Introduction

Schiff bases are widely employed for the design of chromofluorogenic chemosensors due to their potent photophysical characteristics and attractive coordination behavior [1]. Schiff base derivatives are well-known for their antibacterial [2], antifungal [3], anticancer [4], antitumor [5] and cytotoxicity characteristics [6]. Schiff bases act as effective corrosion inhibitors and are also utilized as catalysts in a variety of chemical processes [7]. Schiff base derivatives are employed as active binding receptors to bind various metal ions with the imine-N atom [8]. During complex formation, the electron density is transferred from the imine group to the metal ion [9]. The complexation-induced ligand to metal charge transfer (LMCT) resulted naked-eye detectable color and spectral changes that allowed on-site detection of metal ion [10]. Literature also revealed that Schiff base derived chromofluorogenic chemosensors have gained significant attention in the last few decades due to their potential applications in environmental, biological and industrial processes [11].

Mercury is one of the most toxic heavy metals in the human body [12]. The USEAP and WHO have recommended a permissible limit of 2 ppb of Hg2+ concentration in drinking water [13]. Some microbes turn various forms of mercury into the potent poison methyl mercury [14]. As a non-biodegradable contaminant, mercury can harm the kidney and brain as well as cause serious health conditions like Minamata disease [15]. It may also affect the immune system and the heart [16], resulting in a number of illnesses, including memory loss, the birth of abnormal offspring, a drop in muscular strength, and a decrease in the body’s overall immunity [17]. So, the detection of trace amounts of mercury is so necessary now a day. The fluorescence detection method over other methods has piqued the interest of scientists due to its significant advantages [18], such as real-time detection, high sensitivity [19], high selectivity and non-invasive features [20]. Many attempts have already been made to synthesize fluorescent sensors that could recognize Hg2+ ions by deprotonating thiols [21], ring opening of rhodamine derivatives [22], cyclizing [23], ring opening of thiourea derivatives [24] and, other processes [25]. However, these fluorescent sensors have a number of drawbacks, including a convoluted structure and arduous chemical synthesis, which limit their practical use in environmental monitoring and biological tests [26]. Considering the above facts, this manuscript introduced an easy-to-synthesize chromone-derived Schiff-base (E)-3-(1-(2-(2,4-dinitrophenyl)hydrazineylidene)ethyl)-4 H-chromen-4-one (L) (Scheme 1) for the selective detection of Hg2+. The receptor L exhibited Hg2+ selective turn-on fluorescence at 544 nm in DMSO.

Scheme 1
scheme 1

Synthesis of (E)-3-(1-(2-(2,4-dinitrophenyl)hydrazineylidene)ethyl)-4 H-chromen-4-one ( L )

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Experimental

Materials and Instruments

The chemicals (3-formyl chromone and 2,4-dinitrophenyl hydrazine) and solvents were obtained from Sigma-Aldrich. The chloride salts of Na+, Li+, Mn2+, Cr2+, Ni2+, Ca2+, Mg2+, K+, Co2+, Cu2+, Zn2+, Al3+ and Hg2+ and sulfate salt of Fe2+ were obtained from CDH. UV-Visible spectra were recorded in the wavelength region of 200–800 nm. The FTIR spectrum was recorded using a Bruker Alpha ECO-ATR instrument over a wide range of wave number of 4000 to 400 cm−1. A Perkin-Elmer LS-55 fluorescence spectrophotometer was used to record the fluorescence spectrum with slit width of 5.0 nm. The HRMS data of the receptor was recorded from a SCIX-QTOF Mass spectrometer in DMSO solvent. Using ZEN-3690 Zetasizer device of Malvern Panalytical Ltd., Hyogo, Japan, the hydrodynamic diameters were recorded. All the experiments were performed at ambient temperature. Double distilled water was utilized in all spectral experiments.

Synthesis and Characterization

A formyl chromone-based Schiff base ligand, (E)-3-(1-(2-(2,4-dinitrophenyl)hydrazineylidene)ethyl)-4 H-chromen-4-one (L) was prepared by the 1:1 addition of 2,4-dinitrophenyl hydrazine and 3-acetyl-4 H-chromen-4-one in ethanol based on a previously reported method (Scheme 1) [27].

UV-Vis and Fluorescence Studies

The standard solutions of several metal salts (10−5 M) were made in deionized water. The standard solution of L (10−5 M) was made in a solvent of DMSO. For the UV-Vis and fluorescence titration studies, 2.0 mL solution of L was filled into quartz optical cells with a 1 cm path length. Using a micropipette, required amounts of metal ions were added and spectra were recorded to examine the selectivity, specificity and sensitivity. The spectra were captured within 1 min of the metal salts being added. All measurements are performed at room temperature. For fluorescence measurement of L (λex = 410/λem = 544 nm), the widths of the emission and excitation slits were fixed at 5 nm. Using the fluorescence titration data, the limit of detection (LOD) was calculated using the IUPAC-certified formula, LOD = 3σ/slope [28]. Here σ stands for the standard deviation of a free sample.

The Benesi-Hildebrand plot is used to determine the association constant and association stoichiometry from the fluorescence titrations [29]. The Benesi-Hildebrand equation [30] for a 1:n complex type comprising a ligand and Mn+ is given as follows.

$$\frac{1}{\text{F}-{\text{F}}_{0}}=\frac{1}{{\text{F}}_{{\infty }}-{\text{F}}_{0}}+\frac{1}{\text{K}{\left[{\text{M}}^{\text{n}+}\right]}^{\text{n} }\left(\frac{1}{{\text{F}}_{{\infty }}-{\text{F}}_{0}}\right)}$$
(1)

where, F and F0 are emissions of receptor L in the absence and presence of Hg2+ ions. \({\text{F}}_{{\infty }}\) is the maximum fluorescence intensity of L with metal ions at saturation. [Mn+] is the concentration of Hg2+ ions and K is the association constant.

Results and Discussion

The azomethine group of Schiff bases serves as an active interaction site for metal ions due to its chelating nature [31]. In the above aspect, a novel chromone-based Schiff base L is designed and synthesized in a 1:1 addition of 2,4-dinitrophenyl hydrazine and 3-acetyl-4 H-chromen-4-one in DMSO (Scheme 1). After characterization, the selectivity and sensitivity of L towards Hg2+, among other competitive metal ions were analyzed through various spectrophotometric analyses like UV-Vis, fluorescence and dynamic scattering analysis (DLS) [32].

Colorimetric Analysis

To analyze the potential use of the synthesized chromone derivative L as a colorimetric chemosensor, the colorimetric studies are analyzed by adding various metal ions [33]. The DMSO solution of L was interacted individually with different metal ions, such as Na+, Mn2+, Li+, Cr3+, Fe2+, Ni2+, Ca2+, Mg2+, K+, Co2+, Cu2+, Zn2+, Al3+ and Hg2+, no such color change response was observed except for Hg2+. The incorporation of Hg2+ ions into the orange-colored solution of L results in a change in color to light yellow, which is visible to the naked-eye (Fig. 1a). Also, the non-fluorescent L changed to light blue fluorescent in the presence of Hg2+ when irradiated with UV365 nm light (Fig. 1b).

Fig. 1
figure 1

Colorimetric changes of L (10−5 M, DMSO) in the presence of several metal ions (10−5 M): under day light (a) and UV lamp at 365 nm (b)

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UV-Vis Absorption Spectral Studies

The sensing capabilities of L towards metal ions including Na+, Mn2+, Li+, Cr3+, Fe2+, Ni2+, Ca2+, Mg2+, K+, Co2+, Cu2+, Zn2+, Al3+ and Hg2+ were studied in DMSO medium by UV-Vis absorption spectroscopy [34]. It was astonishing to know that the addition of distinct metal ions to the DMSO solution of L results in an alteration in absorption intensity in the presence of Hg2+ (Fig. 2a). The absorption spectra of L in DMSO solution displayed a strong absorbance peak at 396 nm. The absorption peak of L at 396 nm was due to the n-π* transition between the lone pair electron of the N-atom and the aromatic π electron of the aromatic ring. The incorporation of Hg2+ into the DMSO solution of L results in a decrease in absorption intensity at 396 nm. In the case of other metal ions except Hg2+, no such considerable alteration in absorption spectra was observed.

The incremental addition of Hg2+ to the DMSO solution of L resulted in a gradual decrease in the absorption intensity at 396 nm (Fig. 2b). The change in spectral behavior is associated with a slight blue shift of the absorption peak from 396 nm to 389 nm with a distinct color alteration of orange color to pale yellow which is discernible in the naked-eye (Insert in Fig. 2b). The results clearly delineated that receptor L can be utilized for the selective and sensitive detection of Hg2+. To provide further information regarding the binding affinity of the receptor towards Hg2+, fluorescence selectivity and titration experiments are performed [35].

Fig. 2
figure 2

a UV-Vis absorption spectra of L (2000 µL, 10−5 M) in DMSO upon the incorporation of various metal ions (2 µL in each aliquot); b UV-vis absorption intensity of L that changes when Hg2+ ions are added gradually (0–28 µL, 10−5 M)

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Fluorescence Sensing Behavior of L

To further explore the selectivity of L towards Hg2+, a fluorescence selectivity experiment was carried out in DMSO. The molecular probe L in DMSO solution generates a weak fluorescent emission peak at 544 nm when excited at 410 nm. The weak emission intensity of L at 544 nm may be caused by the intermolecular charge transfer of electrons [36]. With the addition of several metal ions such as Na+, Mn2+, Li+, Ni2+, Fe2+, Cr3+, Ca2+, K+, Mg2+, Co2+, Cu2+, Zn2+, Al3+ and Hg2+ no such noticeable alteration in spectral behavior was observed except for Hg2+ (Fig. 3a). In the presence of Hg2+, the fluorescence intensity of L increased significantly at 544 nm (Fig. 3b). Further titration experiments of Hg2+ against the DMSO solution of L were carried out to provide evidence in favor of the selectivity of L towards Hg2+. The incremental addition of Hg2+ (2 µL in each aliquot, 10−5 M) to the DMSO solution of L results in a gradual enhancement of emission intensity at 544 nm (Fig. 3c). The non-fluorescent L changed to a light blue fluorescent in the presence of Hg2+ (Insert in Fig. 3c). The fluorescence emission intensity of L was increased gradually, with a little red-shift with the incremental addition of Hg2+. The change in spectral behavior and alteration in color provide evidence of interaction between L and Hg2+.

Fig. 3
figure 3

a Fluorescence spectra of L (2000 µL, 10−5 M) in DMSO upon the incorporation of various metal ions (2 µL in each aliquot). b Bar diagram of L fluorescence intensities at 544 nm in the absence and presence of various metal ions. c Fluorescence emission intensity of L that changes when Hg2+ ions are added gradually (0–38 µL, 10−5 M)

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Sensing Mechanism

To comprehend the mechanism of interaction between chromone-based Schiff base L and Hg2+, the Benesi-Hildebrand plot was plotted using the fluorescence titration data [37]. The binding constant Ka for the L-Hg2+ complex was evaluated graphically using the Benesi-Hildebrand plot (Fig. 4a) [38]. The linear fit plot from the Benesi-Hildebrand plot indicates a 1:1 stoichiometry for the complexation occurring between L and Hg2+ [39]. Using the B-H equation [40], the Ka of receptor L with Hg2+ was determined as 1.234 × 107 M−1. The limit of detection for Hg2+ was also calculated to be 1.870 × 10−6 M, which is found to be very much lower than that of the reported probes (Table 1).

To investigate the reversibility behavior of L, the reversibility test of L against Hg2+ was implemented in the presence of the disodium salt of EDTA. The L in DMSO solution results in a weak emission peak at 544 nm (Fig. 4b). The addition of Hg2+ (38 µL) resulted in an enormous increase in the fluorescence emission peak at 544 nm. But it was astonishing to note that the addition of EDTA, even if in excess, didn’t show any alteration in fluorescence intensity. Even in the presence of EDTA, the color of the complex solution didn’t recovered. The results clearly indicated that the complex of L and Hg2+ is irreversible in nature. The irreversibility supported the high stability of the L-Hg2+ complex formed in solution. The effects of pH on the fluorescence sensing of Hg2+ by L was examined from pH 2 to 10 (Fig. 4c). Schiff base L can be employed for the detection of Hg2+ over a wide pH range [41].

Fig. 4
figure 4

a Benesi-Hildebrand plot. b Reversibility of L against Hg2+ in presence of sodium salt of EDTA. c Effect of different pH on the fluorescence changes of L in the absence and presence of Hg2+ ion

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Table 1 Comparison of the characteristics of the proposed ligand with previous reported literature
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The FTIR study was performed to examine the interaction that occurred between L and Hg2+. The FTIR spectrum of L resulted in peaks at 3265 cm−1 and 1606 cm−1 due to –NH stretching vibration and -C═N vibration, respectively (Fig. 5). It is observed that in the FTIR spectrum of the L-Hg2+ complex, the -NH vibration peak and -C═N vibration peak shifted to 3258 and 1609 cm−1, respectively. It clearly signifies that the azomethine group participates in the interaction of L with Hg2+. The other peaks of L at 1316, 1123 and 767 cm−1 of –NO2, -C-O and mono-substituted C-H bending respectively shift to 1321, 1130 and 761 cm−1 in the presence of Hg2+, which also indicates the complexation occurred between L and Hg2+.

Fig. 5
figure 5

FTIR spectra of L in the absence and presence of Hg2+

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The density functional theory (DFT) calculations were performed using B3LYP exchange-correlation functional B3LYP to obtain the 3D structure of the Schiff base L and L-Hg2+ complexes in the gas phase [42]. The basis sets LANL2DZ for the Hg atom whereas 6-31G** for C, H, O and N atoms were used for the calculations by using the computational programme Gaussian 09 W [43]. The computed structures of the Schiff bases L and L-Hg2+ are shown in Fig. 6. In solution, the conformational flexibility at the -C = N linkage, along with the photo-induced electron transfer (PET) process [44], is expected to quench the fluorescence emission of L. Schiff base L provides an ideal bidentate coordination environment through imine-N and formyl chromone-O donor atoms to chelate Hg2+. With the formation of the L-Hg2+ complex, the DFT-computed interaction energy (Eint = Ecomplex − Ereceptor – EHg2+) got lowered by -214.57 kcal/mol, supporting the formation of a stable and favourable complex between L and Hg2+. The formation of the L-Hg2+ complex also lowered the band gap between the lowest unoccupied molecular orbital (LUMO) and the highest occupied molecular orbital (HOMO) [45]. Further analysis of the HOMO and LUMO electron densities of the L and L-Hg2+ complexes revealed that charge transfer occurred from the ligand to the Hg2+ metal ions. Therefore, the complexation-induced inhibition of PET and C = N isomerization is responsible for the fluorescence switch-on response from L upon interaction with Hg2+ [46].

Fig. 6
figure 6

DFT (B3LYP/6-31G**/LANL2DZ) computed 3D structure of L (a) and L-Hg2+ complex with their LUMO and HOMO diagrams

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Real Sample Analysis

The Hg2+ detection ability of Schiff base probe L was investigated under various nutrient soil samples [47]. The soil samples have a profound role in the field of agriculture [48]. The soil samples, like alluvial soil, red soil, sandy soil and loamy soil full of nutrients are collected from nearby agricultural areas of Veer Surendra Sai University of Technology Burla, Sambalpur, and Odisha [49]. All the collected soil samples are poured with Hg2+ solution. The addition of Hg2+ results in a small alteration in the color of the solution to the naked-eye. Further, with the addition of DMSO/H2O (90:10, v/v) solution of L to the mercury-containing soil samples, the color of the solution becomes darkened in the naked-eye [50]. This alteration was also observed under the UV lamp at 365 nm (Fig. 7). The results suggested that the receptor L can detect Hg2+ in real samples.

Fig. 7
figure 7

Recognition of Hg2+ in various soil samples: a in naked-eye without ligand, b in naked-eye with Hg2+, c in naked-eye with Hg2+ and L d in UV-lamp at 365 nm with ligand

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Conclusions

A novel formyl-chromone-based chromofluorogenic sensor L was successfully developed and characterized by various spectral techniques. The receptor L exhibited good sensitivity and selectivity towards Hg2+ over other competing species in DMSO/H2O (10:90, v/v) solution. In the presence of Hg2+, receptor L showed a decrease in the absorption intensity at 396 nm and an increment in the fluorescence emission intensity at 544 nm. In the presence of Hg2+, the receptor L also displayed naked-eye detectable colour change under day and UV light upon complexation with Hg2+ in a 1:1 binding ratio. Using the fluorescence titration data, the detection limit and binding constant of L for Hg2+ were found to be 1.870 × 10−6 M and 1.234 × 107 M−1, respectively. Finally, receptor L was successfully applied for the qualitative detection of Hg2+ ions in various soil samples.