A new fluorimetric and colorimetric dual-mode probe, 4-bromo-2-(hydrazonomethyl) phenol (BHP) has been synthesized and successfully utilized for the recognition of Cd2+/F‒ ions in DMSO/H2O (9:1, v/v) system. The probe displays dual channel of detection via fluorescence enhancement and colorimetric changes upon binding with F‒ and Cd2+ ions respectively. The Job’s plot analysis, ESI-MS studies, Density Functional Theoretical (DFT) calculations, 1H NMR and 19F NMR titration results were confirmed and highly supported the 1:1 binding stoichiometry of the probe was complexed with Cd2+/F‒ ions. Furthermore, intracellular detection of F‒ ions in HeLa cells and fluorescence imaging analysis in Zebrafish embryos results of the probe BHP might be used to reveal their potential applications in a biological living system. Introduction The quantification and detection of toxic metal ions in diverse fields have fascinated more attention in recent years due to their prominent and significant roles in clinical diagnosis and ecological system.1–6 Besides metal ions, anions also play an exclusive role in a variety of chemical and biological processes.7–12 In earlier, analytical methods for the detection of cations/anions has required highly sophisticated and expensive instruments such as atomic absorption spectrometry, inductively coupled plasma mass spectrometry, ion sensitive electrodes, and gas and ion chromatography. Amid, fluorescent techniques have more expedient in terms of rapidness, excellent sensitivity and selectivity, low cost, easy and feasible detection. In addition, optical detection mode analysis is a more appropriate method because of their potential features such as easy handling, real-time analysis and different signal output modes.13–16 Besides, colorimetric assays are more feasible and potent tool as they provide a simple visible authentication for analyte detection in the absence of instruments and tedious techniques. In this perspective, the recent research area has been mainly focused to design the novel multi-functional fluorometric and colorimetric sensors for the detection of ions in the different environments. Cadmium (Cd2+) is one of the important hazardous heavy transition metal ions17 in the environment due its carcinogenic nature. The higher accumulation of Cd2+ ion and inhalation of Cd-dust prompts more awful health issues in human like cancer, cardiovascular diseases, kidneys and liver damage.18 Furthermore, the Cd2+ ion has more advantages in several industries such as pigments in plastics, electroplating and batteries, etc. On the other hand, fluoride ions play an ample role in dental health and in the treatment of osteoporosis.19–22 The excess of fluoride ingestion prompted severe disease in human health like gastric and kidney problems.23 In some remote areas, the high level contamination of fluoride ions in drinking water triggered bone disease such as fluorosis.24–31 Thus, to develop and synthesize novel multifunctional probe for the detection and quantification of both cations and anions is a highly anticipated and imperative task. Scheme 1. Synthesis of probe BHP Herein, we have fabricated and synthesized a novel chromogenic and fluorogenic assay based on bromo substituted salicylhydrazone moiety for the colorimetric and fluorometric detection of F‒ ions and colorimetric detection of Cd2+ ions in DMSO/H2O (9:1, v/v) system. The UV-visible and fluorescence spectral analysis of BHP with Cd2+/F‒ ions exposed an outstanding ratiometric absorbance and colorimetric responses towards F‒ ions and also showed a visible colorimetric response towards Cd2+ ions. The fluorescence enhancement of BHP with F‒ ion was highly evaluated by DFT calculations. As well, the cell viability experimental results of BHP can be used for the detection of F‒ ions in both HeLa cells and Zebrafish embryos via high content analysis system. Experimental Methods 2.1 Materials All the chemicals used in the present study were in the analytical reagent grade and solvents used were of HPLC grade. Reagents were used as such received without any further purification. Metal ions such as K+, Na+, Ca2+, Mg2+, Fe2+, Fe3+, Ag+, Zn2+, Mn2+, Cu2+, Co2+, Ni2+, Cd2+, Al3+, Cr3+, Pb2+ and Hg2+ were purchased from Merck and S.D. Fine chemicals. The anions of Cl-, Br-, I-, SCN-, CN-, H2PO4-, HSO4-, NO3-, AcO- and F- were purchased as their tetrabutylammonium salts from Sigma–Aldrich Pvt. Ltd. Absorption measurements were performed on JASCO V-630 spectrophotometer in 1 cm path length quartz cuvette with a volume of 2 mL at room temperature. Fluorescence measurements were made on a JASCO and F- 4500 Hitachi Spectrofluorimeter with excitation slit set at 5.0 nm band pass and emission at 5.0 nm band pass in 1 cm ×1 cm quartz cell. 1H and 13C NMR spectra were obtained on a Bruker 300 MHz NMR instrument with TMS as internal reference using DMSO-d6 as solvent. Standard Bruker software was used throughout. 19F NMR spectra were recorded at 293K on BRUKER 400 MHz FT-NMR spectrometers using DMSO-d6 as solvent. ElectroSpray Ionisation Mass Spectrometry (ESI-MS) analysis was performed in the positive/negative ion mode on a liquid chromatography-ion trap mass spectrometer (LCQ Fleet, Thermo Fisher Instruments Limited, US). Fluorescence microscopic imaging measurements were determined using Operetta High Content Imaging System (PerkinElmer, US) 2.2. Synthesis of (E)-4-bromo-2-(hydrazonomethyl) phenol, BHP An absolute alcoholic solution (50 ml) of 5-bromosalicylaldehyde (0.5gm, 2.49 mmol) was refluxed under hydrazine hydrate (in excess) for 5 hr and the pale yellow color solid product was collected after recrystallized with ethanol and ethyl acetate mixture (yield, 95 %). 1H NMR (300 MHz, DMSO-d6) δ (ppm): 8.92 (s, 1H), 11.89 (s, 1H), 7.53 (d, J = 8.7 Hz, 1H), 6.94 (d, J = 5.8 Hz, 1H); 13C NMR (75 MHz, DMSO-d6) δ (ppm): 161.36, 158.51, 135.84, 131.82, 120.86, 119.69, 106.72. 2.3 Photophysical analysis of BHP The optical mode analysis of BHP towards various cations/anions in DMSO/H2O (9:1, v/v) system was carried out by using absorbance and fluorescence spectroscopy. UV-visible and fluorescence analysis of BHP with cations were gauged by using their corresponding acetate salts of metal ions. Tetrabutylammonium salts of competing anions were used for the anionic sensing analysis. 2.4 Computational Studies The optimized geometrical and ground state energy level calculations of BHP were obtained by Density functional theoretical (DFT) calculations were executed using Gaussian 09 program 32 with the 6-311G basis set. The optimized geometries and the fluorescence enhancement of probe BHP complexed with Cd2+/F- ions were attained by DFT-B3LYP level theory using 6-311G and LANL2DZ basis sets. 2.5 Cytotoxicity studies HeLa cell lines were procured from the National Center for Cell Science (NCCS), Pune, India. Cell lines are kept in the Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% antimycotic and antibiotic solution was used in this study. The cells were kept in an incubator at 25 °C with humidified atmosphere comprising 5% of CO2 and 95% of air. HeLa cells were loaded over the wells of 96 well-culture plates with a density of 1 x 104 cells/well. After 48 h of incubation, previous DMEM medium was exchanged with new medium and BHP (dissolved in DMSO) was added in the range of 0-200 µM to all the wells and further incubated over 3h. Cytotoxicity of BHP was measured by using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. After incubation of HeLa cells with BHP, the medium was detached. Further, 100 μl of DMSO was added and the resulting formazan crystals were dissolved in DMSO. The cell viability was determined by measuring the absorbance of each well at 540-660 nm (formation of formazan) using a microplate reader. 2.6 In vivo fluorescence analysis in Zebrafish embryos The fluorescence imaging analysis was performed in four days old embryos. The embryos were seeded over F- ion alone for 2 h in the E3 medium. The E3 medium was prepared by dissolving 5.0 mM NaCl, 0.17mM KCl, 0.33mM CaCl2, 0.33mM MgSO4 ingredients in H2O (2L) and the pH 7.2 was adjusted by adding NaOH. The embryos were thoroughly washed with E3 medium. Successively, incubated embryos were sowed over 25 mM of BHP (in DMSO) solution for 3h. Further, embryos were washed again with E3 medium and fixed in 10% methyl cellulose solution for the good oriented images. The fluorescent images of BHP-F- were logged using high content screening microscopy. (Excitation wavelength of 482 nm and emission wavelength range of 500-700 nm). Results and discussion The probe, (E)-4-bromo-2-(hydrazonomethyl) phenol (BHP) has been synthesized by one step condensation between hydrazine and 5-bromosalicylaldehyde in ethanol (yield, 95 %) as shown in Scheme 1. The structure of the probe BHP was confirmed via 1H, 13C NMR analysis (Figure S1-S2, See ESI) 3.1. UV–vis spectral analysis of cations with BHP To investigate the cation sensing events of BHP towards different cations in DMSO/H2O (9:1, v/v) system by using UV-vis and fluorescence titration experiments. Initially, free probe BHP exhibited an absorption band at 367 nm and further addition of mono, di and trivalent cations such as Li+, K+, Ag+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Fe2+, Hg2+, Na+, Mg2+, Ca2+, Pb2+, Fe3+ and Cr3+ exhibited tiny changes in absorption spectr due to their weak interaction towards BHP except Cd2+ ion as shown in Figure 1. Interestingly, upon titrated with Cd2+ ion, a new absorption band appeared at 470 nm due to the highly resonance induced charge transfer ability of bromo substituted salicyl moiety while the solution turns into dark yellow color from pale yellow. Increasing addition of Cd2+ ion results gradual reduction of both higher and lower energy bands at 367 nm and 470 nm respectively as depicted in Figure 2. Figure 1. UV-vis spectra of BHP (10 µM) with different cations (5 × 10-3 M) in DMSO/H2O (9: 1, v/v) system. Figure 2. UV-vis spectra of BHP (10 µM) with Cd2+ (0 – 100 µM) in DMSO/H2O (9: 1, v/v) system Besides, fluorescence response of probe BHP towards various cations such as Li+, K+, Ag+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Fe2+, Hg2+, Na+, Mg2+, Ca2+, Pb2+, Fe3+ and Cr3+ including Cd2+ ion have been inspected in DMSO/H2O (9:1, v/v) system. Initially, the probe BHP displayed low intensed fluorescence band in free state. Addition of other commonly coexistent metal ions including Cd2+ ions exhibited trivial changes in fluorescence spectra. From these results, it is concluded that the probe BHP could serve as an excellent colorimetric assay for the detection of Cd2+ ions. 3.2. The sensing analysis of BHP towards anions Moreover, the anion binding attraction of BHP towards anions have been investigated in DMSO/H2O (9:1, v/v) system via both UV-visible and fluorescence spectral techniques. Initially the probe BHP showed the absorption band at 367 nm. Upon titrated with other anions such as Cl‒, Br‒, I‒, NO3‒, AcO‒, HSO4‒, H2PO4‒ and CN‒ were failed to alter the absorbance of the probe BHP except F‒ ions as shown in Figure 3a. Moreover, the incremental addition of F‒ ions (0-50 µM), the higher energy band at 367 nm was decreased along with the increment in new absorption band at 482 nm results an excellent ratiometric response. The new low energy band observed at 482 nm due to the deprotonation of–OH group present in salicyl moiety initiated by hydrogen bonding [Figure 3b]. At that affair, the solution turns into orange color from pale yellow and it was simply discerned by naked eye [Figure 4]. Besides, under identical condition, the fluorescence titration experiment of BHP was carried out in the presence of different anions. Interestingly, the probe BHP displayed low intensed fluorescence band at 601 nm and the other competing anions were failed to affect the fluorescence intensity except F‒ ions as shown in [Figure 5a]. Further, the incremental addition of F‒ ions triggers the enhancement in intensity results an excellent “turn on” fluorescence response due to the deprotonation and the inhibition of charge transfer state stimulated by resonance around the moiety [Figure 5b]. 3.3. Competitive experiments To gauge the selectivity and recognizing ability of BHP, competitive analysis was performed in the presence of varying concentration of F‒ ion (0-50 µM). Initially, the probe was treated with 5 × 10-3 M of different anions such as, CN-, I-, Br-, Cl-, NO2-, CH3COO-, H2PO4- and HSO4-. The other common competing anions were failed to bind with the probe BHP except F- ion [Figure 6 (a) and (b)]. From these observations, it is ensured that BHP could act as an excellent selective and sensitve chromogenic receptor for F- ions in real time monitoring and different biological applications. Figure 3 (a): UV-vis spectra of BHP with 5 × 10-3 M of other anions in DMSO/H2O (9: 1 v/v) system. (b) UV-visible spectra of BHP (5 µM) with F‒ (0-50 µM) in DMSO/H2O (9: 1 v/v) system. Figure 4. Naked eye detection of F‒ ions with BHP under visible light (top) and UV-lamp (bottom) and BHP with Cd2+ visible light only (bottom). Figure 5 (a): Fluorescence spectra of BHP (5µM) with 5 × 10-3 M of other anions in DMSO/H2O (9: 1, v/v) system. Excitation at 482 nm. Slit width is 5 nm. (b) Fluorescence spectra of BHP (5µM) with F‒ (0-50 µM) in DMSO/H2O (9: 1, v/v) system. Excitation at 482 nm. Slit width is 5 nm. Figure 6 (a): Selectivity analysis of F‒ ion with BHP in the presence of competing anions. Excitation at 480 nm, Slit width = 5 nm. (b) The blue bars represent the change of the fluorescence intensity of BHP with the consequent addition of other anions. The pink bars represent the addition of the competing anions to BHP. Excitation at 480 nm, Slit width = 5 nm. 3.4. Job’s plot analysis and calculation of binding constant of BHP for Cd2+/F‒ ions Furthermore, the Job’s plot [Figure 7(a) and (b)] analysis based on UV-visible and fluorescence titration experiments results confirmed the 1:1 binding stoichiometry of BHP with both Cd2+/F‒ ions respectively. To further support the binding stoichiometry of BHP with Cd2+/F‒ions, ESI-MS spectral analysis were performed. The ESI-MS spectral analysis of BHP-Cd2+/BHP-F‒ disclosed peaks at 327.45/258.28 corresponds to [BHP+Cd2++Na+]/[BHP+F‒+H++Na+] respectively (Figure S3-S4, See ESI). Furthermore, the 1:1 binding stoichiometry of BHP with F− ions was confirmed via 1H NMR titration profile (Figure 8) and 19F NMR. The deprotonation of ‒OH group present in the salicyl moiety was initiated by hydrogen bonding and the plausible binding mode of BHP with Cd2+ and F‒ ion is shown in Scheme 2. Further, the absorbance and fluorescence intensity changes of Cd2+ ions (A472 nm) and F‒ ions (A482 nm, I603 nm) were plotted against [Cd2+] and [F‒] respectively provided a good linear relationship between both BHP and Cd2+/F‒ ions (Figure S5, S6 and S7, See ESI). From absorbance and fluorescence titration profile, the binding constant values of BHP for Cd2+/F‒ ions were calculated using modified Benesi-Hildebrand method ions (Figure S8, S9 and S10, See ESI). The binding constant values of BHP with Cd2+ ions were found to be 4.26 ×10-4 M from UV-visible titration profile. Similarly, the binding constant values of BHP with F‒ ions were estimated to be 6.03 ×10-3 M / and 3.01 × 10-4 M from UV-visible and fluorescence titration profile respectively. The detection limits (LOD) of F‒ were calculated to be 0.05 nM respectively. Moreover, the LOD values of BHP signifies that the probe might be utilized for the quantitative determination of F‒ ions in environment and real system. Figure 7 (a) Job’s plot for BHP with F‒ ion. (b) Job’s plot for BHP with Cd2+ ion Scheme 2. Binding mode of BHP with Cd2+/F‒ ions 3.5. 1H NMR titrations of BHP with F- ions In addition, to confirm and highly supported the 1:1 binding stoichiometry of probe with F- ions, 1H NMR titrations was performed. Upon addition of F- ion (0.5 equiv), the proton signal corresponds to phenolic –OH group at 11.14 ppm was gradually decreased. Further, addition of 1 equiv. of F- ions to BHP showed the complete disappearance of –OH proton signal as depicted in Figure 8. Moreover, the binding stoichiometric ratio of F- ion with BHP was further supported by 19F NMR experiment. The (H2F)- signal appeared at -124.33 ppm (Figure S11-S12, See ESI) confirms the deprotonation process arose from phenolic –OH proton. Figure 8 1H NMR titration of BHP with F- (0-1equiv) in DMSO-d6 3.6. DFT calculations of BHP with Cd2+/F- ion To recognize the fluorescence enhancement of probe BHP after complexation with F-, DFT calculations were accomplished. The optimized structures of BHP, BHP-Cd2+ and BHP-F- were obtained using DFT/B3LYP-6-311G and B3LYP/LanL2DZ basis sets respectively. The frontier molecular orbital diagram obtained from optimized structure of BHP is presented in Figure 9. Upon binding with Cd2+ ion, the HOMO and LUMO are delocalized over the entire salicyl unit and their energy gap was reduced. It is noteworthy that inhibition of charge transfer in probe BHP renders the reduction of absorbance at 367 nm and 470 nm. Moreover, Complexation of F- ion to the probe BHP leads to lowering of HOMO-LUMO energy gap. In the presence of F-, HOMO and LUMO are distributed over the whole molecule of BHP. From these results, the F- ion was efficiently binded and complexed with BHP than Cd2+ ion. Figure 9. Frontier molecular orbital diagram of BHP, BHP-Cd2+and BHP-F‒ 3.7. Live cell Imaging analysis of BHP in HeLa cells / Zebrafish embryos The cell viability or cytotoxicity analysis of BHP (0–200 µM) against Human HeLa cells were performed using MTT assay. In 100 µM of BHP, cell viability was obtained as too high as 98%. (Figure S13, See ESI). Hence, the probe was sucessfully used for live cell imaging analysis of F- ions in Figure 10. Live cell fluorescence imaging analysis of BHP in HeLa cells. (a) Bright field images of HeLa cells incubated with BHP (25 µM) for 3h (b) Fluorescence merged images of HeLa cells incubated with BHP (25 µM) (c) Fluorescence image of HeLa cells incubated with BHP (25 µM) alone (d) Fluorescence image of HeLa cells incubated with BHP (25 µM) and 25 µM of F‒ ions for 1 h HeLa cells. Further, the HeLa cells were pre-treated with 25 µM of BHP alone for 3 h. Then HeLa cells were seaded with 25 µM of F- ions for 1h. In the absence of F- ions, the probe BHP exposed a weak yellow fluorescence. However, addition of F- ions to the probe BHP induced a bright orange fluorescence (Figure 10). These results endorsed that the probe BHP can be successfully utilized for the intracellular fluorescence imaging analysis of F- ions in HeLa cells. Besides, the exceptional cell viability output of BHP has been further explored in four days Zebrafish embryos. Zebrafish has positioned as a well-known vertebrate model in numerous biological applications. From this perspective, we have utilized also zebrafish embryos as a living animal model to expose the excellent imaging potential of BHP for the detection of F‒ ion in the biological environment (Figure 11) . Figure 11. Fluorescence imaging analysis of F‒ ion in 4 days old Zebrafish embryos developed with BHP and various concentrations of F‒ ion (a) bright field images of BHP (25 µM) alone, (b) fluorescence merged images of BHP and F- ion (25 µM) (c) fluorescence image of BHP (25 µM) alone (d) 25 µM of F‒ ion for 2 h continuously incubated with BHP (25 µM) for 3 h. 3.8. Evaluation of BHP with previous reports The probe BHP has valid and multi features such as single step synthesis, dual-mode recognition, turn-on fluorescence response and colorimetric change. The probe BHP displayed unique sensing property among other dual sensors. Table S1 compares the sensing performance of BHP with recently reported F‒ receptors. Amid, BHP exhibits too low limit of detection when compared with other previously reported chemoreceptors cited in table S1. Also, the limit of detection of BHP is within the range of recommended limits set by both EPA and WHO for F‒ Ions. Moreover, the fluorescence imaging experiments inferred that the probe BHP can be utilized as potential tool for mapping F‒ ion distribution in HeLa cells and Zebrafish embryos. Conclusions We have designed and synthesized a new chromogenic and fluorogenic probe based on salicylhydrazone derivative for the selective and sensitive detection of both Cd2+/F- ions by colorimetrically and fluorimetrically respectively. As per our knowledge, it is a novel simple hydrazone receptor for sensing carcinogenic heavy metal Cd2+ via colorimetric method and biologically significant F‒ ion by both colorimetric and fluorimetric methods. The binding constant value of Cd2+ ion was found to be 4.26×10-4 M by UV-visible method where as 6.03×10-3 and 3.01×10-4 M for F- ion by both UV-visible and fluorescence methods respectively. The limit of detection was found to be 0.05 nM for F- ion. The excellent biological potential of BHP has been successfully utilized for the detection of F- ions in Zebrafish embryos and human HeLa cells. Acknowledgments The authors acknowledge the financial support from the Council of Scientific and Industrial Research, Extramural Research, New Delhi, India (Grant No. 01(2901)17/EMR-II. The Department of Science and Technology, SERB, Extramural Major Research Project (Grant No. EMR/2015/000969), Department of Science and Technology, CERI, New Delhi, India (Grant No. DST/TM/CERI/C130(G) and we acknowledge the DST-FIST, DST-PURSE,DST-IRPHA, UPE programme and UGC-NRCBS, SBS, MKU for providing instrumentation facilities. References Jäkle, F. Chem. Rev. 2010, 110, 3985. 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