A dihydrazone based “turn–on” fluorescent probe for selective determination of Al3+ ions in aqueous ethanol

Accepted Manuscript Title: A dihydrazone based “turn–on” fluorescent probe for selective determination of Al3+ ions in aqueous ethanol Author: Divya Pratap Singh Romi Dwivedi Ashish Kumar Singh Biplob Koch Priya Singh Vinod Prasad Singh PII: DOI: Reference:

S0925-4005(16)31078-4 http://dx.doi.org/doi:10.1016/j.snb.2016.07.043 SNB 20541

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

4-5-2016 5-7-2016 12-7-2016

Please cite this article as: Divya Pratap Singh, Romi Dwivedi, Ashish Kumar Singh, Biplob Koch, Priya Singh, Vinod Prasad Singh, A dihydrazone based “turn–on” fluorescent probe for selective determination of Al3+ ions in aqueous ethanol, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2016.07.043 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.

A

dihydrazone

based

“turn–on”

fluorescent

probe

for

selective

determination of Al3+ ions in aqueous ethanol

Divya Pratap Singha, Romi Dwivedia, Ashish Kumar Singhb, Biplob Kochc, Priya Singhc, Vinod Prasad Singha,*

a

Department

of

Chemistry,

Institute

of

Science,

Banaras

Hindu

University,

Varanasi−221005, India. b

School of Material Science and Technology, Indian Institute of Technology (BHU),

Varanasi−221005, India c

Department of Zoology, Institute of Science, Banaras Hindu University, Varanasi 221005

*Corresponding author. Tel.: +919450145060, E-mail address: [email protected] (V.P. Singh).

GRAPHICAL ABSTRACT A dihydrazone based highly selective and sensitive fluorescent probe for detection of Al3+ ions in aqueous–ethanol solution is synthesized and characterized. The binding mode, binding constant and detection limit of the probe for Al3+ were determined by 1H NMR and fluorescence studies.

HIGHLIGHTS 

A dihydrazone based fluorescent probe is synthesized.



The probe serves as a selective fluorescent sensor for Al3+ over competitor cations.



The sensing property is monitored by UV−visible, fluorescence and NMR spectroscopy.



A high binding constant of the receptor with Al3+ is reported here.



H2nmh also act as excellent fluorescence probe for Al3 + in living cell.

ABSTRACT An efficient and highly selective dihydrazone based fluorescent probe N',N'–bis((2– hydroxynaphthalen–1–yl)methylene)malonohydrazide (H2nmh), has been synthesized for selective detection of Al3+ ions and characterized by different physico–chemical and spectroscopic techniques. The probe shows an enhanced fluorescence in the presence of Al3+ ions in ethanol–water (2:3 v/v) solution which is not observed in the presence of other cations (Na+, K+, Mg2+, Ca2+, Mn2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Pb2+, Cd2+ and Hg2+). The binding modes of H2nmh with Al3+ were studied by UV–visible, fluorescence and 1H NMR titrations. The probe act as dibasic hexa–dentate ligand and interacts with two Al3+ ions with a binding constant KB = 5.74 × 109 M−1 and detection limit 5.78 × 10–8 M. Detailed insights of probe– metal interaction mechanism were studied by mean of density functional theory (DFT) as well as time dependent–DFT calculation. MTT assay on live MCF–7 cells has been performed to evaluate the cytotoxicity of the probe which suggests viability of the probe to MCF–7 cells even at higher concentration (100 µM) with no serious cytotoxicity in cells. Live cell imaging study clearly indicates that the accumulation of Al3+ in the cytoplasm of cells can be detected by H2nmh.

Keywords: Acyldihydrazone; Fluorescent probe; Detection limit; Cell imaging.

1

H NMR titration; Binding constant;

1. Introduction The development of new chemosensors for detection of toxic metal ions using UV−visible /fluorescence spectroscopic method is a dynamic area of research in chemical science owing to detrimental effects caused by these metal ions [1–4]. Aluminium and its compounds have been widely used in the water treatment, in food additives, in medicines and in the production of light alloys. Al3+ ions interact with the biological species and leads to harmful effects and is the main source of dementia, myopathy, anemia, alzheimer’s disease, bone and joint diseases [5–8]. Al3+ interferes with the uptake of Ca2+ by plants and causes the retarded growth of plants [9]. Schiff bases are one of the most important and widely explored organic species. Their low-cost, synthetic ease and tremendous applications, inspired us to work on Schiff bases as chemical probes for selective detection of toxic metal ions [10,11]. A Schiff base (5−[{(2−hydroxynaphthalen−1−yl)methylene}amino]pyrmidine−2,4(1H,3H)−dione

(AMN)

developed by our group has lowest detection limit for Al3+ in acetonitrile and water 3.2 × 10−7 M (Kb = 1.5 × 105) and 1 × 10−6 M (Kb = 3.685 × 104 M−1), respectively [12]. Another

Schiff

base,

(E)−N−[(2−hydroxy-naphthalen−1−yl)methylene]thiophene−2−

carbohydrazide (THN), has lowest detection limit 1.35 × 10−9 M (Kb = 7.06 × 106 M−1) for Al3+ in ethanol−water medium which is second highest reported till date in the literature [13]. Although a number of Schiff bases have been utilized as a chemosensor for the detection of various ions [14,15], the dihydrazone Schiff bases are rarely investigated as a fluorescent probe for a metal ion [16]. Here, we are reporting a dihydrazone based efficient fluorescent

probe

N',N'–bis((2–hydroxynaphthalen–1–yl)methylene)malonohydrazide

(H2nmh) for highly selective detection of Al3+ ions. Naphthalene moiety of the selected probe acts as an ideal component of a fluorescent chemosensor due to its short fluorescence lifetime, low fluorescence quantum yield and ability to act as a donor as well as an acceptor [17]. Most of the reported Al3+ sensors suffer from poor water solubility, tedious synthetic methods of preparation and high-cost [12–17]. However, H2nmh is soluble in ethanol water mixture (2:3 v/v), can be synthesized in single step condensation process using inexpensive starting materials and lower detection limit better than most of the reported Al3+ sensors. The low-cost and highly sensitive detection limit may strongly encourage the effective and practical application of H2nmh as a promising chemosensor for the detection of Al3+ ions present in water as well as biological cells.

2. Experimental 2.1 Materials and methods All analytical reagent grade chemicals were obtained from the commercial sources. Metal chloride salts of all cations were purchased from Merck Chemicals, India. 2–Hydroxy– 1–naphthaldehyde, malonic acid dihydrazide (Sigma–Aldrich Chemicals, USA) and solvents (Merck Chemicals, India) were used as such. 2.2 Synthesis of H2nmh The

probe

N,N'–bis((2–hydroxynaphthalen–1–yl)methylene)malonohydrazide

(H2nmh) [18] was synthesized by reacting 50 ml aqueous solution of malonic acid dihydrazide (5 mmol, 0.66 g) with 10 ml ethanolic solution of 2–hydroxy–1–naphthaldehyde (10 mmol, 1.72 g) in a round bottom flask. The product was precipitated by stirring the above solution mixture on a magnetic stirrer for 2 h at room temperature. The precipitate was purified by washing several times with water followed by ethanol. Analytical data: Yield: 85%. M.P. 245 °C. Anal. Calc. for C25H20N4O4 (440.46): C, 68.17; H, 4.58; N, 12.72. Found: C, 68.26; H, 4.55; N, 12.65%. ESI–MS: m/z, 441.1. IR (KBr, cm–1): ν(O–H), 3432; ν(N–H), 3197; ν(C=O), 1678; ν(C=N), 1578; ν(C–OH), 1329; ν(N–N), 955. 1H NMR (δ, ppm (DMSO-d6): 12.46 (s, OH, 2H); 10.75 (s, NH, 2H,); 9.25 (s, CH, 2H); 8.32−7.27 (m, Ar–H, 12H); 3.76 (s, CH2, 2H). 13C NMR (DMSO–d6): 168.2, (C=O); 162.5, (C–OH); 157.9, (C=N); 136.1–108.5, (aromatic carbons); 53.7 (CH2).

2.3 Synthesis of Al(III) complex The Al(III) complex of H2nmh was synthesized by reacting 50 ml ethanolic solution of Al(III) chloride (2 mmol, 0.266 g) with 50 ml suspended ethanolic solution of H2nmh (1 mmol, 0.440 g) in 2:1 (M:L) molar ratio. The reaction solution was stirred for 4 h on a magnetic stirrer and then evaporated slowly at room temperature to get the Al(III) complex as a brown solid product. The product was filtered, washed with ethanol followed by diethyl ether and dried in a desiccator. [Al2(nmh)(H2O)2Cl4]·4H2O

Brown, yield: 60%. M.P. >3000C. Anal. Calc. for C25H30Al2Cl4N4O10 (742.31): C, 40.45; H, 4.07; N, 7.54. Found: C, 40.57; H, 4.06; N, 7.60%. ESI–MS: m/z, 742.7. IR (KBr, cm–1): ν(O–H), 3389; ν(C=N), 1584, 1561; ν(C–OH), 1314; ν(C–O–), 1271; ν(N–N), 989. 2.4 Instrumentation C, H, N contents were determined on an Exeter Analytical Inc. CHN Analyzer (Model CE–440). 1H and

13

C NMR spectra were recorded in DMSO–d6 on a JEOL AL–300 FT–

NMR multinuclear spectrometer. Chemical shifts were reported in parts per million (ppm) using tetramethylsilane (TMS) as an internal standard. Infrared spectra were recorded in KBr on a Perkin Elmer FT–IR spectrophotometer in the 4000–400 cm1 region. UV–visible spectra were recorded on a Shimadzu spectrophotometer, Pharmaspec UV–1700 model in EtOH–H2O (2:3 v/v) solvent. Fluorescence spectra were recorded on a Horiba Jobin–Yvon Fluorolog 3 (model FL3–11) spectrofluorometer with 4 nm slit width. 2.5 General methods UV–visible, fluorescence and 1H NMR titration experiments were carried out at room temperature. For UV–visible titration, 50 µM solutions and for fluorescence titration experiments, 5 µM solutions of H2nmh in EtOH–H2O (2:3 v/v) were used. The solutions of metal chloride salts were prepared in triple distilled water. Donor–acceptor binding ratio was determined by Job’s plot, while binding constant (KB) of the adduct was analyzed by linear fitting of fluorescence titration curve in modified Benesi–Hildebrand equations [19] for spectrofluorometric titration (Eq. 1) Io / I–Io = (a / b–a)2(1 / KB [substrate]2+1)

(1)

where, Io and I are fluorescence intensity of H2nmh at 451 nm in the absence and presence of Al3+; a, b are constants; [substrate] is the concentration of Al3+. The detection limit of H2nmh probe for the analysis of Al3+ was determined from a plot of fluorescence intensity as a function of the concentration of the added metal ions. To determine the S/N ratio, the fluorescence intensity of probe without Al3+ was measured by 10 times and determined the standard deviation (). The detection limit was calculated as three times the standard deviation () from the blank measurement (in the absence of Al3+ ion) divided by the slope of calibration plot between Al3+ ion concentration and fluorescence intensity (Eq. 2). Detection limit = 3/ slope

(2)

The binding affinity of H2nmh with other cations was investigated by UV–visible absorption spectra. 1H NMR titration was performed by treating H2nmh (5 × 10–4 M solution in DMSO–d6) with Al3+ (1 × 10–2 M solution in D2O) on increasing equivalents of Al3+ (0.5, 1.0, 1.5 and 2.0 equivalent). 2.6 Details of Biological Study 2.6.1. Materials Dulbecco Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), were purchased from Gibco, antibiotic solution (Penicillin 1000 IU and Streptomycin 10mg/mL), trypsin and MTT (3–(4,5–dimethylthiazol–2–yl)–2,5-diphenyltetrazoliumbromide dye) were obtained from Himedia, India, and DMSO from Merck, India. 2.6.2. Cell culture MCF–7 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS), 100 units/mL and 100 µg/mL streptomycin solutions. The cells were incubated in a humidified atmosphere at 37 °C in 5% CO2. Stock solution of the probe H2nmh was prepared in DMSO and then diluted with DMEM. The final concentration of DMSO in DMEM was less than 0.1% v/v. 2.6.3. MTT Assay MTT assay has been done to evaluate the cytotoxicity of the probe on live MCF–7 cells. Briefly, 1×104 MCF–7 cells were seeded in DMEM with 10% FBS in 96 well tissue culture plate, incubated and allowed to adhere overnight. The adhered cells were then treated with increasing concentration of H2nmh and incubated in CO2 incubator at 37 °C in a humidified atmosphere for 24h. After incubation, the cells were washed with PBS and MTT (0.5 µg in 100µL) was added to each well and incubated for another 2h. 100 µL DMSO was used to dissolve the purple colored formazan crystals and the plates were analyzed in micro plate reader at 570 nm. The result of MTT assay was expressed in percentage cell viability. 2.6.4. Live cell imaging Briefly, 1×105 MCF−7 cells were seeded in a six well tissue culture plate and allowed to adhere overnight. The cells were then treated and incubated further with different concentrations of H2nmh for 2h in CO2 incubator. For detection of Al3+ ions, pre-treated cells were incubated for 15 min with AlCl3. Subsequently, cells were washed with PBS and images were recorded on inverted fluorescence microscope ((EVOS® FL Cell Imaging System, Life Technologies, Carlsbad, California) in blue channel as well as phase contrast.

2.7. Computational details Theoretical calculations were performed for the H2nmh and its Al(III) complex using Gaussian-09 suit of programs [20]. Both probe and probe-metal complex were treated as spin restricted DFT wave functions (RB3LYP), i.e. the Becke three–parameter exchange functional in combination with the LYP correlation functional of Lee, Yang and Parr with 631G** basis set for all the atoms [21,22]. DFT optimized structures were confirmed to be minima on potential energy surface (PES) by performing harmonic vibration frequency analyses (no imaginary frequency found). No symmetry constraints were applied and only the default convergence criteria were used during the geometric optimizations. Based on the optimized geometries, TDDFT calculations were performed at the same RB3LYP level to calculate the vertical electronic transition energies [23,24]. 3. Results and discussion 3.1 Structural characterization of H2nmh and its Al(III) complex The IR spectrum of H2nmh exhibits broad bands centered at 3432 and 3197 cm–1 assigned as ν(OH) and ν(NH), respectively [16]. In addition, the bands appeared at 1678, 1578 and 1329 cm–1 in H2nmh are assigned as ν(C=O), ν(C=N) and ν(C–OH), respectively. The ν(C=O) and ν(NH) bands disappear in the Al(III) complex due to the enolization of both carbonyl groups [25]. The appearance of a ν(C–O–) at 1271 cm–1 and an additional ν(C=N) band at 1584 cm–1 in Al(III) complex, suggest the deprotonation of enolized cabonyl group and bonding with carbonylate−O. In the complex, ν(C=N) and ν(C–OH) bands shift to lower wave number (17 cm–1 and 44 cm–1, respectively), indicating the coordination of these groups to metal [16]. Further, the shifting of ν(N–N) bands to higher wave number (35 cm–1) in the Al(III) complex as compared to the probe suggest involvement of one of the nitrogen atom of >N−N< in bonding [26]. The 1H NMR signals for –C–OH and >N–H protons in H2nmh (Fig. S1) appear at 12.46 and 10.75 ppm, respectively [16]. The signals observed at 9.25, 8.32–7.27 and 3.76 ppm are assigned for >CH=N, aromatic and CH2 protons, respectively. Signals in

13

C NMR

spectrum of H2nmh appear at 168.2, 162.5, 157.9, 136.1–108.5 and 53.7 ppm for >C=O, –C– OH, >C=N–, aromatic and >CH2 carbons, respectively (Fig. S2). The molecular ion peak observed at m/z = 441.1 in mass spectrum of H2nmh (Fig. S3) corresponds well to [M+H]+ and validates the molecular formula. The ESI-MS spectrum of the Al(III) complex exhibits a molecular ion peak at m/z = 742.7 with a 48% intensity which confirms the molecular formula, [Al2(nmh)(H2O)2Cl4]·4H2O (Fig. S4).

Since, we are unable to get the crystals suitable for single crystal X-ray diffraction study, we have optimized the geometry of H2nmh using density functional theory. Molecular structure of H2nmh with atom labeling scheme is shown in Fig. 1 and selected bond lengths and angles are summarized in Table 1. Imine ‒C=N‒, hydrazinic =N‒N‒, amidic =C‒NH– and carbonyl >C=O bond distances suggest conjugation through these groups. However, due to unavailability of π orbitals at CH2 group, conjugation is not extended throughout the molecule and in fact planes of both naphthalene rings are at 43.55° with respect to each other (Fig. S5). DFT calculations were carried out for the complex in gas phase to fully optimize the ground state structure. Molecular structure of Al(III) complex with atom labeling scheme is shown in Fig. 2 and selected bond lengths and angles are summarized in Table 1. Presence of two chlorides on each Al3+ metal centers indicates that the two negative charges are likely provided by the H2nmh ligand as a result of deprotonation. There is possibility of deprotonation either of phenolic ‒OH or amidic –NH‒. From spectroscopic methods, it is confirmed that amidic –NH‒ group is getting deprotonated. By density functional theory calculations, we can also validate the exact position of deprotonation from the two possible deprotonated structures of nmh2‒ anion (Fig. S6). Out of the two structures, structure 1 is most probable than 2 because of its lower energy (6.29 kcal/mol). Similarly, the Al(III) complex involving nmh2‒ anions (1) has the lower energy than 2+Al3+ (35.77 kcal/mol) as shown in Fig. S6. Upon binding with Al3+, carbonyl >C=O and hydrazinic =N‒N‒ bonds are getting elongated, whereas amidic =C‒NH‒ bond are shortened which clearly indicate the enolization in ligand upon binding. Interestingly, C‒C bonds around methylene group (C3‒C4 and C4‒C5) are also getting shortened, suggesting partial extension of conjugation around methylene group also. Moreover, this extension of conjugation is partial only and hence, metal complex has bent structure (planes of both naphthalene rings are at 84.33° with respect to each other) (Fig. S7). 3.2. Investigation on sensing property 3.2.1 UV-visible studies The solution of H2nmh appears light yellow in EtOH–H2O (2:3 v/v) and its UV– visible spectra exhibit a band at 356 nm. Addition of 100 µM aqueous solution of Al3+ to 50 µM solution of H2nmh in EtOH–H2O (2:3 v/v), results in change of the color of the solution to intense blue which is clearly visible by naked eye. As a result of addition of 2 equivalents of Al3+ ions in H2nmh, the absorption intensity is enhanced and position of the band shifts

from 356 nm to 374 nm with two shoulder peaks at 391 and 412 nm (bathochromic shift of ~18 nm) (Fig. 3a). To further study the binding interactions, UV–visible titrations were performed in 50 µM EtOH–H2O (2:3 v/v) solution of H2nmh after addition of varying amounts (0–5 equivalents) of Al3+ ions, (Fig. 3b). The addition of other transition metal ions viz. Fe3+, Co2+, Ni2+, Cu2+ and Zn2+, Pb2+, Cd2+ and Hg2+ (as chloride) also perturbs the UV–visible spectral pattern of H2nmh to different extents (Fig. S8). The alkali metal ions, alkaline earth metal ions and Mn2+ did not produce significant changes upon their addition in solution of H2nmh. To quantify the binding ratio between H2nmh with Al3+ ion, Job’s plot from UV–visible spectra is constructed by varying the concentration of Al3+ ions (Fig. 4), which clearly indicates 2:1 binding stoichiometry for metal–probe complex. In order to get a deeper understanding of the electronic transitions, TDDFT calculations have been performed. The assignments of the calculated transitions to the experimental bands are based on the criteria of energy and oscillator strength of the calculated transitions. In the description of the electronic transitions, only the main components of the molecular orbitals are taken into consideration [27,28]. The calculated absorption bands for the H2nmh as well as H2nmh–Al(III) complex are shown by the vertical lines in Fig. 5 and band assignments are tabulated in Table 2 with oscillator strengths and energies. The results of time–dependent density functional theory (TDDFT) calculations on [Al2(nmh)(H2O)2Cl4] at the B3LYP level reveal that most of the transitions are ligand centered n→π* or π→π* (ILCT) or ligand to ligand Charge Transfer (LLCT) transitions (Table 2). Intensity of experimental UV–vis spectra is correlated well with the calculated oscillator strength. 3.2.2 Fluorescence studies Since, UV–visible spectroscopic studies were unable to afford exclusive selectivity for sensing to any of the ions under investigation, therefore, the fluorescence emission studies were performed to examine the selectivity of probe H2nmh for various metal ions viz. Na+, K+, Mg2+, Al3+, Ca2+, Mn2+, Fe3+, Co2+ , Ni2+, Zn2+, Pb2+, Cd2+ and Hg2+ (Fig. 6). The probe H2nmh displays weak fluorescence, but becomes highly fluorescent on adding two equivalents of aqueous Al3+ ions. At this stage, a distinct change in color occurs from light yellow–green to intense blue under UV light due to the formation of a strong fluorescent complex with Al3+.

The fluorescence spectra of H2nmh exhibit emission peaks at 451 nm (Fig. 7). On addition of 2 equivalents of aqueous Al3+ ion in 5 µM solutions of H2nmh, a very high intensity emission band appears at 451 nm on exciting at 380 nm. The presence of intra– molecular charge transfer (ICT) in H2nmh is sufficient enough to quench its fluorescence in the absence of Al3+ ions. The cis–trans isomerization across >C=N– bond of dihydrazone probe may also be responsible for their non–fluorescent nature [29]. However, this isomerization is inhibited by the binding of Al3+ ions leading to fluorescence switching of the probe [30]. The chelation of H2nmh with Al3+ not only reduces the ICT effect in H2nmh but also increases the rigidity of the molecular assembly by restricting the free rotations of the azomethine carbon with respect to the naphthalene ring resulting in a significant enhancement of the fluorescence intensity, which is known as chelation–enhanced fluorescence (CHEF) [31–33]. From DFT calculation, we found that bending in metal bonded ligand was more compared to free-ligand. More bending of probe H2nmh may restrict the free rotation around the methylene group and contribute to fluorescence enhancement. Although, Fe3+, Cu2+ and Zn2+ show no significant change in fluorescence intensity of H2nmh individually, but their presence with Al3+ causes inhibition of the fluorescence emission of H2nmh (Fig. 8). Other cations Na+, K+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Pb2+, Cd2+ and Hg2+ show no interference in detection of Al3+ in EtOH–H2O solution. The selectivity of H2nmh probe towards Al3+ may be explained on the basis of smaller ionic radii (0.5A˚) and higher charge density (r = 4.81) of the Al3+ ion. The smaller radii of the Al3+ ion allow appropriate coordination geometry for the chelating probe H2nmh, and the larger charge density allows strong coordination ability between the probe and Al3+ [33]. The fluorescence turn on of H2nmh by Al3+ was further supported by fluorescence titration (Fig. 7b). Upon addition of Al3+, fluorescence response occurs immediately and the maximum fluorescence intensity has been observed on adding up to 6 equivalents of 100 µM aqueous Al3+ in 5 µM EtOH–H2O (2:3 v/v) solution of H2nmh. Accordingly, the binding constant KB (Fig. 9) was determined by linear fitting of fluorescence titration data in Benesi– Hildebrand equation (Eq. 1) [19]. The binding constant values KB = 5.74 × 109 M–1 evaluated for H2nmh–Al3+, indicate 2:1 (M:L) complex stoichiometry, as a result of close agreement of the experimental data to the theoretical fit, respectively. The detection limit was calculated according to the IUPAC definition [34] and found to be 5.78 × 10–8 M in the linearity range of 3.32 × 10–6–6.62 × 10–6 M (R2 = 0.9853) (Fig. S9).

3.2.3 1H NMR titrations In order to further support the UV–visible and fluorescence studies for the interaction of the probe with Al3+, 1H NMR titrations have been performed by the concomitant addition of Al3+ to 5 x 10–4 M solution of the probe in DMSO–d6. On addition of Al3+ to the probe, a significant change in its 1H NMR spectral patterns has been observed (Fig. 10). The sharp intense–NH peak disappears due to its involvement in enolization of >C=O group. The phenolic–OH proton peak becomes less intense and shifts slightly downfield indicating the interaction of Al3+ with phenolic–OH. The overall changes in 1H NMR spectra indicate the complexation between Al3+ and H2nmh through carbonylate–O, azomethine−N and phenolic– OH. 3.3. Bio–imaging To further evaluate the practical applicability of the designed chemosensor probe H2nmh, fluorescent imaging experiments were carried out to demonstrate its value in bio−imaging intracellular Al3 + in MCF–7 cell lines. MCF–7 cells, when incubated with either the probe or Al3+ alone exhibit no fluorescence emission. However, when Al3+ was incubated in MCF–7 cells, pre-treated with the probe, the cells show distinct fluorescence in blue channel as shown in Fig. 11, which indicates that accumulation of Al3+ in the cytoplasm of cells can be detected by the probe. Further, the fluorescence was highly intense when cells were incubated with 20 µM concentration of the probe. In addition, the viability of probe to MCF–7 cells was determined by MTT assay with the probe at various concentrations (Fig. 12). The probe under investigation reveals that it does not induced substantially effect on the viability of MCF–7 cells even at higher concentration (100 µM), indicating that the probe exhibits no serious cytotoxicity in cells at 20 µM concentration. Thus, H2nmh can be used as a biosensor to detect presence of aluminum ions in live biological system and may have potential bio-medical applications. 4. Conclusions This paper describes the synthesis and characterization of a dihydrazone based fluorescent probe for selective determination of Al3+ ion in EtOH–H2O (2:3 v/v) solution. UV−visible and fluorescence studies were performed to determine the selectivity of various metal ions. The probe is weakly fluorescent and enhance its fluorescence property significantly after addition of Al3+ ions due to reduction in ICT and restricted rotation of >C=N– bond. The hexa−dentate probe binds with two Al3+ ions through carbonylate−O, azomethine−N and phenolic−OH. On the basis of job’s plot, 1H NMR titrations and mass

analysis, the 2:1 (M:L) stoichiometry was established for the Al3+ complex with formulation [Al2(nmh)(H2O)2Cl4]·4H2O. The binding constant (KB) and the lowest detection limit for Al3+ were also determined. The structures of probe and its Al3+ complex were optimized by DFT calculations. H2nmh can also be applied as an excellent fluorescent probe for detection of Al3+ in living cell. Acknowledgements The authors thank the Head, S.A.I.F., Central Drug Research Institute, Lucknow, India for recording mass spectra. One of the authors V.P.S. is also grateful to the UGC, New Delhi, for providing financial assistance from the project P–01/700, F. No. 43-204/2014 (SR). Appendix A. Supplementary data Electronic supplementary information (ESI) available: 1H &

13

C NMR; Mass spectra of

H2nmh and its Al(III) complex; UV–visible spectra of H2nmh on addition of various cations.

References [1] K.P. Carter, A.M. Young, A.E. Palmer, Chem. Rev. 114 (2014) 4564–4601. [2] D. Maity, T. Govindaraju, Chem. Commun. 48 (2012) 1039–1041. [3] A. Saini, J. Singh, R. Kaur, N. Singh and N. Kaur, New J. Chem. 38 (2014) 4580–4586. [4] A.D. Arulraj, R. Devasenathipathy, S.-M. Chen, V.S. Vasantha, S.-F. Wang, Sensing and Bio-Sensing Research 6 (2015) 19–24. [5] B. Liu, P.-F. Wang, J. Chai, X.-Q. Hu, T. Gao, J.-B. Chao, T.-G. Chen, B.-S. Yang, Spectrochim. Acta A 168 (2016) 98–103. [6] N. Chatterjee, S.B. Maity, A. Samadder, P. Mukherjee, A. R. Khuda-Bukhsh, P.K. Bharadwaj, RSC Adv. 6 (2016) 17995–18001. [7] L.K. Kumawat, N. Mergu, M. Asif, V.K. Gupta, Sens. Actuators B 231 (2016) 847–859. [8] Z.C. Liao, Z.Y. Yang, Y. Li, B.D. Wang, Q.X. Zhou, Dyes Pigm. 97 (2013) 124–128. [9] J. Kumar, M.J. Sarma, P. Phukan, D.K. Das, Dalton Trans. 44 (2015) 4576–4581. [10] V.K. Gupta, A.K. Jain, S.K. Shoora, Sens. Actuators B 219 (2015) 218–231. [11] S.K. Shoora, A.K. Jain, V.K. Gupta, Sens. Actuators B 216 (2015) 86–104 [12] K. Tiwari, M. Mishra, V.P. Singh, RSC Adv. 3 (2013) 12124–12132. [13] V.P. Singh, K. Tiwari, M. Mishra, N. Srivastava, S. Saha, Sens. Actuators B 182 (2013) 546–554. [14] G.T. Selvan, M. Kumaresan, R. Sivaraj, I.V.M.V. Enoch, P.M. Selvakumar, Sens. Actuators B 229 (2016) 181–189. [15] J.-C. Qin, T.-R. Li, B.-D. Wang, Z.-Y. Yang, L. Fan, Spectrochim. Acta Part A 133 (2014) 38–43. [16] D.P. Singh, V.P. Singh, J. Lumin.155 (2014) 7–14. [17] A. Sahana, A. Banerjee, S. Das, S. Lohar, D. Karak, B. Sarkar, S.K. Mukhopadhyay, A.K. Mukherjee, D. Das, Org. Biomol. Chem. 9 (2011) 5523–5529. [18] R.A. Lal, D. Basumatary, Transition Met. Chem. 32 (2007) 481–493. [19] C.F. Chow, M.H.W. Lam, W.Y. Wong, Inorg. Chem. 43 (2004) 8387–8393. [20] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G.A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J.A.Jr. Montgomery, J.E. Peralta, F. Ogliaro, M. Bearpark, J.J. Heyd, E. Brothers, K.N. Kudin, V.N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J.C. Burant, S.S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J.M.

Millam, M. Klene, J.E. Knox, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J. W.Ochterski, R.L. Martin, K. Morokuma, V.G. Zakrzewski, G.A. Voth, P. Salvador, J.J. Dannenberg, S. Dapprich, A.D. Daniels, O. Farkas, J.B. Foresman, J.V. Ortiz, J. Cioslowski, D.J. Fox, Gaussian, Inc., Wallingford CT, 2009. [21] A.D. Becke, J. Chem. Phys. 98 (1993) 5648–5652. [22] G.A. Petersson and M.A.A. Laham, J. Chem. Phys. 94 (1991) 6081–6090. [23] R.E. Stratmann, G.E. Scuseria, M.J. Frisch, J. Chem. Phys. 109 (1998) 8218–8224. [24] B. Mennucci, C. Cappelli, C.A. Guido, R. Cammi, J. Tomasi, J. Phys. Chem. A 113 (2009) 3009–3020. [25] D.P. Singh, D.S. Raghuvanshi, K.N. Singh, V.P. Singh, J. Mol. Catal. A: Chem. 379 (2013) 21–29. [26] D.P. Singh, B.K. Allam, R. Singh, K.N. Singh, V.P. Singh, RSC Adv. 6 (2016) 15518– 15524. [27] A. Kumar, R. Chauhan, K.C. Molloy, G. Kociok-Köhn, L. Bahadur, N. Singh, Chem. Eur. J. 16 (2010) 4307–4314. [28] P. Singh, D.P. Singh, K. Tiwari, M. Mishra, A.K. Singh, V.P. Singh, RSC Adv. 5 (2015) 45217–45230. [29] J.S. Wu, W.M. Liu, X.Q. Zhuang, F. Wang, P.F. Wang, S.L. Tao, X.H. Zhang, S.K. Wu, S.T. Lee, Org. Lett. 9 (2007) 33–36. [30] S. Goswami, S. Paul, A. Manna, RSC Adv. 3 (2013) 25079–25089. [31] S.S.T. Mukherjee, B. Chattopadhyay, A. Moirangthem, A. Basu, J. Marek, P. Chattopadhyay, Analyst 137 (2012) 3975–3981. [32] M. Arduini, F. Felluga, F. Mancin, P. Rossi, P. Tecilla, U. Tonellato, N. Valentinuzzi, Chem. Commun. (2003) 1606–1607. [33] A. Sahana, A. Banerjee, S. Das, S. Lohar, D. Karak, B. Sarkar, S.K. Mukhopadhyay, A.K. Mukherjee, D. Das, Org. Biomol. Chem. 9 (2011) 5523–5529. [34] G.L. Long, J.D. Winefordner, Anal. Chem. 55 (1983) 712A.

Biographies Vinod P. Singh obtained his Ph.D. degree in Chemistry in 1992 from Banaras Hindu University, India; presently working as Professor at Department of Chemistry, Banaras Hindu University. His current fields of interest are Coordination Chemistry and Bio-inorganic Chemistry. Divya Pratap Singh obtained his Ph.D. degree in Chemistry in 2014 from Banaras Hindu University, India; presently working as Senior Research Scholar at Department of Chemistry, Banaras Hindu University. His current field of interest is Inorganic Chemistry. Ashish Kumar Singh obtained his Ph.D. degree in Chemistry in 2010 from Banaras Hindu University, India; presently working as Young Scientist at School of Material Science and Technology, Indian Institute of Technology (BHU), Varanasi−221005, India. His current field of interest is Inorganic Chemistry and Hydrogen Energy. Romi Dwivedi obtained her Master’s degree in 2012 from CSJM University, Kanpur, India; presently working as Research Scholar at Department of Chemistry, Banaras Hindu University. His current field of interest is Inorganic Chemistry. Biplob Koch received his Master’s degree (2002) and Ph. D. degree (2009) in Zoology from North Eastern Hill University, India. Currently he is an assistant professor in Department of Zoology Institute of Science, Banaras Hindu University, India. His research interest includes cancer biology, genotoxicology and molecular biology. Priya Singh obtained her Master’s degree in 2013 from Banaras Hindu University, India. Her current research interest focus on cancer chemoprevention.

Figure captions: Fig. 1. Optimized molecular structure of H2nmh. (H atoms are omitted for clarity). Fig. 2. Optimized molecular structure of Al(III) complex of H2nmh. Fig. 3. UV–visible spectra of H2nmh (50 µM) in EtOH–H2O (2:3 v/v) solution (a) on addition of 2 equivalents of aqueous Al3+ ions and (b) titration spectra on addition of 0–5 equivalents of aqueous Al3+ ions. Fig. 4. Job’s plot showing 2:1 (M:L) stoichiometry for Al(III) complex formation with H2nmh. Fig. 5. Diagram showing low energy electronic transitions (ILCT and LLCT) in H2nmh (left) and its Al(III) complex (right). (orbital contour value = 0.02). Fig. 6. Fluorescence spectra of H2nmh (5 µM) + metal ions (100 µM) at λem: 451 nm (λex: 380 nm) in EtOH–H2O solution. Fig. 7. Fluorescence enhancement of H2nmh (5 µM) in EtOH–H2O (2:3 v/v) solution (a) upon addition of two equivalents Al3+ ions and (b) titration spectra on addition of 0–6 equivalents Al3+ ions; (λem: 451 nm, λex: 380 nm). Fig. 8. Interference of other metal ions in a binary mixture solution of H2nmh (5 µM) + Al3+ (100 µM) + Mn+ (100 µM), (λem: 451 nm, λex: 380 nm); where Mn+ = Na+, K+, Mg2+, Ca2+, Al3+, Mn2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Pb2+, Cd2+ and Hg2+ in EtOH–H2O (2:3 v/v). Fig. 9. Bensei–Hildebrand plot for H2nmh with Al3+, considering the 2:1 complexation. The goodness of the fit is shown by the R2 value. Fig. 10. 1H NMR titration of H2nmh upon adding 0−2 equivalents of Al3+ in DMSO−d6. Fig. 11. Live cell imaging of probe, phase contrast and fluorescence images of MCF–7 cells incubated with H2nmh (Fig. A and A1), phase contrast and fluorescence images of MCF–7 cells incubated with AlCl3 (B and B1), Phase contrast and Fluorescence images of MCF–7 cell incubated with probe and AlCl3 (C and C1).

Fig. 12. Cytotoxicity of the H2nmh probe against MCF–7 cells at 24 h incubation at various concentrations (µM).

Fig. 1. Optimized molecular structure of H2nmh (H atoms are omitted for clarity).

Fig. 2. Optimized molecular structure of Al(III) complex (H atoms are omitted for clarity).

Fig. 3. UV–visible spectra of H2nmh (50 µM) in EtOH–H2O (2:3 v/v) solution (a) on addition of 2 equivalents of aqueous Al3+ ions and (b) UV–visible titration on addition of 0–5 equivalents of aqueous Al3+ ions.

Fig. 4. Job’s plot showing 2:1 (M:L) stoichiometry for Al(III) complex formation with H2nmh.

Fig. 5. Diagram showing low energy electronic transitions (ILCT and LLCT) in H2nmh (left) and its Al(III) complex (right). (orbital contour value = 0.02).

Fig. 6. Fluorescence spectra of H2nmh (5 µM) + metal ions (100 µM) at λem: 451 nm (λex: 380 nm) in EtOH–H2O solution.

Fig. 7. Fluorescence enhancement of H2nmh (5 µM) in EtOH–H2O (2:3 v/v) solution (a) upon addition of two equivalents Al3+ ions and (b) fluorescence titration on addition of 0–6 equivalents Al3+ ions; (λem: 451 nm, λex: 380 nm).

Fig. 8. Interference of other metal ions in a binary mixture solution of H2nmh (5 µM) + Al3+ (100 µM) + Mn+ (100 µM), (λem: 451 nm, λex: 380 nm); where Mn+ = Na+, K+, Mg2+, Ca2+, Al3+, Mn2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Pb2+, Cd2+ and Hg2+ in EtOH–H2O (2:3 v/v).

Fig. 9. Bensei–Hildebrand plot for H2nmh with Al3+, considering the 2:1 complexation. The goodness of the fit is shown by the R2 value.

Fig. 10. 1H NMR titration of H2nmh upon adding 0−2 equivalents of Al3+ in DMSO−d6.

Fig. 11. Live cell imaging of probe, phase contrast and fluorescence images of MCF–7 cells incubated with H2nmh (Fig. A and A1), phase contrast and fluorescence images of MCF–7 cells incubated with AlCl3 (B and B1), Phase contrast and Fluorescence images of MCF–7 cell incubated with probe and AlCl3 (C and C1).

Fig. 12. Cytotoxicity of the H2nmh probe against MCF–7 cells at 24 h incubation at various concentrations (µM).

Table 1. Selected bond lengths (Å) and angles (°) of H2nmh and Al(III) complex obtained from geometrical optimization H2mhn Bond lengths C1-O1 C7-O4 C2-N1 C6-N4 N1-N2 N3-N4 N2-C3 N3-C5 C3-O2 C5-O3 C3-C4 C4-C5

1.336 1.337 1.290 1.289 1.362 1.361 1.371 1.369 1.220 1.211 1.521 1.541

Bond angles C2-N1-N2 N3-N4-C6 N1-N2-C3 C5-N3-N4 N2-C3-O2 N3-C5-O3 O2-C3-C4 C4-C5-O3 N2-C3-C4 C4-C5-N3 C3-C4-C5

118.5 118.2 120.4 120.0 122.9 125.0 122.9 121.0 114.2 114.0 115.4

Al(III) complex bond lengths C1-O1 C7-O4 C2-N1 C6-N4 N1-N2 N3-N4 N2-C3 N3-C5 C3-O2 C5-O3 C3-C4 C4-C5 O1-Al1 O4-Al2 Cl1-Al1 Cl3-Al2 Cl2-Al1 Cl4-Al2 Al1-O1w Al2-O2w Al1-N1 Al2-N4 Al1-O2 Al2-O3 Bond Angles C2-N1-N2 N3-N4-C6 N1-N2-C3 C5-N3-N4 N2-C3-O2 N3-C5-O3 O2-C3-C4 C4-C5-O3 N2-C3-C4 C4-C5-N3 C3-C4-C5

1.406 1.407 1.291 1.291 1.381 1.379 1.317 1.312 1.294 1.297 1.502 1.507 2.047 2.052 2.242 2.239 2.350 2.339 1.987 1.988 1.997 1.995 1.851 1.861 116.7 116.4 109.4 109.7 124.2 124.4 118.0 117.4 117.8 117.4 113.4

Table 2. TDDFT calculations for UV–Visible transitions in H2nmh and its Al(III) complex with their assignments.a H2nmh λmax (exp.) (nm)

λmax (Calc.) (nm) (f) 402(0.000 5)

Transition HOMO→LUMO 0.70

Assi gnm ents π→π *

Al(III) complex λmax λmax (Calc.) (exp.) (nm) (f) (nm) 402(0.2031) 412

391

391(0.1760) 381(0.2104)

367 356

323 310

361(0.611 5) 349(0.158 0) 312.36(0.0 011) 312.24(0.0 002) 311.03(0.0 997)

HOMO‒1→LUMO 0.52 HOMO→LUMO+1 0.52 HOMO‒2→LUMO 0.70 HOMO‒3→LUMO 0.70 HOMO‒2→LUMO+ 1 0.63

π→π * π→π * π→π * π→π * π→π * π→π *

305 (0.0789)

HOMO‒2→LUMO+ 1 0.61

289 (0.0484)

HOMO→LUMO+3 0.60

π→π *

269 (0.1176) 264 (0.0965)

HOMO‒5→LUMO 0.61 HOMO‒4→LUMO+ 1 0.41

π→π * π→π *

374

329 323

307

f = Oscillator strength, a

Comparable data are places in parallel columns

371(0.3347)

Transition HOMO→LUMO 0.68 HOMO→LUMO+1 0.15 HOMO→LUMO+1 0.57 HOMO‒1→LUMO +1 0.59 HOMO‒1→LUMO 0.46

316.93 (0.0101) 316.08 (0.0151)

HOMO‒2→LUMO +1 0.49 HOMO‒3→LUMO 0.52

307.45 (0.0106) 307.06 (0.0106)

HOMO‒6→LUMO +1 0.54 HOMO‒7→LUMO 0.41

291.32 (0.0127) 290.81 (0.0104)

HOMO‒8→LUMO +1 0.44 HOMO‒9→LUMO 0.56

Assi gnm ents π→π * π→π * π→π * π→π *

π→π * π→π * π→π * n, π→π * π→π * π→π *