A highly effective curcumin analogue as “naked eye” colorimetric and fluorescent sensor for sensitive and selective detection of Hg²⁺ ions and its application on test strips and real sample analysis

Abstract

A thiophene appended curcumin-based colorimetric and fluorescent receptor (TAA) for selective recognition of Hg²⁺ ions was synthesized and characterized using ¹H NMR, ¹³C NMR and LC-MS spectroscopic techniques. TAA facilitates detection of Hg²⁺ by a “naked-eye” color change from yellow to colorless in visible light, and fluorescence ‘turn-off’ in UV light (365 nm). The observed fluorescence quenching is due to the chelation-enhanced fluorescence quenching (CHEQ). TAA exhibited excellent selectivity and sensitivity toward Hg²⁺ ions, even in the presence of competing cations. The binding constant (K_a) for Hg²⁺ ions was found to be 3.4 × 10⁵ M⁻¹, indicating a strong binding affinity. The binding mechanism was elucidated using DFT calculations and supported by LC-MS and FT-IR studies. TAA forms a 1 : 1 complex with Hg²⁺ ions, as confirmed by Job's plot analysis. Additionally, the colorimetric limit of detection was found to be 0.67 μM, while the fluorometric limit of detection was found to be 0.24 μM, which demonstrates the high sensitivity of TAA towards Hg²⁺. Furthermore, TAA probe exhibited successful detection of Hg²⁺ ions in real water samples. Also, it can serve as an effective on-site detection tool for mercury ions by a simple test strip method that requires no additional instrumentation.

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1 Introduction

The environment has been polluted by both biological and non-biological sources, such as harmful bacteria, sewage effluents, animal waste, heavy metals, and both industrial and municipal waste.¹ Due to the rapid pace of industrialisation, heavy metal pollution has become a major global environmental concern. Common heavy metals such as mercury (Hg²⁺), arsenic (As³⁺), lead (Pb²⁺), cobalt (Co²⁺), cadmium (Cd²⁺), nickel (Ni²⁺), and chromium (Cr³⁺) are known for their high toxicity, persistence in the environment, resistance to degradation, and water solubility. Exposure to such toxic metals above permissible levels can pose serious risks not only to human health, but also to aquatic and terrestrial living beings.^2–4

Particularly, the extensive use of mercury ions (Hg²⁺) in batteries, paints, and other electronic goods has resulted in considerable environmental damage and poses a serious threat to both human health and natural systems.⁵ High intake of Hg²⁺ causes serious threats to human health as it can penetrate biological membranes, leading to severe damage to the blood capillaries, brain, kidneys, nervous system, and digestive system. It is a known fact that the accumulation of Hg²⁺ in human bodies can occur through food and drinks, which leads to the importance of detecting Hg²⁺ at the source, especially aqueous level.^6–8 The World Health Organization (WHO) and other national authorities have stated that mercury is recognized as one of the top ten most dangerous chemicals causing serious risks to human health. It is difficult to detect heavy metal ions such as Hg²⁺ in an aqueous environment due to the effective hydration and high stabilization.⁹ Hence, there is a strong demand for the development of efficient, rapid, and on-site methods for the detection of Hg²⁺ in aqueous environments.¹⁰

Various methods are employed for the detection of metal ions, ranging from traditional physicochemical approaches to advanced analytical techniques such as atomic absorption spectrometry (AAS), atomic fluorescence spectrometry (AFS),¹¹ mass spectrometry,¹² liquid chromatography, and gas chromatography.¹³ But these analysis methods are expensive, time-consuming and less sensitive and hence, the development of optical sensors with rapid, low-cost, simple, and sensitive metal ion detection is the need of present world.¹⁴ In order to overcome this, optical sensors like UV-vis and fluorescence sensors have attracted great attention and are well-suited for Hg²⁺ sensing.^15–20 This method is rapid, sensitive, low-cost and selective as well. Using organic molecules in such a process will provide an environmentally friendly approach. Several fluorophores with different moieties such as fluorescein,²¹ rhodamine,²² benzimidazole,²³ anthracene,²⁴ pyrene,²⁵ coumarin,²⁶ quercetin,²⁷ nanoparticles²⁸ and fluorescent nanofibers²⁹ have been reported for the detection of Hg²⁺. Among these, one such fluorophore is curcumin, which is a fluorescent molecule having a large planar structure with more π-conjugation that could be used as a fluorophore for metal ion detection.

Curcumin, a natural yellow pigment isolated from dried turmeric rhizomes, exhibits low cytotoxicity, excellent photostability, and is a popular medicinal herb. Also, curcumin has been popular for its biological activities, such as anti-inflammatory, anti-aging, anti-microbial, antiproliferative, anti-cancer, radioprotective, neuroprotective, and cardioprotective properties.^30–33 Apart from its pharmacological nature, curcumin has been used in food and food packaging³⁴ and also as a fluorescent probe in biomedical applications.³⁵ Curcumin and its derivatives have a large conjugate structure, which aids in the electron delocalization, enabling them to emit long-wavelength fluorescence. It contains 1,3-diketones with keto–enol isomerization, and is a chelating site for various toxic cations and anions based on the Intramolecular charge transfer (ICT) mechanism.³⁶ Due to this, curcumin has great significance in chelation treatment and heavy-metal intoxication. Although several studies reported curcumin-based sensors (Table S1†), there remains great potential for further development by modifying its core structure or employing its analogues in various sensing applications.

Thiophenes are highly reactive organic building blocks that could be used in the field of sensors as a functional group, because of their excellent chemical stability, high carrier mobility, and unique photo-physical properties.³⁷ Curcumin analogue with a thiophene moiety could enhance the fluorescence emission wavelength. Motivated by the above-mentioned characteristics, we developed a thiophene-based curcumin analogue as an Hg²⁺ probe for selective and sensitive detection with a low limit of detection in aqueous medium. This work presents a simple and facile approach for Hg²⁺ detection and provides rapid, non-toxic, naked eye detection.

2 Experimental

2.1 Materials and methods

The synthesis chemicals and solvents were purchased from Avra Chemicals and SD Fine and used without further purification. The NMR spectra were recorded using a Bruker 400 MHz spectrometer using CDCl₃ as the solvent, and tetramethylsaline (TMS) as an internal standard. LC-MS data was recorded using an ESI ionization in the LCMS-8060NX instrument from Shimadzu. The Shimadzu IR Spirit spectrophotometer was used to record Infrared spectra. Absorption measurements were carried out with a Shimadzu UV-1800, and fluorescence measurements were carried out with a RF-6000 Spectrofluorophotometer. Analytical thin-layer chromatography (TLC) was performed on pre-coated plates (Merck, silica gel 60F254).

2.2 General procedure of synthesis of TAA

The preparation of the Curcumin analogue was carried out according to the known method (AN Nurfina et al.³⁸). The reaction was carried out using thiophene-2-carboxaldehyde and acetylacetone as the starting materials. The acetylacetone (0.5 equivalent) was added to the solution of boric anhydride (0.5 equivalent) in ethyl acetate (8 mL). Thiophene-2-carboxaldehyde (1 equivalent) and tri-n-butyl borate (0.75 equivalent) were added to this solution and stirred for 10 minutes at 50 °C. Furthermore, n-butylamine was added dropwise for a prolonged period of 5 minutes, and the reaction was continued for 5 hours at 50 °C. Hydrochloric acid (1 N) was added, and the reaction was stirred for another 60 minutes (Scheme 1). The reaction was monitored using thin-layer chromatography (TLC), and a yellow-coloured fluorescent spot was observed indicating completion of the reaction. The reaction was worked up as explained in the reported method to obtain yellow solid of curcumin analogue TAA with 82% yield.

Scheme 1 Synthesis of (1E,6E)-1,7-di(thiophen-2-yl)hepta-1,6-diene-3,5-dione (TAA).

2.2.1 Experimental procedure and analytical data of the curcumin analogue (3).
2.2.1.1 Synthesis of (1E,6E)-1,7-di(thiophen-2-yl)hepta-1,6-diene-3,5-dione (TAA). The reaction was carried out by treating thiophene-2-carbaldehyde 1 (200 mg, 1 eq.) and acetyl acetone 2 (89 mg, 0.5 eq.) in presence of boric anhydride, tri-butyl borate, and n-butyl amine as a base in ethyl acetate to obtain the desired (1E,6E)-1,7-di(thiophen-2-yl)hepta-1,6-diene-3,5-dione 3 with 82% yield.

(1E,6E)-1,7-di(thiophen-2-yl)hepta-1,6-diene-3,5-dione (TAA, Scheme 1): The compound obtained is a yellow solid. Yield: 421.7 mg (82%); ¹H NMR (400 MHz, CDCl₃) δ: 7.78 (d, J = 10 Hz, 1H), 7.38 (d, J = 2.8 Hz, 1H), 7.26 (d, J = 2.4 Hz, 1H), 7.067 (t, 1H), 6.41 (d, J = 10.4 Hz, 1H), 5.78 (s, 1H).

¹³C NMR (400 MHz, DMSO): δ 183.00, 140.30, 133.72, 132.55, 130.55, 129.24, 123.18, 101.99. Mass spectrometry: LCMS (ESI) m/z: calculated for C₁₅H₁₂O₂S₂ = 288.38, found = 289.

2.3 Photophysical properties

Ten solvents with different polarities i.e. methanol, ethanol, DMSO, DMF, DCM, acetonitrile, tetrahydrofuran, ethyl acetate, toluene and hexane were employed to investigate the excitation and emission behaviour of the synthesized TAA. For this purpose, the absorbance and fluorescence emission spectra of the compound were measured in various solvents using 25 μM solutions of TAA.

2.4 General procedure for spectroscopic measurements

A Stock solution of the probe TAA (1 mM) was prepared using HPLC grade CH₃CN. The stock solutions of other various cations (1 mM) used in the study were prepared by dissolving the following compounds in distilled water: Al(NO₃)₃, BaCl₂, Cd(NO₃)₂, CoSO₄, CrCl₂, AgNO₃, Fe(NO₃)₃, FeCl₂, HgCl₂, Li₂SO₄, MgCl₂, MnSO₄, NiSO₄, Pb(NO₃)₂, ZnSO₄. The other test solutions of anions were prepared using tetra butyl ammonium salts of I⁻, Br⁻, ClO₄⁻; sodium salts of S₂O₃²⁻, F⁻, Cl⁻, NO₃⁻, N₃⁻ and ammonium salt of acetate. All spectroscopic measurements were performed at room temperature using a CH₃CN/H₂O mixture (v/v = 1 : 1). A 25 μM and 50 μM solution of the probe TAA and analytes were used for binding studies. The excitation and emission slit widths were set to 5 nm and 5 nm, respectively, for all measurements.

2.5 Stoichiometry and binding constant calculation

The stoichiometry of the probe TAA and Hg²⁺ was analysed by performing a Job's plot. Stock solutions of the probe TAA and Hg²⁺ were made at a concentration of 25 μM and 50 μM, respectively. The final volume was kept to be 3 ml, and varying volumes of Hg²⁺ (0.3–2.7 ml) and the probe TAA (2.7–0.3 ml) were mixed in a quartz cuvette. A Job's plot was then constructed by plotting the mole fraction against absorbance to determine the mole fraction at which complex formation was maximal.

The binding constant of the complex was evaluated using the Benesi–Hildebrand (B–H) plot. The following equation was used to determine the binding constant.

where A₀ is the absorbance in the absence of Hg²⁺, A is the absorbance in the presence of Hg²⁺, C denotes the concentration of Hg²⁺ added during the titration, and A_max represents the maximum absorbance recorded throughout the experiment.

By absorption and fluorescence titration plot, the limit of detection (LOD) of Hg²⁺ was measured. The standard deviation was achieved by the absorption and fluorescence spectra of the probe, which were calculated from repeated measurements. To attain the slope, the calibration plot between the absorption and fluorescence intensities and the concentration of the analyte (Hg²⁺) was calculated using the following equation:

where σ is the standard deviation of the probe, K is the slope value from the linear calibration plot.

2.6 pH study

To evaluate the influence of pH on the fluorescence sensing performance of probe TAA towards Hg²⁺, solutions of TAA (25 μM) were prepared with and without Hg²⁺ (50 μM) across a pH range of 2–12, using 1 N HCl and 1 N NaOH. The fluorescence spectra of these solutions were then recorded.

2.7 DFT calculations

To validate complex formation, the Gaussian 09 software was used to carry out theoretical calculations. Using the B3LYP/6-311G++ (d, p) basis set, the ground and excited state geometries of the probe TAA were optimized. Further, a B3LYP/LANL2DZ basis set was used to improve the geometry of the TAA-Hg²⁺ complex.

2.8 Real sample analysis

In order to analyse the ability of the probe TAA to detect Hg²⁺ in the environmental samples, real water analysis was carried out. Different water samples, like river water, lake water, pond water, tap water and drinking water samples, were collected and filtered through Whatman filter paper. In the end, the Hg²⁺ solution was injected into the samples and analyzed.

2.9 Fabrication of test paper strip

The square strips of Whatman filter paper were dipped in the solution of TAA and air dried for 10 minutes. Further, these strips were again soaked in a solution of different metal ions for 10 minutes and dried. Then these strips were examined under visible and UV light (365 nm).

3 Results and discussion

3.1 Design and synthesis

In this research, a fluorescent and colorimetric sensor based on curcumin was developed to detect Hg²⁺ ions. Curcumin generally exhibits fluorescence due to its extended π-electrons conjugation, which allows for electron delocalization from one end to the other end of the molecule. Additionally, it can undergo keto–enol tautomerism, contributing to its unique photophysical properties. On the other hand, with the α,β-unsaturated-diketone component as the recognition site for the analyte, the design of a thiophene moiety to the α,β-unsaturated-diketone moiety facilitates the chelation-enhanced fluorescence quenching (CHEQ) process with Hg²⁺ ions. As shown in (Scheme 1), the synthesis of the probe TAA was performed by reacting thiophene-2-carbaldehyde with acetylacetone in a 2 : 1 ratio in ethyl acetate via a condensation reaction. The product was obtained in good yield (82%). The synthesized TAA was characterized by ¹H NMR, ¹³C NMR, LC-MS techniques.

3.2 Photophysical properties of prepared TAA

The screening of the synthesized TAA molecule was done in order to study the Hg²⁺ ion sensing. Based on the position of the excitation and emission peak and also the Stokes shift values of TAA (25 μM) in ten different solvents of varying polarity (Table 1), the highest Stokes shift with better excitation and emission spectra was chosen for the absorption–emission titrations of the Hg²⁺ ion detection study. The excitation and emission peaks were found to be at 417 nm and 522 nm in acetonitrile (CH₃CN), which showed a better and broader intensity peak compared to other solvents with a better Stokes shift value (Fig. 1). Thus, acetonitrile was used as the solvent for TAA in further analysis.

Table 1 Absorption and emission properties of TAA in different solvents

Solvents	λ(ex) nm	λ(em) nm	Stokes shift (cm^−1)
Methanol	428	525	4317
Ethanol	418	521	4730
DMSO	438	514	3376
DMF	436	515	3518
DCM	434	537	4420
Acetonitrile	417	522	4823
Tetrahydrofuran	416	516	4659
Ethyl acetate	445	504	2630
Toluene	418	517	4581
Hexane	410	509	4744

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Fig. 1 Visual change of TAA in comparison with other metal ions: visible light (upper image), UV-light (365 nm) (lower image).

3.3 Visual detection of TAA in response to various metal ions

Colorimetric tests as well as fluorescence change of the probe TAA were performed using a range of metal ions including Al³⁺, Ba²⁺, Cd²⁺, Co²⁺, Cr²⁺, Ag⁺, Fe³⁺, Fe²⁺, Hg²⁺, Li⁺, Mg²⁺, Mn²⁺, Ni²⁺, Pb²⁺ and Zn²⁺. The probe TAA displayed a distinct yellow color in CH₃CN. Upon the addition of these metal ions to a CH₃CN/H₂O mixture (v/v = 1 : 1) there was no significant color change observed, except in the case of Hg²⁺ ions (Fig. 2), which was colorless solution. The probe TAA also showed bright yellow fluorescence under UV light (365 nm) in CH₃CN. Upon addition of Hg²⁺ ion the fluorescence was completely quenched, but there was no significant impact on the fluorescence intensity with the addition of other metal ions. This indicates that the probe TAA exhibits high sensitivity and selectivity specifically toward Hg²⁺. The decolorisation and fluorescence quenching observed upon Hg²⁺ binding is attributed to the preferential coordination of the Hg²⁺ ion with the di-ketone moiety of the sensor, leading to a highly selective detection response.

Fig. 2 (a) UV-vis absorption and (b) fluorescence spectra of TAA (25 μM) in different solvents.

3.4 Spectroscopic behaviour of TAA upon interaction with Hg²⁺

The optical properties of the TAA probe (25 μM) in the absence and presence of Hg²⁺ (50 μM) were investigated using UV-vis absorption and fluorescence spectroscopy. The TAA probe exhibited a prominent absorption band at 417 nm. Upon addition of 50 μM of Hg²⁺ ion, the absorbance at 417 nm decreased, while a new absorption band emerged at 340 nm (hypsochromic shift). This was accompanied by a visible color change from yellow to colorless solution. This indicates the potential of TAA for colorimetric detection of Hg²⁺. Further, fluorescence studies revealed strong emission intensity at 505 nm for the TAA probe in H₂O (v/v = 1 : 1). After introducing Hg²⁺ (v/v = 1 : 1), the fluorescence intensity significantly decreased due to chelation-enhanced fluorescence quenching (CHEQ) (Fig. S4†). The transfer of electrons from the probe to the analyte is attributed to the initiation of the photo-induced electron transfer (PET) process i.e. complexation of probe with Hg²⁺ leads to the non-radioactive decay pathway which in turn results in the quenching of the intrinsic fluorescence of TAA, thereby functioning as a turn-off fluorescence probe for the selective detection of Hg²⁺ ions.³⁹

3.5 Selectivity studies of TAA

The selectivity of the TAA probe for the detection of Hg²⁺ in comparison with other metals was studies. The absorption and fluorescence spectra of the TAA probe after the addition of different analytes, including Al³⁺, Ba²⁺, Cd²⁺, Co²⁺, Cr²⁺, Ag⁺, Fe³⁺, Fe²⁺, Hg²⁺, Li⁺, Mg²⁺, Mn²⁺, Ni²⁺, Pb²⁺ and Zn²⁺ were measured (Fig. 3a and b). After adding these analytes, no significant change was observed in the absorption and emission intensities, except Hg²⁺. The absorption peak blue-shifted to 340 nm, and the fluorescence intensity of the TAA probe was sharply quenched after its reaction with Hg²⁺. These data demonstrated the high selectivity of the TAA probe to Hg²⁺. We further extended the UV-Vis and fluorescence studies of TAA by evaluating its response in the presence of various competing anions including Br⁻, CH₃COO⁻, Cl⁻, ClO₄⁻, F⁻, I⁻, N₃⁻, NO₃⁻ and S₂O₃²⁻. As shown in Fig. S6,† the sensor exhibited no significant interference from any of these anions.

Fig. 3 (a) UV-vis and (b) fluorescence spectra of TAA (25 μM) with various metal ions (50 μM) in CH₃CN/H₂O (v/v: 1/1).

3.6 Anti-interference studies of TAA

To evaluate the selectivity and anti-interference capability of TAA for practical applications, a competitive study was conducted in the presence of various potential interfering metal ions, including Al³⁺, Ba²⁺, Cd²⁺, Co²⁺, Cr²⁺, Ag⁺, Fe³⁺, Fe²⁺, Hg²⁺, Li⁺, Mg²⁺, Mn²⁺, Ni²⁺, Pb²⁺ and Zn²⁺. Initially, absorption studies at 417 nm were carried out using solutions containing 25 μM of TAA along with 50 μM of various other interfering metal ions, which displayed no significant change in absorption in comparison with TAA. When Hg²⁺ was introduced with other metal ions, decrease in the absorbance of the TAA-Hg²⁺ complex was observed (Fig. 4a). Similarly, in fluorescence spectroscopy (Fig. 4b), TAA solutions with competing ions exhibit significant fluorescence at 505 nm, which are quenched after the addition of Hg²⁺. These findings confirm the outstanding selectivity of TAA for Hg²⁺ detection, even in complex environments. As Hg²⁺ is a soft metal, the selective affinity of Hg²⁺ towards TAA over other competing metal ions may be attributed to its strong preference for binding with soft bases, particularly favoring sulfur-containing ligands. In this study, the presence of a sulfur atom in the ligand likely plays a crucial role in facilitating the specific interaction with Hg²⁺ over other metal ions.⁴⁰

Fig. 4 Competitive study of TAA (25 μM) with Hg²⁺ upon addition of other interfering metal ions (50 μM): (a) absorption studies, (b) emission studies.

3.7 Sensitivity studies of TAA

To investigate the sensitivity of TAA (25 μM) towards Hg²⁺ (50 μM) ions, UV-vis and fluorescence titration experiments were conducted in a CH₃CN/H₂O (v/v, 1 : 1) medium. Upon incremental addition of Hg²⁺ ions, decrease in absorption and fluorescence intensity at 417 nm and 505 nm was observed (Fig. 5a and b). This indicates that TAA exhibits a specific response towards Hg²⁺ due to complex formation.

Fig. 5 (a) UV-vis and (b) fluorescence titration plot of TAA (25 μM) upon addition of Hg²⁺ (50 μM), detection limit of TAA by (c) UV-vis spectroscopy, (d) fluorescence spectroscopy.

Furthermore, using UV-vis and fluorescence titration curves the limit of detection (LOD) for Hg²⁺ ions was calculated (using eqn (2)) and were determined to be 0.67 μM and 0.24 μM, respectively (Fig. 5c and d), indicating the use of TAA in simulated water samples. The LOD value was found to be quite low compared to the recent sensor reports (Table 3). The colorimetric and fluorometric linear detection range was found within 0–10 μM (R = 0.98) and 0–6μM (R = 0.9943), respectively. The fluorescence quantum yield was determined using quinine sulphate as a reference standard, which has a known quantum yield (Φ_R) of 0.54 in distilled water. The calculated quantum yield values were 0.0229 for TAA and 0.0029 for the TAA-Hg²⁺ complex. The photo image of colorimetric linear detection from yellow to colorless is been noticed proportional to the concentration of Hg²⁺ (Fig. S8†). Therefore, TAA demonstrates strong potential as a visual colorimetric probe for Hg²⁺ detection under environmental conditions.

3.8 Binding stoichiometry ratio analysis of TAA with Hg²⁺

To gain deeper insight into the binding affinity of TAA towards Hg²⁺, the stoichiometry and binding constant were further evaluated using Job's and Benesi–Hildebrand (B–H) plots. In Job's plot, the [TAA]/([TAA] + [Hg²⁺]) = 0.5 mole fraction, indicates the stoichiometry ratio of TAA to Hg²⁺ is 1 : 1 (Fig. 6a). Binding affinity was further supported by the linearity of the B–H plot (Fig. 6b), which yielded a high correlation coefficient (R² = 0.9833). The binding constant (K_a) was calculated to be 3.4 × 10⁵ M⁻¹. Upon complexation, the core moiety gets the charge density, which in turn induces a chelation-enhanced fluorescence quenching (CHEQ) effect. Additionally, a stable photo-induced electron transfer (PET) mechanism is triggered.⁴¹

Fig. 6 (a) Job's plot of TAA (25 μM) for Hg²⁺ (50 μM), (b) Benesi–Hildebrand (B–H) plot of TAA (25 μM) for Hg²⁺ (50 μM).

In order to know the binding mechanism, infrared (IR) spectroscopy was performed. The IR spectrum showed C=O stretching vibration at 1625 cm⁻¹ in the free ligand. But, the IR spectrum of the complex revealed that there was no C=O stretching vibration (Fig. S5†). These spectral changes provide strong evidence of Hg²⁺ coordination through the di-ketone moiety (–C=O) of the ligand. To further demonstrate the binding mechanism, mass spectrum of TAA-Hg²⁺ complex was taken, that indicates a peak at m/z = 568 was assignable to [TAA + Hg²⁺ + Cl₂⁻ + ½H₂O] (Fig. S7†).

3.9 Time response of TAA towards Hg²⁺

The time-dependent behavior of probe TAA (25 μM) towards Hg²⁺ (50 μM) was performed, the absorption and the emission intensities of TAA at 417 nm and 505 nm remained nearly constant for up to 30 minutes. Furthermore, a rapid response time of approximately 4–5 seconds was observed for the interaction between the TAA-Hg²⁺ complex, the absorption and emission peaks appeared when the recognition process completed, which remained stable for up to 30 minutes as shown in Fig. 7a and b.

Fig. 7 Time evolution of TAA (25 μM) towards Hg²⁺ (50 μM), (a) UV-vis spectroscopy, (b) fluorescence spectroscopy.

3.10 Density functional theory (DFT) calculations

The optimized geometry of TAA and TAA-Hg²⁺ complex through DFT is shown in (Fig. 8a and b). The TAA-Hg²⁺ complex, containing the Hg²⁺ metal centre, exhibits a tetrahedral geometry formed through coordination with a neutral ligand possessing an O₂ donor system of the TAA and two mononegative chloride counter ions, resulting in the formation of a mononuclear complex.

Fig. 8 Geometry optimized structures of: (a) TAA, (b) TAA-Hg²⁺ complex, (c) frontier molecular orbital with energy difference of TAA and TAA-Hg²⁺ complex.

The theoretical evidence of the binding mechanism of TAA and Hg²⁺ was confirmed by DFT calculations with B3LYP/6-311G++ (d, p) as a basis set for the TAA probe and B3LYP/LANL2DZ basis set for the complex using the Gaussian 09 program.^42,43 As illustrated in (Fig. 8c) the frontier molecular orbitals of the TAA-Hg²⁺ complex are lower in energy (ΔE = 3.37 eV) compared to the frontier molecular orbitals of the TAA probe alone (ΔE = 3.78 eV). Thus, the complex is electronically more stable compared to the probe. The HOMO of the complex indicates that the probe significantly binds to the molecule with possible charge transfer between the metal ion and the probe upon excitation. This is a PET process, providing an explanation for the change in absorbance and fluorescence quenching.

3.11 pH studies

To evaluate the practical applicability of TAA in environmental and agricultural contexts, its fluorescence behaviour was analysed across a range of pH conditions with and without the Hg²⁺. The solutions CH₃CN/H₂O (v/v, 1 : 1) were prepared with pH values ranging from 2 to 12 by gradually adding 1 M hydrochloric acid and 1 M sodium hydroxide, with pH monitored using a pH meter. As illustrated in Fig. 9, the fluorescence intensity of probe TAA remained stable between pH 2–9, but showed a gradual decline from pH 10–12, which was likely due to degradation of its molecular structure. The degradation of TAA structure in a basic medium is may be due to the nucleophilic attack by hydroxide ions leading to the cleavage of the heptadienone moiety, resulting in the loss of the active methylene group. Additionally, upon introducing Hg²⁺ to the TAA solution, there was a slight quenching in fluorescence intensity within the pH range of 2–6 in which the Hg²⁺ detection may not be significant. But at pH 7–9, significant fluorescence quenching was observed. Therefore, the optimal pH range for effective application of TAA for Hg²⁺ detection was between pH 7–9.

Fig. 9 Effect of pH on TAA (25 μM) and TAA-Hg²⁺ in fluorescence spectroscopy.

3.12 Reversibility of TAA

Reversibility and reusability are key attributes of an effective chemosensor. In this context, the reversible sensing ability of TAA was evaluated. Initially, TAA displayed an emission intensity at 505 nm. Upon the addition of Hg²⁺ ions, complete decrease in the fluorescence intensity was observed, indicating complex formation. To assess reversibility, a well-known chelating agent ethylenediaminetetraacetic acid (EDTA) (50 μM), was introduced to the TAA-Hg²⁺ complex (Scheme 2). Upon addition of EDTA, the fluorescence intensity was restored. This fluorescence recovery confirmed the displacement of Hg²⁺ ions by EDTA, demonstrating the reversible binding interaction. Furthermore, this cycle of switching between fluorescence on–off was successfully repeated over 2 cycles with alternating additions of Hg²⁺ and EDTA (Fig. 10), affirming the ability of the TAA probe to reproduce and reuse.

Scheme 2 Probable binding mode in the solution phase.

Fig. 10 Reversibility cycle of TAA (25 μM) with EDTA (50 μM).

4 Practical application

4.1 Real water sample analysis

To assess the practical utility of TAA for detecting Hg²⁺, real-water sample experiments were conducted using five types of water: river water, pond water, distilled water, lake water and tap water from nearby places. Since these environmental samples were not fully purified, trace amounts of heavy metal ions could still be present. Using such water samples, Hg²⁺ detection is more challenging. Each sample was first filtered using Whatman filter paper. No significant fluorescence quenching was observed in the pretreated distilled water, tap water, pond water, lake water and river water samples, indicating minimal background interference. Subsequently, known concentrations of Hg²⁺ were introduced into each sample. Fluorescence intensity values were recorded and used to calculate Hg²⁺ concentrations using the linear calibration curve. The recovery rates of Hg²⁺, shown in Table 2, ranged from 99.0% to 102.0%. These results clearly demonstrate the credibility and practical applicability of the TAA sensor for detecting Hg²⁺ in real environmental water samples.

Table 2 Determination of Hg²⁺ in different real water samples

Samples	Hg^2+ spiked (μM)	Hg^2+ found (μM)	Recovery (%)
River water	5.00	5.10	102.0
Lake water	5.00	5.06	101.2
Pond water	5.00	5.03	100.6
Tap water	5.00	4.98	99.6
Drinking water	5.00	4.99	99.8

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Table 3 Comparison of previously reported Hg²⁺ sensors with the present work

S no.	Ligand	Sensing method	Probe type	LOD	Application	Ref.
1		Colorimetric & fluorescence	Turn-on	2.57 μM	Bioimaging	44
2		Colorimetric & ratiometric fluorescence	Turn-on	0.451 μM	Bioimaging	45
3		Fluorescence	Turn-off	0.47 μM	Logic gate & test kits	46
4		Fluorescence	Turn-on	4.92 μM	Bioimaging	47
5		Fluorescence	Turn-on	0.501 μM	Bioimaging & soil sample test	48
6		Fluorescence	Turn-off	4.68 μM	Real water sample test	49
7		Ratiometric fluorescence	Turn-off	2.91 × 10^−5 M	—	50
8		Colorimetric & fluorescence	Turn-on	Colorimetric 7.83 μM	Biological, logic gate & real sample tests	51
				Fluorometric 1.04 μM
9		Colorimetric & fluorescence	Turn-off	1.87 μM	Real soil sample test	52
10		Colorimetric & fluorescence	Turn-off	Colorimetric 0.67 μM	Real water analysis & test paper strips	This work
				Fluorometric 0.24 μM

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4.2 Test strip analysis

Portable test strip-based detection provides a real-time, simple and efficient method for the visual recognition of target analytes. To evaluate the practical applicability of TAA, portable test strip experiments were carried out. Filter paper strips were coated with the solution of TAA (1 mM) and exposed to various metal ion solutions (1 mM) including Al³⁺, Ba²⁺, Cd²⁺, Co²⁺, Cr²⁺, Ag⁺, Fe³⁺, Fe²⁺, Hg²⁺, Li⁺, Mg²⁺, Mn²⁺, Ni²⁺, Pb²⁺ and Zn²⁺. In the visible light, all the test strips dipped in various metal ion solutions were yellow in color, except the one dipped in Hg²⁺ solution, which was colorless. This proves that TAA can be used as a naked eye sensor for Hg²⁺ detection using a test strip as a medium (Fig. 11a). Similarly, under 365 nm UV light, TAA exhibited a highly selective response to Hg²⁺ ions, evidenced by a clear quench in fluorescence. But not with other metal ions in the TAA-coated strips (Fig. 11b). These results demonstrate that TAA can function as a portable fluorometric sensor with excellent selectivity for the visual detection of Hg²⁺ ions.

Fig. 11 TAA-coated (1 mM) test strips for Hg²⁺ (1 mM) in comparison with other metal ions: (a) visible light, (b) UV-light (365 nm).

5 Conclusion

We present an easy-to-use curcumin-based fluorescent probe (TAA) that functions as a colorimetric and fluorescence “turn-off” probe, demonstrating high sensitivity and selectivity for the detection of Hg²⁺ ions in a CH₃CN/H₂O (1 : 1, v/v) medium. The multifunctional sensing behaviour of TAA was confirmed by absorption and emission studies, which revealed hypsochromic shifts in absorption and quenching of emission intensity upon complexation with Hg²⁺. In order to understand the sensitivity of TAA for Hg²⁺, both absorption and emission studies were performed which revealed the colorimetric detection limit to be 0.67 μM and the fluorometric detection limit to be 0.24 μM. The Job's plot indicated 1 : 1 stoichiometry, and the B–H plot produced a binding affinity of 3.4 × 10⁵ M⁻¹. The complex formation of Hg²⁺ onto TAA was confirmed by LC-MS, IR and DFT analysis. Nature of TAA and TAA-Hg²⁺ complex in different pH^2–12 was also studied, in which pH 7–9 was a suitable environment for Hg²⁺ detection. Reusability of TAA was studied using EDTA. Finally, the evident practicability of the TAA was validated through its high sensitivity in detecting low concentrations of Hg²⁺ in real water sample analysis. Its applicability on test strip applications and in the evaluation of various water samples highlights its exceptional property. The key attributes of TAA, such as reversibility, reusability, high selectivity, and practical applicability, highlight its significance in advancing colorimetric and fluorescent sensors for diverse analytical applications.

Data availability

The data supporting this article have been included as part of the ESI.†

Author contributions

Ramesh D. K.: conceptualization, methodology, investigation, visualization, writing – original draft, writing – review & editing. Prasad Pralhad Pujar: supervision, funding acquisition, writing – review & editing.

Conflicts of interest

There are no conflicts of interest to declare.

Acknowledgements

This study was financially supported by the Major Research Project Grant (MRPDSC-1616) awarded by Christ University, Bangalore.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5ra03656a

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