Multifunctional Fluorescent Schiff Base Probe Derived From Chlorosalicylaldehyde for Selective Detection of Proteins, Nitroexplosives, Metal Ions and Antibiotics

ABSTRACT

Multifunctional fluorescent chemosensors capable of detecting chemically diverse analytes using a single molecular platform are increasingly valuable for environmental monitoring and biomedical analysis due to reducing analytical cost, time, and complexity. However, most Schiff base probes are limited to single‐analyte detection or operate through a single sensing mechanism. In this study, a new Schiff base probe, ClSNP, synthesized from 3,5‐dichlorosalicylaldehyde and N‐phenyl‐o‐phenylenediamine, exhibited a fluorescence “turn‐off” response toward Cu^{2 +} and Co^{2 +} ions in a CH₃CN/H₂O medium with high selectivity even in the presence of competing metal ions. Job's plot analysis confirmed a 1:1 binding stoichiometry, with detection limits of 1.79 and 3.40 µM for Cu^{2 +} and Co²⁺ ions, respectively, both below WHO‐permitted levels. Additionally, it selectively detected picric acid (PA) in THF with a detection limit of 2.49 µM and responded to antibiotics, including nitrofurantoin and metronidazole. It showed a “turn‐on” fluorescence enhancement at 460 nm upon binding with bovine serum albumin (BSA) with a detection limit of 1.28 µM, attributed to restricted intramolecular rotation, supported by a molecular docking study. The probe demonstrated high sensitivity, good selectivity, and real‐sample applicability, offering a versatile, cost‐effective, and mechanism‐switchable sensing platform.

A Schiff base probe, ClSNP, synthesized via the condensation of 3,5‐dichlorosalicylaldehyde with N‐phenyl‐o‐phenylenediamine, was developed for multifunctional sensing applications. The probe showed fluorescence quenching (turn‐off) responses toward Cu^{2 +} and Co^{2 +} ions, demonstrating excellent selectivity over other metal cations. Conversely, it displayed a fluorescence turn‐on response toward bovine serum albumin (BSA) with a detection limit of 1.28 µM, exhibiting high selectivity over other competing proteins. Furthermore, the probe ClSNP effectively recognized picric acid (PA) and various antibiotics, underscoring its potential as a versatile, sensitive, and selective analytical tool for diverse chemical and biological sensing applications.

1. Introduction

Schiff bases are a prominent class of compounds in coordination chemistry owing to their ability to coordinate through multiple donor atoms, and they find a wide range of applications in analytical chemistry, catalysis, biological systems, and so forth [1, 2, 3, 4, 5]. Due to their low cost, ease of synthesis, biodegradability, favorable photophysical properties, and strong interactions with metal ions [6], Schiff bases are frequently employed as molecular chemosensors. They have been widely explored for the detection of heavy metals and the removal of environmental pollutants, functioning as efficient spectrophotometric and fluorescence probes [7, 8]. In addition, Schiff bases have attracted considerable attention for their photochromic behavior, which arises from excited‐state intramolecular proton transfer (ESIPT). Their metal complexes are also recognized as a promising class of luminescent materials with potential applications across various fields, including electrical conductors, organic light‐emitting diodes (OLEDs), and molecular optoelectronics [9, 10, 11, 12].

Nitroaromatic compounds are not only key constituents of explosives but also significant environmental pollutants. Consequently, their detection is crucial for military security, public safety, and environmental protection [13]. In recent years, fluorescent Schiff base compounds have been explored as effective sensors for nitroaromatic explosives and related hazardous pollutants [14, 15]. Among these, picric acid (PA) is one of the most potent and toxic nitroaromatic explosives, posing severe risks to human health due to its poisonous effects. Molecular sensors typically detect PA through a fluorescence turn‐off mechanism, which is generally attributed to host–guest interactions and electron transfer from the electron‐rich fluorophore to the electron‐deficient PA. However, structural evidence supporting the formation of stable PA–fluorophore complex remains limited [16, 17].

Antibiotics can be harmful at high concentrations and ineffective at sub‐therapeutic levels; therefore, detecting their presence and concentration is essential for (i) pharmaceutical quality control, (ii) environmental monitoring, and (iii) biomedical diagnosis. Schiff base compounds can directly interact with antibiotic molecules through multiple noncovalent interactions, including hydrogen bonding, pi–pi stacking, and, in some cases, coordination involving their metal‐binding sites. These interactions often lead to noticeable alterations in the characteristics of Schiff base compounds, such as colorimetric or fluorescence responses, thereby enabling the sensitive detection of antibiotics [18, 19, 20].

The interaction of Schiff base compounds with proteins or nucleic acids plays a vital role in regulating biological processes that are essential for proper physiological function. Serum albumin is the predominant protein circulating in the bloodstream, playing key roles in transport and physiological regulation [21, 22]. Bovine serum albumin (BSA) is a well‐characterized globular protein, made up of a single 583 amino‐acid polypeptide chain, featuring 17 disulfide linkages and one accessible thiol (─SH) group. Structurally, it comprises three major domains (I–III), and each domain is further partitioned into subdomains, A and B. The protein includes two intrinsic tryptophan residues, Trp‐134 and Trp‐213, both of which function as native fluorescent centers. Trp‐134 is located on the protein surface in a hydrophilic environment, while Trp‐213 resides in the largest hydrophobic cavity [23, 24, 25]. Serum albumins readily bind to a wide range of small organic molecules with diverse functional groups, making them key transport proteins in drug delivery. Schiff bases, which feature azomethine functional groups, find broad use in biological and industrial fields because they are easy to synthesize, offer high yields, and exhibit strong coordination properties. Their interactions with albumins such as BSA and HSA provide valuable insights into pharmacological applications [26, 27].

Metal ions represent an active research area due to their ubiquitous presence in natural and biological systems. In the human body, they play crucial roles in physiological and biochemical processes, while in the environment, they occur commonly in food, soil, and water. However, concentrations exceeding or failing below permissible limits can pose serious risks to both human health and the ecosystem [28]. Consequently, the rapid and quantitative detection of metal ions has garnered significant scientific interest. Various analytical techniques have been employed for this purpose, including gas chromatography (GC) [29], high‐performance liquid chromatography (HPLC) [30], cyclic voltammetry (CV) [31, 32, 33, 34], ion mobility spectrometry (IMS) [35], surface Plasmon resonance [36], and Raman or surface‐enhanced Raman scattering methods [37]. While these methods offer high sensitivity, they are often expensive, complex and instrument‐dependent. In contrast, fluorometric detection has emerged as a superior optical sensing strategy. Its simplicity, high sensitivity, and versatility across solution, solid, and vapor phases enabled by measurable changes in fluorophore emission upon metal‐ion interactions make it particularly appealing [38].

Multifunctional chemosensors are attractive for practical applications as they reduce both cost and analysis time. Nevertheless, the development of a facile chemosensor designed to selectively detect and differentiate multiple analytes via unique signal outputs continues to pose a significant challenge [39, 40, 41]. Here, we describe the rational design and facile one‐step preparation of a new Schiff base, CISNP, as a chemosensor, based on a dichlorosalicylimine derivative, followed by detailed characterization using multiple spectroscopic methods. A distinctive feature of the dichlorosalicylimine derivative chemosensor is its ability to integrate multiple sensing functions within a single molecular scaffold. By exploiting the peculiar photoluminescent property of the CISNP, we expanded and executed various sensing applications. The novelty of this work is demonstrated by the development of a single Schiff Base probe ClSNP, capable of selectively detecting a diverse range of chemically distinct analytes including metal ions such as Cu² and Co^{2 +} ions, nitroexplosives including PA, antibiotics, and proteins through multiple fluorescence response mechanisms Unlike most reported Schiff Base sensors which typically target a single analyte or employ a single sensing mechanism, ClSNP exhibited turn‐off responses via metal chelation‐enhanced quenching and photoinduced electron transfer toward Cu^{2 +} and Co^{2 +} metal ions as well as turn‐on response toward BSA due to restricted intramolecular rotation. This analyte‐specific modulation of excited‐state relaxation pathways represents a significant advancement over conventional ESIPT‐active or dichloroaldehyde‐based Schiff Base sensors. By demonstrating that a single, easily synthesized Schiff base ClSNP can serve as a mechanism‐switchable, multifunctional chemosensor, this work makes a meaningful contribution to the rational design of next‐generation fluorescent probes for environmental, biological, and security‐related sensing applications.

2. Experimental Methods

2.1. Materials and Methods

3,5‐dichloro salicylaldehyde, N‐phenyl‐o‐phenylenediamine, and metal nitrate salts were sourced from Alfa Aesar, Thermo Fisher Scientific India Pvt Ltd, Mumbai, India. BSA was supplied by GENEI Laboratories Private Limited in Bengaluru, India. Ethanol (EtOH), acetonitrile (CH₃CN), and tetrahydrofuran (THF) were purchased from commercial suppliers and purified prior to use. Double‐distilled water was employed whenever purified water was needed. All reagents were used as received without any further purification.

FT‐IR measurements were performed on a Shimadzu 8400S spectrometer operating over the 4000‐400 cm⁻¹ range, was used to record the infrared spectra using the KBr pellet method. Fluorescence and UV–visible absorption spectra were collected using a JASCO FP‐8300 spectrofluorometer and a JASCO variant‐650 spectrophotometer. ¹H NMR spectra were obtained on a 400 MHz Bruker spectrometer in CDCl₃, taken from Gandhigram Rural Institute‐Deemed to be University, India. ESI–MS spectra were acquired using an Agilent HP 5973 instrument, IIT Madras, India. Excited‐state lifetime studies of compound CISNP were performed using a FLUOROLOG—FL3‐11 single photon‐counting system available at Pondicherry University, with a nano‐LED source for excitation at 295 nm. The computational studies are carried out by using b3lyp/Aug‐cc‐pvdz in Gaussian 16 in vacuo. The pre‐optimized structure was drawn using GaussView 6.

2.2. Preparation of a Schiff Base, ClSNP

An ethanolic solution of 3,5‐dichlorosalicylaldehyde (0.0766 g, 0.1 mmol) was allowed to react with N‐phenyl‐o‐phenylenediamine (0.406 g, 0.1 mmol) and a few drops of acetic acid, followed by refluxing for 3 h. The resulting mixture was permitted to cool and it was subsequently filtered to collect the solid product. The crude product was washed thoroughly with hot ethanol and dried under vacuum to yield (70%) the light yellow colored Schiff base, ClSNP [48]. The synthesis of ClSNP is depicted in Scheme 1. It has the following characteristics: m.p: 150°C; ESI‐MS (m/z): C₁₉H₁₄ Cl₂N₂O [M + H⁺]: Calcd. 356, found 357, (Figure S1); Anal. Calc. (%) for C₁₉H₁₄Cl₂N₂O: C, 63.9; H, 3.95; Cl, 19.9; N, 7.8;O, 4.5; Found (%):C, 63.2; H, 3.8; Cl, 19.1; N, 7.5; O, 4.1; FT‐IR (KBr, cm ⁻¹): ν 1596 (stretch, ─CH═N─), 3750 and 1373 (stretch and bend, ─OH), 3063 (stretch, N─H), 2333 (stretch, C─N), 1255–1011 (stretch, ─C─C─ aromatic), 555 (stretch, ─C─Cl) (Figure S2); ¹H‐NMR (CDCl₃, ppm): δ 7.6 (s, 1H, ─CH═N─), 7.8 (s, 1H, N─H), 14.6 (s, 1H, O─H) (Figure S3); UV–vis (THF, λ _max/nm (ε/M⁻¹cm⁻¹)): 240 (136626) and 340 (58942) (Figure S4).

SCHEME 1.

Synthesis of the chemosensor, ClSNP.

2.3. Spectral Titration of ClSNP Toward Metal Ions

The chemosensor ClSNP dissolves readily in THF, CH₃CN, DMSO, DCM, DMF, hot ethanol, and methanol. A CH₃CN/H₂O mixture (9/1, v/v) was employed in this study for the detection of cations. A 1 mM stock solution of ClSNP was prepared by dissolving 1.78 mg of the compound in 5 mL of CH₃CN. A 10 µM working solution was obtained by diluting 0.05 mL of the stock solution with CH₃CN. Standard solutions of various cations (1 mM) were prepared in H₂O medium using the corresponding nitrate salts of Na⁺, K⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Pb²⁺, Hg²⁺, Sr²⁺, Cd²⁺, Ca²⁺, Al^3+, and Cr³⁺ ions. The sensing performance of ClSNP (10 µM) toward these cations was examined in CH₃CN─H₂O (1/9, v/v) across a concentration range of 10–150 µM. To assess the cation recognition capabilities of ClSNP, UV–vis absorption and emission titration experiments were conducted under ambient conditions [42].

The binding constants of ClSNP with different guest molecules (metal ions or other analytes) were calculated from absorption spectral titration data via the Benesi–Hildebrand Equation (1) [43].

$$\frac{A_0}{A-A_0}=\frac{{\epsilon }_G}{{\epsilon }_V}+\left(\frac{{\epsilon }_G}{{\epsilon }_C{K}_{\mathrm{a}}}\right)\frac{1}{\left[G\right]},$$ (1)

where A ₀ and A represent the absorption intensity of ClSNP without and with metal ions, respectively. The term [G] denotes the [analytes]. The molar extinction coefficient of the analytes is represented by ε _G, while ε _v and ε _c refer to the molar extinction coefficients of the free and bound forms of the ClSNP, respectively. A linear double reciprocal plot of 1/(A − A ₀) versus 1/[G] was used to determine the binding constant, K _a.

Additionally, the modified form of the Benesi–Hildebrand Equation (2) was employed to calculate the binding constant obtained from the emission titration data [43].

$$\frac{I_o}{I-I_o}=\frac{b}{\left(a-b\right)}+\left(\frac{1}{K_{\mathrm{b}}\left[G\right]+1}\right),$$ (2)

where I ₀ and I denote the emission intensity of ClSNP without and with a guest molecule, respectively. The constants “a” and “b” are the fitting parameters, and [G] again denotes the [analytes]. Using this equation, the binding constant (K _b) was derived from the emission titration data. Detection limits of the target ions and molecules were determined according to Equation (3).

$$\mathrm{Detection}\mathrm{limit}=3S_{\mathrm{d}/\rho }$$ (3)

Where S _d represents the standard deviation of the blank ClSNP emission (n = 5) and ρ is the calibration sensitivity derived from the fluorescence intensity versus [analyte] plot [24, 25].

2.4. Spectral Titration of ClSNP Toward Nitro Compounds

Absorption and fluorescence techniques were employed to investigate the sensing ability of ClSNP toward a series of nitro compounds, including nitrobenzene (NB), dinitrobenzene (DNB), nitrobenzoic acid (NBA), 1‐chloro‐2‐nitrobenzene (1‐Cl‐2‐NB), 1‐chloro‐4‐nitrobenzene (1‐Cl‐4‐NB), 2‐nitrotoluene (2‐NT), 4‐nitrotoluene (4‐NT), p‐nitrophenol (PNP), and PA in THF solvent. For the sensing experiments, the concentration of ClSNP was fixed at 10 µM, while the concentrations of nitro compounds were varied from 10 to 150 µM. The mixtures were stirred for 1 min, and the absorbance and fluorescence spectra were recorded at room temperature [26, 27]. The Stern–Volmer equation (Equation 4) was used to calculate binding efficiencies from the decrease in ClSNP fluorescence with increasing [nitro compound] [43].

(4)

where F ₀ and F denote the emission intensity of ClSNP without and with nitro compounds, respectively, [Q] is the concentration of nitro compounds, and K _SV is the Stern–Volmer quenching constant.

2.5. Spectral Titration of ClSNP Toward Antibiotics

The sensing ability of ClSNP toward antibiotics such as nitrofurantoin and metronidazole was evaluated using absorption and fluorescence spectral techniques in THF solvent. For the sensing experiments, 10 µM of each drug solution was added to 10 µM of ClSNP in 5 mL of THF solution, and the mixtures were stirred for 1 min. Subsequently, the absorption and fluorescence spectra were measured under ambient conditions [28, 29]. The binding constants of ClSNP with the antibiotics were determined from absorption titration studies via the Benesi–Hildebrand equation (Equation 1). The Stern–Volmer (Equation 4) and modified Benesi–Hildebrand (Equation 2) equations were employed to obtain binding efficiencies from the decrease in ClSNP fluorescence with increasing [drug].

2.6. Spectral Titration of ClSNP Toward Biomolecules

The interactions between ClSNP with biomolecules, including BSA, Ovalbumin, and Lysozyme, have been investigated through UV–vis absorption and emission studies. The UV–vis absorption titration experiments were performed using a fixed concentration (10 µM) of the ClSNP, while the [biomolecules] were incrementally varied from 10 to 150 µM in phosphate buffer solution (pH 7.4). The binding constant (K _b) values for the ligand–protein interactions were determined using the Benesi–Hildebrand equation (Equation 1). Similarly, fluorescence‐quenching titrations were carried out with 10 µM ClSNP in DMSO, to which biomolecules (10–150 µM in phosphate buffer) were added stepwise. The excitation wavelength was set at 365 nm. The binding constants were determined via the modified Benesi–Hildebrand equation (Equation 2) [22, 23].

2.7. Molecular Docking Studies

AutoDock Vina was employed for molecular docking to study ClSNP interactions with biological macromolecules. The hydrogen‐bonding interactions were examined using Discovery Studio 4.0, while hydrophobic interactions were visualized with PyMOL. The 3D crystal structure of BSA (PDB ID: 4F5S) was retrieved from the Protein Data Bank, and all crystallographic water molecules, bound ligands, and ions were removed using PyMOL to prevent interference during docking [44]. The ClSNP structure was energy‐minimized and converted into the required PDBQT format. The docking grid box was defined around the active site region of BSA to allow flexible sampling of possible binding conformations. AutoDock Vina was then executed with default parameters, and the top‐ranked binding poses were selected based on binding affinity scores.

3. Results and Discussion

3.1. Synthesis and Structure of CISNP

The Schiff‐base ClSNP was prepared via a one‐step condensation of 3,5‐dichlorosalicylaldehyde with N‐phenyl‐o‐phenylenediamine in ethanol (Scheme 1). The obtained light‐yellow compound, ClSNP, was stable under ambient conditions, and was highly soluble in acetonitrile, chloroform, DCM, DMF, DMSO, THF, and hot alcohols. The structure of CISNP was confirmed by several spectral techniques, verifying its successful formation.

The Schiff‐base CISNP possesses an azomethine (─CH═N─) unit, an ortho‐positioned phenolic ─OH group, an ─NH group, and dichloro substituents. These functionalities enable ClSNP to act as a bidentate ligand, coordinating with cations through the imine nitrogen and phenolic oxygen, while also allowing interactions with anions via hydrogen bonding through the phenolic ─OH and ─NH groups. This dual coordination ability toward both cations and anions enables electronic modulation, resulting in distinct and readily observable color changes. The dichloro substituents further enhance the reactivity of CISNP, enabling substitution reactions that yield a variety of structurally diverse chloro‐containing Schiff bases. Furthermore, chloro substituents are capable of covalent interactions with reactive protein residues, such as the thiolate of cysteine‐34 in BSA [45, 46]. Guided by these features, we designed CISNP by integrating these functional moieties and subsequently investigated its potential as a versatile chemosensor.

3.2. Absorption and Emission Spectra of ClSNP

The absorption spectrum of ClSNP featured three prominent bands at 240, 295, and 340 nm along with a shoulder at ∼385 nm in THF, corresponding to π–π* and n–π* transitions, respectively (Figure S4). When excited at 340 nm, compound ClSNP exhibited a structured emission profile with a peak at 490 nm and a shoulder at 520 nm (Figure S5). Owing to its inherent emissive nature, ClSNP is suitable for fluorometric titration studies with various ions and macromolecules [47].

3.3. Metal Ions Sensing Study

To investigate the interactions between chemosensor ClSNP and various metal ions, including Na⁺, K⁺, Sr²⁺, Ca²⁺, Zn²⁺, Ni²⁺, Cu²⁺, Pb²⁺, Co²⁺, Hg²⁺, Cd²⁺, Cr³⁺, and Al³⁺ ions, absorption and emission analysis were employed (Figure 1). The chemosensor ClSNP exhibited a π→π* absorption band at 305 nm and an n→π* charge‐transfer band at 345 nm in CH₃CN/H₂O (9:1, v/v) mixture [48, 49, 50].

FIGURE 1.

Absorption spectra of ClSNP (10 µM) upon addition of different metal ions, including Na⁺, K⁺, Sr²⁺, Ca²⁺, Zn²⁺, Ni²⁺, Cu²⁺, Pb²⁺, Co²⁺, Hg²⁺, Cd²⁺, Cr³⁺, and Al³⁺ ions (10 µM) in CH₃CN─H₂O (9/1, v/v) mixture.

Upon the incremental addition of Cu²⁺ and Co²⁺ ions (10–150 µM), the absorption band observed at 345 nm underwent a pronounced redshifts, moving from 345 to 395 nm in the presence of Cu²⁺ ion, and to 360 nm in the presence of Co²⁺ ion, while other band at 305 nm showed a moderate redshifts in the presence of Cu²⁺ and Co²⁺ ions (Figure S6). This spectral shift was attributed to ligand‐to‐metal charge transfer (LMCT) interactions. As illustrated in Figures 2a and 3a, distinct spectral changes were observed upon the gradual addition of Cu²⁺/Co²⁺ ions, while minor significant alterations were noted in the presence of other metal ions. This specificity highlights the potential of chemosensor ClSNP for applications in detecting these particular metal ions in various environments. The binding constants (K _a) for the interaction between ClSNP and the metal ions were determined via the Benesi–Hildebrand equation (Equation 1). It exhibited a binding constant of 3.50 × 10⁴ M⁻¹ toward Cu²⁺ ion (Figure S7), while for the Co²⁺ ion, it was determined to be 1.98 × 10⁴ M⁻¹. These values reflected the affinity of the chemosensor ClSNP for the respective metal ions, confirming its selective binding characteristics.

FIGURE 2.

Changes in the absorption (a), emission (b), and intensities of ClSNP (10 µM) recorded upon progressive addition of Cu²⁺ ions (10−150 µM) in CH₃CN─H₂O mixture (9/1, v/v).

FIGURE 3.

(a) Absorption and (b) emission responses of ClSNP (10 µM) by increasing Co²⁺ ion concentrations (10–150 µM) in a CH₃CN/H₂O mixture (9/1, v/v).

Upon excitation at 340 nm, chemosensor ClSNP showed a well‐defined emission band at 490 nm accompanied by a shoulder peak at 520 nm in THF, which are associated with the combination of π→π* and n→π* charge‐transfer transitions. The fluorescence intensity of the chemosensor ClSNP decreased progressively with increasing concentrations of Cu²⁺ and Co²⁺ ions in the range of 10–150 µM. (Figures 2b and 3b) This quenching effect was attributed to the formation of a coordination complex between the chemosensor ClSNP and the metal ions. Notably, the addition of other metal ions did not result in any appreciable changes in color or spectral characteristics. The observed fluorescence quenching behavior aligns with the chelation‐enhanced quenching (CHEQ) mechanism, wherein coordination of the metal ions with ClSNP results in a reduction of its emission intensity [51]. This mechanism is supported by the rapid formation of a nonfluorescent complex ClSNP + M²⁺ (M²⁺ = Cu²⁺ and Co²⁺ ions), highlighting the selective binding properties of ClSNP toward Cu²⁺ and Co²⁺ ions. From the modified Benesi–Hildebrand equation (Equation 2), ClSNP exhibited binding constants of 2.11 × 10⁵ and 1.83 × 10⁵ M⁻¹ toward Cu²⁺ (Figure S8) and Co²⁺ ions, respectively. The corresponding Stern–Volmer quenching constants were 3.38 × 10⁵ and 1.43 × 10⁵ M⁻¹ for Cu²⁺ (Figure S9) and Co²⁺ (Figure S10) ions, respectively. Job's plot analysis confirmed the formation of a 1:1 coordination complex between ClSNP and Cu²⁺/Co²⁺ ions. (Figure S11 and S12) The detection limits for Cu²⁺ and Co²⁺ ions were calculated as 1.79 and 3.40 µM, respectively (Figure S13 and S14).

The selectivity of ClSNP toward Cu^{2 +} and Co²⁺ ions originates from the suitable ionic radii and strong coordination affinity for N,O‐donor ligands with flexible coordination geometries. The Cu²⁺ (d⁹) and Co²⁺ (d⁷) are paramagnetic ions, and upon coordination with ClSNP, they induce efficient spin–orbit coupling and enhance nonradiative decay pathways, leading to suppression of radiative fluorescence of CISNP and resulting in pronounced CHEQ [52]. In contrast, diamagnetic ions such as Na⁺, K⁺, Ca²⁺, Zn²⁺, and Cd²⁺ ions have all their electrons paired, and therefore, possess no net magnetic moment; as a result, they do not facilitate the spin‐orbit interactions or nonradiative decay pathways required for efficient fluorescence quenching [53]. Meanwhile, trivalent ions like Al³⁺ and Cr³⁺ ions possess high charge density and act as hard Lewis acids, which lead to strong hydration effects and charge mismatch, which hinder effective coordination with ClSNP [54]. These results demonstrate that chemosensor ClSNP functions as a selective and irreversible emission “turn‐off” probe capable of effectively detecting Cu²⁺ and Co²⁺ ions [55, 56, 57, 58, 59, 60, 61]. Table S1 presents a comparative overview of the sensing performance of previously reported Schiff base probes for analyte detection. The emission decay profile of ClSNP exhibited a biexponential decay with lifetime (τ) values of 2.1 and 1.4 ns. After the addition of Cu²⁺ ion (100 µΜ), the lifetime decreased to 1.8 and > 1 ns, respectively (Figure S15). Anions have weak interactions (hydrogen bonding/electrostatic), no paramagnetic activity, and can not induce a strong electronic effect such as LMCT or CHEQ (Figure 4), so there are no changes in fluorescence relative to Cu²⁺ and Co²⁺ ions.

FIGURE 4.

Fluorescence recognition mechanism of Cu²⁺ /Co²⁺ ions by the ClSNP.

Competitive studies with various cations in CH₃CN─H₂O (1/9, v/v) solution were conducted to further assess the practical application of ClSNP as a receptor of Cu²⁺ and Co²⁺ ions selective sensor. This produced a bar chart, as seen in Figure 5. Cu²⁺ and Co²⁺ ion selectivity was clearly depicted as a bar graph at 490 nm. The findings demonstrated that the detection of Cu²⁺ and Co²⁺ ions was not significantly hampered by the presence of other cations. These findings further confirm that variations in fluorescence intensity are only caused by the addition of Cu²⁺ and Co²⁺ ions. Thus, the ClSNP sensor can be utilized as an effective fluorescent chemical sensor for Cu²⁺ and Co²⁺ ions for future studies [62].

FIGURE 5.

Bar graph for the behavior of ClSNP (10 µM) toward Cu²⁺ (a) and Co²⁺ ions (b) (10 µM) and other cations measured by fluorescence in CH₃CN–H₂O (1/9, v/v) solution at 490 nm.

3.4. Sensing of PA

Nitroaromatic compounds are well‐known potent fluorescence quenchers, while their influence on other measurable physicochemical properties is minimal. Consequently, fluorescence‐based detection has emerged as the primary and most effective strategy for identifying nitroaromatic analytes using organic and inorganic sensing materials. In this study, the ClSNP Schiff base probe was evaluated for its response toward a series of nitroaromatic compounds, including NB, DNB, 2‐NT, 1‐Cl‐2‐NB, 4‐NT, 1‐Cl‐4‐NB, NBA, PNP, and PA. Among the tested analytes, only PA produced significant spectral changes: an enhancement in the absorbance intensity at 345 nm (Figure S16) and pronounced fluorescence quenching of the ClSNP emission at 490 nm. In contrast, the addition of other nitroaromatics under identical conditions produced negligible variations in fluorescence spectra and no observable color changes under UV illumination (Figure S17). These results clearly demonstrate the high selectivity of ClSNP toward PA over other structurally related nitroaromatic species [63, 64, 65, 66]. Using the Benesi–Hildebrand method (Equation 1), the binding constant (Ka) for the ClSNP–PA interaction was calculated to be 2.20 × 10⁴ M^{− 1}.

The quantitative fluorescence response of ClSNP was analyzed under optimized conditions with varying concentrations of PA. Both the absorbance enhancement (Figure 6a) and fluorescence quenching (Figure 6b) of ClSNP were found to depend on the PA concentration. A gradual decrease in fluorescence intensity at 490 nm was observed with successive additions of PA. These findings indicate that the introduction of PA alters the surface states of ClSNP. The fluorescence‐based sensing assay exhibited excellent sensitivity, simplicity, and selectivity for PA detection in aqueous solution [67]. The quenching interaction between PA and ClSNP was analyzed using the Stern–Volmer equation (Equation 4). Analysis of the Stern–Volmer plot (Figure S18) yielded a Stern‐Volmer constant (K _sv) of 5.02 × 10⁴ M^{− 1} for ClSNP. The detection limit for PA using ClSNP was calculated to be 2.49 µM. (Figure S19) The linear region of the Stern–Volmer plot indicates that the quenching mechanism follows a static quenching process (Figure S20). Furthermore, application of the modified Benesi–Hildebrand equation (Equation 2) provided a binding constant (K _b) of 8.76 × 10⁵ M^{− 1}.

FIGURE 6.

Absorption (a) and emission (b) responses of ClSNP (10 µM) to rising PA concentrations (10–150 µM) in THF.

3.5. Binding of Antibiotics

Antibiotics can be sensitively detected by Schiff base through the formation of complexes that exhibit measurable changes in their optical properties, such as absorbance and fluorescence. This strategy enables selective identification of specific antibiotics, supporting applications in environmental monitoring and biomedical diagnostics [68]. To evaluate the detection selectivity of ClSNP, its absorbance and fluorescence responses were recorded in the presence of different antibiotics. These findings imply that ClSNP exhibits strong interaction with target antibiotics even in competitive environments. Nitrofurantoin and metronidazole (10–150 µM) prepared in THF were employed to investigate the binding behavior of ClSNP via UV–vis absorption and emission techniques. It showed characteristic absorbance bands at 295 and 340 nm in THF. Upon incremental addition of nitrofurantoin, the band at 295 nm shifted to 273 nm (blueshift), while the band at 345 nm shifted to 375 nm (redshift), accompanied by increased absorption intensities (hyperchromism). (Figure 7a) Similarly, when metronidazole (10 µM) was added, the absorption bands merged at 317 nm with a notable increase in intensity (Figure 8a). These spectral changes confirm the strong binding affinity of ClSNP toward both nitrofurantoin and metronidazole [69]. The binding constant (K _a) for the ClSNP–antibiotic complexes was obtained from the double reciprocal Benesi–Hildebrand plot (Equation 1), further supporting the high binding propensity of ClSNP toward these antibiotics.

FIGURE 7.

Absorption (a) and emission (b) responses of ClSNP (10 µM) to rising nitrofurantoin concentrations (10–150 µM) in THF.

FIGURE 8.

Absorption (a) and emission (b) responses of ClSNP (10 µM) to rising metronidazole concentrations (10–150 µM) in THF.

Excess residues of nitrofuran antibiotics are known to exhibit carcinogenic potential, underscoring the need for sensitive detection of trace‐level contaminants to mitigate risks to human health and the environment [70]. To quantitatively evaluate the sensing ability of ClSNP toward antibiotics, fluorescence titration experiments were performed by gradually adding nitrofurantoin (Figure 7b) and metronidazole (Figure 8b) to a THF solution of ClSNP. In THF, ClSNP displayed fluorescence bands at 490 and 520 (sh) nm. Upon addition of 10 µM of either antibiotic, the emission at 490 nm was significantly quenched, and the fluorescence intensity progressively decreased with increasing analyte concentration (10–150 µM), indicating strong quenching interactions. Notably, this change was clearly observable to the naked eye. (Figure S25). The modified Benesi–Hildebrand equation (Equation 2) gave binding constants (K _b) of 4.13 × 10⁴ and 8.19 × 10⁴ M⁻¹ for nitrofurantoin and metronidazole, respectively, confirming their high affinity toward ClSNP. Quenching behavior was further analyzed via the Stern–Volmer equation (Equation 4). The Stern–Volmer constants (K _sv) were determined to be 5.21 × 10⁴ and 3.42 × 10⁴ M⁻¹ for nitrofurantoin and metronidazole, respectively (Figures S21 and S22). The observed nonlinear Stern–Volmer plots are indicative of a static quenching mechanism. The detection limits for nitrofurantoin and metronidazole, calculated from the corresponding calibration plots (Figure S23 and S24), were found to be 2.22 and 2.60 µM, respectively, demonstrating the high sensitivity of ClSNP toward these clinically relevant nitrofuran antibiotics [71].

3.6. BSA Sensing Study

BSA was chosen as the model protein because it is a well‐characterized, stable, and widely used transport protein, and the structural and binding properties closely resemble those of human serum albumin (HSA). Its availability, cost‐effectiveness, and strong intrinsic fluorescence make it particularly suitable for probing ligand–protein interactions using spectroscopic techniques. Absorption titration experiments were performed to evaluate the sensitivity of ClSNP toward BSA. Free ClSNP in DMSO displayed characteristic absorption peaks at 295 and 345 nm. Upon the stepwise addition of BSA in phosphate buffer, a gradual increase in absorbance at both wavelengths was observed, as shown in Figure 9a [45]. Emission titration studies were performed to investigate the sensitivity of ClSNP in DMSO toward BSA, among other proteins such as Ovalbumin and Lysozyme in phosphate buffer. The fluorescence intensity of ClSNP progressively increased at 490 nm with incremental addition of BSA, whereas other proteins caused a decrease in fluorescence intensity, accompanied by a notable change in emission wavelength (Figure S26). In DMSO, compound CISNP exhibited moderate emission due to its conformational flexibility and ─CH═N─ isomerization in the excited state, which facilitates nonradiative relaxation. Using this fluorescence‐based sensing approach, BSA was effectively detected by the Schiff base ClSNP. Upon excitation at 365 nm, ClSNP in DMSO displayed a broad emission peak centered at 490 nm. With the gradual addition of BSA dissolved in phosphate buffer, the fluorescence intensity of ClSNP increased significantly, accompanied by a blueshift from 490 to 460 nm, as illustrated in Figure 9b. This spectral behavior suggests that ClSNP interacts with the hydrophobic cavity of BSA through multiple noncovalent interactions with amino acid residues, restricting the intramolecular rotation of the ─CH═N─ group of the ClSNP. The formation of the BSA–ClSNP complex thus enhances molecular rigidity in the excited state, suppressing C─N isomerization and favoring radiative decay [46, 47]. The Benesi–Hildebrand (B–H) plot revealed a linear relationship consistent with a 1:1 binding stoichiometry between BSA and ClSNP. The binding constants were calculated to be 2.70 × 10⁴ M⁻¹ from absorption data, and 2.30 × 10⁴ M⁻¹ from fluorescence data. Studies conducted under physiologically relevant conditions confirmed that ClSNP interacts with BSA at a single high‐affinity binding site, forming a stable ClSNP–BSA complex. A detection limit of 1.28 µM was achieved for BSA [72] (Figure S27). A visual comparison of ClSNP and the ClSNP–BSA complex under UV illumination is presented in Figure 10. The sensing performance of ClSNP with various analytes is presented in Table 1.

FIGURE 9.

(a) Absorbance spectral response of ClSNP (10 µM) in DMSO upon rising BSA concentrations (10−150 µM) in a phosphate buffer. (insert) A linear relationship between 1/∆A at 345 nm and [BSA]. (b) The fluorescence spectral response of ClSNP (10 µM) in DMSO upon increasing BSA concentrations (10–150 µM) in a phosphate buffer. (insert) modified Benesi–Hildebrand plot.

FIGURE 10.

Photograph of (left) ClSNP (10 µM) alone in DMSO and (right) the combination of CISNP and BSA (100 µM) under UV light (365 nm).

TABLE 1.

Absorbance and emission spectral data of ClSNP with various analytes.

		Binding constant		Emission
S. no.	Analytes	Absorption (K a, M−1)	Emission (K b, M−1)	Stern–Volmer constant (K SV, M−1)	LOD (µM)
1.	Cu2+	3.50 × 104	2.11 × 105	3.38 × 105	1.79
2.	Co2+	1.98 × 104	1.83 × 105	1.43 × 105	3.40
3.	PA	2.20 × 104	8.76 × 104	5.02 × 104	2.49
4.	Nitrofurantoin	1.68 × 10 4	4.13 × 104	5.21 × 104	2.22
5.	Metronidazole	2.54 × 104	8.19 × 104	3.42 × 104	2.60
6.	BSA	2.70 × 104	2.30 × 104	—	1.28

Molecular docking studies were carried out to evaluate the binding conformations and interactions between the drug‐like molecule and the target protein. In this study, the Schiff base ClSNP was docked with BSA to elucidate its binding behavior [73]. The docking results revealed that ClSNP interacts strongly with BSA, forming stable contacts with key amino acid residues such as Leu‐182, Arg‐153, and Val‐183. The calculated binding energy was −13.31 kcal/mol, indicating a favorable and stable interaction.

In this study, molecular docking was carried out to explore the interaction of Schiff base ClSNP with BSA [74]. The interaction of ClSNP with BSA involved key residues such as Leu‐182, Arg‐153, and Val‐183, with a calculated binding energy of –13.31 kcal/mol, indicating a strong binding affinity. A proposed binding mode of ClSNP with the BSA active site is illustrated in Figure 11.

FIGURE 11.

Docking studies of ClSNP with BSA.

Overall, the analyte‐dependent fluorescence responses of ClSNP originate from distinct interaction mechanisms governing excited‐state relaxation pathways. The chemosensor ClSNP distinguishes itself from ESIPT‐active [75 ^– 77] or dichlorosalicylaldehyde‐based Schiff base sensors as follows: ESIPT‐active Schiff bases typically exhibit dual emission peaks, including a short‐wavelength emission from the enol form and a long‐wavelength emission (large Stokes shift) from the tautomeric keto form. ClSNP does not rely on ESIPT‐driven fluorescence, which is a crucial process due to the presence of an intramolecular hydrogen bond between the phenolic hydroxyl (─OH) and the imine nitrogen (C═N) moiety as its primary sensing signal, despite the intrinsic presence of ESIPT character in its excited state. Instead, ESIPT is largely suppressed in the free probe, serving primarily as a non‐radiative decay pathway that enables other analyte‐dependent excited‐state processes to dominate. Previously reported dichlorosalicylaldehyde‐based Schiff base sensors, including thioether‐based system [78] showed fluorescence enhancement upon the addition of Zn²⁺ and CN⁻  ions, respectively In contrast, ClSNP sensor underwent pronounced fluorescence quenching in the presence of Cu²⁺ and Co²⁺ ions. Unlike conventional ESIPT‐ or dichlorosalicylaldehyde‐based Schiff‐based sensors, which typically employ a single sensing mechanism to target a specific analyte, ClSNP distinguishes chemically diverse species through distinct fluorescence response pathways. These include CHEQ for Cu²⁺ and Co²⁺ ions, static quenching and photoinduced electron transfer for nitroexplosives and antibiotics, and restricted intramolecular rotation‐induced fluorescence enhancement upon protein binding. This multifunctional, mechanism‐switchable behavior within a single dichlorosalicylaldehyde‐derived scaffold clearly sets ClSNP apart from earlier ESIPT‐active Schiff base sensors.

3.7. DFT Study

The optimized molecular geometry of the ClSNP (Figure 12) revealed a stable and well‐conjugated framework with effective electron delocalization throughout the aromatic backbone. The frontier molecular orbital (FMO) analysis showed that the HOMO is predominantly localized over the electron‐rich donor region, whereas the LUMO is distributed over the acceptor moiety, indicating efficient intramolecular charge transfer characteristics. The calculated HOMO and LUMO energies were found to be −5.31 and −2.40 eV, respectively, with an energy band gap of 2.91 eV, suggesting moderate chemical stability and enhanced electronic transition probability. The relatively narrow HOMO–LUMO gap confirms the potential applicability of the ligand in optoelectronic, nonlinear optical, and molecular photovoltaic applications [79].

FIGURE 12.

(a) Optimized structure of ClSNP. (b) HOMO–LUMO energy level diagram of ClSNP.

3.8. Application in Water Samples

ClSNP for the measurement of Cu²⁺/Co²⁺ ions (Figures S28 and S29) was tested in a real water sample to ensure its practical application. Water samples were obtained on our college campus. Cu²⁺/Co²⁺ ions were added to water samples at varying concentrations (15–20 µM). The ClSNP fluorescence responses for sensing Cu²⁺/Co²⁺ ions were observed at 490 nm. The results revealed that as the concentration of Cu²⁺/Co²⁺ ions in the tested samples increased, the fluorescence intensity gradually dropped. The fluorescence intensity showed good linearity with Cu²⁺/Co²⁺ ions over the concentration range of 15–20 µM. The desirable recovery and relative standard deviation (RSD) values (Tables S2 and S3) showed that ClSNP [80].

4. Conclusion

A fluorometric molecular probe containing a ClSNP Schiff base was prepared through a simple condensation of dichloro‐substituted salicylaldehyde with N‐phenyl‐o‐phenylenediamine. The structure of ClSNP was thoroughly characterized using various spectroscopic techniques. Owing to its rational design, ClSNP functioned as an efficient and versatile chemosensor for the fluorometric detection of various analytes. For instance, it exhibited selective fluorescence quenching (turn‐off) responses toward Co^{2 +} and Cu^{2 +} ions. Chelation of these metal ions with ClSNP led to the formation of 1:1 metal–ligand complexes (ClSNP–Co^{2 +}/Cu^{2 +} ions), as confirmed by Job's plot analysis. The observed decrease in fluorescence intensity was attributed to the chelation‐enhanced quenching (CHEQ) phenomenon. The probe exhibited detection limits of 1.79 µM for Cu²⁺ and 3.40 µM for Co²⁺ ions. In addition, ClSNP displayed a pronounced quenching response toward PA compared to other nitroaromatic compounds, which was attributed to intermolecular hydrogen‐bonding interactions, facilitating effective recognition. This probe also exhibited a fluorescence enhancement (turn‐on) response toward BSA, with a detection limit of 1.28 µM. Job's plot and molecular docking analyses confirmed the specific binding interactions between ClSNP and BSA. Furthermore, spectroscopic studies revealed that ClSNP interacted distinctly with antibiotics such as metronidazole and nitrofurantoin, demonstrating its applicability as a versatile fluorescent sensor in environmental and biological systems.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: asia70829‐sup‐0001‐SuppMat.doc.

Data Availability Statement

The data that support the findings of this study are available in the Supporting Information.