Thiazole-Formulated Azomethine Compound for Three-Way Detection of Mercury Ions in Aqueous Media and Application in Living Cells.

Heavy metal ions are extremely poisonous and cause long-term harm to living organisms. Among these ions, mercury is the most toxic metal and has no notorious purpose in the human body. In this regard, an elegant azomethine thiazole compound AM1 was synthesized, and it was found to be highly sensitive to three-way detection of mercury ions with detection limits of 0.1126 × 10-9 M (FL) and 0.64 × 10-6 M (UV-vis). AM1 highlighted the capability to detect mercury ions through the colorimetric method, the fluorometric method, and via the naked eye in three-way detection. In addition, the structure of AM1 was confirmed by single-crystal X-ray diffraction studies and crystallized in a monoclinic crystal system with a P21/c space group, and it shows numerous noncovalent interactions in the crystal packing. The high sensitivity of AM1 to Hg2+ ions was imputed to the quenching mechanism and was estimated by Fourier transform infrared (FT-IR) spectroscopy, nuclear magnetic resonance (1H-NMR), high-resolution mass spectrometry (HRMS), ultraviolet-visible (UV-vis) absorbance, fluorescence (FL) emission, Job's plot, B-H plot, and DFT calculation. Naked eye color change of AM1 solution to yellow and turn-off FL by the addition of mercury ion is due to complex formation. In addition to mercury ions, the sensor displayed a new absorption peak at around 240 nm. Furthermore, an AM1-coated test strip is used as the solid support sensor, and real-time detection of Hg2+ ions in the HeLa cell line by fluorescence microscopy is performed.

Introduction

Because of the increasing human population throughout the world, the environment gets polluted by the contamination of heavy metal ions. This will lead to the intake and absorption of heavy metal ions that cause disturbance of biogeochemical cycles.[1−4] Thus, the detection of heavy metal ion concentration in various analytes becomes significant. Among these heavy metal ions, mercury is highly toxic even at low concentrations. Mercury is a naturally occurring metal that mainly affects the marine environment. The inorganic mercury compound is less toxic than the organic mercury compound.[5,6] This hazardous mercury also has various applications in the field of lamp-producing factories and in industrial processes as a catalyst, for extraction of gold, and in pharmaceutical medicines; they are also used in paper industry.[7,8] Contamination of mercury metal in the form of liquid is not identified because of its colorless and odorless nature and solubility in water.[9] This causes severe damage to the liver, renal function especially in female, and damage to the function of any organ that initially causes malfunction and finally death.[10] According to the WHO requirement, the maximum limits of Hg2+ in drinking water are less than 6.0 μg L–1 (∼30.0 nM).[11]

In past decades, several methods have been developed for the detection of heavy metal ions in environmental and biological samples. Various methods like atomic absorption spectroscopy (AAS),[12] ion chromatography (IC),[13] inductively coupled plasma spectroscopy (ICP),[14] laser-induced breakdown spectroscopy (LIBS),[15] neutron activation analysis (NAA),[16] and X-ray fluorescence (XRF)[17] techniques were used. These analytical techniques suffer from severe disadvantages like highly expensive equipment, requiring skilled operators, and difficulty distinguishing different ions.

Therefore, simple chemical sensors turned the attention of several researchers to this field and took it to a great extent. In recent years, several azomethine compounds have been reported for the sensing of metal ions by applying fluorometric and colorimetric methods. Nitrogen- and sulfur-containing heterocyclic compounds play an important role in many biological applications, used as “off–on” sensors to detect metal ions, and they also have excellent chelation properties with metal ions. Among them, thiazole is a five-membered heterocyclic compound that has nitrogen and sulfur in its ring structure, which is naturally present in vitamin B. It acts as an acceptor because of the occurrence of electron-accepting nitrogen (C=N) in its structure.[18−21] Hence, thiazole-based azomethine compounds are employed in the development of chemosensors with colorimetric, fluorometric, and naked-eye detection of metal ions.

In this work, a novel thiazole formulated azomethine (AM1) was developed for the selective detection of Hg2+ ions. Herein, we report the synthesis, characterization, and sensing properties of AM1.

Experimental Section

Reagents and Solvents

The starting materials were purchased from Sigma-Aldrich and TCI. Metal salts were purchased from Sd-fine Ltd. and used without further purification. All solvents were dried and distilled before using as per standard procedures.

General Structural Characterization

The NMR spectra were recorded on a Bruker (400 MHz) spectrometer, and chemical shifts were reported in δ (ppm). Fourier transform infrared (FT-IR) spectra were obtained using KBr disks with a Shimadzu FT-IR spectrometer (400–4000 cm–1). High-resolution mass spectrometry (HRMS) spectra were recorded on WATER-XEVO G2-XS-QT. Absorption spectra were recorded using an Agilent 8453 UV–vis instrument, and diffuse reflectance spectra were obtained on a Jasco-v-670 spectrophotometer. All steady-state photoluminescence (PL) measurements were performed using an Edinburg FLS 980 spectrometer.

General Procedure for UV–vis and Fluorescence Spectroscopy

Preparation of Stock Solution

AM1 (12 mg) was dissolved in 50 mL of tetrahydrofuran (THF) solution in standard volumetric flasks to prepare the stock solution of the AM1 compound. In other standard volumetric flasks, 5 mg of HgCl2 was dissolved in 20 mL of water to prepare the stock solution of HgCl2. Using these stock solutions, UV–vis and FL spectroscopy was performed.

Measurement of UV–vis and Fluorescence Spectroscopy

An appropriate volume of stock solution of AM1 was taken in 2 mL quartz cuvette, and an appropriate amount of HgCl2 solution was added to the AM1 solution (2:1). The UV–vis absorption spectra and FL emission spectra (excitation wavelength = 350 nm) were recorded before and after the addition of mercury chloride solution.

The stock solution of AM1 was prepared using THF and the concentration of the working solution. All cationic solutions are prepared using the respective salts in ultrapure water (1 × 10–3 M). The solution of metal ions such as Ag+, Co2+, Cr3+, Cu2+, Cd2+, Ga3+, Fe2+, Hg2+, Mn2+, Ni2+, Pb2+, Na+, Mg2+, Eu3+, Fe3+, Zn2+, Nb5+, and Al3+ is used for the selectivity and interference studies.

Computational Studies

The density functional theory (DFT)-based calculations were performed for understanding the structural and functional features. The selection of DFT functional and combination of the basis set helps define bonding patterns, electronic charge, and molecular orbital energy distribution. The geometries of the AM1 and AM1-Hg2+ in gas and liquid phases were optimized using Becke’s three-parameter and Lee–Yang–Parr functional (B3LYP) and Los Alamos National Laboratory 2-double-z (LANL2DZ). The basis sets 6-311G (d, p) is employed for the N, O, H, and C atoms, and LANL2DZ is applied for the metal complex.[22,23] The highest occupied molecular orbital–lowest unoccupied molecular orbital (HOMO–LUMO) energy band gap values were also computed for AM1 and AM1-Hg2+. Computational studies were performed using the G16 package.[24] The electronic geometries and frontier molecular orbital structures were taken using the Gauss View 6.1.1 molecular visualization program.[25]

Determination of the Crystal Structure

The crystal structure of the azomethine AM1 was obtained by slow evaporation in ethyl acetate, which resulted in a brownish-yellow crystal. The crystal was stored in paraffin oil, mounted in a MiTeGenloop, and measured at 296 K. The crystal measurements were performed on a Bruker Kappa Apex II coupled with a CCD area detector with graphite monochromated Mo Ka radiation (0.71073 Å). The crystal, with an approximate size of 0.300 × 0.200 × 0.200 mm, was mounted on a glass loop. It was solved by direct methods and expanded using Fourier techniques. All the calculations were performed using the crystallographic software package X shell.[26] The Apex2 package was used for cell refinements and data reductions. The structure was solved by direct methods using the SHELXS-9727 program with the Olex2 graphical user interface. The structural refinements were carried out using Shelxl-2018.[27] The positions of all the atoms were obtained using direct methods, and the crystallographic details are summarized in Table S1.

MTT Assay Protocol

The human cervical cancer cell line (HeLa) cells were coated discretely in 96-well plates

(1 × 104 cells/well) with 10% fetal bovine serum, 1× antibiotic antimycotic solution, and 5% CO2 in MEM media at 37 °C. The cells were eroded with 200 μL of 1× PBS, treated with different concentrations (10, 20, 30, 40, and 50 μg/mL) of AM1 incubated for 24 h in serum-free media. Furthermore, 0.5 mg/mL MTT in 1× PBS was injected and incubated for 4 h. Then, MTT was removed, washed using 200 μL of PBS, and the formed crystals were mixed with 100 μL of dimethylsulfoxide (DMSO) solution.[28]

Analytical Application

Small strips of Whatman filter paper were dipped in the AM1 solution (1 × 10–5 M) and dried. After some time, the aqueous solution of metal ions (1 × 10–3 M) was dropped over the coated strips and dried using an air dryer and used. Twenty milliliters of AM1 stock solution were purged into the 60-120 mesh of silica gel (2 g), after air-drying for about 5–10 min and testing against mercury ions.

Synthesis of Compound AM1

Thiazole-formulated azomethine was synthesized by reacting an equal ratio of 3,5-dimethyl aniline (300 mg; 1.0 mmol) and 4-methylthiazole-5-carboxaldehyde (314 mg; 1.0 mmol) in methanol (20 mL). Then, triethylamine (0.25 mL) was added to the reaction mixture and refluxed at 70 °C overnight. Then, the reaction mixture was cooled to room temperature, poured into ice water, and then extracted with ethyl acetate (50 mL). The organic layer was dried over sodium sulfate, and the solvent was evaporated in a vacuum. The crude product was purified by column chromatography (hexane/ethyl acetate, 20%).

Results and Discussion

Synthesis of thiazole-formulated azomethine AM1 was synthesized by the condensation reaction of 3,5-dimethyl aniline and 4-methylthiazole-5-carboxaldehyde in methanol under reflux to remove water (Scheme S1). There is a good yield with brownish-yellow crystal formation. The structure was confirmed using analytical, spectral techniques and further with a single-crystal X-ray diffraction study. The compound was readily soluble in all organic solvents.

Spectral Characterization

In 1H NMR (in CDCl3), there was a disappearance of the amine peak around 5 ppm and the aldehyde peak around 10 ppm with the formation of a new imine peak in the region of 8.6 ppm. The compound AM1 shows two singlets between 8.79–8.6 ppm for the thiazole proton between nitrogen and sulfur and one imine proton. The doublet between 6.83–6.89 ppm integrated for two aromatic protons and a pair of singlets obtained at 2.67 ppm for three methyl protons in the thiazole ring and 2.34 ppm for six methyl protons in the phenylene ring is shown in Figure S1. The 13C NMR spectra of AM1 show that the peak at 156.3 ppm corresponds to thiazole ring carbon between nitrogen and sulfur, 155.1 ppm to imine carbon, four peaks at 151.3, 150.3, 138.9, and 118.66 ppm for phenylene carbon, two peaks at 131.6 and 128.1 ppm correspond to thiazole ring carbon, 21.23 ppm for phenylene ring methyl carbon, and 15.87 ppm for thiazole ring methyl carbon (Figure S1).

FT-IR spectra were obtained for the compound AM1 and a strong peak appeared in the region of 1648.60 cm–1 for imine (C=N−) and 3000–2915.63 cm–1 for aromatic C–H stretching mode. Additionally, strong absorption was obtained at the region of 1600.44 cm–1 for C=C stretching, 1242.73 cm–1 for C–N stretching, and 746.71 for C–H bending modes (Figure S2). The theoretical mass of the compound AM1 is the well-matched experimental value, which was observed from HRMS (Figure S2).

Crystal Study

The complete structure of the compound AM1 was concluded by single-crystal X-ray diffraction studies. The brownish-yellow colored crystal of AM1 was grown in ethyl acetate, which is photographed and shown in Figure a. It is crystallized in a monoclinic crystal system with a P21/c space group and also it shows numerous noncovalent interactions in the crystal packing. The atomic coordination, equivalent isotropic displacement parameters, bond length, bond angle, anisotropic displacement parameters, hydrogen coordination, and isotropic displacement parameters of the unit cells of AM1 are given in Table S1. The bond lengths of new imine N1-C7 [1.247 (3)] were observed. In addition, we have observed noncovalent interactions with the neighboring molecules in the crystal packing of AM1. The C–H−π interactions were mainly observed between thiazole C–H and the neighboring molecule of the phenyl ring, C13–H13−π (3.246 Å); C11–H11−π (3.605 Å); C1–H1−π (3.566 Å); and C5–H5–N1 (2.511 Å) (shown in Figure b).

Figure 1

(a) Crystal structure of AM1 and (b) unit cell packing pattern and C–H−π interactions of AM1.

Optical Properties

The optical properties of both AM1 and AM1-Hg2+ in the powder form and solution form (1 × 10–5 in THF) were obtained through UV–vis spectroscopy, as shown in Figure S3. The AM1 solution shows two different absorption peaks. The first peak with a higher energy band due to π–π* transition at 242 and 292 nm (lower wavelength). The next peak at 332 nm (higher wavelength) with a lower energy absorbance peak that denotes the intraligand charge transfer (ILCT).[29] When mercury metal ions are added to the AM1 solution, the higher energy band gets decreased, and the lower energy band gets enhanced (THF: H2O).

The solid-phase absorbance spectra of both the ligand and the metal ion complex were blue-shifted (∼45 nm) when correlated with the liquid-phase absorbance peak. Both AM1 and AM1-Hg2+ show a broad single absorbance peak because of C-C restricted rotation with the disappearance of π–π* transition (Figure S3).

Colorimetric Study

The naked eye detection of metal ions is the simplest, most economical, and appropriate method through color change. The compound AM1 was found to show an instant color change and turn-off FL (colorless to brownish-yellow) with Hg2+ in the semi-aqueous medium at room temperature immediately. No significant changes were observed upon the addition of other metal ions such as Ag+, Co2+, Cr3+, Cu2+, Cd2+, Ga3+, Fe2+, Mn2+, Ni2+, Pb2+, Na+, Mg2+, Eu3+, Fe3+, Zn2+, Nb5+, and Al3+metal ions in THF: H2O (9:1). This ratio is suitable to undergo effective internal charge transfer.[30] The observed selective detection with color change can be used for the colorimetric recognition of Hg2+. The color changes were abruptly seen through the naked eye (Figure a) and also FL quenching under a UV lamp (Figure b). This color change is due to the complex formation of the ligand with metal ions.[31] As shown in Figure a,b, the addition of the Hg2+ ion solution to the AM1 solution exhibits 48 nm blueshift in the UV–vis absorbance and FL quenching. Mercury is selectively sensed by the AM1 compound because of the metal-mediated ILCT. The color change of mercury alone shows a vast shift as compared to other metal ions in UV–vis absorbance and FL intensity. Thus, this shows that the thiazole-formulated azomethine AM1 was selective to hazardous mercury metal ions.

Figure 2

(a) Visual detection of AM1 with various metal ions under normal light. (b) Fluorescence visual detection of AM1 with different cations under a 365 nm UV–vis lamp.

Figure 3

(a) UV–vis spectra. (b) Emission spectral response of AM1 in the absence and presence of 10 equiv. of various metal ions in THF: H2O (9:1).

Sensitivity and Selectivity Studies of AM1-Hg2+

UV–vis titration of mercury ions to the AM1 compound determines the associate constant between AM1 and Hg2+ metal ions. The serial addition of mercury ions to the AM1 solution was recorded. The hump peak at 242 nm gradually rises and the peak at 289 nm declines with the addition of mercury metal ions to the AM1 solution. Overlapping of the absorption peak leads to the isosbestic point, which indicates the formation of the metal complex. In addition to mercury ions, the isosbestic point appeared at 254 nm (Figure a). The slope value of the straight line, the associate constant using Benesi–Hilderbrand standard formula, the binding mode using Job’s plot, and the limit of detection and quantification using Kσ/s standard formula, where K = 3 (LOD) and 10 (LOQ), were determined.[32,33] The stoichiometric ratio from Job’s plot is 1:1 between AM1 and Hg2+ ions. (Figure b) The detection and quantification limits of AM1 for hazardous Hg2+ ions were 0.64 × 10–6 M and 0.022 × 10–6 M, respectively, which shows a less limit of detection as compared to the previously stated compound. The serial addition of mercury ions to AM1 solution was recorded in FL spectra, which gradually decrease (Figure S4a). The binding constant of the metal complex from the linear equation of the BH plot were 1.6022 × 105 M–1, which shows the binding efficacy of AM1 with Hg2+ ions (Figure S4b). Among the known probe, the AM1 compound shows three responses: naked eye detection, colorimetry, and fluorometry for the detection of Hg2+ ions. The AM1 compound is highly stable, easy to synthesize, and soluble in organic solvents. Furthermore, the sensing ability of AM1 with Hg2+ was compared with the recently reported literature in Table S2.

Figure 4

(a) UV spectral titration of AM1 (2 equivalent) with various concentrations of Hg2+ ions, (b) Job’s plot method, and (c) calibration plot of AM1 with various concentrations of Hg2+.

To check the selectivity of the AM1 compound to Hg2+ ions, the metal complex AM1-Hg2+ was subjected to competitive metal ions. To reveal the selectivity of AM1 for the detection of Hg2+ ions, the interference study was investigated. In the presence of mercury ions in the AM1 solution, other metal ions such as Ag+, Co2+, Cr3+, Cu2+, Cd2+, Ga3+, Fe2+, Mn2+, Ni2+, Pb2+, Na+, Mg2+, Eu3+, Fe3+, Zn2+, Nb5+, and Al3+ ions were added to perform the interference study (Figure a). The interference studies were conducted with UV and FL spectroscopy. The absorbance peaks corresponding to ligands with mercury metal ions remain at the same range while adding other competitive metal ions. This exposed that there was no interference of other metal ions in the presence of Hg2+ ions in the AM1 solution. The above study shows that the compound AM1 has good anti-interference ability and high selectivity to mercury metal ions.

Figure 5

(a) Interference studies in the bar chart: blue bar represents the absorbance of AM1+ Hg2+ with various metal ions and black with pale blue represent the absorbance of AM1 with various metal ions. (b) Emission spectra for the reversibility response of AM1 upon the alternate addition of Hg2+ metal ions and EDTA solution.

Reversibility Studies and Response Time of AM1 to Hg2+ Ions

Binding reversibility of AM1 (10–5 M) with Hg2+ ions was done to reveal the dynamic response of the AM1-Hg2+ interaction. In addition to EDTA to AM1-Hg2+ complex solution, it retains the color from brownish-yellow to the colorless solution of the AM1 compound. As shown in Figure b, AM1 exhibited turn on–turn off fluorescence, color changes from colorless to color in the naked eye, and red/blue shift in absorbance in the function of added metal ions. When AM1 is treated with Hg2+ ions, the FL intensity of AM1 decreased from 21.59 to 10.21 a.u. along with FL quenching because of AM1-Hg2+ complex formation. Further addition of EDTA solution to AM1-Hg2+ solution results in the reclamation of color and intensity, which indicates that the binding of AM1 with Hg2+ is chemically reversible (Figure b). The reversible ability of AM1 with metal ions was confirmed by performing the same technique for up to three cycles.[34] Thus, AM1 can be used as an “off:on” chemosensor. From the above study, it was confirmed that the AM1 compound was ecologically recyclable and reusable.

Favorably, the color change and FL quenching response of AM1 for Hg2+ is reproducible and reversible through the addition of EDTA. The reversibility nature of AM1 was tested repeatedly and confirmed. Therefore, AM1 exhibited excellent color change in 13 s correspondingly to imply their rapid response time (Figure ).

Figure 6

Time response curve toward Hg2+ions.

Binding Mechanism of AM1-Hg2+

The binding interaction of AM1 with Hg2+ metal ions was studied by 1H-NMR spectral titration studies in DMSO-d6. The AM1 compound shows the respective peaks at δ ppm = 8.79 (1H, s, thiazole H between N and S) and 8.6 (1H, s, imine H). During the titration of the Hg2+ metal ion with several equivalents, the respective peaks get diminished. The addition of 1 equiv. of Hg2+ metal ion solution to the same solution will lead to the complete disappearance of the respective peak and disturbance of the aryl proton. It is abruptly known that the S atom of thiazole and the N atom of the imine act as the binding site for Hg2+ metal ions and the formation of coordination bonds (Figure ). The above 1H-NMR titration proved that the binding ratio of AM1-Hg2+ is 1:1 according to the previous statement given under the Job’s plot. In addition, the complex formation of AM1-Hg2+ was confirmed by the FT-IR spectrum, and HRMS spectra are shown in Figure S5. The FT-IR spectrum of the AM1 compound shows peaks at 1648.60 cm–1 (imine, N=CH) and 1242.73 cm–1 (C–N stretch). In complex formation, the peaks get decreased energy and change in frequencies.[35] After the interaction of Hg2+ with AM1, the molecular weight calculated was 400.09, and the obtained mass was 400.19162.[36] Thus, this shows the formation of complex formation and leads to the chelation-enhanced fluorescence quenching (CHEQ) effect.[37]

Figure 7

(a) 1H NMR spectra (400 Hz, DMSO-d6) of sensor AM1 in the absence and presence of Hg2+ ions (1.0 equiv). (b) Suggested response mechanism for the sensor AM1 through the coordination bond.

Theoretical Studies

The structure of the AM1 compound and the AM1-Hg2+ complex was optimized through the density functional theory (DFT) study with Becke’s three parameters Lee–Yang–Parr (B3LYP)/6-31 + G** and the Los Alamos National Laboratory 2-double-z (LANL2DZ) basis set under the gas phase. The 6-31 + G** basis set is applied for C, H, N, and S atoms, and LANL2DZ is the special basis set for the metal complexes.[38,39] The 3D-computed structure and the energy level of the AM1 compound and the AM1-Hg2+ complex was studied theoretically by DFT studies (Figure a). The band lengths of HC=N–Hg and HC–S–Hg are 4.1414 and 4.4324 Å, respectively. The calculated interaction energy of the AM1-Hg2+ complex was −345.1884 Hartree; this indicates that AM1 complexed with Hg2+ ions is stable via the coordination bond.

Figure 8

(a) Optimized structure and (b) frontier molecular orbitals of AM1 and AM1 + Hg2+ using the DFT/B3LYP method.

The optical band gap is also calculated through the following equation:where Eg is the energy band gap, A is the constant, h is plank’s constant, hν is photon energy, and ν is the frequency of the incident photon. The energy gap is obtained via Tauc’s plot through plotting (αhν)2 versus hν. The compound AM1 shows a low band gap, which is apt for optical applications. Thus, the optical band gap of the AM1 compound and AM1-Hg2+ complex is 4.3 and 3.9 eV, respectively. The experimental optical band gap was proved through analyzing the Fortier molecular orbital for the AM1 compound. The AM1-Hg2+ complex is shown in Figure b. The AM1 compound shows HOMO and LUMO orbitals in their molecular system with a band gap of 4.3 eV. In complex formation with Hg2+ metal ions, the HOMO orbitals are in the AM1 compound and the LUMO orbitals are present near the metal center with a decreased band gap of 3.8 eV (Figure ). Compared with metal complex AM1-Hg2+, the AM1 compound shows a slightly higher LUMO energy level, certainly caused by the low-lying LUMO energy level (Table ). Thus, this shows that there was a charge transfer between AM1 and Hg2+ metal ions.

Figure 9

Tauc’s plots of (αhν)2 versus photon energy (hν) of the AM1 compound and the AM1 + Hg2+ metal complex.

Table 1

Experimental and Theoretical HOMO–LUMO Energy and Band Gap Using the B3LYP/6-31 + G** Level of Theory in the Gas Phase

	HOMO energy level (eV)a	LUMO energy level (eV)a	band gap (eV)a	optical band gap (eV)
AM1	6.140	1.806	4.3	4.3
AM1 + Hg2+	6.040	2.232	3.8	3.9

Theoretical.

Analytical Application

To extend their practical application, dropping of mercury ion solution gradually turns the colorless coated strip into brown color. The qualitative analysis of mercury ions by the AM1 compound was also succeeded in the solid state (Figure a). Same as the previous test, the mercury ion solution was added to the AM1 devised silica gel and dried. Speedy color changes from colorless to brown were observed (Figure b). Therefore, this outcome tells that chemosensor AM1 can be utilized productively and conveniently to sense mercury ions in those practical applications.

Figure 10

(a) Test strip images of AM1 + Hg2+ and other metal ions coated in filter paper and (b) solid-state color change of AM1 upon the addition of Hg2+ in aqueous solution.

Cytotoxicity of AM1 to Hela Cells and Bioimaging Application

The cytotoxicity test was performed using the MTT method against the growth of HeLa cells. The results of the cytotoxic nature of the AM1 compound are shown in Table S3. The value of IC50 on HeLa cells showed that the AM1 compound does not inhibit the growth of HeLa cells (Figures S6 and S7).

We examined the bioimaging capability of AM1 using an Optika IM-3FL4 fluorescent microscope (Optika, Germany) using a UV filter. In addition, the cytotoxicity of AM1 to the HeLa cells in different concentrations (10, 20, 30, 40, and 50 μg/mL) was obtained using the MMT assay test. When AM1 is treated with different concentrations, more than 95% of cells retained their viability. This shows the low toxicity of AM1 against HeLa cells and is further utilized to image mercury ions in HeLa cells to observe FL after the cells incubated in AM1 for 15 min.[40] Blue FL of AM1 incubated to HeLa cells turn off their fluorescence after the addition of Hg2+ ions (Figure ). Thus, the AM1 compound is nontoxic and an efficient moiety to detect Hg2+ ions under a biological state.

Figure 11

Brightfield and FL image of HeLa cells incubated with (i) AM1 (10 μg/mL, blue fluorescence) and (ii) then treated with Hg2+ ions (30 μg/mL, turn-off blue fluorescence).

Conclusions

In summary, we have designed and synthesized an elegant azomethine compound using thiazole AM1 for the selective detection of hazardous mercury metal ions from the synthetic water sample. The synthesized AM1 compound achieved fluorescent turn-off and shows optical response toward Hg2+ ions, highly selective and sensitive. Complex formation displayed a new absorption peak at around 240 nm with an LOD of 0.1126 × 10–9 M, which is lesser than the WHO permissible limit in drinking water. AM1 was retained from the metal ion coordinated system when EDTA solution was added. Besides, the detection of mercury metal ions using provision methods like strips and silica gel through the naked eye are quick, feasible, and real-time sensing. Thus, AM1 is the promising moiety for real-time monitoring of Hg2+ ions in HeLa cells through bioimaging. Therefore, thiazole-based azomethine compound AM1 is helpful to sense poisonous mercury metal ions from waste and environmental samples.

Experimental Content

Characterization of AM1, quantum yield calculation, binding constant, and calculating LOD and LOQ are provided in SI-1 to SI-4.