Selective and sensitive turn-on chemosensor for Al(III) ions applicable in living organisms: nanomolar detection in aqueous medium

Abstract

A highly sensitive and selective fluorescent reporter L for Al(III) ions was synthesized and characterized by physicochemical and spectroscopic tools along with single crystal X-ray crystallographic study. This is so far the first report of a crystallographically established fluorescence probe having two rhodamine units which make this probe highly sensitive towards Al(III) ions. L, with a high binding affinity towards Al(III) ions of 3.33 × 10⁸ M⁻², selectively detects Al(III) ions with almost no interference among various competitive, biologically relevant ions by a strong fluorescence (250 times) as well as colour change in HEPES buffer (1 mM, pH 7.4; acetonitrile/water: 1/3, v/v). The quantum yields (Ф) and molar extinction coefficient (ε) of [Al₂(L)₂(CH₃CN)(H₂O)(NO₃)₄](NO₃)₂ (complex-1) were significantly greater than the sensor L, meaning this probe (L) can detect Al(III) ions at concentrations as low as 3.26 nM, which is comparable with the lowest LOD available in the literature. This non-cytotoxic probe (L) is also an efficient candidate to detect the intercellular distribution of aluminium ions in human lung cancer cells (A549) and Al(III) ions in matured tea leaves.

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Introduction

The development of fluorescent chemosensors for selective detection and imaging of trace levels of metal ions is an attractive research area because of their simplicity, high sensitivity and real-time monitoring with a short response time.¹ For this purpose, the assessment of biologically active ions through fluorescence turn-on sensors still remains a challenging task.

Aluminium, being the third most abundant element in the Earth’s crust, has long been proven to be neurotoxic, and the abnormal accumulation of aluminium in all body tissues in humans and animals can cause many health hazards such as Parkinson’s disease and Alzheimer’s disease, colic and gastrointestinal problems, osteomalacia, rickets, interference with the metabolism of calcium, anaemia, and the risk of breast and lung cancer, it can even damage the brain, liver and kidneys.² Several relevant aluminium compounds are extensively used in the paper, dye, textiles and food industry and as a component of many cosmetic preparations.³ Additionally, almost 40% of soil acidity is caused by aluminium; moreover, high concentrations of aluminium in the ecosystem are toxic to plants, fish, algae and other species, and can enter into the human body; and can enter into the human body through the diet by bio-cycle to cause other relevant diseases.⁴ The WHO (World Health Organization) described the average human intake of aluminium as around 3–10 mg per day with a weekly dietary intake of 7 mg kg⁻¹ body weight.^4b,c

Due to the potential impact of Al(III) ions on human health and the environment, the detection and estimation of trace levels of aluminium ions is mandatory. Available standard techniques for Al(III) ion detection with moderate sensitivity, such as chromatographic and spectrophotometric techniques, or atomic absorption or inductively coupled plasma atomic emission spectrometry are expensive and time-consuming in practice.³ Owing to the weak coordination and strong hydration of Al(III) ions in water, the coexistence of interfering ions is a problem.⁵ Therefore, highly selective and sensitive ‘‘turn-on’’ type fluorescence chemosensors for Al(III) ions are highly necessary.

To the best of our knowledge, various fluorescent chemosensors have been reported for the detection of Al(III) ions with moderate sensitivity to date⁶ including a very few reports of Al(III) ion sensors with two fluorophore units.^6g,h However, the majority of these probes have limited solubility, require tiresome synthetic efforts⁷ and lack practical applicability in aqueous solutions.⁸ Thus, there is still a high demand for new and superior methods for the selective estimation of Al(III) ions with high sensitivity and efficiency. Herein, we will report a highly sensitive, non-cytotoxic and cell permeable Al(III) ion selective chemosensor (L) with two identical fluorophore units for the enhancement of fluorescence through the selective chelation of Al(III) ions in an aqueous system. This probe (L) is quite useful to detect Al(III) ions in the presence of huge amounts of several competitive ions and to facilitate the mapping of the distribution of Al(III) ions in living cells.

Experimental

Materials and physical measurements

The analytical grade solvents and the other reagent grade chemicals used in this work were purchased from commercial sources and used as received. Here, throughout the experiments Milli-Q 18 Ω water was employed. A Shimadzu (model UV-1800) spectrophotometer was used for recording UV-vis spectra. IR spectra were recorded using a Prestige-21 SHIMADZU FTIR spectrometer. A Perkin-Elmer 2400 CHN elemental analyzer was utilized for elemental analyses (C, H and N). ¹H NMR spectra were collected from a JEOL 400 spectrometer using DMSO-d₆ solution and ¹³C NMR spectra were collected from a Bruker Avance DPX 500 MHz spectrometer using CDCl₃ solution. All solution pH values were measured by a Systronics digital pH meter (model 335) and adjusted using either 50 mM HCl or NaOH solution. Electrospray ionization (ESI) mass spectra were recorded on a Qtof Micro YA263 mass spectrometer. Steady-state fluorescence emission and excitation spectra were recorded with a Hitachi-7000 spectrofluorimeter. Time-resolved fluorescence lifetime measurements were performed using a HORIBA JOBIN Yvon picosecond pulsed diode laser-based time-correlated single-photon counting (TCSPC) spectrometer from IBH (UK) at λ_ex = 340 nm and MCP-PMT as a detector. Emission from the sample was collected at a right angle to the direction of the excitation beam maintaining magic angle polarization (54.71). Maintaining the resolution of 28.6 ps per channel, the full width at half-maximum (FWHM) of the instrument response function was 250 ps. IBH DAS 6.2 data analysis software was used to fit the data to multiexponential functions after deconvolution of the instrument response function by an iterative reconvolution technique and here, reduced w2 and weighted residuals served as parameters for goodness of fit.

The fluorescence properties of L were checked in HEPES buffer (1 mM, pH 7.4; acetonitrile/water: 1/3, v/v) at 25 °C. The pH study was carried out in 100 mM HEPES buffer solution by adjusting the pH using HCl or NaOH. In vivo study was performed at biological pH ∼ 7.4 with 100 mM HEPES buffer solution. The stock solutions (∼10⁻² M) for the selectivity study of L towards different metal ions were prepared by taking the nitrate salts of sodium, potassium, copper(II), chromium(III) and silver(I); acetate salts of manganese(II) and zinc(II); chloride salts of nickel(II), cobalt(II), mercury(II), calcium(II), magnesium(II) and iron(III); and iron(II) sulphate in HEPES buffer (1 mM, pH 7.4; acetonitrile/water: 1/3, v/v) solvent. In this selectivity study the amount of these metal ions was fifty times greater than that of the probe (L) used. Fluorimetric titration was performed with aluminium nitrate in HEPES buffer (1 mM, pH 7.4; acetonitrile/water: 1/3, v/v) solvent while varying the metal concentration from 0 to 150 μM, the probe concentration was 50 μM.

Preparation of L

Isophthalaldehyde (402.39 mg, 3.0 mmol) dissolved in methanol was added to the methanolic solution of rhodamine-B hydrazide^13,14b (2.79 mg, 6.1 mmol) with stirring. The resulting mixture was refluxed for 8 h. It was then evaporated to a small volume and cooled, from which white colored precipitate was filtered. The pure recrystallized product including the single crystals suitable for X-ray crystallographic study were isolated from acetonitrile/methanol (3 : 1) mixed solvents on slow evaporation.

C₆₄H₆₆N₈O₄. MP: 250 ± 2 °C. Anal. found: C, 75.92; H, 6.39; N, 11.21; calc: C, 76.01; H, 6.58; N, 11.08. ESI-MS in methanol: [M + H]⁺, m/z, 1011.9662 (100%) (calcd: m/z, 1011.5215), where M = molecular weight of L (Fig. S1, ESI†). IR (KBr, cm⁻¹): ν_C=C (aromatic), 2970; ν_C=O, 1686; ν_CH=N, 1613 (Fig. S2, ESI†). ¹H NMR (400 MHz, DMSO-d₆): 8.59 (s, 2H, CH=N); 7.90 (s, 1H); 7.86 (d, 1H, J = 6.84); 7.61–7.49 (m, 6H); 7.41 (s, 1H); 7.29–7.27 (m, 1H); 7.05 (d, 2H, J = 7.64); 6.39 (s, 4H); 6.34 (d, 4H, J = 9.16); 6.26 (d, 4H, J = 8.4); 3.46–3.12 (m, 16H, 8CH₂); 1.02–0.84 (m, 24H, 8CH₃) (Fig. S3, ESI†). ¹³C NMR (δ, 76.99 MHz, ppm in CDCl₃): 165.13, 152.89, 152.22, 148.97, 146.23, 135.38, 135.37, 128.61, 128.22, 128.13, 127.99, 127.89, 127.63, 123.67, 123.39, 107.99, 105.75, 98.14, 65.85 (spirolactam carbon), 44.29, 29.67, 12.62 (Fig. S4B, ESI†). Yield: 78%.

Synthesis of aluminium(III) complex, [Al₂(L)₂(CH₃CN)(H₂O)(NO₃)₄](NO₃)₂ (complex-1)

To a solution of L in acetonitrile (1.0110 g, 1.0 mmol), aluminum(III) nitrate nonahydrate (in acetonitrile, 1125 mg, 3.0 mmol) was gently added dropwise and then the reaction mixture was stirred for 3.0 h at room temperature. A dark red precipitate was obtained after evaporation of the solvent by a rotary evaporator. It was then filtered, thoroughly washed with acetonitrile, and then dried in vacuum.

[C₆₆H₇₁Al₂N₁₃O₁₇](NO₃)₂. Anal. found: C, 52.48; H, 4.44; N, 14.26; calc: C, 52.98; H, 4.78; N, 14.04. ESI-MS in acetonitrile: [M]⁺ = [Z²⁺]/2, m/z, 685.7311 (obsd with 25% abundance) (calcd: m/z, 685.7361) where [Z]²⁺ = [Al₂(L)₂(CH₃CN)(H₂O)(NO₃)₄]²⁺ (Fig. S5†). ¹H NMR (400 MHz, DMSO-d₆): 9.35 (2H broad); 8.41–8.32 (s, 2H, CH=N); 7.86 (broad, 2H); 7.58–7.52 (m, broad, 6H); 7.34 (s, 1H); 7.27–7.22 (m, 1H); 7.01 (broad, 2H); 6.37 (s, 4H); 6.30 (broad, 4H); 6.27 (broad, 4H); 3.69–3.22 (m, 16H, 8CH₂); 2.82 (s, CH₃ of acetonitrile); 0.97 (t, 24H, J = 8.4, 8CH₃) (Fig. S6 ESI†). ¹³C NMR (δ, 76.99 MHz, ppm in CDCl₃): 165.33, 152.92, 152.62, 149.37, 146.53, 136.42, 135.37, 133.40, 128.24, 127.66, 123.69, 123.40, 108.12, 105.87, 98.24, 44.36, 29.66, 12.56 (Fig. S4A, ESI†). Yield: 57%.

X-ray data collection and structural determination

X-ray data were collected on a Bruker Apex-II CCD diffractometer using Mo Kα (λ = 0.71069). The data were corrected for Lorentz and polarization effects and empirical absorption corrections were applied using SADABS software from Bruker. A total of 14 411 reflections were measured out of which 7333 were unique [I > 2σ(I)]. The structure was solved by direct methods using SIR-92 (ref. 9) and refined by full-matrix least squares refinement methods based on F², using SHELX-97.¹⁰ All non-hydrogen atoms were refined anisotropically. All calculations were performed using the Wingx package.¹¹ Important crystal and refinement parameters are given in Table S1.†

Preparation of cells and in vitro cellular imaging with HL

Cell culture. A549 human lung cancer cell lines were obtained from the National Center for Cell Science, Pune, India, and used throughout the experiments. Cells were grown in DMEM (Himedia) supplemented with 10% FBS (Himedia), and an antibiotic mixture (1.0%) containing PSN (Himedia) at 37 °C in a humidified incubator with 5.0% CO₂ and cells were grown to 80–90% confluence, harvested with 0.025% trypsin (Himedia) and in phosphate-buffered saline (PBS), plated at the desired cell concentration and allowed to grow overnight before any treatment.

Cell imaging study. A549 cells were rinsed with PBS and incubated with DMEM containing the Al-sensor making the final concentration up to 10 μM in DMEM (the stock solution (3 mM) was prepared by dissolving the Al-sensor into DMSO) for 30 min at 37 °C. After incubation, bright field and fluorescence images of A549 cells were taken by a fluorescence microscope (Model: LEICA DM4000B, Germany) with an objective lens of 20× magnification. Similarly, fluorescence images of A549 cells (pre-incubated with 10 μM Al-sensor) were taken with the addition of different concentrations (10–40 μM) of Al(NO₃)₂ salt at 10 minute intervals. A merged image between phase contrast and fluorescence images at 40 μM salt concentration was taken.

Cell cytotoxicity assay. In order to determine the cytotoxicity of the Al-sensor, the 3-(4,5-dimethylthiazol-2-yl)-2,S-diphenyltetrazolium bromide (MTT) assay was performed on the A549 cells according to the standard procedure.^12b Briefly, after treatment of an overnight culture of A549 cells (10³ cells in each well of 96-well plate) with the Al-sensor (1.0, 10.0, 20.0 and 50.0 μM) for 6.0 h, 10.0 μL of a MTT solution (1 mg ml⁻¹ in PBS) was added in each well and incubated at 37 °C continuously for 3.0 h. All media were removed from wells and 100 μL of acidic isopropyl alcohol was added into each well. The intracellular formazan crystals (blue-violet) formed were solubilized with 0.04 N acidic isopropyl alcohol and the absorbance of the solution was measured at 595 nm wavelength with a microplate reader (Model: THERMO MULTI SCAN EX). The cell viability was expressed as the optical density ratio of the treatment to control. Values are the mean ± standard deviation of three independent experiments. The cell cytotoxicity was calculated as % cell cytotoxicity = 100% − % cell viability.¹²

Results and discussion

Synthesis and characterization

The organic moiety (L) was synthesized by condensing a methanolic solution of isophthalaldehyde and rhodamine-B hydrazide in 1 : 2 ratio (Scheme 1). The data obtained from the physico-chemical and spectroscopic tools (ESI†), and the detailed structural analysis using single crystal X-ray crystallography are in accordance with the formulation of L as shown in Scheme 1. L is soluble in common polar organic solvents and sparingly soluble in water. The ESI mass spectrum of the compound in methanol shows a peak at m/z 1011.9662 with 100% abundance assignable to [M + H]⁺ (calculated value at m/z, 1011.5215) where M = molecular weight of L (Fig. S1, ESI†). The peaks obtained in the ¹H NMR spectrum of L have been assigned and these are in good agreement with the structural formula of L in the solution state (Fig. S3, ESI†). An ORTEP view and the packing arrangement of the probe L with the atom numbering scheme are illustrated in Fig. 1 and S7 (ESI†), respectively. The crystallographic data and the bond parameters (selected bond distances and angles) are listed in Tables S1 and S2,† respectively .

Scheme 1 Schematic representation of the synthesis of the probe (L).

Fig. 1 Molecular views of L with atom numbering scheme. Hydrogen atoms are omitted for clarity.

To establish the formation of complex-1 in a solid state, it was isolated from the reaction of aluminium(III) nitrate with L respectively in a 2 : 1 mole ratio in an acetonitrile medium (Scheme 2). The complexes are soluble in methanol, DMSO, chloroform and acetonitrile etc. The peaks obtained in ¹H NMR of the Al(III) complex have been assigned and are in accordance with the structural formula of complex-1 as [Al₂(L)₂(CH₃CN)(H₂O)(NO₃)₄](NO₃)₂ (Fig. S6 and S8, ESI†).

Scheme 2 Schematic representation of the synthesis of complex-1.

Spectral characteristics

Absorption study

The electronic spectrum of L (50 μM) recorded in HEPES buffer (1 mM, pH 7.4; acetonitrile/water: 1/3, v/v) exhibited absorption bands at higher energy below 400 nm, corresponding to π → π* (274 nm, ε = 1.34 × 10⁴) and n → π* (311 nm, ε = 9.8 × 10³) transitions. With the incremental addition of aluminium(III) ions (0–150 μM) to the solution of L a new absorption peak at ca. 559 nm gradually developed due to the formation of an aluminium(III) complex with L. A visual colour change from colourless to red appeared due to the spirolactam ring opening of L in complex-1 (Fig. 2). The molar extinction coefficient (ε) at 569 nm increased from 8.5 (L) to 10⁴ (complex-1) which clearly indicated that probe L is an efficient colorimetric sensor for Al(III).

Fig. 2 UV-vis titration spectra of L with Al(III) ions in HEPES buffer (1 mM, pH 7.4; acetonitrile/water: 1/3, v/v) at 25 °C. Inset shows the visual color change of L and L + Al(III) (1 : 2).

Emission study

The fluorescence spectra of L displayed no characteristic band emission at around 586 nm (λ_ex = 555 nm) (Fig. 3) in HEPES buffer (1 mM, pH 7.4; acetonitrile/water: 1/3, v/v). Stepwise addition of Al(III) ions (0–150 μM) to the solution of L (50.0 μM) caused a gradual increase (250 times) in the fluorescence intensity at ca. 586 nm with a slight red shift (Fig. 3) which gives considerable support for L–Al complex formation. Fluorescence quantum yields (Ф) were estimated (at λ_ex = 555 nm) by integrating the area under the fluorescence curves with the reported method.^13,14 The quantum yield (Ф) of complex-1 was nearly 9 times greater than that of probe L, which clearly demonstrates the chelation-enhanced fluorescence (CHEF) process through spirolactam ring opening.^13,14b

Fig. 3 Fluorescence titration of L with gradual addition of Al(III) ions in HEPES buffer (1 mM, pH 7.4; acetonitrile/water: 1/3, v/v) at 25 °C. Inset shows 1 : 2 (L : Al) stoichiometry by Job’s plot and the fluorescence colour change of L and L + Al(III) (1 : 2).

Job’s plot analysis (Fig. 3 inset) showed that complex-1 formed in solution state in a 1 : 2 [L : Al(III)] stoichiometric ratio. To evaluate the affinity of the probe towards Al(III) ions, the binding constant (K, 3.33 × 10⁸ M⁻²) was determined from the plot of (F_max − F₀)/(F_x − F₀) vs. 1/[M]² (Fig. S9†) using the modified Benesi–Hildebrand equation corresponding to 1 : 2 [L : Al(III)] stoichiometry.^15,16

(F_max − F₀)/(F_x − F₀) = 1 + (1/K) (1/[M]ⁿ)

where F₀, F_x and F_∞ are the emission intensities of the organic moiety considered in the absence of Al(III) ions, at an intermediate Al(III) ion concentration, and at a concentration of complete interaction, respectively, and [C] is the concentration of Al(III) ions (here n = 2).

In addition to this dependency of the occurrence of CHEF, the selectivity of L towards Al(III) ions has also been verified by recording the fluorescence due to Al(III) ions even in the presence of 50 equivalent concentration of alkali and alkaline earth metal ions [Na(I), K(I), Mg(II) and Ca(II)], and 50 equivalent concentration of several transition and other metal ions [Hg(II), Cr(III), Mn(II), Fe(III), Co(II), Ni(II), Cu(II), Zn(II), Cd(II) and Pb(II)] (Fig. S10 and S11, ESI†). This study reasonably shows that L suffers almost no interference towards the detection of Al(III) ions, with an excellent specificity to Al(III) ions over other cations.

To study the role of pH in the fluorescence of L, the fluorescence intensities were measured at various pH values by adjusting the pH using HEPES buffer in the presence and absence of Al(III) ions. In the absence of Al(III) ions, the weak fluorescence intensity of L is almost independent of pH over the range of 6.0 to 10.0 (Fig. S12, ESI†); but at lower pH range (4.0 to 6.0), fluorescence intensity dramatically increased due to spirolactam ring opening. Again, in the presence of Al(III) ions the enhanced fluorescence intensity is also independent of the variation of pH over the range of pH 6.0 to 9.5. Here it is also noteworthy that L–Al species have a much higher fluorescence intensity than only the ring opened probe L, which clearly indicates the chelation-enhanced fluorescence (CHEF) process.

The fluorescence average lifetime measurement (at λ_em = 586 nm) of the organic moiety (L) in the presence and absence of Al(III) ions in HEPES buffer (1 mM, pH 7.4; acetonitrile/water: 1/3, v/v) medium showed a considerable increase in lifetime with increase of Al(III) ion concentration (Fig. 4). The average lifetimes were calculated to be 0.625 ns for only L and 1.189 ns for the mixture of L : Al(III) (1 : 2). The radiative rate constant k_r and total non-radiative rate constant k_nr of the organic moiety, L and Al(III) complex calculated from the equations: τ⁻¹ = k_r + k_nr and k_r = Φ_f/τ,¹⁷ were tabulated in Table S3.† The data supported the fluorescence enhancement due to the increase of the ratio of k_r/k_nr from 0.091 for L to 3.16 for the L–Al complex which indicates the more radiative decay pathway for the L–Al complex.

Fig. 4 Fluorescence lifetime decay profiles of L and L : Al(III) (1 : 2) at 586 nm with a nano-LED of 550 nm as the light source.

NMR spectral study

To confirm the above fact of the formation of complex-1 and the bonding pathway, we analysed the peaks in the observed ¹H NMR and ¹³C NMR spectral data of L and complex-1. The characteristic peaks in the ¹H NMR spectrum of L are almost identical with those in the spectra of complex-1 with a slight change in some typical signals (Fig. S8, ESI†). On complexation with Al(III), the peaks due to the proton of –CH=N (δ = 8.59 ppm) and the proton marked as ‘a’ on Fig. S8† (δ = 7.42 ppm) obtained in the ¹H NMR spectrum of L shifted upfield by 0.18 ppm and 0.15 ppm respectively. All other peaks due to the hydrogens in the ¹H NMR spectrum of L are present in that of complex-1 with some slight changes for some protons. The peak at ca. 65.85 ppm attributable to spirolactam carbon (sp³) observed in the ¹³C NMR spectrum of L remarkably shifted to δ = 136.42 ppm in the L–Al(III) complex due to the spirolactam ring opening. For this opening of the ring during complex formation, the sp³ carbon converted to sp² hybridised carbon (Fig. S8, ESI†). This study is in good agreement with the spirolactam ring opening mechanism during the coordination of L with Al(III) ions in the proposed fashion (Scheme 3).

Scheme 3 Plausible mechanistic pathway of L for sensing Al(III).

Analytical figure of merit

The detection limit (LOD) was calculated from the calibration curve based on the fluorescence enhancement at 586 nm (Fig. 5) magnifying on the lower concentration region of Al(III) ions using the equation 3σ/S, where the slope of the curve is S and σ_zero is the standard deviation of seven replicate measurements of the zero level,^14a,c and it was found to be 3.26 nM. This observation clearly indicates the efficiency of this probe towards the detection of trace levels of Al(III) ions.

Fig. 5 Calibration curve for the nanomolar range, with error bars for calculating the LOD of Al(III) by L in HEPES buffer (1 mM, pH 7.4; acetonitrile/water: 1/3, v/v) at 25 °C.

Aluminium detection in tea leaves

To explore the efficacy of this probe (L) for the detection of Al(III) ions in real samples, we examined the presence of aluminium in matured tea leaves. First the leaves were boiled in Milli-Q 18 Ω water and the leaf extract was diluted by HEPES buffer (1 mM, pH 7.4). As the Al(III) ions present in tea leaves are mainly in the fluoride form,¹⁸ citric acid was added to the extract to solubilise it into aqueous form while keeping the pH fixed at 7.4 using buffer. Then 10⁻⁴ M probe (L) in acetonitrile was added into the extract. Visual and fluorescence color changes were observed and this surveillance clearly indicated the presence of Al(III) ions in tea leaves (Fig. S13†).¹⁹

Cell imaging

The cytotoxicity study (MTT assay) in human lung cancer cells treated with various concentrations of L for up to 6.0 h (as shown in Fig. S14†) showed that L concentrations up to 50.0 μM did not show significant cytotoxic effects on human lung cancer cells for at least up to 6.0 h of treatment. This study on A549 cells showed 91.52% viability in presence of L compared to the control i.e. the sensor showed only 8.48% cytotoxicity at 50.0 μM concentrations. So this probe has been employed to detect the distribution of Al(III) ions in living creatures by acquiring the image by fluorescence microscopy.

In fluorescence imaging studies, L did not show any fluorescence itself in the absence of Al(III) ions (Fig. 6B). However, addition of Al(III) ions to the cells (pre-incubated with 10 μM of L) showed red fluorescence (Fig. 6C). Interestingly, the intensity of the fluorescence exhibited was a function of the concentration of Al(III) ions (Fig. 6C–E). The intracellular Al(III) ion imaging behavior of L was studied in the A549 human lung cancer cell line by fluorescence microscopy. After incubation with L (10 μM) at 37 °C for 30 min, the cells displayed no intracellular fluorescence (Fig. 6B). However, cells displayed light fluorescence with the addition of a low concentration of aluminum ions (10 μM) (Fig. 6C) and exhibited gradually more intense fluorescence when more exogenous Al(III) ions were introduced into the cell via incubation with solutions of aluminium nitrate (Fig. 6D and E). The fluorescence responses of L with various concentrations of added Al(III) ions proves that its fluorescence intensity can be used as a clear signature of selective sensor response, as is clearly evident from the cellular imaging. Hence, these results indicate that L is an efficient candidate for monitoring changes in the intracellular Al(III) ion concentration under biological conditions.

Fig. 6 (A) Phase contrast and (B) fluorescence image of A549 cells incubated with 10 μM L for 30 min at 37 °C. L (10 μM) incubated cells were washed with PBS and were exposed to the presence of sequentially increasing concentrations of added extracellular Al(III) ions: (C) 10 μM, (D) 20 μM and (E) 40 μM. (F) is a merged image of the phase contrast and fluorescence images. For all imaging, the samples were excited at 555 nm.

Conclusion

In conclusion, a newly designed and structurally characterized rhodamine–isophthaldehyde conjugate Schiff base (L) has been synthesized and characterised by physico-chemical and thorough spectroscopic analysis. L behaves as an aluminium ion selective chemosensor through a CHEF process in HEPES buffer (1 mM, pH 7.4; acetonitrile/water: 1/3, v/v) over other competitive ions. The processes have been nicely established by electronic, fluorimetric and NMR titration. This probe is also a useful marker for Al(III) ions in human lung cancer cell lines (A549 cells) as L has no cytotoxicity. This is the first report so far of a crystallographically established probe having two rhodamine-fluorophore units to enhance the sensitivity parameter, consequently L showed a very low detection limit (LOD = 3.26 nM) towards Al(III) ions comparable with previous reports.^5c,20 Compared to the reported probes,^5c,20 this probe is however advantageous in terms of the visible light excitation (λ_ex = 555 nm) and red emission (λ_em = 586 nm).

Acknowledgements

Council of Scientific and Industrial Research (CSIR) New Delhi, India is gratefully acknowledged for financial support. We deeply acknowledge Mr Ajay Das and Prof. Samita Basu, Chemical Science Division, SINP, Kolkata, for allowing use of the TCSPC instrument. S. Pal wishes to thank to state fund, West Bengal, India for offering the fellowships.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. CCDC 1403869. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c5ra13478a

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