition of excited state intramolecular proton transfer. Spectrochim Acta, Part A 2015;138:67-72.
AC C
[39] Kim HM, Cho BR. Two-Photon Probes for Intracellular Free Metal Ions, Acidic Vesicles, And Lipid Rafts in Live Tissues. Acc Chem Res 2009;42(7“:863-72. [40] Kim D, Singha S, Wang T, Seo E, Lee JH, Lee S-J, et al. In vivo two-photon fluorescent imaging
of
fluoride
with
a
desilylation-based
reactive
probe.
Chem
Commun
2012;48(82“:10243-5.
[41] Hu W, Zeng L, Wang Y, Liu Z, Ye X, Li C. A ratiometric two-photon fluorescent probe for fluoride ion imaging in living cells and zebrafish. Analyst 2016;141:5450-5. [42] Zhang JF, Lim CS, Bhuniya S, Cho BR, Kim JS. A highly selective colorimetric and ratiometric two-photon fluorescent probe for fluoride ion detection. Org Lett 2011;13(5“:11903. [43] Zhu X, Wang J, Zhang J, Chen Z, Zhang H, Zhang X. Imaging of fluoride ion in living cells and tissues with a two-photon ratiometric fluorescence probe. Sensors 2015;15(1“:1611-
ACCEPTED MANUSCRIPT 22. [44] Albota MA, Xu C, Webb WW. Two-photon fluorescence excitation cross sections of biomolecular probes from 690 to 960 nm. Appl Opt 1998;37(31“:7352-6. [45] Shang H, Chen H, Tang Y, Guo R, Lin W. Construction of a two-photon fluorescent turnon probe for hydrogen persulfide and polysulfide and its bioimaging application in living mice. Sens Actuators, B 2016;230:773-8.
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[46] Yuan L, Lin W, Chen H, Zhu S, He L. A unique family of rigid analogues of the GFP chromophore with tunable two-photon action cross-sections for biological imaging. Angew Chem, Int Ed 2013;52(38“:10018-22.
[47] Wang Y, Zhao Q, Zang L, Liang C, Jiang S. Discriminating detection of multiple analytes (F and CN “ by a single probe through colorimetric and fluorescent dual channels. Dyes ‒
‒
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Pigm 2015;123:166-75.
[48] Mahapatra AK, Maiti K, Sahoo P, Nandi PK. A new colorimetric and fluorescent mode. J Lumin 2013;143:349-54.
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bis(coumarin“methylene probe for fluoride ion detection based on the proton transfer signaling [49] Bineci M, Baglan M, Atilgan S. AIE active pyridinium fused tetraphenylethene: Rapid and selective fluorescent "turn-on" sensor for fluoride ion in aqueous media. Sens Actuators, B 2016;222:315-9.
[50] Zang L, Shang H, Jiang S. A multi-stimuli-responsive organogel based on salicylidene Schiff base. Sensor Acutators B 2013;185:389-97.
[51] Hijji YM, Barare B, Kennedy AP, Butcher R. Synthesis and photophysical characterization
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of a Schiff base as anion sensor. Sensor Acutators B 2009;136:297-302. [52] Dalapati S, Alam MA, Jana S, Karmakar S, Guchhait N. Test kit for detection of biologically important anions: a salicylidene-hydrazine based Schiff base. Spectrochim Acta
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Part A 2013;102:314-8.
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Captions to figs:
Scheme1. Proposed imaging mechanism of the TP fluorescent F‒ probe TXS in liver tissue.
Fig. 1. Fluorescence spectra changes of the probe TXS (10 µM) upon gradual addition of fluoride in the reaction system incubated for 50 min. Inset: Concentrationdependent fluorescence intensity change at 512 nm of the probe (10 µM) in the presence of increasing fluoride. λex = 444 nm. Fig. 2. Change in 1H NMR spectra in DMSO-d6 of A) TXS, B) TXS+F‒ (20 equiv.)
ACCEPTED MANUSCRIPT and C) isolated TX from the reaction of TXS+F‒. Fig. 3. Fluorescence responses of probe TXS (10 µM) to various analytes (30 equiv.) in HEPES buffer (pH = 7.4,10 mM). From left to right: Br‒, AcO‒, Cl‒, CO32‒, I‒, N3‒, NO3‒, PO43‒, SO32‒, SO42‒, CN‒, Cys, Hcy, GSH, blank, F‒. Inset: Fluorescence
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response of the probe to various analytes (30 equiv.) in HEPES buffer ( pH = 7.4, 10 mM). 1) Ag+, 2) Al3+, 3) Ba2+, 4) Ca2+, 5) Cd2+, 6) Cr3+, 7) Cu2+, 8) Fe2+, 9) Fe3+, 10) Hg2+, 11) K+, 12) Li+, 13) Mn2+, 14) Na+, 15) Ni2+, 17) Pb2+, 18) Zn2+, 19) F‒. All data
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were obtained after the reaction was conducted for 50 min. λex = 444 nm.
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Fig. 4. OPM and TPM imaging in HeLa cells. a) Bright-field image of untreated HeLa cell in one-photon mode; b) Fluorescence image of (a); c) the merged image of (a) and (b); d) Fluorescence image of HeLa cell in two-photon mode; e) Bright-field image of HeLa cells treated with sodium fluoride (100 µM) in one-photon mode; f) Fluorescence image of (e); g) The merged image of (e) and (f); h) Fluorescence
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image of HeLa cells treated with sodium fluoride (100 µM) in two-photon mode; i) Bright-field image of HeLa cells treated with the probe (20 µM) in one-photon mode;
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j) Fluorescence image of (i); k) The merged image of (i) and (j); l) Fluorescence image of HeLa cells treated with the probe (20 µM) in two-photon mode; m) Bright-
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field image of HeLa cells treated with the probe (20 µM) and sodium fluoride (100 µM) in one-photon mode; n) Fluorescence image of (m); o) The merged image of (m) and (n); p) Fluorescence image of HeLa cells treated with the probe (20 µM) and sodium fluoride (100 µM) in two-photon mode. Scale bar: 20 µm. Fig. 5. Two-photon fluorescence imaging of liver tissue slices. A) Fluorescence images of liver slices. B) Fluorescence images of liver slices incubated with 100 µM sodium fluoride for 30 min. C) Fluorescence images of liver slices incubated with TXS (20 µM) for 30 min. D) Fluorescence images of liver slices incubated with TXS
ACCEPTED MANUSCRIPT (20 µM) for 30 min, then treated with fluoride (20 µM) for 30 min. TPEF images were recorded after excitation at 800 nm with femtosecond laser, and the emission collection was from 500-550 nm. Scale bar: 20 µm. Labels from 0-60 µm indicate
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scanning depths of the tissue slices.
Scheme 1
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Fig. 1.
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Fig. 2.
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IN NO3 PO 3 3SO4 2SO3 24 CN Cy s Hc y GS H Bla nk
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Fig. 3. Cl CO 2-
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Fig. 4.
Fig. 5.
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Research highlights we design a new two-photon fluorescent probe TXS for the detection of fluoride ions
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in aqueous solution with turn-on signal. The cleavage of the Si-O bond of TXS based on tetrahydroxanthenone guarantees the excellent selectivity and sensitivity for fluoride ions.
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The probe TXS is successfully applied to image fluoride in living cells and tissues
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with a depth of 60 µm.
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1
S0143-7208(16)31265-7
DOI:
10.1016/j.dyepig.2017.01.038
Reference:
DYPI 5737
To appear in:
Dyes and Pigments
Received Date: 25 November 2016 Revised Date:
10 January 2017
Accepted Date: 16 January 2017
Please cite this article as: Shi X, Fan W, Fan C, Lu Z, Bo Q, Wang Z, Black CA, Wang F, Wang Y, A two-photon fluorescent probe for imaging aqueous fluoride ions in living cells and tissues, Dyes and Pigments (2017), doi: 10.1016/j.dyepig.2017.01.038. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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A Two-Photon Fluorescent Probe for Imaging Aqueous Fluoride Ions in Living Cells and Tissues
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Xiaomin Shi, a Wenlong Fan, a Chunhua Fan, a Zhengliang Lu, *,a and Qibing Bo, *,a Zhuo Wang,b,* Cory A. Black, c Fangfang Wang a, and Yanqing Wang a a
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Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong (University of Jinan), Shandong Provincial Key Laboratory of Fluorine Chemistry and Chemical Materials, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China. E-Mail:[email protected] b College of Science, Beijing University of Chemical Technology, Beijing, 100029, China. BDG Synthesis, PO Box 38627, Wellington Mail Centre, Wellington 5045, New Zealand.
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c
Corresponding Author: [email protected], [email protected],
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[email protected]
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Abstract
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Fluoride ions are extensively found in aqueous solution, body fluids and tissues, and play an important function in life, so the detection and stable imaging of fluoride ions
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in living organs is of considerable importance. Herein, we designed and synthesized a new two-photon probe TXS to detect fluoride ions in aqueous solution with turn-on fluorescence. The chemical probe was constructed from tetrahydroxanthenone with a Si-O bond to provide excellent selectivity for fluoride ions. Based on the emission
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properties of tetrahydroxanthenone, two-photon microscopy (TPM) was applied to image fluoride ions in the organs, and the results showed good images in liver tissue at a depth of 60 µm. TPM images of TXS supported a feasible application in
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biological analyses.
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1.Introduction
Fluoride, the smallest and most electronegative anion, has attracted considerable attention due to its essential role in chemical and biological processes of living things, from bacteria to animals [1]. Appropriate levels of fluoride are conducive to
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preventing teeth caries and osteoporosis [2, 3]. High doses of fluoride anion, however, may cause dental and skeletal fluorosis, nephrotoxic changes and urolithiasis in humans [4-7]. Even higher exposure to fluoride ions might cause mitochondrial
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dysfunction due to damage of mitochondria by elevated oxidative stress and decrease of mitochondrial respiratory chain efficiency, and finally lead to serious late-onset
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neurodegenerative diseases [8, 9]. Therefore, sensitive and selective recognition of fluoride is an urgent need for the medical community. In this regard, molecular probe-based fluorescent methodology is a more
convenient technique towards imaging in cells and in vivo than other traditional techniques such as ion-selective electrode and ion chromatography. Furthermore, molecular probe fluorescence has simple sample preparation, high selectivity and sensitivity, noninvasive visibility and a limited requirement for expensive equipment
ACCEPTED MANUSCRIPT [10-17]. Therefore, in recent years, numerous turn-on fluorescent probes for fluoride containing common fluorophores including BODIPY, 6-hydroxybenzothiazole, and luciferase, have been developed according to various mechanisms, especially excitedstate intramolecular proton (ESIPT), photoinduced electron transfer (PET), and
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intramolecular charge transfer (ICT) [18-35]. Unfortunately, some fluoride probes have long reaction times, high detection limits, or non-aqueous working conditions possibly because of the recognition mechanism such as reaction-based fluoride-
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hydrogen bonding, stibonium-fluoride complex formation and boron-fluoride bond formation, which limit their applications for biomedical science [36-38]. Moreover,
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these probes were based on one-photon microscopy (OPM), which showed only very weak penetration ability in tissues with potential damage to cells and tissues from the short one-photon (OP) excitation wavelength. These disadvantages restrict applications of OP probes for fluoride ions on living samples.
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By contrast, two-photon microscopy (TPM) can more effectively bioimage cells and tissues than OPM due to the low energy near-infrared excitation wavelength, with deep penetration ability and weak autofluorescence, lowered phototoxicity and
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photobleaching, and reduced photo damage [39]. Furthermore, TPM not only
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provides the possibility of three-dimensional temporal/spatial localization, but it also can extend the observation window. Therefore, a straightforward direction of effort would be to combine the advantages of fluorescent probes with two-photon microscopy imaging for precise recognition and tracking of target fluoride in complex biological systems. To the best of our knowledge, two-photon (TP) probes for visualization of fluoride ions in tissue have not been demonstrated previously, although a few two-photon fluorescent probes for fluoride have been reported and applied in buffers or cells [30, 40-43], in which only two of them were developed and
ACCEPTED MANUSCRIPT applied for bioimaging fluoride in tumor tissue or zebrafish. Zhang and coworkers developed a probe (Z2) by anchoring tert-butyldiphenylsilane to 1,8-naphthalimide, which successfully detected fluoride in 30% DMSO solution. The high amount of DMSO limited the application in biological analysis because of the obvious toxicity.
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Very recently, another probe (HQB) based on quinoline-2-benzothiazole was reported to sense fluoride in zebrafish with low cytotoxicity. Currently there is no probe capable of detecting fluoride ions in mammalian animals as OP probes cannot
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effectively penetrate tissues to image the target. We have designed a TP probe to image fluoride ions in opaque tissues with greater penetration than has been observed
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previously with OP probes. Generally, the OP probes have a lower two-photon absorption cross section, which is one fundamental parameter of TPM. For example, the fluorophores of both fluorescent probes (Z2 and HQB) with donor-π-acceptor (Dπ-A) structure have a low two-photon absorption cross section (50 GM for Z2 and
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121 GM for HQB) [30, 44]. In this work we have applied new TP fluorophores in order to construct the TP probe.
The tetrahydroxanthenone fluorophore has been shown to display excellent two-
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photon properties with a very big two-photon absorption cross section up to 810 GM,
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good solubility and low cytotoxicity and biocompatibility [45, 46]. We envisioned that the introduction of a Si-O bond into tetrahydroxanthenone would conceivably produce a new two-photon fluorescent F‒ probe. Herein, we report a turn-on F‒specific two-photon fluorescent probe (TXS) based on a tetrahydroxanthenone fluorophore with high selectivity and sensitivity. For the first time, TXS is applied for imaging fluoride ions in liver tissues. The Si-O bond between the tertbutyldimethylsilyl (TBDMS) group and TXS could specifically be cleaved by fluoride ions, which granted the probe TXS with the ability to penetrate live cell
ACCEPTED MANUSCRIPT membranes and rapidly and selectively detect and image the fluoride anions. TXS not only is readily prepared, but also shows excellent F‒-sensing properties in living cells and deep tissues.
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2.Experimental section Materials and instruments. All reagents were purchased from commercial suppliers and used without further purification. Solvents were purified following standard
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methods prior to use. TX was synthesized using a revised procedure [46]. NMR spectra were recorded on a Bruker AVANCE III 400 MHz Digital NMR
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Spectrometer, using TMS as an internal standard. MS spectra were collected from a Bruker apex Ultra 7.0 T FTMS mass spectrometer in electrospray ionization mode (ESI). UV/Vis absorption spectra were obtained on a Shimadzu UV-2600 UV-vis spectropotometer. Fluorescent spectra were recorded at room temperature with an F4600 fluorescence spectrophotometer with the excitation and emission slit widths at
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10.0 and 10.0 nm, respectively. OP and TP images were obtained with an Olympus FV1000-IX81 confocal-laser scanning microscope and Olympus FV1000 two-photon
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fluorescence microscope, respectively. TLC analysis was performed on silica gel plates and column chromatography was conducted over silica gel from the Qingdao
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Ocean Chemicals.
Synthesis of TXS. A mixture of TX (0.1 g, 2 mmol), tert-butyldimethylsilyl chloride (0.33 g, 2.2 mmol), and imidazole (0.16 g, 2.4 mmol) in 10 mL of anhydrous CH2Cl2 was stirred overnight at room temperature. At the end of the reaction, the solvent was removed in vacuo. After purification by silica gel flash chromatography using petroleum ether/EtOAc, the pure title compound as a light yellow solid was obtained in moderate yield (0.232 g, 69%).1H NMR (400 MHz, DMSO-d6) δ (ppm): 0.21 (d, J = 1 Hz, 6H), 0.94 (s, 9H), 1.61-1.73 (m, 1H), 1.86-1.98 (m, 2H), 2.33-2.40 (m, 3H),
ACCEPTED MANUSCRIPT 4.98-5.03 (m, 1H), 6.37-6.38 (d, J = 2.2 Hz, 1H), 6.38-6.46 (dd, J1 = 8.2 Hz, J2 = 2.4 Hz, 1H), 7.09 (d, J = 8.2 Hz, 1H), 7.42 (s, 1H);
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C NMR (400 MHz, DMSO-d6): ‒
4.11, ‒4.07, 17.79, 18.44, 25.93, 29.47, 38.72, 74.94, 107.51, 114.62, 116.50, 128.56,
m/z calcd. 331.17295; Found 331.17245.
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130.58, 131.75, 157.27, 159.10, 196.75; HR-MS: calculated for C19H26O3Si, [M+H]+
Cell culture and imaging. The HeLa cells were washed with PBS buffer three times and first loaded with F- at 37oC for 30 min. After removal of excess F‒ by washing the
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cells with PBS buffer, TXS was added to stain the cells for 30 min. The fluorescence
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images were collected with an Olympus FV1000-IX81 confocal-laser scanning microscope.
Liver slices and TP fluorescence imaging. Slices were prepared from the liver of 4week-old mice, cut to 400 µm thickness and washed with PBS buffer. One set of the
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liver tissue slices was treated with 30 µM TXS for 40 min, and then the culture medium was removed and washed three times with PBS. The other set of the liver slices in the culture medium was treated with 30 µM TXS for 40 min, and then
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incubated with F‒ for 1 h. The residual probe was removed and washed three times using PBS before imaging with an Olympus FV1000 two-photon fluorescence
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microscope equipped with 40× objective lens. The two-photon fluorescence emission was collected between 500 and 550 nm upon excitation at 800 nm with a femtosecond pulse.
3.Results and discussion TXS was obtained in moderate yield by an elimination reaction between tertbutyldimethylsilyl chloride and hydroxyl tetrahydroxanthenone, the latter of which was synthesized according to a previously reported procedure. All compounds were
ACCEPTED MANUSCRIPT characterized by 1H NMR,
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C NMR, and MS (ESI, Fig. S1-S3). Detailed synthetic
procedures are described in the ESI. With TXS in hand, the optical properties of TXS were determined in 1% EtOH aqueous solution. In the absence of fluoride ions, the solution of probe TXS (10 µM)
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in H2O (1% EtOH) is colorless and exhibits a major absorption band centered at 368 nm with slight negligible fluorescence (Fig. S4). The absorption band of probe TXS bathochromically shifted to 377 nm upon addition of 10 equiv. of fluoride ions. A
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strong fluorescence emission at 512 nm was also observed upon exposure to UV light after adding fluoride ions to the solution, with a maximum intensity observed after the
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addition of 30 equiv. of fluoride (Fig. 1). A plot of fluorescence intensity at 512 nm vs. the concentrations of fluoride ions ranging from 0 to 100 µM showed a good linear relationship (R2 = 0.995) in 1% EtOH aqueous solution (HEPES 20 mM, pH 7.4) (Inset of Fig. 1). The detection limit was calculated to be 0.74 µM based on a signal-
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to-noise (S/N = 3) ratio, which is much lower than those previously reported and favorable for direct imaging of intracellular fluoride in a submicromolar range [18, 24, 38, 47-49]. The absorption and fluorescence results indicated that the probe TXS
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possessed high sensitivity for fluoride ions over a large dynamic range concentration.
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The proposed reaction mechanism is shown in Scheme 1. With the anchored TBDMS moiety, intra-molecular charge transfer (ICT) cannot take place in TXS due to lack of an electron-donor group. The presence of fluoride ions selectively cleaves the Si-O bond of TXS, which releases the phenolic hydroxylate group ultimately fulfilling the requirement of a donor-π-acceptor structure. Therefore, ICT from the phenolic hydroxylate group to the xanthenone moiety is activated, leading to enhanced fluorescence. This hypothesis was further confirmed by HRMS (Fig. S5), which displayed a m/z 215.07124 peak for the deprotonated TX (agreeing well with
ACCEPTED MANUSCRIPT the calcd. 215.07082 value) existing in 10 µM TXS solution in HEPES buffer containing 1% EtOH upon addition of fluoride ions (2 mM). Another direct evidence is from the 1 H NMR titration experiments of TXS with F‒. As shown in Fig. 2, when F- in D2O was added to the DMSO-d6 solution of TXS, Ha-f chemical shifts from
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TXS, especially tert-butyldimethylsilyl group all shifted upfield, which evidenced cutting down of Si-O bond (Fig. 2A, B). The phenolic hydroxyl proton signal from the reaction product TX disappeared probably because of the formation of O-H…F
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hydrogen bond with the hydroxyl group and deprotonate the phenolic proton [50-52]. The downfield shift of proton signal of D2O further indicated this phenomena (Fig.
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2B) which was confirmed by the broad OH proton signal at 10.18 ppm from isolated TX from the mixture of TXS + F‒ (Fig. 2C).
In order to investigate the utility of TXS in biology, the pH influence on fluorescence response of TXS to F‒ was examined at different pH values in 1% EtOH
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aqueous solution. As shown in Fig. S6, no fluorescence emission of probe TXS was observed in the absence of fluoride in the pH range 2-7.8. In the presence of fluoride, fluorescence emissions were detected in weakly acidic or near neutral environment
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(pH 5.5-7.8). These experimental results indicated that both probe TXS and TX was
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not sensitive and could be applied for the detection of fluoride ions in a physiological pH range.
As one of the most important fundamental parameters for reaction-based probes, response time of TXS when reacted with various concentrations of fluoride was further examined (Fig. S7). A range of fluoride concentrations (1 equiv., 5 equiv. and 30 equiv.) in 1% EtOH aqueous solution (HEPES 10 mM, pH 7.4) resulted in remarkable enhancement of fluorescence intensity. An apparent plateau was reached upon addition of 30 equiv. of F‒ after 50 min with a pseudo-first-order rate constant
ACCEPTED MANUSCRIPT (k) for F‒ calculated to be 0.044 min‒1 (Fig. S8). The above results therefore indicated that the probe TXS could be used for detecting F‒ at biologically relevant pH. To clarify the selectivity of fluoride, competition experiments to study the selectivity of TXS with various metal cations and anions were performed and the
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respective fluorescence intensities are displayed in Fig. 3. When the titration was conducted in 1% EtOH aqueous solution (HEPES 20 mM, pH 7.4), no competitive metal ions including Na+, K+, Ag+, Hg2+, Cd2+, Cr3+, Mn2+, Cu2+, Ca2+, Ni2+, Li+, Ni2+,
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Ba2+, Al3+, Zn2+, Fe2+, Fe3+, Pb+ induced any obvious fluorescence emission change, even at a concentration of 50 equiv. under physiological conditions. By contrast, only
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fluoride led to a significant fluorescence enhancement at 512 nm compared to other representative anions (Br‒, CH3COO‒, Cl‒, CO32‒, I‒, N3‒, NO3‒, PO43‒, SO32‒, SO42‒ and CN‒) as well as important biothiols (Cys, Hcy and GSH). This data demonstrate
analytes.
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that the probe TXS has exceptional selectivity in vitro towards fluoride against other
To ascertain the biocompatibility of probe TXS, first cytotoxicity experiments were
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carried out using standard cell viability protocols for bioimaging in living HeLa cells by MTT assay. At least 95% of HeLa cells remained in good condition over 12 h,
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after treatment with TXS at concentrations up to 50 µM (Fig. S9), indicating that probe TXS has good cytotoxicity and biocompatibility. To further corroborate the potential biological applications of this newly developed probe with these excellent fluorescence properties, we performed a series of cellbased experiments using confocal microscopy to study whether the probe TXS would selectively bioimage fluoride in living cells in both one-photon (444 nm) and twophoton (800 nm) modes. As shown in Fig. 4a-d, as expected, the untreated living
ACCEPTED MANUSCRIPT HeLa cells did not show any fluorescence with either excitation mode. In fact, regardless of the type of excitation (OP or TP), the incubation of the HeLa cells with either fluoride (40 µM) or TXS (10 µM) did not result in any observable fluorescence emission over 30 min at 37oC in PBS (Fig. 4e-l). Another control experiment was
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carried out to determine whether fluoride selectively triggers the cleavage of the Si-O bond from TXS to emit fluorescence in the living biological samples. First, the Hela cells were incubated with 20 µM TXS for 30 min, then 1 mL of the culture medium
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containing sodium fluoride (100 µM) for another 30 min. Finally, the residual fluoride was removed by washing three times using PBS before imaging with a two-photon
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fluorescence microscope equipped with a 40×objective lens. The incubation of Hela cells with TXS and then fluoride afforded a remarkable OP excited fluorescence enhancement under one-photon excitation (Fig. 4m-o). The fluorescence image collected from the green channel for the same samples under two-photon excitation
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demonstrated a strong fluorescence, which is in excellent agreement with those obtained in the one-photon mode (Fig. 4p). It should be noted that bright-field images of the cells treated with fluoride or TXS, and TXS+F‒ showed that the cells
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maintained good morphology, implying that TXS did not induce cytotoxicity and cell
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death. These results established that TXS is a promising turn-on fluorescent probe for fluoride in living HeLa cells in both the one- and two-photon modes. The favorable performance of the living cell studies inspired us to further explore the probe’s ability to tracking fluoride inside living tissues. Thick tissues slices (1.0 mm) were obtained and treated with 100 µM fluoride and the probe TXS for 30 min, respectively. After washing with PBS, imaging was accomplished using a two-photon fluorescence microscope with a femtosecond laser of 800 nm. As shown in Fig. 5A-C no fluorescence emission was observed in either the untreated liver slices, or liver
ACCEPTED MANUSCRIPT slices incubated with fluoride or the probe, respectively. However, liver tissue slices first treated with fluoride for 30 min and then with the probe for another 30 min exhibited strong green fluorescence, which was enhanced and then decreased gradually with increasing imaging depth (Fig. 5D). The penetration depth of the
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signals from the green channel was up to 60 µm. These studies strongly verified that TXS is able to image basal levels of fluoride deep inside liver tissues under twophoton excitation.
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4.Conclusions
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In summary, we have demonstrated the use of the first two-photon excited fluorescent probe based on the tetrahydroxanthenone fluorophore for sensing fluoride in aqueous buffer. This probe, TXS, displayed highly favorable two-photon fluorescence properties as well as low cytotoxicity and low detection limit. TXS has been successfully applied to image and track fluoride in HeLa cells. Moreover, the
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probe is also applicable for fluoride visualization in liver tissues. Thus, we anticipate that the probe will help to disclose both the physiological and pathological roles of
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fluoride and understand the therapeutic mechanism pertinent to fluoride in biomedical science. Moreover, the rational strategy of TXS for fluoride will promote the
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development of more fluorescent fluoride probes for better imaging performance with other two-photon fluorophores. Acknowledgements
We thank the national Natural Science Foundation of China (grant No. 21101074, 21575032 and 21403088), Shandong Provincial Natural Science Foundation of China (grant No. ZR2013BQ009 and ZR2016BL17), the Doctor’s Foundation of University of Jinan (Grant No. XBS1320), and the startup fund of University of Jinan, Research
ACCEPTED MANUSCRIPT Foundation for Advanced Talents of Beijing University of Chemical Technology. Appendix A. Supplementary information Supporting Information (ESI) including detailed procedures, characterization data,
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and additional plots associated with this article can be found in the online version at.
References:
[1] Newbrun E. What we know and do not know about fluoride. J Public Health Dent 2010;70:227-33.
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[2] Farley JR, Wergedal JE, Baylink DJ. Fluoride directly stimulates proliferation and alkaline phosphatase activity of bone-forming cells. Science 1983;222(4621“:330-2.
[3] Yang Z, Zhang K, Gong F, Li S, Chen J, Ma JS, et al. A new fluorescent chemosensor for
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fluoride anion based on a pyrrole-isoxazole derivative. Beilstein J Org Chem 2011;7:46-52. [4] Kleerekoper M. The role of fluoride in the prevention of osteoporosis. Endocrinol Metab Clin North Am 1998;27(2“:441-52.
[5] Upadhyay KK, Mishra RK, Kumar V, Chowdhury PKR. A coumarin based ICT probe for fluoride in aqueous medium with its real application. Talanta 2010;82(1“:312-8. [6] Singh PP, Barjatiya MK, Dhing S, Bhatnagar R, Kothari S, Dhar V. Evidence suggesting 2001;29(4“:238-44.
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that high intake of fluoride provokes nephrolithiasis in tribal populations. Urol Res [7] Kirk KL. Biochemistry of the elemental halogens and inorganic halides. Plenum Press: New York1991.
[8] Mahaboob Basha P, Saumya SM. Suppression of Mitochondrial Oxidative Phosphorylation
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and TCA Enzymes in Discrete Brain Regions of Mice Exposed to High Fluoride: Amelioration by Panax ginseng (Ginseng“ and Lagerstroemia speciosa (Banaba“ Extracts. Cell Mol Neurobiol 2013;33(3“:453-64.
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[9] Li P, Xue Y, Zhang W, Teng F, Sun Y, Qu T, et al. Sodium fluoride induces apoptosis in odontoblasts via a JNK-dependent mechanism. Toxicology 2013;308:138-45. [10] Breadmore MC, Palmer AS, Curran M, Macka M, Avdalovic N, Haddad PR. On-column ion-exchange
preconcentration
electrochromatography
with
of
elution
inorganic using
anions
in
open
tubular
transient-isotachophoretic
capillary
gradients.
3.
implementation and method development. Anal Chem 2002;74:2112-8. [11] De Marco R, Clarke G, Pejcic B. Ion-selective electrode potentiometry in environmental analysis. Electroanalysis 2007;19(19-20“:1987-2001. [12] Gong Q, Shi W, Li L, Wu X, Ma H. Ultrasensitive fluorescent probes reveal an adverse action of dipeptide peptidase IV and fibroblast activation protein during proliferation of cancer cells. Anal Chem 2016;88(16“:8309-14.
ACCEPTED MANUSCRIPT [13] Li X, Gao X, Shi W, Ma H. Design strategies for water-soluble small molecular chromogenic and fluorogenic probes. Chem Rev 2014;114(1“:590-659. [14] Wan Q, Chen S, Shi W, Li L, Ma H. Lysosomal pH rise during heat shock monitored by a lysosome-targeting near-infrared ratiometric fluorescent probe. Angew Chem, Int Ed 2014;53(41“:10916-20. [15] Hu F, Huang Y, Zhang G, Zhao R, Yang H, Zhang D. Targeted bioimaging and
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photodynamic therapy of cancer cells with an activatable red fluorescent bioprobe. Anal Chem 2014;86(15“:7987-95.
[16] Huang Y, Zhang G, Hu F, Jin Y, Zhao R, Zhang D. Emissive nanoparticles from pyridinium-substituted tetraphenylethylene salts: imaging and selective cytotoxicity towards cancer cells in vitro and in vivo by varying counter anions. Chem Sci 2016;7:7013-9.
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[17] Jiang T, Guo D, Wang Q, Wu X, Li Z, Zheng Z, et al. Developing a genetically encoded green fluorescent protein mutant for sensitive light-up fluorescent sensing and cellular imaging of Hg(II“. Anal Chim Acta 2015;876:77-82.
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[18] Ke B, Wu W, Wei L, Wu F, Chen G, He G, et al. Cell and in Vivo Imaging of Fluoride Ion with Highly Selective Bioluminescent Probes. Anal Chem 2015;87(18“:9110-3. [19] Hu R, Feng J, Hu D, Wang S, Li S, Li Y, et al. A Rapid Aqueous Fluoride Ion Sensor with Dual Output Modes. Angew Chem, Int Ed 2010;49(29“:4915-8.
[20] Xu S-Y, Sun X, Ge H, Arrowsmith RL, Fossey JS, Pascu SI, et al. Synthesis and evaluation of a boronate-tagged 1,8-naphthalimide probe for fluoride recognition. Org Biomol Chem 2015;13(14“:4143-8.
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[21] Zhou Y, Zhang JF, Yoon J. Fluorescence and Colorimetric Chemosensors for FluorideIon Detection. Chem Rev 2014;114(10“:5511-71. [22] Li Y, Cao L, Tian H. Fluoride ion-triggered dual fluorescence switch based on naphthalimides winged zinc porphyrin. J Org Chem 2006;71(21“:8279-82.
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[23] Vazquez M, Fabbrizzi L, Taglietti A, Pedrido RM, Gonzalez-Noya AM, Bermejo MR. A colorimetric approach to anion sensing: A selective chemosensor of fluoride ions, in which color
is
generated
by
anion-enhanced
delocalization.
Angew
Chem,
Int
Ed
AC C
2004;43(15“:1962-5.
[24] Wei G, Yin J, Ma X, Yu S, Wei D, Du Y. A carbohydrate modified fluoride ion sensor and its applications. Anal Chim Acta 2011;703(2“:219-25. [25] Liu XY, Bai DR, Wang S. Charge-transfer emission in nonplanar three-coordinate organoboron compounds for fluorescent sensing of fluoride. Angew Chem, Int Ed 2006;45(33“:5475-8. [26] Ke B, Chen W, Ni N, Cheng Y, Dai C, Dinh H, et al. A fluorescent probe for rapid aqueous fluoride detection and cell imaging. Chem Commun 2013;49(25“:2494-6. [27] Zhou X, Lai R, Li H, Stains CI. The 8-silyloxyquinoline scaffold as a versatile platform for the sensitive detection of aqueous fluoride. Anal Chem 2015;87(8“:4081-6. [28] Kim SY, Park J, Koh M, Park SB, Hong J-I. Fluorescent probe for detection of fluoride in water and bioimaging in A549 human lung carcinoma cells. Chem Commun 2009(31“:4735-7.
ACCEPTED MANUSCRIPT [29] Qiu B, Zeng Y, Cao L, Hu R, Zhang X, Yu T, et al. A colorimetric and ratiometric fluorescence sensor for sensitive detection of fluoride ions in water and toothpaste. RSC Adv 2016;6(54“:49158-63. [30] Wu Z, Tang X. Visualizing fluoride ion in mitochondria and lysosome of living cells and in living mice with positively charged ratiometric probes. Anal Chem 2015;87(17“:8613-7. [31] Fan G, Lin Y-X, Yang L, Gao F-P, Zhao Y-X, Qiao Z-Y, et al. Co-self-assembled
RI PT
nanoaggregates of BODIPY amphiphiles for dual colour imaging of live cells. Chem Commun 2015;51(62“:12447-50.
[32] Wu X, Chen X-X, Song B-N, Huang Y-J, Li Z, Chen Z, et al. Induced helical chirality of perylenebisimide aggregates allows for enantiopurity determination and differentiation of
-
hydroxy carboxylates by using circular dichroism. Chem - Eur J 2014;20(37“:11793-9.
SC
[33] Wu X, Chen X-X, Song B-N, Huang Y-J, Ouyang W-J, Li Z, et al. Direct sensing of fluoride in aqueous solutions using a boronic acid based sensor. Chem Commun 2014;50(90“:13987-9.
M AN U
[34] Zhang S, Fan J, Zhang S, Wang J, Wang X, Du J, et al. Lighting up fluoride ions in cellular mitochondria using a highly selective and sensitive fluorescent probe. Chem Commun 2014;50(90“:14021-4.
[35] Yoo M, Park S, Kim H-J. Activatable colorimetric and fluorogenic probe for fluoride detection
by
oxazoloindole-to-hydroxyethylindolium
2016;6(24“:19910-5.
transformation.
RSC
Adv
[36] Wade CR, Ke I-S, Gabbai FP. Sensing of aqueous fluoride anions by cationic stibine-
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palladium complexes. Angew Chem, Int Ed 2012;51(2“:478-81. [37] Zhang G, Wang L, Cai X, Zhang L, Yu J, Wang A. A new diketopyrrolopyrrole (DPP“ derivative bearing boronate group as fluorescent probe for fluoride ion. Dyes Pigm 2013;98(2“:232-7.
EP
[38] Gupta AS, Paul K, Luxami V. Ratiometric fluorescent chemosensor for fluoride ion based on inhibition of excited state intramolecular proton transfer. Spectrochim Acta, Part A 2015;138:67-72.
AC C
[39] Kim HM, Cho BR. Two-Photon Probes for Intracellular Free Metal Ions, Acidic Vesicles, And Lipid Rafts in Live Tissues. Acc Chem Res 2009;42(7“:863-72. [40] Kim D, Singha S, Wang T, Seo E, Lee JH, Lee S-J, et al. In vivo two-photon fluorescent imaging
of
fluoride
with
a
desilylation-based
reactive
probe.
Chem
Commun
2012;48(82“:10243-5.
[41] Hu W, Zeng L, Wang Y, Liu Z, Ye X, Li C. A ratiometric two-photon fluorescent probe for fluoride ion imaging in living cells and zebrafish. Analyst 2016;141:5450-5. [42] Zhang JF, Lim CS, Bhuniya S, Cho BR, Kim JS. A highly selective colorimetric and ratiometric two-photon fluorescent probe for fluoride ion detection. Org Lett 2011;13(5“:11903. [43] Zhu X, Wang J, Zhang J, Chen Z, Zhang H, Zhang X. Imaging of fluoride ion in living cells and tissues with a two-photon ratiometric fluorescence probe. Sensors 2015;15(1“:1611-
ACCEPTED MANUSCRIPT 22. [44] Albota MA, Xu C, Webb WW. Two-photon fluorescence excitation cross sections of biomolecular probes from 690 to 960 nm. Appl Opt 1998;37(31“:7352-6. [45] Shang H, Chen H, Tang Y, Guo R, Lin W. Construction of a two-photon fluorescent turnon probe for hydrogen persulfide and polysulfide and its bioimaging application in living mice. Sens Actuators, B 2016;230:773-8.
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[46] Yuan L, Lin W, Chen H, Zhu S, He L. A unique family of rigid analogues of the GFP chromophore with tunable two-photon action cross-sections for biological imaging. Angew Chem, Int Ed 2013;52(38“:10018-22.
[47] Wang Y, Zhao Q, Zang L, Liang C, Jiang S. Discriminating detection of multiple analytes (F and CN “ by a single probe through colorimetric and fluorescent dual channels. Dyes ‒
‒
SC
Pigm 2015;123:166-75.
[48] Mahapatra AK, Maiti K, Sahoo P, Nandi PK. A new colorimetric and fluorescent mode. J Lumin 2013;143:349-54.
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bis(coumarin“methylene probe for fluoride ion detection based on the proton transfer signaling [49] Bineci M, Baglan M, Atilgan S. AIE active pyridinium fused tetraphenylethene: Rapid and selective fluorescent "turn-on" sensor for fluoride ion in aqueous media. Sens Actuators, B 2016;222:315-9.
[50] Zang L, Shang H, Jiang S. A multi-stimuli-responsive organogel based on salicylidene Schiff base. Sensor Acutators B 2013;185:389-97.
[51] Hijji YM, Barare B, Kennedy AP, Butcher R. Synthesis and photophysical characterization
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of a Schiff base as anion sensor. Sensor Acutators B 2009;136:297-302. [52] Dalapati S, Alam MA, Jana S, Karmakar S, Guchhait N. Test kit for detection of biologically important anions: a salicylidene-hydrazine based Schiff base. Spectrochim Acta
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Part A 2013;102:314-8.
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Captions to figs:
Scheme1. Proposed imaging mechanism of the TP fluorescent F‒ probe TXS in liver tissue.
Fig. 1. Fluorescence spectra changes of the probe TXS (10 µM) upon gradual addition of fluoride in the reaction system incubated for 50 min. Inset: Concentrationdependent fluorescence intensity change at 512 nm of the probe (10 µM) in the presence of increasing fluoride. λex = 444 nm. Fig. 2. Change in 1H NMR spectra in DMSO-d6 of A) TXS, B) TXS+F‒ (20 equiv.)
ACCEPTED MANUSCRIPT and C) isolated TX from the reaction of TXS+F‒. Fig. 3. Fluorescence responses of probe TXS (10 µM) to various analytes (30 equiv.) in HEPES buffer (pH = 7.4,10 mM). From left to right: Br‒, AcO‒, Cl‒, CO32‒, I‒, N3‒, NO3‒, PO43‒, SO32‒, SO42‒, CN‒, Cys, Hcy, GSH, blank, F‒. Inset: Fluorescence
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response of the probe to various analytes (30 equiv.) in HEPES buffer ( pH = 7.4, 10 mM). 1) Ag+, 2) Al3+, 3) Ba2+, 4) Ca2+, 5) Cd2+, 6) Cr3+, 7) Cu2+, 8) Fe2+, 9) Fe3+, 10) Hg2+, 11) K+, 12) Li+, 13) Mn2+, 14) Na+, 15) Ni2+, 17) Pb2+, 18) Zn2+, 19) F‒. All data
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Fig. 4. OPM and TPM imaging in HeLa cells. a) Bright-field image of untreated HeLa cell in one-photon mode; b) Fluorescence image of (a); c) the merged image of (a) and (b); d) Fluorescence image of HeLa cell in two-photon mode; e) Bright-field image of HeLa cells treated with sodium fluoride (100 µM) in one-photon mode; f) Fluorescence image of (e); g) The merged image of (e) and (f); h) Fluorescence
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image of HeLa cells treated with sodium fluoride (100 µM) in two-photon mode; i) Bright-field image of HeLa cells treated with the probe (20 µM) in one-photon mode;
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j) Fluorescence image of (i); k) The merged image of (i) and (j); l) Fluorescence image of HeLa cells treated with the probe (20 µM) in two-photon mode; m) Bright-
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field image of HeLa cells treated with the probe (20 µM) and sodium fluoride (100 µM) in one-photon mode; n) Fluorescence image of (m); o) The merged image of (m) and (n); p) Fluorescence image of HeLa cells treated with the probe (20 µM) and sodium fluoride (100 µM) in two-photon mode. Scale bar: 20 µm. Fig. 5. Two-photon fluorescence imaging of liver tissue slices. A) Fluorescence images of liver slices. B) Fluorescence images of liver slices incubated with 100 µM sodium fluoride for 30 min. C) Fluorescence images of liver slices incubated with TXS (20 µM) for 30 min. D) Fluorescence images of liver slices incubated with TXS
ACCEPTED MANUSCRIPT (20 µM) for 30 min, then treated with fluoride (20 µM) for 30 min. TPEF images were recorded after excitation at 800 nm with femtosecond laser, and the emission collection was from 500-550 nm. Scale bar: 20 µm. Labels from 0-60 µm indicate
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scanning depths of the tissue slices.
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Fig. 5.
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Research highlights we design a new two-photon fluorescent probe TXS for the detection of fluoride ions
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in aqueous solution with turn-on signal. The cleavage of the Si-O bond of TXS based on tetrahydroxanthenone guarantees the excellent selectivity and sensitivity for fluoride ions.
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The probe TXS is successfully applied to image fluoride in living cells and tissues
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with a depth of 60 µm.
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