A differentially selective probe for trivalent chemosensor upon single excitation with cell imaging application: potential applications in combinatorial logic circuit and memory devices

Abstract

A new rhodamine 6G-benzylamine-based sensor (L¹), having only hydrocarbon skeletons in the extended part, was synthesized and characterized by single-crystal X-ray crystallographic study. It exhibited excellent selective and sensitive recognition of trivalent metal ions M³⁺ (M = Fe, Al and Cr) over mono- and di-valent and other trivalent metal ions. A large enhancement of the fluorescence intensity for Fe³⁺ (41-fold), Al³⁺ (31-fold) and Cr³⁺ (26-fold) was observed upon the addition of 3.0 equivalent of these metal ions into the probe in H₂O/CH₃CN (4 : 1, v/v, pH 7.2) with naked eye detection. The corresponding K_f values were evaluated to be 9.4 × 10³ M⁻¹ (Fe³⁺), 1.34 × 10⁴ M⁻¹ (Al³⁺) and 8.7 × 10³ M⁻¹ (Cr³⁺). Quantum yields of the L¹, [L¹–Fe³⁺], [L¹–Al³⁺] and [L¹–Cr³⁺] complexes in H₂O/CH₃CN (4 : 1, v/v, pH 7.2) were found to be 0.012, 0.489, 0.376 and 0.310, respectively, using rhodamine-6G as standard. LODs for Fe³⁺, Al³⁺ and Cr³⁺ were determined by 3σ methods and found to be 1.28, 1.34 and 2.28 μM, respectively. Cyanide ion scavenged Fe³⁺ from the [Fe³⁺–L¹] complex and quenched its fluorescence via its ring-closed spirolactam form. Advanced level molecular logic devices using different inputs (2 and 4 inputs) as advanced level logic gates and memory devices were constructed. The large enhancement in fluorescence emission of L¹ upon complexation with M³⁺ metal ions makes the probe suitable for the bio-imaging of M³⁺ (M = Fe, Al and Cr) in living cells.

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Introduction

With the increase in urbanization and socioeconomic activities, unlike other pollutants like petroleum hydrocarbons and domestic and municipal litter, which may visibly build up in the environment, the traces of heavy metal ions increase the toxicity level to a higher extent in the environment and also cause harmful effects on human health. Nowadays, contamination by toxic metal ions is increasing due to leather tanning, electroplating, pigments, emissions from vehicular traffic gas exhausts, energy and fuel production, intensive agriculture and sludge dumping and from mining industries.

As a result, drinking water and food, especially in developing countries, with metal contamination is very much unsafe. So many researchers have tried to detect toxic metal ions, such as iron,¹ chromium,² aluminium,^3,4 lead,⁵ silver,⁶ cadmium,⁷ zinc^8,9 and mercury,¹⁰ in water and foods.

Among these trivalent metal ions, Fe³⁺, Al³⁺and Cr³⁺ have biological as well as environmental importance.^11–24 Fe³⁺ is not only the most abundant transition metal in cellular systems but also plays an important role in many metabolic pathways, such as oxygen transport processes in tissues, nerves signal conduction, cellular growth and tissue formation.²⁵ On the other hand, the excess accumulation of Fe³⁺ can lead to a variety of diseases, such as cell damage and organ dysfunction through the abnormal production of reactive oxygen species (ROS),^26,27 leading to Alzheimer's, Huntington's, Parkinson's etc. diseases.²⁸ Moreover, disruption of iron homeostasis can lead to a number of disease, such as cancer,²⁹ hepatitis³⁰ and neurodegenerative diseases.³¹

Cr³⁺ is an effective nutrient and gives immunity power to the human body. Cr³⁺ overdose is known to inflict a negative effect on normal enzymatic activities, and the cellular structure and function causing a disturbance in glucose levels and lipid metabolism, while a deficiency of Cr³⁺ in humans can cause maturity-onset diabetes and cardiovascular disease and nervous system disorders.^32,33 The Cr³⁺ ion, present in the cytoplasm, is known to bind non-specifically to DNA at an elevated level, affecting the cellular structures and damaging the cellular components, which can lead to mutation and cancer.³⁴ Chromium deficiency can cause a risk of diabetes, cardiovascular diseases and nervous system disorders.³⁵

Al³⁺ is the third most abundant metal in the Earth's crust and also one of the most common species of metal cations that are mostly found in the +3 oxidation state in most kinds of animal and plant tissues and in natural waters everywhere.^36–40 It has been found that aluminium accumulates in various mammalian tissues, such as the brain, bone, liver and kidney,^41,42 which causes renal failure⁴³ and problems associated with age.⁴⁴ Aluminium toxicity damages the central nervous system and it is surmised to play a role in neurodegenerative Alzheimer's and Parkinson's diseases. It is also responsible for intoxication in haemodialysis patients.⁴⁵ Moreover, aluminium toxicity may cause gastrointestinal problems and interference with Ca²⁺ metabolism.^46–48 Again, increasing free Al³⁺ due to acid rain and human activities in the environment and surface water is detrimental to growing plants.⁴⁹

Various methods, such as inductively coupled plasma emission spectrometery (ICP),⁵⁰ X-ray photoelectron spectrometry (XPS) and atomic fluorescence spectroscopy (AFS) have been used for heavy metal ion detection.^51,52 Compared with these complicated methods, optical probes are inexpensive, simple and rapid.

Thus, there is an urgent need to design single fluorogenic probes, displaying changes in optical properties through a “turn-on” response that are capable of detecting the presence of Fe³⁺, Al³⁺ and Cr³⁺ ions simultaneously and in the presence of large number of monovalent, divalent and other trivalent metal cations^53–55 in biological samples.

As Cr³⁺ and Fe³⁺ are paramagnetic in nature, they function as fluorescent quenchers,⁵⁶ which makes it a challenging task to develop a turn-on fluorescent sensor for these ions. Very few turn-on sensors for Cr³⁺ and Fe³⁺ have been reported with cell-imaging applications.^57–59

Although, Al³⁺ functions as a turn-on fluorescent sensor, due to its strong hydration in water, most of the reported dye-based Al³⁺ sensors require organic solvents or mixed solvents, with very few being suitable for Al³⁺-imaging applications.⁶⁰

Given our interest in developing new chemosensors, we report herein a rhodamine 6G-based probe (Scheme 1), characterized by X-ray single-crystal diffraction analysis (Fig. 1) and by other common spectroscopic analysis, for the detection of trivalent cations, like Fe³⁺, Al³⁺ and Cr³⁺, in an aqueous medium over monovalent, divalent and other trivalent metal ions. Though there are a few^61,62 reports on trivalent sensors, where in all cases external coordinating atom(s) are present along with the basic amidic moiety of rhodamine, however, in our reported probe it is absent. Herein, we disclose a benzylamine-rhodamine-6G (L¹) conjugate (Scheme 1) that selectively senses these trivalent metal ions in a mostly aqueous medium (4 : 1, H₂O : CH₃CN, v/v) with very high fluorescence enhancement.

Scheme 1 Tentative binding mode of L¹ with M³⁺.

Fig. 1 The molecular view of ligand L¹. All H-atoms are omitted for clarity.

Experimental section

Materials and methods

All solvents used for synthesis were of reagent grade (Merck). For the spectroscopic (UV/Vis and fluorescence) studies, HPLC-grade MeCN and double-distilled water were used. Rhodamine 6G hydrochloride and metal salts, such as perchlorates of Na⁺, Fe²⁺, Co²⁺, Ni²⁺, Zn²⁺, Pb²⁺, Cd²⁺, Hg²⁺, Cu²⁺, Al(NO₃)₃·9H₂O, Cr(NO₃)₃·9H₂O, Fe(NO₃)₃·9H₂O were purchased either from Sigma–Aldrich or Merck and used as received. All other compounds were purchased from commercial sources and used without further purification.

Physical measurements

¹H-NMR spectra were recorded in CDCl₃ and DMSO-d₆, on a Bruker 300 MHz NMR spectrometer using tetramethylsilane (δ = 0) as an internal standard. Infrared spectra (400–4000 cm⁻¹) were recorded in the liquid state using a Nickolet Magna IR 750 series-II FTIR spectrometer. ESI-MS⁺ (m/z) of the ligand and complexes were recorded on a Waters’ HRMS spectrometer (Model: XEVO G2QTof). UV-Vis spectra were recorded on an Agilent diode-array spectrophotometer (Model, Agilent 8453). Steady-state fluorescence measurements were performed on a PTI QM-40 spectrofluorometer. Lifetimes were measured using an Horiba Jobin–Yvon Hamamatsu MCP photomultiplier (R3809) and data were analyzed using IBH DAS6 software. The pH of the solutions were recorded using a digital pH meter 335, calibrated using pH 4, 7 and 10 buffers in the range pH 2–12.

Synthesis of rhodamine 6G conjugate (L¹)

Rhodamine 6G (5.0 mmol) and benzylamine (10.0 mmol) were dissolved in EtOH and refluxed for 10 h with continuous stirring, whereupon a white crystalline solid of the probe (L¹) was deposited (Scheme 1). The solid was filtered and washed several times with ethanol and dried in air (75% yield). The compound (L¹), was dissolved in MeOH and refluxed for 2 h with constant stirring and filtered. After 2 days, single crystals suitable for X-ray diffraction studies were obtained. ¹H NMR (300 MHz, DMSO-d₆) (ppm): 1.18 (t, J = 6.8 Hz, 6H (–CH3)), 1.69 (s, 6H (–Ar–CH₃)), 2.49 (s, 2H, (–CH2)), 3.09 (t, J = 6.3 Hz, 4H (–Ar–CH2)), 4.96 (s, 2H, (–NH)), 5.87 (s, 2H, (–Ar–H)), 6.18 (s, 2H, (–Ar–H)), 6.85 (s, 2H, (–Ar–H)), 6.95 (d, J = 5.4 Hz, 4H (–Ar–H)), 7.48 (m, 1H,(–Ar–H)), 7.50 (m, 1H, (Ar–H)), 7.80 (m, 1H, (–Ar–H)) (Fig. S1†). ¹³C NMR: 14.63, 17.32, 37.90, 43.69, 65.08, 95.90, 104.76, 118.44, 122.84, 124.09, 126.53, 127.75, 128.39, 128.68, 130.92, 133.16, 138.07, 147.90, 151.54, 153.95, 167.38 (Fig. S2†). ESI-MS⁺ (m/z): 504.26 (L¹ + H⁺) (Fig. S3†). IR spectrum: 1684 cm⁻¹ (–C=O), 1378 cm⁻¹ (–C–N) (Fig. S4†).

Solution preparation for UV-Vis and fluorescence studies

For both the UV-Vis and fluorescence titrations, a stock solution of 1.0 × 10⁻³ M of the probe L¹ was prepared by dissolving 12.58 mg in 25 mL CH₃CN. Analogously, 1.0 × 10⁻³ M stock solutions of Fe³⁺, Al³⁺ and Cr³⁺ were prepared in MeOH. A solution of 20 mM HEPES buffer (4 : 1, H₂O : CH₃CN) was prepared and the pH was adjusted to 7.2 by using HCl and NaOH. For the UV-Vis spectra, a 60 μM probe was taken in a cuvette containing 2.5 mL of buffer solution and then Fe³⁺ salt solution was added incrementally starting from 0 to 240 μM in a regular interval of time, and the absorption spectra were recorded. Similar experiments were performed for Al³⁺ and Cr³⁺. Again 2.5 ml of this buffer solution was pipetted into a cuvette to which 60 μM of the probe (L¹) solution was added and Fe³⁺ salt solution was then added incrementally starting from 0 to 140 μM in a regular interval of time, and the fluorescence spectra were recorded, setting the excitation wavelength at 502 nm. Similar titrations were conducted with Al³⁺ and Cr³⁺. Path lengths of the cells used for absorption and emission studies were 1 cm. Fluorescence measurements were performed using a 2 nm × 2 nm slit width.

Cell culture

Human hepatocellular liver carcinoma (HepG2) cell lines (NCCS, Pune, India), were grown in DMEM supplemented with 10% FBS and antibiotics (penicillin, 100 μg ml⁻¹; streptomycin, 50 μg ml⁻¹). Cells were cultured at 37 °C in a 95% air/5% CO₂ incubator.

Cell cytotoxicity assay

To assess if there was any cytotoxic effect of the ligand (L¹), a cell viability assay was performed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT).⁶³ HepG2 cells (1 × 10⁵ cells per well) were cultured in a 96-well plate with incubating at 37 °C, and were treated with increasing concentrations of L¹ (1, 10, 20, 40, 60, 80 and 100 μM) for 24 h. After the incubation, 10 μl of MTT solution [5 mg ml⁻¹, dissolved in 1× phosphate-buffered saline (PBS)] was added to each well of the 96-well culture plate, and then incubated at 37 °C for 4 h. Media were decanted from the wells and 100 μL of 0.04 N acidic isopropyl alcohol was added into each well to solubilize the intracellular formazan crystals (blue-violet) formed, and the absorbance of the solutions was measured at 595 nm wavelength (EMax Precision Micro Plate Reader, Molecular Devices, USA). Values were calculated as the mean ± standard errors of three independent experiments. The cell viability was expressed as the optical density ratio of the treatment to control.

Cell-imaging study by fluorescence microscopy

HepG2 cells were cultured in a 35 × 10 mm culture dish on a coverslip for 24 h at 37 °C. The cells were treated with 10 μm solutions of L¹, prepared by dissolving L¹ into the mixed solvent DMSO : water = 1 : 9 (v/v) and incubated for 1 h at 37 °C. To study the complex formation of L¹ with the three metal ions (Fe³⁺, Cr³⁺, Al³⁺), HepG2 cells were pre-incubated separately with 10 μM, 20 μM and 40 μM of each of the metal ions for 60 min at 37 °C, followed by washing them twice with 1× PBS and subsequent incubation with 10 μM L¹ for 60 min at 37 °C. Fluorescence images of HepG2 cells were taken using a fluorescence microscope (Leica DM3000, Germany) with an objective lens of 40× magnification.

Job's plot

This method is based on the measurement of the fluorescence of a series of solutions in which molar concentrations of the probe (L¹) and M³⁺ vary but their sum remains constant. Here, the fluorescence of each solution was measured at 558 nm and plotted against the mole fraction of M³⁺. The maximum fluorescence occurred at the mole ratio corresponding to the combined ratio of the two components. The composition of the complex was determined by Job's method and found to be 1 : 1 with respect to L¹ for the Fe³⁺, Al³⁺ and Cr³⁺ complexes.

Results and discussion

As depicted in Scheme 1, receptor L¹ was synthesized from the reaction of rhodamine-6G with benzylamine in EtOH in reflux conditions for 10 h. The final crystallized product (L¹) was well characterized by ¹H NMR (Fig. S1†), ¹³C NMR (Fig. S2†), HRMS (Fig. S3†), IR (Fig. S4†) and a single-crystal X-ray diffraction method (Fig. 1). The receptor L¹ was found to be a very sensitive and highly selective colorimetric and fluorogenic chemosensor for trivalent metal ions, M³⁺ (M³⁺ = Fe³⁺, Al³⁺ and Cr³⁺), while in the absence of M³⁺, the solution of L¹ was colourless and very weakly fluorescent.

X-ray crystallography study

Single-crystal X-ray diffraction studies revealed that the compound L¹ crystallizes in a triclinic system of space group P1̄ (no. 2). The crystallographic details are depicted in Table 1. A molecular view of L¹ is shown in Fig. 1, with H atoms are removed to get better clarity.

Table 1 Crystallographic data and details of the structure determination

Molecular formula	C33H33N3O2
Formula weight	503.62
Crystal system	Triclinic
Space group	P1̄ (no. 2)
a/Å	9.0397(8)
b/Å	12.5823(11)
c/Å	12.8196(11)
α/°	86.214(2)
β/°	73.428(2)
γ/°	72.357(2)
V/Å^3	1331.5(2)
Z	2
D(calc)/g cm^−3	1.256
μ(MoKα)/mm^−1	0.079
F(000)	536
T/K	273
θ min, max/°	2.3, 27.5
Dataset	−11: 11; −16: 16; −16: 16
Tot., Uniq. data, R(int)	13 172, 6036, 0.023
Observed data [I > 2σ(I)]	4637
N ref, Npar	6036, 356
R, wR2, S	0.0527, 0.1522, 1.05

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UV-Vis absorption studies

The UV-Vis spectrum of L¹ (60 μM) was recorded in a mixed aqueous solvent of H₂O/CH₃CN (4 : 1, v/v, pH 7.2, 20 mM HEPES buffer). The gradual addition of Fe³⁺, Al³⁺ and Cr³⁺ separately to a solution of L¹ revealed that there was a development of two absorption peaks at 350 nm and 530 nm (Fig. 2 and Fig. S5, S5a†), with a sharp visual colour change of the representative solution from colourless to orange-red, whereas no such peaks appeared in the presence of other monovalent, divalent or trivalent metal ion solutions (Fig. S5b†). Between these two peaks, the second one is very important as it exhibits a greater increase in absorbance. The appearance of this peak clearly manifested the opening of the spirolactam ring due to the coordination of Fe³⁺, Al³⁺ and Cr³⁺ with the probe L¹. The probable coordination mode of L¹ towards M³⁺ (Fe³⁺, Al³⁺ and Cr³⁺) is demonstrated in Scheme 1. UV-Vis titrations were carried out by varying the trivalent metal ion Fe³⁺, Al³⁺ and Cr³⁺ concentration (0–240 μM) keeping the probe concentration fixed at 60 μM in H₂O/CH₃CN (4 : 1, v/v, pH 7.2, 20 mM HEPES buffer). Plots of absorbance vs. [M³⁺] yielded linear curves, which were analyzed by linear curve-fitting of the titration data according to eqn (1) (where a, b and c have the usual meaning) under the conditions 1 ≫ c × x with n = 1, giving apparent association constant K_f values as 1.19 × 10⁴ M⁻¹, 1.09 × 10⁴ M⁻¹ and 1.04 × 10⁴ M⁻¹ for Fe³⁺, Al³⁺ and Cr³⁺, respectively.

y = (a + b × c × xⁿ)/(1 + c × xⁿ) (1)

Fig. 2 (a) UV-Vis absorption spectra of L¹ (60 μM) in H₂O/CH₃CN (4 : 1, v/v, pH 7.2, 20 mM HEPES buffer) solutions with the increase in concentration of Fe³⁺ solution (0–240 μM); (b) linear fit of absorbance vs. [Fe³⁺] plot.

The absorbance intensity of the [L¹–Fe³⁺] complex was found to be selectively quenched in the presence CN⁻ ion (Fig. S6†).

Fluorescence studies

The emission spectra of L¹ and its fluorescence titration with M³⁺ (Fe³⁺, Al³⁺ and Cr³⁺) were performed in H₂O/CH₃CN (4 : 1, v/v, pH 7.2, 20 mM HEPES buffer) with the fixed concentration of L¹ at 60 μM. A significant turn-on fluorescence response was observed in the presence of Fe³⁺, Al³⁺ and Cr³⁺ with a fluorescent maximum at 558 nm. For example, upon the gradual addition of Fe³⁺ (0–3 equivalent) to the non-fluorescent solution of L¹, a 41-fold enhancement in fluorescence intensity at 558 nm was observed following excitation at 502 nm, which also suggests the opening of the spirolactam ring in L¹ upon coordination to the Fe³⁺ ion⁶⁴ (Fig. 3). A 31-fold and 26-fold enhancement of fluorescence intensity was observed during the titration of L¹ with Al³⁺ and Cr³⁺ respectively (Fig. S7 and S7a†). Fascinatingly, this change was also accompanied with a naked-eye colour change from colourless to orange-red after the addition of Fe³⁺, Al³⁺ and Cr³⁺, indicating that the probe L¹ is a highly sensitive colorimetric chemosensor for these trivalent metal cations.

Fig. 3 (a) Fluorescence spectra of L¹ (60 μM) in H₂O/CH₃CN (4 : 1, v/v, pH 7.2, 20 mM HEPES buffer) solutions upon the addition of Fe³⁺ (0–3.0 equivalent), each spectrum was taken after 3 min of Fe³⁺ addition; λ_ex = 502 nm, λ_em = 558 nm; (b) linear curve fitting of the titration curves with K_f values.

Plots of FI vs. [M³⁺] give linear curves. Linear curve-fitting of the titration data according to eqn (1) (where a, b and c have the usual meaning) gave an apparent association constant K_f = (0.94 ± 0.01) × 10⁴ M⁻¹ for Fe³⁺ under the conditions 1 ≫ c × x with n = 1. Similarly, binding constants for Al³⁺ and Cr³⁺ were calculated and found to be (1.34 ± 0.1) × 10⁴ M⁻¹, K_f = (0.87 ± 0.01) × 10⁴ M⁻¹, respectively (Fig. 4). There was an excellent agreement between the values of K_f obtained from the absorption and fluorescence titration data, manifesting the self-consistency of our results. Using these fluorescence data, the detection limits of the probe L¹ for Fe³⁺, Al³⁺ and Cr³⁺ were calculated to be 1.28, 1.34 and 2.28 μM, respectively (Fig. S8, S8a and S8b†). These results strongly indicate that this probe L¹ is sensitive enough to detect trace levels of Fe³⁺, Al³⁺ and Cr³⁺. In this context, we must highlight that the quantum yield of the ligand (L¹) was very less. The quantum yields of L¹ and [L¹–Fe³⁺], [L¹–Al³⁺] and [L¹–Cr³⁺] complexes in H₂O/CH₃CN (4 : 1, v/v, pH 7.2) were found to be 0.012, 0.489, 0.376, 0.310 respectively using rhodamine-6G as a standard. The comparatively higher values of quantum yields for complexes compared to the free ligands indicate the higher stability of the complexes in the excited states.

Fig. 4 Linear fitting of the fluorescence titration curves for Fe³⁺, Al³⁺ and Cr³⁺ with K_f values.

Job's method was again employed to determine the composition of the complex, which was found to be 1 : 1 (Fig. S9†) and was further supported by the mass spectrometric analysis results (m/z = 724.18 [Fe(L¹)(NO₃⁻)₂(CH₃CN)]⁺); (m/z = 198.06 [Al(L¹)(MeOH)₂]³⁺); 721.52 [Cr(L¹)(NO₃⁻)₂(CH₃CN)]⁺ (Fig. S3a and S3b†).

Moreover, a conspicuous reddish-orange fluorescence response of the probe upon interaction with M³⁺ (Fig. 5a) provides the scope for naked eye detection. The possibility of using the chemosensor L¹ in the development of paper test strips was examined and it was found that the turn-on fluorescence response of L¹ towards M³⁺ is also visually detectable in test paper strips (Fig. 5b).

Fig. 5 (a) Visual fluorescent response of L¹ towards Fe³⁺, Al³⁺ and Cr³⁺ (under 365 nm UV light). (b) Paper strip for the fluorescent sensing of Fe³⁺, Al³⁺ and Cr³⁺ towards the probe L¹.

Selectivity studies

Selectivity is an important and essential requirement for an excellent chemosensor. Selectivity experiments were carried out by taking 60 μM of probe L¹ in a cuvette containing 2.5 mL of 20 mM buffer solution and then different metal ion solutions of about 5 equivalent were added separately. Surprisingly, L¹ could selectively recognize the trivalent metal ions Cr³⁺, Fe³⁺ and Al³⁺ in a mixed aqueous medium over other biologically abundant divalent 3d transition metal cations, like Mn²⁺, Fe²⁺, Co²⁺, Ni²⁺, Cu²⁺ and Zn²⁺, hazardous heavy metal ions, like Pb²⁺, Cd²⁺ and Hg²⁺, alkali and alkaline earth metal ions, like Na⁺, K⁺, Ca²⁺, Mg²⁺ (Fig. 6 and Fig. S10†) and also in the presence of Ga(III), Y(III), Sm(III), Dy(III), Au(III), Ru(III), Co(III) and Cr(VI) ions (Fig. S17†), which were taken in 5 equivalent with respect to the probe (1 : 5 ratio).

Fig. 6 (a) Fluorescence bar diagram for the selective response of L¹ (60 μM) towards M³⁺ (M = Fe, Al, Cr) over other di- and monovalent metal ions (5 equivalent) in H₂O/CH₃CN (4 : 1, v/v, pH 7.2, 20 mM HEPES buffer), λ_ex = 502 nm, λ_em = 558 (nm); (b) fluorescence response of L¹ (60 μM) upon the addition of 3.0 equivalent of Fe³⁺, Al³⁺, Cr³⁺.

It was also found that not a single anionic species among OAc⁻, HCO₃^–, CO₃^2–, S₂O₃^2–, SCN⁻, N₃^–, NO₃^–, NO₂^–, H₂PO₄^–, SO₄^2–, ClO₄⁻, F⁻, Cl⁻, Br⁻, I⁻, PO₄^3– and CN⁻ could enhance the fluorescence intensity of the probe L¹ (Fig. 7) when taken in 5 equivalent with respect to the probe (1 : 5 ratio). However, the fluorescence intensity of the [L¹–M³⁺] complex was found to be selectively quenched in the presence CN⁻ ions (Fig. S11 and S12†). An excellent reversible fluorescence ON–OFF property of L¹ was observed through fluorescence study with the sequential addition of M³⁺ and CN⁻ ions in 20 mM HEPES buffer in H₂O/CH₃CN (4 : 1) (pH 7.2) solution at room temperature (Fig. 8). The addition of cyanide ions to the solution containing L¹–M³⁺ complex quenched the emission of the probe with the disappearance of the orange-red colour of the solution. The fluorescence quenching of the complexes was characterized by a linear Stern–Volmer (SV) plot and analyzed using the classical Stern–Volmer (SV) eqn (2) ⁶⁵

where F₀ and F are the steady state fluorescence intensities at the maximum wavelength in the absence and presence of a quencher (Q), respectively, [Q] is the quencher concentration and K_SV is the Stern–Volmer constant. The Stern–Volmer quenching constant (K_SV) of [L¹–Fe³⁺], [L¹–Al³⁺] and [L¹–Cr³⁺] complexes with CN⁻ ion were calculated and found to be (5.60 ± 0.20) × 10² M⁻¹, (2.42 ± 0.24) × 10² M⁻¹ and (1.88 ± 0.11) × 10² M⁻¹, respectively (Fig. S18†). The reason behind this observation is that the interaction of M³⁺ with the probe results in opening of the spirolactam ring, thereby producing strong fluorescence. Then treatment with CN⁻ results in the abstraction of a metal ion and regeneration of the spirolactam ring, leading to quenching of the emission. The mechanism between L¹–M³⁺ with CN⁻ ion sensing was confirmed by the HRMS study (Fig. S13†). It is clear from the HRMS study that the ligand is in a ring-closed structure exactly the same with the L¹. This reversibility test suggests the reusability of this chemosensor.

Fig. 7 (a) Histogram of the fluorescence responses of different anions (5 equivalent) towards L¹ (60 μM) in 4 : 1 v/v, water/MeCN in HEPES buffer at pH 7.2 with λ_ex = 502 nm, λ_em = 558 nm. (b) The fluorescence response of L¹ towards Fe³⁺, Al³⁺, Cr³⁺ with respect to different anions (300 μM).

Fig. 8 Fluorescence experiment to show the reversibility and reusability of the receptor for sensing Fe³⁺ by the alternate addition of CN⁻: (a) Fluorescence intensity obtained during the titration of L¹–Fe³⁺ with CN⁻ followed by the addition of Fe³⁺. (b) Visual fluorescent colour changes after each addition of CN⁻ and Fe³⁺ sequentially.

pH studies

For practical application, the appropriate pH condition for the sensor was evaluated. At pH > 4.0, no obvious ring opening of the probe was observed, thereby satisfying the usefulness of the probe in biological systems over a wide pH range (4–8) for the detection of Fe³⁺ (Fig. 9), Al³⁺ and Cr³⁺ (Fig. S14†). However, upon the addition of 3.0 equivalent of Fe³⁺, the FI jumps to a very high value and remains almost unchanged in the range pH 3–7, but then on a further increase in pH, the FI gradually falls. At pH > 8, no FI was observed in the case of Fe³⁺, Al³⁺ and Cr³⁺ due to the precipitation of hydroxides of these metal ions.

Fig. 9 pH dependence of fluorescence responses of L¹ and its [L¹–Fe³⁺] complex.

Spectral studies

The mechanistic pathway proposed for the formation of the L¹–M³⁺ complex by the opening of the spirolactam ring was established through IR and ¹H-NMR studies. The IR studies revealed that the characteristic stretching vibrational frequencies of the amidic ‘C=O’ of the rhodamine moiety at 1684 cm⁻¹ and azomethine group (C=N) at 1636 cm⁻¹ were shifted to lower wavenumbers 1636, 1637, 1635 cm⁻¹ and 1605, 1604, 1604 cm⁻¹ in the presence of 3.0 equivalent of Fe³⁺, Al³⁺ and Cr³⁺, respectively (Fig. 10). These large shifts in IR frequencies signify a strong polarization of the C=O and C=N bonds upon efficient binding to the M³⁺ (M = Al, Fe and Cr) ion. The coordination mode of L¹ towards Al³⁺ was supported by ¹H-NMR studies (Fig. S1a†), which showed a downfield shift of azomethine proton in L¹ and also of the protons on the benzene ring of the benzylamine moiety in the L¹–Al³⁺ complex. The broadening of the –NH proton at 4.9 was due to opening of the spirolactam ring and it bearing a positive charge on it. HRMS study (Fig. S1a†) also confirmed the formation of a complex with M³⁺ (M = Al, Fe and Cr).

Fig. 10 IR spectra of (L¹), [L¹ + Fe³⁺], [L¹ + Al³⁺] and [L¹ + Cr³⁺] complexes in MeOH.

Selective sensing of Fe³⁺, Al³⁺ or Cr³⁺

It is desirable for the probe to be selective towards one trivalent metal ion over the other two. The probe was sensitive towards all the three trivalent metal ions, but in the presence of ppi (inorganic pyrophosphate), the fluorescence of L¹–Al³⁺ and L¹–Cr³⁺ were quenched significantly while the fluorescence of L¹–Fe³⁺ remained almost unchanged, making L¹ selective towards Fe³⁺ in the presence of ppi (Fig. S19†). Again in the presence of I⁻, the fluorescence of L¹–Fe³⁺ and L¹–Al³⁺ undergoes quenching, while that of L¹–Cr³⁺ remains unchanged, making L¹ selective towards Cr³⁺ in the presence of I⁻ (Fig. S20†). Now if L¹ is fluorescent towards an unknown solution, but remains non-fluorescent in the presence of both ppi and I⁻, then the initial fluorescence is due to the presence of Al³⁺. Thus, L¹ can be made selective towards Fe³⁺, Cr³⁺ or Al³⁺ in an unknown solution.

Molecular logic operations

The spectroscopic properties of the probe L¹ encouraged us to apply it for multiple logic operations with the sequential addition of inputs like cations, such as Al³⁺, Fe³⁺, Cr³⁺, and CN⁻ anions and to then monitor their emission as the output. An INHIBIT logic gate was constructed with a particular combination of logic operations, like NOT and AND functions, which was important due to its non-commutative behaviour, i.e. its output signal is inhibited by only one type of input. To demonstrate this INHIBIT logic function, first we chose two inputs, namely Fe³⁺ (Input 1) and CN⁻ (Input 2), and used its emission intensity at 558 nm as the output. A high value of emission intensity (>5 × 10⁵ at 558 nm) has been designated as 1 (ON) and a low value (≤5 × 10⁵) as 0 (OFF). In the absence of both the 1st input (Fe³⁺) and 2nd input (CN⁻), the emission intensity was low, indicating the OFF state; whereas when only input 1 was present, then a significant enhancement of the emission (at 558 nm) took place, indicating the 1 (ON) state, while, on the other hand, in the presence of input 2, the output emission value became very weak indicating the OFF state. Therefore, it was necessary to apply NOT gate with Input 2. Additionally, it is noteworthy that L¹ displayed the emission output signal in such a way that it seemed to understand the requirements of the AND operation. In the presence of both inputs, the output emission value was again low, indicating the OFF state, in accordance with the truth table (Fig. 11(a)). Thus, by the sequential addition of these two inputs, an INHIBIT function logic gate could be achieved.

Fig. 11 (a) Corresponding truth table of the logic gate. (b) Output signals (at 558 nm) of the logic gate in the presence of different inputs. (c) Corresponding bar diagram at 558 nm in the presence of different inputs. (d) General representation of an INHIBIT logic gate-based circuit.

Advanced level OR-INHIBIT gate based 4 input logic gate

A combination of OR and INHIBIT logic functions was used for the construction of the 4 inputs 1 output logic circuit. Now to imitate an OR logic gate function, the emission intensity at 558 nm was used as the output response similar to the earlier 2 input logic gate, and the inputs were Al³⁺, Fe³⁺, Cr³⁺ and CN⁻ (Fig. S15†). When the 1st (Al³⁺) and 2nd (Fe³⁺) inputs were both absent, the output response, i.e. the emission intensity, was very low, indicating the 0 (OFF) state. However, when only any one of the two inputs was present, the output signal was high, indicating the 1 (ON) state. Again in the presence of both the input Al³⁺ and Fe³⁺, the output response was 1 (ON). Thus, according to its truth table (Fig. 12a), an OR function logic gate could be contracted by the sequential addition of these two inputs. Then we verified the nature of the output signal in the presence of a 3rd ionic input (Cr³⁺) in the presence of the first two ionic inputs. Here, any one of these three inputs or the presence of two of these three inputs caused a high intensity emission, output indicating the ON state (1). Thus, the probe behaved like an OR logic function. On the other hand, when only a 4th input (CN⁻) was present or in the presence of all other inputs (Al³⁺, Fe³⁺ and Cr³⁺) in the system, the output emission was very weak, indicating the 0 (OFF) state. Therefore, we applied a NOT logic function with a 4th input. As the probe functions parallel with the output signal, so we could imply another AND logic function. Thus, from an INHIBIT logical function and following its corresponding truth table, an advanced level 4 input logic gate circuit could be constructed (Fig. 12b).

Fig. 12 (a) Truth table of an advanced level 4 input logic gate. (b) Schematic representation of a combined logic circuit of INHIBIT and OR logic gates.

Molecular memory device

Molecular memory devices are used for data storage technologies that use molecular species as the data storage element and can be constructed by sequential logic circuits. One of the output signals acts as the input of the memory device and it is memorized as a “memory element”. So by using binary logic function, we developed a sequential logic circuit showing a “write–read–erase–read” property. For our system, we chose a strong emission output at 558 nm as the ON state (1) and a weak emission output as the OFF state (0). Now to construct this memory device, we chose two inputs, namely Fe³⁺ and CN⁻, for the SET and RESET processes, respectively. In this memory function, the system writes when it gets input A (Fe³⁺), i.e. a high emission value, and it memorizes the binary number 1. However, in the presence of input B (CN⁻), which is a reset input, it erases the data and then memorizes the binary number 0 (Fig. 13). The properties of the material allow for a much greater capacitance per unit area than with conventional DRAM (dynamic random-access memory), thus potentially leading to smaller and cheaper integrated circuits.

Fig. 13 (a) Schematic demonstration of the reversible logic operation for the memory element with a “write–read–erase–read” kind of behaviour. (b) Sequential logic circuit showing a memory unit with two inputs (In A and In B) and one output, and (c) the corresponding truth table.

The most important thing is that this write–erase–write cycle could be repeated many times (Fig. 8) using the same concentration of the system without any significant change in emission intensity.

Cell-imaging studies

As L¹ showed extensive selective complex formation with trivalent metal ions (namely Fe³⁺, Cr³⁺, Al³⁺ ions), it was further checked for its ion-sensing ability in living cells (Fig. 14). A cell viability assay using MTT^66–68 was done to find out whether L¹ had cytotoxic effects, with calculating the % cell viability on HepG2 cells (Fig. S16†). As found from the result, no significant decrease in formazan production occurred up to 40 μM concentration of L¹, thus reflecting that a below 40 μM ligand concentration for L¹ would be much more effective for the analysis of its complex formation with trivalent metal ions in vitro. More than 95% cell viability was observed for L¹ at 10 μM, after which the viability of the HepG2 cells decreases slightly. Hence, further experiments were carried out with 10 μM for L¹ for treatment.

Fig. 14 Phase contrast and fluorescence images of HepG2 cells captured (40×) after cells were incubated with L¹ for 30 min at 37 °C and pre-incubated with Al³⁺, Cr³⁺ and Fe³⁺ for 30 min at 37 °C, followed by washing with 1× PBS and treatment with L¹ for 30 min at 37 °C. The cytoplasmic complex formation was confirmed by nuclear stain DAPI.

Upon incubation with 10 μM of the ligand L¹ for 1 h, it exhibited weak intracellular fluorescence on HepG2 cells due to the presence of intracellular Fe³⁺ (Fig. 14). However, a distinct red fluorescence was observed inside the cells when the HepG2 cells were incubated with 10 μM of the trivalent metal ions for 1 h at 37 °C, washed twice with PBS buffer and then reincubated with 10 μM of the ligand L¹ for 30 min at 37 °C. At 10 μM concentration of the Fe³⁺ ions, the ligand L¹ showed more intense fluorescence emission than Al³⁺ and Cr³⁺ ions.

Keeping the ligand L¹ concentration constant (10 μM) and increasing the concentration of metal ions (from 10 μM, to 20 μM, 40 μM) showed a concentration-dependent increase in the intracellular red fluorescence, caused by the formation of the complex of L¹ with either of the trivalent metal ions. Highly enhanced fluorescence was observed due to complex formation between the ligand L¹ and the metal ions nearly at 40 μM of metal ion concentration. These results suggest that the ligand L¹ with low cytotoxicity and biocompatibility has a high potential for in vitro application as an ion sensor of trivalent Fe³⁺, Cr³⁺, Al³⁺ ions as well as for live cell imaging for their detection in biological samples.

Fig. 15 represents some trivalent sensors reported so far and Table S1† displays some important parameters. A closer inspection of Table S1† reveals that our probe is superior to all the probes listed here in the sense that it provides higher excitation wavelength (502 nm). There is one report (probe 6) where CH₃OH–H₂O (6 : 4, v/v) was used, but the serious drawback of this system was that the excitation wavelength was in UV region (330 nm), which is not desirable for bioimaging applications.

Fig. 15 Some representative trivalent sensors.

Conclusion

In summary, we reported herein a new rhodamine-6G based fluorogenic probe, which showed a selective colorimetric as well as “turn-on” fluorescence response towards trivalent metal ions M³⁺ (M = Al, Fe and Cr) over mono- and divalent metal ions. A large enhancement of fluorescence intensity of L¹ [Fe³⁺ (41-fold), Al³⁺ (31-fold) and Cr³⁺ (26-fold)] was observed upon the addition of 3.0 equivalent of these metal ions in H₂O/CH₃CN (4 : 1, v/v, pH 7.2), which clearly indicate the feasibility of the naked eye detection of these metal ions. Take-off values were evaluated from the fluorescence titration data at variable concentrations of metal ions and a fixed concentration of ligand and were found to be 9.4 × 10³ M⁻¹ (Fe³⁺); 8.7 × 10³ M⁻¹ (Cr³⁺) and 13.4 × 10³ M⁻¹ (Al³⁺). The higher values of quantum yields (0.489, 0.376, 0.310 for [L¹–Fe³⁺], [L¹–Al³⁺] and [L¹–Cr³⁺], respectively) over the free ligand (0.012) indicate the higher stability of the complexes in the excited states. An excellent reversible fluorescence ON–OFF property of L¹ was observed through fluorescence study with the sequential addition of M³⁺ and CN⁻ ions at room temperature, which suggests the reusability of this chemosensor. The very low detection limit for Fe³⁺, Al³⁺ and Cr³⁺ were 1.28 μM, 1.34 μM and 2.28 μM, respectively, which could make it have a potential application in real water samples for trivalent ion detection. Advanced level molecular logic devices using different inputs (2 and 4 inputs) in advanced level logic gates and memory device were constructed. A suitably large increase in fluorescence intensity of L¹ upon complexation with M³⁺ suggests the probe may be used for bioimaging applications in living cells.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

Financial supports from DST (Ref. SR/S1/IC-20/2012) New Delhi are gratefully acknowledged.

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Footnote

† Electronic supplementary information (ESI) available. CCDC 1836133. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c8pp00381e

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