Nanomolar Zn(II) sensing and subsequent PPi detection in physiological medium and live cells with a benzothiazole functionalized chemosensor

Abstract

A new fluorescent chemosensor (L₁) exhibits relay recognition of Zn²⁺ and pyrophosphate anion in a mixed buffer solution at a physiological pH. The probe exhibits excellent Zn²⁺ induced turn-on fluorescence, even as low as 1 nM (LOD = 71 ppb). Furthermore, addition of the pyrophosphate ion led to quenching of the fluorescence signal of the L₁–Zn²⁺ ensemble. The sensitive fluorescence behavior of the L₁ rendered a useful probe for in vitro assays of the intracellular Zn²⁺ and PPi ions in a model human cell line.

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Introduction

The development of molecular fluorescence-based chemosensors for metal ions has emerged as an important field of research owing to the central role of the metal in biology, environmental and chemical processes.¹ Fluorescent chemosensors are widely preferred over other optical sensors based on their high sensitivity, ease of handling, robustness and the possibility of real-time monitoring with fast response times.^2,3 Amongst the metal ions, zinc ion sensing in aqueous environments is assuming considerable importance due to the fundamental role of the metal in a plethora of biological processes, such as gene transcription, regulation of metalloenzymes, neural signal transmission and apoptosis.^4–9 Zinc is acknowledged as the second most abundant metal ion in human body after iron and is commonly associated with proteins where it bears structural and catalytic implications.¹⁰ Besides the fixed form of cellular zinc, pools of labile zinc is found in the pancreas,¹¹ central nervous system,¹² prostate,^13,14 intestine^15,16 and retina.¹⁷ Deficiency of zinc ion has been implicated in grave ailments such as acrodermatitis enteropathica, impaired cognition, immune dysfunction and diarrhea, while an excess concentration has been known to cause superficial skin diseases, prostate cancer, diabetes and brain diseases.^18,19 The paucity of knowledge with regard to the physiological implications of cellular zinc homeostasis and the increasing need to unravel the role of Zn²⁺ in disease-related pathways has triggered the development of analytical tools for rapid and sensitive detection of the metal.^20,21 However, the inherent traits of zinc such as absence of any spectroscopic signature as well as neutral magnetism are considered as bottlenecks to apply common analytical techniques for detection. Furthermore, the toxic cadmium ion, which is analogous to zinc in electronic configuration mostly interferes in the detection of zinc ion. Given this predicament, selective, sensitive and rapid detection of zinc ions in complex milieu and in biological samples is in great demand.

In recent times, zinc chemosensing ensembles have emerged as secondary sensors towards various anions,^22–24 small molecule such as biothiols,²⁵ nitro aromatic compounds,^26,27 and amino acids.²⁸ Amongst the anion, sensing of biologically important pyrophosphate (P₂O₇⁴⁻, PPi) is desirable as it plays a germane role in various biological process.^29,30 PPi is the product of ATP hydrolysis under cellular conditions and is a by-product of DNA replication and DNA sequencing.^31,32 Most importantly, the detection of PPi has also been considered to be important in cancer research.³³ Further, PPi detection has become an important issue for rheumatological disorder that originates due to the accumulation of crystals of calcium pyrophosphate dehydrates in the connective tissues.^34–37 The high solvation energy of PPi in water (ΔG° = −584 KJ mol⁻¹)³⁸ and the presence of other competitive anions is a formidable challenge for detection of PPi in aqueous medium using receptors, which are based only on H-bonding interaction. Thus, certain transition metal chemosensor ensembles including Zn²⁺, Cd²⁺ and Cu²⁺, are being deployed to surmount this problem.^39–42 It is envisaged that such ensembles can provide the geometry and suitable orientation of the binding site for the anions, which have an inherent proclivity for binding the positive centred metal ions. Apart from this colorimetric/fluorimetric type sensors, bioluminescence⁴³ electrochemical⁴⁴ and ISFET⁴⁵ based sensors are also known in the literature.

Our research group has a longstanding interest in the design and development of chemosensors for the molecular recognition and sensing of various analytes.^46–49 Based on the aforementioned premise, herein we report the synthesis and photophysical properties of a benzothiazole based dipodal Schiff base chemosensor L₁ (2,6-bis((E)-(2-(benzothiazol-2-yl)hydrazono)methyl)-4-methylphenol).

The benzothiazole unit acts as chelating ligand⁴⁶ and the dipodal Schiff base framework provide efficient binding for metal ion.^48,50–53 The new chemosensor is highly selective towards Zn²⁺ ion with a detectable fluorescence response even with 1 nM Zn²⁺. Furthermore, the chemosensor L₁ rendered Zn²⁺ sensing in the physiological milieu and the resultant L₁–Zn²⁺ ensemble responded specifically to pyrophosphate (PPi) anion through fluorescence quenching.

Experimental section

General information and materials

All the materials used for synthesis were procured from commercial suppliers. The absorption spectra were recorded on a Perkin-Elmer Lamda-750 UV-Vis spectrophotometer using 10 mm path length quartz cuvettes in the range 250–700 nm wavelength, while fluorescence measurements were conducted on a Horiba Fluoromax-4 spectrofluorometer using 10 mm path length quartz cuvettes with a slit width of 3 nm at 298 K. Mass spectra of L₁ was obtained using Waters Q-ToF Premier mass spectrometer. NMR spectra were recorded on a Varian FT-400 MHz instrument as well as on a BRUKER-600 MHz and the chemical shifts were presented in parts per million (ppm) on the scale. The following abbreviations are used to describe spin multiplicities in ¹H NMR spectra: s = singlet; d = doublet; t = triplet; m = multiplet. IR spectra were recorded on a Perkin-Elmer-Spectrum One FT-IR spectrometer with KBr disks in the range 4000–450 cm⁻¹.

Synthesis of L₁

Condensation of freshly prepared 2,6-diformyl-4-methylphenol with 2-hydrazino benzothiazole for 4 hours yielded a yellowish precipitate, which was then filtered and washed with cold methanol to obtain pure L₁ (Scheme 1). % yield = 85. ¹H NMR of L₁ (400 MHz, DMSO-d₆): 12.267 (bs, 1H), 8.484 (s, 2H), 7.727–7.753 (d, 2H), 7.561 (s, 2H), 7.354–7.229 (m, 4H), 7.128–7.091 (t, 2H), 2.319 (s, 3H). ¹³C NMR (600 MHz): 166.58, 153.78, 133.41, 128.43, 126.28, 121.92, 121.66, 120.16, 20.12. ESI-MS (positive mode, m/z) calculated [L₁ + H⁺] = 459.1062, found mass: 459.1042.

Scheme 1 Synthetic scheme of the receptors with the binding sites for guest metals.

Similarly, condensation of 2,6-diformyl-benzene with 2-hydrazino benzothiazole for 4 hours gives gave a yellowish type precipitate of L₂. % yield = 80. ¹H NMR of L₂ (400 MHz, DMSO-d₆): 12.353 (bs, 1H), 8.181 (s, 2H), 7.981 (s, 1H), 7.801–7.603 (m, 4H), 7.528–7.454 (m, 3H), 7.310 (t, 2H), 7.125 (t, 2H). ¹³C NMR: 167.180, 134.94, 129.12, 127.386, 126.059, 124.125, 121.765, 120.94. ESI-MS (positive mode, m/z) calculated [L₂ + H⁺] = 429.1004, found mass: 429.0948.

UV-Vis and fluorescence spectral studies

Stock solutions of various ions (1 × 10⁻³ M) were prepared in deionized water. Perchlorate or nitrate salts were used for metal ions while tetrabutyl/tetraethyl or sodium salts of the corresponding anions and nucleotides were used for the preparation of anion stock solutions. The stock solution of L₁ and L₂ (5 × 10⁻³ M) were prepared in DMSO. For the titration experiments, a 1 × 10⁻³ M solution of L₁ taken in a quartz optical cell of 1 cm optical path length was titrated with incremental concentration of anion stock solutions in an ethanolic buffer medium (1 : 1 v/v EtOH : HEPES buffer at pH 7.2). For the competitive selectivity experiment, fluorescence emission of the L₁–Zn²⁺ ensemble was collected in the absence and presence of the competitive metal ions in excess (10 equivalent).

Evaluation of the apparent binding constant

Probe L₁ at a concentration of 10 μM in buffered ethanol was used for the titration studies with Zn²⁺ solution. The effective Zn²⁺ concentration was varied between 0 and 70 μM and the pH of the solution was adjusted to 7.2 using an aqueous HEPES buffer solution of effective concentration of 10 mM. The apparent binding constant for the formation of the respective zinc complex was evaluated using the Benesi–Hildebrand (B–H) plot (eqn (1)).^54,55

1/(I − I₀) = 1/{K(I_max − I₀)C} + 1/(I_max − I₀) (1)

I₀ is the emission intensity of L₁ at maximum (λ = 510 nm), I is the observed emission intensity at that particular wavelength in the presence of a certain concentration of the analyte (C), I_max is the maximum emission intensity value that was obtained at λ = 510 nm during titration with varying analyte concentration, K is the apparent binding constant (M⁻¹) and was determined from the slope of the linear plot. Similarly, the PPi binding constant was also evaluated.

Detection limit

The detection limit was calculated on the basis of the fluorescence titration. The fluorescence emission spectrum of L₁ was measured 10 times, and the standard deviation of blank measurement was determined. To gain the slope, the ratio of the fluorescence emission at 510 nm was plotted against the concentration of Zn²⁺ or PPi. The detection limit was calculated using the following equation:

Detection limit = 3σ/k (2)

where σ is the standard deviation of blank measurement, and k is the slope between the ratio of fluorescence emission versus respective analyte concentration.

Sensing of Zn(II) in physiological milieu

To investigate the potential of L₁ as a probe for intracellular detection of Zn²⁺, the subsequent endeavour was to study its interaction in milieu replicating the conditions relevant in an in vivo system. To that end, two proteins were chosen, bovine serum albumin (BSA) and human serum albumin (HSA), and the interaction study between L₁ and Zn²⁺ was studied. Stock solutions of the proteins were prepared at a final concentration of 4 mg mL⁻¹. Proteins were then titrated from the stock solution in a cuvette with preformed L₁–Zn²⁺ complex. To further validate the biological importance of L₁, the interaction of L₁ with Zn²⁺ was also studied in simulated body fluid (SBF), whose ionic composition is similar to that of human extracellular fluid. SBF was prepared as reported previously.⁵⁶

Cytotoxic effect of L₁, L₁–Zn²⁺ and L₁–Zn²⁺-PPi ensemble

The cytotoxic effect of L₁ and L₁–Zn²⁺ complex and L₁–Zn²⁺-PPi ensemble on HeLa cells (human cervical carcinoma cells) were determined by an MTT assay as per the manufacturer instruction (Sigma-Aldrich, MO, USA). HeLa cells were initially cultured in a 25 cm² tissue culture flask in DMEM medium supplemented with 10% (v/v) FBS, penicillin (100 μg mL⁻¹) and streptomycin (100 μg mL⁻¹) under a humidified atmosphere of 5% CO₂ until the cells were approximately 90% confluent. Prior to MTT assay, cells were passaged and seeded into 96 well tissue culture plates at a cell density of 10⁴ cells per well and incubated with varying concentrations of L₁, L₁–Zn²⁺ complex, L₁–Zn²⁺-PPi ensemble, Zn²⁺ and PPi solution (15, 30, 60, 90 and 120 μM) and incubated for a period of 24 h under 5% CO₂. Following incubation, the growth media was carefully aspirated, and fresh DMEM containing MTT solution was added to the wells. The plate was incubated for 4 h at 37 °C. Subsequently, the supernatant was collected and the insoluble colored formazan product was solubilized in DMSO and its absorbance was measured in a microtiter plate reader (Infinite M200, TECAN, Switzerland) at 550 nm. The assay was performed in six sets for each concentration of the test samples. Data analysis and determination of standard deviation was performed with Microsoft Excel 2013 (Microsoft Corporation). In the MTT assay, the absorbance for the control cells (solvent control) was considered as 100% cell viability and the absorbance for the treated cells was compared to determine % cell viability with respect to the solvent control.

Detection of intracellular Zn²⁺ and PPi by imaging

HeLa cells were initially cultured in a 25 cm² tissue culture flask containing DMEM medium supplemented with 10% FBS, penicillin (100 μg mL⁻¹) and streptomycin (100 μg mL⁻¹) in a CO₂ incubator. Prior to imaging studies, HeLa cells were seeded into a 6 well plate and grown in DMEM medium at 37 °C till 80% confluency. Subsequently, the cells were washed thrice with sterile phosphate buffered saline (PBS), incubated with 15 μM L₁ in DMEM at 37 °C for 1 h in a CO₂ incubator and their images were acquired using a fluorescence microscope (Eclipse Ti-U, Nikon, USA) with a filter that allowed green light emission. The cells were further washed with sterile PBS in order to remove excess L₁ and then incubated for 1 h with 30 μM Zn(ClO₄)₂ prepared in sterile PBS. The images of the cells were acquired with a fluorescence microscope. The cells were subsequently washed with sterile PBS and then incubated further with 30 μM PPi for 1 h. Following incubation, the images of the cells were again acquired with a fluorescence microscope as mentioned earlier.

Result and discussion

Selective sensing of Zn(II) in aqueous medium by L₁

The photophysical properties of the chemosensor (L₁) was ascertained by UV-visible absorbance and fluorescence emission changes upon addition of various analytes. Absorption spectrum of L₁ (10 μM) in buffered ethanol (1 : 1 EtOH : 10 mM HEPES, pH ∼ 7.2) exhibited an intense band at 333 nm and a weak band around 386 nm, which may be ascribed to intramolecular π–π* transition (Fig. 1a). However, addition of Zn²⁺ ion resulted in the formation of new red shifted absorption peaks at 376 nm and 431 nm with a distinct change in the colour of the solution from colorless to yellowish (Inset, Fig. 1a). Incremental addition of Zn²⁺ ion (0–70 μM) to L₁ resulted in a decrease in intensity at 333 nm accompanied by blue shifting of the 386 nm peak to 377 nm, which emerged with increasing intensity (Fig. 1b). Furthermore, higher Zn²⁺ concentration also lead to the manifestation of a broad peak at 431 nm. The isosbestic points at 297 nm, 355 nm and 414 nm indicated the formation of a new zinc complex with 1 : 1 stoichiometry (Fig. S9†). Interestingly, none of the other tested cations, which included biologically relevant metal ions like Na⁺, Mg²⁺ and Ca²⁺, toxic heavy metal ions like Hg²⁺ and Cd²⁺ affected the absorption spectra of L₁. It may also be mentioned that the absorption spectra of the chemosensor L₁ remained unaffected even in presence of other cations such as K⁺, Cu²⁺, Mn²⁺, Fe²⁺, Co²⁺, Ni²⁺, Ag⁺, Pb²⁺ and Al³⁺ (Fig. 1a).

Fig. 1 (a) Change in absorption spectra of L₁ (10 μM) with different metal ions (50 μM) in ethanol buffer mixed buffered solution. Inset: change in the colour of L₁ solution after the addition of Zn²⁺ and Cd²⁺. (b) UV-Vis titration spectra of L₁ with increasing Zn²⁺ ion concentration.

To acquire a nuanced understanding of the binding mechanism, we have also performed fluorescence emission studies and ¹H NMR titrations in presence of Zn²⁺ ion. The naked probe L₁ (10 μM) was weakly fluorescent at 510 nm perhaps due to rapid rotation around single bond adjacent to azo methine group (λ_ex = 430 nm, slit = 3/3 nm). Interaction with Zn²⁺ resulted in a significant Stokes shift of around 130 nm and a remarkable enhancement of the fluorescence emission peak at around 510 (Fig. 2a). Interaction of L₁ with the other tested metal ions failed to impart any change in the fluorescence emission of the probe (Fig. 2a). Interestingly, under a UV lamp (λ = 365 nm) the solution displayed a strong yellowish green fluorescence (Inset, Fig. 2a) and thus rendered rapid naked eye detection of Zn²⁺. Collectively the results are encouraging as chemosensors, which exhibit large stokes shift and manifold enhancement in fluorescence upon binding of target analyte are envisaged as potential candidates for fabrication of sensing devices that minimize background interference.

Fig. 2 (a) Emission intensities of L₁ (10 μM) in presence of various metal ions in mixed buffered solution. Inset: change in colour of L₁ solution observed under a UV lamp following addition of Zn²⁺. (b) Fluorescence titration of L₁ with varying concentration of Zn²⁺ (0–20 nM) (λ_ex = 430 nm, slit = 3/3 nm).

To explore the binding mechanism, we have titrated a fixed concentration of the probe (10 μM) with varying concentration of Zn²⁺ ion. With increasing concentration of Zn²⁺ there was a systematic enhancement in the fluorescence emission at 510 nm. Following addition of 0.1 equivalent of Zn²⁺ (1.0 μM of Zn²⁺), there was nearly 27 fold increase in the fluorescence intensity of the probe (Fig. S10†). It may also be mentioned that a noticeable enhancement in the fluorescence intensity of L₁ was even observed with 1.0 nM Zn²⁺, which indicated the excellent sensitivity of the probe (Fig. 2b). The binding constant for Zn²⁺ ion was 1.25 × 10⁵ M⁻¹ as calculated by Benesi–Hildebrand equation using the fluorescence titration reading (Fig. S11a†). The calculated lowest detection limit for Zn²⁺ was 71 ppb on the basis of signal : noise = 3 : 1 (Fig. S11b†). In comparison with the reported examples of fluorescent sensors for Zn²⁺ (Table S1 and S2†), we have achieved better detection limit. Again, such level of detection limit bears significant implications to sense Zn²⁺ in biological systems.

Subsequently, interference studies were conducted to ascertain the affinity of L₁ for Zn²⁺ in presence of other competitive metal ions. For this, fluorescence emission spectra were recorded after the addition of different metal ion solution to a mixture of 1 : 1 L₁ and Zn²⁺. Interestingly, the sensing capability of L₁ for Zn²⁺ was not affected in presence of any of the competing species (Fig. S12†).

Plausible mechanism of Zn²⁺ sensing

To gain an insight of the chelation mode of zinc ion to L₁, ¹H NMR titration were conducted. However, due to the poor solubility of the probe in CD₃OD, titrations were performed in DMSO-d₆ (Fig. 3). There are three possible binding sites in L₁ viz. OH, Schiff base N and N of benzothiazole group; and Zn²⁺ coordination through this functionalities are expected to change the nearby electronic environment. Just Following addition of the first aliquot of Zn²⁺ ion, the OH peak was obliterated, which suggested the strong involvement of the OH functionality in the binding event (Fig. 3). Interaction with the Zn²⁺ ion also caused de-shielding of the Schiff base CH(Hb) and the aromatic CHs of the benzothiazole ring.

Fig. 3 ¹H NMR stack plot of L₁ with different concentration of Zn²⁺ in DMSO-d₆ and the plausible Zn²⁺ binding on top.

A control receptor L₂ which was devoid of OH group also revealed the importance of the same, as it failed to produce any selectivity under the same experimental condition (Fig. S13†). Further, the 1 : 1 complex mass (L₁ + Zn²⁺ + NO₃⁻ = 584.0234, Fig. S14†) also suggested the involvement of OH as oxide.

Thus, guided by the phenolic OH, Schiff base N and benzothiazole N are also coordinated to Zn²⁺ ion, and thereby rigidify the molecular assembly by restricting the free rotation of the azomethine carbon, which resulted in significant fluorescent enhancement through the process of chelation-enhanced fluorescence (CHEF).

To test the sensing potential of the developed probe in biological system, it was important to ascertain the performance of the probe in presence of serum proteins such as albumin.⁵⁷ Thus, the Zn²⁺ sensing by L₁ was analyzed in presence of human serum albumin (HSA) and bovine serum albumin (BSA) in mixed solvent system as well as in simulated body fluid (SBF) by tracking the fluorescence emission at 510 nm (Fig. 4). Interestingly, even in presence of higher concentration of HSA, BSA and SBF the fluorescence emission intensity of L₁–Zn²⁺ at 510 nm remained unaffected. These results reiterated the selectivity of the developed probe and demonstrated its application potential in a physiologically relevant milieu.

Fig. 4 Histogram showing the fluorescence response of L₁–Zn²⁺ ensemble towards various concentrations of BSA and HSA in (a) buffered ethanol (1 : 1 EtOH : 10 mM HEPES, pH ∼ 7.2) and (b) simulated body fluid (SBF).

Given that chemosensors can exhibit proton-induced fluorescence, the zinc ion induced ‘turn-on’ fluorescence of L₁ was also checked in a wide pH range. It was observed that L₁ rendered Zn²⁺ sensing in the pH range 5.0 to 11 (Fig. S15†). In the pH range <5.0, there was no fluorescence response to Zn²⁺ which may perhaps be attributed to the weak coordination capability of zinc ions due to the protonation of the co-ordination sites of L₁.

For practical application of the chemosensor, paper test strips were used for facile and rapid qualitative detection of Zn²⁺ ion. The ligand coated test strips were prepared by immersing the filter papers into a DCM solution of L₁ and then air dried. An intense ‘turn-on’ fluorescence could be observed under the UV lamp (λ = 365 nm) on spraying a Zn²⁺ solution over the test strips (Fig. S16†).

Selective PPi sensing by L₁–Zn²⁺ ensemble

The next endeavour was to ascertain the anion sensing aptitude of the L₁–Zn²⁺ ensemble. To this end, the L₁–Zn²⁺ ensemble was treated with various anions such as F⁻, Cl⁻, Br⁻, I⁻, CN⁻, CO₃²⁻, HCO₃⁻, CH₃CO₂⁻ (OAc⁻), NO₃⁻, PPi, SO₄²⁻, PO₄³⁻, H₂PO₄⁻, AMP, ADP and ATP etc. Among the aforesaid anions, only PPi induced a conspicuous change in the emissive behaviour of the ensemble (Fig. 5a). Again, the addition of dNTP (N = A, G, C, T) also produce no change in the emissive behavior of the ensemble. Addition of 1.0 equivalent of PPi (10 μM) solution resulted in complete quenching of the initial fluorescence emission of the L₁–Zn²⁺ ensemble. Titration of the metal ensemble with sequentially added PPi solution lead to a continuous decrease in emission intensity at 510 nm. The ‘turn-off’ fluorescence behaviour of the L₁–Zn²⁺ ensemble could be explained by considering the strong binding affinity of PPi towards Zn²⁺. The sequestration of Zn²⁺ ions by PPi and formation of stable ZnPPi adduct releases the free probe in the solution, which renders the strong emission of the L₁–Zn²⁺ ensemble.

Fig. 5 Change in fluorescence emission upon addition of (a) different anions and (b) increasing concentration of PPi (0–10 μM) to the L₁–Zn²⁺ ensemble ion in mixed buffered solution (λ_ex = 430 nm, slit = 3/3 nm). Inset: change of fluorescence emission intensity of the L₁–Zn²⁺ ensemble at 510 nm with increasing concentrations of PPi.

The UV-Vis spectral pattern further validated our conjecture, as absorption maximum at 380 nm for naked L₁ was regained and the characteristic 431 nm peak for the zinc ensemble diminished after PPi addition (Fig. S17†). Concurrently, the yellowish color solution was also transformed to the original colorless solution. However, neither any visual color change nor any development of new peak around 380 nm was observed in the UV-visible absorbance spectra after addition of any of the anions viz. F⁻, Cl⁻, Br⁻, I⁻, CO₃²⁻, HCO₃⁻, OAc⁻, NO₃⁻, SO₄²⁻, PO₄³⁻, H₂PO₄⁻ even at higher mole ratio. This indicated the high selectivity of the L₁–Zn²⁺ ensemble for PPi ion in the physiological pH. It may also be mentioned that the aforementioned anions could not be detected by the free probe L₁ (Fig. S18†). Thus the L₁–Zn²⁺ ensemble can be employed both as colorimetric as well as fluorometric sensor for PPi.

The Job's plot obtained from the titration experiment suggested a 1 : 1 binding of PPi with metal ensemble and the calculated binding constant was 1.4 × 10⁵ M⁻¹ (B–S equation, Fig. S19†). The LOD of the zinc ensemble for PPi anion was 480 pM (Fig. S20†). To establish the practical applicability of the L₁–Zn²⁺ ensemble as selective PPi sensor, competitive fluorescence experiment was carried out with the competing anions. Addition of PPi caused a prominent fluorescence quenching in every case (Fig. S21†), which suggested excellent selectivity and sensitivity of L₁–Zn²⁺ ensemble towards PPi in the buffer medium even in the presence of other interfering anions.

Detection of Zn²⁺ in live cells

The results obtained from the fluorescence experiments were encouraging and we sought to investigate the potential of L₁ as a probe for detection of intracellular Zn²⁺. However, prior to ligand-mediated zinc detection in live cells, it was important to determine its cytotoxic potential. To that end, an MTT assay was performed. It was observed that that probe alone failed to impart any adverse effect on the viability of HeLa cells at a concentration of 60 μM (Fig. S22†). Even at high concentrations of 90 μM and 120 μM, cell viability was around 80%. Akin to the results obtained with L₁, HeLa cells treated with L₁–Zn²⁺ and L₁–Zn²⁺-PPi ensembles also displayed significant viability, which suggested high biocompatibility of the ensembles.

The non-toxic nature of the developed chemosensor L₁ and its Zn²⁺-ensemble suggested that the probe could perhaps be deployed for fluorescence-based intracellular detection of Zn²⁺ in live cells. Therefore, to ascertain the potential of L₁ in intracellular sensing of Zn²⁺, HeLa cells were incubated with 15 μM L₁ followed by 30 μM of Zn(ClO₄)₂ to promote the formation of intracellular L₁–Zn²⁺ complex. It was observed that L₁ alone failed to elicit any fluorescence signals as evidenced by the absence of any intracellular fluorescence (Fig. 6). However, addition of zinc led to the manifestation of bright green fluorescence in HeLa cells (Fig. 6). The fluorescence microscopic analysis strongly suggested that compound L₁ could readily cross the cell membrane, infuse into HeLa cells, and sense Zn²⁺ through the formation of intracellular L₁–zinc complex. It must be also mentioned that bright field images suggested that HeLa cells retained their characteristic morphology, which reiterated the biocompatibility of L₁ and L₁–Zn²⁺ ensemble (Fig. 6). Interestingly, subsequent incubation of the cells with excess PPi resulted in a dramatic loss of fluorescence (Fig. 6), perhaps originating from fluorescence quenching behavior of PPi on L₁–Zn²⁺ ensemble as evidenced from previous solution-based studies (Fig. 5).

Fig. 6 Fluorescence microscopic images of HeLa cells after adding 15 μM of L₁ (row A) and after subsequent treatment with 30 μM Zn²⁺ (row B) and 30 μM PPi (row C). Scale bar for the images is 100 μm.

Conclusions

A novel benzothiazole containing Schiff base was developed which rendered relay recognition of Zn²⁺ and PPi in physiological medium. The Zn²⁺ sensing was evidenced in mixed buffered medium and also in the physiological milieu in presence of HSA, BSA and SBF. In presence of Zn²⁺, the colorless chemosensor solution displayed a strong greenish fluorescence and thus enabled naked eye detection of the metal. The chemosensor exhibited a strong affinity (K = 1.25 × 10⁵ M⁻¹) for Zn²⁺ even in the presence of the other interfering metal ions and was ultrasensitive as it could respond even to 1 nM Zn²⁺ (LOD for Zn²⁺ = 71 ppb). A facile application of the chemosensor could be demonstrated through successful detection of Zn²⁺ using the sensor-coated paper strips. The Zn²⁺-chemosensor ensemble also responded to PPi in the same experimental medium through fluorescence quenching. On the basis of the selectivity of L₁ for Zn²⁺ in physiological medium and its biocompatible attribute, the probe could facilitate fluorescence-based sensing of intracellular Zn²⁺ in live HeLa cells. It is envisaged that the developed probe holds considerable potential as a Zn²⁺ sensor for future environmental and biomedical applications.

Acknowledgements

The authors thank CSIR (01/2727/13/EMR-II), Science & Engineering Research Board (SR/S1/OC-62/2011) and Department of Biotechnology (BT/01/NE/PS/08), India for financial support and CIF, IIT Guwahati for providing instrument facilities. AG and SM acknowledge IIT Guwahati for research fellowship.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: NMR, UV-Vis and fluorescence changes. See DOI: 10.1039/c5ra09150k

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