Description:FIELD OF THE INVENTION
[0001] The present disclosure relates to a Schiff base ligand. Specifically, the present disclosure relates to a Schiff base fluorescent ligand and to a method of preparing the same. The present disclosure also relates to the use of the Schiff base fluorescent ligand as a chemical sensor for Hg2+ ion detection.

BACKGROUND OF THE INVENTION
[0002] Background description includes information that may be useful in understanding the present disclosure. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[0003] The rapid industrialization, driven by the global expansion of chemical industries and mining projects, has resulted in substantial environmental degradation, posing serious threats to various living organisms. While heavy metals play an essential role in various human metabolic processes at controlled levels, excessive exposure can lead to severe health complications.
[0004] 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.
[0005] 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 humans. 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 acrodynia.
[0006] 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 others.
[0007] 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 selectivity.
[0008] 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 methods.
[0009] 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.
[0010] Schiff base fluorescence-based compounds exhibit strong chemical interactions with fingerprint residues, ensuring superior adhesion, prolonged stability, and well-defined ridge pattern visualization. Their selective affinity for sweat components, fatty acids, and amino acids enhances detection precision, while their non-destructive nature enables repeatable analysis an essential feature in forensic science. By integrating fluorescence with exceptional adhesion properties, Schiff base compounds redefine latent fingerprint detection, providing superior clarity and adaptability for more accurate forensic investigations.
[0011] Accordingly, the present disclosure provides a new, simple, and robust thiol-based Schiff base ligand as a chemical sensor for Hg2+ ion detection and a method of preparation thereof.
[0012] The present disclosure satisfies the existing needs, as well as others, and generally overcomes the deficiencies found in the prior art.

OBJECTS OF THE INVENTION
[0013] Objects of the present disclosure are to provide a thiol-based Schiff base ligand.
[0014] An object of the present disclosure is to provide a thiol-based Schiff base ligand having formula (I).
[0015] An object of the present disclosure is to provide a thiol-based Schiff base ligand for use as a chemical sensor for Hg2+ ion detection.
[0016] Another object of the present disclosure is to provide a method of preparing the thiol-based Schiff base ligand.
[0017] Yet another object of the present disclosure is to provide a sensor system comprising the compound of formula (I) for Hg2+ ion detection.

SUMMARY OF THE INVENTION
[0018] This summary is provided to introduce a selection of concepts in a simplified form that is further described below in the detailed description section. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.
[0019] Aspects of the present disclosure relate to a Schiff base ligand. Specifically, the present disclosure relates to a Schiff base fluorescent ligand and to a method of preparing the same. The present disclosure also relates to the use of the Schiff base fluorescent ligand as a chemical sensor for Hg2+ ion detection.
[0020] In an aspect of the present disclosure, the Schiff base fluorescent ligand comprises a formula of (E)-3-(((2-mercaptophenyl) imino) methyl) pyridin-2-ol (PMP).

(E)-3-(((2-mercaptophenyl) imino) methyl) pyridin-2-ol (PMP)
[0021] In an aspect of the present disclosure, the PMP comprises an imine (>C=N) functional group, a phenolic hydroxyl (–OH) functional group, and a thiol (–SH) functional group.
[0022] In an aspect of the present disclosure, the PMP exhibits chelation-enhanced fluorescence quenching upon exposure to Hg²⁺, as coordination with the sulfur and nitrogen atoms, which results in the formation of a simple "turn-off" sensor. In some embodiments, the PMP forms stable chelate complexes with Hg²⁺, exhibiting outstanding selectivity, and upon binding, they often induce fluorescence changes, enabling rapid and highly sensitive detection of Hg²⁺.
[0023] In an aspect of the present disclosure, the PMP binds with Hg²⁺ in a ratio of 1:1.
[0024] In an aspect of the present disclosure, the PMP has a detection limit of 5.2×10-8 M for Hg²⁺ ions.
[0025] In another aspect, the present disclosure provides a method of preparing the PMP as disclosed herein, comprising the steps of:
a) dissolving 2-hydroxynicotinaldehyde and 2-aminobenzenethiol in an organic solvent to obtain a reaction mixture;
b) stirring the reaction mixture to obtain a crude product of the compound;
c) purifying the crude product by medium-pressure liquid chromatography (MPLC) to yield a partially pure compound; and
d) recrystallizing the partially pure compound in an organic solvent, followed by cooling the same at room temperature, filtering, and drying at vacuum to obtain PMP as a pale-yellow solid.
[0026] In an aspect of the present disclosure, the 2-hydroxynicotinaldehyde and the 2-aminobenzenethiol are used in a ratio of 1.5:2.
[0027] In an aspect of the present disclosure, the stirring is effected at 90 °C for 16 hours.
[0028] In an aspect, the present disclosure provides a turn-off sensor comprising PMP, for imaging Hg²⁺ ions.
[0029] In another aspect, the present disclosure provides a method for sensing and quantifying mercury ions (Hg²⁺) in a sample using a smartphone. The smartphone-based approach is particularly notable for its accessibility and convenience, providing a portable and cost-effective solution for field-based applications.
[0030] In an aspect of the present disclosure, the sample is selected from various water sources including but not limited to borewell, canal, dam water, soil samples, vegetables and green leaves highlighting its potential for proactive and quantitative mercury monitoring. In addition, this can also be employed to detect mercury in the.
[0031] In an aspect of the present disclosure, the method disclosed hereinabove has a detection limit of 0.2 µM of Hg²⁺ ions.
[0032] In an aspect of the present disclosure, the PMP is applied for fluorescence-based imaging of Hg²⁺ ions in live cells. The compound is cell-permeable and exhibits a strong intracellular fluorescence signal in the absence of mercury. Upon exposure to Hg²⁺, the intracellular fluorescence is quenched, providing a visual and quantitative measure of mercury accumulation in cellular compartments.
[0033] In yet another aspect of the present disclosure, the PMP is effective in detecting latent fingerprints on various non-porous surfaces, paving the way for advanced forensic display devices.
[0034] In some aspect, the non-porous surfaces include, but are not limited to glass, steel, plastic, mobile screen guard, TV panel, security safe box.
[0035] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIG. 1 relates to the schematic representation of preparing the synthesis of the PMP ligand.
[0037] FIG. 2 illustrates 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.
[0038] FIG. 3 relates to the 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.
[0039] FIG. 4 relates to the 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 431nm, upon the excitation 371 nm.
[0040] FIG. 5 relates to the 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.
[0041] FIG. 6 relates to the (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.
[0042] FIG. 7 relates to the (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 (365nm).
[0043] FIG. 8 relates to the 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.
[0044] FIG. 9 relates to the (a) Emission spectra of PMP in different homogeneous solvents (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.
[0045] FIG. 10 relates to the (a) Fluorescence intensity plot of PMP (b) Fluorescence intensity of PMP-Hg2+ by varying pH in DMSO:H2O ( v/v, 6:4) solution.
[0046] FIG. 11 relates to the 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+.
[0047] FIG. 12 relates to the Fluorescence emission spectrum of real water sample analysis for the detection of Hg2+ ion in Borewell water (a-c), Dam water (d-f), Canal water (g-i).
[0048] FIG. 13 relates to the relative fluorescence image of HeLa cells treated with PMP in the absence and presence of Hg2+ ions.
[0049] FIG. 14 relates to the image of fingerprints on different surfaces (a) Lens (b) Stainless steel (c) Plastic and (d) Mobile screen guard.
[0050] FIG. 15 relates to the analysis of different types of levels for forensic investigation of fingerprint (a) White light (b) UV-light (365 nm).
[0051] FIG. 16 relates to the Gray value analysis plot provides greater understanding of the chosen fingerprint region's ridges and furrows.

DETAILED DESCRIPTION OF THE INVENTION
[0052] The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure.
[0053] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
[0054] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
[0055] In some embodiments, numbers have been used for quantifying weight percentages, ratios, and so forth, to describe and claim certain embodiments of the invention and are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
[0056] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.
[0057] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
[0058] Unless the context requires otherwise, throughout the specification, which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”
[0059] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.
[0060] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[0061] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified.
[0062] The description that follows, and the embodiments described therein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present disclosure. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.
[0063] It should also be appreciated that the present disclosure can be implemented in numerous ways, including as a system, a method or a device. In this specification, these implementations, or any other form that the invention may take, may be referred to as processes. In general, the order of the steps of the disclosed processes may be altered within the scope of the invention.
[0064] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
[0065] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements a, b, and c, and a second embodiment comprises elements b and d, then the inventive subject matter is also considered to include other remaining combinations of a, b, c, or d, even if not explicitly disclosed.
[0066] Embodiments of the present disclosure relate to a Schiff base ligand. Specifically, the present disclosure relates to a Schiff base fluorescent ligand and to a method of preparing the same. The present disclosure also relates to the use of the Schiff base fluorescent ligand as a chemical sensor for Hg2+ ion detection.
[0067] In an embodiment of the present disclosure, the Schiff base fluorescent ligand comprises a formula of (E)-3-(((2-mercaptophenyl) imino) methyl) pyridin-2-ol (PMP).

(E)-3-(((2-mercaptophenyl) imino) methyl) pyridin-2-ol (PMP)
[0068] In an embodiment of the present disclosure, the PMP comprises an imine (>C=N) functional group, a phenolic hydroxyl (–OH) functional group, and a thiol (–SH) functional group.
[0069] In an embodiment of the present disclosure, the PMP exhibits chelation-enhanced fluorescence quenching upon exposure to Hg²⁺, as coordination with the sulfur and nitrogen atoms, which results in the formation of a simple "turn-off" sensor. In some embodiments, the PMP forms stable chelate complexes with Hg²⁺, exhibiting outstanding selectivity, and upon binding, they often induce fluorescence changes, enabling rapid and highly sensitive detection of Hg²⁺.
[0070] In an embodiment of the present disclosure, 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.
[0071] In an embodiment of the present disclosure, the PMP binds with Hg²⁺ in a ratio of 1:1.
[0072] In an embodiment of the present disclosure, the PMP has a detection limit of 5.2×10-8 M for Hg²⁺ ions.
[0073] In another embodiment, the present disclosure provides a method of preparing the PMP as disclosed herein, comprising the steps of:
e) dissolving 2-hydroxynicotinaldehyde and 2-aminobenzenethiol in an organic solvent to obtain a reaction mixture;
f) stirring the reaction mixture to obtain a crude product of the compound;
g) purifying the crude product by medium-pressure liquid chromatography (MPLC) to yield a partially pure compound; and
h) recrystallizing the partially pure compound in an organic solvent, followed by cooling the same at room temperature, filtering, and drying at vacuum to obtain PMP as a pale-yellow solid.
[0074] In an embodiment of the present disclosure, the 2-hydroxynicotinaldehyde and the 2-aminobenzenethiol are used in a ratio of 1.5:2.
[0075] In an embodiment of the present disclosure, the stirring is effected at 90 °C for 16 hours.
[0076] In an embodiment of the present disclosure, the organic solvent in step a) is selected from but not limited to acetonitrile, methanol, ethanol, isopropanol, acetone, methyl ethyl ketone, tetrahydrofuran, dimethylformamide, dimethyl sulfoxide, ethyl acetate, dichloromethane, chloroform, 1,4-dioxane, benzene, toluene, xylene, n-hexane, cyclohexane, diethyl ether, methyl tert-butyl ether, and N-methyl-2-pyrrolidone. Preferably, ethanol.
[0077] In an embodiment of the present disclosure, the organic solvent in step d) is selected from but not limited to acetonitrile, methanol, ethanol, isopropanol, acetone, methyl ethyl ketone, tetrahydrofuran, dimethylformamide, dimethyl sulfoxide, ethyl acetate, dichloromethane, chloroform, 1,4-dioxane, benzene, toluene, xylene, n-hexane, cyclohexane, diethyl ether, methyl tert-butyl ether, and N-methyl-2-pyrrolidone. Preferably, acetonitrile.
[0078] In an embodiment, the present disclosure provides a turn-off sensor comprising PMP, for imaging Hg²⁺ ions.
[0079] In an embodiment of the present disclosure, the PMP exhibits significant fluorescence in its unbound state, owing to the extended π-conjugation and electron-donating characteristics of the phenolic and thioaryl moieties. Upon coordination with Hg²⁺ ions, the fluorescence intensity is markedly reduced (“turn-off”), which is attributed to a combination of factors, including the heavy atom effect of Hg²⁺, enhanced intersystem crossing, and possible photoinduced electron transfer (PET) suppression.
[0080] In an embodiment of the present disclosure, the PMP exhibits high selectivity for Hg²⁺ ions over other common mono- and divalent metal ions, including Na⁺, K⁺, Ca²⁺, Mg²⁺, Zn²⁺, Cu²⁺, Pb²⁺, and Cd²⁺. This selectivity is ascribed to the unique affinity of the soft sulfur donor atom (from the mercaptophenyl group) and nitrogen/oxygen donors in the Schiff base structure for the soft Lewis acid character of Hg²⁺, forming a stable chelate complex.
[0081] In another embodiment, the present disclosure provides a method for sensing and quantifying mercury ions (Hg²⁺) in a sample using a smartphone. The smartphone-based approach is particularly notable for its accessibility and convenience, providing a portable and cost-effective solution for field-based applications.
[0082] In another embodiment, the present disclosure provides a method for sensing and quantifying mercury ions (Hg²⁺) in a sample, comprising:
(a) preparing a test solution from the sample comprising in a mixture of dimethyl sulfoxide (DMSO) and water in a volume ratio of 6:4;
(b) adding the PMP prepared as a standard solution to the test solution from step (a) to form a PMP- Hg²⁺ mixture;
(c) placing the PMP- Hg²⁺ mixture in a container suitable for optical imaging;
(d) illuminating the container containing the PMP- Hg²⁺ mixture with a 40-watt ultraviolet (UV) LED light source;
(e) capturing a digital image of the illuminated the PMP- Hg²⁺ mixture using a smartphone camera positioned at a fixed distance from the container;
(f) extracting red, green, and blue (RGB) intensity values from the captured image using a mobile application; and
(g) analyzing the red channel intensity from the RGB values to quantify the concentration of Hg²⁺ ions in the test solution based on the fluorescence response of the PMP, wherein the decrease in fluorescence of PMP is inversely proportional to the presence and level of Hg²⁺ ions.
[0083] In an embodiment of the present disclosure, the standard solution comprises 10 mM of PMP.
[0084] In an embodiment of the present disclosure, the sample is selected from various water sources including but not limited to borewell, canal, dam water, soil samples, vegetables and green leaves highlighting its potential for proactive and quantitative mercury monitoring.
[0085] In an embodiment of the present disclosure, the method disclosed hereinabove has a detection limit of 0.2 µM of Hg²⁺ ions.
[0086] In an embodiment of the present disclosure, the PMP is applicable to aqueous media over a physiologically relevant pH range (approximately pH 6.0–8.0), making it compatible with biological fluids and cell culture conditions.
[0087] In an embodiment of the present disclosure, the colorimeter mobile application is used to calculate the RGB values.
[0088] In an embodiment of the present disclosure, the PMP is applied for fluorescence-based imaging of Hg²⁺ ions in live cells. The compound is cell-permeable and exhibits a strong intracellular fluorescence signal in the absence of mercury. Upon exposure to Hg²⁺, the intracellular fluorescence is quenched, providing a visual and quantitative measure of mercury accumulation in cellular compartments.
[0089] In yet another embodiment, the present disclosure provides a method for detecting intracellular Hg²⁺ ions in a cell comprising the steps of:
(a) staining the cells using the PMP standard solution for 30 min, followed by washing the same with phosphate-buffered saline (PBS); and
(b) visualizing the cells using a fluorescence inverted microscope, wherein the absence of fluorescence indicating the presence of intracellular Hg²⁺ ions.
[0090] In yet another embodiment of the present disclosure, the PMP is effective in detecting latent fingerprints on various non-porous surfaces, paving the way for advanced forensic display devices.
[0091] In some embodiments, the non-porous surfaces include, but are not limited to glass, steel, plastic, mobile screen guard, TV panel, security safe box..
[0092] In still another embodiment, the present disclosure provides a method for detecting latent fingerprints on a non-porous surface, comprising:
(a) applying a finely ground PMP, onto a clean and dry non-porous surface suspected of bearing latent fingerprints using a powder dusting technique;
(b) removing excess powder from the surface to reveal the ridge patterns of the latent fingerprints;
(c) lifting the developed fingerprints using an adhesive medium;
(d) illuminating the lifted fingerprints under both white light and ultraviolet (UV) light at approximately 365 nm wavelength; and
(e) capturing high-resolution images of the developed fingerprints using a digital single-lens reflex (DSLR) camera to document the ridge detail and fluorescence-based contrast of the developed fingerprints.
[0093] While the foregoing description discloses various embodiments of the disclosure, other and further embodiments of the invention may be devised without departing from the basic scope of the disclosure. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.

EXAMPLES
[0094] The present disclosure is further explained in the form of following examples. However, it is to be understood that the foregoing examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the invention.
Materials:
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⁺, Co³⁺, Pb²⁺, Hg²⁺, Cu²⁺, Al³⁺, K⁺, Ag⁺, Na⁺, Ni²⁺, Ce³⁺, and Fe²⁺, as well as metal anions such as F⁻, Br⁻, Cl⁻, I⁻, and SCN⁻, were prepared from tetrabutylammonium salts and also 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.
[0095] Example 1: Synthesis of PMP
FIG. 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 hours, with reaction progress monitored by thin-layer chromatography (TLC) using a 9:1 hexane/ethyl acetate mobile phase. 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 minutes, 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 ppm 12.57 (bs, 1H), 8.72 (dd, J=7.26, 2.13 Hz, 1H), 8.11 (d, J=8.2 Hz, 1H), 8.01 (d, J=8.07 Hz, 1H), 7.76 (d, J=5.28 Hz, 1H), 7.46 - 7.57 (m, 1H), 7.36 - 7.46 (m, 1H), 6.55-6.58 (m, 1H).
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 different metal ions. As shown in Fig. 2, a range of metal ions (Cd²⁺, Mn²⁺, Zn²⁺, Co³⁺, Pb²⁺, Hg²⁺, Cu²⁺, Al³⁺, K⁺, Na⁺, Ni²⁺, Ce³⁺, Ag⁺, Fe²⁺, F⁻, Br⁻, Cl⁻, I⁻, and SCN⁻) at a concentration of 0.01 mM were introduced into a 4 mL solution of DMSO/H₂O (v/v = 6:4) containing PMP (0.025 mM) in HEPES buffer (0.01 M, pH = 7.4) at room temperature. Under UV light, PMP 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. 3. 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:
QYs = QYR × × ×
Where R and S denote the reference and sample, respectively, I represents 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 Al³⁺ ions to the PMP solution, the quantum yield decreased to 0.02.
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⁺, Co³⁺, Pb²⁺, Hg²⁺, Cu²⁺, Al³⁺, K⁺, Na⁺, Ni²⁺, Ce³⁺, Ag⁺, Fe²⁺, F⁻, Br⁻, Cl⁻, I⁻, and SCN⁻, 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⁺ solution. 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. 4).
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 behavior between PMP and Hg²⁺. As shown in Fig.5, 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.
Stern-Volmer 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 constant. 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. 5a. The association constant was determined using the modified Stern–Volmer equation.
-------- (1)
Where F0 and F represent the fluorescence intensity of PMP in the absence and presence of varying metal ion concentrations. 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.
Reversible nature of PMP
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. As shown in Fig. 7a-c, 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. 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).
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 PMP. As shown in Fig. 8, 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:
Limit of Detection (LOD) = 3σ/k --------------------(2)
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.
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. 9a-b, 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 spectrum. The solvent effect was further explored by preparing a mixture of the PMP (40μM, 10.0 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). However, as the water content further increased, the fluorescence intensity began to decrease, as shown in Fig. 8. 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). This phenomenon signifies the typical response of AIE active compounds in polar conditions.
pH studies
To assess the practical applicability of PMP as a fluorescent probe, the optimal pH conditions for PMP alone and in the presence of Hg²⁺ were evaluated using fluorescence spectroscopy. As illustrated in Fig. 10, the fluorescence intensity of PMP and the PMP-Hg²⁺ complex was measured across a pH range of 3 to 14. Notably, the fluorescence intensity of PMP significantly increases at pH values below 10, while in the presence of Hg²⁺, the fluorescence is quenched at all pH values below 10 and increases at pH 12 and 14. These results suggest that the fluorescence variation of PMP and the PMP-Hg²⁺ complex is regulated by a two-step ON-OFF mechanism influenced by pH. 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.
[0096] Example 2: 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 hours 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 minutes, followed by washing with phosphate-buffered saline (PBS). The stained cells were then visualized using an Invitrogen EVOS M5000 fluorescence inverted microscope (Washington, WA, USA).
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 minutes at 37 °C with 5% CO₂. As shown in Fig. 13a and 13b (top row), the treated cells exhibited green fluorescence. In contrast, cells exposed to the PMP-Hg²⁺ complex showed no fluorescence (Fig. 13a and 13b, bottom row). These findings indicate that PMP exhibits high membrane permeability, making it a practical and biocompatible sensing probe for detecting Hg²⁺ ions in live cells.
[0097] Example 3: 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 Pro. 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.
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 changes. 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. 11a). 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. 11b. 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.
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. 12a, d, and g, the results demonstrated a gradual quenching of the fluorescence emission peak of PMP as the Hg²⁺ concentration increased. Notably, Fig. 12b, e, and h show that in the absence of Hg²⁺, the fluorescence intensity of PMP remained consistent despite the presence of other metal ions. However, Fig. 12c, f, and i clearly indicate that PMP effectively detected Hg²⁺ in all three water samples, even in the presence of competing metal ions, by quenching its fluorescence intensity. These findings highlight the high sensitivity of the PMP probe for detecting Hg²⁺ in water samples, demonstrating its potential for real-world applications.
[0098] Example 4: 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 method.
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. 14). 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. 15). To enhance visualization, the gray value, which represents light absorption and reflection across ridge patterns, was analyzed. The fingerprint ridges and furrows were successfully distinguished by plotting the gray value against distance, allowing for high-precision forensic analysis (Fig. 16).

ADVANTAGES OF THE PRESENT DISCLOSURE:
[0099] The major advantage of this work is that using the single chemical sensor, multifunctional applications such as
(a) Detection of mercury ion in water, soil, vegetables and green leaves can be studied,
(b) By using smart-phone application, on spot mercury detection is possible using this chemical sensor. Further, limit of detection can be determined using this mobile application,
(c) This molecule is promising in the forensic latent fingerprint application, as this molecule can be used both in the white and UV light, and
(d) Furthermore, this molecule can be utilized to detect mercury in the live cells.
, Claims:1. A Schiff base fluorescent ligand of formula (E)-3-(((2-mercaptophenyl) imino) methyl) pyridin-2-ol (PMP), wherein the ligand comprises an imine (>C=N–) functional group, a phenolic hydroxyl (–OH) functional group, and a thiol (–SH) functional group, and wherein the ligand has a structure of:

(E)-3-(((2-mercaptophenyl) imino) methyl) pyridin-2-ol (PMP)

2. The ligand as claimed in claim 1, wherein the PMP binds with mercury ions (Hg²⁺ ions) in a ratio of 1:1.

3. The ligand as claimed in claim 2, wherein the PMP has a detection limit of 5.2×10-8 M for Hg²⁺ ions.

4. A method of preparing the PMP as claimed in claim 1 comprises the steps of:
i) dissolving 2-hydroxynicotinaldehyde and 2-aminobenzenethiol in an organic solvent to obtain a reaction mixture;
j) stirring the reaction mixture to obtain a crude product of the compound;
k) purifying the crude product by medium-pressure liquid chromatography (MPLC) to yield a partially pure compound; and
l) recrystallizing the partially pure compound in an organic solvent, followed by cooling the same at room temperature, filtering, and drying at vacuum to obtain the PMP as a pale-yellow solid.
5. The method as claimed in claim 4, wherein the 2-hydroxynicotinaldehyde and the 2-aminobenzenethiol are used in a ratio of 1.5:2.

6. The method as claimed in claim 4, wherein the stirring is effected at 90 °C for 16 hours.

7. The method as claimed in claim 4, wherein the organic solvent in step a) is selected from acetonitrile, methanol, ethanol, isopropanol, acetone, methyl ethyl ketone, tetrahydrofuran, dimethylformamide, dimethyl sulfoxide, ethyl acetate, dichloromethane, chloroform, 1,4-dioxane, benzene, toluene, xylene, n-hexane, cyclohexane, diethyl ether, methyl tert-butyl ether, and N-methyl-2-pyrrolidone.

8. The method as claimed in claim 4, wherein the organic solvent in step d) is selected from acetonitrile, methanol, ethanol, isopropanol, acetone, methyl ethyl ketone, tetrahydrofuran, dimethylformamide, dimethyl sulfoxide, ethyl acetate, dichloromethane, chloroform, 1,4-dioxane, benzene, toluene, xylene, n-hexane, cyclohexane, diethyl ether, methyl tert-butyl ether, and N-methyl-2-pyrrolidone.

9. A turn-off sensor for imaging Hg²⁺ ions comprising the PMP as claimed in claim 1.

10. A method for sensing and quantifying mercury ions (Hg²⁺) in a sample, comprising:
(a) preparing a test solution from the sample comprising in a mixture of dimethyl sulfoxide (DMSO) and water in a volume ratio of 6:4;
(b) adding the PMP as claimed in claim 1 prepared as a standard solution to the test solution from step (a) to form a PMP- Hg²⁺ mixture;
(c) placing the PMP- Hg²⁺ mixture in a container suitable for optical imaging;
(d) illuminating the container containing the PMP- Hg²⁺ mixture with a 40-watt ultraviolet (UV) LED light source;
(e) capturing a digital image of the illuminated the PMP- Hg²⁺ mixture using a smartphone camera positioned at a fixed distance from the container;
(f) extracting red, green, and blue (RGB) intensity values from the captured image using a mobile application; and
(g) analyzing the red channel intensity from the RGB values to quantify the concentration of Hg²⁺ ions in the test solution based on the fluorescence response of the PMP, wherein the decrease in fluorescence of PMP is inversely proportional to the presence and level of Hg²⁺ ions.

11. The method as claimed in claim 10, wherein the method has a detection limit of 0.2 µM of Hg²⁺ ions.

12. The method as claimed in claim 10, wherein the sample is selected from water sources, soil samples, vegetables and green leaves.

13. A method for detecting intracellular Hg²⁺ ions in a cell comprising the steps of:
(a) staining the cells using the PMP standard solution for 30 min, followed by washing the same with phosphate-buffered saline (PBS); and
(b) visualizing the cells using a fluorescence inverted microscope, wherein the absence of fluorescence indicating the presence of intracellular Hg²⁺ ions.

14. A method for detecting latent fingerprints on a non-porous surface, comprising:
(a) applying a finely ground PMP, onto a clean and dry non-porous surface suspected of bearing latent fingerprints using a powder dusting technique;
(b) removing excess powder from the surface to reveal the ridge patterns of the latent fingerprints;
(c) lifting the developed fingerprints using an adhesive medium;
(d) illuminating the lifted fingerprints under both white light and ultraviolet (UV) light at approximately 365 nm wavelength; and
(e) capturing high-resolution images of the developed fingerprints using a digital single-lens reflex (DSLR) camera to document the ridge detail and fluorescence-based contrast of the developed fingerprints.