A FRET-based ‘off–on’ molecular switch: an effective design strategy for the selective detection of nanomolar Al³⁺ ions in aqueous media

Abstract

A new water-soluble rhodamine-based Al³⁺ ion-selective probe (L¹) was synthesised and characterized by physico-chemico and spectroscopic tools. In the presence of a large excess of other competing ions, L¹ specifically binds Al³⁺ ions with a concurrent visually observable change from colorless to pink in electronic spectral behavior, making it possible to detect the presence of Al³⁺ ions with the naked eye. The addition of Al³⁺ ions to a solution of L¹ in HEPES buffer (1 mM, pH 7.4, 2% EtOH) at 25 °C, results in a decrease in the weak fluorescence intensity at λ_em = 470 nm, while a new peak (at λ_em = 588 nm) increases gradually through a fluorescence resonance energy transfer process. This ratiometric enhancement helps to detect Al³⁺ ions at a very low concentration of 33 nM. The detection limit of L¹ for Al³⁺ ions was estimated to be 6.19 × 10⁻⁹ M using the 3σ method. This probe is also useful for imaging Al³⁺ ions in HeLa cells.

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Introduction

Chemosensors for the selective detection of various biologically and environmentally relevant metal ions have attracted great attention because of their potential use in medicine and in environmental research. Aluminum is the third most abundant element on the Earth after oxygen and silicon and there is widespread exposure of humans to aluminum due to its use in food additives, aluminum-based pharmaceuticals and cooking utensils.^1,2 After absorption, aluminum is distributed throughout all living tissues in humans and animals and accumulates in bone. Iron-binding proteins (e.g. transferrin C1 and C2, ferritin) are the main carriers of Al³⁺ ions in plasma and Al³⁺ ions can enter into the brain and reach the placenta and fetus. Al³⁺ ions may persist for a very long time in various organs and tissues before excretion through urine. Aluminum salts are neurotoxic and may be associated with Parkinson's disease³ and senile dementia (commonly known as Alzheimer's disease⁴), microcytic anemia, dialysis dementia and osteomalacia, and have even been shown to increase the risk of lung and bladder cancer.^5–8 Aluminum should therefore be regarded as a toxic metal and its concentration in the environment should be monitored.

In 1989 the WHO listed aluminum as a source of food pollution and limited its concentration in drinking water to 200 mg L⁻¹ (7.41 mM). The FAO/WHO Joint Expert Committee on Food Additives recommended a maximum daily intake of aluminum of 3–10 mg per day per kg body mass. It is believed that almost 40% of the world's acid soils are affected by aluminum toxicity, which is a key factor affecting plant (i.e. crop) performance on acid soils.^9,10 The detection of Al³⁺ ions is therefore crucial in controlling its concentration levels in environmental monitoring and its impact on human health.

Several conventional methods with moderate sensitivity for Al³⁺ ions have been developed with detection based on atomic absorption spectrometry (AAS) and chromatographic and spectrophotometric techniques.¹¹ Of these methods, AAS requires expensive instrumentation and complicated sample preparation processes. Although other techniques have been developed for detecting trace amounts of Al³⁺ ions, most of these involve the use of harmful chemicals and the analytical procedures are easily interfered with by variations in pH and the coexistence of interfering ions. However, spectrofluorimetric methods have received considerable attention in recent years due to their simplicity, high sensitivity and real-time monitoring with a low response time.^12–14

There are several fluorescent sensors for Al³⁺ ions with good selectivity, but this approach has several disadvantages, including complicated synthetic procedures, poor water solubility, insensitivity to biological systems, interference from other ions and variations with pH.¹⁵ The sensitive bioimaging of Al³⁺ ions in cells is a prerequisite for understanding the underlying mechanisms of how Al³⁺ ions cause aluminum-induced human diseases. Few reports have been published on chemosensors for the detection of Al³⁺ ions in aqueous media and most of these are based on chelation-enhanced fluorescence/photo-induced electron transfer.¹⁵ Very few published methods have used the fluorescence resonance energy transfer (FRET) mechanism.¹⁶ The sensors with fluorescence enhancement (a ‘turn-on’ response) through FRET are of considerable interest as FRET is a distance-dependent radiation-less transfer of energy from an excited donor fluorophore to a suitable acceptor fluorophore which can be used to investigate molecular level interactions.¹⁷ Therefore the development of new ratiometric FRET-based sensors for Al³⁺ ions with improved detection limits in the presence of water is desirable.

We report here a newly designed ratiometric FRET-based fluorescent sensor (L¹) which is highly selective for Al³⁺ ions in HEPES buffer (1 mM, pH 7.4, 2% EtOH) at 25 °C. On excitation at 350 nm, the L¹ probe exhibits a fluorescence maximum at 470 nm, which decreases along with a gradual increase in a new peak at 588 nm due to the addition of Al³⁺ ions. This phenomenon is a result of the ring opening of the spirolactam system of rhodamine, which gives rise to a strong fluorescence emission and a visual color change of the solution from colorless to pink. Ratiometric responses are attractive because the ratio between the two emission intensities can be used to measure the analyte concentration and the sensor molecule concentration, providing a built-in correction for environmental effects and giving stability under illumination.^14,18 Interestingly, the presence of an excess of other metal ions, e.g. alkali (Na⁺, K⁺), alkaline earth (Mg²⁺, Ca²⁺), and transition metal ions (Cr³⁺, Mn²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Hg²⁺) and Pb²⁺, does not affect the ‘switch-on’ behavior of the receptor L¹ observed in the presence of Al³⁺ ions. Fluorescence microscopic studies confirmed that L¹ could also be used as an imaging probe for the detection of the uptake of Al³⁺ ions in HeLa cells.

Experimental section

Materials and methods

High-purity HEPES, 8-aminoquinoline, 2-chloroacetyl chloride and aluminum nitrate nonahydrate were purchased from Sigma Aldrich (India) and rhodamine B and ethylene diamine (en) from E. Merck. The solvents used were of spectroscopic grade. All metal salts were used as either their nitrate or chloride salts. Other chemicals were of analytical-reagent grade and used without further purification except where specified. Milli-Q, 18.2 MΩ cm⁻¹ water was used throughout all experiments. A Shimadzu (Model UV-1800) spectrophotometer was used for recording electronic spectra. FTIR spectra were recorded using a Perkin Elmer FTIR Model RX1 spectrometer using a KBr disk. ¹H-NMR spectra were obtained on a Bruker Avance DPX 500 MHz spectrometer and a Geol 400 MHz spectrometer using CDCl₃ solution. Electrospray ionization (ESI) mass spectra were recorded on a Qtof Micro YA263 mass spectrometer. A Systronics digital pH meter (Model 335) was used to measure the pH of the solution and the pH was adjusted using either 50 mM HCl or NaOH solution. Steady-state fluorescence emission and excitation spectra were recorded with a Hitachi 4500 spectrofluorimeter. Time-resolved fluorescence lifetime measurements were obtained using a HORIBA JOBIN Yvon picosecond-pulsed diode laser based time-correlated single-photon counting spectrometer from IBH (UK) at λ_ex = 377 nm with a micro-channel plate photomultiplier tube (MCP-PMT) as a detector. Emission from the sample was collected at right angles to the direction of the excitation beam maintaining magic angle polarization (54.71). The full width at half-maximum of the instrument response function was 250 ps and the resolution was 28.6 ps per channel. Data were fitted to multi-exponential functions after deconvolution of the instrument response function by an iterative reconvolution technique using IBH DAS 6.2 data analysis software in which reduced w² and weighted residuals served as parameters for goodness of fit.

Synthesis of the L¹ probe

The L¹ probe was synthesised using a three-step reaction process (Scheme 1). Rhodamine B-en carboxamide (1) was first prepared using a previously published method.¹⁹ Ethylene diamine (en, 4 mL) was added to a solution of rhodamine B (1 g, 2.09 mmol) in ethanol (40 mL). The solution was refluxed for 6 h. The reaction mixture was then evaporated under reduced pressure to give orange oil, which was subsequently recrystallized from methanol–water to give rhodamine B-en carboxamide (1) as a light orange crystal (77%).

Scheme 1 Synthesis of L¹.

2-Chloro-N-(quinol-8-yl)acetamide (2) was then prepared from the reaction of 2-chloroacetyl chloride and 8-aminoquinoline. 2-Chloroacetyl chloride (5.31 mL) was dissolved in chloroform (5 mL) and then added dropwise to a cooled, stirred solution of 8-aminoquinoline (2.88 g, 20 mmol) and Et₃N (3.0 mL) in chloroform (10 mL) within 1 h. After stirring for 2 h at room temperature the mixture was removed under reduced pressure to obtain a white solid, which was then filtered out and extracted with dichloromethane to give compound 2 (ref. 20) with a yield of 82% (m.p. 133 ± 2 °C).

In the final step, rhodamine B-en carboxamide (1) was taken in dry acetonitrile with anhydrous K₂CO₃. 2-Chloro-N-(quinolin-8-yl)acetamide (2) in dry acetonitrile was then added dropwise with stirring. The resulting reaction mixture was refluxed for 6 h. The volume of the solution was reduced to obtain a solid and then extracted with dichloromethane and finally purified by silica gel column chromatography using dichloromethane as the eluent. The yield was 73% (m.p. 103 ± 2 °C).

C₄₁H₄₄N₆O₃. Anal. found: C, 73.39; H, 6.46; N, 12.83. Anal. calc.: C, 73.63; H, 6.63; N, 12.57. IR (cm⁻¹): ν_NH, 3441; ν_C=C, 2970; ν_C=O, 1681; ν_C=N, 1618. ¹H-NMR (400 MHz, CDCl₃): 10.91 (s, 1-NH–CO), 8.43 (dd, 1H), 8.21 (dd, 1H), 8.12 (dd, 1H), 7.45 (m, 2H), 7.08 (dd, 2H), 7.07 (m, 1H), 6.61 (dd, 2H), 6.06–6.14 (m, 7H), 3.56 (s, 2H, –CH₂–CO), 3.31 (q, 8H, 4CH₂), 2.84 (t, 2H), 2,49 (t, 2H), 1.06 (t, 12H, 4CH₃). ¹³C-NMR (CDCl₃): 166.18, 165.02, 154.18, 153.72, 149.53, 149.26, 148.99, 136.39, 133.69, 132.96, 131.60, 130.50, 129.32, 128.74, 127.59, 124.1, 123.57, 122.03, 116.90, 109.01, 108.48, 103.98, 98.19, 67.11, 44.80, 41.67, 40.54, 40.13, 13.04. ESI-MS m/z 669.0028 [M + H⁺, 60%], calc.: 669.347 [M + H⁺].

Synthesis of L–Al complex as [Al(L)(NO₃)₂]

A solution of aluminum nitrate was added dropwise to a 10 mL ethanolic solution of L¹ (0.01 mmol) and stirred for 4 h. The solvent was removed using a rotary evaporator and a blood red solid was obtained (Scheme S1†).

[Al(L)(NO₃)₂]. Anal. found: C, 59.89; H, 5.16; N, 13.91. Anal. calc.: C, 60.14; H, 5.29; N, 13.68. IR (cm⁻¹): ν_NH, 3437; ν_C=C, 2845; ν_C=O, 1691; ν_C=N, 1612. ¹H-NMR (500 MHz, CDCl₃): 11.18 (s, 1-NH–CO), 8.76 (dd, 1H), 8.52 (dd, 1H), 8.11 (dd, 1H), 7.93 (dd, 1H), 7.87 (dd, 1H), 7.46 (dd, 2H), 7.39 (m, 1H), 7.08 (dd, 2H), 6.36 (t, 1H), 6.22 (d, 1H), 6.19 (d, 1H), 6.15 (d, 1H), 3.89 (s, 2H, –CH₂–CO), 3.33 (q, 8H, 4CH₂), 2.94 (t, 2H), 2.59 (t, 2H), 1.09 (t, 12H, 4CH₃). ¹³C-NMR (DMSO-d6): 169.73, 169.10, 154.89, 154.50, 153.49, 150.12, 149.42, 134.44, 134.17, 130.23, 129.42, 129.11, 128.91, 127.88, 124.46, 124.15, 123.53, 123.23, 117.10, 109.15, 104.88, 104.68, 98.12, 47.02, 44.58, 38.64, 38.19, 13.20. ESI-MS in methanol: [M + CH₃OH + H]⁺, m/z, 851.4242 (obs. with 11% abundance) (calc. m/z 851.85, where M = [Al(L)(NO₃)₂]).

General method of UV-visible and fluorescence titration

The path length of the cells used for absorption and emission studies was 1 cm. For UV-visible and fluorescence titrations, a stock solution of L¹ was prepared in HEPES buffer (1 mM, pH 7.4, 2% EtOH) at room temperature. Working solutions of L¹ and Al³⁺ ions were prepared from their respective stock solutions. Fluorescence measurements were performed using a 5 nm × 5 nm slit width. All the fluorescence and absorbance spectra were taken after 30 minutes of mixing the Al³⁺ ions and L¹ to acquire the optimized spectra.

A series of solutions containing L¹ and Al(NO₃)₃ was prepared such that the total concentration of L¹ (10 μM) remained constant in all the sets. The mole fraction (X) of Al³⁺ ions was varied from 0.1 to 0.6. The absorbance at 561 nm was plotted against the mole fraction of the probe for stoichiometric determination.

Emission study

The organic moiety (L¹) shows a very weak emission at 588 nm in HEPES buffer (1 mM, pH 7.4, 2% EtOH) at 25 °C when excited at 550 nm considering the absorption at 561 nm. Fluorescence quantum yields (Φ) were estimated by integrating the area under the fluorescence curves using the equation:
where A is the area under the fluorescence spectral curve and OD is the optical density of the compound at the excitation wavelength of 550 nm and η is the refractive index of the solvent used. The standard used for the measurement of fluorescence quantum yield was rhodamine B (Φ = 0.7 in ethanol).

Calculation of Förster distance (R₀)

The Förster distance (R₀) for the FRET process was calculated from the following simplified equation:^21,22
where η is the refractive index (η = 1.33 in water),²³ Φ_D is the quantum yield of the donor, k denotes the average squared orientational part of a dipole–dipole interaction, typically k² = 2/3,²⁴ J_DA is the degree of spectral overlap between the donor emission and the acceptor absorption, I_D(λ) is the normalized fluorescence spectra of the donor and ε_A(λ) is the molar absorption coefficient of the acceptor.

Preparation of cell and in vitro cellular imaging with L¹

Human cervical cancer cells (HeLa cell line) were purchased from the National Center for Cell Science, Pune, India and used throughout the study. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco BRL) supplemented with 10% fetal bovine serum (FBS) (Gibco BRL) and a 1% antibiotic mixture containing penicillin, streptomycin and neomycin (PSN, Gibco BRL) at 37 °C in a humidified incubator with 5% CO₂. For the experimental study, cells were grown to 80–90% confluence, harvested with 0.025% trypsin (Gibco BRL) and 0.52 mM EDTA (Gibco BRL) in phosphate-buffered saline (PBS, Sigma Diagnostics), plated at the desired cell concentration and then allowed to re-equilibrate for 24 h before treatment. Cells were rinsed with PBS and incubated with DMEM containing L¹ (10 μM, 1% DMSO) for 30 min at 37 °C. All experiments were conducted in DMEM containing 10% FBS and 1% PSN antibiotic. The imaging system consisted of a fluorescence microscope (ZEISS Axioskop 2 plus) with an 10× objective lens.

Cell cytotoxicity assay

To test the cytotoxicity of L¹, an MTT [3-(4,5-dimethyl-thiazol-2-yl)-2,S-diphenyl tetrazolium bromide] assay was performed using a previously published procedure.²⁵ After treatment of the probe (5, 10, 20, 50 and 100 μM), 10 μL of MTT solution (10 mg mL⁻¹ PBS) was added to each well of a 96-well culture plate and incubated continuously at 37 °C for 6 h. All media were removed from the wells and replaced with 100 μL of acidic isopropanol. The intracellular formazan crystals (blue-violet) formed were dissolved with 0.04 N acidic isopropanol and the absorbance of the solution was measured at 595 nm with a microplate reader. Data are given as the mean ± S.D. of three independent experiments. The cell cytotoxicity was calculated based on a cell viability of 100%.

Results and discussion

Synthesis and characterization

Rhodamine B-en carboxamide (1) was obtained from rhodamine B using a previously published method¹⁹ and 2-chloro-N-(quinolin-8-yl)acetamide (2) was prepared from the reaction of 2-chloroacetyl chloride and 8-aminoquinoline in chloroform in the presence of NEt₃ (Scheme 1). Probe L¹ was then isolated from the reaction of rhodamine B-en carboxamide and 2-chloro-N-(quinolin-8-yl)acetamide in a dry acetonitrile solution in the presence of anhydrous K₂CO₃. The formulation of L¹ was confirmed by physico-chemico and spectroscopic methods (Fig. S1A–D†). The L–Al complex was obtained when aluminum nitrate was mixed with an ethanolic solution of L¹ with stirring. After removing the solvent, a blood red solid was obtained (Scheme S1†). The formulation of the L–Al complex was confirmed by physico-chemico and spectroscopic tools (Fig. S2A–D†).

UV-visible spectroscopic studies of L¹

The UV-visible spectra of L¹ recorded in HEPES buffer (1 mM, pH 7.4, 2% EtOH) at 25 °C show an absorption maximum at 318 nm, which may be attributed to the intramolecular π–π* charge transfer transition. On the stepwise addition of Al³⁺ ions (0–30 μM) to the solution of L¹ in HEPES buffer (1 mM, pH 7.4, 2% EtOH), the absorption intensity at 318 nm increased gradually and a new peak at 561 nm (Fig. 1) was generated due to the formation of a pink color from the colorless solution (Fig. 2). Considering the complexity of the intracellular environment, an additional experiment was performed with the probe to determine whether other ions were potential interferents. Metal ion selectivity assays were therefore performed while keeping the other experimental conditions unchanged. No significant change in the UV-visible spectral pattern was observed with the addition of 10 equivalents excess of the relevant metal ions, i.e. Na⁺, K⁺, Ca²⁺, Mg²⁺, Cr³⁺, Mn²⁺, Fe³⁺, Co²⁺, Ni²⁺, Zn²⁺, Cd²⁺, Hg²⁺, Cu²⁺ and Pb²⁺.

Fig. 1 UV-visible titration spectra of L¹ (10 μM) with the incremental addition of Al³⁺ ions (0–30 μM) in HEPES buffer (1 mM, pH 7.4, 2% EtOH).

Fig. 2 Naked eye visual and fluorescence color changes in (A) probe L¹ only and (B) in the presence of Al³⁺ ions in HEPES buffer (1 mM, pH 7.4, 2% EtOH).

The complex formed between L¹ and Al³⁺ was found to be of a 1 : 1 stoichiometry, as established using the absorbance data with the help of Job's plot (Fig. S3†). Further confirmation of a 1 : 1 stoichiometry with the probable formulation of an L–Al complex was established by the physico-chemical and spectroscopic data of the L–Al complex isolated in the solid form. The molecular ion peak in the ESI-MS spectra of L–Al³⁺ was observed at m/z 851.4242, evidence of the 1 : 1 stoichiometric species (Fig. S2B†).

Fluorescence spectroscopic studies of L¹

To optimize the pH of the experimental conditions, a pH study was performed to control the efficiency of the probe (L¹). In the absence of Al³⁺ ions, L¹ showed a weak intensity fluorescence and an interesting independence of pH in the range 6.0–10.0 (Fig. S4†). At low pH the probe showed a high emission intensity because, at these pH values, the spirolactam ring opens irrespective of the species of metal ion added.^18a,26 It was also noticed that the presence of Al³⁺ ions enhances the emission intensity of L¹ significantly at pH 4.0–10.0. The emission spectrum of L¹ excited at 350 nm shows a fluorescence maximum at 470 nm in HEPES buffer (1 mM, pH 7.4, 2% EtOH) at 25 °C. The intensities at 470 nm were significantly decreased with a concomitant increase in the intensities at 588 nm through an isoemissive point at ca. 553 nm when various concentrations of Al³⁺ ions (0–30 μM) were added (Fig. 3).

Fig. 3 Emission spectra of L¹ (10 μM) in the presence of Al³⁺ ions (0–30 μM) at λ_ex = 350 nm in HEPES buffer (1 mM, pH 7.4, 2% EtOH) at 25 °C.

Ratiometric signaling of the fluorescence output at two different wavelengths plotted as a function of the concentration of Al³⁺ ions indicates that the fluorescence intensity ratio of wavelengths 470 and 588 nm (I₅₈₈/I₄₇₀) gradually increases with an increase in the concentration of Al³⁺ ions (Fig. 4). L¹ showed an almost 75-fold increase in its fluorescence intensity with the addition of only 3.0 equivalents of Al³⁺ ions.

Fig. 4 Ratiometric signaling of fluorescence output at two different wavelengths plotted as a function of the concentration of Al³⁺ ions in aqueous HEPES buffer.

From the plot of the fluorescence intensity at 588 nm (I₅₈₈) versus [Al³⁺] it is suggested that L¹ could be used to detect Al³⁺ ions as low as ca. 33 nM (Fig. S5†).²⁷ To calculate the detection limit, a calibration graph (Fig. S6†) was obtained in the lower region. From the slope (S) of the graph and the standard deviation of seven replicate measurements at the zero level (σ_zero), the detection limit was estimated using the equation 3σ/S.^17,28 This indicates that the detection limit of L¹ for Al³⁺ ions is 6.19 × 10⁻⁹ M, which is comparable with the previously reported FRET-based Al³⁺ ion-selective fluorescent sensor,^16b although our sensor is superior to the one reported previously because our FRET process takes place in aqueous solution.

A metal ion selectivity study was performed using L¹ under identical experimental conditions to understand this phenomenon. Interestingly, the introduction of other metal ions causes the fluorescence intensity to be either unchanged or weakened. Fluorescence enhancement of L¹ (10 μM) was not observed with the addition of an excess of 50 equivalents of biologically relevant metal ions, i.e. Na⁺, K⁺, Ca²⁺ and Mg²⁺ and 10 equivalents excess of several competitive metal ions (Cr³⁺, Mn²⁺, Fe³⁺, Co²⁺, Ni²⁺, Zn²⁺, Cd²⁺, Hg²⁺, Cu²⁺ and Pb²⁺) (Fig. S7†); also no color change was seen with visual naked eye detection (Fig. S8 and S9†). Almost no adverse effect on intensity was observed in the presence of a 10 times excess of various tested ions with L¹ and Al³⁺ ions (Fig. S10†).

As the receptor L¹ has two different fluorophore units, we considered it appropriate to study the metal binding event of L¹ at the two different excitation wavelengths corresponding to the xanthene unit (550 nm) and the quinoline unit (350 nm). Fig. 5 shows that the excitation of L¹ at 550 nm in the absence of Al³⁺ did not initially show any significant emission in the range 550–700 nm, with a quantum yield of only 0.03. This supports the fact that the receptor remains in the spirolactam form in the absence of metal ions; the non-existence of the highly conjugated xanthene form results in the suppression of emission in this range of wavelengths. However, the addition of Al³⁺ ions to L¹ induces a significant switch to an ON fluorescence response near 588 nm, showing a visual display of reddish fluorescence with a quantum yield of 0.61 (∼20-fold).

Fig. 5 (A) Fluorimetric titration spectra of L¹ with Al³⁺ ions (0–30 μM) at λ_ex = 550 nm in HEPES buffer (1 mM, pH 7.4, 2% EtOH) at 25 °C. (B) Fluorescence intensity as a function of Al³⁺ ion concentration at λ_ex = 550 nm in HEPES buffer (1 mM, pH 7.4, 2% EtOH) at 25 °C.

The switch to an ON response for the absorption spectral band at 561 nm and the emission band at 588 nm with binding to Al³⁺ ions suggests the opening of the spirolactam ring of L¹ when metal ion coordination occurs (Scheme 2). It is observed that Al³⁺ ions bind to L¹, inducing the ring opening of L¹ and the generation of the xanthene moiety which is selective towards Al³⁺ ions. No noticeable spectral change was seen for the other metal ions tested.

Scheme 2 Probable mechanism of FRET process induced by Al³⁺ ions.

The binding of Al³⁺ ions induces the opening of the spirolactam ring of L¹ with an associated switch in the UV-visible spectral response in the range 380–650 nm, which has a significant spectral overlap with the emission spectrum of the N-(quinol-8-yl)-acetamide fragment (Fig. S11†). This suggests a plausible route for the non-radiative transfer of excitation energy from the donor quinoline to the acceptor xanthene moiety within the Förster critical distance (R₀), calculated to be 20.03 Å, and initiates an intramolecular FRET process (viz. Scheme 2). In the free state of L¹ the FRET pathway is totally suppressed and only an emission maximum near 470 nm was observed when L¹ was excited at 350 nm. Binding of the receptor to Al³⁺ ions induces the FRET process to produce an intense rhodamine-based reddish emission, i.e. energy transfer from the N-(quinol-8-yl)-acetamide moiety to xanthene is due to the ring opening,²⁹ resulting in an increase in the overlap integral between N-(quinol-8-yl)-acetamide and the xanthene moiety. Therefore, when titrated with Al³⁺ ions, the emission band at ca. 470 nm begins to decrease, along with a concomitant generation of a new fluorescence band at ca. 588 nm. This change in fluorescence was clearly visualised and the fluorescence color is significantly red (see Fig. 2).

The apparent binding constant (K) was determined using the modified Benesi–Hilderbrand method³⁰ and was found to be 5.81 × 10⁶ M⁻¹ (Fig. S12†). The ring-opening phenomenon is also supported by the ¹³C-NMR analyses of L¹ and the L–Al complex. In the ¹³C-NMR spectrum of L¹ the signal at δ = 67.114 ppm, assignable to the tertiary carbon (sp³ hybridized) of the spirolactam ring in L¹ (C₆), is absent in the spectrum of the L–Al complex, but appears at δ = 134.44 ppm due to the conversion of C₆ from sp³ to sp² hybridized carbon; this feature supports the opening of the spirolactam ring (Fig. 6).³¹

Fig. 6 ¹³C-NMR spectra of L¹ in CDCl₃ and the L–Al complex in DMSO-d₆.

In the ¹H-NMR titration, a shift in the corresponding characteristic peaks into the downfield region was observed in support of the chelation of L¹ in the solution state (Fig. S13†). The peaks at δ = 3.56 ppm assignable to –CH₂–CO protons and at δ = 10.91 ppm assignable to NH–CO protons in the spectrum of L¹ (Fig. S1C†) are significantly shifted to δ = 3.89 ppm and 11.18 ppm, respectively (Fig. S2C†).

The occurrence of the FRET process also agrees with the fluorescence lifetime data (Fig. 7 and Table 1). In the fluorescence lifetime experiments (λ_em = 470 nm), the average lifetime of L¹ was 12.87 ns. After the addition of Al³⁺ ions to the solution of L¹, the average lifetime (λ_em = 470 nm) of the complex species decreased to 11.15 and 8.37 ns, respectively, when the concentration of Al³⁺ ions was increased from 0.5 equivalent to 1.0 equivalent with respect to L¹. In contrast, the average lifetime of the probe L¹ increased from 1.60 to 9.77 ns (λ_em = 588 nm) when Al³⁺ ions were added to the solution of L¹, which resembles the FRET process induced by Al³⁺ ions (Fig. S14†).

Fig. 7 Time-resolved fluorescence decay of L¹ (10 μM) only and in the presence of added Al³⁺ ions in HEPES buffer (1 mM, pH 7.4, 2% EtOH) at 25 °C using a nano-LED of 377 nm as the light source at λ_em = 470 nm.

Table 1 Fluorescence lifetime (ns) of the corresponding of L¹ and L–Al complexes at λ_em = 470 nm

λem = 470 nm	τav (ns)	χ^2	φ	kr (10^8 s^−1)	knr (10^9 s^−1)
L^1	12.87	1.1	0.26	0.2020	0.0575
L^1 + Al^3+ (1 : 0.5)	11.15	1.2	—	—	—
L^1 + Al^3+ (1 : 1)	8.37	1.05	0.14	0.1673	0.1028

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The values of k_r and k_nr for the organic moiety L¹ and the L–Al species are listed in Tables 1and 2 according to the equations³² τ⁻¹ = k_r + k_nr and k_r = Φ_f/τ, where k_r = the radiative rate constant and k_nr = total non-radiative rate constant. The data in Table 2 suggest that k_r has only changed slightly; the factor that induces fluorescent enhancement is mainly ascribed to the decrease in k_nr.

Table 2 Fluorescence lifetime (ns) of the corresponding L¹ and L–Al complexes at λ_em = 588 nm

λem = 588 nm	τav (ns)	χ^2	φ	kr (10^8 s^−1)	knr (10^9 s^−1)
L^1	1.6013	1.1	0.03	0.1873	0.60576
L^1 + Al^3+ (1 : 0.5)	2.065	1.00	—	—	—
L^1 + Al^3+ (1 : 1)	9.7729	1.05	0.61	0.6242	0.03990

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Biological studies of L¹ in the presence of Al³⁺

To examine the utility of the probe in biological systems, it was applied to the human cervical cancer HeLa cell line. In these experiments, both Al³⁺ ions and L¹ were taken up by the cells and images of the cells were recorded by fluorescence microscopy following excitation at ∼550 nm. After incubation with L¹ (10 μM) for 30 min, the cells displayed very faint intracellular fluorescence. However, the cells exhibited intensive fluorescence when exogenous Al³⁺ ions were introduced into the cell via incubation with an Al salt (Fig. 8). The fluorescence responses of the probe with various added concentrations of Al³⁺ ions are clearly evident from the cellular imaging. In addition, the in vitro study showed that 10 μM of L¹ showed no cytotoxic effect on cells for up to 6 h (Fig. S15†). These results indicate that the probe has a huge potential as a sensor for Al³⁺ ions in both in vitro and in vivo applications.

Fig. 8 Fluorescence image of HeLa cells after incubation with L¹ for 30 min followed by treatment with (1) 0 μM; (2) 3 μM; (3) 5 μM; and (4) 10 μM Al³⁺ ions at 37 °C. The samples were excited at ∼550 nm.

Conclusions

In summary, we conclude that this newly designed fluorescent chemosensor (L¹) behaves as a highly specific and selective FRET-based ratiometric fluorescence probe towards Al³⁺ ions, which can also be detected by the naked eye. This fluorescence is due to the ring opening of the rhodamine spirolactam system. The detection limit of this L¹ probe is very low (6.19 × 10⁻⁹ M) and it is comparable with the previously reported FRET-based Al³⁺ ion-selective fluorescent sensor. In this regard it may be considered as a superior FRET-based chemosensor for Al³⁺ ions in aqueous solution.^16b This probe may be used as a biomarker for the imaging of Al³⁺ ions in living cells.

Acknowledgements

Financial assistance from CSIR, New Delhi, India is gratefully acknowledged. B. Sen thanks UGC, New Delhi, India for offering him a fellowship. We sincerely acknowledge Professor Samita Basu and Mr Ajay Das, Chemical Science Division, SINP, Kolkata for enabling us to use the TCSPC instrument and we also thank Mr Samya Banerjee, IISC, Bangalore and Dr B. Mukherjee, IISER, Kolkata for recording the ¹H-NMR, ¹³C-NMR and ESI–MS spectra.

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Footnote

† Electronic supplementary information (ESI) available: Schemes, characterization data, tables, figures, and some spectra. See DOI: 10.1039/c4ra02378a

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