Imino–phenolic–azodye appended rhodamine as a primary fluorescence “off–on” chemosensor for tin (Sn⁴⁺⁾ in solution and in RAW cells and the recognition of sulphide by [AR–Sn]

Abstract

A new azo-rhodamine based species, AR was developed to act as an ‘off–on’ reversible luminescent probe for Sn⁴⁺ detection. The chemosensing behavior of the AR has been demonstrated through fluorescence, absorption, visual fluorescence color changes, ESI MS and ¹H NMR titrations. This chemosensor AR shows a significant visible color change and displays a remarkable luminescent switch on (>2300 fold) in the presence of Sn⁴⁺ ions. The chemosensor can be used as a ‘naked eye’ sensor. The roles of the fluorophore–photochrome (azodye) dyad platform as well as the iminophenolic binding core in AR's selective recognition of tin have been demonstrated by studying appropriate control molecules. Importantly, AR can selectively recognize Sn⁴⁺ in organo-aqueous media in the presence of other cations. The biological applications of AR were evaluated in RAW cells and it was found to exhibit low cytotoxicity and good membrane permeability for the detection of Sn⁴⁺. The development of practically viable colorimetric test strips of the chemosensor AR to detect Sn⁴⁺ was also reported. It has been possible to build an INHIBIT logic gate for two binary inputs viz., Sn⁴⁺ and S²⁻ by monitoring the fluorescence emission band at 582 nm as output.

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Introduction

The development of highly sensitive and selective fluorescent chemosensors for heavy and transition-metal ions has attracted tremendous interest because of their importance in chemistry, biology, and environmental science.^1–4 The development of tin (Sn⁴⁺) fluorescent chemosensors has attracted intense attention due to the concern over the adverse effect of tin on the environment and human health due to excess accumulation. Tin, widespread in the air, water and soil, is one of the most commonly used heavy metals in agricultural, industry,⁵ including food container, food processing equipment, toothpaste, perfumes, soaps, food additives and dyes. Again organotin compounds are used to make plastics, plastic pipes, PVC stabilizer, pesticides, paints, and pest repellents.⁶ Inorganic tin compounds are used as pigments in the ceramic and textile industry.⁷ However, human and various animal studies show that excess accumulation of tin can cause eye and skin irritation, headaches, stomachaches, sickness and dizziness, breathlessness, urination problems, liver damage, malfunctioning of immune systems, chromosomal damage, gastrointestinal effects (abdominal cramps, nausea, diarrhoea, vomiting).⁸ Thus, there is a strong need for tin selective chemosensors that rapidly detect Sn⁴⁺ in aqueous media by simple spectroanalysis.

A promising way is to develop optical chemosensors for detecting Sn⁴⁺ ions, which are based on an indicator that is capable of reporting on the selectivity of Sn⁴⁺ ions recognition through a variety of optical responses, mainly due to their distinct advantages in sensitivity, selectivity and fluorescence imaging in living cells. Among numerous indicators, rhodamine-based dyes are a kind of excellent candidate for the construction of an off–on-type fluorescent chemosensor due to their excellent spectroscopic properties of large molar extinction coefficients, high fluorescence quantum yields, and long absorption and emission wavelengths elongated to the visible region.⁹ The metal ion sensing mechanism of these sensors is based on the change in structure between the spirocyclic and open-cycle forms. Typically, these sensor molecules prefer their spirolactam ring-closed state, which shows little absorption or fluorescence, whereas ring-opening of the corresponding spirolactam induced by metal ions gives rise to orange fluorescence and a clear color change from colorless to pink. Therefore, most of the reported rhodamine-based chemosensors for metal ions¹⁰ are of colorigenic or fluorogenic type.

However, Sn⁴⁺ responsive fluorescent probes are barely explored. To date, very few rhodamine B-based probe for Sn⁴⁺ has been reported, whose application is limited by its low sensitivity and water solubility.¹¹ Recently, our group developed a oxo-chromene–rhodamine B-derivative acts as a selective colorimetric and fluorometric Sn⁴⁺ chemosensor in water–organic two phase detection system.¹² The colorless spirolactam form is converted into the pink colored ring opening form upon binding with Sn⁴⁺, which shows a distinctive absorption band at 555 nm. The addition of Sn⁴⁺ resulted in a prominent enhancement (14 fold) of fluorescence at 580 nm, which allows detection limit of Sn⁴⁺ (2.58 μM) by a fluorescent analysis.

However, in the present case, we report the design and synthesis of a new rhodamine B-based probe operating in turn-on mode for selective and sensitive detection of Sn⁴⁺ ions in a water–organic solvent mixture. Here, the change in spirocycle to open-ring form of the rhodamine fragment in AR result in the remarkable enhancement of emission intensities (>2300 fold), and these offer us the possibility of studying the Sn⁴⁺ recognition process through the switch-on optical response-a criterion that is important for developing an in-field detection reagent. The chemosensor is composed of a rhodamine dye, a hydroxyl group, and an azo moiety. We reasoned that Sn⁴⁺ could coordinate to the novel receptor formed by the hydroxyl group and an imine unit due to its selective coordinating character.¹³ This coordination may induce the transformation of the rhodamine dye from the non-fluorescent spirocyclic form to the highly fluorescent opened-ring form for a significant fluorescence enhancement signal. To achieve high selectivity, we designed a new reversible and selective chemosensor (AR) for tin ions by simple structural modifications of our previously reported receptors.¹² Obviously, the structural modifications should: (1) introduction of nonfluorescent azodye quencher; (2) significantly improve the sensitivity for tin (>2300 fold) over other ions; (3) taking model compounds by the combination of azo and fluorophore moieties. It is noteworthy that this design concept has not been previously employed in the construction of Sn⁴⁺ fluorescent probes.

Experimental section

Experimental details corresponding to the materials and methods used, synthesis, characterization, solution preparation for absorption and fluorescence titrations, determination of association constant, calculation of detection limit, effect of p^H, cellular and computational studies are given in the ESI† (pp. S3–S8).

Synthesis of AR

A 326 mg portion of 5-(4-carboethoxyphenylazo)salicylaldehyde (1.1 mmol) was dissolved in 15 ml dry chloroform with continuous stirring and then 500 mg (1.1 mmol) of rhodamine B hydrazide in 15 ml dry methanol was added to the solution and heated to reflux for 12 hours. A yellow precipitate was appeared. After that the reaction mixture was cooled to room temperature and then the precipitate was collected through filtration. The residue was washed several times with ethanol to isolate AR in pure form with 90% yield. M.P. > 250 °C. ¹H NMR (400 MHz, CDCl₃, Si(CH₃)₄, J (Hz), δ(ppm)): 11.57(1H, s, –OH), 9.14(1H, s, –CH=N), 8.15 (2H, d, J = 8.44 Hz), 7.99 (1H, d, J = 6.92 Hz), 7.84 (3H, d, J = 8.24 Hz), 7.76 (1H, d, J = 2.08 Hz), 7.53(2H, m), 7.18 (1H, d, J = 7.04 Hz), 6.98 (1H, d, J = 8.84 Hz), 6.49 (4H, dd, J = 3.36 & 9.36 Hz), 6.27(2H, dd, J = 2.24 & 2.28 Hz), 4.39 (2H, q, J = 7.16 Hz), 3.32 (8H, q, J = 7.04 Hz, –NCH₂CH₃), 1.41(3H, t, J= 7.12 Hz), 1.15 (12H, t, J= 7.00 Hz, –NCH₂CH₃).¹³ C NMR (CDCl₃, 400 MHz) δ(ppm): = 12.58, 14.10, 44.35, 61.17, 66.34, 98.01, 105.01, 108.21, 118.00, 118.76, 122.27, 123.48, 124.13, 125.26, 127.93, 128.02, 128.63, 129.12, 129.34, 129.85, 130.52, 131.54, 133.74, 145.59, 149.17, 153.43, 155.19, 162.03, 164.43, 166.14. TOF MS ES⁺, m/z = 736.9504, calc. for C₄₄H₄₄N₆O₅ = 736.8686.

Results and discussion

The receptor AR was prepared according to Scheme 1. Initially the intermediate azo compound 5-(4-carboethoxyphenylazo)salicylaldehyde was synthesized according to our reported procedure.¹⁴ The Schiff base chemosensor AR was prepared in 90% yield as yellow solid from the condensation of 5-(4-carboethoxyphenylazo)salicylaldehyde with rhodamine B hydrazide in an equal molar ratio in methanol and chloroform mixture under refluxing condition. A control molecule contain naphthylazo platform, NAR has been synthesized by similar diazocoupling and condensation reactions starting from 2-formyl-1-naphthol instead of salicylaldehyde. AR and NAR were well characterized by ¹H NMR, ¹³C, FTIR and mass analyses (Fig. S1–S3, S9A and S5–S6 ESI†).

Scheme 1 Synthetic approach for the chemosensor AR and control molecule NAR.

Fluorescence titrations of AR with Sn⁴⁺ in aqueous EtOH (HEPES, 10 mM, pH = 7.4, 1 : 4, v/v) were performed. Probe AR exhibited almost no fluorescence in aqueous EtOH (HEPES, 10 mM, pH = 7.4, 1 : 4, v/v), indicating that the spirocyclic form is retained in solution. However, with the addition of increasing concentrations of Sn⁴⁺, a excellent enhancement (up to 2428-fold) in fluorescence intensity at 582 nm was noticed, which was much higher than the result obtained with the early reported Sn⁴⁺ sensors.¹⁵ The large fluorescence turn-on was corroborated by the observation that the emission color of the sensor solution turned from dark to orange (Fig. 1a, inset), which indicates that probe AR is an excellent turn-on sensor for Sn⁴⁺.

Fig. 1 (a) Change in the emission spectra of ligand AR [c = 4 × 10⁻⁵ M], EtOH : H₂O = 4 : 1, v/v, 10 mM HEPES buffer, pH = 7.4, λ_ext = 563 nm) upon addition of Sn⁴⁺ ions (c = 4 × 10⁻⁴ M). Inset: change of emission intensity at 582 nm with incremental addition of Sn⁴⁺ [λ_ext = 563 nm] and fluorescence photographs of AR before and after addition of Sn⁴⁺ ions. (b) Change in the fluorescence emission of AR in presence of different metal ions such as Li⁺, Na⁺, K⁺, Ca²⁺, Mg²⁺, Sr²⁺, Ba²⁺, Sn⁴⁺, Cr³⁺, Al³⁺, Mn²⁺, Fe²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Hg²⁺, Pb²⁺, Sn⁴⁺ and Ag⁺ in aq. EtOH (EtOH : H₂O = 4 : 1, v/v, 10 mM HEPES buffer, pH = 7.4) (λ_ext = 563 nm).

A more than thousand-fold increase observed in the quantum yield (QY) of [AR + Sn⁴⁺] (0.216 ± 0.004) as compared to that of AR (1.16 × 10⁻⁴) supports the fluorescence enhancement of AR observed in presence of Sn⁴⁺, suggesting a sensitive and selective detection of Sn⁴⁺ by AR compared to other metal ions. The recognition interaction was completed immediately after the addition of Sn⁴⁺ within 2 min and hence, AR could be used in real-time determination of Sn⁴⁺ in environmental and biological conditions. The formation of an intense absorption peak at 563 nm in the absorption profile of probe AR upon the addition of Sn⁴⁺ is in good agreement with the fluorescence enhancement (Fig. 1a).

Importantly, the sensor showed a nice linear relationship between the fluorescence intensity at 582 nm and the concentrations of Sn⁴⁺ from 3.96 to 112 μM (Fig. 1a, inset), suggesting that sensor AR is potentially useful for quantitative determination of Sn⁴⁺ with a large dynamic range. Similar fluorescence studies were carried out with the control molecule such as NAR and found very small amount of change (only 3.7 fold) in the emission intensity compare to AR, suggesting that fluorophore (naphthyl moiety)–photochrome (azodye part) dyad activates intercomponent electron or energy transfer pathways and weakens the emission of the fluorophore (Fig. S14B, ESI†).¹⁶

The Job plot¹⁷ shows that sensor AR forms a 1 : 1 complex with Sn⁴⁺ ions (Fig. S8A, ESI†). The 1 : 1 binding mode of the sensor with Sn⁴⁺ was also confirmed by the ESI MS mass spectrum of the complex (Fig. S4, ESI†), which showed an intense peak at m/z = 943.3710 (calc. 943.5153), assigned to the 1 : 1 complex [AR + Sn⁴⁺ + 2Cl⁻ + NH₄⁺]⁺. Therefore, we suggest that probe AR coordinates with Sn⁴⁺ with 1 : 1 stoichiometry. Based on a 1 : 1 binding mode, apparent association constant (K_a) of the AR–Sn⁴⁺ interaction was calculated to be (1.414 ± 0.41) × 10⁴ M⁻¹ (Fig. S8B, ESI†) from the data of the fluorescence titration experiments using nonlinear curve fitting procedure.^18,19d The detection limit for Sn⁴⁺ ion with AR was estimated to be 7.1 μM. Fig. S9 (ESI†) shows the partial IR spectra of sensor AR in the absence or presence of 1 equiv. of Sn⁴⁺. The peak at 1617.32 cm⁻¹, assigned to the characteristic amide carbonyl absorption, was shifted to 1592.61 cm⁻¹ in the presence of Sn⁴⁺, indicating that the amide carbonyl group is involved in the interactions with Sn⁴⁺. This is key to the spiro ring-opening and fluorescence turn-on of the rhodamine dye. Taken together, a likely sensing mechanism based on the Sn⁴⁺-triggered spiro ring-opening process is proposed in Scheme 2. We then proceeded to examine the selectivity of the sensor. The selectivity of compound AR to the various metal ions was tested as selectivity is an important characteristic feature of an ion-selective chemosensor. We tested our chemosensor with possible interferences including metal ion salts of Li⁺, Na⁺, K⁺, Ca²⁺, Mg²⁺, Sr²⁺, Ba²⁺, Cr³⁺, Al³⁺, Mn²⁺, Fe²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Hg²⁺, Pb²⁺ and Ag⁺ in aqueous EtOH (HEPES, 10 mM, pH = 7.4, 1 : 4, v/v) (Fig. 1b). Remarkably, only Sn⁴⁺ elicited a large fluorescence enhancement. By contrast, alkali metals ions (Li⁺, Na⁺, K⁺) and alkali-earth metals ions (Ca²⁺, Mg²⁺, Sr²⁺, Ba²⁺) even in the presence of large excess (100 equiv.) have no observable fluorescence response.

Scheme 2 Schematic presentation showing the possible binding mechanism of AR with Sn⁴⁺.

Furthermore, sensor AR gave only a minimal response to transition metal ions such as Al³⁺ and Cr³⁺, indicating that the sensor is highly selective. To explore the utility of AR as an ion-selective chemsensor, the competition experiments was carried out by adding Sn⁴⁺ to AR solution in presence of other competitive metal ions (Fig. S10, ESI†). As shown in Fig. 1b, Sn⁴⁺ induced fluorescent responses were not hardly influenced by these common coexistent metal ions (Fig. S12, ESI†). Thereby, sensor AR appears to be useful for selectively sensing Sn⁴⁺ even in the presence of other relevant metal ions.

UV-vis spectra recorded for AR in EtOH : H₂O HEPES buffer (10 mM, pH 7.4; 4 : 1, v/v) indicated three spectral bands at 267 nm, 276 nm and 320 nm respectively, which may possibly be attributed to intraligand charge transfer (CT) transition.

Upon addition of increasing concentrations of Sn⁴⁺ ions to the solution, a new absorption band centered at 563 nm appeared with increasing intensity and other three bands decrease gradually resulted in an isosbestic point at 473 nm (Fig. 2). Switch on responses for the absorption spectral band at 563 nm and the luminescence band at 582 nm on binding to Sn⁴⁺ suggest opening of the spirolactam ring in AR on metal ion coordination. This type of enhancement was not observed when the analogous experiments were conducted with other cations (Fig. S11, ESI†). On the other hand, the control molecule, NAR did not show any significant change in the absorbance as well as necked eye response when titrated with Sn⁴⁺, suggesting that AR is sensitive and selective toward Sn⁴⁺ (Fig. S14A, ESI†). Interestingly, a solution of AR in optimized EtOH : H₂O solution (4 : 1, v/v, HEPES buffer, pH = 7.4) is colorless and emits no orange fluorescent light, but during the fluorometric titration of AR with Sn⁴⁺ ions the colorless solution of the receptor became deep orange (Fig. 1a, inset). This orange fluorescent color is attributed to the opening of the spirolactam ring and generation of the delocalized xanthenes moiety.¹⁹ Whereas compound AR shows obvious pink color in buffered ethanol–HEPES solution upon addition of Sn⁴⁺ under visible light (Fig. 2, inset). This was not observed with other metal ions except Cu²⁺, Cr³⁺ and Al³⁺ ions. In case of Cu²⁺, Cr³⁺ and Al³⁺ ions, the colorless solution of AR turned into a red and faint pink color respectively (Fig. S12†). The results support our expectation that AR could serve as a sensitive naked-eye probe for Sn⁴⁺.

Fig. 2 Change in the absorption spectrum of receptor AR [c = 4 × 10⁻⁵ M], EtOH : H₂O = 4 : 1, v/v, 10 mM HEPES buffer, (pH = 7.4) upon addition of increasing amounts of Sn⁴⁺ ions (c = 4 × 10⁻⁴ M). Inset showing the change in color of AR solution before and after addition of Sn⁴⁺.

The reversibility is an important aspect of any receptor to be employed as a chemical sensor for detection of specific metal ions. To examine whether the process is reversible, an excess amount of Na₂S was added into the solution of sensor AR pre-incubated with Sn⁴⁺. The bright fluorescence immediately turned off (inset, Fig. 3a). We also studied fluorometric titration experiments with a series of different anions such as F⁻, Br⁻, I⁻, NO₃⁻, NO₃⁻, SCN⁻, SO₄²⁻, SO₃²⁻, ClO₄⁻, CH₃COO⁻, HSO₄⁻, and H₂PO₄⁻. Among these only addition of S²⁻ to the complex solution of AR brought the reverse change in the emission spectra due to the regeneration of AR (Fig. S13A, ESI†). This is attributable to the binding of S²⁻ ions to the [AR + Sn⁴⁺] followed by the removal of Sn⁴⁺ in the form of SnS₂, thus releasing the free AR (Scheme 3). Interestingly, while Na₂S diminished emission significantly, further addition of excess Sn⁴⁺ ions could recover emission signals with less intense color in a reversible manner (Fig. 3b). This result implies the reversible character of the binding of sensor AR with Sn⁴⁺ (Fig. 3). Furthermore, like Na₂S, addition of aq. solutions of Na₂EDTA brought about similar change in emission spectra (Fig. S13B, ESI†) and further confirmed the reversibility in the binding process. Thus, fluorescent chemosensors are reversible in general.²⁰

Fig. 3 (a) Change in the emission spectra of ligand AR [c = 4 × 10⁻⁵ M], EtOH : H₂O = 4 : 1, v/v, 10 mM HEPES buffer, pH = 7.4, (λ_ext = 563 nm) with 4 equiv. of Sn⁴⁺ upon addition of sodium sulfide (c = 4 × 10⁻⁴ M). Inset: fluorescence photographs of AR–Sn complex after addition of S²⁻ ions. (b) Fluorescence experiment showing on–off reversible visual fluorescent color changes after each addition of Sn⁴⁺ and S²⁻ sequentially.

Scheme 3 Schematic presentation for fluorescence quenching.

To get insight into the binding mode, the ¹H NMR titration experiments were carried out. As shown in Fig. 4, the ring protons of rhodamine and salicylaldehyde moieties were moved to apparent downfield region (Δδ_c = 0.60, Δδ_d,e = 0.37, Δδ_f = 0.19, Δδ_g,h = 0.05 and Δδ_i = 0.13 ppm) in the presence of Sn⁴⁺. This downfield movement is ascribed to the decrease in electron density arising from an intramolecular charge transfer from NEt₂ group to spirolactam ring opening during metal ion complexation. Specially, the imine proton (H_b) at around δ 9.11 ppm was considerably shifted downfield toward δ 9.22 ppm upon Sn⁴⁺ addition, indicating that a decrease in electron density at imine nitrogen resulting from direct coordination with Sn⁴⁺.

Fig. 4 Partial ¹H NMR of (a) AR (3 × 10⁻³ M) and with (b) 0.5 equiv. (c) 1 equiv. amounts of Sn⁴⁺ in D₂O.

To explore potential and analytical applications of the chemosensor AR for Sn⁴⁺ ions tested, the test strips was carried out.²¹ The strips was prepared by using a TLC plates which were further immersed into the solution of AR (4 × 10⁻⁴ M) in ethanol and then drying it by two way (i) exposure to air and (ii) in vacuum.

After that, we immersed the TLC plate in Sn⁴⁺ (3 × 10⁻³ M) solution and then exposed to air and in vacuum to evaporate the solvent. The TLC plates turned pink from light yellow color and also give orange fluorescent in fluorescence light. This experiment exhibits steady colorimetric and fluorimetric changes in presence of Sn⁴⁺ by showing different colors and fluorescent changes which can be detect by our naked eye (Fig. 5). Therefore, this experiment gives a real time monitoring without using any sophisticated instrumentation.

Fig. 5 Colorimetric and fluorometric test kit. Photographs of the TLC plate coated with AR used for the detection of Sn⁴⁺ ions in solution: AR (I & III); AR + Sn⁴⁺ (II & IV).

To better understand the photophysical properties of chemosensor AR and the complex AR–Sn⁴⁺, they were examined by density function theory (DFT) calculations at the B3LYP²² level of the Gaussian 09 program. The 6-31 G (d) basis set was used for the H, C, N, and O atoms; the exception was for the Sn atom, where the LanL2DZ basis set with effective core potential was employed. The optimized structure is shown in Fig. 6, which shows that the Sn⁴⁺ ion binds to AR very well through five coordination sites. The molecular orbital plots of compound AR and AR–Sn⁴⁺ are shown in Fig. S16 and S17 (ESI†) respectively. For chemosensor AR, the π electrons on both the HOMO and HOMO-1 are essentially distributed in the entire rhodamine backbone, but the LUMO and LUMO-1 are mostly positioned at the electron-withdrawing phenylazo-salicylaldehyde part. This indicates that AR bears efficient electron transfer from the rhodamine dye part to the phenylazo group, thus rendering the fluorescence relatively weak.

Fig. 6 B3LYP optimized geometries of rhodamine derivative AR (a) and its complexes with Sn⁴⁺ ion (b).

In contrast, the π electrons on the HOMO of AR–Sn complex are mainly located on the phenylazo-salicylaldehyde part, but the LUMO is mostly positioned at the rhodamine framework. Moreover, the HOMO–LUMO energy gap of complex becomes much smaller relative to that of probe AR. The energy gaps between HOMO and LUMO in the probe AR and AR–Sn complex are 3.06 eV and 2.57 eV respectively (Fig. 7). The interaction of the salicylaldehyde OH group with Sn⁴⁺ can change the orbital energy level, realizing the optical detection. The optimized complex of Sn⁴⁺ with AR at this stage showed a nearly planar pentacoordinated²³ geometry around Sn⁴⁺ where all five bonds (2 × Sn–O, Sn–N and 2 × Sn–Cl) are bonded to the central ion with their distances of 1.99, 2.04, 2.14, 2.32 and 2.33 Å respectively. The result clearly suggest that the Sn⁴⁺ ion binds to AR very well through five coordination sites, and the whole molecular system forms a nearly planar structure. To get detailed insight into the changes in electronic spectra of AR and AR–Sn complex TDDFT calculations have been carried out by TDDFT/CPCM method in methanol solvent. The calculated vertical electronic excitations well matched with the experimentally observed bands both for the AR and AR–Sn complex and have intra-ligand charge transfer (ILCT) origin (Table S1 and S2, ESI†).

Fig. 7 HOMO and LUMO orbitals energy diagram of AR and AR–Sn complex calculated by DFT/B3LYP/LANL2DZ/6-31 + G(d) method.

Chemosensor AR exhibited favorable features including fast response, reversibility, high sensitivity with a large fluorescence (up to 2384-fold) enhancement and a low detection limit of 7.1 μM, high selectivity for Sn⁴⁺, and working well at physiological pH. These desirable attributes render the sensor suitable for fluorescent imaging of tin ions in living cells. Relying on the promising properties of chemosensor AR, we next questioned whether chemosensor AR could be used for monitoring the accumulation of tin ions in living cells. RAW cells were incubated with chemosensor AR (10 μM) for 30 min, followed by the addition of Sn⁴⁺ and incubation for another 30 min. The fluorescence images were recorded before and after the addition of Sn⁴⁺ (20 μM) (Fig. 8).

Fig. 8 Confocal microscopic images (Andor Spinning Disk Confocal microscope, 40× objective lens) of chemosensor in Raw 264.7 cells. Cells pretreated with SnCl₄ (a) Bright field image of the Raw 264.7 cells. (b) After addition of Sn⁴⁺ (20 μM), nuclei counterstained with DAPI (1 μg ml⁻¹) (c) stained with chemosensor AR (10 μM) (in green filter) (d) overlay image of Raw 264.7 cells in dark field.

RAW cells incubated with chemosensor AR exhibited no fluorescence, whereas a bright fluorescence signal was observed in the cells stained with chemosensor AR and Sn⁴⁺, which in good agreement with the fluorescence turn-on profile of the sensor in the presence of Sn⁴⁺ in the solution.

Moreover, bright red colored fluorescence cells obtained from the incubation of the receptor AR followed by treatment with Sn⁴⁺ became invisible in fluorescence upon addition of Na₂S (30 μM) (Fig. 9). The results establish that chemosensor AR is cell membrane permeable and can be efficiently used for in vitro imaging of tin ions in living cells. Moreover, there were no indications of cell damage. Cells were intact and showed healthy spread and adherent morphology during and after the labeling process with chemosensor AR, indicating an absence of cytotoxic effects (Fig. S18, ESI†).

Fig. 9 Confocal microscopic images of Raw 264.7 cells. (a) After treating with probe AR in presence of SnCl₄. (b) After adding Na₂S (30 μM) solution to the (AR + Sn⁴⁺) treated cells.

The properties exhibited by AR as reported in this paper allows the design of molecular logic gate²⁴ using two binary input signals, while one is Sn⁴⁺ and the other is S²⁻, and output monitored as a fluorescence emission of AR at 582 nm. From the logic gate functions, the emission of AR is observed only in the presence of single input, viz., Sn⁴⁺ and not with S²⁻ anion is insensitive to AR. Inputs of Sn⁴⁺ and S²⁻ have been considered as zero when they are not present and one when they are added. When both the inputs are zero, the output signal is zero and the gate is OFF.

The AR treated with S²⁻ alone do not show any fluorescence change and thus upon adding S²⁻ the output signal is zero. On the other hand if AR is treated with Sn⁴⁺ alone as input, then the fluorescence enhances significantly (2368 fold at 582 nm), and the output signal is read out as one and the gate is ON. However, if all these two inputs (Sn⁴⁺ and S²⁻) are present together, then the fluorescence of AR is quenched with the output read out as zero and the gate is OFF. From these studies, it has been clearly demonstrate that AR can be used as INHIBIT (INH) logic gate using Sn⁴⁺ and S²⁻ as inputs and the fluorescence emission at 582 nm as output. The truth table and the pictorial representation for the corresponding INH logic gate are given in Fig. 10.

Fig. 10 Logic diagram and Truth table of INH logic circuit.

Conclusion

In conclusion, we have synthesized and characterized 5-(4-carboethoxyphenylazo) salicylaldehyde–rhodamine B-derivative of AR exhibits high selectivity towards Sn⁴⁺. Its utility as a highly selective fluorescence switch ON (>2300-fold) chemosensor that responds stoichiometrically and rapidly to Sn⁴⁺ under physiological conditions. This azo appended chemosensor for Sn⁴⁺ ions is a new example among the few reports in the literature. Interaction of Sn⁴⁺ with AR enhances the fluorescence emission at 582 nm and induces a turn on response in electronic and fluorescence spectra in the visible region. AR is sensitive and selective toward Sn⁴⁺ over other biologically important ions studied, viz., Li⁺, Na⁺, K⁺, Ca²⁺, Mg²⁺, Sr²⁺, Ba²⁺, Cr³⁺, Al³⁺, Mn²⁺, Fe²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Hg²⁺, Pb²⁺ and Ag⁺ ions, as demonstrated by individual as well as competitive metal ion titrations. Thus, these chemosensors could be used as a dual probe for visual detection through change in color and fluorescence via Sn⁴⁺-promoted reversible ring opening i.e. coordination/disconnection reactions. Comparison of the fluorescence results of AR with those of the control molecule, NAR clearly suggest that covalently bonded fluorophore–photochrome dyad (NAR) is less sensitive towards selective sensing of Sn⁴⁺ ion. It has to be mentioned that the in situ prepared tin complex AR–Sn⁴⁺, was able to detect S²⁻ exactly reverse manner and this experiment could serve as experimental evidence to support this reversible spiro ring-opening mechanism. Additionally, the chemosensor is also noted to be efficient probe for intracellular detection of Sn⁴⁺ by confocal microscope. Preliminary confocal microscopy experiments showed that because of the nature of its good membrane permeability, and its low toxicity, chemosensor AR performed well as a sensing probe for the detection of intracellular Sn⁴⁺ and S²⁻ in RAW cells. The displacement of Sn⁴⁺ from the binding core of the [AR + Sn⁴⁺] complex upon addition of S²⁻ has been demonstrated by molecular logic gate operation. Thus the results obtained in the present studies help to construct the molecular level INH logic gate.

Acknowledgements

We thank the DST-New Delhi [Project file no. SR/S₁/OC-44/2012] for financial support. SKM thanks UGC, New Delhi and DS thanks CSIR, New Delhi, India for providing a fellowship.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: See DOI: 10.1039/c4ra05729e

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