Introduction

Researchers are designing a new chemical sensor to identify the significant transition and heavy metal ion with high sensitivity and selectivity, in concern with industrial, health and environmental related issues. Metal toxication leads to serious health complication and cause ecosystem hazards. Hence, there is need for the development of least complicated, modest, and cost-effective sensors to detect metals1,2,3,4.

Heavy metal ions cause high toxicity due to their ability to co-ordinate with other biomolecules in the body even at extremely low quantities5. Lead ion is a second most toxic heavy metal, it is considered to be malleable, pervasive and non-degradable affecting the human health by accumulating the body, facing difficulty to excrete and seriously contaminates the ecosystem leading to pollution of air, water and soil6,7,8,9,10,11. Lead is renowned for its versatility in industrial applications due to its widespread usage in epoxy paints, radiation shielding, petroleum refining and ammunition12,13. The use of lead cannot be controlled in modern life due to its usage in cosmetic items and food preservatives14. Lead enters the environment through its use in dyes, waste plastics, pigments, batteries, gasoline and jewelry15,16. The prolonged exposure to Pb2+ even with minimal amount can cause high risks such as anemia, memory loss, digestive, neurological and cardiovascular disorders, ataxia, hypo immunity, muscle paralysis, nerve disorder, kidney disorders and mental retardation, hearing loss and Alzheimer’s disease5,17,18,19,20. It causes decreased IQs and hypertension in children11,21. According to the World Health Organization, drinking water can contain up to 10 µg of lead per liter22. Drinking lead contaminated water can cause long-term health issues including kidney problems and high blood pressure for adults, retardation of physical and mental development in infants and children23. Lead contamination in water has attracted significant attention around the world, to monitor the fast and sensitive methods for water quality assessment for the protection of human and animal health is intensified all over the world. Hence, it is very imperative to explore methods for the detection of lead that are highly convenient, reliable, good selective, rapid, quantitative and high sensitivity.

Iron is the most essential trace element and most imperative bio-active transition metal involved in living systems24,25,26. Iron exists in the form of ferrous ion (Fe2+) and ferric ion (Fe3+), which are necessary to the metabolic and other biological processes27,28,29. Iron owing a special place among variety of transition metals due to their integral role in the reactive centres of the metallo-enzymes like haemoglobin, cytochromes and biological processes. However, deficiency of iron makes serious symptoms like abnormal heart rhythms, organ damages and diseases including anaemia, Alzheimer’s and Parkinson’s disease30,31,32,33. Lack of iron weakens the immune system, allowing deadly viruses such as pneumonia and typhoid to affect the human body34. In cosmetic industry, products such as kajal, lipstick, sindoor, nail polish, Fe2+ are extensively used as colorant35. This metal ion is hazardous and toxic at higher concentrations. Iron levels in drinking water must not exceed 0.3 mg/L, as reported by China’s drinking water sanitation agencies36. Greater significance has been renowned for monitor the iron content. Although there are numerous optical and spectroscopic techniques such as atomic absorption spectroscopy (AAS), neutron activation analysis (NAA), atomic emission spectroscopy (AES), inductively coupled plasma-optical emission spectrometry (ICP-OES), inductively coupled plasma mass spectroscopy (ICP-MS), x-ray fluorescence spectrometry (XRF) is some of the methods used to detect trace levels of iron in complex matrices37,38,39,40,41,42. However, these techniques do have certain drawbacks, though, such as high costs, the requirement for skilled operators, and challenging procedures because of the preparation of several samples43. As a result, it is inevitable that low-cost, dependable, and straightforward techniques that can conduct quick and real-time analysis will be developed44. Due to its exceptional benefits, colorimetric iron chemosensors have drawn a lot of attention up to this point. However, there aren’t many reports in the literature on the colorimetric naked-eye Fe2+ detection techniques. Hence, the development of colorimetric chemosensors for ferrous ion (Fe2+) detection is taking the limelight.

The creation of chemosensors for identifying metal ions such as Pb2+ and Fe2+ has garnered significant interest because of their crucial roles in medicine, biological, and environmental systems. Among the various types of sensors, fluorescence and chromogenic “naked-eye” chemosensors are being recognized lately for metal ion detection, owing to their high sensitivity, exceptional selectivity, ease of use, quick response times, and overall simplicity. Further, single probe with numerous targets in environment and biological samples has gained increased attention due to low-cost, and high efficiency lately. The review of the literature for the detection of various metal ions using single probe by different detection methods as presented in the Table 1. Furthermore, the literature showed least detection of iron, and lead ions using the colorimetric, and fluorescence methods. In this concern the authors explored an efficient method for the detection of such metal ions using naked eye and quenching fluorescent probes.

In this study, we designed and synthesised a chemosensor utilising eugenol-based fluorophore and chromophore compound and detected for two analytes. The effectiveness of the ligand and its Fe2+ and Pb2+ complexes was examined using DFT, and the designed chemosensor was assessed for the biological practicability with real water sample analysis. Furthermore, EABH has the competence of detecting the two chief metal ions in the milieu and acts as colorimetric and fluorometric sensor response to Fe2+ and Pb2+ ions.

Table 1 Represents the previously reported methods in the detection of transition and heavy metal ions.
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Experimental

Materials and methods

2-hydroxy benzohydrazide, 5-allyl-2-hydroxy-3-methoxybenzaldehyde (Eugenol-5- aldehyde), Lead (II), iron (II) and other metal salts were procured from (Merck chemicals Pvt Ltd, India). pH of the solutions was maintained by HEPES buffer. DMSO, Ethanol was procured from S.D-fine chemical Pvt Ltd. DMSO solvent system is used through-out the colorimetric and fluorescence experiments. The ligand’s (EABH) melting point and their metal complexes C1 and C2 were determined using open capillary digi apparatus. The course of the reaction is monitored using TLC technique performed with [n-hexane-ethyl acetate 8:2] medium. The m/z values of ligand (EABH) and its complexes were determined using EV LC-MS Shimadzu mass spectrometer in order to differentiate in terms of their molecular mass. The coordination admittance of the ligand (EABH) with metal ions can be captured by recording IR spectrum (400–4000 cm− 1) using Perkin-Elmer 783 spectrophotometer. 1H and 13C spectra was recorded with Bruker Advance DPX spectrometer at 400 MHz, in an ambient temperature in d6-DMSO using TMS. Sensory action of the ligand was detected using a Shimadzu spectrofluorometer (RF-6000) equipped with 150-W xenon lamp and 1 cm path length quartz cell used. Electronic absorption measurement was performed using a 3100-XE UV/vis spectrophotometer in the range 200–800 nm (Lab India).

Chemical synthesis

Synthesis of ligand EABH

An equimolar solution of 2- hydroxy benzohydrazide (0.05 g, 0.0003 mmol) and 5-allyl-2-hydroxy-3-methoxybenzaldehyde (0.063 g, 0.0003 mmol) in ethanol were refluxed for 8 h at 60 ℃. The reaction was facilitated by adding 1 ml of glacial acetic acid. The progress of the reaction was monitored using TLC plate and UV chamber to visualize the spots. The lime yellow solid product was filtered off using Whatman filter paper. The obtained product was dried and recrystallized using hot ethanol. The pure product was subjected for further analysis. General synthetic scheme of ligand (EABH) is depicted in Fig. 1.

Yield − 86%; m. p. 215 °C, 1H-NMR(DMSO-d6, 400 MHz, ppm)-δ 12.0(br, s, 1H -OH),11.74(s,1H - OH), 10.57 (br, s, 1H -NH), 8.66 (s, 1H = C-H), 7.88 (d, J = 8 Hz, 1H, Ar-H), 7.48 (t, J = 8 Hz, 1H, Ar-H), 7.0- 6.97 (m, 3 H), 6.88 (d, J = 16 Hz, 1H), 5.98 (m, 1H), 5.13–5.08 (d, J = 20.4 Hz, 2 H), 3.81 (s, 3 H), 3.33 (d, J = 6.8 Hz, 2 H) (Figure S1 and S1a). (13C-NMR (DMSO-d6, 100 MHz, ppm)-δ164.98, 159.45, 148.84, 148.32, 145.90, 138.25, 134.39, 131, 129.01, 120.08, 119.45, 119.18, 117.73, 116.24, 116.17, 114.82 56.03, 40.08) (Figure S2). LC-MS calculated for C18H18N2O4: m/z = 326.13 and found m/z = 327.58 [M + 1] peak (Figure S3). FT-IR spectrum (v, cm− 1) 3216 − 2918 (broad, O-H), 1570 (C = N), 1624 (C = O).

Fig. 1
figure 1

General synthetic route for obtaining EABH.

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Synthesis of metal complexes (C1 and C2)

To the ethanolic solution of ligand (EABH), an ethanolic solution of FeSO4.7H2O and Pb(CH3COO)2.3H2O were added separately. The reaction mixture was refluxed for 10 h at 80 ℃, precipitate was formed, the solution was filtered off and washed with ethanol for several times and kept aside for drying. The complexes of Fe(II) and Pb(II) were obtained as a grey and yellow colored solid, respectively (Fig. 2).

C1: [Fe(EABH)2], Yield-76%, m.p. 280 °C. LC-MS: m/z = 706.17 and found m/z = 707.03 [M + 1] (Figure S4). 1H NMR (400 MHz, DMSO-d6) δ 11.81 (s, 2 H), 10.58 (s, 1H), 8.68 (s, 1H), 7.89 (s, 1H), 7.46 (s, 1H), 6.94 (d, J = 18.2.0 Hz, 4 H), 5.97 (s, 1H), 5.08 (dd, J = 18.4, 12.7 Hz, 2 H), 3.81 (s, 3 H), 3.37(m, 2 H) (Figure S5 and S5a).(13C-NMR (DMSO-d6, 100 MHz, ppm)-δ 165.0, 159.4, 148.9, 148.3, 145.8, 138.2, 134.4, 131.0, 129.02, 120.06, 119.5, 119.1, 117.7, 116.2, 116.1, 114.8, 56.31, 40.47 (Figure S6).

C2: [Pb(EABH)2], Yield-72%, m.p. 260 °C. LC-MS: m/z = 858.21 and found m/z = 858.30 [M+] (Figure S7). 1H NMR (400 MHz, DMSO-d6) δ 13.89 (s, 1H), 8.41 (s, 1H), 7.84 (d, J = 7.8 Hz, 1H), 7.25 (t, J = 8.1 Hz, 1H), 6.84 (d, J = 8.1 Hz, 1H), 6.79 (d, J = 7.1 Hz, 1H), 6.74 (d, J = 2.1 Hz, 1H), 6.70 (s, 1H), 5.94 (d, J = 6.8 Hz, 1H), 5.09 (d, J = 1.9 Hz, 1H), 5.06 (m, 1H), 3.73 (s, 3 H), 3.24 (d, J = 6.7 Hz, 2 H) (Figure S8 and S8a). (13C-NMR (DMSO-d6, 100 MHz, ppm)-δ 172.10, 159.952, 157.31, 156.71, 152.28, 138.98, 131.85, 129.19, 124.96, 123.94, 120.57, 120.21, 118.21, 116.49, 115.58, 114.47, 55.84, 40.46 (Figure S9).

Fig. 2
figure 2

Structure of Fe(II) and Pb(II) complexes.

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Cytotoxicity assay and biosensing studies

MTT (5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) assay was used to assess the cytotoxic effect of EABH59. HeLa cells procured from BioScienceClix laboratory (Mysuru, India) were cultured in Dulbecco’s modified eagle’s medium (DMEM) containing streptomycin (100 µg/mL) under 5% CO2 atmosphere at 37 °C. The cells were then cultivated on a 36-well plate so must reach about 75% confluence before the treatment. EABH was added (25 µg/mL) to the cells in a 36-well plate and incubated for additional 24 h. To this, 25 µL of MTT (2.5 mg/mL) was added into each well and cultured for another 4 h. UV-Visible absorbance was measured at 316 nm after adding 200 µL of DMSO into the each well. In a similar way, this was performed in the case of Fe2+ and Pb2+ complexes. After the incubation period (30 min), the staining of cells was carried out. The cells were then cleaned with PBS buffer solution. The fluorescence bioimaging of cells was then viewed and captured using an Invitrogen EVOS M5000 fluorescent inverted microscope.

Density functional theory (DFT) calculations

DFT calculations for EABH, EABH-Fe2+ and EABH-Pb2+ molecules were performed using Gaussian09 software60. Geometry optimization was conducted at the B3LYP level with 6-311 + + G(d, p) and LanL2DZ basis sets.

Results and discussion

FT-IR spectral studies

Bonding nature and functional group present in EABH and its Fe (II) and Pb (II) complexes were studied by FT-IR analysis. Figure 3 shows the FTIR spectra of EABH and its Fe2+ and Pb2+ complexes. The characteristic band of stretching frequency of carbonyl (C = O) and imine group (C = N) in EABH was seen at 1624 cm− 1 and 1570 cm− 1. After complexation, the bands of the carbonyl C = O and imine (C = N) moieties were shifted to 1606 cm− 1, 1601 cm− 1 and 1549 cm− 1 and 1520 cm− 1 for Fe2+ and Pb2+, respectively. The phenolic OH stretching band in the region between 3216 and 2918 cm− 1 in free EABH were disappeared in respective complexes indicating the deprotonation of the Schiff bases due to its involvement in the coordination61. These characteristic changes in the IR spectra suggested that the imine nitrogen, carbonyl oxygen and phenolic oxygen atoms of EABH have taken part in the coordination with Fe2+ and Pb2+ ions.

Fig. 3
figure 3

FT-IR spectrum of (a) EABH, (b) EABH-Fe2+ complex and (c) EABH-Pb2+ complex.

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UV-Visible absorption studies

To better understand the sensing ability of EABH as an efficient probe for the detection of Fe2+ ion, UV-vis absorption studies were conducted. Test solution of 60 µM of EABH (HEPES, 0.01 M pH = 7.4) in DMSO was used to carry out the experiments at room temperature. The UV-vis spectra of EABH shows a characteristic absorption peak at 281 nm due to π→π* transition of C = N group (Fig. 4a), whereas the peak at 322 nm reflected intra-molecular charge transfer (ICT) resulting due to n→π* transition, involving donor–acceptor conjugation systems47,62,63. The absorption peaks at 281 and 322 nm were blue shifted to 267 and 302 nm for Fe2+ metal ion. Furthermore, the complex formation between EABH and Fe2+/Pb2+ ions was confirmed by performing the absorbance titrations experiment, which were carried by incremental addition of Fe2+/ Pb2+ ions in a DMSO solution of EABH. It is interesting to note that the change in absorption spectra was observed at the isobestic points at 360 nm for the Fe2+ ion (Fig. 4b), and at 373 nm for Pb2+ ion (Fig. 4c). Thus, the change in the absorbance wavelength and the appearance of isobestic point indicates the formation of stable complex of EABH with Fe2+/Pb2+ ions64,65,66,67. On the other hand, the absorption band for Fe2+ ion demonstrates a strong complex formation between the EABH and Fe2+ ion. With the addition of Fe2+, Pb2+, Zn2+, Cd2+, Co2+, Fe3+, K +, Ni2+, Cu2+, Al3+ and Na+, F−, Br−, I−, HSO4− and CN− ions to the EABH solution, only Fe2+ shows brown colouration from lime yellow on addition of two equivalence of distinct metal ions, and other metal ions did not exhibit colour changes (Fig. 5). The transformation in the colour of EABH demonstrate high selectivity to Fe2+ ions due to ligand–metal change transfer (LMCT) of EABH with Fe2+68. According to the above-described experiment, the EABH in DMSO solution was used to determine Fe2+ using naked eye analysis.

Fig. 4
figure 4

UV–visible spectra of EABH (60µM) [0.01 M, HEPES buffer at pH 7.4] in DMSO solution with various metal ions (40 µM). Absorption spectra of EABH on incremental addition of (b) Fe2+ and (c) Pb2+ ions indicating the interaction between EABH and Fe2+ / Pb2+.

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Fig. 5
figure 5

Colorimetric and fluorescence photographic observations of the developed EABH chemosensor with different metal ions under (a) white light (b) UV light.

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Fluorescence studies

The selectivity of probe was determined by fluorescence emission spectra in the range of 200–900 nm. The addition of Fe2+, Pb2+, Zn2+, Cd2+, Co2+, Fe3+, K +, Ni2+, Cu2+, Al3+, Na+, F−, Br−, I−, HSO4− and CN− (40 µM) metal ions to the sensor EABH (60 µM, HEPES 0.01 M pH = 7.4) in DMSO with an excitation wavelength 326 nm (Fig. 6a). Only Pb2+ showed a significant fluorescence quenching at 498 nm, while the remaining metal ions did not show discernible changes in the spectra of EABH. Further, a decrease in fluorescence intensity upon the addition of Pb2+ ion due to the complexation between EABH and Pb2+ ion69. “Turn-off” mechanism was noticed for Pb2+ ion addition into the EABH, while the sensor failed to sense other metal ions (Fig. 6b). The above results indicated that the probe EABH is highly selective for Pb2+ and the sensor EABH functions as an exceptional selective fluorescence “on-off” sensor. The prepared complex follows static quenching, since the fluorescence intensity diminishes on increasing the metal ion concentration. Further, the metal complex acting as non-fluorophore on coordination with metal ion, due to the electrostatic interaction stabilizing the complex and the ligand-metal ions interaction. Hence, the complex does not exhibit fluorescence70. However, the quantum yield (QY) of EABH was calculated using the standard reference Quinine sulphate (Methanol, QY = 0.54) at 326 nm excitation wavelength using below mentioned in Eq. (1):

$$\:{QY}_{S}={QY}_{R\:\:}\times\:\:\frac{{I}_{S}}{{A}_{S}}\:\times\:\:\frac{{A}_{R}}{{I}_{R}}\:\times\:\:\frac{{\left({}_{R}\right)}^{2}}{{\left({}_{S}\right)}^{2}}\:$$
(1)

Where, R and S represent reference and sample, I is the intensity, A is the absorbance value and η is refractive index of the solvent. The relative quantum yield of EABH in DMSO was evaluated to be 0.04 However, addition of Pb2+ ions to EABH solution obtained quantum yield was 0.00959.

Fig. 6
figure 6

EABH (60 µM) (a) Fluorescence spectra and (b) fluorescence intensity bar graph (0.01 M, HEPES buffer at 7.4 pH in DMSO solution) with various metal ions (40 µM) excited at 326 nm and emitted at 498 nm respectively.

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Competitive titration

To verify the practicability of EABH as a selective probe for the specific recognition of Pb2+, metal cation competition experiment was conducted. The effect of competing ions on the coordination relationship between the probe EABH and Pb2+ was investigated by adding Pb2+ to EABH solutions containing different metal ions (Fe2+, Zn2+, Cd2+, Co2+, Fe3+, K +, Ni2+, Cu2+, Al3+, Na+, F−, Br−, I−, HSO4− and CN− ions), respectively. By comparing the fluorescence intensity of EABH-Pb2+ complexes after the addition of competing ions, from the experiments we determined that the addition of competing ions doesn’t alter the selectivity of the sensor EABH with Pb2+ and the probe EABH was still quenched by Pb2+ and remained unchanged on adding competing metal ions. The results indicate that the specific response of the probe EABH with Pb2+ is largely uninterrupted by the competing metal ions. The response of Pb2+ ions and the probe EABH can be attributed to the chelation of EABH with Pb2+71 and the fluorescence is quenched but in the case of other competing ions, there is no significant changes in the fluorescence spectra because they did not form stable complexes. The fluorescence intensity of EABH responds only to Pb2+ ions, this indicates the high sensitivity and selectivity of EABH towards Pb2+ detection, while the latter metal ions offered no- interference as presented in bar diagram (Fig. 7).

Fig. 7
figure 7

Competitive results of EABH with 60 µM and HEPES 0.01 M, pH = 7.4 in the solution of DMSO. EABH (black coloured bar graph), EABH with various metal ions (40 µM) (red coloured bar graph), and addition of Pb2+ to EABH consisting various metal ions (blue coloured bar graph).

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Binding stoichiometric studies (Job’s plot)

The EABH with the Fe2+ and Pb2+ binding ratios were obtained with Job’s plot method of continuous variation operated at ambient temperature72. The plot of absorbance/fluorescence vs. mole fraction of ferrous ions at 326 nm/ Pb2+ at 498 nm were used to assess the binding ability of complex with EABH (60 µM) and metal ions at 40 µM. From jobs plot, maximum absorbance and fluorescence intensity for Fe2+ and Pb2+ were observed at a molar ratio 0.3, which suggests that the binding stoichiometry between the sensor EABH and the metal ions Fe2+ and Pb2+ is 1:2 [M: L], which was further confirmed by LC-MS, stoichiometry with respect to the [probe + M2+]73. Accordingly, we proposed the possible structures shown in (Fig. 2) between the sensor EABH and the metal ions (Fe2+/Pb2+) (Fig. 8).

Fig. 8
figure 8

Job’s plot depicting the EABH stoichiometric ratio with metal ions (40 µM) in DMSO solution, (a) Fe2+ and (b) Pb2+ ions.

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Solvent effect

The solvent dependency of the EABH was assessed to estimate the fluorescence property to comprehend its interaction with Fe2+/Pb2+ ions. Which was examined in various solvents viz., DMF, DMSO, acetonitrile, THF, ethanol, and methanol. Figure 9 depicts the experiment performed by using EABH (60 µM) solution in different solvents. EABH displayed maximum fluorescence intensity in DMSO, compared to various solvents.

Fig. 9
figure 9

EABH (60 µM) emission spectrum of fluorescence with several solvents.

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Stern-Volmer analysis

The equivalence relationship between EABH, and Pb2+ and the metal quenching efficiency of fluorophore and quencher was investigated by fluorescence titration up to 40 mM concentrations of Pb2+ with 5 mM intervals and maintaining the constant EABH concentration (60 µM) in DMSO, 0.01 M HEPES buffer at 326 nm excitation (Fig. 10a). The EABH intensity in DMSO at 498 nm decreases continuously with increasing Pb2+ concentration (Fig. 10b). Fluorescence quenching mechanism of the sensor probe EABH can be understood by applying Stern-Volmer Eqs.2,74.

$${{\text{F}}_0}/{\text{F}}\,=\,{{\text{K}}_{{\text{sv}}}}\left[ {\text{Q}} \right]\,+\,{\text{1}}$$
(2)

The fluorescence intensities of the sensor in the absence and presence of different concentrations of Pb2+ ion is denoted as F0 and F, respectively. Ksv is the Stern-Volmer constant, and the total concentration of Pb2+ is denoted by [Q]. The plot of F0 /F against [Q], gives 2.1 × 105 M− 1 Ksv of the Pb2+ (Fig. 10c).

Fig. 10
figure 10

(a) Fluorescence spectrum of EABH (60 µM), (b) Changes in Fluorescence intensity in DMSO solution (HEPES 0.01 M, pH 7.4) with different concentration of Pb2+ (0–40 µM), λexc, and emission at 326 nm, 498 nm, respectively. The intensity bar graphs for EABH (pink bar) and with Pb2+ (blue bar), (c) stern volmer plot of EABH with different Pb+ 2 ion concentration.

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Limit of detection

The limit of detection of EABH was determined by maintaining a constant concentration of EABH and altering the concentrations of Fe2+ and Pb2+ ions. The absorption and fluorescence spectra were obtained using 60 µM of EABH in DMSO the results show a linear rise in absorbance for Fe2+ and decrease in fluorescence intensity for Pb2+ ions. LOD of the sensor molecule for Fe2+ and Pb2+ ion was calculated using Eq. (3):

$${\text{Detection limit}}\,={\text{ 3}}.{\text{3}}s/{\text{k}}$$
(3)

where σ is the standard deviation and k is the slope of the calibration curve. The limit of detection for Fe2+ and Pb2+ ions was found to be 0.166 × 10− 6 M and 0.095 × 10− 6 M based on plotting the absorbance against their concentrations of Fe2+, and fluorescence intensity vs. Pb2+ concentration, respectively as shown in Fig. 11 are lower than the WHO guidelines for drinking water. Furthermore, the comparison of EABH with the reported chemosensors is shown in Table 2. The key characteristics of the developed chemosensor was its simplicity, usefulness, and affordable synthesis along with its ease in high sensitivity, selectivity, and naked eye detection of Pb2+/Fe2+ ions with lower detection limit compared to reported chemosensors. Similarly, the previously reported hydrazide-based Schiff base and their LOD are tabulated in Table 3.

Table 2 Comparison of chemosensor EABH with previously reported ligands for the detection of Pb2+ /Fe2+ ions.
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Table 3 Sensing performance and LOD of previosulsy reported hydrazide-base schiff bases sensor.
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Fig. 11
figure 11

Calibration curve depicting the absorption and fluorescence spectrum of EABH in DMSO solution (60 µM, HEPES 0.01 M, pH 7.4) with different concentrations of (a) Fe2+ and (b) Pb2+ solution.

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Reversible EDTA Titration

Amplified its regeneration and reversible properties of the developed sensor are the substantial features for its real time applicability using sodium salt of EDTA86. The solutions of EABH-Fe2+ and EABH-Pb2+ with the chelating ligand EDTA acting as complexing agent (Fig. 12a and b). Addition of one equivalence of Pb2+ ion solution, exhibts decreased fluorescence emission intensity at (498 nm), and the regeneration of free EABH was noticed when 1.0 eq of EDTA was added to the EABH-Pb2+ complex. When EDTA was added to EABH-Fe2+ complex, the initial brown colour of the solution changes to lime yellow indicating the formation of Fe-EDTA complex, affirming the release of EABH as a free ligand and reversal of colour change was noticed (Fig. 12c and d). The above results greatly potential for its use in logic gate construction.

Fig. 12
figure 12

(a) Fluorescence, and (b) absorption graphs depicting EABH reversible nature with EDTA. The photographs showing colour changes on addition of Fe2+ and Pb2+ with EDTA under (c) white light and (d) UV light.

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10. Logic gate application

Molecular logic gate was constructed based on the photochemical behaviour of fluorescence probes under consideration87. The reversibility of fluorescence study for the EABH probe was estimated by constructing the logic gate of INHIBIT type, based on the ON/OFF characteristics of the fluorescence probe88,89, as shown in (Fig. 13). The reversibility of EABH’S detection of Pb2+ ions by adding EDTA was framed using the Boolean logic function. EABH emission at 498 nm was used as the output of the logic gate function90. Addition of Pb2+ to strong fluorescent solution of free ligand, weak fluorescence was observed, whereas EDTA added to EABH-Pb2+ shows strong fluorescence. The “ON” (output = 1) for strong and “OFF” (output = 0) for weak emission, respectively. For this particular truth table, the presence and absence of the Pb2+ or EDTA corresponds to the signals ‘1’ and ‘0’, respectively. Addition of Pb2+ (input 1) to EABH, quenching of fluorescence was observed. With input 2, mere addition of EDTA had no effect on the intensity of EABH. The addition of Pb2+ and EDTA turned on the EABH fluorescence. The output signal was noted with the shift in emission at 498 nm. The 2 input can be credited to Pb2+ and EDTA, showing outputs state fluorescence “OFF” for A and B (Table 4). The input of Pb2+ was represented by a NOT gate. The OR gate function was used to represent the input – output combination of EABH-Pb2+-EDTA.

Table 4 Truth table showing the input and output values.
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Fig. 13
figure 13

Logic gate of EABH, Pb2+ and EDTA.

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Practical applications in real water sample

Figure 14 depicts UV-visible and fluorescence spectra for the detection of the Fe2+/Pb2+ in real specimens using EABH probe in both tap water and lake water is a crucial aspect. Real water samples were gathered, and standard addition approach is used to analyse the samples using UV-visible and Fluorescence spectroscopic techniques91,92,93. Lake water from kukkaralli lake, Mysuru and laboratory tap water was collected. The different sample concentrations of Fe2+/Pb2+ metal ions (0–25 µM) were added to the probe EABH (30 µM), and spiked samples were obtained. The results represent sensitive, and selectiveness monitored in environmental samples of the probe towards the Fe2+/Pb2+ ions.

Fig. 14
figure 14

Detection of Pb2+ in (a, c) tap water and (b, d) lake water through fluorescence and Fe2+ ions through colorimetric assays.

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Cell viability and bio-imaging studies

The cell viability was determined using MTT assay to assess the cytotoxicity of EABH. The HeLa cells were treated with the different concentrations of EABH (5, 10, 15, 20 and 25 µM) and incubated for 24 h. More than 91% of cells remained viable, indicating their non-toxic nature. Further, the sensing property of EABH was further studied in the biological system using in vitro cell imaging experiments. The biosensing property of EABH was performed in the presence of Fe2+ and Pb2+ ions. As shown in Fig. 15, in the presence of Fe2+ and Pb2+ ions, no fluorescence was observed. Thus, it is evident that EABH can be utilized in the biomedical applications.

Fig. 15
figure 15

Live-cell imaging of HeLa cells treated with (a) before and (b) after incubation with 25 µg/mL of EABH. (c) Before and (d) after incubation with 25 µg/mL of EABH-Pb2+ complex. (e) Before and (f) after incubation with 25 µg/mL of EABH-Fe2+ complex. (g) Cell viability assay.

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Frontier molecular orbitals and reactivity indices

Frontier Molecular Orbitals (FMOs) theory is essential for understanding chemical reactivity and various physical parameters94. It involves the molecular orbital energies of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO). These orbitals affect molecular interactions, with HOMO indicating electron donation ability and LUMO representing electron acceptance. The HOMO-LUMO energies and the energy gap (ΔE) play a key role in determining molecular stability95. A small ΔE signifies high polarizability, reduced kinetic stability, and increased chemical reactivity, categorizing the molecule as soft. The ΔE values for EABH and its complexes, EABH-Fe2+ and EABH-Pb2+, were found to be 4.275 eV, 2.004 eV, and 3.040 eV, respectively. Figure 14 illustrates the encapsulation of the HOMO and LUMO maps in the molecules. The significant reduction in the HOMO-LUMO gap upon EABH complexation with Fe2+ (2.004 eV) and Pb2+ (3.040 eV), compared to free EABH (4.275 eV), is due to metal-ligand interactions. The stronger d-orbital hybridization and ligand-to-metal charge transfer (LMCT) in EABH-Fe2⁺ leads to greater gap reduction, while weaker p-orbital interactions and relativistic effects in EABH-Pb2+ result in a smaller decrease96. This electronic modulation influences fluorescence properties, making these complexes potential candidates for sensing applications97.

Figure 16 demonstrates that the HOMOs are localized on the methoxybenzylidene and acetohydrazide moieties, and partially localized on the methyl moieties, excluding the phenol ring in the EABH structure. In contrast, LUMOs are mainly distributed across the entire molecule, except for the methyl moiety with the allyl group in the EABH structure. In the bidentate structures of EABH-Fe2+ and EABH-Pb2+, the HOMO is primarily localized on the methoxybenzylidene and acetohydrazide moieties, with additional localization on the Fe atom in EABH-Fe2+, while excluding the phenol ring and allyl group in both complexes. The LUMO is distributed across the entire molecule, with the exception of the allyl group in EABH-Fe2+ and both the allyl group and Pb atom in EABH-Pb2+. The chemical reactivity parameters of the compounds were evaluated based on the HOMO and LUMO energy values (Table 5). EABH has an ionization potential (I) of 5.812 eV, which indicates its strong electron-donating ability. Hardness (η), which reflects resistance to changes in the electronic environment, was calculated as 2.137 eV for EABH, and 1.002 eV and 1.520 eV for EABH-Fe2+ and EABH-Pb2+, respectively, suggesting a soft character along with strong polarizability and reactivity98. The chemical potential (µ), which represents the tendency of electrons to escape from an equilibrium system, was calculated as -3.674 eV for EABH, -2.988 eV for EABH- Fe2+, and − 3.263 eV for EABH-Pb2+. The global electrophilicity index (ψ) measures how stable the energy is when the system gets an extra electronic charge from the environment. The calculated values were 3.157 eV for EABH, and 4.455 eV and 3.502 eV for the EABH-Fe2+ and EABH-Pb2+ complexes, respectively.

Fig. 16
figure 16

FMO orbital surfaces of EABH, EABH-Fe2+ and EABH-Pb2+.

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Table 5 The calculated FMOs and GCRDs of the EABH, EABH-Fe2+ and EABH-Pb2+ at B3LYP/LanL2DZ level of theory.
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Molecular electrostatic potential

The Molecular Electrostatic Potential (MEP) reflects electron density and serves as a crucial tool for identifying electrophilic and nucleophilic interaction sites, as well as hydrogen bonding regions99. In the MEP map, red and yellow represent negatively charged areas associated with electrophilic reactivity, while blue indicates positively charged areas linked to nucleophilic reactivity, as shown in Fig. 17. The MEP visualization indicates that the EABH molecule has several possible sites for electrophilic attack, primarily around oxygen and nitrogen atoms. The negative regions are concentrated near these electronegative atoms, suggesting electrophiles would preferentially target these positions. The MEP map further reveals that the negative potential sites correspond to oxygen and nitrogen atoms, while the positive potential sites are located around hydrogen atoms. These regions provide information about possible noncovalent interactions within the molecule.

Fig. 17
figure 17

Molecular electrostatic charge distribution plots for EABH, EABH-Fe2+, and EABH-Pb2+.

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Sensing mechanism

Figure 18 depicts the sensing mechanism of the chemosensor EABH towards the detection of Fe2+ and Pb2+ ions. In the case of metal complexes, the metal ion form coordination bonds with imine nitrogen, carbonyl oxygen and phenolic oxygen atoms of EABH. Addition of Fe2+ to the sensor EABH, Fe2+ ion establishes ligand–metal change transfer (LMCT) with EABH ligand, thereby hinders photoinduced electron transfer (PET). On the other hand, the addition of lead centrals quenching in the fluorescence intensity of the EABH due to the excited-state intramolecular proton transfer (ESIPT) process explains the fluorescence quenching mechanism100,101.

Fig. 18
figure 18

Proposed sensing mechanism of EABH with Fe2+ and Pb2+ ions.

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Test-kit application

To find the practical applicability of the probe, cotton swabs and a paper strip test applications were performed for Fe2+/Pb2+ determination in DMSO solution. The test-kit studies were performed with the cotton swabs and Whatman filter strips saturated with the EABH solution. When different amounts of Fe2+/Pb2+ (8.33–125µM) were dipped with the cotton swab distinct brown coloration changes for Fe2+ were clearly seen under daylight and becoming non- fluorescent for Pb2+ under irradiation with UV under 365 nm (Fig. 19a and b). Whereas, for paper strip experiment test Whatman filter strips were immersed into solution of EABH for duration of 5 min and then left to dry. the dried strips were immersed into Fe2+/Pb2+ ion solution, for Fe2+ change in brown coloration was noticed and become non-fluorescent on paper strips after addition of Pb2+ ion (Fig. 19c and d) hence, these applications could be used for semi-quantitative identifying Fe2+/Pb2+ ions102,103.

Fig. 19
figure 19

The photographs cotton swabs dipped in different concentration of Fe2+ under day light (a) for Pb2+ under UV light (b) colorimetric (c) and fluorometric test paper strip test of EABH for the detection of Fe2+ and Pb2+ ions.

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Conclusion

Herein, we have synthesized and characterized a new and robust colorimetric and fluorimetric sensor, EABH based on eugenol moiety, which shows a good sensitivity and selectivity towards both Fe2+ and Pb2+ ions in DMSO (0.01 M HEPES buffer, pH = 7.4). The imine nitrogen, phenolic oxygen and carbonyl oxygen atom of the EABH co-ordinated with lead and iron ions, forming a Pb2+ and Fe2+ complex with the EABH ligand, which was confirmed by FTIR. The job’s plot and LC-MS techniques, signifies the co-ordination at the ratio of 2:1 (L: M) stoichiometry. The chemosensor EABH detected Fe2+ by “naked eye” colour change from lime yellow to brown, this confirms the effective and selective sensor for Fe2+ detection by UV-visible spectroscopic method and displayed fluorescence quenching for Pb2+. The chemosensor EABH can detect the Fe2+and Pb2+ ions with the LOD of 0.166 µM and 0.095 µM, respectively. The established chemosensor acts as an intriguing compound for the quantification of Fe2+/Pb2+ ions even in the live cells, revealing its biosensor potential. In addition, the frontier molecular orbitals, reactivity indices and molecular electrostatic potential calculations revealed that the electronic modulation influences fluorescence properties, thus making the ligand as a potential candidate for sensing applications. Furthermore, the significant sensing nature of EABH was demonstrated by employing it in test-kit applications.