Introduction

The rapid industrialization of 21st century, driven by the global expansion of chemical industries and mining projects, has resulted in substantial environmental degradation, posing serious threats to various living organisms1,2,3,4,5. While heavy metals play an essential role in various human metabolic processes at controlled levels, excessive exposure can lead to severe health complications. Due to their significant environmental and health impacts, heavy metals and transition metal ions, including mercury, copper, lead, cadmium, and selenium, have become a focus of research. Among these, mercury is particularly concerning as one the most hazardous and widespread pollutants, attracting considerable attention in recent studies.

Mercury pollution in industrial settings arises from its ability to form amalgams with other metal ions, which, upon heating, release mercury oxide into the environment. The chlor-alkali industry is a major contributor to mercury contamination, significantly impacting aquatic ecosystems and drinking water quality. Mercury’s strong affinity for thiol groups enables it to readily bind to biological ligands such as proteins, DNA, and enzymes, leading to mercury poisoning in humans6,7,8,9,10,11,12. Prolonged atmospheric interaction with mercury causes extensive damage to biological and environmental systems. Human exposure to mercury primarily affects the endocrine and central nervous systems, leading to severe health complications, including kidney failure, brain damage, and disorders such as Minamata disease and acrodynia13,14,15,16.

Currently, these metal ions can be accurately quantified using several highly sensitive analytical techniques, including atomic absorption spectrometry (AAS), inductively coupled plasma mass spectrometry (ICP-MS), and inductively coupled plasma-atomic emission spectrometry (ICP-AES), among others17,18. However, these techniques are not well-suited for on-site field studies due to their high maintenance costs and expensive instrumentation. Consequently, significant attention has been directed toward developing novel Schiff base chemosensors that utilize fluorometric response detection. These sensors offer several advantages, including ease of use, simplicity, low detection limits, high sensitivity, and improved selectivity19,20.

Additionally, latent fingerprint (LFP) detection technology plays a crucial role in forensic science by enabling the identification of individuals based on the unique ridge patterns of their fingerprints. While traditional nanoparticle-based techniques for fingerprint visualization offer high sensitivity and strong adhesion, their effectiveness is limited by their reliance on visibility under white light. This constraint reduces their applicability on complex surfaces with low contrast, necessitating the development of more advanced detection methods21,22,23. To overcome this limitation, Schiff base fluorescence-based compounds offer a sophisticated solution by enabling dual-mode visibility under both ultraviolet (UV) and white light. The incorporation of Schiff base moieties imparts fluorescence properties, enhancing fingerprint visualization with improved contrast under UV illumination. This dual-mode capability significantly advances forensic imaging, facilitating the detection of faint or partial fingerprints that might otherwise go unnoticed under standard conditions.Thus, in the present study, a Schiff base ligand containing thiol group is synthesized, which demonstrate exceptional effectiveness in detecting mercury ions due to their strong affinity for Hg2+, facilitated by the highly reactive sulfur donor in the thiol group. These Schiff bases form stable chelate complexes with Hg2+, exhibiting outstanding selectivity. Upon binding, they often induce fluorescence changes, enabling rapid and highly sensitive detection. Furthermore, their fast and predictable interactions make them ideal for real-time sensing applications. Consequently, the development of chemosensors for Hg2+ detection is of significant interest due to the critical roles of mercury in biological systems and the environment. To date, reports on Hg2+ detection using fluorometric methods remain limited. A summary of existing literature is provided in Table 124,25,26,27,28. The ligand reported in this study has demonstrated promising results, achieving a lower detection limit for Hg2+ ions in real-world applications compared to previously published Schiff base sensors. The novelty of the present work lies in the fact that although there are a very limited reports on Schiff bases of aminobenezethiol, the chemical sensor synthesized in this study acts as highly sensitive and selective towards Hg2+ ion with low detection limit. Further, PMP also shows the significant practical applicability. Thus, herein we carried out the synthesis and characterization of the PMP ligand, which enables fluorometric detection of Hg2+ with high selectivity, sensitivity, stable reusability, and a broad response range, without interference from competing ions. The PMP ligand successfully detects Hg2+ through visual detection in borewell, canal, and dam water, highlighting its potential for proactive and quantitative mercury monitoring. Additionally, its applicability in forensic science is demonstrated by its effectiveness in detecting latent fingerprints on various non-porous surfaces, paving the way for advanced forensic display devices.

Table 1 Previously reported schiff base ligands as chemical sensor for Hg2+ ion detection.
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Experiments

Materials and methods

The chemicals, including 2-hydroxynicotinaldehyde, 2-aminobenzenethiol, acetic acid, and ethanol, were purchased from Avra Synthesis (Bengaluru, India) and used without further purification. Metal chlorides and nitrates of Cd2+, Mn2+, Zn2+, Co2+, Pb2+, Hg2+, Cu2+, Al3+, K+, Ag+, Na+, Ni2+, Ce3+, and Fe2+ were obtained from Avra Synthesis (Bengaluru, India). The characterization of the synthesized compounds was carried out using various analytical techniques. The proton nuclear magnetic resonance (¹H NMR) spectra were recorded on a Bruker Avance 400 MHz spectrometer. Photoluminescence (PL) spectra were obtained using a Shimadzu RF-6000 spectrofluorophotometer, while UV-Vis absorption spectra were recorded using a Shimadzu UV-3101 spectrophotometer. All glassware used in the experiments was oven-dried prior to use. Thin-layer chromatography (TLC) was performed on precoated silica gel plates to monitor reaction progress, with spot detection conducted under UV light. HeLa cells were obtained from Life Technologies (India) Pvt Ltd., Delhi, India and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) and grown in a humidified incubator at 5% CO2.

Synthesis of PMP

Figure 1 illustrates the synthesis of the PMP ligand following a standard procedure. In an oven-dried 100 mL sealed tube, 2-hydroxynicotinaldehyde (1.5 g, 1.0 eq., 0.012 mmol) was dissolved in ethanol (30 mL), and 2-aminobenzenethiol (2.0 g, 1.2 eq., 0.014 mmol, 1.7 mL) was added at room temperature. The reaction mixture was stirred at 90 °C for 16 h, with reaction progress monitored by thin-layer chromatography (TLC) using a 9:1 hexane/ethyl acetate mobile phase29,30. Upon completion, the reaction mixture was concentrated to obtain the crude product, which was then purified via medium-pressure liquid chromatography (MPLC) to yield a partially pure compound. Further purification was achieved by recrystallization in acetonitrile (30 mL) at 70 °C for 15 min, followed by cooling to room temperature. The resulting solid was filtered and dried under vacuum, yielding the target compound as a pale-yellow solid. Yield: 0.5 g (18%); 1H NMR (400 MHz, DMSO-d6) δ 12.56 (s, 1H), 8.72 (d, J = 5.1 Hz, 1H), 8.11 (d, J = 7.4 Hz, 1H), 8.01 (d, J = 7.9 Hz, 1H), 7.76 (t, J = 5.2 Hz, 1H), 7.52 (t, J = 7.0 Hz, 1H), 7.41 (t, J = 6.9 Hz, 1H), 7.20 (s, 1H), 6.55 (t, J = 6.8 Hz, 1H), 3.38 (s, 1H) (Fig. S1); LCMS (M+): m/z = 230.38 (Fig. S2).

Fig. 1
figure 1

General synthetic route for synthesizing Schiff base ligand, PMP.

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Synthesis of metal complex

A solution of (E)-3-(((2-mercaptophenyl)imino)methyl)pyridin-2-ol (115.05 mg, 0.5 mmol) was dissolved in ethanol under constant stirring. To the above ligand solution, ethanolic solution of mercury chloride (67.8 mg, 0.25 mmol) was added dropwise with continuous stirring at 60 °C for 6 h in an oil bath. Initially, when metal salt was added, colour of the solution was intense yellow in colour. As the reaction proceeds near the completion, the colour of the reaction mixture changed to pale yellow. The solution thus obtained was evaporated under vacuum to get a solid product31. The overlay of 1H NMR spectra of PMP and PMP-Hg2+ complex is depicted in Figure S4.

Stock solution Preparation

Stock solutions of various metal cations, such as Cd2+, Mn2+, Zn2+, Co2+, Pb2+, Hg2+, Cu2+, Al3+, K+, Na+, Ni2+, Ce3+, Ag+, Fe2+ were prepared by dissolving their nitrite and chlorides salts in double distilled water (10 mM) and ligand solution was prepared in DMSO: H2O (v/v, 6:4 ) mixture (10 mM). To perform the fluorescence experiments, the stock solution was diluted to 40 µM.

Solvent effect

The solvent effect on the fluorescence behaviour of the ligand PMP (40 µM) was studied in various solvents such as DMF, EtOAc, ACN, THF, DCM, DMSO, EtOH, and MeOH. The PMP (40 µM) was soluble in all the solvents and form a homogenous solution. Further, PMP showed different fluorescence emission based on polarity of the solvent. However, based on the significant changes in the fluorescence intensity, PMP ligand (20 µM) solution in DMSO: H2O mixture with varying ratios were used to assess polarity and aggregation effects32.

Fluorescence studies

To perform the fluorescence studies, the stock solution was diluted to 40 µM using DMSO: H2O (v/v, 6:4) (HEPES 0.01 M, pH = 7.4) at room temperature and the fluorescence spectra was recorded between 200 and 900 nm. The UV–visible absorbance was determined to get an excitation wavelength, which was further used for fluorescence emission studies to figure out the selectivity and sensitivity of ligand PMP towards various metal ions33. Furthermore, the fluorescence intensity was monitored for both the free ligand PMP (40 µM) and PMP + Hg2+ complex at regular intervals ranging from 5 to 50 min34.

Competitive studies

In the competitive binding studies, 40 µM of PMP solution in DMSO: H2O (v/v, 6:4) was added to various metal ion solutions including Hg2+ (40 µM) and maintained at pH = 7.4 using HEPES buffer. At room temperature, the fluorescence emission studies were performed in order to determine the ligand interaction with Hg2+ ion in the presence of competing metal ions35.

Jobs plot

Job’s plot method was performed to determine the stoichiometric ratio between the PMP ligand and Hg2+ metal ion. Equal concentration (40 µM) of Hg2+ and PMP were prepared. A series of mixtures were made by varying the volumes of Hg2+ and PMP ligand to obtain mole fractions ranging from 0.1 to 0.9 while keeping the total volume constant at 5 mL. Each solution was mixed thoroughly, and the fluorescence intensity of each mixture was then measured using a spectrofluorophotometer36.

Fluorescence quenching studies

At a fixed concentration of PMP ligand (40 µM), an incremental addition of Hg2+ ion solution was carried out. The fluorescence emission was recorded at a constant excitation wavelength (371 nm) and the association constant was calculated from the Stern-Volmer plot37.

Reversible EDTA Titration

To determine the reusability of PMP ligand, reversible EDTA titration were performed. Here, stock solutions of Hg2+ metal ions (10 mM) and EDTA solution (10 mM) were prepared. 40 µM of each of Hg2+ ion and EDTA solution were mixed and to this the PMP ligand (40 µM) was added and monitored the changes using fluorescence spectroscopy. All measurements were conducted in triplicate to ensure reproducibility38.

Detection limit evaluation

The limit of detection of PMP ligand was determined by varying the concentration of Hg2+ ions (2–10 µM) in DMSO: H2O (HEPES 0.01 M, pH = 7.4) solvent mixture. The fluorescence response emission was recorded at room temperature and the detection limit was calculated from the calibration curve.

pH effect analysis

DMSO: H2O mixture solutions were prepared at different pH ranging from 1 to 12 by adding HCl or NaOH in presence of HEPES buffer solution. When the desired pH was achieved, 40 µM of PMP ligand was added from the stock solution. The fluorescence emission of PMP ligand was recorded in a solution having different pH. To the each of the solution, 40 µM of Hg2+ metal ion solution was added and mixed well. The fluorescence spectra at each pH were recorded using fluorescence spectra39.

Quantification of Hg2+ in real samples

To study the real sample application of the PMP ligand, water samples from three different regions collected from Odisha district (India) were tested. The analysis was carried out in three different ways. Firstly, by adding 40 µL of PMP ligand solution to 4 mL of real water sample and incremental addition Hg2+ alone. Secondly, interacting PMP ligand with competitive metal ions. Finally, combining PMP ligand with Hg2+ ion and other competitive metal ions. The fluorescence analysis was done at room temperature40.

Cytotoxicity assay and cell imaging

To determine the cytotoxicity of the PMP ligand, the MTT assay (5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) was performed. HeLa cells were plated in a 36-well plate and cultured for 24 h to reach approximately 75% confluence before treatment. Following the addition of 50 µL of MTT solution (5 mg/mL) to each well, cells were incubated for four hours. Subsequently, 200 µL of dimethyl sulfoxide (DMSO) was added to each well, and the absorbance was measured at 316 nm. HeLa cells were maintained at 37 °C in a 5% CO2 atmosphere using Dulbecco’s Modified Eagle Medium (DMEM). For fluorescence imaging, PMP staining was performed for 30 min, followed by washing with phosphate-buffered saline (PBS)41. The stained cells were then visualized using an Invitrogen EVOS M5000 fluorescence inverted microscope (Washington, WA, USA).

Smart-phone based detection of Hg2+

Samples were prepared in a DMSO: H2O mixture (v/v; 6:4) containing varying concentrations of Hg2+ (0–10 µM). Each sample was mixed with a standard solution of the PMP sensor. The prepared solutions were placed in a square box and illuminated using a 40-watt UV LED chip, positioned at an optimal distance from an iPhone. RGB (Red, Green, Blue) values were extracted from digital images of the samples using the ColorDetector application on an iPhone 13 Pro42. The red intensity of each pixel was analyzed by determining the R (red), G (green), and B (blue) values, enabling quantitative assessment of Hg2+ detection based on fluorescence response.

Density functional theory calculations

Gaussian 16 software, along with GaussView 05, was employed for computational studies. The structure of the synthesized PMP probe was optimized using the B3LYP/6-311G(d, p) level of theory. For the Hg2+ complex, optimization was performed using the B3PW91 exchange functional and the LanL2DZ basis set. The frontier molecular orbitals and electrostatic potential energy surface were visualized using GaussView 05, providing insights into electronic distribution and binding interactions43.

Latent fingerprint visualization

Latent fingerprint detection was performed using the powder dusting method with finely ground PMP compound on clean and dry non-porous surfaces. Excess powder was carefully removed to reveal the ridge patterns of the latent fingerprint. The developed prints were then lifted using adhesive tape and observed under both white light and UV light (365 nm). High-resolution fingerprint images were captured using a DSLR camera, ensuring clear visualization and documentation of the fluorescence-based detection method44.

Results and discussion

Solvent effect

The solvent effect on the fluorescence properties of the PMP probe was investigated using a fluorometric method at room temperature. The PMP probe was dissolved in various organic solvents, maintaining a fixed concentration of 40 µM, and its fluorescence intensity was recorded. As shown in Fig. 2a, upon excitation at the absorption maxima corresponding to each solvent, the emission spectrum was obtained. In nonpolar solvents, the fluorescence intensity remained low; however, as the solvent polarity increased, the fluorescence intensity also increased, accompanied by a noticeable redshift in the emission spectrum45. The solvent effect was further explored by preparing a mixture of the PMP (20 µM, 10 mL) in DMSO/H2O solution of constant concentration and volume while varying the DMSO/H2O ratio (v/v = 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9). As water act as a poor solvent for the probe PMP, since it promotes the formation of aggregates in DMSO/H2O solution.

In pure DMSO, the PMP probe exhibited minimal fluorescence. Initially, the intensity of blue fluorescence at 431 nm increased with increasing water content, reaching a maximum at a DMSO/H2O ratio of 6:4 (v/v) (Fig. 2c). However, as the water content further increased, the fluorescence intensity began to decrease, as shown in Fig. 2b. This reduction in fluorescence emission is likely due to the formation of larger molecular aggregates of PMP, which precipitate rapidly. Such aggregation-induced fluorescence quenching is a commonly observed characteristic of aggregation-induced emission luminogens (AIEgens)43. This phenomenon signifies the typical response of AIE-active compounds in polar conditions.

Fig. 2
figure 2

(a) Emission spectra of PMP in different homogeneous solvents and (b) Fluorescence emission spectra of probe PMP in different proportions of DMSO: H2O (v/v, 6:4) (HEPES 0.01 M, pH = 7.4) solution. (c) Image depicting the range of fluorescence in the mixtures of DMSO and H2O in various proportions (v/v).

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Fluorescence Titration studies

The fluorescence response of the PMP probe to various metal ions was investigated to gain insight into its sensing behavior. The π-conjugated structure of PMP, which includes imine (> C = N-), phenol (–OH), and thiol (-SH) functional groups, provides potential binding sites for metal ions. Based on this property, we examined the interaction of PMP with one equivalent of different metal ions. As shown in Fig. 3, a range of metal ions (Cd2+, Mn2+, Zn2+, Co2+, Pb2+, Hg2+, Cu2+, Al3+, K+, Na+, Ni2+, Ce3+, Ag+, Fe2+) at a concentration of 10 mM were introduced into a 4 mL solution of DMSO/H2O (v/v = 6:4) containing PMP ligand (40 mM) in HEPES buffer (0.01 M, pH = 7.4) at room temperature. Under UV light, PMP ligand exhibited intense fluorescence in the presence of most metal ions. However, upon the addition of Hg2+ ions, the fluorescence was completely quenched. Fluorescence spectroscopy analysis was performed using a spectrofluorophotometer. The PMP probe displayed a strong emission peak at 431 nm when excited at 371 nm. Fluorescence intensity measurements revealed that only Hg2+ ions induced significant quenching, whereas other metal ions had negligible effects on the fluorescence intensity, as shown in Fig. 4. This finding demonstrates that PMP serves as a highly selective fluorescence “turn-off” sensor for detecting Hg2+ ions. To further evaluate the sensitivity of PMP toward Hg2+ ions, competitive testing was conducted. Additionally, the quantum yield (QY) of PMP was calculated using quinine sulfate in methanol (QY = 0.54) as the standard reference at an excitation wavelength of 431 nm, according to the following equation:

$$\:\text{Q}\text{Y}\text{s}\:=\:\text{Q}\text{Y}\text{R}\:\times\:\:\frac{{\text{I}}_{\text{S}}}{{\text{A}}_{\text{s}}}\:\times\:\:\frac{{\text{A}}_{\text{R}}}{{\text{I}}_{\text{R}}}\:\times\:\frac{{\left({{\upeta\:}}_{\text{R}}\right)}^{2}}{{\left({{\upeta\:}}_{\text{S}}\right)}^{2}}$$

Where R and S denote the reference and sample, respectively, I represent the fluorescence intensity, A is the absorbance value, and η is the refractive index of the solvent. The relative quantum yield of PMP in a DMSO: H2O mixture (v/v = 6:4) was determined to be 0.15. However, upon the addition of Hg2+ ions to the PMP solution, the quantum yield decreased to 0.02.

Fig. 3
figure 3

Ilustration of how distinct metal ions (40 µM) affect the fluorescence of PMP (40 µM) in DMSO: H2O (v/v; 6:4) (HEPES 0.01 M, pH = 7.4) under UV- light (365 nm) at room temperature.

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Fig. 4
figure 4

Fluorescence spectra of PMP (40 µM) in DMSO: H2O ( v/v, 6:4) (HEPES 0.01 M, pH = 7.4) solution in the presence of various metal ions (40 µM) upon the excitation 371 nm.

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Effect of time

The fluorescence response of PMP ligand and PMP + Hg2+ complex was analyzed by measuring the intensity at 431 nm over a period of 5 to 60 min. For this study, the fluorescence intensity of PMP ligand and PMP + Hg2+ complex was measured separately. The fluorescence intensity for the free probe and the Hg2+ bound complex remained constant throughout the time course (Fig. 5).

Fig. 5
figure 5

The graph depicting the effect of time on the fluorescence intensity.

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Competitive interference studies

Selectivity and sensitivity are critical characteristics of an effective receptor, particularly in the presence of competing species. In this study, we evaluated the sensitivity of PMP toward various competing metal ions, including Cd2+, Mn2+, Zn2+, Co2+, Pb2+, Hg2+, Cu2+, Al3+, K+, Na+, Ni2+, Ce3+, Ag+, Fe2+ in a DMSO/H2O (v/v = 6:4) solution buffered with HEPES (0.01 M, pH 7.4). The experiment was conducted by adding these metal ions to a PMP-containing solution while maintaining their concentrations equal to that of the Hg2+ solution46,47. The results demonstrated that PMP exhibits exceptional selectivity toward Hg2+ ions, with minimal interference from other metal ions. The fluorescence intensity remained largely unchanged in the presence of competing metal ions, whereas a significant quenching effect was observed specifically with Hg2+, as illustrated in the bar graph (Fig. 6).

Fig. 6
figure 6

Fluorescence intensity of PMP to Hg2+ and other competing ions in DMSO: H2O (v/v, 6:4) (HEPES 0.01 M, pH = 7.4) solution at 431 nm, upon the excitation 371 nm.

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Stoichiometry determination

To determine the stoichiometry between PMP and Hg2+, a Job plot analysis was conducted. The total concentration of PMP and Hg2+ was maintained at 40 µM in a 5 mL solution, while the mole ratio of Hg2+ to PMP was systematically varied. The emission intensity was recorded to evaluate the coordination behaviour between PMP and Hg2+48,49. As shown in Fig. 7a, the Job plot was constructed by plotting fluorescence intensity against the mole fraction of Hg2+ in the PMP-Hg2+ complex. The inflection point appeared at a molar ratio of [Hg2+]/([Hg2+] + [PMP]) = 0.5, indicating that PMP and Hg2+ interact in a 1:1 stoichiometric ratio. This was further confirmed by recording mass spectrum of PMP-Hg2+ complex, which showed the presence of molecular ion peak at m/z = 453.15 [M + Na] (Fig. S3).

The Benesi–Hildebrand method was used to analyse the interaction between PMP and Hg2+ in order to calculate the apparent binding constant and the stoichiometric ratio using Eq. 150,51.

$$\:\frac{1}{{F - F_{0} }} = \frac{1}{{F\alpha \: - F_{0} }} + \:\frac{1}{{K(F\alpha \: - F_{0} \:)}}\: \times \:\frac{1}{{[Hg^{{2 + }} ]}}$$
(1)

F₀ is the fluorescence intensity measured without Hg2+, whereas F is the intensity recorded after adding Hg2+. The term “Fα” refers to the fluorescence intensity measured when an excessive level of Hg2+ is present. Hence, in the case of 1:1 metal-complex formation, the graph of 1/(F - F₀) versus 1/[Hg2+] should yield a straight line. The binding constant (K) can be calculated from the intercept and slope of the linear plot. The Fig. 7b illustrates a specific linear fit based on Eq. (1), indicating a 1:1 stoichiometry for the metal complexation between PMP and Hg2+, with a binding constant of 4.7 × 10⁴ M⁻¹. Furthermore, the mechanism of binding in 1:1 stochiometric ratio of metal complex formation of PMP-Hg2+ is depicted in Fig. 852, which was further supported by the of Job’s plot analysis.

Fig. 7
figure 7

(a) Job’s plot for determining the stoichiometry for PMP and Hg2+ in DMSO: H2O (v/v, 6:4) (HEPES 0.01 M, pH = 7.4) solutions upon the excitation 371 nm. (b) Inset demonstrates the double reciprocal plot for 1:1 complex formation.

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Fig. 8
figure 8

The mechanism of sensing of PMP towards Hg2+ ion in 1:1 stoichiometric ratio.

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Fluorescence quenching analysis

The fluorescence titration method was employed in a Stern–Volmer analysis to investigate the ligand-quenching process with Hg2+ ions and to determine the association constant53,54. The sensitivity of PMP toward Hg2+ ions was evaluated by varying the Hg2+ concentration from 0 to 100 µM. The PMP sensor exhibited significant fluorescence quenching as the concentration of Hg2+ ions increased. The fluorescence intensity steadily decreased with increasing Hg2+ ion concentrations, as shown in Fig. 9a. The association constant was determined using the modified Stern–Volmer Eq. 

$$\:\frac{F0}{F}=Ksv\left[Q\right]+1$$
(2)

Where F0 and F represent the fluorescence intensity of PMP in the absence and presence of varying metal ion concentrations (Fig. 9b). The Stern–Volmer constant is denoted by Ksv, and the overall concentrations of Hg2+ ions are represented by [Q]. A plot of fluorescence intensities versus varying Hg2+ concentrations yielded a linear fit, unveiling a KSV value for the sample was 4.5 × 104 M− 1.

Fig. 9
figure 9

(a) Fluorescence emission spectrum of PMP upon addition of Hg2+ in DMSO: H2O (v/v, 6:4) (HEPES 0.01 M, pH = 7.4) upon the excitation 371 nm and (b) linear fitted graph of fluorescence intensity versus concentration of Hg2+ ions.

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Reversible nature of PMP ligand

Reversibility is a desirable property in fluorescence probes; thus, it is essential to evaluate whether the interaction between PMP and Hg2+ is reversible. This was investigated by adding an equimolar (1 equiv.) solution of EDTA to the PMP-Hg2+ complex solution (40 µM)55,56. As shown in Fig. 10a, the addition of an equimolar concentration of EDTA led to a significant increase in the fluorescence emission intensity of the PMP-Hg2+ complex, restoring it to its original value. This indicates the dissociation of the PMP-Hg2+ complex and the regeneration of free PMP. The subsequent sequential addition of Hg2+ and EDTA resulted in alternating fluorescence quenching and enhancement. This reversible cycle was repeated at least five times with only slight variations in fluorescence intensity (Fig. 10b). These findings confirm that PMP functions as a reversible fluorescent probe for the selective detection of Hg2+ in a DMSO/H2O solvent system (6:4, v/v) (Fig. 10c).

Fig. 10
figure 10

(a) and (b) Fluorescence emission spectrum showing the reversible nature of PMP (40 µM) with Hg2+(40 µM) in the presence of EDTA (40 µM) in DMSO: H2O (v/v, 6:4) (HEPES 0.01 M, pH = 7.4) upon the excitation 371 nm. (c) Showing the colour change of PMP when alternative addition of Hg2+ and EDTA observed under UV light (365 nm).

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Limit of detection

The limit of detection (LOD) was estimated using fluorescence titration, where the sensitivity of PMP toward Hg2+ ions was assessed by varying the concentration of Hg2+ while maintaining a constant concentration of PMP57. As shown in Fig. 11, the fluorescence intensity of PMP in a DMSO/H2O (v/v = 6:4) solution decreases progressively with increasing concentrations of Hg2+ ions. The limit of detection (LOD) for the sensor molecule was calculated using the following formula:

$${\text{Limit of Detection }}\left( {{\text{LOD}}} \right)\,=\,{\text{3s}}/{\text{k}}$$
(3)

Where, σ is the standard deviation of the response curve and k is the slope of the calibration curve. A plot of fluorescence intensities against varying Hg2+ concentrations produced a linear fit, and the calculated LOD of PMP for Hg2+ was determined to be 5 × 10− 8 M.

Fig. 11
figure 11

Fluorescence intensity calibration curve of PMP in DMSO: H2O (v/v, 6:4) (HEPES 0.01 M, pH = 7.4) upon addition of different micro molar concentration of Hg2+ ions upon the excitation 371 nm.

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pH effect analysis

To assess the practical applicability of PMP ligand as a fluorescent probe, the optimal pH conditions for PMP ligand alone and in the presence of Hg2+ were evaluated using fluorescence spectroscopy. As illustrated in Fig. 12, the fluorescence intensity of PMP ligand and PMP-Hg2+ complex was measured across a pH range of 3 to 1439,58,59. Notably, the fluorescence intensity of PMP ligand significantly increases at pH values below 9, while in the presence of Hg2+, the fluorescence intensity was quenched at all pH values below 9, explicitly due to the fluorescence intensity of PMP ligand alone. These results suggest that the fluorescence variation of PMP and PMP-Hg2+ complex is regulated by a two-step ON-OFF mechanism influenced by pH (Fig. 13). The ability of PMP to effectively detect Hg2+ across a broad pH range enhances its practical applicability. Furthermore, its excellent turn-off detection at physiological pH underscores its potential for biological applications.

Fig. 12
figure 12

(a) Fluorescence intensity plot of PMP (b) Fluorescence intensity of PMP-Hg2+ by varying pH in DMSO: H2O (v/v, 6:4) solution.

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Fig. 13
figure 13

Proposed mechanism of effect of pH (above and below pH = 9) on the fluorescence behavior of PMP.

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Smart-phone based detection of Hg2+

To evaluate the feasibility of using PMP for Hg2+ detection without a spectroscope, a smartphone-based image analysis was conducted. Samples were prepared in a DMSO: H2O mixture (v/v; 6:4) with varying concentrations of Hg2+ (0–10 µM) and subsequently mixed with a standard solution of the PMP sensor. Each solution was placed in a square box illuminated by a UV LED chip and positioned at an optimal distance from the smartphone to capture fluorescence changes45,60. Digital photographs of the illuminated samples were captured, and the RGB (Red, Green, Blue) values were extracted using the Color Detector application on a smartphone. The red intensity of each pixel was analyzed based on the three primary color components: R (red), G (green), and B (blue). The measurement area was kept consistent across all samples to ensure accuracy. The obtained RGB values were then plotted against the Hg2+ concentration (Fig. 14a). The lowest detectable concentration using this RGB-based analysis was 0.2 × 10⁻⁶ M, highlighting the high sensitivity of this method for quantifying trace amounts of the analyte. Equation (2) was utilized to determine the detection limit of PMP for Hg2+, which was estimated to be 0.2 µM. This estimation was based on the slope of the linear regression plot of the blue value against the Hg2+ concentration, as illustrated in Fig. 14b61. This analytical method offers a significant advantage by reducing the cost of analysis and simplifying conventional analytical procedures, making it a more practical and accessible alternative to expensive and complex instrument-based techniques.

Fig. 14
figure 14

Schematic representation of the application of PMP ligand in smartphone based RGB detection. (a) Plot of red, green and blue colour channel level of signal images obtained from smartphone using colorimeter app (b) Plot of blue colour channel level versus concentration of Hg2+.

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Probe PMP detect Hg2+ in real water sample

To explore the potential applicability of the PMP probe for Hg2+ detection, preliminary tests were conducted using three water samples—borewell water, canal water, and dam water—collected from Odisha. The study employed three distinct approaches to examine variations in fluorescence intensity in the presence of Hg2+ and other metal ions. In the first approach, all three water samples were spiked with varying concentrations of Hg2+ (0–10 µM), and 4 mL of each real water sample was treated with 4 µM of the PMP ligand solution. In the second approach, the PMP probe was exposed to various metal ions except Hg2+. The third approach assessed the ability of PMP to detect Hg2+ even in the presence of competing metal ions. As illustrated in Fig. 15a, the results demonstrated a gradual quenching of the fluorescence emission peak of PMP as the Hg2+ concentration increased. Notably, Fig. 15b show that in the absence of Hg2+, the fluorescence intensity of PMP remained consistent despite the presence of other metal ions. However, Fig. 15c clearly indicate that PMP effectively detected Hg2+ in the water sample, even in the presence of competing metal ions, by quenching its fluorescence intensity. In addition, the recovery percentage was calculated in all the three water samples (Table 2). These findings highlight the high sensitivity of the PMP probe for detecting Hg2+ in water samples, demonstrating its potential for real-world applications62,63,64.

Fig. 15
figure 15

Fluorescence emission spectrum of real water sample analysis for the detection of Hg2+ ion in Borewell water.

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Table 2 Illustrate the recovery percentage of Hg2+ in three different type of water samples.
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Cytotoxicity results and cell imaging investigations

Fluorescence imaging was employed to detect Hg2+ ions in live cells, leveraging PMP’s low cytotoxicity and strong fluorescence intensity. The cytotoxicity of PMP and its Hg2+ complexes was assessed in viable HeLa cells using the MTT assay at various concentrations. The results demonstrated that neither the ligand nor its complexes induced harmful effects on the cells. To facilitate fluorescence imaging, HeLa cells were cultured with 50 µg and 100 µg of PMP and incubated for 30 min at 37 °C with 5% CO2. As shown in Fig. 16a and b (top panel), the treated cells exhibited green fluorescence. In contrast, cells exposed to the PMP-Hg2+ complex showed no fluorescence (Fig. 16a and b, bottom panel). These findings indicate that PMP exhibits high membrane permeability, making it a practical and biocompatible sensing probe for detecting Hg2+ ions in live cells.

Fig. 16
figure 16

Relative fluorescence image of HeLa cells treated with PMP in the absence and presence of Hg2+ ions.

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DFT investigation

Figure 17 presents the optimized molecular structures and frontier molecular orbitals (HOMO and LUMO). The Hg2+ ion interacts with a nitrogen lone pair of electrons, forming a bond with a length of 2.475 Å. Additionally, the oxygen and sulfur atoms exhibit significant coordination with Hg2+, with bond lengths of 2.187 Å and 2.547 Å, respectively. The electron-donating and electron-accepting capacities of a molecule can be determined by its highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energies, respectively. The HOMO, which contains a high electron density, is primarily localized over the molecule, with a notable concentration on the sulfur-linked phenyl ring. In contrast, the LUMO exhibits a lower electron density distributed across the molecule, with a particularly reduced density on the sulfur atom. Upon coordination with the Hg2+ ion, the distribution of high and low electron densities extends throughout the molecule, except for the metal center. This suggests that Hg2+ does not contribute significantly to the primary HOMO or LUMO orbitals. Further analysis revealed that Hg2+ exhibits lower electron density in the secondary LUMO + 1 orbital. The electron transfer mechanism involves the migration of electrons from the PMP primary HOMO orbital to the Hg2+ secondary LUMO + 1 orbital.

The frontier molecular orbital energy gap (ΔE) was analyzed to assess the absorption properties of PMP and PMP + Hg. The calculated energy gaps (ΔE) for PMP and PMP + Hg were 3.6762 eV and 1.3698 eV, respectively. The lower energy gap of PMP + Hg compared to PMP suggests that less energy is required to promote electrons in the metal complex. The energy difference between the HOMO and LUMO + 1 orbitals of the complex was found to be 3.5266 eV, which is approximately equivalent to the HOMO–LUMO energy gap of the free PMP probe. This similarity explains why PMP and PMP + Hg exhibit comparable absorption wavelengths. Additional global reactivity parameters, summarized in Table 3, were determined based on literature-reported methodologies65. The interaction energy was calculated to be -56.47 kcal/mol by subtracting optimized complex’s total energy from the sum of metal ion and ligand energies. The system’s negative interaction energy implies a good binding contact between the PMP ligand and Hg2+ ions. The ionization potential and electron affinity values increased following complex formation, indicating stronger interactions between the probe and the metal orbitals. Moreover, the higher hardness and lower softness values of PMP suggest that the molecule is less polarizable, which enhances its ability to form stable bonds with metal ions, further supporting its sensing behavior. The negative chemical potential values for both PMP and the metal complex confirm the system’s stability. Additionally, the higher electronegativity of the metal relative to the probe suggests strong interactions between the two species. Thus, it suggests that the selected probe has the potential to be used as a sensitive and selective Hg2+ ion sensor.

Fig. 17
figure 17

Optimized geometries and FMO orbital surfaces of PMP and PMP + Hg2+ complex.

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Table 3 Global reactivity parameters values of PMP and PMP + Hg2+.
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The molecular electrostatic potential (MEP) graphs of PMP and PMP + Hg2+ are presented in Fig. 18. These three-dimensional representations provide insight into the chemical reactivity of the molecules, highlighting their electrophilic and nucleophilic regions. Additionally, the MEP maps illustrate dipole moments and variations in electronegativity, further elucidating the electronic distribution and potential interaction sites within the molecules66,67. Different colors in the MEP surface representation indicate varying electrostatic potential values. Green and blue correspond to regions of positive and zero electrostatic potential, representing electron-deficient centers. In contrast, yellow and red depict regions of negative electrostatic potential, signifying electron-rich areas. Due to their high electron density, the oxygen (O), pyridine nitrogen (N), and sulfur (S) atoms serve as ideal sites for metal ion interactions. However, the nitrogen in the pyridine ring is less favorable for coordination due to its structural orientation. In contrast, the sulfur and oxygen atoms are optimal candidates for metal ion coordination, with the interaction further stabilized by the nitrogen lone pair of electrons. This structural feature makes PMP a highly suitable probe for Hg2+ detection. Upon complex formation, the MEP map shows regions of positive and zero electrostatic potential, indicating that the ligand’s electron density has been neutralized by the electron-deficient Hg2+ ion. This observation further supports the strong binding affinity of PMP for Hg2+, reinforcing its potential as an effective sensor for mercury detection.

Fig. 18
figure 18

Molecular electrostatic potential (MEP) mapped on the electron density surface of (a) PMP and (b) PMP + Hg2+ complex.

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Latent fingerprint visualization

Rapid crime scene analysis depends on the quick detection of latent fingerprints (LFPs), which are unique biometric markers left by sweat and oils. Since LFPs degrade over time, prompt collection is essential for preserving forensic integrity. In this study, a volunteer rubbed their fingerprints across their forehead before imprinting them on four different substrates: glass, steel, plastic, and mobile screen guard. A finely powdered PMP ligand was applied using the powder dusting method, with any excess powder removed using a heat gun. The prints were then captured using a DSLR camera under both white light and 365 nm UV-Vis illumination. The resulting images exhibited strong contrast, enabling accurate fingerprint imaging (Fig. 19). Recent advancements in forensic science have emphasized the critical importance of distinguishing three distinct levels of detail in latent fingerprint analysis on non-porous surfaces: Type-I, Type-II, and Type-III. Type-I refers to the overall ridge flow and pattern configurations, such as whorls and islands. Type-II involves finer details, including ridge endings and bifurcations. Type-III encompasses intricate features like sweat pores and ridge contours, providing the highest level of specificity for fingerprint identification (Fig. 20a and b). To enhance visualization, the gray value, which represents light absorption and reflection across ridge patterns, was analyzed (Fig. 20c). The fingerprint ridges and furrows were successfully distinguished by plotting the gray value against distance, allowing for high-precision forensic analysis44,68.

Fig. 19
figure 19

Image of fingerprints on different surfaces (a) Lens (b) Stainless steel (c) Plastic and (d) Mobile screen guard.

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Fig. 20
figure 20

analysis of different types of levels for forensic investigation of fingerprint (a) White light (b) UV-light (365 nm). (c) Gray value analysis plot provides greater understanding of the chosen fingerprint region’s ridges and furrows.

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Conclusion

In summary, we have successfully synthesized a novel Schiff base ligand, PMP, which has been identified as a promising fluorometric sensor for the detection of Hg2+ in a DMSO: H2O (v/v; 6/4) solution. Job’s plot analysis suggested that the stoichiometric binding between PMP and Hg2+ ions occur in a 1:1 ratio, indicating specificity of binding. The ligand exhibited a low level of detection limit, 5.2 × 10⁻⁸ M for sensing of Hg2+ ions, demonstrating the high sensitivity towards of the probe towards Hg2+ ion. Additionally, it was observed that the binding of PMP with Hg2+ is reversible, which provides a key advantage in its reuse, as the sensor can be regenerated by using EDTA, making it suitable for repeated applications. On the other hand, PMP exhibited a broad functional pH range, indicating its versatility and robustness in different environmental conditions. The probe was successfully applied for the detection of Hg2+ in different environmental samples under various experimental settings, demonstrating its practical applicability. To validate the practical use of the sensor, we further explored its performance in real-life scenarios. Smartphone-based imaging, cell-imaging studies, and analyses of real-water samples were carried out to assess the probe’s efficacy in detecting harmful Hg2+ ions. The smartphone-based approach is particularly notable for its accessibility and convenience, providing a portable and cost-effective solution for field-based applications. Additionally, the ligand’s utility was extended to forensic science, where it was employed in latent fingerprint detection. The image-based analysis of fingerprints revealed the potential of PMP as a reliable tool for forensic fingerprint analysis, underscoring its versatility in both environmental and criminal investigation settings. These findings highlight versatility of PMP for forensic investigations as well as environmental monitoring, thus offering a practical, efficient, and reusable method for environmental monitoring and forensic applications.