Fluorogenic selective detection of Zn²⁺ using a pyrazole-ortho-vanillin conjugate: insights from DFT, molecular docking, bioimaging and anticancer applications

Abstract

A fluorescent sensor, (E)-N′-(2-hydroxy-3-methoxybenzylidene)-3,5-dimethyl-1H-pyrazole-1-carbohydrazide (HMPC), was designed and synthesized for the selective fluorescence recognition of Zn²⁺ in semi-aqueous media. Notably, HMPC exhibited a red-shifted, two-fold fluorescence “turn-on” enhancement in response to Zn²⁺ at 490 nm, with a detection limit of 1.68 μM, which is significantly lower than the WHO guideline (76.0 μM). The binding constant of HMPC with Zn²⁺ was calculated to be 5 × 10⁴ M⁻¹. The fluorescence enhancement of HMPC in the presence of Zn²⁺ is attributed to the suppression of the PET process and the enhancement of ICT, leading to fluorescence via the CHEF mechanism. The sensing mechanism was demonstrated through UV-vis, fluorescence spectroscopy, Job plots, ESI-MS, and DFT calculations. For biological applications, cytotoxicity and cell imaging studies were performed using MCF-7 cells. Molecular docking studies revealed a high binding energy of HMPC (ΔG = −7.1 kcal mol⁻¹) with the 4,5-diaryl isoxazole HSP90 chaperone protein, suggesting its potential as an anticancer agent. Additionally, its binding energy of −6.5 kcal mol⁻¹ with the HDAC8 protein indicates greater efficacy than suberoylanilide hydroxamic acid (SAHA) in inhibiting HDAC, as it binds more strongly to the HDAC8 protein than SAHA (−7.4 kcal mol⁻¹). Furthermore, due to its favorable ADME profile, HMPC may be suitable for oral administration, enhancing its potential as an anticancer drug.

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1. Introduction

A chemosensor is an abiotic molecule that produces a measurable signal when interacting with matter or energy. Fluorescent chemosensors, a common type, emit a fluorescence signal upon binding with an analyte. These systems consist of two components: a fluorophore (signaling unit) and a receptor (recognition unit). Fluorescence occurs when a molecule absorbs photons from UV-visible light, transitioning to a higher energy state, and then emits photons while returning to its ground state, though alternative de-excitation pathways also exist.¹ In 1867, F. Goppelsröder introduced the first fluorescent chemosensor, which was designed to detect aluminum ions (Al³⁺) by forming a highly fluorescent morin chelate. This pioneering work paved the way for the creation of numerous fluorescent chemosensors for detecting various metal ions over the following decades, contributing significantly to the emergence of modern analytical chemistry.² Among the metal ions, zinc ion (Zn²⁺) has garnered significant attention due to its biological importance as the second most abundant transition metal in the human body after iron. Zinc influences a variety of physiological processes, including DNA synthesis,³ apoptosis,^4,5 gene expression,^6,7 neurotransmission,^8–11 and signal transduction.¹² Additionally, Zn²⁺ modulates several ion channels¹³ and serves as a crucial component in enzymes and transcription factors such as carbonic anhydrase and zinc finger proteins, where it plays both structural and catalytic roles.¹⁴ Zn²⁺ is vital for many biological processes, including brain function, gene transcription, immune response, and reproduction,¹⁵ and is implicated in pathological conditions such as Alzheimer's disease, epilepsy, ischemic stroke, and infantile diarrhea.¹⁶ While most Zn²⁺ is tightly bound to enzymes and proteins, free zinc pools exist in tissues such as the brain, intestine, pancreas, and retina. Given its biological significance, optical imaging using fluorescent sensors for Zn²⁺ has drawn considerable interest.¹⁷

The design and synthesis of molecular receptors for the selective detection of biologically important Zn²⁺ ions through fluorescence changes have gained considerable attention in recent years. This is because fluorescence signals are highly sensitive to alterations in the fluorophore's surrounding environment. Due to its 3d¹⁰ 4s⁰ electronic configuration, Zn²⁺ lacks intrinsic spectroscopic or magnetic properties, making it incompatible with common analytical methods like UV-vis spectroscopy, Mössbauer spectroscopy, nuclear magnetic resonance (NMR), and electron paramagnetic resonance (EPR) spectroscopy.¹⁸ Although techniques such as atomic absorption spectroscopy offer highly accurate quantitative measurements of Zn²⁺, they typically destroy the sample being analyzed.¹⁹ In contrast, a fluorophore-based sensor that exhibits changes in fluorescence intensity or wavelength upon binding Zn²⁺ can provide not only the location of Zn²⁺ but also a semi-quantitative estimate in both in vivo and in vitro samples.²⁰ The ability of Zn²⁺ to form complexes with various organic structures that can be linked to a fluorophore makes it possible to develop fluorescent probes for biological applications.^21,22 Thus, target ligand was synthesized by normal Schiff base condensation reaction between 5-bromo-2-hydroxy-3-methoxybenzaldehyde with 3,5-dimethyl-1H-pyrazole-1-carbohydrazide to give the expected ligand (E)-N′-(2-hydroxy-3-methoxybenzylidene)-5-methyl-1H-pyrazole-3-carbohydrazide (HMPC) which has been shown in the Scheme 1. The target ligand HMPC has been characterized by ¹H-NMR and mass spectra (Fig. S4–S6†).

Scheme 1 Synthesis of HMPC.

2. Experimental section

2.1 General

The chemicals and solvents were purchased from Sigma-Aldrich Chemicals Private Limited and were used without further purification. Melting points were determined on a hot-plate melting point apparatus in an open-mouth capillary and were uncorrected. ¹H-NMR spectra were recorded on Brucker 400 MHz instruments. NMR titration was carried out in DMSO-D₆ solvent on 400 MHz instrument with TMS as an internal standard. Chemical shifts are expressed in δ units and ¹H–¹H and ¹H–C coupling constants in Hz. UV-vis and fluorescence titration experiments were performed on UV-spectrophotometer: PerkinElmer, Lambda 30 and Shimadzu spectrofluorophotometer RF-6000 using a fluorescence cell of 10 mm path respectively.

2.2 Synthesis of the ligand HMPC

5-Methyl-pyrazole-3-carbohydrazide was synthesized according to reported literature method.²² To the methanolic solution of 5-methyl-pyrazole-3-carbohydrazide (140 mg, 1.0 mmol), 5-bromo-2-hydroxy-3-methoxybenzaldehyde (152 mg, 1.0 mmol) was added and refluxed for 6 hours over oil bath (Scheme 1). The resulting solution is then concentrated to ∼15 mL, cooled and a pale yellow precipitated was separated out. The yellow precipitate was filtered and washed with chilled ethanol and dried over CaCl₂ in a vacuum desiccator to get dry solid ligand as HMPC.

Yield: 150 mg, 64%. ¹H NMR (DMSO-d_6, 400 MHz): δ (ppm): 13.14 (s, 1H, –OH), 12.03 (s, 1H, –NH), 11.08 (s, 1H, –NH), 8.34 (s, 1H, =C–H), 7.29 (s, 1H), 7.15 (s, 1H), 6.52 (s, 1H) 3.84 (s, 3H), 2.30 (s, 3H). Mass (MS): M⁺ calculated for C₁₃H₁3BrN₄O₃ is 352.0171; found: 354.9801 (M + 2H⁺).

3. Results and discussion

3.1 Binding study with Zn²⁺

The binding study of HMPC (c = 5 × 10⁻⁵ M) with various metal ions (c = 10⁻⁵ M) by UV-vis and fluorescence methods were carried out in an acetonitrile/aqueous (9 : 1, v/v) HEPES-buffered (1 mM, pH 7.4) solution. The UV-vis spectrum of HMPC showed a major absorption peak at 318 nm. Upon the gradual addition of Zn²⁺, a gradual increase in absorbance at 318 nm was observed, indicating complexation between HMPC and the Zn²⁺ ion (Fig. 1). However, this absorption characteristic did not result in any visible color change.

Fig. 1 (a) UV-vis absorption spectra of HMPC (c = 5 × 10⁻⁵ M) in CH₃CN–HEPES buffer (9 : 1, v/v, pH = 7.4) upon addition of 0 to 5 equiv. of Zn²⁺ (c = 10⁻⁵ M). (b) Changes of absorbance as a function of Zn²⁺ concentration including error bars (error amount, 1%; Y error bar for both [±] deviation).

In the fluorescence experiment, the receptor HMPC exhibited two emission peaks centered at 400 nm and 427 nm (λ_ex = 318 nm). However, with the gradual addition of Zn²⁺ to the HMPC solution, a new red shifted emission peak generated at 490 nm with strong fluorescence enhancement by the appearance of naked eye blue color. As expected, HMPC initially exhibited weak fluorescence at 490 nm when excited at 318 nm, with a low quantum yield (Φ₀ = 0.1415). Upon addition of increasing amounts of Zn²⁺, about a 2-fold increase in fluorescence quantum yield (Φ/Φ₀ = 0.259/0.142 = 2, λ_em = 490 nm) was observed (Fig. 2).

Fig. 2 (a) Fluorescence spectra of HMPC (c = 5 × 10⁻⁵ M) in CH₃CN–HEPES buffer (9 : 1, v/v, pH = 7.4) upon addition of 0 to 5 equiv. of Zn²⁺ (c = 10⁻⁵ M). Inset: changes of emission color on addition of Zn²⁺ ions (b) changes of emission intensity of HMPC as a function of Zn²⁺ concentration including error bars (error amount, 5%; Y error bar for both [±] deviation).

One of the challenges for Zn²⁺ probes is to develop systems that are selective over a wide range of potentially competing ions, such as Mn²⁺, Cr³⁺, Pb²⁺, Fe²⁺, Co²⁺, Ni²⁺, Hg²⁺, Cd²⁺, Fe³⁺, and Cu²⁺. HMPC showed almost negligible effects on its fluorescence behavior in the presence of these competing ions, thereby maintaining its selectivity toward Zn²⁺ by naked eye as well as in the fluorescence spectra (Fig. 3).

Fig. 3 (a) Fluorescence color changes of HMPC in presence of various cations. (b) Fluorescence titration spectra of HMPC (c = 5 × 10⁻⁵ M) in HEPES buffered aqueous CH₃CN (9 : 1, v/v) upon addition of 15 equiv. of different metal ions. (c) A comparative fluorescence response of HMPC by bar representation in presence of different cations.

However, Cd²⁺ exhibits slight interference because both ions share similar chemical and physical properties. Zn²⁺ and Cd²⁺ are in the same group (Group 12) of the periodic table and have comparable ionic radii, electronegativities, and coordination geometries resulting a slight interference in the fluorescence spectra of HMPC with Cd²⁺. Despite this, the sensor HMPC demonstrated higher selectivity and sensitivity toward Zn²⁺ compared to Cd²⁺.²³

Competition studies were also carried out in the presence of other interfering cations to further investigate the selectivity of HMPC for Zn²⁺. The fluorescence enhancement of HMPC for Zn²⁺ was unaffected by the presence of other interfering cations. This indicates that the receptor HMPC is highly sensitive and selective for cation Zn²⁺ eventually in presence of other interfering analytes (Fig. 4). From the fluorescence titration experiments, the detection limit of HMPC for Zn²⁺ was calculated to be 1.68 μM using the formula DL = K × Sb₁/S, where K = 3, Sb₁ is the standard deviation of the blank solution, and S is the slope of the calibration curve, (Fig. S2†).²⁴ Job plot analysis indicated 1 : 1 binding stoichiometry between HMPC with Zn²⁺ (Fig. S3†) and from the fluorescence titration experiment, the association constant (K_a) of HMPC with Zn²⁺ was calculated as 5 × 10⁴ M⁻¹ (error < 10%) (Fig. S1†).²⁵

Fig. 4 (Maroon bars) variation of fluorescence intensity of HMPC + 15 equiv. Mⁿ⁺; (blue bars) fluorescence intensity of HMPC + 15 equiv. Mⁿ⁺, followed by 10 equiv. Zn²⁺.

The reversible binding of HMPC with the metal ion Zn²⁺ has been proved by the reversibility experiment carried out by adding an aqueous solution of excess Na₂EDTA (0–5.0 equiv.) in situ to the solution of HMPC-Zn²⁺ complex (Fig. 5). And it has been observed that the fluorescence intensity of HMPC-Zn²⁺ complex is quenched steadily in presence of excess Na₂EDTA which indicates that Na₂EDTA possibly strips away Zn²⁺ from the metal binding cavity.

Fig. 5 (a) Fluorescence titration spectra of HMPC-Zn²⁺ complex (c = 5 × 10⁻⁵ M) with Na₂EDTA (0–5.0 equiv.) in CH₃CN–HEPES buffer (9 : 1, v/v, pH = 7.4). Inset: bluish white colored emission observed with Zn²⁺ was disappeared in presence of Na₂EDTA. (b) Change of emission intensity as a function of Na₂EDTA concentration including error bars (error amount, 5%; Y error bar for both [±] deviation.

3.2 Probable binding mode in solution phase

Probable binding of HMPC with Zn²⁺ was demonstrated by UV-vis, fluorescence and mass spectrometry. Initially HMPC alone showed weak fluorescence exhibiting two emission signals at 400 nm and 427 nm but in the presence of Zn²⁺, a notable red shifted fluorescence enhancement was observed at 490 nm. This remarkable fluorescence enhancement of HMPC in presence of Zn²⁺ can be attributed due to the suppression of PET process within the molecule and enhancement of ICT for the charge transfer between ligand and metal, which results also generation of the fluorescence enhancement by chelation enhanced fluorescence (CHEF) mechanism. Before binding with Zn²⁺ (off-state), the fluorescence of free HMPC is very weak due to operating PET from electron donor phenolic hydroxy group to electron acceptor hydrazide coupled imine moiety and weak ICT (Scheme 2). In the presence of Zn²⁺, the ligand HMPC forms four coordination via the bond formation between O₂ donors from hydroxy and hydrazide moieties and one nitrogen from imine nitrogen and one negative chloride counter anion resulting in fluorescence enhancement (on-state) by CHEF.²⁶ On the other hand, the emission intensity of HMPC is notably very weak due to the radiationless deactivation of the excited state caused by conformational changes via the free rotation around the imine (C=N) bond, a phenomenon commonly observed in Schiff-base dyes. But, co-ordination with Zn²⁺ inhibits the isomerization which results the increase of fluorescence intensity of the ligand for enhanced ICT.²⁷ The formation of a complex between HMPC and Zn²⁺ has been verified through the emergence of a mass peak at m/z = 450.6280, attributed to [HMPC + Zn²⁺ + Cl⁻] (Fig. S6† and Scheme 2), which has been justified also by 1 : 1 binding stoichiometric ratio in the Jobs plot analysis and theoretical calculation.

Scheme 2 Probable binding mode in solution phase.

3.3 DFT study

The optimized geometry of HMPC and HMPC + Zn²⁺ complex through DFT is shown in Fig. 6. The HMPC + Zn²⁺ complex having metal centre Zn²⁺ are tetra-coordinated with neutral ligand with O₂ donor system, one nitrogen and one negative chlorides counter anion, satisfy the tetrahedral geometry to form the mononuclear complex. The theoretical Zn–Cl and Zn–N (of imine) bond distances are as 2.175 Å and 2.003 Å respectively and Zn–O bond lengths are 2.199 Å (amide oxygen) and 1.947 Å (phenolic oxygen) for HMPC + Zn²⁺ complex. Moreover, it is found that the HOMO–LUMO energy gap are ΔE = 7.652 eV and 6.984 eV for HMPC and HMPC + Zn²⁺ complex respectively which is responsible for the stabilization of the complex formation (Fig. 7).

Fig. 6 Geometry optimized molecular structure of (a) HMPC and (b) HMPC-Zn²⁺ complex.

Fig. 7 Frontier molecular orbital with energy difference of HMPC and complex.

Experimental absorbance of ligand HMPC at 328 nm at room temperature was increased by the gradual addition of Zn²⁺. The theoretical electronic spectra shows the corresponding calculated absorption band at 317 nm, demonstrating excellent agreement with the experimental data and these spectral bands can be attributed to the S₀ → S₇ transition.

3.4 Biological application

MCF-7 cells at sub-confluent volume were cultured in DMEM for 24 hours under standard conditions in a CO₂ incubator. Following this, HMPC was administered to the cells at its determined IC₅₀ concentration. After an appropriate incubation period, fluorescence images of the treated MCF-7 cells were captured at 40× magnification using a ZEISS fluorescence microscope (Germany). In parallel, the cells were also exposed to a 10 μM Zn²⁺ solution for one hour, and additional fluorescence images were captured at excitation wavelengths of 582 nm and 581 nm for the cells pre-treated with HMPC at the IC₅₀ concentration, as described by Mabhai et al. (2019).²⁸

3.4.1 Cell viability study on MCF 7 and human lymphocyte cells (HLCs). The cytotoxic effects of HMPC on MCF-7 and HCT-116 cells were assessed using the MTT assay, which revealed a significant dose-dependent reduction in cell viability, indicating potent cytotoxicity. The IC₅₀ value of HMPC was found to be 19.26 μg mL⁻¹ for MCF-7 cells and 21.31% μg mL⁻¹ for HCT-116 cells, which are comparable to the IC₅₀ of the standard anticancer drug 5-FU (16.14 μg mL⁻¹ for MCF-7 and 10.21 μg mL⁻¹ for HCT-116). These results further substantiate the potent cytotoxic activity of HMPC (Fig. 8A and B). A time-dependent study demonstrated that HMPC treatment resulted in a 48.47% reduction in cell viability for MCF-7 cells and a 51.49% reduction for HCT-116 cells at 24 hours. At 48 hours, the reduction in cell viability was 30.89% for MCF-7 cells and 38.23% for HCT-116 cells (Fig. 8C). These results underscore the strong cytotoxic potential of HMPC against MCF-7 and HCT-116 cell lines. Furthermore, HMPC exhibited minimal toxicity to human lymphocytes at concentrations up to 50 μg mL⁻¹, with a slight reduction in cell viability, as the viability at 50 μg mL⁻¹ was recorded at 52.32%. Overall, these findings suggest that HMPC could be a promising anticancer agent for targeting MCF-7 and HCT-116 cells (Fig. 8D, HLC).

Fig. 8 MTT assay: cytotoxic effects of HMPC on MCF-7 cells at different concentrations, with an IC₅₀ value of 19.26 μg mL⁻¹ (A). Cytotoxic effects of HMPC on HCT-116 cells at different concentrations, with an IC₅₀ value of 21.31 μg mL⁻¹ (B). Cell viability was assessed at 24 h and 48 h following treatment with HMPC at its IC₅₀ concentrations (C). Cytotoxic study on HLC: a slight decrease in lymphocyte viability was observed at much higher concentration of HMPC (50 μg mL⁻¹) (D). Data are presented as mean ± SEM of three independent experiments; **p < 0.01, ***p < 0.001, compared to the control group.

3.4.2 Fluorescence cell imaging study. During 1 hour incubation at 37 °C with HMPC at its IC₅₀ concentration, less significant intracellular fluorescence was observed in fluorescent cell imaging on MCF-7 and HCT-116 cell lines. However, when the cells were incubated with an exogenous Zn²⁺ ion solution, a strong intracellular blue fluorescence was detected, with the fluorescence intensity of the HMPC-Zn²⁺ + complex being significantly higher (Fig. 9). This indicates that HMPC effectively penetrates the cell membrane and binds to intracellular Zn²⁺ ions, forming the HMPC-Zn²⁺ complex, as demonstrated by the fluorescence imaging. The fluorescence generation mechanisms of HMPC, Density Functional Theory (DFT) calculations were employed to analyze the electronic structure and frontier molecular orbitals of HMPC and the HMPC-Zn²⁺ complex. The results suggest that the fluorescence arises from the ligand-to-metal charge transfer mechanism facilitated by the coordination of Zn²⁺ ions with the HMPC molecule. In its free state, HMPC exhibits a less efficient intramolecular charge transfer due to the absence of metal ion coordination, which explains the low fluorescence intensity observed. Upon binding with Zn²⁺, the electronic transitions are significantly altered, as evidenced by leading to enhanced fluorescence.

Fig. 9 Intercellular biosensor cell imaging study was investigated on MCF 7 and HCT 116 cells. Cells were incubated alone with free HMPC and brighter blue fluorescence was observed HMPC-Zn²⁺ treated groups other than HMPC. The image was captured by a fluorescence microscope (ZEISS, Germany).

The strong blue fluorescence of the HMPC-Zn²⁺ complex highlights the specificity and efficiency of the Zn²⁺ binding. These findings demonstrate that HMPC could serve as a selective sensor for Zn²⁺ ions in bio-imaging applications under the given concentrations and incubation conditions, without inducing cytotoxic effects. By combining experimental fluorescence imaging and computational analysis, we provide a comprehensive understanding of HMPC have potential as a Zn²⁺ ion sensor against MCF-7 and HCT-116 cells.

3.5 Molecular docking study

The link between the mark protein (PDB-ID: 2VCJ) and the ligands is assessed by molecular docking.^28–34 The designs reveal the free energy change of ligand HMPC for the interactions with protein 2VCJ, ΔG = −7.1 kcal mol⁻¹ (Table 1) inside a grid box of 28.6 × 12.7 × 19.82 Å with dimensions 25 × 25 × 25 Å along three axes (Fig. 10a). The 2D diagram shows that ligand HMPC forms one hydrogen bonding interaction with the 51st asparagine residue, along with a pi–sulfur bond with the 98th methionine residue of the A chain in the binding site of the protein 2VCJ (Fig. 10b).

Table 1 Results of the docking of ligands and target protein 2VCJ

Name of ligands	Docking score (kcal mol^−1)
HMPC	−7.1

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Fig. 10 Docking poses of HMPC with 2VCJ, (a) at active site, (b) 2D diagram of the docked complex.

The link between the mark protein (PDB-ID: 1T69) and the ligand HMPC is assessed by molecular docking. The designs reveal the free energy change of ligand HMPC for the interaction with protein 1T69, ΔG = –6.5 kcal mol⁻¹ (Table 2) inside a grid box of 30.09 × −3.16 × −13.64 Å with dimensions 22 × 22 × 22 Å along three axes (Fig. 11a). The 2D diagram (Fig. 11b) shows that ligand HMPC forms two hydrogen bonding interactions with the 33rd lysine residue and 180th histidine residue of chain A in the binding site of the protein 1T69, along with van der Waals and pi–pi stacked interactions as shown in the Fig. 11b.

Table 2 Results of the docking of ligands and target protein 1T69

Name of ligands	Docking score (kcal mol^−1)
HMPC	−6.5

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Fig. 11 Docking poses of HMPC with 1T69, (a) at active site, (b) 2D diagram of the docked complex.

3.6 ADME prediction results

The result from SWISS-ADME³⁵ is agreeable as HMPC offers no violation of Lipinski's, Veber's, and Egan's rule of drug-likeness properties and it has high gastrointestinal absorption (Fig. S7a†), no blood–brain barrier (BBB) permeant property (Fig. S7a†), it has a good bio-radar (Fig. S7b†) which makes it orally admissible to people. HMPC does not inhibit any of the cytochrome P50 enzymes except the CYP1A2 inhibitor, and is not a P-gp substrate. It has a good log P value of 1.97, essential for showing drug-like properties. It has a Total Polar Surface Area (TPSA) of 99.60 Ų (Table S1†).

4. Conclusion

In conclusion, the fluorescent sensor HMPC has demonstrated high selectivity and sensitivity for Zn²⁺ detection, with a detection limit well below the WHO guideline. Its fluorescence enhancement is driven by a CHEF mechanism through the suppression of PET and enhancement of ICT, as validated by comprehensive spectroscopic analyses and computational studies. Additionally, HMPC shows promising biological applications, including low cytotoxicity and potential anticancer activity, as supported by molecular docking studies with HSP90 and HDAC8 proteins. With a favorable ADME profile and strong binding affinities, HMPC presents potential not only as a fluorescent probe for Zn²⁺ but also as a candidate for anticancer drug development. Furthermore, the cytotoxicity and cell imaging studies conducted with MCF-7 cells indicate the potential of HMPC for biological applications, paving the way for further exploration in the field of chemosensing and biomedicine.

Data availability

The data supporting this article have been included as part of the ESI.†

Conflicts of interest

The authors declare no competing financial interest.

Acknowledgements

MD acknowledges to Science & Engineering Research Board (SERB), Govt. of India (ref. No. PDF/2016/000334). AHS thankful to the Researchers Supporting Project number (RSP2025R373) at King Saud University, Riyadh, Saudi Arabia for supporting this research. AKD specially acknowledges State University Research Excellence (SERB-SURE) of the Science and Engineering Research Board (SERB) (File Number: SUR/2022/ 002461) under Anusandhan National Research Foundation (ANRF) and Department of Science and Technology (DST), Government of India, for the financial support by the research grant. The authors express their gratitude to Christ University, Bangalore, for providing research facilities, and Center for Research, Christ University for the seed money grant (grant approval number CU-ORS-SM-24/09). Malavika S. Kumar extends her appreciation to the University Grants Commission (UGC), Government of India, for the Savitribai Jyotirao Phule Fellowship for Single Child (SJSGC), F. No. 82-7/2022(SA-III).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ay02218a

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