Polymer-based biocompatible fluorescent sensor for nano-molar detection of Zn²⁺ in aqueous medium and biological samples

Abstract

Chemosensors are prepared through condensation reactions between polymeric amines and three different aldehydes; the activities of the three products were evaluated in DMF–H₂O (7 : 3, v/v) solvent system. Chemosensor 2c demonstrated selectivity for Zn²⁺ enhancement of the fluorescence intensity at 420 nm. Upon binding of 2c with Zn²⁺, an approximately 9 fold enhancement in fluorescence intensity was observed due to the combined effect of ESIPT, C=N isomerisation and the two-channel quenching of PET. The investigation of the photophysical properties of 2c revealed that the sensor at low pH led to a shift in the emission profile because of the presence of two types of nitrogen i.e. sp² and sp³ nitrogens and the pH dependent fluorescence profile is due to the protonation at sp³ hybridised nitrogen that influences the charge transfer transitions of 2c. However, the salt effect has not much influence on the photophysical properties of 2c; and 2c was found to form organic nano-particles (ONP) in a DMF–H₂O (1 : 99, v/v) solvent system and this material was found to detect a minimum of 40 nM concentration of Zn²⁺. The receptor 2c was found to be biocompatible through MTT assay and potent to detect Zn²⁺ in real biological conditions of microbial cells. The compounds 2a, 2b and 2c while immobilized in PVC based membrane, exhibited their selectivity for zinc ions as ion selective electrodes. These electrodes responded linearly in a wide concentration range with a lower detection limit of 10 μM. The electrodes have been successfully used as indicator electrodes in potentiometric titration of zinc ions with EDTA.

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Introduction

The selective recognition of trace levels of transition metal ions is an in demand and developing research area.¹ These metal ions play important roles in biology and environment.² The design, synthesis and development of metal chemosensors with high sensitivity, selectivity, applicability in aqueous or semi-aqueous medium is highly desirable.³ Zn²⁺ is the second most abundant transition metal ion in the human body and its concentration varies from sub μM to mM.⁴ For proper physiological functions, it is vital to maintain Zn²⁺ homeostasis; which is maintained by the import/export between extracellular space and cell organelles.⁵ Hence, to understand the physiological function; it is crucial to monitor the Zn²⁺ levels in living cells and body fluids. Numerous scientific endeavours have been focused on the development of chemosensors for in vitro and in vivo detection of Zn²⁺.^6,7 However, for metal ion detection in body fluids and in the cellular environment, the chemosensor must be able to operate in an aqueous/semi-aqueous medium;⁸ while most of the reported Zn²⁺ sensors have poor water solubility and are susceptible to the interference of other metal ions.⁹ This poor solubility is due to the presence of hydrophobic moieties in the design of the chemosensor such as anthracene,^9a,b naphthalimide,^9c,d pyrene^9fetc. Contrary to these types of chemosensors, the binding units constituted by the polyamine¹⁰ scaffolds may offer good solubility in aqueous medium and the polyamine derivatives can form stable metal chelates.¹¹ However, the selective binding and sensing of metal cations can be achieved by functionalization of polyamines with fluorescent/chromogenic moieties. For cellular applications, the biocompatibility of the sensor for living cells is another important concern. In a continuation of our interest in sensor development,¹² the present manuscript is focused on developing a water soluble, biocompatible, selective, sensitive sensor with direct cellular applications. The polyamine (1) on condensation with different aldehydes gave substituted polyamines 2a–c, which were evaluated as fluorescent chemosensors; secondly these compounds are used in an ion selective electrode (ISE). Generally, ISEs have the problems of receptor leaching, short lifetimes and suffer from interference by species with chemical behaviour similar to primary ions like Cd²⁺ and Hg²⁺ interfering with a Zn²⁺ selective electrode.¹³ The advantage of receptors made up of polymeric scaffolds may help to stop the leaching process with retained properties of chemical sensing with a longer lifetime.

Results and discussion

The present investigation deals with the development of solution phase and matrix bound (ISE) fluorescent chemosensors for analysis of Zn²⁺ in neutral aqueous/semi-aqueous medium. It is well documented that the imine linkages provide effective binding sites for Zn²⁺.¹⁴ We envisioned that the imine linked receptors appended on the polyamine backbone will improve the (semi)aqueous solubility of the receptor to afford a selective, sensitive fluorescence response to Zn²⁺. Further, this interaction will help in the determination of Zn²⁺ concentration using the polymer appended with an ionophore as an electroactive component in the ISE as well.

The synthesis of compounds 2a–c is detailed in Scheme 1 and these compounds were synthesized by the condensation reaction between the polymeric polyamine 1 and the corresponding aldehydes. The resultant imine linked product was characterized by spectroscopic methods i.e. a singlet for –CH=N– in the ¹H NMR spectra at 8.43, 8.96 and 7.84 ppm and a characteristic band for –CH=N– in the IR spectra at ν_max: 1629, 1664 and 1611 cm⁻¹ corresponding to the structures of 2a, 2b and 2c respectively (Fig. S1–9†). A 10 μM concentration of 2a–c in HEPES buffered (10 mM, pH = 7.0) DMF–H₂O (7 : 3, v/v) solvent system exhibits absorption bands between 310–425 nm (Fig. S10†); these bands are assigned to charge transfer transitions due to imine linkages.¹⁵ The fluorescence spectra recorded with 10 μM concentration of 2a–c in HEPES buffered (10 mM, pH = 7.0) DMF–H₂O (7 : 3, v/v) solvent system have unique fluorescence signatures for each compound (Fig. 1). The probe 2a exhibits dual channel emission with weak emission at λ_max = 345 nm and moderate emission at λ_max = 480 nm. The receptor 2a exists in keto and enol tautomers through an excited state intramolecular proton transfer (ESIPT) mechanism.¹⁶ The emission at lower wavelength is due to the enol form; while the keto form emits at a higher wavelength. The fluorescence signature of 2b was recorded under the same conditions as that used for 2a and appears as an intense fluorescence emission band at λ_max = 350 nm. The receptor 2b is unable to exhibit the ESIPT mechanism involving keto–enol tautomerism; this may be due to geometrical restrictions. The fluorescence profile of receptor 2c depicted the fluorescence spectra with two equal intensity emission bands at λ_max = 416 and 480 nm. The emission bands are explained on the basis of the ESIPT mechanism through keto–enol tautomerism involving the –OH and sp² nitrogen of the imine linkage. The longer wavelength band found at 480 nm is because of the keto form and the fluorescence emission band observed at 416 nm is attributed to fluorescence emission from the excited enol state. The compounds 2a–c differ only in the substitution at the end group; otherwise every compound is fabricated with –CH=N– and –OH groups. To gain more insights about the modulation of the fluorescence signatures of 2a–c due to structural features, DFT calculations were performed using Becke's three parameterized Lee–Yang–Parr (B3LYP) exchange functional with 6-31G* basis sets, on Gaussian-09 programs¹⁷ and fragments of each receptor were optimised by taking a small segment of the polymer (Fig. S22†). The fully optimised structures of fragments of receptors 2a–c indicate the energy equivalence for the enol and keto states (Table S1†). The enol form of all the receptors has approximately the same type of symmetry to show H-bonding between –OH groups and the –sp² hybridised nitrogens. Therefore, both the tautomeric forms should exist in equilibrium and the receptors 2a–c may exhibit the ESIPT phenomenon. DFT calculations predict the keto form of each receptor to be energetically more stable than the enol form. Since we could optimize a part of each of the receptors 2a–c, the practical inability of 2b to offer ESIPT seems to be the consequence of some structural features of the polymer backbone. All the receptors (2a–c) bear the proton acceptor site i.e. sp² hybridised nitrogen and also bear an –OH, ionisable moiety under basic conditions. Thus the fluorescence properties of these compounds are expected to modulate with the change in the pH of the solution. Compound 2a was dissolved readily in DMF–H₂O (7 : 3, v/v) and fluoresces with λ_max at 345 nm and 480 nm under neutral conditions; however the lowering in pH from 8.96 to 2.0 led to an enhancement in the fluorescence intensity by two times (Fig. S12A†). The phenomenon can be explained as: the receptor under neutral conditions may undergo cis–trans isomerism at the –C=N– linkage and thus is under the influence of non-radiative decay.¹⁸ The protonation of the sp² nitrogen at lower pH restricts the cis–trans isomerism and also retards the non-radiative decay; thus the fluorescence intensity is increased (Fig. S12A†). The increase in pH to 12.0 leads to switch “ON” the fluorescence intensity of 2a due to deprotonation of –OH; which restricts the non-radiative decay due to vibronic coupling in –OH (Fig. S12B†).¹⁹ Under neutral conditions, receptor 2b shows a fluorescence maximum at λ_max = 350 nm. The effect of pH on receptor 2b was studied in DMF–H₂O (7 : 3, v/v) solvent system. A two-fold increase in fluorescence intensity is observed with a decrease in pH from 10.68 to 2.0 (Fig. S13A†). However, with a slight increase in pH from 10.68 to 10.96, a decrease in the fluorescence intensity at 350 nm is observed with the simultaneous appearance of a new band at 440 nm (Fig. S13B†). With a further increase in the pH to 12.0; an abrupt increase in fluorescence of the latter band is observed. Interestingly, the lowering in pH of the solution of 2c from 8.65 to 2.0 leads to a shift in the emission profile (Fig. S14A†). In fact, compound 2c bears two types of nitrogens i.e. sp² and sp³ nitrogens and the pH dependent fluorescence profile seems to be due to the protonation at the sp³ hybridised nitrogen, because a close binding of H⁺ to the fluorophore may lead to affect the ring current and consequently influence the charge transfer positions.²⁰ A comparison of the effect of base on the fluorescence profile of 2a and 2c leads to conclude that relatively 2c is less prone to be influenced under basic conditions due to the different acidity of the –OH group in the two compounds. The perturbation of high ionic strength was ruled out by comparison of the fluorescence spectra of 2a–c (recorded with 10 μM concentration of sensor in DMF–H₂O (7 : 3, v/v) solvent system) with the respective fluorescence spectrum recorded upon addition of 100 equiv. of tetrabutylammonium nitrate under the same concentration of sensor and solvent system.

Scheme 1

Fig. 1 (A) Changes in fluorescence intensity of receptor 2a (10 μM) upon addition of 50 μM of a particular metal nitrate salt in HEPES buffered (10 mM, pH = 7.0) DMF–H₂O (7 : 3, v/v) with λ_ex = 310 nm. (B) Changes in fluorescence intensity of receptor 2b (10 μM) upon addition of 50 μM of a particular metal nitrate salt in HEPES buffered (10 mM, pH = 7.0) DMF–H₂O (7 : 3, v/v) with λ_ex = 309 nm. (C) Changes in fluorescence intensity of receptor 2c (10 μM) upon addition of 50 μM of a particular metal nitrate salt in HEPES buffered (10 mM, pH = 7.0) DMF–H₂O (7 : 3, v/v) with λ_ex = 370 nm.

It was found that the fluorescence profiles of 2a–c are influenced by the change in pH of the solution; thus all the further studies were conducted under HEPES buffered (10 mM, pH = 7.0) DMF–H₂O (7 : 3, v/v) solvent system. To evaluate the extent to which Zn²⁺ will fit in the proposed design concept of 2a; 50 μM of different metal nitrate salts such as (Na⁺, K⁺, Mg²⁺, Ca²⁺, Ba²⁺, Sr²⁺, Al³⁺, Cr³⁺, Mn²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Ag⁺, Pb²⁺, Cd²⁺ and Hg²⁺) were added to HEPES buffered (10 mM, pH = 7.0) DMF–H₂O (7 : 3, v/v) solution containing 10 μM of 2a (Fig. 1A). A significant increase of fluorescence is observed for complex (2a·Zn²⁺) formed upon addition of Zn²⁺ and relatively smaller enhancements were observed upon addition of Cd²⁺, Al³⁺ and Mg²⁺. The binding of Cd²⁺ and Al³⁺ with 2a may not bring much interference in bio-analytical methods due to the scarcity of both these metal ions in biological systems. However, Mg²⁺ seems to be a major interfering cation for zinc estimation in biological systems. The [Zn²⁺] dependent fluorescence response of 2a was observed in HEPES buffered (10 mM, pH = 7.0) DMF–H₂O (7 : 3, v/v) solution and, as per expectations, the enhancement in fluorescence intensity of 2a is directly proportional to [Zn²⁺] within the concentration range of 0–50 μM (Fig. 2). An enhancement factor of 325% is observed when the mole ratio (2a/Zn²⁺) is 1 : 1 and higher [Zn²⁺]_total results in further evident fluorescent change, suggesting the formation of multicavities for the encapsulation of Zn²⁺. The encapsulation of Zn²⁺ by the sensor 2a has engaged the lone pairs of –OH and the sp² nitrogen; thus restricting the electron donating ability of the groups, which results in a decrease in PET efficiency. Besides, Zn²⁺ could be detected up to 10 μM; a concentration found in many biological systems. The association constant (K_a) of Zn²⁺ with 2a is determined by the equation: log[I_max − I]/[I − I_min] = log[cation] − log β and is found to be 2.6 × 10⁴.²¹ The sensor 2a seems to be sensitive for Zn²⁺ along a fairly good concentration range of Zn²⁺ with a detection limit of 60 nM making it a well suited method for bioanalytical applications. Since, for a sensor, selectivity is an important criteria for practical applicability, so competitive metal binding experiments were performed to estimate Zn²⁺ (10 μM) in the presence of any of Na⁺, K⁺, Mg²⁺, Ca²⁺, Ba²⁺, Sr²⁺, Al³⁺, Cr³⁺, Mn²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Ag⁺, Pb²⁺, Cd²⁺ and Hg²⁺ (50 μM) to check 2a as a selective fluorescent sensor for Zn²⁺ (Fig. S23†). The sensor activity of 2a for Zn²⁺ is influenced by the presence of Ca²⁺, Mg²⁺, Cu²⁺, Al³⁺ and Cd²⁺; this interference will limit the application of 2a as a sensor for Zn²⁺. However, when structural features of 2a are compared with those of 2b and 2c, it was envisaged that they may show improved photophysical properties. This can be due to: (a) insertion of bulkier group, i.e., naphthyl (design of 2b) may provide rigidity to the receptor and hence rule out the possibility of a flexible coordination sphere for many metal ions; (b) recently, Prof. A. P. de Silva has reported the “path selective PET” where the molecule needs to possess a minimum two paths for PET.²² These paths are arranged by building a “receptor1–spacer1–fluorophore–spacer2–receptor2” system and this system is realized as a high generation version of the basic “fluorophore–spacer–receptor” format.²³ The advantage of this approach lies in the fact that only strongly binding metal ions switch “OFF” the PET process from two channels and binding of metal ions to one donor group may not be able to block two channels so they are not potent to switch “ON” the fluorescence intensity. The design of 2c is fabricated from the basic design of 2a; however with another PET channel from the sp³ nitrogen. The cation binding properties of 2b (10 μM) are evaluated from the change in its fluorescence intensity upon addition of a particular metal nitrate salt (50 μM) in HEPES buffered (10 mM, pH = 7.0) DMF–H₂O (7 : 3, v/v) solvent system, when excited at 309 nm (Fig. 1B). However, the fluorescence profile of 2b is perturbed by the addition of many of the tested metal ions. Thus receptor 2b is found to be non-selective towards any of the tested metal ions. With the envisaged two paths PET phenomenon in 2c, the metal binding studies were conducted to further validate the concept. The probe 2c (10 μM) was tested against a series of metal ions Na⁺, K⁺, Mg²⁺, Ca²⁺, Ba²⁺, Sr²⁺, Al³⁺, Cr³⁺, Mn²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Ag⁺, Pb²⁺, Cd²⁺ and Hg²⁺ (50 μM) in HEPES buffered (10 mM, pH = 7.0) DMF–H₂O (7 : 3, v/v) solvent system, when excited at 370 nm (Fig. 1C). The primary metal binding studies suggested improvement in binding selectivity towards Zn²⁺ by probe 2c as compared to 2a. None of the other metal ions i.e. Na⁺, K⁺, Mg²⁺, Ca²⁺, Ba²⁺, Sr²⁺, Al³⁺, Cr³⁺, Mn²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Ag⁺, Pb²⁺, Cd²⁺ and Hg²⁺ could show a high enhancement of fluorescence intensity w.r.t. Zn²⁺. This may be due to the strongly fitting capacity of Zn²⁺ in the coordination sphere of the binding pocket laced with –C=N– and –OH groups of pendant arms in the polymeric chain and extra stability being provided by the sp³ nitrogen present on the aromatic ring. The quantum yields for 2c and 2c·Zn²⁺ were found to be 0.21 ± 0.01 and 0.66 ± 0.01 respectively depicting high quantum efficiency for the complex (Table S4†). In order to identify the working range of this polymeric sensor (2c), fluorescence titrations were performed between 2c and Zn²⁺ (Fig. 3). The fluorescence changes in the probe 2c were recorded by adding aliquots of Zn²⁺ and interpreted to be linear in the range 0–60 μM. It has been found that the addition of Zn²⁺ from nM to μM led to a tremendous increase in the fluorescence intensity of the probe i.e. 9 fold with binding constant of 2.7 × 10⁴. The minimum concentration at which polymeric sensor 2c can detect the presence of Zn²⁺ in the given media has been found to be 40 nM. The addition of EDTA quenches the fluorescence of the receptor 2c·Zn²⁺ complex (Fig. S15†), making receptor 2c a reversible chemosensor. The kinetics of the quenching of the fluorescence intensity due to the 2c·Zn²⁺ complex (20 μM) upon the addition of EDTA (40 μM) was determined by the initial rate method by monitoring the quenching of the band at 418 nm due to the 2c·Zn²⁺ complex as a function of time. A plot of log[A_∞/A_∞ − A_t] versus time was plotted over a time interval of 0–20 min and the value of the rate constant was determined to be 3.90 × 10⁻² min⁻¹ (Fig. S16 and 17†). The competitive metal ion binding studies were conducted to find whether 2c can detect Zn²⁺ selectively even in the presence of other metal ions. To the solution of 2c·Zn²⁺ (10 μM) fixed amounts of different metal ions (50 μM) were added and their fluorescence profiles were studied in HEPES buffered (10 mM, pH = 7.0) DMF–H₂O (7 : 3, v/v) solvent system (Fig. S24†). The data revealed high selectivity of 2c for Zn²⁺ as none of the added metal ions could displace Zn²⁺ from the binding pocket of the polymeric sensor. To investigate the binding behaviour of Zn²⁺ with 2c, the UV-Vis spectrum was recorded. The UV-Vis absorption profile exhibited λ_max at 370 nm, which can be ascribed to intramolecular charge transfer transitions with a clear isosbestic point at 341 nm (Fig. S10C†). In order to understand the role of the polymer backbone, a linear receptor (3) was synthesized by a literature method²⁴ (Scheme S1†) and this material was found to be non-selective for any metal ion (Fig. S25†). Although, 2c and 3 bear similar types of binding sites, this experiment highlights the importance of the polymer backbone to induce selectivity in the sensor system. The effect of pH on the binding affinity of 2c for Zn²⁺ was also recognised (Fig. S26†). A decrease in pH enhances the fluorescence intensity, which may be due to cancellation of PET channels; however, with an increase in pH we observed the formation of some white precipitates and the analysis of these precipitates revealed the formation of ZnO nanoparticles.

Fig. 2 Fluorescence emission spectra of 2a (10 μM) upon successive addition of Zn²⁺ (0–50 μM) in HEPES buffered (10 mM, pH = 7.0) DMF–H₂O (7 : 3, v/v).

Fig. 3 Fluorescence emission spectra of 2c (10 μM) upon successive addition of Zn²⁺ (0–50 μM) in HEPES buffered (10 mM, pH = 7.0) DMF–H₂O (7 : 3, v/v).

To evaluate the maximum water content up to which the sensor can exhibit its activities, the photophysical properties were evaluated in different solvent combinations of DMF–H₂O and it was found that an excess of water fraction (i.e. 99% of H₂O in DMF–H₂O combination) led to the formation of organic nano-particles (ONP) of 2c (Fig. 4A). Thus it was decided to evaluate Zn²⁺ recognition studies with a solvent system of DMF–H₂O (1 : 99, v/v). The complexation of 2c(ONP) with Zn²⁺ triggered fluorescence with an emission band at 418 nm for 2c(ONP)·Zn²⁺. Complete addition of 6 equiv. of Zn²⁺ to the 10 μM solution of 2c(ONP) leads to a significant enhancement of fluorescent intensity of 2c(ONP) (13 times).

Fig. 4 (A) Particle size analysis of organic nano-particles (ONP) of 2c as determined with DLS. (B) Fluorescence emission spectra of 2c (10 μM) upon successive additions of Zn²⁺ (0–60 μM) in HEPES buffered (10 mM, pH = 7.0) DMF–H₂O (1 : 99, v/v).

The cytotoxicity of 2c was determined using the MTT assay with HeLa cells cultured in nutrient mixture. The cells were incubated at 37 °C with 5% CO₂ environment for 24 h. Only marginal cell death (∼95% cell survival) was caused by 2c when the experiments were performed at probe concentration, which is used for recognition studies (10 μM). To investigate the potential cytotoxic effects of 2c at higher concentrations, the dose dependent culture studies were performed. This is important because sometimes higher concentrations of probe may be needed for certain biological applications. The results indicate that 2c is not very toxic to HeLa cells if probe concentrations remain below 18 μM, i.e. with ∼85% cell survival over 24 h for 18 μM concentration of 2c. At much higher concentrations of 2c, the probe becomes more toxic. For example, the IC₅₀ value was found to be 43 μM over 24 h (Fig. S17†). To explore the real application of 2c for Zn²⁺ recognition in a real biological system such as microbial cells (Saccharomyces cerevisiae), the microbe cells were cultured separately in normal medium, and also microbes were cultured in a medium enriched with Zn²⁺. The culture content of both cases was divided into two parts and one part of each was treated with probe 2c (incubated for 3 h at 37 °C). The content of all the four cultures was centrifuged and then washed with a DMF–H₂O (7 : 3, v/v) solvent mixture. All the four slides were investigated under a fluorescence microscope and the results are presented in Fig. 5. Thus, in the presence of probe 2c in Zn²⁺ enriched microbe cells, the cells were illuminated under the fluorescence microscope. Furthermore, the level of Zn²⁺ in spiked tap water was estimated and compared with AAS. The analysis results obtained by using 2c and by AAS are summarized in Table S5.†

Fig. 5 Microscopic images of: (A) microbe cells cultured in normal medium; (B) microbe cells cultured in medium enriched with Zn²⁺; (C) microbe cells cultured in normal medium and treated with 2c; (D) microbe cells cultured in medium enriched with Zn²⁺ and treated with 2c. Before performing microscopy, the microbe cells were washed with a DMF–H₂O (7 : 3, v/v) solvent mixture.

The ion-selective electrodes (ISE) were prepared with the development of membranes with different amounts of ionophore and were tested for better response towards Zn²⁺ ions. The membrane with the composition: ionophore : PVC : plasticiser : lipophilic additive in the ratio of 10 : 29 : 56 : 5 gave the best results. The presence of lipophilic additives generally improves the performance characteristics of various cation selective electrodes with the minimisation of ohmic resistance. Experiments were conducted to study the electrode response with different ionophores 2a–c. Potentiometric readings were recorded with the electrode immersed in different concentrations of Zn²⁺ ions in the concentration range of 10⁻⁸ to 10⁻¹ M. The representative plots of electrode potential (E) for the ISE of ionophore 2a against the logarithm of primary ion concentration are shown in Fig. S18.† Slopes of the calibration curves for Zn²⁺ ion using electrodes with ionophores 2a–c are shown in Table S2.† It can be seen (Fig. S18†) that the electrode exhibited a near Nernstian slope in the working range 10⁻⁵ to 10⁻¹ M with the detection limit as 10⁻⁵ M. The effect of interfering ions on the potentiometric response of the electrode was measured by a fixed interference method. Keeping the concentration of the interfering ion fixed at 10⁻³ M, the concentration of the primary ion (Zn²⁺) was varied from 10⁻⁸ M to 10⁻¹ M. Selectivity coefficient values of the Zn²⁺ selective electrodes prepared with ionophores 2a–c for different interfering ions is shown in Table S3.† It can be seen from the values of selectivity coefficients that the electrode has limited interference from a large number of metal ions. Electrode response in aqueous environments with different pH in the range 2–11 was also studied. Adjustment of the desired pH was done by the dropwise addition of HNO₃ (0.1 M) or NaOH (0.1 M) solution. Plots of electrode potentials (E) against pH of the electrode with ionophore 2a are shown in Fig. S19.† Mostly, for a given solution of the Zn²⁺ ion, the electrode response remains stable in the pH range 4–8. At lower pH, electrodes start responding to H⁺ ions and at higher pH, the Zn²⁺ ions are precipitated due to the formation of Zn(OH)₂. The electrodes were used as indicator electrodes for the titration of Zn²⁺ solution against EDTA. The pH of the solution was adjusted to 5–6 with buffer. The representative plots between electrode potentials of electrodes against volume of EDTA used for the titration are shown in (Fig. S20†) which show that with all the ionophores 2a–c sharp end points were obtained corresponding to a 1 : 1 stoichiometry of the Zn²⁺–EDTA complex.

Experimental section

All the starting materials including polymer 1 were purchased from Sigma Aldrich and were used without any further purification. ¹H NMR and ¹³C NMR spectra were recorded in DMSO-d₆ on a Bruker Avance II 400 spectrometer (which operated at 400 MHz for ¹H NMR and at 100 MHz for ¹³C NMR with TMS as internal standard; chemical shifts are expressed in ppm). IR spectra were recorded on a Bruker Tensor 27 spectrometer for the neat samples. The absorption spectra were recorded on a Specord 250 Plus Analytikjena spectrometer spectrophotometer. The fluorescence measurements were performed on a Perkin Elmer L55 Fluorescence spectrophotometer. ISE measurements were carried out using Equiptronics potentiometer (EQ-602) with accuracy ±0.1 mV.

Synthesis of 2a

Compound 2a was prepared by dissolving 1 (650 mg) and salicylaldehyde (488 mg) in dry methanol separately. The two solutions were mixed and the reaction mixture was refluxed with stirring for 6 h. Upon completion of reaction, the semi solid material was separated out. The material was washed with CH₃OH (50 mL) and dried under vacuum. Yield = 80%; IR (ν_max, cm⁻¹): 1629 (–CH=N–); ¹H NMR (400 MHz, DMSO-d₆) δ: 8.43 (brs, CH=N), 7.13–7.35 (m, ArH), 6.69–6.84 (m, ArH), 3.57 (s, CH₂), 3.19–3.24 (m, CH₂), 2.48–2.73 (m, CH₂); ¹³C NMR (100 MHz, DMSO-d₆) δ: 166.1, 160.8, 131.7, 129.2, 118.4, 116.2, 58.5, 56.7, 54.4, 52.6, 49.7, 48.7, 47.2, 41.3.

Synthesis of 2b

To synthesize compound 2b, polymer 1 (650 mg) and 2-hydroxynaphthaldehyde (688 mg) were dissolved separately in dry methanol. The two solutions were then mixed and the reaction mixture was refluxed with stirring for 8 h. Upon completion of reaction, the semi-solid material was separated out. The material was washed with CH₃OH (50 mL) and dried under vacuum. Yield = 86%; IR (ν_max, cm⁻¹): 1664 (–CH=N–); ¹H NMR (400 MHz, DMSO-d₆) δ: 8.95 (brs, CH=N), 7.99 (d, ArH), 7.54–7.72 (m, ArH), 7.26 (d, ArH), 7.23 (d, ArH), 7.09 (t, ArH), 6.70 (s, ArH), 3.21–3.62 (m, CH₂), 2.82 (s, CH₂), 2.52 (s, CH₂); ¹³C NMR (100 MHz, DMSO-d₆) δ: 160.7, 139.8, 134.5, 132.8, 132.7, 131.3, 131.1, 127.8, 123.9, 113.8, 62.4, 62.0, 57.8, 56.4, 54.1, 53.8, 52.3.

Synthesis of 2c

Compound 2c was prepared by dissolving polymer 1 (650 mg) and 4-(diethylamino)salicylaldehyde (772 mg) in dry methanol separately. The two solutions were mixed and the reaction mixture was refluxed with stirring for 8 h. The semi solid material was separated out upon completion of reaction. The material was washed with CH₃OH (50 mL) and dried under vacuum. Yield = 76%; IR (ν_max, cm⁻¹): 1611(–CH=N–); ¹H NMR (400 MHz, DMSO-d₆) δ: 7.84 (brs, CH=N), 6.62–6.75 (m, ArH), 5.81–5.84 (m, ArH), 5.63 (s, ArH), 3.00–3.22 (m, CH₂), 2.76–2.98 (m, CH₂), 2.23–2.45 (m, CH₂); ¹³C NMR (100 MHz, DMSO-d₆) δ: 168.9, 163.6, 156.7, 153.9, 138.6, 134.8, 60.0, 59.0, 57.9, 55.0, 53.7, 52.2, 48.9, 48.8.

Metal recognition studies

The recognition studies were performed at 25 ± 1 °C, and the solutions were shaken for a sufficient time before recording the spectrum to ensure uniformity of solutions. The cation binding ability of receptors 2a–c (10 μM) in a HEPES buffered (10 mM, pH = 7.0) DMF–H₂O (7 : 3, v/v) solvent system was determined by preparing standard solutions of receptors 2a–c (10 μM) along with fixed amounts of a metal nitrate (50 μM). For titrations, volumetric flasks each containing a standard solution of receptor 2a and 2c (10 μM each) along with varied amounts of a zinc nitrate (0–50 μM) for receptor 2a and 2c under HEPES buffered (10 mM, pH = 7.0) DMF–H₂O (7 : 3, v/v) solvent system were used. However under the DMF–H₂O (1 : 99, v/v) solvent system for 2c the Zn²⁺ concentrations were varied up to 60 μM. To evaluate any possible interference due to different cations for the estimation of Zn²⁺, solutions were prepared containing 2a and 2c (10 μM each) and Zn²⁺ (10 μM) along with both with and without other interfering metal ions (50 μM) in DMF–H₂O (7 : 3, v/v) HEPES buffered solution (10 mM, pH = 7.0).

Kinetics studies

The rate constant for the quenching of the fluorescence intensity of the complex was determined by treating a 20 μM solution of the zinc complex of receptor 2c with 40 μM of EDTA at 25 ± 1 °C in HEPES buffered (10 mM, pH = 7.0) DMF–H₂O (7 : 3, v/v) solvent system. The fluorescence spectra of solutions were recorded directly after the addition and successively after regular intervals of 2 min over a period of 20 minutes. The fluorescence intensity at 418 nm was measured as a function of time.

Cytotoxicity of 2c

The cytotoxicity of 2c was determined through an MTT assay, using HeLa cells seeded in a 96-well flat-bottomed microplate in growth medium (100 μL) and incubated at 37 °C under a 5% CO₂ atmosphere for 24 h. The 2c as test compound and blank analysis were performed and 10 μL of MTT in PBS was added to each well. The microplate was incubated at 37 °C under a 5% CO₂ atmosphere for another 3 h. The medium was then removed, and DMSO (100 μL) was added to each well. The absorbance spectrum of each solution was measured at 570 nm. The dose dependent cytotoxicity of 2c was determined using different concentrations of 2c; however, using the same conditions as mentioned above.

Recognition of Zn²⁺ in microbe cells

To investigate the Zn²⁺, the culture medium was taken in culture tubes guarded with plug. For inoculation, the dry microbes (Saccharomyces cerevisiae) were dissolved in water and one drop of this suspension was added to each tube of medium. The cultures were a clear solution (to the naked eye) at the beginning and with the growth of microbes, cloudiness appeared. The culture tubes were marked as A, B, C, D; where tubes A and C contains normal culture medium; while tubes B and D were enriched with Zn²⁺ (0.01 M). All the four tubes were placed in an incubator at 27 °C. After incubation, the content of each tube was centrifuged and washed three times with double distilled water. The content of tubes C and D was treated with sensor 2c and again content was centrifuged and washed with DMF–H₂O (7 : 3, v/v) solvent combination. Finally, the microbes of all the tubes were submitted for fluorescence microscopy.

ISE fabrication with 2a–c

Membranes with each of 2a–c were prepared by mixing poly vinyl chloride (PVC), ortho-nitrophenyl octyl ether (o-NPOE), and sodium tetraphenyl borate (TPB) in appropriate amounts in tetrahydrofuran (THF) as a solvent. The solvent was evaporated slowly and the viscous solution was poured in glass rings placed on glass plates. Solutions were further evaporated slowly at room temperature overnight. The membranes so prepared were removed from the glass plate and fixed to one end of the glass tubes 5 cm in length and 1.8 cm in diameter, flattened on one side by using an epoxy resin as adhesive. Each membrane was equilibrated with Zn²⁺ solution (0.1 M) for about 5 to 6 days by putting Zn²⁺ solution (0.1 M) on both sides of the membrane. After equilibration, the electrodes were washed thoroughly with distilled water to remove excess salt and kept immersed in distilled water when not in use. Membrane electrodes were filled one-fourth with zinc nitrate solution and immersed in a beaker containing the test solution of varying concentrations of zinc. The potentiometric measurements were made with a digital potentiometer having an accuracy of ±0.1 mV. All the measurements were done through using the following cell assembly:

SCE|Zn²⁺ (0.1 M)||Membrane||Zn²⁺ (test solution)|SCE

Buffer solutions were not used to avoid the presence of any unwanted ions that might affect the potential. The effect of interfering ions on the potentiometric response of the electrode was measured in terms of selectivity coefficients (K_A,B) determined by the fixed interference method (FIM). EMF values were measured by changing the activity of the primary ions but the activity of interfering ions remains fixed throughout the experiment. Intersection of the extrapolated linear portions of the curves drawn between EMF and the primary ion concentration was taken as the value of a_A, and is applied in the Nikolsky–Eisenmann equation to find the selectivity coefficient values.

K_A,B = a_A/(a_B)^z_A/z_B

Here, a_A is the activity of the primary ion A and a_B is the activity of interfering ion B. z_A and z_B are charges on the ions A and B, respectively. The concentration of the primary ion was varied from 10⁻⁸ M to 10⁻¹ M while that of the interfering ion was fixed at 10⁻³ M for all the membranes.

Conclusion

In summary, we have developed three polymer based Schiff base fluorescent sensors 2a–c. Receptor 2c is acting as an “on–off” fluorescent chemosensor for the biocompatible, operative in real microbe cell, selective, and sensitive detection of Zn²⁺. On binding with Zn²⁺, a 9 fold enhancement in fluorescence intensity is observed due to the combined effect of ESIPT, C=N isomerisation and the two-channel quenching of PET. The polymeric sensor is able to detect zinc in (1 : 99) DMF–H₂O to a minimum concentration of 40 nM. Receptors 2a, 2b and 2c while immobilized in a PVC based membrane, exhibited their selectivity for Zn²⁺ ions as ion selective electrodes. These electrodes responded linearly in a wide concentration range with a lower detection limit of 10 μM. The electrodes have been successfully used as indicator electrodes in potentiometric titration of Zn²⁺ ions with EDTA.

Acknowledgements

This work was supported with research grant (SR/FT/CS-97/2010(G) from Department of Science and Technology (DST), Government of India.

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Footnote

† Electronic supplementary information (ESI) available: NMR, IR spectra, UV-vis absorption, fluorescence profiles and graphs for the fabrication and operation of ISE based upon 2a–c. See DOI: 10.1039/c3qi00031a

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