Al³⁺ selective coumarin based reversible chemosensor: application in living cell imaging and as integrated molecular logic gate

Abstract

An efficient coumarin based fluorescent ‘turn-on’ receptor (H₂L) for the detection of Al³⁺ has been synthesized following simple Schiff base condensation of 4-hydroxy-3-acetylcoumarin with 2-amino-4-methylphenol. The receptor H₂L shows about 21 fold increase in fluorescence intensity upon addition of Al³⁺ than in the case of other metals. The limit of detection is 0.39 μM. H₂L is efficient in detecting Al³⁺ in the intracellular region of human cervical cancer cells and also exhibits an INHIBIT logic gate with Al³⁺ and EDTA as chemical inputs by monitoring both the absorption as well as emission mode. Theoretical calculations (DFT and TDDFT) are applied to interpret the sensing mechanism of the synthesized receptor.

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Introduction

Aluminium being the third most abundant element in the earth's crust¹ has tremendous utility in the food packaging industry, electrical industry, food processing, water purification and clinical drugs, etc.² However free Al³⁺ formed from leaching due to acid rain can be fatal to growing plants.³ Al³⁺ has neurotoxic activities⁴ and been identified as a major cause of Alzheimer's disease⁵ and Parkinson's disease.⁶ Moreover Al³⁺ is also biologically very toxic causing osteomalacia, breast cancer and also intoxication in haemodialysis patients.⁷ According to WHO, the permissible weekly intake of Al³⁺ should not exceed 7 mg Kg⁻¹ body weight.⁸ Thus detection of Al³⁺ in environmental and biological samples have gained a lot of importance.⁹ Among the several detection techniques of Al³⁺,¹⁰ fluorescence technique is popularly used due to its simplicity in operation, high sensitivity, rapidity and non-destructive nature.¹¹ Till date, various ‘turn-on’ fluorescent chemosensors have been reported where the basis fluorophore used are hydrazone, pyrollidine, 8-hydroxyquinoline, oxazoline, imidazoline etc.¹² Recently, Goswami et al. reported a molecular switch for Al³⁺ based on spiropyran platform.¹³ Very few Al³⁺ sensor based on coumarin framework has been reported so far but most of them suffer from the problem of cost of starting material, irreversibility, low limit of detection.¹⁴ However in our present work we report herein a coumarin based chemosensor for the detection of Al³⁺ which has excellent selectivity, very low limit of detection and can be synthesized easily using a very economically cheap route. On top of that the developed sensor is reversible i.e. in presence of EDTA the receptor (H₂L) gets completely free from H₂L–Al³⁺ complex and hence can be used over again. Gradual addition of Al³⁺ (10 μM) to the receptor H₂L (10 μM) in MeOH–H₂O, 1 : 1, v/v (at 25 °C) shows an excellent fluorescence emission intensity enhancement of 21 fold. Again H₂L represents an INHIBIT logic gate with Al³⁺ and EDTA as inputs through both the absorption and emission mode.¹⁵ The receptor H₂L can also act as Al³⁺ sensor in living cells. Further theoretical calculation using DFT/B3LYP method has been used to interpret the sensing mechanism as well as electronic structure of the synthesized receptor H₂L.

Results and discussion

Synthesis and spectral characterisation

Synthetic route towards H₂L involves a very facile and economically cheap route using Schiff base condensation of 3-acetyl-4-hydroxycoumarin with 2-amino-4-methylphenol in 1 : 1 molar ratio in methanolic medium under refluxing condition (Scheme 1).

Scheme 1 Synthesis and keto–enol tautomerism of chemosensor H₂L.

IR spectrum taken in KBr disk shows stretching at 1710 cm⁻¹ corresponding to lactone C=O, the keto C=O and C=C appears at 1619 cm⁻¹ and 1571 cm⁻¹ respectively. ¹H NMR spectra are recorded in CDCl₃ which shows band at around δ 15.43 which is due to the hydrogen bonded NH proton (Fig. S1†). This peak vanishes in the H₂L–Al³⁺ complex indicating co-ordination to the metal centre through N donating site in the enol form. The aromatic protons in H₂L appear as expected in the region δ 8.06–6.90. The –OH proton appears as a singlet at δ 5.95. The –COCH₃ protons appear at δ 2.65 as singlet and the Ph-CH₃ appear at δ 2.32. In the H₂L–Al³⁺ complex the –OH peak also vanishes indicating co-ordination to Al³⁺ using O centre (Fig. S1†). All aromatic protons appear at a bit downfield position compared to that of H₂L, which can be clearly explained due to the co-ordination of Al³⁺ with H₂L. Mass spectrum shows m/z peak corresponding to Na⁺[H₂L] at 332.1 along with a peak at 310.1 corresponding to H⁺[H₂L] for H₂L (Fig. S2†). For H₂L–Al³⁺ complex the strong peak at 419.3 correspond to Na[Al(L-2H)NO₃]⁺ along with a weak peak at 437.3 corresponding to Na⁺[Al(L-2H)(NO₃)(H₂O)] species (Fig. S3†) supporting 1 : 1 complex formation.

Cation sensing studies of H₂L

UV-Vis study. Receptor H₂L (10 μM) shows a strong absorbance band at 326 nm, in 1 : 1, v/v MeOH–H₂O using HEPES buffered solution at pH = 7.2. Gradual addition of Al³⁺ (10 μM) shows a slight red shift of this band to 330 nm and a new band appears at 414 nm. Distinct isosbestic point appears at 369 nm (Fig. 1). This formation of new band at 414 nm indicates the co-ordination of the receptor to Al³⁺. Interestingly when to this solution 10 μM EDTA solution is gradually added the band at 414 nm again gets depressed with the formation of new band at 326 nm (Fig. 2). This clearly indicates that the synthesized receptor H₂L shows reversibility in binding with Al³⁺. In presence of EDTA, Al³⁺ gets free from the receptor, thus it can again be used for the detection of Al³⁺. UV-Vis spectrum of H₂L is also studied in presence of other metals i.e. Na⁺, K⁺, Ca²⁺, Mg²⁺, Mn²⁺, Fe³⁺, Cr³⁺, Co²⁺, Ni²⁺, Cu²⁺, Cd²⁺ and Hg²⁺ but no significant changes are observed except for Zn²⁺ and Cu²⁺ (Fig. S4†). The change in colour of H₂L in presence of Al³⁺ compared to other metals is also visible under naked eye (Fig. S5†).

Fig. 1 Change in UV-Vis spectrum of H₂L (10 μM) upon gradual addition of 10 μM Al³⁺ in 1 : 1, v/v MeOH–H₂O. Inset shows the visual effect of addition of Al³⁺ to H₂L in ambient light.

Fig. 2 Change in UV-Vis spectrum of H₂L–Al³⁺ (10 μM) upon gradual addition of EDTA (10 μM) in 1 : 1, v/v MeOH–H₂O.

Fluorescence study. In the absence of metal ions the emission spectrum of the synthesized chemosensor H₂L shows a very weak emission band with maxima (F₀) at 371 nm (λ_excitation, 326 nm). The fluorescence quantum yield (ϕ = 0.006) is very poor. Gradual addition of Al³⁺ to the above solution shows an excellent fluorescence enhancement of 21 fold (ϕ = 0.076) and the maxima at 371 nm vanished with the formation of new emission maxima at 398 nm (Fig. 3). This red shift of 27 nm is due to co-ordination of the metal centre to the receptor. This fluorescence enhancement reflects a strong selective OFF–ON fluorescent signaling property of H₂L for Al³⁺. On addition of EDTA, fluorescent intensity at 398 nm gradually decreases (Fig. 4). This indicates the reversible nature of the receptor and thus our synthesized receptor can be used over and over again making it very economically useful. Thus H₂L basically shows an OFF–ON–OFF signally pattern in presence of Al³⁺ and EDTA.

Fig. 3 Change in emission spectrum of H₂L (10 μM) upon gradual addition of 10 μM Al³⁺ 1 : 1, v/v MeOH–H₂O. Inset shows the visual effect of addition of Al³⁺ to H₂L under UV light.

Fig. 4 Change in emission spectrum of H₂L–Al³⁺ (10 μM) upon gradual addition of EDTA (10 μM) in 1 : 1, v/v MeOH–H₂O.

Jobs plot of emission intensity shows a maxima in the plot corresponds to ∼0.5 mole fraction indicating 1 : 1 complex formation of H₂L with Al³⁺ (Fig. S6†). From emission spectral change, limit of detection of the chemosensor for Al³⁺ is determined using the equation LOD = K × SD/S where SD is the standard deviation of the blank solution and S in the slope of the calibration curve (Fig. S7†). The limit of detection for Al³⁺ is 0.393 μM from fluorescent spectral titration. This result clearly demonstrates that the chemosensor is highly efficient in sensing Al³⁺ even in very minute level. From fluorescent spectral titration the association constant of H₂L with Al³⁺ is found to be 4.8 × 10⁵ and stoichiometry of the reaction n = 1.17 indicating 1 : 1 complex formation (Fig. S8†).

Fluorescence emission intensity of H₂L (10 μM) is studied in presence of other metals i.e. Na⁺, K⁺, Ca²⁺, Mg²⁺, Mn²⁺, Fe³⁺, Cr³⁺, Co²⁺, Ni²⁺, Cu²⁺, Cd²⁺ and Hg²⁺ (10 μM) in MeOH–H₂O (1 : 1, v/v, pH = 7.2) but there is hardly any increase in emission intensity of H₂L (Fig. 5). Then to these solutions Al³⁺ is added which then shows an obvious fluorescent enhancement (Fig. S9†). Thus the synthesized receptor H₂L is highly efficient in detection of Al³⁺ even in presence of other metals and thus it can detect Al³⁺ in biological or environmental samples where other metals usually co-exist with Al³⁺.

Fig. 5 Change in emission spectrum of H₂L (10 μM) upon addition of Na⁺, K⁺, Ca²⁺, Mg²⁺, Mn²⁺, Fe³⁺, Cr³⁺, Al³⁺, Co²⁺, Ni²⁺, Cu²⁺, Cd²⁺ and Hg²⁺ (10 μM) in MeOH–H₂O (1 : 1, v/v, pH = 7.2).

The effect of pH on the emission intensity of the receptor (H₂L) in absence and presence of Al³⁺ is studied. In case of H₂L there is hardly any change in fluorescence intensity in the pH range 5–10 (Fig. 6). Below pH 5 sharp increase in fluorescence intensity is observed due to protonation of imine N and hydroxy O atoms preventing the excited state intramolecular proton transfer (ESIPT) process, which is responsible for the quenching of fluorescence intensity.¹⁶ On addition of 1.2 equivalents of Al³⁺ the fluorescence intensity remains almost unchanged in the pH < 4, while there is a sharp increase in fluorescence intensity in the pH range 5–8. But, on further increase in pH fluorescence intensity drops drastically due to the formation of Al(OH)₃ at pH > 8. Thus the receptor (H₂L) is efficient in detection of Al³⁺ in the biologically relevant pH range (6.0–7). However at low pH values (pH < 4) receptor tends to combine with protons and hence becomes ineffective in detection of Al³⁺.

Fig. 6 pH dependence of fluorescence intensity of H₂L and its complex with Al³⁺.

Electronic structure and sensing mechanism

To interpret the electronic structure of H₂L geometry optimization has been performed by DFT/B3LYP method in singlet ground state (S₀) and first excited state (S₁) by TDDFT/B3LYP method. The potential energy scans (Fig. S10†) in S₀ state reveals that the keto form is more stable by an amount of energy of 7.308 kcal mol⁻¹ than the corresponding enol form which is consistent with the X-ray structure of this type of molecules.¹⁷ The geometry of H₂L–Al³⁺ has been optimized and the energy minimized structures are shown in Fig. 7. In the complex the chemosensor H₂L binds to Al³⁺ through two phenolic-O atoms and imine-N. In an octahedral geometric environment other three coordination site are satisfied by NO₃⁻ and two water molecules and the proposed geometry is supported by mass spectral analysis of H₂L–Al³⁺ complex. Contour plot of selected molecular orbitals of H₂L and its complex with Al³⁺ are given in Fig. S11 and S12† respectively. The HOMO–LUMO gap of H₂L is significantly decreased from 4.17 eV to 2.82 eV in Al³⁺ complex.

Fig. 7 Optimized structure of H₂L–Al³⁺ complex in DFT/B3LYP/6-311G(d) method.

To interpret the changes in electronic spectra TDDFT calculation by DFT/B3LYP method has been carried out in MeOH. The intense band at 326 nm for chemosensor H₂L corresponds to HOMO → LUMO transition (Table 1). The new band at 414 nm along with peak at 330 nm for Al³⁺ complex correspond to HOMO → LUMO and HOMO-1 → LUMO transitions respectively.

Table 1 Vertical electronic transitions calculated by TDDFT/CPCM method and experimental λ_max (nm)

Compds.	Excitation	λexcitation (nm)	Oscillator strength (f)	λexpt. (nm)
H2L	HOMO → LUMO	353	0.3188	326
H2L–Al^3+	HOMO → LUMO	430	0.3151	414
	HOMO-1 → LUMO	343	0.3139	330

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In the absence of Al³⁺, H₂L shows a weak emission band centered around 371 nm. Upon gradual addition of Al³⁺, the receptor H₂L shows an excellent fluorescence intensity enhancement of 21 fold and a new emission band appears at 398 nm. To interpret whether the excited state intramolecular proton transfer (ESIPT)¹⁶ is responsible for the quenching of fluorescence intensity for H₂L, theoretical calculations are carried out. The possible intramolecular proton transfer process both in ground (S₀) and excited (S₁) state have been considered (Fig. 8). The energy difference between S₀ and S₁ states is only 14.63 kcal mol⁻¹ and the hydrogen transfer can proceed very easily both in ground and excited state with a energy barrier of 6.42 and 6.10 kcal mol⁻¹ respectively. Thus DFT calculations suggest that H₂L exists in the form of S₀-a and S₁-a in the ground- and excited state respectively. The hydrogen transfer takes place easily both in ground and excited state resulting in quenching of fluorescence for H₂L. On coordination with Al³⁺ this ESIPT process is inhibited resulting in fluorescence intensity enhancement.

Fig. 8 The hydrogen transfer processes for H₂L in ground (S₀) and excited state (S₁), along with key bond lengths (Å) and relative energies (kcal mol⁻¹) at the DFT/B3LYP/6-311G(d) for the S₀ and TDDFT/B3LYP/6-311G(d) for S₁ state.

Application as logic function

Arithmetic operations performed by several combination of logic gates are widely implemented in semiconductor technology¹⁸ as well as for computation in nano scale level.¹⁹ Several molecular function systems²⁰ are reported recently. Now molecular logic function was studied with our synthesized chemosensor H₂L along with Al³⁺ as well as the chelating agent EDTA as inputs. As discussed earlier absorption band at 414 nm emerged in presence of Al³⁺ and again in presence of EDTA the absorption band at 414 nm decreased along with decrease in emission band at 398 nm. Thus with two inputs as Al³⁺ and EDTA, H₂L has the ability to exhibit INHIBIT function via both absorption as well as emission output. Only when Al³⁺ is present the absorption as well as emission at 414 nm and 398 nm respectively is 1 while the values of all other functions are 0. Actually it represents an AND gate with an inverter²¹ in one of its input. Thus the absorption change at 414 nm and emission change at 398 nm with Al³⁺ as well as EDTA as inputs can be interpretated as a monomolecular circuit showing an INHIBIT logic function (Fig. 9).

Fig. 9 Truth table and the monomolecular circuit based on Al³⁺ and EDTA.

Biological application study

To explore the biological application of the synthesized receptor H₂L, Human cancer cell line HeLa are treated with the receptor and receptor–Al³⁺ complex separately for 24 h. The cells are able to take up both the receptor as well as Al³⁺. The cells treated with ligand at a dose of 10 μM have a slight green fluorescence at a range of 370 nm. This clearly indicates that the receptor has some autofluorogenic properties when applied to biological systems. Upon addition of equimolar Al³⁺ (10 μM), increase in intensity of fluorescence emission is observed (Fig. 10). Thus the synthesized receptor H₂L has the potential for live cell imaging and can be used in detection of Al³⁺ in the intracellular region.

Fig. 10 (A) Fluorescence image of HeLa cells after incubation with 10 μM Al³⁺ and 10 μM H₂L and respective bright field (B). (C) Fluorescence image of HeLa cells after incubation with 10 μM H₂L and its respective bright field (D).

HeLa cells are treated with Al³⁺, H₂L and H₂L–Al³⁺ complex at various concentrations (5 μM–80 μM). But it is observed that H₂L has slight effect on survivability of cells at higher dosage (40 μM) (Fig. 11).

Fig. 11 MTT assay of Al³⁺, H₂L and H₂L–Al³⁺ complex on HeLa cells. (p > 0.05 as compared with respective controls).

Experimental

Material and methods

4-Hydroxycoumarin and 2-amino-4-methylphenol were purchased from Aldrich. All other organic chemicals and inorganic salts were available from commercial suppliers and used without further purification.

Elemental analysis was carried out in a 2400 Series-II CHN analyzer, Perkin Elmer, USA. HRMS mass spectra were recorded on Waters (Xevo G2 Q-TOF) mass spectrometer. Infrared spectra were taken on a RX-1 Perkin Elmer spectrophotometer with samples prepared as KBr pellets. Electronic spectral studies were performed on a Perkin Elmer Lambda 25 spectrophotometer. Luminescence property was measured using Perkin Elmer LS 55 fluorescence spectrophotometer at room temperature (298 K). NMR spectra were recorded using a Bruker (AC) 300 MHz FTNMR spectrometer in CDCl₃.

The luminescence quantum yield was determined using carbazole as reference with a known ϕ_R of 0.42 in MeCN. The complex and the reference dye were excited at the same wavelength, maintaining nearly equal absorbance (∼0.1), and the emission spectra were recorded. The area of the emission spectrum was integrated using the software available in the instrument and the quantum yield is calculated according to the following equation:

ϕ_S/ϕ_R = [A_S/A_R] × [(Abs)_R/(Abs)_S] × [η_S²/η_R²].

Here, ϕ_S and ϕ_R are the luminescence quantum yield of the sample and reference, respectively. A_S and A_R are the area under the emission spectra of the sample and the reference respectively.

(Abs)_S and (Abs)_R are the respective optical densities of the sample and the reference solution at the wavelength of excitation, and η_S and η_R are the values of refractive index for the respective solvent used for the sample and reference.

Synthesis of 3-(1-(2-hydroxy-5-methylphenylimino) ethyl)-4-hydroxy-2H-chromen-2-one (H₂L)

3-Acetyl-4-hydroxy-2H-chromen-2-one (L)²² (0.184 g, 0.9 mmol) and 2-amino-4-methylphenol (0.111 g, 0.9 mmol) were refluxed for 6 hours in methanolic medium. Excess solvent was evaporated under reduced pressure and then dissolved in dichoromethane which is then further subjected to silica gel (60–120 mesh) column chromatographic separation. The desired light yellow solid product was obtained by elution with 20% ethylacetate–petether (v/v) mixture. Yield was, 0.243 g, 88%.

Anal. Calc. for C₁₈H₁₅NO₄ (H₂L): calc. (%) C 6.89, H 4.89, N 4.53. Found (%), C 6.97, H 4.91, N 4.51. IR data (KBr, cm⁻¹): 1710 ν(lactone C=O); 1619 ν(keto C=O), 1571 ν(C=C). ¹H NMR data (CDCl₃, 300 MHz): δ 15.44 (1H, s), 8.05 (1H, d, J = 7.5 Hz), 7.57 (1H, t, J= 7.1 Hz), 7.24–7.21 (2H, m), 7.09(1H, d, J = 8.27 Hz), 6.94–6.90 (2H, m), 5.95 (1H, s), 2.65 (3H, s), 2.51 (3H, s).

General method for UV-Vis and fluorescence titration

Stock solution of the receptor H₂L (10 μM) in [(MeOH–H₂O), 1 : 1, v/v] (at 25 °C) using HEPES buffered solution at pH = 7.2 was prepared. The solution of the guest cations using their chloride salts in the order of 100 μM were prepared in deionized water. Solutions of various concentrations containing host and increasing concentrations of cations were prepared separately. The spectra of these solutions were recorded by means of UV-Vis methods. EDTA solution of 100 μM was added to the same solution where Al³⁺ was added gradually to H₂L and UV spectra recorded. The spectra of all these solutions were also recorded by means of fluorescence methods.

Job's plot by fluorescence method

A series of solutions containing H₂L (10 μM) and Al(NO₃)₃ (10 μM) were prepared in such a manner that the sum of the total metal ion and H₂L volume remained constant (4 mL). MeOH–H₂O (1 : 1, v/v) was used as solvent at pH 7.2 using HEPES buffer. Job's plots were drawn by plotting ΔF versus mole fraction of Al³⁺ [ΔF = change of intensity of the emission spectrum at 398 nm (for Al³⁺) during titration and X_g is the mole fraction of the guest in each case].

In vitro cell imaging

Cell cytotoxicity assay. HeLa cells were evaluated for cytotoxicity with aluminium nitrate (Al³⁺), H₂L and H₂L–Al³⁺ complex by the following protocol as described by Shi et al. (2012).²³ Cells were seeded in 96-well plates at a density of 1 × 10⁴ cells per well and cultured for 24 h. Al³⁺ was treated in aqueous medium while ligand was dissolved in DMSO but final concentration of DMSO while treatment of cells was maintained below 1%. After treatment for 24 h, Methyl tetrazolium dye (MTT) was used to determine the cell viability and absorbance of MTT formazan was determined at 595 nm in spectrophotometer (Epoch Micro-plate Spectrophotometer, USA). Untreated cells were served as 100% viable.

Cell bio-imaging. HeLa cells were seeded for overnight. Further cells were treated with ligand and complex respectively for 45 min at a dose less than LD₅₀ (10 μM). After treatment cells were washed with 1× phosphate buffer saline and observed under fluorescent microscope at excitation of 326 nm and bright field.

Data analysis. We repeated these experiments six times and the data were expressed by calculating the standard deviation of all the six experiments. Comparisons of the mean of experiments were made by a model I ANOVA test (using a statistical package, Origin 6, Northampton, MA) with multiple comparison-tests, p > 0.05 as a limit of significance.

Computational method

All calculations were carried out at the B3LYP²⁴ level using Gaussian 09 software.²⁵ The 6-311G(d) basis set was assigned for the elements. All the ground state (S₀) stationary points were fully optimized at the B3LYP/6-311G(d) and the excited states at TD-B3LYP/6-311G(d) method.^26,27 Vertical electronic excitations based on B3LYP optimized geometries were computed using the time-dependent density functional theory (TDDFT) formalism²⁸ in methanol using conductor-like polarizable continuum model (CPCM).²⁹

Conclusions

Thus we have successfully developed a new coumarin based reversible chemosensor for the selective detection of Al³⁺ over other metal ions. Fluorescence intensity enhancement of 21 fold upon addition of 10 μM Al³⁺ to H₂L (10 μM) is observed. More importantly the developed chemosensor can also detect Al³⁺ in the intracellular region of human cervical cancer cells. H₂L can also function as an INHIBIT logic gate with Al³⁺ and EDTA as inputs.

Acknowledgements

Financial supports received from the Department of Science and Technology, New Delhi, India is gratefully acknowledged. D. Sarkar is thankful to CSIR, New Delhi, India, S. Biswas is thankful to UGC, New Delhi for fellowship. A. Pramanik is thankful to DBT, India for his fellowship.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Association constant determination, detection limit determination, ¹H NMR, HRMS, UV-Vis titration spectra of HL with different metal ions etc. See DOI: 10.1039/c4ra04318a

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