Letter pubs.acs.org/ac
Quaternary Ammonium Promoted Ultra Selective and Sensitive Fluorescence Detection of Fluoride Ion in Water and Living Cells Long Li, Yuzhuo Ji, and Xinjing Tang* State Key Laboratory of Natural and Biomimetic Drugs, The School of Pharmaceutical Sciences, Peking University, Beijing 100191, China State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, China S Supporting Information *
ABSTRACT: Highly selective and sensitive fluorescent probes with a quaternary ammonium moiety have been rationally designed and developed for fast and sensitive fluorescence detection of fluoride ion (F− from NaF, not TBAF) in aqueous solution and living cells. With the sequestration effect of quaternary ammonium, the detection time was less than 2 min and the detection limit of fluoride ion was as low as 0.57 ppm that is among the lowest detection limits in aqueous solutions of many fluoride fluorescence probes in the literature. (TBAF) rather than inorganic fluoride (such as NaF) in organic solution or aqueous solution with a high content of organic solvent. In addition, a high concentration of probes or long detection time was required. Until recently, several watersoluble fluorescence probes for fluoride detection were developed. The attachment of sugar moiety greatly enhanced the solubility of fluoride probes which were used to sense fluoride ion in the aqueous environment and cells.11,14 The addition of surface active reagent (CTAB) was sometimes applied to solubilize hydrophobic fluorescent probes and increase the kinetics of desilylation in aqueous solution.12 Our preliminary experiments also indicated that desilyation by NaF aqueous solution was greatly enhanced with the addition of tetrabutylammonium bromide (TBAB) (data not shown). On the basis of these observations, we rationally designed our fluoride fluorescence probes with the attachment of quaternary ammonium. Two concerns were considered: (1) Positively charged quaternary ammonium will greatly increase the solubility of fluorescence probes in water and promote their uptake by cells. (2) Quaternary ammonium itself could sequester fluoride anion due to charge attraction and promote the desilylation of chemodosimeters.
F
luoride anion, as one of the most important ions in biological systems, has received an increasing interest due to its significant role in health science. For dental health, fluoride is added into drinking water in many countries and the maximum limit of fluoride ion in drinking water should be 1.5 ppm, as suggested by the World Health Organization (WHO).1 Fluoride intake is always regarded as a double-edged sword. Appropriate fluoride ingestion can prevent dental cavities and osteofluorosis,2 while excess fluoride intake may result in dental and skeletal fluorosis,3 kidney and gastric disorders, and urolithiasis in humans, which may lead to death.4 Hence, there is a need to sensitively and accurately detect and monitor fluoride ion in drinking water and biological samples. However, common analytic methods, such as ion-selective electrode and ion chromatography,5 require expensive equipment and facilities. Those limitations hinder the rapid and convenient detection of fluoride ion. Fluorescent detection of fluoride ion offers a highly convenient and sensitive approach in authentic sample testing. The development of chemodosimeters or sensors for the recognition and detection of fluoride ion is of growing interest for many years,6 and several fluoride probes have been reported in recent years based on various sensing mechanisms, such as fluoride-hydrogen bonding,7 boronfluoride complexation,8 and desilylation.9−14 Because of the smallest size and high hydration enthalpy of fluoride ion and/or hydrophobic fluorescence probes, most of the fluorescent fluoride probes based on fluoride-hydrogen bonding and boron-fluoride complexation could only work in organic solution or aqueous solution with a high content organic solvent, which are not suitable for fluoride detection in aqueous solution or biological samples. Fluoride induced desilylation is a chemically selective cleavage of the Si−O bond by fluoride ion, originally discovered by Kim and Swager.9 However, most of the fluorescent fluoride probes based on desilylation still required fluoride anion from tetrabutylammonium fluoride © XXXX American Chemical Society
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RESULTS AND DISCUSSION Rational Design and Synthesis of Fluorescence Probes for Fluoride Anion. We reported the design, synthesis of water-soluble fluorescent fluoride probes for rapid and sensitive detection of fluoride ion in aqueous solution. 6-Hydroxybenzothiazole was first chosen as the fluorophore for rational design of quaternary ammonium promoted fluoride probes due to its small size and relative Received: August 24, 2014 Accepted: October 3, 2014
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biological compatibility (Scheme 1).11 Ethyl 6-hydroxybenzothiazole-2-carboxylate was synthesized in two steps according Scheme 1. Synthetic Routes to the Probes L1, L2, and L3 from Intermediate 2
Figure 1. (a) Time-dependent increase in fluorescence intensity change (λex = 380 nm and λem= 520 nm) of 10 μM probe L1, L2, and L3 in PBS (10 mM, pH = 7.2) at 25 °C in the presence of 20 mM NaF. The spectra were performed on a Cary Eclipse fluorometer. (b) Fluorescence intensity of probe L3 in the presence of F− (from NaF) at various concentrations in PBS buffer (10 mM, pH = 7.2). The final F− concentration was 0, 0.19, 0.57, 0.95, 1.43, 1.90, 3.80, and 5.70 ppm, and the data were obtained after 20 min incubation time with Molecular Devices Flex Station III microplate reader (λex = 380 nm and λem= 520 nm). (c) Direct observation of the difference in fluorescence emission of the probe L3 (10 μM) under UV irradiation (365 nm) with various concentration F− (F− final concentration: 0, 0.19, 0.57, 0.95, 1.43, 1.90, 3.80, 5.70 ppm) after 20 min incubation time.
to the previous literature.15 After ester hydrolysis, further coupling between the 6-hydroxybenzothiazole-2-carboxylic acid and corresponding ethanol amine, N,N-dimethylamino ethyl amine and trimethylammonium ethyl amine chloride afforded the intermediates 3, 4, and 5. Probes L1, L2, and L3 were then synthesized through selective silylation of phenol moiety of intermediates 3, 4, and 5 with tert-butyldiphenylsilyl chloride (TBDPS-Cl). (See the Supporting Information for details.) The fluorescence quantum yields were 0.066, 0.036, and 0.062 for L1, L2, and L3 and 0.28, 0.24, and 0.26 for their corresponding desilylated products. Evaluation of Sensitivity and Selectivity. With the hydrophilic moieties (hydroxyl, tertiary amine, and quaternary ammonium), all three probes (L1, L2, and L3) were soluble in PBS buffer (pH = 7.2) at a working concentration (10 μM) and organic solvents or CTAB were not necessarily needed. All three probes in PBS buffer showed almost no fluorescence emission. As shown in Figure 1a, the addition of 20 mM NaF desilylated the TBDPS and induced more than a 30-fold increase in fluorescence intensity for probe L3 and almost reached to the plateau in 2 min, which is almost the fastest fluoride detection among fluorescence fluoride probes based on the desilylation mechanism. However, only a 1.5-fold and 6-fold increase in fluorescence intensity were observed for probes L1 and L2 under the same conditions. Figure 1 also indicated that L2 showed better kinetics in detection of fluoride ion than L1. It took 15 min for L2 to reach the similar plateau of L3. However, less than half of fluorescence intensity was achieved for L1 even after 20 min incubation with NaF under the same conditions. In comparison to time dependence of increase in fluorescence intensity for L1, L2, and L3; L3 showed the best kinetics of fluoride-triggered desilylation, probably due to the fluoride sequestration effect of quaternary ammonium. L2 with tertiary amine showed better kinetics than L1 with a hydroxyl moiety, as shown in Figure 1a. This may be due to the fact that the tertiary amine group of probe L2 in aqueous solution could be partially protonized to form ammonium salt, which might sequester fluoride ion in the same way as probe L3 did. Among these three probes, L3 showed excellent kinetics for rapid detection of fluoride, we then evaluated its sensitivity and selectivity. Since ideal standards of fluoride anion in drinking water are only several ppm, we further evaluated the sensitivity
of L3 in detecting an extremely low concentration of fluoride aqueous solution. Concentration-dependent study shows an excellent linearity between fluorescent intensity (λem = 520 nm) and F− (from NaF) concentrations (0−5.70 ppm) with the regression equation y = 173.9x + 55.8 in PBS (10 mM, pH 7.2) at 25 °C (Figure 1b). In addition, we can also visibly find the fluorescence difference of L3 aqueous solution in the presence of fluoride anion as low as 0.57 ppm by direct observation of fluorescence emission under 365 nm light irradiation (Figure 1c), which is among the lowest detection limits in aqueous solutions of many fluoride fluorescence probes in the literature.12,13 To further evaluate fluorescence fluoride detection of the probe L3, we then investigated the selectivity of probe L3 in the presence of common anions (1 mM in the form of sodium salts and 1 mM TBAF), such as F−, Cl−, Br−, I−, HCO3−, NO2−, N3−, HPO42−, H2PO4−, SO42−, and TBAF. As shown in Figure 2, only F− induced the strong fluorescence emission, while other anions did not. A large increase in fluorescence intensity in the presence of F− was observed with an enhancement ratio up to 121- and 143-fold, respectively, for NaF and TBAF solutions (Figure 2c), which further demonstrated that the quaternary ammonium of probe L3 functioned as the sequestration moiety for enriching the fluoride anion close to the surrounding of the hydrophobic Si− O bond. These results indicated that the quaternary ammonium of L3 not only played an important role in improving solubility but also had a significant sequestration effect for the fluoride ion and enhanced the sensitivity and selectivity of the probe for fluoride detection. A short linker of positively charged ammonium showed effective cleavage of the Si−O bond of L3. To achieve the distance effect on Si−O bond desilylation, we extended the distance of the Si−O band and positive charge by using a 6hydroxybenzothiazole derivative, dehydroluciferin moiety, B
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quaternary ammonium (Figure 3b). It is worth noting that L5 showed slower desilylation of the probe and slow generation of the fluorescent chromophore than that of probe L1. This slower kinetics was due to the longer distance between the Si− O bond and amide bond of probe L5 than that of probe L3, and the shorter linker (CH2CH2) limited the quaternary ammonium to enrich the fluoride ion around the surrounding of the Si−O bond. This also explained the previous report in which the benzothiazolium cation moiety of the fluorescent fluoride probe had no sequestration effect on fluoride detection due to the probe rigid structure.13 In addition, the fluorescence quantum yields were 0.036 and 0.065 for L4 and L5 and 0.20 and 0.35 for their corresponding desilylated products. Even though the response of L5 to fluoride ion in comparison to that of probe L3 was slower, its detection time was still among the fastest fluorescent fluoride probes in the literature and a large increase in triggered fluorescence intensity was observed, as shown in Figure 3b. In addition, the linear dependence of triggered fluorescence intensity on fluoride ion was essential and important for quantitative determination of fluoride concentrations. The probe L5 was treated with NaF aqueous solution in PBS buffer at various concentrations from 0 to 9.5 ppm. The fluorescence enhancement of L5 aqueous solutions (10 μM) was monitored at 25 °C. As seen in Figure 3c, standard curves for L5 was gained between fluorescence signal at 550 nm and the concentrations (ppm) of NaF in 10 min incubation. A good linear relationship between the concentration of fluoride ion and the triggered fluorescence intensity was also observed and the regression equations y = 24.0x + 10.6. Similar to L3, the probe L5 also showed high selectivity for fluoride anion over other common anions. Only fluoride ion can induce a significant increase in fluorescent intensity at 550 nm with the decrease in fluorescence intensity at 450 nm. Without the assistance of organic solvents or a high concentration of external quaternary ammoniums (such as CTAB, TBAB), F− from NaF triggered fluorescence emission close to the addition of F− from TBAF. Cell Studies. Since dehydroluciferin derivative has been widely applied in biological systems, we then tested the turn-on fluorescence response of fluoride ion in HeLa cells using the probe L5. Cell viability was first measured and almost no cellular toxicity under 10 μM of probe L5 was observed (Figure S1 in the Supporting Information). We then incubated HeLa cells with NaF (0.2 mM) for half an hour, followed by washing with PBS buffer three times. The cells were then incubated in DMEM with 10 μM L5 for an hour. Immediate visualization of turn-on fluorescence response of the probe L5 to fluoride ion in HeLa cells was carried out. As shown in Figure 4, only blue fluorescence at 450 nm was observed and almost no green fluorescence emission at 550 nm was visible in cells without fluoride ion preincubation. In contrast, a strong intracellular fluorescence emission at 550 nm was observed for HeLa cells preincubated with NaF solution. The ratiometric images constructed from 410 to 480 nm and from 530 to 590 nm fluorescence collection windows using Image-Pro Plus software gave an average emission ratio value of 0.02 to 0.88, suggesting 44-fold enhancement of ratiometric imaging in the presence of F−. These results confirmed that fluorescent probe L5 can be successfully used to detect and image fluoride ion in cells.
Figure 2. (a) Fluorescence spectra of probe L3 (10 μM) and (b) increase ratios (λex = 380 nm and λem= 520 nm) in fluorescence intensity of probe L3 after the addition of different anion (1 mM in the form of sodium salts) 60 min in PBS (10 mM, pH = 7.2) at 25 °C. (c) Direct observation of fluorescence emission difference of the probe L3 (10 μM) under UV irradiation (365 nm) in the presence of different cations (1 mM). Left to right: F−, Cl−, Br−, I−, HCO3−, NO2−, N3−, HPO42−, H2PO4−, SO42−, and TBAF.
which was widely used in biological systems. Two probes, L4 and its quaternary ammonium form L5, were synthesized (Figure 3a, see the Supporting Information for the synthetic details). We then first examined the kinetic of fluorescence emission upon the addition of fluoride ion (NaF). Similar to the observations of L2 and L3, L5 with a quaternary ammonium moiety exhibited much faster generation of fluorescence emission than that of L4 with a tertiary amine, which further confirmed the sequestration effect of the
Figure 3. (a) Structures of two fluoride fluorescence probes (L4 and L5) based on dehydroluciferin; (b) time-dependent increase in fluorescence intensity (λex= 380 nm, λem = 550 nm) of the probe L4 and L5 in the presence of 50 mM NaF in PBS (10 mM, pH = 7.2) at 25 °C. (c) Fluorescence intensity of probe L5 in the presence of F− at various concentrations in water. (F− final concentration: 0, 1.9, 3.8, 5.7, 7.6, 9.5 ppm). (d) Fluorescence spectra of probe L5 (10 μM) and (e) direct observation of fluorescence emission of the probe L5 (10 μM) under UV irradiation (365 nm) after the addition of each anion (1 mM in the form of sodium salts) 30 min in PBS (10 mM, pH = 7.2−7.4) at 25 °C. Left to right: AcO−, SO42−, H2PO4−, NO2−, Br−, N3−, Cl−, TBAF, F−, HPO42−, I−, ClO4−, and CO32−. C
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CONCLUSIONS We have successfully designed and synthesized quaternary ammonium functionalized fluorescent probes for rapid and sensitive detection of fluoride ion based on desilylationtriggered turn-on fluorescence. The quaternary ammonium moiety of probes not only played an important role in improving solubility, but also had a significant sequestration effect for fluoride ion which enhanced the sensitivity and selectivity of the probes for fluoride detection. With help of the quaternary ammonium moiety, the fluoride ion can be visibly detected at the concentration as low as 0.57 ppm that is among the lowest detection limits by fluorescent probes in the literature. In addition, fluoride ion sensing is rapid and takes only about 2 min and showed large differentiation from many common anions without the need of organic solvents. Further investigation in HeLa cells indicated that probe L5 can be used for the sensitive detection of fluoride ion in cells by both green channel and ratiometric imaging. On the basis of this rational design, fluorescence sensors for different anions can be achieved and these studies are on the way. ASSOCIATED CONTENT
S Supporting Information *
Experimental details, synthesis procedures, and NMR spectra. This material is available free of charge via the Internet at http://pubs.acs.org.
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Figure 4. Cell images taken with high content analyzer. (a) HeLa cells without the treatment of NaF and probe, (b) HeLa cells treated only with probe L5, (c) HeLa cells treated first with NaF, followed by probe L5. Images were recorded as bright field, green channel and blue channel. Ratiometric images were constructed from 410 to 480 nm (blue channel) and from 530 to 590 nm (green channel) fluorescence collection windows. Scale bar is 200 μm.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Fax: 86-10-82805635. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program o f China (“973” P r o g r a m ; G r a nt No . 2013CB933800), the National Natural Science Foundation of China (Grant No. 21372018), Innovation Team of Ministry of Education (Grant No. BMU20110263), and the State Key Laboratory of Pharmaceutical Biotechnology (Grant KF-GN201305). D
dx.doi.org/10.1021/ac503177n | Anal. Chem. XXXX, XXX, XXX−XXX
