Fluorescent sensor for in-vivo bio-imaging, precise tracking of Fe³⁺ ions in Zebrafish embryos and visual measuring of Cu²⁺ ions in pico-molar level

2.1 Materials and methods

All the solvents and reagents (analytical and spectroscopic grade) were obtained from Sigma-Aldrich and used without further purification. Metal chloride and nitrate salts were purchased from Merck and used as such. NMR spectra were recorded using Bucker 300 MHz spectrophotometer. The chemical shifts were shown in parts per million (ppm) downfield from tetramethylsilane (reference chemical shift is zero ppm) using residual DMSO (internal standard). Multiplicities were reported as follows: s (singlet), d (doublet), t (triplet), m (multiplet), and br (broadened). All the absorption measurements were recorded using Jasco 630 spectrophotometer and fluorescence measurements were performed in Agilent Cary Eclipse fluorescence spectrophotometer at room temperature. Electrospray ionization mass spectra (ESI-MS) were collected on liquid chromatography-ion trap mass spectrometer at room temperature (LCQ Fleet, Thermo Fisher instruments limited, USA.

2.2 UV–visible and fluorescence titrations

An acetonitrile solution of RS (2.5 µM) has been used for the absorption and fluorometric spectral titrations. Slit width of 5/5 and 375 nm as an excited wavelength for all fluorescent measurements. Freshly prepared probe solution has been used in each experiment, and the corresponding metal ions were added using micropipette. The UV–visible and fluorescence spectral experiments were performed with high concentration of chemosensor RS (10⁻³M) in DMSO as a stock solution to avoid the dilution error. 10 equivalents of all the biologically important metal ions were used for the selectivity experiment with respect to the probe and μM/10⁻⁷ M of Fe³⁺/Cu²⁺ ions were used for the sensitivity experiments respectively. In the jobs plot analysis, 10 μM of analyte and chemosensor RS were used respectively in both spectroscopic techniques.

2.3 Fluorescence imaging on embryos

Three days old embryos were pre-treated with Fe³⁺ ions for 2 h, and washed with phosphate buffer. These iron pre-treated embryos were treated with 2.5 µM of RS solution for 1 h and they were again washed with phosphate buffer solution. Finally, these were fixed in 10% methylcellulose solution for secured orientation and then fluorescent images were recorded by fluorescence microscopy.

2.4 pH studies

The medium adopeted to proced the experiments under variying pH gradients have been carried out using 0.1 M of hydrochloric acid and triethylamine to adjust the pH. The individual pH solution of acetonitrile has been prepared in the range of 2–12. Before recording the absorbance and fluorescence spectra, the invivisual pH of the chemosensor, RS and Cu²⁺/Fe³⁺ ion solutions were examined to the respective pH gradient at room temperature.

3.1 Synthesis of chemosensor, RS

Rhodamine hydrazine was synthesised by the previously reported procedure (Ma et al., 2013) with slight modifications. Rhodamine 6G (1 mmol) and hydrazine monohydrate (5 mmol) was dissolved in methanol and this reaction mixture was refluxed for 2 h, a pinkish white precipitate was obtained, which was filtered and washed with cold methanol then dried at room temperature. This rhodamine hydrazide was re-dissolved in hot methanol and 1 equivalent of 3,5-diiodo salicylaldehyde is added to it and refluxed for 3 h, it was cooled to room temperature yielding the desired chemosensor RS, as pale yellow solid which was filtered and washed with cold methanol (yield 72%) (Scheme 1). It was characterized by NMR and ESI-MS analysis. ¹H NMR (300 MHz, DMSO) δ 11.87 (s, 1H), 8.68 (s, 1H), 7.95 (d, J = 6.3 Hz, 2H), 7.77–7.49 (m, 3H), 7.06 (d, J = 7.1 Hz, 1H), 6.35 (s, 2H), 6.20 (s, 2H), 5.16 (t, J = 5.2 Hz, 2H), 3.23–3.03 (m, 4H), 1.84 (s, 6H), 1.20 (t, J = 7.1 Hz, 6H) (Fig. S1). ¹³C NMR (75 MHz, DMSO) δ 164.62, 157.04, 152.35, 151.91, 148.94, 148.78, 147.73, 139.75, 135.24, 129.80, 128.53, 127.56, 124.68, 124.13, 121.25, 119.49, 104.62, 96.85, 88.52, 82.82, 66.64, 41.38, 41.10, 40.82, 40.54, 40.26, 39.98, 39.71, 38.36, 17.71, 15.01 (Fig. S2). Calculate Mass = 784.04. Mass obtained from ESI-MS: m/z = 785.05 (M + 1) (Fig. S3). Obtain mass from HR-MS: 785.0434 (M + 1) (Fig. S4).

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Scheme 1

Chemical synthesis of the chemosensor RS.

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3.2 Naked eye and fluorescence sensor

A stock solution was prepared with the synthesised chemosensor RS (3.9 mg) in DMSO (5 mL). This stock solution has been diluted to 2 mL in acetonitrile and cover up concentration of 2.5 μM, which was used for all sensor studies. To evaluate its naked eye sensor abilities, 10 equivalents of various biologically and environmentally important metal ions have been used (Fe³⁺, Fe²⁺, Cu²⁺, Al³⁺, Cr³⁺, Pb²⁺, Cd²⁺, Hg²⁺, Zn²⁺, Ni²⁺, Co²⁺, Mn²⁺, Mg²⁺, Ba²⁺ and Ca²⁺). The non-fluorescent RS did not show any significant fluorescence changes upon the addition aforementioned metal ions with the exception of Fe³⁺ and Fe²⁺ ions. The addition of Fe³⁺ ions leads to an intense fluorescence change in the UV cabinet along with a puny fluorescence due to Fe²⁺ ions, because of the Lewis acidic nature of Fe³⁺ ions, and it’s tendency to cleave the lactam ring present in the RS. Interestingly, the occurred colour changes of RS with Fe³⁺ (pale pink) and Cu²⁺ (intense pink) ions in CH₃CN could also be monitored by the naked eye. These observed colorimetric and fluorescence sensing studies could indicate that the probe can be efficiently utilized for the selective detection of Fe³⁺/Cu²⁺ ions (Fig. 1).

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Fig. 1

Photograph of chemosensor, RS (2.5 μM) with various metal ions in (a) normal day light (RS, Fe³⁺, Fe²⁺, Cu²⁺, Al³⁺, Cr³⁺, Pb²⁺, Cd²⁺, Hg²⁺, Zn²⁺, Ni²⁺, Co²⁺, Mn²⁺, Mg²⁺, Ba²⁺ and Ca²⁺ left to right) and (b) fluorescence lamp (RS, Fe³⁺, Fe²⁺, Cu²⁺, Al³⁺, Cr³⁺, Pb²⁺, Cd²⁺, Hg²⁺, Zn²⁺, Ni²⁺, Co²⁺, Mn²⁺, Mg²⁺, Ba²⁺ and Ca²⁺ left to right).

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3.3 Fluorescence studies

Generally, acids (organic/inorganic) can be used to stimulate and open the existing locked lactam ring in the chemosensor RS, which could enhance the fluorescence/absorbance (Bao et al., 2019; Karakus et al., 2013; Queirós et al., 2014). As like, inorganic Lewis acid, Fe³⁺ ions can also trigger the opening of the existing lactam ring in the proposed probe RS, which leads to excellent photo-physical properties at 554 nm. On the addition of 10 equivalents of various biologically important metal ions didn’t show any new band in a emission profile except Fe³⁺ ions, shows an bright fluorescence at 554 nm (Fig. 2a). Similarly, the existing unpaired electron in Fe²⁺ ion, it also showed a trivial fluorescence enhancement over the Fe³⁺ ions. In order to check out the sensing propensity of RS analysis between static concentration of RS and increasing concentrations (0–22.5 μM) of Fe³⁺ ions was performed (Fig. 2b). The incremental addition of the Fe³⁺ ions yields a gradual appearance of a new band at 554 nm with clear linearity (7.5–17.5 μM, Fig. S5), further addition of excess Fe³⁺ ions leads to saturation. These studies demonstrated that the chemosensor RS tends to recognize Fe³⁺ ions quantitatively. Such accurate quantity of limit of detection (2 nM) could be calculated by using the formulae 3σ/slope. Chemosensor RS has also showed a strong binding constant (3.4 × 10⁻¹ M⁻¹, Fig. S6) with Fe³⁺ ions. The stoichiometry of RS-Fe³⁺ complex was determined using Job's method by the plot between the intensity of conjugate, RS-Fe³⁺ with respect to the mole-fraction of RS, which clearly exposed that its efficient and intense fluorescence band was observed at 0.5 mol-fraction. This observation indicated that the occurrence of 1:1 mode of binding between RS and Fe³⁺ ions (Fig. 2c). Further ahead, RS showed a good sensing knack towards Fe³⁺ ions even in the presence of high concentrations of the other metal ions except Cu²⁺ ions, which may be due to the Cu²⁺ scavenging on the excited-state electron from RS-Fe³⁺ complex. Moreover, the addition of Fe²⁺ ions also causes a small interference in the fluorescence of RS-Fe³⁺ conjugate.

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Fig. 2

(a) Fluorescence selectivity spectral analysis of RS (2.5 μM) with 10 equivalents of other relevant metal ions in CH₃CN:H₂O (9:1 v/v) mixture, (b) Fluorescence sensitivity spectral analysis of RS (2.5 μM) with incremental addition of Fe³⁺ ions (0–22.5 μM) in CH₃CN:H₂O (9:1 v/v) mixture, (c) Job’s plot analysis of Fe³⁺ ions with respect to the mole-fraction of RS at 554 nm in fluorescence spectroscopy.

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3.4 Absorption spectroscopy

Chemosensor RS showed two absorption bands at 300 and 350 nm. These bands are attributed to its π-π* and n-π* transitions, and there is no absorbance was appeared in the range of 500–600 nm. Interestingly, addition of 10 equivalents of Cu²⁺ and Fe³⁺ ions, showed an excellent chromo-isomerism with rigid absorption bands at 521 and 529 nm respectively as a consequence of the cleaved spirolactam ring (Lewis acidic nature of these two paramagnetic metal ions) exist in the RS, while, other metal ions did not show any significant colour or spectral changes (Fig. 3a).

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Fig. 3

(a) Electronic absorbance selectivity spectra of RS (2.5 μM) with 10 equivalents of other relevant metal ions in CH₃CN:H₂O (9:1 v/v) mixture, (b) absorption spectral sensitivity analysis of RS (2.5 μM) with incremental additions of the Fe³⁺ ions in CH₃CN:H₂O (9:1 v/v) mixture, (c) Job’s plot analysis of Fe³⁺ ions with respect to mole-fraction of the RS in absorbance spectroscopy.

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The cation-sensing mechanism of RS is based on the change in its structure between spirocyclic locked-form to ring-opened form due to the chelation and lewis acidity of metal ions, which leads to the appearance of intense pink colour as shown in Scheme 2. The chemosensor RS is colourless and non-fluorescent in aqueous CH₃CN solution. While, upon addition of Cu²⁺, leads its chelation with chemosensor RS may simultaneously open the spirolactam ring and convert the RS into its ring-opened form along with a remarkable colour change of experimental solution. Such changes enhanced the electronic absorption responses at 521 nm performed as substantial colorimetrically off/on sensing probe. Hence, the addition of Fe³⁺ ions enhanced the chemosensor RS absorbance at 300 and 350 nm, but such spectral changes were lacking in the presence of Cu²⁺ ions. Instead, decrement of RS absorbance along with a rigid band was observed at 401 nm due to ligand to metal charge transfer.

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Scheme 2

Proposed sensing mechanism of the chemosensor RS and its Cu²⁺/Fe³⁺ complexes.

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These dissimilar observations of Cu²⁺ and Fe³⁺ ions may due to their lewis acidic nature and chelation effect. Eventhough Cu²⁺ ions are paramagnetic in nature, it is highly unstable consequently more reactive may due to its d⁹ system, while Fe³⁺ ions have five unpaired electrons and highly stable due to its half-filled d⁵ electronic state. Hence, Fe³⁺ ion has more lewis nature than the Cu²⁺ ions, moreover, Cu²⁺ ion has a great tendency to involved in chelation with ligand/probe than Fe³⁺ ions. Therefore, Fe³⁺ ions could be able to open the spiro-lactam ring very quickly, later it interacts with the donor site of the chemosensor RS formed week complex, instead Cu²⁺ ions are rapidly involved in chelation leads to complexation, simultaniously this complex may lead to open the lactam ring. Due to this rapid formation of chelation with chemosensor RS, the absorption bands at 300 and 350 nm responsible for RS underwent decrement in its intensity, and appearance of new band at 401 nm may occurred due to Ligand to Metal Charge Transfer (LMCT).

Sensitivity titrations were performed to find out the sensing ability of RS towards Fe³⁺ ions. Upon the incremental additions of Fe³⁺ ions (0–30 μM) to RS solution a gradual generation of a new band at 529 nm and linear absorption enhancements at 300 and 350 nm along with a small blue shift were observed (Fig. 3b) until the concentration range of 5–12.5 μM (Fig. S7). Further addition of Fe³⁺ ions leads to their saturation, indicating that the locked spirolactam has been opened which is reliable with that of fluorescence spectra, and demonstrating RS can aid as a “naked-eye” chemosensor for Fe³⁺ ions in aqueous CH₃CN solution. In addition, the detection limit for Fe³⁺ ions was found to be 3.1 nM with a binding constant of 5.2 × 10⁻² M⁻¹, which was determined by B-H equation (Fig. S8). The stoichiometry between RS and Fe³⁺ ions were determined by Jobs plot method, it clearly indicates that the probe will interact with Fe³⁺ ions in 1:1 binding fashion (Fig. 3c).

The addition of Cu²⁺ ions with RS solution, a gradual appearance of band at 521 nm along with three enhancements at 332, 270, and 401 nm was observed. In addition, gradual decrease in absorbance at 300 and 350 nm were also observed along with four isosbestic points at 374, 351, 321, and 290 nm, which leads to a visible colour change from colourless RS to intense pink which may be due to the attained equilibrium between free copper ions and in-situ formed RS-Cu complex at corresponding wavelengths (Fig. 4a). Such spectral changes were not observed for Fe³⁺ ions, due to its spirolactam ring opening behaviour rather than stable complex formation as like RS-Cu²⁺. Sensitivity experiment showed a good linear relationship up to the concentration of 55 × 10⁻⁷ M (Fig. S9) which was used to find out the lowest detection limit in aqueous CH₃CN (1:1, v/v) of 263 pM and binding constant of 1.1 × 10⁻² M⁻¹ (Fig. S10). Job’s plot has exposed a maximum absorbance at 521 nm with 0.5 mol fraction of the RS (Fig. 4b). The stoichiometry of the Fe³⁺ and Cu²⁺ ions has been confirmed by the ESI-MS spectrometer. It shows that the m/z value of 863.6 (M + Na) and 847.10 (M + 1) for Fe³⁺ and Cu²⁺ ions respectively (Fig. S11,S12).

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Fig. 4

(a) Absorption spectral sensitivity analysis of RS (2.5 μM) with incremental additions of the Cu²⁺ ions in CH₃CN:H₂O (9:1 v/v) mixture, (b) Job’s plot analysis of Cu²⁺ ions with respect to mole-fraction of RS in absorbance spectroscopy.

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3.5 Interference experiment

The competitive experiments have been carried out between 10 equivalents of Cu²⁺/Fe³⁺ ions and 15 equivalents of other relevant metal ions towards RS. The observed results showed that the chemosensor RS could be able to selectively recognize Fe³⁺/Cu²⁺ ion couple rather than other metal ions (Fig. 5a and b). The competition between Cu²⁺ and Fe³⁺ ions towards RS was also analysed using electronic absorption and fluorescence spectroscopic techniques. In the presence of both Fe³⁺/Cu²⁺ metal ions, chemosensor has expressed its great tendency of attraction towards Fe³⁺ ions than Cu²⁺ ions, due to the quick spectrolactam ring cleavage existing in RS by Fe³⁺ ions which leads to more resonance of xanthyl moiety rather than the stable chelation of Cu²⁺ ions, which is also agreed with the aforementioned greater binding constant value of Fe³⁺ ions than Cu²⁺ ions. The observed fluorescence spectral changes showed only partial quenching during the addition of Cu²⁺ ions, while absorbance was enhanced, which may be due to scavenging of the excited state electron of iron complex by copper ions (Fig. 5c). This crucial process can only be detected in fluorescence spectroscopy due to its higher sensitivity, while not in the absorbance spectroscopy. This partial quenching effect could be reduced by the addition of cysteine. Upon addition of cysteine, leads the formation of cysteine copper complex initially (Yang et al., 2019; Lee et al., 2015; Senthil Murugan et al., 2020) and it creates free environment around Cu²⁺ ions, it causes for a significant enhancement in the fluorescence of iron complex (Fig. S13). Moreover, this competitive experiment has also been done in ESI-MS spectroscopy. The obtained m/z value of 845.4 and 863.9 which is attributed to the Cu²⁺ and Fe³⁺ complexes respectively and the peak for the copper complex has less intensity than the iron complex, these observations concluded that the chemosensor has more affinity towards Fe³⁺ than the Cu²⁺ ions (Fig. S14).

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Fig. 5

Absorption spectral interference analysis of RS with 10 equivalents of Fe³⁺ in the presence of 15 equivalents of other metal ions, (b) Absorption spectral interference analysis of RS with 10 equivalents of Cu²⁺ in the presence of 15 equivalents of other metal ions, (c) Fluorescence spectral interference analysis of RS with 10 equivalents of Fe³⁺ in the presence of 15 equivalents of other metal ions.

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3.6 NMR studies

NMR spectroscopy is also used to understand the binding nature of metal ions with chemosensor RS. For the NMR studies, 8 mg of the RS in 0.4 mL of deuterated DMSO was treated with one equivalent of each metal ion (Fe³⁺/Cu²⁺) individually in 0.1 mL of deuterated water. Frist, we performed RS alone and then metal ions were added to the RS solution in individual NMR tubes and shaken well before the each experiment. The chemosensor RS showed two singlet peaks at deshield region which is attributed to phenolic —OH of salicylaldehyde (11.87 ppm) and azomethine (8.68 ppm) proton of imine bond (Fig. 6a). The addition of one equivalent Cu²⁺ ions on RS, its existing peak at 11.87 ppm was utterly vanished along with shielded imine peak (8.65 ppm) were observed (Fig. 6b). On the other hand, addition of one equivalent Fe³⁺ ion, the peak at 11.87 ppm became broaden with a decrement in its intensity and a slight deshielding (11.90 ppm) asobserved. This study obviously indicates that the Cu²⁺ ions are involved in deprotonation of phenolic –OH due to its strong chelation through oxygen and imine nitrogen atoms present in the chemosensor. While addition of Fe³⁺ ions, does not deprotonate its phenolic —OH, however it interacts strongly with imine peak leads to deshielding which means the double bond character of the C⚌N has reduced, as a consequence the spirolactam ring has been opened (Fig. 6c). These studies clearly show that the metal ions are involved in typical chelation interaction rather than acid hydrolysis.

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Fig. 6

H¹-NMR spectra of the probe RS (a) with one equivalent of copper ions (b) one equivalent of copper ions and (c) one equivalent of iron ions.

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3.7 Theoretical studies

To comprehend the ICT mechanism underlying the experimentally observed photophysical properties of RS upon its spirolactam ring open due to Fe³⁺ and chelation with Cu²⁺, we had explored the structural, electronic, and optical properties using Gaussian 09 package. Here we have derived HOMO, LUMO, and LUMO+1 of the chemosensor RS and its complexes (Cu²⁺/Fe³⁺) as shown in Fig. 7. The energy difference between the HOMO and LUMO/LUMO + 1 for RS are 3.69 and 3.73 eV respectively. This energy gap has been significantly quenched by complex formation with Cu²⁺/Fe³⁺ ions. The Cu²⁺-RS complex showed 2.43 eV for HOMO-LUMO and 2.84 eV for HOMO-LUMO + 1, while the Fe³⁺-RS has shown 1.35 eV for HOMO-LUMO and 2.40 eV for HOMO-LUMO + 1. Such energy difference clearly confirms the occurred strong interaction between RS and Fe³⁺/Cu²⁺ cations. The existing xanthyl moiety in RS acts as HOMO, and the electrons were distributed over the whole Schiff’s base moiety at LUMO and LUMO + 1. It illustrated that the chemosensor has an intra charge transfer process while the lactam ring is closed. Cu²⁺-RS complex has more electron cloud on the copper centre in HOMO, indicating that the copper ions are strongly bound with RS at the ground state level. Since the spiro-lactam ring has opened during the course of addition of Fe³⁺ ions, the electrons were moved towards the xanthyl moiety at LUMO leads to appear as strong pink colour, which could be observed even by the naked eye. This obtained HOMO-LUMO energy difference in DFT calculation was observed at 510 nm, which is closely agreed to the early obtained absorbance (521 nm) of RS by the addition of Cu²⁺ ions in the electronic spectroscopic studies.

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Fig. 7

DFT analysis of the chemosensor RS, RS-Cu and RS-Fe (left to right).

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More interestingly, RS-Fe complex showed a very low energy difference between HOMO and LUMO than RS-Cu complex, which may be due to the formation of excited state RS-Fe complex like LUMO + 1 rather than at ground state, as a consequence a significant enhancement was observed in the fluorescence. RS-Fe complex also has electron densities on xanthyl moiety at HOMO, and on Schiff’s base portion at LUMO and LUMO + 1, respectively. However, the electron transition may occur from HOMO to LUMO + 1 at 516 nm and is practically firmed the absorbance (529 nm) observed in the UV–vis spectroscopy. Here, the existing dihalo substituted salicylaldehyde group in this chemosensor RS is having the electron deficiency on the Schiff base moiety. Due to this, the imine nitrogen carries a slightly positive charge and it has pulled the electron from the xanthyl moiety. Thus, Schiff base part acts as an acceptor and xanthyl moiety as a donor site. Based on the aforementioned observation, the chemosensor RS showed a clear ICT from xanthyl group to Schiff’s base moiety, while this ICT process has been arrested in the presence of copper ions due to Cu²⁺ ions are involved in strong chelation at the ground state so that imine nitrogen has provided electron to the Cu²⁺ ions and its plugged in the whole electron towards itself. This happened only at the ground state HOMO level whereas the excited state has more electron density on the xanthyl moiety because the Cu²⁺ ions only interact at ground state and are not disturbing the excited state electron distribution. In addition, its counterpart (iron complex, RS-Fe) has a very low energy gap between HOMO and LUMO (than the RS and its copper complex), illustrated that the Fe³⁺ ions induced the fluorescence of the xanthyl group rather than the formation of ground state complex. Based on theoretical and spectral studies, the mechanism has been proposed in Scheme 2.

3.8 Reversibility and stability

Reversibility is one of the significant criteria for chemosensor. Henceforth, we performed reversibility experiments between RS and EDTA, and it showed good reversibility with EDTA complexes up to five cycles. As we mentioned earlier, the colourless RS did not show any absorbance in the range of 500–600 nm, while the addition of Cu²⁺/Fe³⁺ ions generates new peaks at their respective characteristic wavelengths. The subsequent additions of EDTA leads to de-metalation of individual (Cu²⁺-RS and Fe³⁺-RS) complexes and retained the absorbance and fluorescence of probe RS alone. These observations clearly indicate that RS could potentially be able to sense Fe³⁺/Cu²⁺ ions and also has tendency to reproduce RS by the addition of EDTA (Fig. S15-S17).

In general, the light-sensitive rhodamine in solution state, which exhibited excellent colour and fluorescent changes due to its spirolactam ring induced to open by photons. Hence, it is also essential to find out the stability of RS in solution state. In order to carry out this, we prepared 10⁻³ M DMSO solution of RS as a stock solution and examined its stability by using absorbance spectroscopy. The required dilution has been done with this stock solution suitable for the present sensor system and recorded the absorbance spectra. Once every 3 days, we have monitored its absorbance and observed that the chemosensor has shown excellent stability for up to 30 days (Fig. S18).

3.9 pH studies

The absorbance and fluorescence spectra were recorded for RS and with Cu²⁺/Fe³⁺ metal ions at various pH (2–12) gradients. Fig. 8 shows that the probe has opened the ring at higher pH on higher absorbance and fluorescence. This may due to the protonation of lactam carbonyl group, which triggered the opening of the spirolactam ring. The absorbance and fluorescence of the chemosensor RS were inactive at pH of greater than 5. This clearly notified that the spirolactam ring has been induced to open only at acidic pH gradients of 2 to 4, it sustained its closed form at the higher pH (>5). On the other hand, the sensing of the Cu²⁺ and Fe³⁺ also performed at the same pH range. Interestingly, RS is more active at the biological pH (5–7) with both metal ions. Moreover. Fe³⁺ ions lead to a small dwindle in both absorbance and fluorescence at higher pH (8–12) gradients, due to the possible interaction between trimethylamine (base) and Fe³⁺ ions, consequently the lewis acidic character of Fe³⁺ ions gets decrease.

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Fig. 8

(a) pH studies of RS (2.5 µM) with 10 equiv. of Cu²⁺ and Fe³⁺ ions in various pH solutions in absorbance spectroscopy and (b) pH studies of RS (5 µM) with 10 equiv. of Fe³⁺ ions in various pH solutions in fluorescence spectroscopy at excitation wavelength of 375 nm. the absorbance were taken at 521 and 529 nm for Cu²⁺/Fe³⁺ respectively and emission at 554 nm were taken for the fluorescence spectroscopy.

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3.10 Live cell imaging studies on zebra fish embryos

To investigate the applicability of this chemosensor RS with excellent sensing properties by fluorescence microscopic imaging studies were conducted by incubating Zebrafish embryos with Fe³⁺ ions. Since zebrafish embryos have thin skin which could easily convey its fluorescence, hence researchers mostly have chosen zebrafish embryos as a prominent animal model vertebrate for in-vivo live-cell imaging applications. In addition, recent reports reveal that these zebrafish embryos canbe used as a model for various human diseases (Zon, 1999). Initially, the toxicity of the proposed chemosensor RS and Fe³⁺ ions on zebrafish embryos was examined for four days, and then the mortality rate of the zebrafish embryos was monitored. It exposed that the RS and Fe³⁺ ions showed very minor toxicity towards them (Fig. S19). This lower mortality result induced us to carry out the in-vivo imaging studies. There were two sets of embryos were treated with Fe³⁺ ions and chemosensor RS, then monitored under fluorescence microscopy. As we mentioned earlier, the non-fluorescent RS did not show any significant response in spectroscopic technique as well as in zebrafish embryos. Fig. 9a, b and c refers to bright, fluorescence and merge field images of the RS alone. While the embryos were pre-treated with Fe³⁺ ions exhibited an excellent yellow fluorescence with chemosensor RS. Fig. 9e clearly shows that most of the Fe³⁺ ions are accumulated at the abdomen part of the embryos and exhibit a bright fluorescence with RS. These observed results exactly revealed that the RS could be efficiently utilized for the detection of the Fe³⁺ ions in biological species.

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Fig. 9

Fluorescence microscopic imaging of RS (a) bright field (b) fluorescence image of chemosensor RS and (c) merged field images of bright and fluorescence field (d) bright field (e) fluorescence and (f) merged field images of Fe³⁺ ions and RS.

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3.11 Molecular logic gates

Lately, researchers are infatuated to develop molecular logic gates by using the supra-molecular system. That will be achieved mainly with a system consisting of chemically encoded information as inputs and its emission or absorbance properties as output. As the previous observations illustrated that this chemosensor RS could be able to recognize Fe³⁺/Cu²⁺ ions via two different analytical modes (UV–visible and fluorescence methods) with different wavelengths, it can also be utilized as a molecular-scale logic device. In the absence of chemical inputs (Fe³⁺/Cu²⁺), RS did not show any band in the range of 500–600 nm in electronic absorption and fluorescence spectral techniques. The addition of 1 equivalent of Cu²⁺ or Fe³⁺ions generates new bands at 521 and 529 nm in the electronic absorption spectroscopy. Likewise, a new fluorescent band was also observed at 554 nm in the presence of Fe³⁺ions. If it is active in the electronic absorption and fluorescence spectra, it is denoted as 1 and at the inactive stage, it is denoted as 0. In the same way, it is denoted as 1 in the presence of analytes and 0 in their absence. Based on the absorbance and emission spectroscopic observations, the truth table (Table 1) could be constructed and a new molecular logic circuit is developed, which exactly mimics the present sensor system (Fig. 10).

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Fig. 10

Molecular logic circuit for the chemosensor RS.

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3.12 Comparison with recent reports

Since the existing few literature reports as dual sensors of two paramagnetic metals using different analytical tool, have several demerits such as sensitivity, limit of detection and having some interference with others, etc (Ding et al., 2016; Tang et al., 2018; Tao et al., 2017; Bhowmick et al., 2017; Zhu et al., 2019; Park et al., 2018; Huang et al., 2018; Rao et al., 2019; Qiao et al., 2018; Jo et al., 2017). In order to insist on the improvement of quality and novelty of our work, here we have compared with some recent reports. The simple and facile synthesized our chemosensor RS could be able to detect similar kind of two different analytes (cations) in different wavelengths for the first time ever. In addition, we have achieved ultra lowlevel sensitivity towards Fe³⁺ and Cu²⁺ ions. Though some probes existing only with rhodamine moiety were reported, such kind of distinguished detection of two paramagnetic cations by a single chemosensor is infrequent. Moreover, such previous reports did not illustrate their several applicabilities in biological aspects (Table S1). Since our chemosensor, RS is the novel one; it may be applicable as a potential sensing device for detecting Fe³⁺ and Cu²⁺ metal ions in the future.