Bis-thiosemicarbazone functions as a selective chemosensor with the lowest LOD for Hg ions in water-rich medium: implications for on-site detection and logic gate experiments

Abstract

In this investigation, a mesitylene-anchored bis-thiosemicarbazone (TOMH), with the potential to chelate a metal ion via S,S donor atoms, was used as a chemosensor for the selective detection of mercury ions in an aqueous medium from mixtures with several metal ions (Ag, Al, Ba, Bi, Ca, Cd, Co, Cr, Cs, Cu, Fe, K, Li, Mg, Mn, Na, Ni, Pb, Th, and Zr). Electronic absorption spectroscopy revealed the composition of the identified species to be Hg(S,S-TOMH)·(NO₃)₂ (TOMHN). The spectroscopic (¹H NMR, ¹³C NMR, and FT-IR), theoretical (DFT and TD-DFT) and structural techniques confirmed the bonding of TOMH to Hg through S donor atoms (Hg–S: 2.484–2.490 Å and S–Hg–S bond angle: 160.1°), while the nitrates are non-bonded. The TOMH chemosensor has the lowest limit of detection (LOD: 10.97 nM) for Hg ions in a water-rich medium (90% H₂O : 10% DMSO) among thiosemicarbazones or closely related S donor chemosensors. It exhibits multiple applications, such as on-site detection of Hg using filter paper strips, formation of TOMH–Hg–EDTA-supported INHIBIT logic gate, and enhanced antimicrobial activity of TOMHN relative to TOMH or Hg(NO₃)₂ alone.

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1 Introduction

Environmental pollution is among the most challenging issues confronting human society today. One cause of pollution is linked to the presence of heavy metals, which may threaten various living organisms owing to their toxic nature.¹ For instance, mercury, as its inorganic salts or organomercury derivatives,^2–5 disrupts protein and enzyme functions and affects the kidneys and brain.^6–15 The acceptable limit of mercury in drinking water is 10 nM as per the US Environmental Protection Agency.^16,17 The burning of fossil fuels, incineration of garbage, mining, and natural volcanic eruptions all contribute to the rise of Hg contamination.¹⁸ Thus, in order to monitor mercury in the environment, several analytical techniques, such as chromatography, high-performance liquid chromatography (HPLC), inductively coupled plasma-mass spectrometry (ICP-MS), flow injection technologies, and atomic absorption spectroscopy (AAS), have been used.^19–28 Further, colorimetric techniques, being easily accessible due to their low cost, have played a significant role as chemosensors in the selective detection of metal ions.^29–35 Our group has used colorimetric techniques for the detection of analytes consisting of metal ions (Cu and Al), neutral molecules (picric acid) or anions (F⁻ and CN⁻) with the help of chemosensors based on a variety of aromatic platforms containing imine/hydroxyl, hydrazone, thiourea and other functional groups.^36–44

It is known that thiosemicarbazones, a distinct class of organic compounds, can chelate metal ions through their N,S or S donor atoms and thus serve as cost-effective chemosensing materials.^45–49 In the literature, there are several reports of mono-thiosemicarbazone-based chemosensors for detecting mercury at nanomolar levels in aqueous media. However, in these cases, the aqueous medium generally requires a higher proportion of organic solvent with higher LODs (approx. 31 to 900 nM).^50–53 There is another class of chemosensors based on pyrazole-, thiourea-, and napthothiazole-type organic compounds used for the detection of mercury, and their LODs range from 21 nM to 69.02 µM.^54–70 In these cases, it is also observed that the aqueous medium requires a higher proportion of organic solvent. Limited studies of Hg detection in water medium alone have been reported in the literature.^55,71 This survey (Table S1) prompted us to seek alternative chemosensors with the primary goal of detecting Hg at lower LODs in predominantly water-containing media. For this purpose, a bis-thiosemicarbazone (Fig. 1, TOMH) with multiple electron-rich donor centers and two arms positioned in cis-orientation was selected for the present studies.

Fig. 1 2D structure of TOMH.

In this investigation, TOMH is utilized to detect Hg(II) ions in aqueous medium, and the studies include stoichiometry, bonding modes, and structural aspects of the detected species, characterized through electron absorption spectroscopy, NMR spectroscopy, theoretical techniques (DFT, TD-DFT), and crystallography. The findings indicate successful detection of Hg at the nano level in predominantly water medium, on-site Hg detection using sensor-coated paper strips, and enhanced antimicrobial activity of the detected species (Hg(S,S-TOMH)·(NO₃)₂) compared to that of TOMH or Hg salt alone.

2 Experimental section

2.1. Materials and techniques

All the commercially available chemicals were purchased from Aldrich and used without further purification. Standard methods were used to dry all of the solvents. TLC was carried out on glass sheets pre-coated with silica gel. The ¹H and ¹³C NMR spectra were carried out in DMSO-d₆ with TMS as an internal reference on a Bruker Topspin-FTNMR-500 MHz spectrometer. The infrared spectra (KBr pellet) were recorded using a PerkinElmer FT-IR C92035 spectrophotometer in the range 400–4000 cm⁻¹. An SDFCL digital pH meter was used to measure the pH of the solutions. The electronic absorption spectra were recorded on a Shimadzu Pharmaspec UV-1900 UV-Vis spectrophotometer with a quartz cuvette (path length 1 cm). The absorption spectra were recorded between 1100 and 200 nm. The cell holder of the spectrophotometer was thermostatic at 25 °C for consistency in the recordings. Microwave reactions were carried out using the Anton-Par Monowave 200 microwave synthesis reactor operating at 200 W.

2.2. Synthetic procedures

2.2.1. Synthesis of (2Z,2′Z)-2,2′-(((((2,4,6-trimethyl-1,3-phenylene) bis(methylene))bis(oxy))bis(2,1-phenylene))bis(methaneylylidene))bis(N-methylhydrazine-1-carbothioamide) (TOMH) (Scheme S1). A previously documented method^42,72 was modified to synthesize TOMH to improve yield and reduce reaction time. A mixture of 0.0108 g (1.027 mmol) of 3-methylthiosemicarbazide and 0.2 g (0.5152 mmol) of aldehyde (2,2′-(((2,4,6-trimethyl-1,3-phenylene)bis-(methylene))bis(oxy))dibenzaldehyde) suspended in 7 mL of ethanol was placed in a microwave vial and irradiated for ten minutes. After filtration and multiple washings with ethanol, the precipitate was vacuum dried for an hour. Yield: 0.280 g (97%). The product is a very pale yellow solid with a melting point of 150–152 °C (Fig. S1–S6).

2.2.2. Synthesis of Hg(NO₃)₂(TOMH) complex (TOMHN). A 10 mL solution of Hg(NO₃)₂ (0.746 g, 2.30 mmol) in methanol was added as a suspension to a 10 mL solution of TOMH (0.647 g, 1.15 mmol) in dichloromethane. The mixture was then refluxed for an hour. The reaction mixture was stirred for eight hours and then concentrated. Pale yellow precipitates were collected on a Buchner funnel. These precipitates were recrystallized from a mixture of CH₂Cl₂, EtOH, and CH₃CN. Yield: 0.853 g (82%). The product is a very pale yellow solid with a melting point of 188–190 °C. IR (KBr, cm⁻¹) 3202, 2919, 2838, 1596, 1445, 1300, 1233, 1156, 1111, 1073, 1026, 987, 845, 749, 562; ¹H NMR (500 MHz, DMSO-d₆) (δ): 2.29 (t, 3H, –CH₃, J = 10.0), 2.34 (s, 6H, –CH₃), 3.05 (s, 6H, –CH₃), 5.16 (s, 4H, –CH₂), 6.96 (s, 1H, –Ar), 7.02 (s, 2H, –Ar), 7.08 (t, 2H, –Ar, J = 10.0), 7.37 (m, 2H, –Ar), 7.55 (t, 2H, –Ar, J = 10.0), 8.09 (d, 2H, J = 5), 8.39 (s, 2H, –CH=N), 9.39 (s, 2H, –NH), 11.76 (s, 2H, –NH); ¹³C NMR (500 MHz, DMSO-d₆) (δ): 15.45 (–CH₃), 19.83 (–CH₃), 32.06 (–N–CH₃), 65.65 (–CH₂), 113.45 (–Ar), 121.27 (–Ar), 122.20 (–Ar), 126.88 (–Ar), 130.41 (–Ar), 131.31 (–Ar), 132.71 (–Ar), 138.82 (–CH=N), 139.68 (–Ar), 141.93 (–Ar), 158.33 (–C–O), 173.42 (–C=S) (Fig. S7–S11).

2.2.3. Synthesis of HgI₂(TOMH) (TOMHI). A 10 mL solution of HgI₂ (1.0451 g, 2.30 mmol) in MeOH was added slowly to a 10 mL solution of TOMH (0.647 g, 1.15 mmol) in CH₂Cl₂, followed by refluxing for an hour. The reaction mixture was stirred overnight and allowed to evaporate. Pale yellow precipitates were collected on a Buchner funnel. These precipitates were recrystallized from a mixture of CH₂Cl₂, EtOH, and CH₃CN and formed fine crystals. Yield: 0.821 g (70%). The product is a white solid with a melting point of 193–195 °C. IR (KBr, cm⁻¹) 3287, 3207, 2914, 1572, 1238, 1087, 992, 855, 751; ¹H NMR (500 MHz, DMSO-d₆) (δ): 2.31 (s, 3H, –CH₃), 2.34 (s, 6H, –CH₃), 3.07 (d, 6H, –CH₃, J = 4.2), 5.15 (s, 4H, –CH₂), 7.04 (m, 2H, –Ar), 7.37 (d, 2H, –Ar, J = 5.0), 7.49 (t, 2H, –Ar, J = 5.0), 8.11 (d, 2H, J = 10.0), 8.38 (s, 2H, –CH=N), 8.99 (s, 2H, –NH), 11.54 (s, 2H, –NH); ¹³C NMR (500 MHz, DMSO-d₆) (δ): 15.34 (–CH₃), 19.77 (–CH₃), 31.98 (–N–CH₃), 65.71 (–CH₂), 113.54 (–Ar), 121.11 (–Ar), 122.20 (–Ar), 126.84 (–Ar), 130.62 (–Ar), 131.27 (–Ar), 132.67 (–Ar), 134.29 (–Ar), 138.75 (–CH=N), 139.65 (–Ar), 158.31 (–C–O), 174.25 (–C=S) (Fig. S12–S15).

2.3. Spectral analysis of TOMH using UV-vis studies

A stock solution of TOMH (1 mM) was prepared in DMSO solvent. Similarly, stock solutions of various metal ions (also 1 mM), including Ag(I), Al(III), Ba(II), Bi(III), Ca(II), Cd(II), Co(II), Cu(II), Cr(III), Fe(III), Hg(II), K(I), Mg(II), Mn(III), Ni(II), Na(I), Pb(II), Zn(II), etc., were prepared by dissolving the corresponding salts in double-distilled water. The interaction of TOMH with these metal ions was investigated using UV-vis spectroscopy in 10⁻⁵ M solutions in DMSO.

2.4. X-ray crystallography

The crystals of the complex HgI₂(TOMH) (MF: C₃₃H₃₇HgI₂N₈O₂S₂) were grown by slow evaporation from a mixture of dichloromethane, acetonitrile, and ethanol. A suitable crystal was selected and mounted on a Bruker APEX4 diffractometer. The crystal was maintained at 296.15 K during data collection. Using Olex2,⁷³ the structure was solved with the SHELXT⁷⁴ structure solution program utilizing Intrinsic Phasing and refined with the SHELXL refinement package using least squares minimization.

2.5. Computational methodology

The 3D structures of the TOMH ligand and its complexes, TOMHN (nitrate complex) and TOMHI (iodide complex) were optimized in gas phase followed by frequency analysis of the same at the B3LYP/gen level of quantum theory. All calculations were carried out using the Gaussian16 suite of programs.^75–79 C, H, N, S, and O atoms were optimized using a 6-311++G(d,p) basis set, and the def-2tzvpp basis set with the effective core potential (ECP) function was used to optimize heavier atoms Hg and I. TD-DFT was carried out to predict the UV-Vis energy spectra of the TOMH and TOMHN complexes using the B3LYP/6-311++G(d,p)/def-2tzvpp method. The energies of the highest occupied molecular orbital (HOMO, ΔE_H), lowest unoccupied molecular orbital (LUMO, ΔE_L), and their associated HOMO–LUMO gap (ΔE_H–L) were estimated using the same basis set. Chemical descriptors such as chemical potential (µ), electronegativity (χ), hardness (η), softness (S), and electrophilicity (ω) were estimated for electronic transitions between ligand TOMH and associated complexes (TOMHN and TOMHI) as seen in Schemes S2–S6.^80–84 The molecular electrostatic potentials (MEP) of TOMH, TOMHN, and TOMHI were generated using the iop (6/7 = 3) function, and Frontier molecular orbital (FMO) diagrams of TOMH, TOMHN and TOMHI were obtained in implicit solvent (IEFPCM, DMSO and H₂O) model.

2.6. Antimicrobial activity

The antimicrobial activity^85–88 of the Hg(NO₃)₂·(TOMH) complex was evaluated against two Gram-negative (E. coli and P. syringae) and two Gram-positive bacteria (B. subtilis and S. aureus). An inoculum of 100 µL of bacterial suspension (∼1 × 10⁶ CFU mL⁻¹) was inoculated into 2 mL of Luria-Bertani (LB) broth, followed by the addition of TOMHN (5 µM), TOMH (5 µM), and Hg(NO₃)₂ (5 µM). The cultures were incubated overnight under specific conditions: 37 °C for E. coli and S. aureus, 30 °C for B. subtilis, and 28 °C for P. syringae. Kanamycin (1 mg mL⁻¹) was taken as the reference antibiotic for the study. The optical density (OD) was measured at 600 nm, and antimicrobial activity was calculated using the following formula:

All experiments were performed in triplicate, and results are presented as mean ± standard error. Statistical significance was analyzed using one-way ANOVA, with a significance threshold of p < 0.05.

3 Results and discussion

In this section, the interaction of TOMH with mercury salt is described. The discussion covers (a) a variety of photophysical properties, including the effects of pH, water content, selectivity towards Hg(II), interference from other metal ions under study, stoichiometry of interaction, stability constant, detection limit, and reversible nature of the sensor, and (b) spectroscopic, structural, and computational studies.

3.1. Photophysical studies of TOMH–Hg interaction

To develop a versatile sensor, the tolerance of TOMH towards water and the effect of water on its detection efficiency for Hg(II) ions in a Hg(NO₃)₂ solution were examined. Titration experiments involving water were performed using UV-Vis spectroscopy in DMSO solvent spanning water fractions from 0% to 90% (Fig. 2a). The appearance of a new peak at 372 nm across the water fraction range indicated the formation of a new species, probably TOMHN (Fig. 3a). After reaching 90% water content, the absorbance sharply decreases, ruling out the formation of the TOMHN complex beyond this level. Thus, it is concluded that the sensor properties of TOMH are operative in a DMSO medium with water tolerance from 0% to 90%.

Fig. 2 Effect of (a) water fraction and (b) pH on the absorbance of TOMH and its solution with Hg(NO₃)₂ at 372 nm.

Fig. 3 (a) Changes in the absorbance spectrum of TOMH in DMSO : HEPES (1 : 9) (10 µM) upon the gradual addition of Hg(II). (b) Color changes in the sensor solution with Hg(II) under a UV lamp.

In the next phase, the effect of pH changes on the sensitivity of the TOMH sensor was examined in the pH range 2–12 using UV-visible spectroscopy (Fig. 2b) (the pH of the medium was altered using HCl or NaOH). The analysis of the pH-induced changes of the diagnostic 372 nm band, presumably of TOMHN complex, showed that TOMH was pH-insensitive between 2.07 and 10.30. In contrast, there was a notable increase in TOMHN intensity at pH 4.05, and thereafter the intensity remained relatively stable until pH 11.03. Significantly, this is the accepted range for studies of several biological systems. Therefore, the water content was maintained at 90% in the DMSO–water solvent system at pH 7.4 (DMSO : H₂O, 1 : 9, v/v) throughout the experiment.

The electronic absorption spectrum of TOMH in HEPES buffer (pH 7.4, containing 10% DMSO as a co-solvent) exhibits three absorption bands, centred at λ_max = 246 nm (ε_max = 81 800 M⁻¹ cm⁻¹) assigned to a π → π* electronic transition; λ_max = 305 nm (ε_max = 94 100 M⁻¹ cm⁻¹) due to n → π* (–C=N–) transition, and λ_max = 335 nm (ε_max = 124 500 M⁻¹ cm⁻¹) ascribed to another n → π* (–C=S–) transition^89,90 (Fig. S16). With the gradual addition of Hg(II), the electronic environment of TOMH changed, thereby altering its optical properties. The high-energy band at λ_max 246 nm increased in intensity, with the appearance of an isobestic point at 358 nm and a new band at λ_max 372 nm (ε_max = 88 200 M⁻¹ cm⁻¹) (Fig. 3a). The appearance of an isobestic point supported the formation of a 1 : 1 complex, Hg(NO₃)₂(TOMH). This optical density change was accompanied by a color change from yellow to colorless (Fig. 3b).

Further, screening of TOMH with various metal ions under study, such as Al(III), Ag(I), Ba(II), Bi(III), Ca(II), Cd(II), Co(II), Cr(III), Cs(I), Cu(II), Fe(III), K(I), Li(I), Mg(II), Mn(II), Na(I), Ni(II), Pb(II), Th(IV), and Zr(IV), did not show any visible effect. However, there was a small effect with Zn(II), indicating a weak interaction, as it belongs to the same d¹⁰ group (Fig. 4a). TOMH reliably detects Hg(II) ions without interference from other metal ions, as demonstrated by tests in which 100 µM solutions of various metal ions under study were added to a mixture of 10 µM TOMH and 50 µM Hg(II) which showed no significant spectral changes (Fig. S17). A Job's plot⁹¹ determined the stoichiometry of the interaction of Hg(II) with TOMH. For solutions with mole fractions from 0.1 to 0.9 of Hg(II), the absorption values are shown in Fig. 4b. From this curve, it can be seen that, at the highest absorbance, the mole fraction is 0.5, which suggests the formation of a 1 : 1 complex, namely, Hg(NO₃)₂(TOMH).

Fig. 4 (a) Effect of the addition of different metal ions (100 µM) on the spectral changes at λ_max = 246 nm of TOMH (10 µM) in a HEPES buffer (pH 7.4, containing 10% DMSO as a co-solvent). (b) Job's plot of TOMH with Hg(NO₃)₂.

The Benesi–Hildebrand plot,⁹² obtained by titrating TOMH with Hg(II), was used to calculate the binding constant, which was 7.65 × 10⁴ M⁻¹ (Fig. 5a). The limit of detection (LOD) of the TOMH sensor, which indicates its sensitivity, was calculated from the standard deviation (σ) of absorbance in the TOMH solution without analyte, followed by titrations with varying Hg(II) concentrations (Fig. 5b). Using the formula 3σ/k, where k is the slope of absorbance vs. concentration, the LOD was determined. The World Health Organisation (WHO, 2011) states that drinking water typically contains about 0.001 mg L⁻¹ of mercury.⁹³ Interestingly, TOMH can detect Hg levels as low as 0.01097 µM (10.97 nM), which is less than the WHO maximum permissible level.

Fig. 5 (a) Benesi–Hildebrand plot for the stability constant of TOMH with Hg(II) from the absorbance data. (b) Calibration plot of TOMH with Hg(II) in a HEPES buffer (pH 7.4, containing 10% DMSO as a co-solvent).

To demonstrate reversibility, a 1 : 10 mixture of TOMH and Hg(II) was titrated with 100 µM EDTA, showing increased absorbance at 335 nm upon EDTA addition, indicating TOMH regeneration (Fig. 6). This reproducibility was further confirmed by reintroducing Hg(II) ions. Investigations into logic gates⁹⁴ confirmed TOMH's reversible chromogenic response, enabling the construction of an INHIBIT logic gate with two inputs: EDTA and Hg(II) ion. An “OFF” or “0” state occurs when both inputs are absent or when only EDTA is present. An “ON” or “1” state occurs with a notable decrease in absorbance at 335 nm when only Hg(II) is introduced, which is consistent with the INHIBIT logic gate architecture. For environmental testing, TOMH demonstrated strong selectivity for Hg(II), as indicated by colorimetric analysis, which is corroborated by a solid-state study of filter paper strips. This experiment shows a color change (Fig. S18) from yellow to nearly colorless upon interaction with aqueous Hg(II). The strips demonstrated excellent stability, showing no color change even after two weeks.

Fig. 6 (a) Schematic of an INHIBIT logic gate and (b) truth table. (c) UV-vis spectra and (d) visual color outputs of TOMH with Hg(II) and EDTA solutions.

3.2. Spectroscopic, structural, and computational studies

In order to further understand the interaction of the TOMH sensor with Hg(II) ions, NMR titrations were performed in DMSO-d₆ (Fig. 7, 8, and S19). The hydrazinic (=N–NH^o–) and thioamide protons {–NH^q–C(=S)–} of free TOMH appeared at δ 11.42 and δ 8.40 ppm and shifted to the low field region at δ 11.76 and δ 9.39 ppm, respectively. This result clearly supported the coordination of TOMH to Hg(II) ion through S donor atoms (Fig. 7). Signals for other protons of TOMH, namely –CH=N group (δ 8.30 ppm) and methyl protons {(CH₃)–NH^q–C(=S)–} (δ 2.99 ppm), showed small low field shifts to δ 8.39 and 3.05 ppm when TOMH binds to Hg(II). The methyl protons on the mesitylene ring appear as two distinct signals, in both the TOMH and TOMHN complex, with a 2 : 1 intensity ratio. The single methyl group (C6) of the TOMHN complex shifts upfield, from δ 2.39 ppm to δ 2.28 ppm, compared to the other two methyl groups (C5 at δ 2.36 ppm). This upfield shift of the C(6) protons probably results from the diamagnetic effect of the neighbouring mesitylene ring in the complex (Fig. 8a). The aromatic protons in the range of δ 8.13–δ 7.00 ppm also shift downfield, while the methylene protons remain almost unchanged (Fig. 8b). In the ¹³C NMR of TOMH, the –C=S peak at δ 177.77 ppm shifted to δ 173.04 in the TOMHN complex (Fig. S19). Both the ¹H and ¹³C NMR data very clearly support the coordination of TOMH to Hg forming the complex Hg(S,S-TOMH)·(NO₃)₂.

Fig. 7 Overlay of the partial ¹H NMR spectra of TOMH and TOMHN in DMSO-d₆ (range: δ 12.2–δ 6.9 ppm).

Fig. 8 Overlay of the partial ¹H NMR spectra of TOMH (5 mM) with increasing amounts of Hg(NO₃)₂ and the solid TOMHN complex in the ranges of (a) δ 3.10–δ 2.25 ppm and (b) δ 12.2–δ 6.9 ppm.

The significant IR spectral bands (Fig. S20) of the TOMHN complex and TOMH are listed in Table S2. The interaction of mercury with TOMH affects several bands. In the TOMH-mercury nitrate complex, viz. TOMHN, the coordination of Hg to two sulfur functional moieties of TOMH shifts the diagnostic ν(C=S) band from 1082 cm⁻¹ to 1026 cm⁻¹. Additionally, other groups, such as ν(N–H) and ν(C–H), which fall in the vicinity of the HgS₂ coordination core are also affected. The hydrazinic ν(N–H) stretching mode (=N–NH^o–) of TOMH around 3329 cm⁻¹ shifts to a lower energy, appearing at 3202 cm⁻¹ in TOMHN. Similarly, the ν(C–H) bands shift to lower energy in the TOMHN complex. The appearance of an IR band due to ν(N–O) at 1300 cm⁻¹ suggests the ionic character of the complex Hg(TOMH)·(NO₃)₂. Other bands, including ν(C=N), δ(N–H), ν(C–N), δ(C–H), and ν(C–O), appear at different positions in the TOMHN complex compared to in the free ligand.

When establishing the formation of Hg–S and Hg–O bonds (if any from NO₃) in the TOMHN complex, the lack of single-crystal formation of Hg(TOMH)(NO₃)₂ due to the amorphous nature of mercury nitrate (Fig. S11) prevented an X-ray structural study. Nonetheless, a computational study of both the chemosensor TOMH and its nitrate complex, TOMHN, was conducted to understand the bonding modes of TOMH with mercury(II) nitrate. The 3D optimized geometry of TOMH is shown in Fig. 9a, while that of its nitrate complex, TOMHN, is shown in Fig. 9b. Although not coplanar, both thiosemicarbazone arms of TOMH are in cis-orientation, enabling effective metal binding.

Fig. 9 3D optimized geometries of (a) TOMH and (b) TOMHN.

The observed –C=S bond length of 1.676 Å indicates the nearly double-bond character of this moiety of TOMH. In the nitrate complex, TOMHN, mercury is bonded to two sulfur donor atoms with S–Hg bond lengths of 2.484–2.490 Å and an effective coordination number of two, with an S–Hg–S bond angle of 160.1°. The –C=S bond lengths in TOMHN, between 1.735 and 1.745 Å, are longer than in TOMH, suggesting a weakening of the pπ–pπ bonding of the –C=S groups when sulfur binds to Hg(II). Additionally, one –NO₃ group is near the –CH and –NH protons, participating in intermolecular H-bonding, which helps stabilize the nitrate complex (Fig. 9b, average O⋯H bond lengths ranging from 1.815 to 2.969 Å). The Hg⋯O (NO₃) bond distances, 3.439 Å and 3.767 Å, are beyond the sum of the van der Waals radii for Hg and O (3.00 Å),⁹⁵ indicating that uncoordinated nitrate ions remain close to Hg as H-bonded species (Fig. 9b).

The crystal structure of the related iodide complex, TOMHI, confirms Hg–S bond formation (Fig. 10 and S21–S23; see Table S3 for crystal parameters). Here, Hg binds to two sulfur atoms at equal distances of 2.5676(10) Å and to iodine atoms at distances of 2.7249(6) Å (Hg–I2), 2.854(5) Å (Hg1–I1), and 2.860(3) Å (Hg1–I1A). It is noted that I1 is spatially distorted, occupying positions I1 and I1A, and the minor difference in bond lengths is attributed to thermal distortion of the iodine atom. The complex exhibits a distorted tetrahedral geometry with bond angles between 101° and 115°. The S1–Hg1–S1 bond angle is 106.20(5)°, and the I2–Hg–I1 angle is 101.48(17)°, with the Hg–S bond distances being shorter in the nitrate complex than in the iodide one due to differences in coordination geometry. The Hg–S distance of 2.860 Å aligns with the sum of covalent radii for Hg and S. Hence, the strong Hg–S interaction in the nitrate complex is crucial for sensing Hg at nanomolar levels in aqueous media amidst various metal ions.

Fig. 10 ORTEP diagram of the TOMHI complex drawn in 25% thermal probability ellipsoids showing the atomic numbering scheme. The solvent acetonitrile has been removed for clarity.

Finally, TD-DFT calculations were used to analyze the absorption spectra and charge-transfer properties of TOMH, TOMHN, and TOMHI (Fig. S24–S26). The HOMO–LUMO energy gap in TOMH is 3.71 eV, decreasing to 0.78 eV upon Hg(II) coordination in TOMHN, indicating energy release during binding (Fig. 11). The C=S bond lengths are 1.676 Å in TOMH, 1.735–1.745 Å in TOMHN, and 1.716 Å in TOMHI, positioned between typical single (1.81 Å) and double bonds (1.69 Å), confirming the partial double-bond character in the complexes. Molecular electrostatic potential surface (MESP) analysis^96,97 (Fig. 12) reveals strong sulfur-Hg(II) interactions, with TOMH showing electron-rich S centers and TOMHN indicating a more pronounced S–Hg(II)–S site.

Fig. 11 HOMOs and LUMOs of TOMH and its nitrate complex TOMHN.

Fig. 12 ESP diagrams of (a) TOMH, (b) TOMHN complex, and (c) TOMHI complex (the red region indicates an electron-rich site, and the light blue region indicates an electron-deficient site).

3.3. Antimicrobial studies

Thiosemicarbazones and related compounds have a variety^98–104 of applications; mercury-based thiosemicarbazones exhibit antimicrobial properties.^105–114 In light of our research on the sensing properties of TOMH for Hg(II) ions, we were interested in studying the antimicrobial activity of TOMH and its nitrate complex, Hg(NO₃)₂(TOMH). Fig. 13 shows a plot of the growth inhibition percentages for both Gram-positive and Gram-negative bacteria. Kanamycin, used as a positive control, demonstrated the highest antibacterial activity against E. coli, P. syringae, B. subtilis, and S. aureus, with growth inhibition values exceeding 80%. As a negative control, DMSO showed very little inhibition. The sensor TOMH exhibited less than 1% growth inhibition, while Hg(NO₃)₂ displayed 8–12% bacterial growth inhibition. Interestingly, the nitrate complex, TOMHN, showed high antibacterial activity, ranging from 73.3% (E. coli) to 76.9% (S. aureus). The minimal inhibitory concentration (MIC) of TOMHN was determined to be 14.57 µM for E. coli and 12.85 µM for S. aureus, indicating effective antimicrobial activity at low concentrations.

Fig. 13 Antimicrobial activity analysis of TOMHN against Gram-positive and Gram-negative bacteria. Bars represent mean ± SE (n = 3). Different letters (a, b, c, d, and e) represent values that were significantly different among different samples (Fisher LSD, p ≤ 0.05).

The time-kill analysis revealed a progressive decline in antimicrobial activity when tested against E. coli and S. aureus. This suggested that the TOMHN complex exhibited bactericidal activity over time (Fig. 14).

Fig. 14 Time-kill analysis of the TOMHN complex against (a) E. coli and (b) S. aureus.

4 Conclusion

In the present investigation, the TOMH chemosensor exhibits the lowest LOD for Hg ions (10.97 nM) in a water-rich medium among thiosemicarbazones or closely related S donor chemosensors (Table S1). It exhibits multiple application possibilities, such as in on-site detection of Hg using filter paper strips, formation of an INHIBIT logic gate, and enhanced antimicrobial activity of TOMHN relative to that of TOMH or Hg(NO₃)₂ alone. In literature, mono-thiosemicarbazone and other related sulfur-containing chemosensors exhibited high LODs (21 nM to 69.02 µM) for Hg detection in a relatively organic solvent-rich medium of study. The sensing activity of TOMH remains unaffected in the pH range 2.07 to 10.30, supporting its working ability even in a biological medium. The low detection limit of TOMH may be attributed to both the cis-orientation of the sulfur-containing arms of the dipodal chemosensor and stronger S,S-chelation by TOMH to Hg ions, keeping in mind the HSAB principle.

Author contributions

Sanyog Sharma (conceptualization, experimentation, data analysis and manuscript drafting); Nabajyoti Patra (DFT studies and concerned data analysis); Navdeep Kaur (antimicrobial activity and concerned data analysis); Pratap Kumar Pati (antimicrobial data analysis, and supervision); Sanjay Mandal (X-ray data curation); P. V. Bharatam (DFT data curation, and supervision); and Tarlok Singh Lobana (conceptualization, review and editing). All authors have approved the final version of the manuscript.

Conflicts of interest

There are no conflicts to declare.

Data availability

CCDC 2470765 (TOMHI) contains the supplementary crystallographic data for this paper.¹¹⁵

Supplementary information (SI): spectral characterization of TOMH, TOMHN, and TOMHI; UV-vis responses; literature comparison; NMR titrations; IR analysis, crystal data and structure refinement for TOMHI; powder diffractogram; DFT studies of TOMH, TOMHN, and TOMHI. See DOI: https://doi.org/10.1039/d6ra02561g.

Acknowledgements

SS is grateful for the funding received under the DST-Women Scientist-A (WOS-A) program {DST/WOS-A/CS-64/2021} from the Department of Science and Technology (DST), Government of India. SS is also thankful to Dr Geeta Hundal for the crystal data analysis. Tarlok Singh Lobana thanks the National Academy of Sciences of India for the ‘NASI Honorary Scientist Fellowship’. NK is thankful to the DST for the fellowship.

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