Water-soluble triazole-based azo-Schiff base fluorescent–colorimetric dual chemosensor for selective detection of sulfide ions: analytical and biological applications

Abstract

A triazole-based azo-Schiff base ligand (L) has been designed and synthesized as a dual-mode fluorescent–colorimetric chemosensor for the selective and sensitive detection of sulfide ions (S²⁻) in aqueous media. The probe exhibits a distinct color change from light yellow to deep orange and a significant fluorescence enhancement upon interaction with S²⁻, enabling both naked-eye detection and spectroscopic monitoring. UV-vis titration studies revealed a red shift from 346 nm to 403 nm, while fluorescence emission was enhanced up to 21-fold at 428 nm. The sensing mechanism is not governed solely by basicity-induced deprotonation. The selective response toward S²⁻ may arise from its higher polarizability, stronger nucleophilicity, and favorable host–guest interaction with the phenolic-azomethine framework of the receptor, supported by ¹H NMR, FTIR, and DFT analyses. The sensor demonstrates high selectivity over competing anions, excellent anti-interference capability, rapid response time, and a low detection limit (3.3 × 10⁻⁷ M). Job's plot confirms a 2 : 1 binding stoichiometry with a strong association constant (5.0 × 10⁵ M⁻¹). The probe operates effectively over a wide pH range and exhibits reversible sensing behavior in the presence of Hg²⁺ ions, enabling molecular logic gate applications. Practical applicability was validated through successful detection of sulfide ions in spiked water samples with high recovery. Additionally, molecular docking studies revealed strong binding affinity of L with VEGFR-2 (−9.4 kcal mol⁻¹), indicating potential biological relevance. The ligand also exhibited significant antimicrobial activity, particularly against Gram-negative bacteria, as confirmed by resazurin-based MIC assays. These results highlight L as a multifunctional platform for environmental monitoring, biological sensing, and antimicrobial applications.

A triazole-based azo-Schiff base ligand (L) has been designed and synthesized as a dual-mode fluorescent–colorimetric chemosensor for the selective and sensitive detection of sulfide ions (S²⁻) in aqueous media.

1. Introduction

In recent years, the development of chemical sensors that can monitor biologically and environmentally important anions has been of special interest, because of the widespread use of these ions in industry, medicine, biology and the environment.^1–4 Sulfide (S²⁻) has been long known to be highly toxic with a rotten egg smell.^5,6 As one of the abundant and important anions in biological systems, the sulfide ion (S²⁻) has received attention due to its significant roles in many physiological processes, including regulation of inflammation and neuromodulation, angiogenesis, vasodilation and apoptosis.^7–9 S²⁻ exhibits significant effects on both the cardiovascular and neuronal systems and is produced endogenously from cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate sulfur transferase (3MST), as well as from nonenzymatic processes.^10–12 On the other hand, exposure to high concentrations of S²⁻ could lead to pathological disturbances in the form of hypertension, atherosclerosis, heart failure, diabetes, cirrhosis, and neurodegenerative diseases.^13–16 Abnormal production of S²⁻ is linked to human diseases such as Alzheimer's disease, Down's syndrome and hyperglycemia.^17,18 Thus, the development of selective and sensitive chemosensors for S²⁻ is greatly necessary because of its significant roles in environmental and health problems.

In order to detect S²⁻, various traditional analytical techniques have been developed such as gas chromatography, precipitation, and inductively coupled plasma spectroscopy, polarography and sulfide-selective electrodes.^19–22 However, most of these methods require expensive equipment and trained operators. Therefore, other detection methods, especially those with low instrumentation requirements, are needed.²³ Optical signaling is attractive due to its simplicity, sensitivity, and ease of quantification. In contrast, colorimetric probe method has short response time, naked-eye observation, and low cost.^24–26 The fluorescence probe method has high sensitivity, low detection limit and simple operation.^27–30 So these two methods have attracted more and more attention.

Fluorescence and colorimetric probes reported for sulfides detection are mainly based on the following four mechanisms: (1) azide reduction to amine; (2) nucleophilic reactions; (3) copper sulfide precipitation and (4) based on deprotonation of acidic hydrogen atom of some organic receptors.^31–34 Among them, azide is dangerous and reaction conditions are relatively harsh. The nature of the leaving group can influence the reaction in case of nucleophilic reactions. Copper sulfide precipitation method is achieved indirectly through the replacement of organic metal complex. This method is complicated to operate and generally has a high detection limit.

Therefore, the design of Schiff base fluorescent–colorimetric probe for the selective and sensitive detection of S²⁻ has become an active research focus. In this context and in continuation of our ongoing research,^35–37 we have reported here a, 1,2,4-triazole based Schiff base ligand of an azo-dye from sulphanilic acid, which successfully detect sulfide ion both visually and spectro-photometrically. The presence of –SO₃H group makes the probe L perfectly water soluble. The Schiff bases of substituted 1,2,4-triazole ligands and their metal complexes are well known microbial agents and having promising biological activities.^38,39 Triazole based Schiff bases are used in drug discovery, as catalysts in chemical transformations, and as biological and pharmaceutical active agents.^40,41 Triazoles have shown promise in cancer therapy, infectious diseases, and central nervous system disorders, making them valuable tools in developing targeted therapies.^42,43 Their ability to interact with a wide range of biomolecules and their stability in various chemical conditions make them ideal for use in medicinal chemistry and pharmaceutical research. Moreover, these ligands can bridge transition metal ions through an N1,N2-coordination mode to afford polynuclear compounds with unique properties.^44,45

2. Experimental

2.1. General information

All of the chemicals used in this study were provided by Sigma-Aldrich and Merck Chemical, and no further purification was required. The fluorescence spectra were recorded using a Hitachi spectrophotometer. A Shimadzu UV 1800 spectrophotometer was used to record UV/visible spectra using a quartz cuvette with a route length of 10 mm. An electro-thermal melting point equipment was used to measure all of the uncorrected melting points. Infrared spectra were recorded using a Shimadzu 8400 FT-IR spectrophotometer. The proton nuclear magnetic resonance (¹H and ¹³C-NMR) spectra were obtained using the FT-NMR (500 MHz) Brucker equipment spectrometer. The chemical changes in δ ppm were represented using TMS as an internal standard triple-distilled water and methanol were utilized in a mixed solvent HPLC to record ESI-MS spectra using a waters mass spectrometer. The pH was measured using a digital pH meter (Merck).

2.2. Synthesis of the 4-((E)-(3-((E)-((4H-1,2,4-triazol-4-yl)imino)methyl)-4-hydroxyphenyl)diazenyl)benzenesulfonic acid (L)

L was prepared by the condensation reaction in 1 : 1 ratio between (E)-4-((3-formyl-4-hydroxyphenyl)diazenyl)benzenesulfonic acid (1) and 4H-1,2,4-triazol-4-amine in methanol under dry condition.

Sulfanilic acid's acidic –SO₃H group was changed into –SO₃Na group, and its diazonium salt was produced in good yield using the previously mentioned procedures⁴⁶ with slight modifications. Following the diazotization reaction, salicylaldehyde was dissolved in water that contained sodium hydroxide and sodium carbonate in a different flask, which was then cooled in an ice bath to 0 to −50 °C. By keeping the pH between 7.5 and 8.5 for 30 minutes, the phenolate ion solution in basic medium was gradually mixed with the ice-cold diazonium solution. The resulting solution was stirred in an ice bath for 30 minutes and then left at room temperature for an hour. The precipitate was gathered and repeatedly washed with cold water after diluted HCl was added to acidify the solution (pH = 5.5–6.5). The precipitate was recrystallised in MeOH/H₂O to produce azo dye (1). Another methanolic solution of 4H-1,2,4-triazol-4-amine (1 mmol in 25 mL) was mixed with the azo-dye 1 methanolic solution (1 mmol in 25 mL). For 8 hours, the reaction mixture was refluxed while being kept dry. Then, it was gradually allowed to cool to ambient temperature. A solid with a deep crimson color separated out, was filtered off, and allowed to dry in the air. Yield, 0.290 g (78%); m.p. >200 °C. Anal. found (calc. for C₁₅H₁₂N₆O₄S): C, 48.49 (48.38%); H, 3.31 (3.25%); N, 22.67 (22.57%). ¹H NMR (500 MHz, DMSO-d₆, TMS): δ 11.40 (s, 1H), δ 10.38 (s, 1H, –OH), δ 9.25 (s, 2H), 8.39 (s, 1H, –CH N), 8.05 (d, 2H), 7.84 (m, 3H), 7.21 (d, 2H) (Fig. S1). ¹³C NMR (125 MHz, DMSO-d₆, TMS): δ 183.62, 165.25, 161.45, 153.99, 152.14, 150.77, 145.49, 140.75, 139.56, 128.89, 127.21, 122.35, 122.33, 119.52,118.11, (Fig. S2). ESI-MS: 373.07 (MH⁺, 100%) (Fig. S3). HR-MS: 373.0129 (MH⁺, 100%) (Fig. S4). FTIR/cm (KBr): 3478 (br, –OH, SO₃H), 2931 (m), 1620 (vs.) (C N), 1491 (vs., N N), 1377 (vs.), 1174 (vs., S O), 1030 (vs.), 844 (vs.), 699 (s), 575 (s) (Fig. S5). UV-vis: λ_max/nm (H₂O): 240; 285; 346.

2.3. UV-vis titrations

To reach a final concentration of 10 µM, 30 µL of the chemosensor L (3.72 mg, 0.01 mmol) was diluted to 3 mL with the solvent mixture after it had been dissolved in 10 mL of triple-distilled water. After dissolving sodium sulphide nonahydrate (0.1 mmol) in 10 mL of triple-distilled water, 1.5–90 µL of the anion solutions (10 µM) was added to the previously made solution of L (10 µM). At room temperature, UV-vis spectra were taken after 30 seconds of mixing.

2.4. Job plot measurements

L (3.72 mg, 0.01 mmol) was dissolved in 10 mL of triple-distilled water. The L solution was divided into vials with 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, and 0 µL. Each vial was diluted with 2.9 milliliters of distilled water. Sodium sulphide nonahydrate (0.01 mmol) was dissolved in 10 mL of triple-distilled water. 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 µL of the sodium sulfide solution were combined with each diluted L solution. Each vial has a 3 mL total capacity. After 30 seconds of shaking, absorbance spectra were measured at room temperature.

2.5. Fluorescence titrations

Following the dissolution of 24.1 mg of sodium sulphide nonahydrate in 10 mL of triple-distilled water, 1.5–90 µL of this sulphide solution (10 µM) was added to receptor solution (10 µM) to yield 0.5–20 equiv. L (3.72 mg) was dissolved in 10 mL of triple-distilled water to prepare a solution of 1 × 10⁻³ M. A final concentration of 10 µM was obtained by diluting 30 µL of this solution with 2.97 mL of the solvent mixture. The fluorescence spectra were acquired at room temperature following 30 seconds of mixing period.

2.6. Competition with other anions

To get a final concentration of 10 µM for S²⁻, L (3.72 mg, 0.01 mmol) was dissolved in 10 mL of water, and 30 µL of this solution was diluted to 3 mL with water. Ten milliliters of triple-distilled water were used to dissolve sodium salts of CO₃²⁻, N₃⁻, HSO₃⁻, CN⁻, F⁻, S²⁻, OAc⁻, Cl⁻, PO₄³⁻, SO₄²⁻, NO₃⁻, Br⁻ and OH⁻ (0.1 mmol). The mixture of each anion and L was then combined with 30 µL of S²⁻ solution (10 µM) to make 10 equiv. After mixing them for 30 seconds, absorbance spectra were recorded at room temperature.

2.7. pH effect test

100 mM HEPES buffer was used to prepare a range of buffers with pH values between 4 and 12. Once the pH of the solution was reached, receptor L (3.72 mg, 0.01 mmol) was dissolved in 10 mL of methanol. Thirty microlitres of this solution (1 µM) were diluted to three millilitres using the previously specified buffers, yielding a final concentration of 10 µM. Ten millilitres of HEPES buffer (pH 7.00) were used to dissolve 24.1 milligrams of sodium sulphide nonahydrate. Each receptor solution (10 µM) received 30 µL of the S²⁻ solution (10 µM). Absorption spectra were acquired at room temperature following 30 seconds of mixing period.

2.8. ¹H NMR titrations

Four NMR tubes containing receptor L (3.72 mg, 0.01 mmol) dissolved in DMSO-d₆ (700 µL) were prepared for ¹H NMR titrations of receptor L. Each receptor solution was subsequently treated with four different doses of sodium sulfide dissolved in D₂O (0, 0.01, 0.02, and 0.03 mmol). ¹H NMR spectra were collected at room temperature following a minute of shaking.

2.9. Computational details

All computations were made using the GAUSSIAN-09 Revision C.01 program package.⁴⁷ Using the gradient-corrected DFT level in conjunction with the hybrid exchange–correlation functional that employs the Coulomb-attenuating method B3LYP.⁴⁸ Basis set 6-31++G was found to be appropriate for the entire molecule, and the gas phase geometries of the compound were fully optimised without any symmetry restrictions in singlet ground state. Materials Studio 2020 used the PWC exchange–correlation functional under the local density approximation (LDA) to conduct molecular docking investigations.^49,50

2.10. Molecular docking study

2.10.1. Protein preparation

The 3D crystal structure of the target protein, Vascular Endothelial Growth Factor Receptor (PDB ID: 2OH4), was taken from the Protein Data Bank. Before starting the docking study, the protein was prepared by using AutoDock Tools and Biovia Discovery Studio Visualizer software. All the water molecules in the crystal structure were deleted.⁵¹ The co-crystallized ligand and other unwanted heteroatoms were also removed because they can affect or disturb the binding of the test ligand, L. After that, polar hydrogen atoms were added to the protein structure and Kollman charges were given. Finally, the prepared protein was saved in PDBQT file format, which is required for further molecular docking analysis.

2.10.2. Ligand preparation

The triazole-based ligand, L was first optimized before performing the docking study. After that, hydrogen atoms were added correctly to the structure, and Gasteiger charges were given to the ligand.⁵² The rotatable bonds were also selected so that the ligand can move freely during docking process. Finally, the optimized ligand file was saved in PDBQT format by using AutoDock Tools, because this format is needed to run the docking in AutoDock Vina.

2.10.3. Molecular docking procedure

Molecular docking was done by using AutoDock Vina to check how the ligand, L binds with the active site of the protein. The grid box was adjusted at the coordinates x = 2.46, y = 34.81 and z = 16.28, and the grid size was fixed at 30 × 30 × 30 Å so that the full binding pocket area can be covered properly.⁵³ The exhaustiveness parameter was kept as 8 to get good and acceptable docking results.

After docking, many different poses were obtained. From those poses, the best one was chosen mainly based on the lowest binding energy value and also by seeing the correct binding direction inside the active site. The final docked complexes were then checked by visualization software to observe hydrogen bonding, π–π interactions and hydrophobic contacts between ligand, L and nearby amino acid residues of the protein.

2.11. Determination of minimum inhibitory concentration (MIC) by a resazurin-based assay

2.11.1. Standardization of MIC assay reagents and sample solutions

A resazurin working solution was prepared by dissolving 0.01 gram of resazurin sodium salt (HiMedia) in 100 mL of distilled water. The test compound, L, was prepared as a 1000 µg mL⁻¹ stock solution in methanol and stored at 4 °C until use.

2.11.2. Preparation of bacterial inoculum

Fresh cultures of Escherichia coli, Klebsiella sp., Pseudomonas sp., Bacillus sp., and Staphylococcus aureus were grown on nutrient agar plates and incubated at 37 °C for 24 hours. From each plate, five morphologically distinct colonies were aseptically transferred into 10 mL of Mueller–Hinton broth (MHB) and incubated overnight at 37 °C. The resulting bacterial suspension was adjusted to a 0.5 McFarland standard using sterile 0.85% NaCl, ensuring uniform inoculum density. A working inoculum of approximately 5 × 10⁴ CFU per mL was prepared by performing a 1 : 20 dilution of the standardized culture (0.5 mL inoculum added to 9.5 mL MHB).

2.11.3. Preparation of the microtiter plate for minimum inhibitory concentration (MIC)

In the present study, MIC was determined using the resazurin-based 96-well plate assay, a rapid and accurate method in which the blue resazurin dye is reduced to pink resorufin by metabolically active bacterial cells. A sterile 96-well microtiter plate was used for all experiments. Each well received 100 µL of Mueller–Hinton broth (MHB), and the test sample concentrations prepared were 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, and 100 µg mL⁻¹. Subsequently, 20 µL of overnight-grown bacterial culture was added to each well, except those in column 11 (sterility control). The growth control consisted of drug-free wells containing only the bacterial suspension, whereas the negative control comprised wells containing sterile Mueller–Hinton broth (MHB) without bacteria.^54,55

2.11.4. Resazurin-based viability detection

The microtiter plate was prepared, sealed with parafilm, and incubated statically at 37 °C for 24 hours. Following incubation, 30 µL of freshly prepared 0.01% resazurin solution was added to each well, and the plates were incubated for 2 hours. Colour change was then observed, a pink colour indicated bacterial growth, whereas wells that remained blue or purple indicated inhibition. The MIC was recorded as the lowest concentration of the test sample that showed no colour change, confirming the absence of bacterial growth.^56,57

3. Results and discussion

3.1. Synthesis and structure of L

The condensation reaction in 1 : 1 ratio between (E)-4-((3-formyl-4-hydroxyphenyl)diazenyl)benzenesulfonic acid and 4H-1,2,4-triazol-4-amine in methanol under dry condition produced the 4-((E)-(3-((E)-((4H-1,2,4-triazol-4-yl)imino)methyl)-4-hydroxyphenyl)diazenyl)benzenesulfonic acid (L) chemosensor with 78% yield (Scheme 1). L has been characterized by ¹H NMR, ¹³C NMR, FTIR, ESI-mass spectrometry, and elemental analyses. In the IR absorption spectra of the mono-Schiff base ligand L showed a sharp bands 3478, 1620, 1491, and 1174 cm⁻¹, which might be assigned as –OH, –C N, –N N and S O stretching frequencies respectively. In the proton NMR spectra of L, the –SO₃H, –OH, triazole protons and –CH N– proton resonate at 11.40, 10.38, 9.25 and 8.39 ppm respectively.

Scheme 1. Synthetic procedure of the receptor L.

The molecule of L was subjected to DFT calculations. The geometry optimisations begin with the Gauss-view structure of L and end with a global minimum as the stationary level. Fig. 1a depicts the L's optimised structure. Fig. 1b shows the outlines of the chosen HOMO and LUMO orbitals of L as well as a schematic representation of MO energy. For L, the HOMO and LUMO orbitals differ in energy by 2.20 eV.

Fig. 1. (a) Geometry optimized structure and (b) FMO HOMO–LUMO diagram of the molecule L.

3.2. Colorimetric and UV-vis spectral studies

The chromogenic sensing ability of the synthesized chemosensor L towards a variety of biological and environmentally affected ions, including F⁻, Cl⁻, Br⁻, HSO₃⁻, N₃⁻, PO₄³⁻, SO₄²⁻, NO₃⁻, OAc⁻, CO₃²⁻, S²⁻, CN⁻ and OH⁻ was assessed by visually checking the color change by adding 10 equivalents of each of these anions as their inorganic sodium salts to the pure aqueous solution of L at room temperature. In naked-eye experiment addition of only S²⁻ ion induced a significant colour change from light yellow to deep orange in MeOH–water mixture (1 : 1, v/v), whereas the remaining anions, including OH⁻ can cause negligible colour change at the same condition (Fig. 2). This initial finding suggests that the chemosensor L is capable of colorimetrically identifying the sulphide ion in a mixed aqueous solution. Alkali metals, alkaline earth metals, and certain transition metal cations are unable to significantly alter colour, which suggests that there is no binding interaction with them.

Fig. 2. Color changes of L after addition of 10 equiv. of different anions in methanol–water (1 : 1, v/v).

In order to correlate the visual observation spectrophotometrically, a number of selected anions were monitored in an aqueous–methanol (1 : 1) solution after the UV-vis absorption studies of receptor L were carried out. Although the UV-vis spectra of L were taken in different solvents, e.g., H₂O, CH₃OH and 1 : 1 MeOH–H₂O (Fig. S6) better colour and spectral changes have been observed in the solvent mixture 1 : 1 MeOH–H₂O. The absorption bands at 240 and 285 nm are attributed to π–π* transitions of the aromatic framework, whereas the band centered at 346 nm originates from the n–π* transition and intramolecular charge transfer (ICT) associated with the azomethine chromophore (Fig. 3). Upon addition of S²⁻ ions, the absorption band at 346 nm gradually decreased with the simultaneous appearance of a new broad band at 403 nm, accompanied by a visible colour change. In contrast, no significant spectral shift was observed in the presence of other competing anions, including OH⁻. The pronounced spectral response toward S²⁻ suggests that the sensing process is not governed solely by simple basicity-induced deprotonation. Although hydroxide ion is a strong base, its strong hydration in aqueous medium may suppress effective interaction with the receptor L. In contrast, the higher polarizability and nucleophilicity of sulfide ion enable stronger interaction with the phenolic–azomethine framework of L, resulting in efficient modulation of the ICT process and consequent colour and spectral changes. The observed bathochromic shift therefore originates from selective host–guest interaction between receptor L and S²⁻ ions.

Fig. 3. UV-vis spectral responses of L before and after addition of 10 equiv. of different anions in methanol–water (1 : 1, v/v).

To find out how sensitive receptor L is to sulphide ions, a UV-titration experiment was carried out in a pure aqueous medium. The n–π* transition and intramolecular charge transfer band of the receptor's azo moiety caused the probe L to exhibit an absorption peak at 346 nm, while the π–π* transition of the aromatic group induced absorption bands at 240 and 285 nm (Fig. 4). As the concentration of sulphide ions are increased, the absorption peak at 346 nm progressively decreased, and a new absorption peak appeared at 403 nm. Two different isosbestic points at 256 nm and 374 nm indicated that only one stable complex with a certain stoichiometric ratio was prepared upon interaction of the sulphide ion. A distinct absorption band is now produced by the redshift of the absorption peak at 346 nm to 403 nm, denoting an improvement in the efficiency of the ICT process.

Fig. 4. UV-vis titration of L with sulfide ion (0–10 equiv.) in methanol–water (1 : 1, v/v).

The chemosensor L's selectivity for the sulphide ion in the presence of other interfering ions was further investigated using colorimetric and spectral studies in an aqueous–methanol (1 : 1) solution. In order to accomplish this, many receptor solutions containing extra anions (F⁻, Cl⁻, Br⁻, HSO₃⁻, N₃⁻, PO₄³⁻, SO₄²⁻, NO₃⁻, OAc⁻, CO₃²⁻, S²⁻, CN⁻ and OH⁻) were prepared. The intensity of the absorption band produced by the deprotonation activity at 403 nm is completely unaffected by the addition of additional anions (Fig. 5). The colour intensity only slightly decreases due to dilution effects.

Fig. 5. UV-vis spectral responses of L before and after addition of 10 equiv. of different anions in 100% water in methanol–water (1 : 1, v/v).

3.3. Fluorescence spectroscopic studies

The emission properties of receptor L in the presence of several anions (F⁻, Cl⁻, Br⁻, HSO₃⁻, N₃⁻, PO₄³⁻, SO₄²⁻, NO₃⁻, OAc⁻, CO₃²⁻, S²⁻, CN⁻ and OH⁻) were also examined in a methanol–water combination (1 : 1 v/v). When excited at a wavelength of 330 nm (charge transfer envelope), L displayed remarkably low fluorescence intensity (quantum yield Φ = 0.0016). On the addition of S²⁻ ions to the solution of receptor L resulted in significant enhancement of the emission intensity (quantum yield Φ = 0.034) at 428 nm along with slight spectral modulation, indicating strong interaction between S²⁻ and the receptor framework (Fig. 6). The fluorescence enhancement may be attributed to effective perturbation of the ICT process after interaction of sulfide ions with the phenolic–azomethine binding site of L. The interaction likely restricts non-radiative decay pathways and stabilizes the excited-state electronic distribution, leading to enhanced fluorescence emission.

Fig. 6. Fluorescence spectra of L before and after addition of 10 equiv. of various anions in methanol–water (1 : 1, v/v).

On the other hand hydroxide ions did not induce comparable fluorescence changes under identical experimental conditions. This result indicates that the sensing process is not solely dependent on basicity-induced deprotonation. Instead, the selective fluorescence response toward S²⁻ is likely associated with its higher polarizability, stronger nucleophilic character, and favorable host–guest interaction with receptor L in aqueous medium.

A titration experiment was carried out by gradually adding S²⁻ ions in order to obtain the details of the emission spectra. The emission intensity steadily increased up to 21 times when the concentration of S²⁻ ion in the receptor solution was increased, and it became saturated up to 10 equiv. of S²⁻ ion (Fig. 7). Emission intensity increased as a result of the deprotonations from the phenolic proton of L, which likely limited both the non-radiative PET and >C N isomerisation processes.

Fig. 7. Fluorescence titration of L in methanol–water (1 : 1, v/v).

The fluorescence interference tests carried out under the same experimental conditions were used to examine the particular S²⁻ ion selective responses of the chemosensor L. When S²⁻ ions coexist with other major competing anions at equivalent concentrations, independently, the fluorescence intensity of the L–S²⁻ adduct at 428 nm remains unchanged (Fig. S7). Even at background cation concentrations up to two times higher than those of the analytes, the anti-interference behaviour persisted.

3.4. Detection limit and binding constant of L

The UV titration studies revealed the chemosensor L's ratiometric sensing behaviour towards sulphide ions, which increases a chemosensor's dynamical strength by allowing signal rationing between two variable regions. A linear dependency in the range of 10–110 µM was found when the ratio of absorption intensity at (A₄₀₃/A₃₄₆) was plotted as a function of S²⁻ concentration (Fig. 8). Using the formula LOD = 3SD/S, where S is the slope of the calibration curve and SD is the standard deviation of the blank solution, the detection limit of the chemosensor L was also ascertained from this calibration curve. The probe L exhibits a comparatively low detection limit among reported Schiff base-based sulfide chemosensors. The predicted detection limit for S²⁻ ion was determined to be 3.3 × 10⁻⁷ M, which is far below the WHO recommended limits. As a result, the chemosensor L can detect S²⁻ ions at extremely low concentrations.

Fig. 8. Detection limit of L for S²⁻ by ratiometric method.

3.5. Stoichiometry and association constant

Using Job's continuous variation method, the stoichiometric ratio of the receptor L and sulphide ion was found at 403 nm. A 2 : 1 binding relationship between L and the sulphide ion is indicated by the maximum absorbance intensity at the 0.33 mole-fraction of S²⁻ ion (Fig. S8).

Eqn (1) was used to get the association constant using the Hill's plot.

log(A − A₀/A_m − A) = n log[S²⁻] + log K_a 1

A₀ is the absorbance of free receptor L; A is the absorbance at 403 nm at intermediate concentration of sulphide ion and A_m is the highest absorbance obtained at saturation conditions. n is the binding coefficient that offers information about the binding stoichiometry while K_a is the association constant.

The slope and intercept of the Hill plot were 0.0759 ± 0.0053 and −0.0400 ± 0.0124, with acceptable linearity (R² = 0.9372). Plot analysis further confirms the 2 : 1 binding mode between receptor L and sulphide ions. The association constant obtained from Hill analysis was 5.0 × 10⁵ M⁻¹ (Fig. S9), which suggested a robust binding of receptor L to S²⁻.

3.6. pH and time dependence

The pH of the medium plays an important role in determining the sensing performance of receptor L because protonation–deprotonation processes can strongly influence the electronic distribution and intramolecular charge-transfer (ICT) behavior of the probe. Therefore, the absorbance intensity of receptor L at 403 nm was investigated over a pH range of 2–12 in the absence and presence of S²⁻ ions using HEPES buffer solution.

In the absence of S²⁻ ions, receptor L exhibited negligible spectral variation throughout the studied pH range, indicating good stability of the probe under different pH conditions. Notably, even under moderately basic conditions, no significant colour change or absorption band corresponding to the sulfide-bound form was observed, suggesting that hydroxide ions alone are insufficient to induce the spectral response of the receptor.

In the presence of S²⁻ ions, however, a distinct absorption band at 403 nm appeared together with a visible colour change from yellow to reddish orange, particularly in the pH range 4–10 (Fig. 9). At lower pH values (<4), the sensing response was suppressed because sulfide ions predominantly exist in protonated forms (HS⁻/H₂S), reducing their effective interaction with the receptor. The enhanced response observed in near-neutral and mildly basic media indicates favorable interaction between S²⁻ and the phenolic–azomethine framework of L under the experimental condition.

Fig. 9. pH dependence of L.

3.7. Binding mechanism

A ¹H NMR titration experiment was carried out to better understand the binding mechanism (Scheme 2) of probe L with S²⁻ ions. The free receptor, L underwent a chemical shift around 10.38 ppm due to the phenolic proton. When the phenolic groups interacted with sulphide ions, they deprotonated, L, causing this proton to eventually disappear. The negative charge produced when S²⁻ ions deprotonated L was distributed across the entire L molecule, as seen by the aromatic protons moving towards upfield. The position of the proton signals remained unchanged when more than two equivalents of S²⁻ were added to L (Fig. 10).

Scheme 2. Probable sensing mechanism.

Fig. 10. ¹H NMR titration of L in DMSO-d₆/D₂O.

The observed spectral changes suggest that S²⁻ ion interacts strongly with the phenolic–azomethine binding site of the receptor, leading to effective modulation of the intramolecular charge transfer (ICT) pathway. The enhanced ICT process is responsible for the pronounced bathochromic shift and fluorescence enhancement observed experimentally.

No comparable colour or spectral response was observed in the presence of OH⁻ ions under identical experimental conditions. This observation suggests that the sensing process is not controlled solely by simple base-induced deprotonation. Although hydroxide ion possesses strong basicity, its strong hydration in aqueous medium likely suppresses effective interaction with the receptor framework. In contrast, sulfide ion, owing to its higher polarizability and nucleophilicity, can interact more efficiently with the phenolic–azomethine moiety of L, thereby inducing substantial perturbation in the ICT process.

FTIR spectroscopy has also verified this de-protonation mechanism. After isolating the double deprotonated molecule and analysing its FTIR spectrum, we found that the L–S²⁻ adduct's FTIR spectra showed a decrease in the IR absorption peaks of –OH group (3440 cm⁻¹) (Fig. S10). L–H⁺ formed by the deprotonation of L was confirmed to exist using HRMS mass spectrometric analysis (peak obtained at 371.0941, Fig. S11). Thus, it is suggested that the observed colorimetric and fluorescence responses result from a selective host–guest interaction between receptor L and S²⁻ ions, together with partial charge redistribution within the conjugated system. Scheme 2 provides an illustration of the suggested mechanism.

Similar spectral responses were observed when sulfide ions were introduced in the form of different sulfide salts such as K₂S and (NH₄)₂S, confirming that the sensing response originates specifically from S²⁻ ions. The highly selective response of receptor L toward sulfide ions cannot be explained solely on the basis of anion basicity. Instead, the higher polarizability and nucleophilicity of S²⁻, together with its favorable interaction with the receptor binding site, are likely responsible for the observed sensing behavior. DFT analysis further supports the electronic perturbation occurring in the receptor framework after interaction with S²⁻ ions (Fig. S12). Compared to L, the HOMO–LUMO energy gap in L–H⁺ is substantially less (the HOMO–LUMO energy gap = 1.63 eV).

3.8. Reversibility test

Since Hg²⁺ has a very excellent thiophilic character, the reversibility of probe L to S²⁻ ions was assessed using Hg(NO₃)₂ solution. When Hg(NO₃)₂ was added, the orange red-colored L–S²⁻ complex solution turned yellow, indicating the regeneration of receptor L. As soon as a prominent peak developed at 346 nm, the absorption band at 403 nm vanished. Both colour and absorptivity returned once S²⁻ was added again (Fig. 11). The colour changes and switching absorption intensity at 404 and 346 nm were nearly reversible with the subsequent addition of S²⁻ and Hg²⁺ ions, even after several cycles.

Fig. 11. Reversibility study of L for S²⁻ by (a) absorption and (b) emission spectroscopy.

Fluorescence experiments utilising L + S²⁻ solution and Hg(NO₃)₂ as the working media have also confirmed the reversibility. The characteristic emission bands of the host–guest combination at 428 nm disappeared as soon as the L + S²⁻-adduct interacted with two equivalents of Hg(NO₃)₂ (Fig. 11). After the addition of S²⁻, the emission intensity at 426 nm nearly entirely recovered, indicating that the sensing process is more reversible than any irreversible reaction.

4. Application of the chemosensor L

4.1. Molecular logic gate application

Our synthesised chemosensor L can be used as a molecular logic device since distinct absorbance bands were seen when S²⁻ and Hg²⁺ ions were present. Molecular logic gates operate at the nanoscale using spectroscopy for output signals and chemical processes for input inputs. As an output signal, the analyte S²⁻ ion interacted with L to produce absorption band maxima at 346 nm and fluorescence amplification at 428 nm (Fig. 12). When Hg²⁺ is added to the receptor solution, both the fluorescence at 428 nm and the amount of absorption at 403 nm drop once more. As shown in the truth table (Table 1), the chemosensor L can thus display the INHIBIT logic gate function with two inputs, S²⁻ and Hg²⁺ (Fig. 12). The output at 428 nm is only 1 when S²⁻ is present; otherwise, it is 0.

Fig. 12. Logic scheme for the proposed INHIBIT type logic gate.

Table 1. The truth table for the proposed INHIBIT type logic gate.

4.2. Application of chemosensor L in real samples

Since sulphide ions from waste water from the food and chemical industries can contaminate conventional water supplies, one of the most significant applications for the chemosensor L is the detection of S²⁻ ions in water samples. This was accomplished by creating artificial sulfide-contaminated samples by spiking several known concentration levels. The concentrations of these samples were then detected using the recommended sensing technique. Table 2 demonstrates a strong recovery and extremely low standard deviation for each sample used in the experiment three times.

Table 2. Determination of sulfide in different water samples.

Anion	Spiked amount (µM)	Recovered amount (µM)	% Recovery ± SD (n = 3)
S2−	5	4.87	97.4 ± 0.78
	10	10.18	101.8 ± 1.44
	20	19.93	99.65 ± 1.7

4.3. Molecular docking studies

4.3.1. Binding affinity analysis

The docking study showed that the ligand can fit properly inside the active site of the protein. The best docking pose gave a binding energy of −9.4 kcal mol⁻¹, which means the interaction between ligand and protein is strong and stable. Because the binding energy value is low (more negative), it indicates that the complex formed is stable and the compound may show good biological activity. Other docking poses also gave similar binding energy values, between −9.1 and −7.2 kcal mol⁻¹. These close values suggest that the docking results are reliable and the ligand consistently interacts well with the protein active pocket.

4.3.2. Protein–ligand interaction analysis

The study of the best docked structure shows that the ligand properly enters into the binding pocket of the protein. It fits inside the cavity in a stable manner. The stability of the ligand–protein complex mainly comes from hydrogen bonds, π-interactions and hydrophobic contacts. One conventional hydrogen bond was found between the ligand and the amino acid Phe1045, and the bond distance was around 2.5–2.6 Å, which is considered good for a strong hydrogen bond. The ligand also forms π–π stacking interaction with the aromatic residue Phe1045. Along with this, π–alkyl type interactions were observed with Leu1033, Leu838, Val914 and Val846 residues. There is also a π–cation interaction with Arg1049 and a π–sulfur interaction with Cys1043, which further helps to stabilize the complex. All these interactions together suggest that the ligand is strongly held inside the active site region of the protein and does not move easily.

4.3.3. Structure–activity relationship

The ligand shows strong binding energy with the target protein. The hydrogen bond distance is also in a suitable range, and there are several hydrophobic and π–π interactions present. The hydrogen bond distances between 2.5 to 3.6 Å suggest that proper electrostatic interaction is happening, which is important for good biological activity. Overall, from the docking study, it can be said that the ligand fits well inside the protein receptor binding site and may have good bioactive potential. The molecular docking interactions of the ligand with VEGFR-2 are illustrated in Fig. 13. Detailed docking parameters and interaction profiles are summarized in Table 3.

Fig. 13. Molecular docking interaction of ligand (L) with vascular endothelial growth factor receptor (VEGFR-2, PDB ID: 2OH4), showing (a) hydrogen bonding and (b) key π-interactions within the active site.

Table 3. Summary of molecular docking parameters and key interactions of ligand (L) with VEGFR-2 (PDB ID: 2OH4).

Parameter	Observation
Protein (PDB ID)	2OH4
Best binding energy (kcal mol−1)	−9.4
Docking software	AutoDock Vina & Biovia discovery studio visualizer
Key hydrogen-bonding residue	Phe1045
H-bond distance (Å)	2.5–2.6 Å
π–π stacking interactions	Phe1045
π–cation interaction	Arg1049
π–sulfur interaction	Cys1043
π–alkyl interactions	Leu1033, Leu838, Val914, Val846, Ala864
Overall interaction type	Hydrogen bonding + π + hydrophobic
Bioactivity inference	Strong (low binding energy and optimal H-bonding)

4.4. Minimum inhibitory concentration

Minimum inhibitory concentration (MIC) provides a quantitative measure of the lowest concentration of an antimicrobial agent required to inhibit visible microbial growth. In this study, the triazole based ligand sample, L was evaluated for its antimicrobial activity against Escherichia coli, Klebsiella sp., Pseudomonas sp., Bacillus sp., and Staphylococcus aureus using the resazurin-based microdilution assay. The results demonstrated that L inhibited bacterial growth in a dose-dependent manner (Fig. 14 and Table 4).

Fig. 14. Resazurin colorimetric response in a 96-well MIC assay demonstrating the antimicrobial activity profile of the test sample.

Table 4. Dose-dependent growth inhibition of Gram positive and Gram negative bacteria by L^a.

Concentration	Types of microbes
	E. coli		Klebsiella		Pseudomonas		Bacillus		S. aureus		NC	GC
GC	+	+	+	+	+	+	+	+	+	+	−	+
1.50 µg mL−1	+	+	+	+	+	+	+	+	+	+	−	+
3.12 µg mL−1	+	+	+	+	+	+	+	+	+	+	−	+
6.25 µg mL−1	−	−	−	−	+	+	+	+	+	+	−	+
12.5 µg mL−1	−	−	−	−	−	−	+	+	+	+	−	+
25 µg mL−1	−	−	−	−	−	−	+	+	+	+	−	+
50 µg mL−1	−	−	−	−	−	−	−	−	−	−	−	+
100 µg mL−1	−	−	−	−	−	−	−	−	−	−	−	+

^a(+) growth bacteria at give concentration, (−) no growth bacteria/inhibition of growth at give concentration.

For E. coli, wells containing 1.56–3.12 µg mL⁻¹ appeared pink, indicating active bacterial growth. Wells with 6.25–100 µg mL⁻¹ remained purple/blue, reflecting complete inhibition. Therefore, the MIC for E. coli was 6.25 µg mL⁻¹. For Klebsiella, wells with 1.56–3.12 µg mL⁻¹ showed a pink colour (growth), whereas wells with 6.25–100 µg mL⁻¹ retained a purple/blue colour, indicating inhibition. The growth and sterility controls performed as expected, validating the assay. For Pseudomonas, pink coloration was observed in wells containing 3.12–6.25 µg mL⁻¹, confirming bacterial growth. Complete inhibition occurred at 12.5–100 µg mL⁻¹ (blue/purple wells). Thus, the MIC for Pseudomonas was 12.5 µg mL⁻¹. For Bacillus, bacterial growth (pink colour) was observed at 1.56–25 µg mL⁻¹, while complete inhibition (blue wells) occurred at 50–100 µg mL⁻¹. Hence, the MIC for Bacillus was determined to be 50 µg mL⁻¹. For Staphylococcus aureus, wells containing 1.56–25 µg mL⁻¹ were pink, indicating growth, whereas those at 50–100 µg mL⁻¹ remained blue, indicating complete inhibition. The MIC for S. aureus was thus 50 µg mL⁻¹.

Based on the resazurin-based MIC analysis, the L sample demonstrated substantial antimicrobial activity against both Gram-negative and Gram-positive bacteria. The compound exhibited the highest efficacy against Escherichia coli and Klebsiella sp. (MIC: 6.25 µg mL⁻¹), along with moderate activity against Pseudomonas sp. (MIC: 12.5 µg mL⁻¹). In contrast, higher MIC values (50 µg mL⁻¹) were observed for the Gram-positive strains Bacillus sp. and Staphylococcus aureus, indicating comparatively lower susceptibility. Overall, the dose-dependent inhibitory pattern confirms the antimicrobial potential of the probe L, particularly against Gram-negative bacterial pathogens.

5. Comparison of the performance of the proposed chemosensor L with other previously reported chemosensors for S²⁻ ions

In this work, a novel fluorescent–colorimetric probe L was developed and validated to detect and quantify S²⁻ ions in an aqueous-methanol media. This study contributes to our continuous efforts to develop practical analytical methods that may be used to track the growing number of analytes of environmental concern as fast, cheaply, and sensitively as possible. S²⁻ ions have been selectively detected in a number of studies using optical techniques and fluorescent colorimetric sensors. The reported chemosensors and our reported probe L are compared in Table 5. While previous chemosensors have shown some advantages, such high sensitivity and selectivity, the main characteristics of the fluorescent–colorimetric chemosensor, L, are its simple, useful, and affordable synthesis, selectivity, and sensitivity.

Table 5. Comparison with some previously reported chemosensors for S²⁻ ions.

S. no.	Molecular structure	Sensor type	Probe	Method	Solution media	LOD	Stoichiometry	Ref.
1		Fluorescent	Salicylaldehyde based		Aqueous solution	18 nM	1 : 1	58
2		Fluorescent	Coumarin based	On–off–on		52.2 nM	1 : 1	59
3		Colorimetric and fluorometric	N-squaraines based	Turn-on	1 v/v 105 M DMSO–H2O (0.1 M NaOH) solution	3σ	1 : 2	60
4		Fluorescent	Thiadiazol based		Deep eutectic solvent (DES)	1.76 × 10−4 M	1 : 2	61
5		Colorimetric and fluorometric	Salicylaldehyde based	Turn-on	Ethanol	8.48 µM	1 : 2	62
				Turn-off
6		Fluorescent	Chlorosalicylaldehyde based		CH3 OH : H2O	10−5 M	1 : 1	63
7		Fluorescent	2-Hydroxybenzylidene based	On–off–on	DMF/H2O tris buffer	1.20 × 10−8 M	1 : 2	64
8		Colorimetric and fluorometric	Triazole based	Off–on–off	CH3 OH : H2O (1 : 1)	3.3 × 10−7 M	2 : 1	Present work

6. Conclusions

In summary, a novel triazole-based azo-Schiff base chemosensor (L) has been successfully developed for the selective and sensitive detection of S²⁻ ions in aqueous methanol medium through dual colorimetric and fluorescence responses. The probe exhibited a rapid and distinct optical response toward S²⁻ ions with excellent sensitivity, good reversibility, and favorable pH tolerance under near physiological conditions. UV-vis, fluorescence, FTIR, and ¹H NMR and DFT studies collectively suggest that the sensing process involves selective interaction of sulfide ions with the phenolic-azomethine framework of receptor L, resulting in modulation of the intramolecular charge transfer (ICT) pathway. Importantly, other competing anions, did not induce comparable spectral or colour changes under identical conditions, demonstrating the remarkable selectivity of the probe toward sulfide ions.

The practical applicability of the chemosensor, L was validated through successful detection of S²⁻ ions in real water samples with high recovery efficiency and low standard deviation. In addition, the reversible switching behavior observed in the presence of Hg²⁺ ions demonstrates the potential utility of the system in molecular logic gate applications and recyclable sensing. Moreover, the probe L, displays promising biological potential, supported by strong molecular docking interactions with VEGFR-2 and notable antimicrobial activity against both Gram-negative and Gram-positive bacteria. The integration of sensing capability, biological activity, and logic gate behavior establishes L as a multifunctional material with significant potential in environmental analysis, biomedical research, and smart sensing systems.

Author contributions

Devanand Sahu: conceptualization, investigation, methodology, Abhilash Pandey: formal analysis, investigation, Srishti Dutta: visualization, Dishen Kumar: investigation, Annpurna Sahu: validation, Goutam Kumar Patra: writing the manuscript, supervision, conceptualization and funding.

Conflicts of interest

Authors declare no conflicts of interest.

Data availability

The data supporting this article have been included as part of the supplementary information (SI). Supplementary information: Fig. S1–S12. See DOI: https://doi.org/10.1039/d6ra03741k.