KR102361653B1 - Dual-mode chemosensor and heavy metal detection method using thereof - Google Patents

Abstract

The present invention relates to a composition for detecting heavy metal ions containing a novel Schiff base, and the chemical sensor using the Schiff base can determine whether a sample contains heavy metals with the naked eye, which is convenient, and heavy metal ions to be detected Since it is less affected by other metal ions and has a very low limit of detection (LOD) for metal ions, selective and precise detection is possible.

Description

Dual-mode chemosensor and heavy metal detection method using thereof

The present invention relates to a composition and the like for detecting heavy metal ions including a novel Schiff base.

Due to rapid industrialization, the toxicity of heavy metals such as Ni ²⁺ , Cu ²⁺ , Cd ²⁺ , Co ²⁺ is emerging worldwide. When these toxic metals are exposed to the natural environment for a long period of time, they cause various diseases to the human body, and when used industrially, they act as a major source of pollution in the ecological environment. Accordingly, in order to protect human health, it is important to monitor heavy metals and their content in nature. Accordingly, various analytical methods and various molecular probes are widely used today to detect heavy metal ions in aqueous solution, but each of these methods has unique advantages and disadvantages, so it is a field that still has a lot of room for research.

Meanwhile, the development of fluorescence-based analytical probes that provide colorimetric detection of one or more heavy metal ions with the naked eye remains an enthusiastic research task in biology, environmental sampling, and pharmaceutical chemistry. However, studies on selective and sensitive probes capable of recognizing multiple ions in aqueous solution through such an optical approach are insufficient.

Republic of Korea Patent Registration 10-2053750

The present invention provides a compound represented by the following formula (1).

[Formula 1]

The present invention provides a composition for detecting heavy metal ions selected from the group consisting of Ni ²⁺ , Cu ²⁺ , Cd ²⁺ and Co ²⁺ , including the compound represented by Formula 1 above.

The present invention provides a method for detecting heavy metal ions selected from the group consisting of Ni ²⁺ , Cu ²⁺ , Cd ²⁺ and Co ²⁺ , comprising reacting the compound represented by Formula 1 with a sample.

The present invention provides a method for preparing a compound represented by Formula 1 above.

In order to solve the above object of the present invention, the present invention provides a compound represented by the following formula (1).

[Formula 1]

In addition, the present invention provides a composition for detecting heavy metal ions selected from the group consisting of Ni ²⁺ , Cu ²⁺ , Cd ²⁺ and Co ²⁺ , including the compound represented by Formula 1 above.

In one embodiment of the present invention, the pH of the composition may be 6 to 8.

In addition, the present invention provides a method for detecting heavy metal ions selected from the group consisting of Ni ²⁺ , Cu ²⁺ , Cd ²⁺ and Co ²⁺ comprising reacting the compound represented by Formula 1 with a sample.

In addition, the present invention comprises the steps of (a) reacting a compound represented by the following formula (2) and a compound represented by the following formula (3) to prepare a compound represented by the following formula (4);

(b) preparing a compound represented by Formula 1 by reacting the compound represented by Formula 4 obtained in step (a) with a compound represented by Formula 5 below to prepare a compound represented by Formula 1 provides

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

The chemical sensor using the novel Schiff base of the present invention is convenient to use because it can determine whether a sample contains heavy metals with the naked eye, is less affected by metal ions other than heavy metal ions to be detected, and Since the limit of detection (LOD) is very low, it has the advantage of enabling selective and precise detection of heavy metal ions.

1 shows the fluorescence intensity response of chemical sensor SB in different pH solutions.
2 shows a digital photograph when various metal ions are added to a solution containing the chemical sensor SB.
3 shows absorption spectra when various metal ions are added to a solution containing the chemical sensor SB.
FIG. 4 shows absorption titration profiles when Ni ²⁺ (a), Cd ²⁺ (b), Cu ²⁺ (c) and Co ²⁺ (d) were treated in chemical sensor SB at various concentrations.
Fig. 5 shows a digital photograph when various amounts of metal ion solutions are added to the chemical sensor SB.
6 shows fluorescence emission spectra when various metal ion solutions (10 nM) were treated on the chemical sensor SB (measured at λ _ex = 340 nm).
7 shows the effect of external metal ions on metal ion detection of Cd ²⁺ (a), Co ²⁺ (b), Ni ²⁺ (c) and Cu ²⁺ (d), respectively.
8 shows Job's plots derived from fluorescence and absorption outputs for each metal ion.
9 shows a modified Benesi-Hildebrand (BH) plot for a representative metal ion.
10 shows the IR spectrum of the chemical sensor SB with or without addition of each metal ion (1 : 1 equivalent).
11 is a ¹ H NMR titration result of a chemical sensor SB with each metal ion (1 : 1 equivalent) in DMSO-d ₆ .
12 shows the observation of fluorescence quenching of the chemical sensor SB by adding Ni ²⁺ (a), Cu ²⁺ (b), Co ²⁺ (c) and Cd ²⁺ (d) in a concentration range of 0-10 nM. will be.
13 shows a Stern-Volmer plot for each metal ion studied with the chemical sensor SB.
14 shows electron distribution and complexation with each metal ion of HOMO and LUMO of chemical sensor SB.
15 shows the IR spectrum of SB.
16 shows a ¹ H-NMR spectrum of SB.
17 shows the ¹³ C-NMR spectrum of SB.
18 shows the mass spectrum of SB.
19 shows the effect of pH on the fluorescence intensity of the chemical sensor SB in the presence of 10 nM of Ni ²⁺ , Cd ²⁺ , Cu ²⁺ and Co ²⁺ metal ions.
20 shows a ¹ H NMR spectrum when Cu ²⁺ is treated (1: 1 equivalent) in the chemical sensor SB.
21 shows a ¹ H NMR spectrum when the chemical sensor SB is treated with Ni ²⁺ (1: 1 equivalent).
Figure 22 shows the ¹ H NMR spectrum when the chemical sensor SB is treated with Co ²⁺ (1: 1 equivalent).
23 shows a ¹ H NMR spectrum when Cd ²⁺ treatment (1 : 1 equivalent) was applied to the chemical sensor SB.
24 shows a correction plot based on absorption titration of chemical sensor SB to which Ni ²⁺ (a), Cd ²⁺ (b), Cu ²⁺ (c) and Co ²⁺ (d) are added.

The present invention provides a compound represented by the following formula (1).

[Formula 1]

The compound represented by Formula 1 is a novel compound, and the name is (2E,NE)-N-(2,4-dihydroxybenzylidene)-2-

(quinolin-2-ylmethylene)hydrazinecarbothioamide, and is referred to herein as SB.

SB can be prepared through the following two-step synthesis.

In addition, the physicochemical properties of SB are as follows.

M.P.: 247-249℃

IR: 3750, 3393, 2969, 1738, 1606, 1526, 1501, 1360, 1322, 1260, 1230, 925, 902, 866, 838, 769, 749 cm ^-1

¹ H-NMR (400 MHz, DMSO-d ₆ ): δ 11.80 (s, 1H, -NH), 8.47 (s, 1H, -OH), 8.45 (s, 1H,=CH), 8.44 (s, 1H) , -OH), 8.37-8.36 (s, 2H, Ar-H, J = 4 Hz), 8.32 (s, 1H, Ar-H), 8.24 (s, 1H,=CH), 7.98-8.02 (q, 2H, Ar-H), 7.76-7.79 (t, 2H, Ar-H, J=4 & 8 Hz), 7.61-7.64 (t, 2H, Ar-H, J=8 & 4 Hz) ppm

¹³ C-NMR (100 MHz, DMSO-d ₆ ): δ 179.11, 153.98, 147.98, 143.27, 136.46, 130.60, 129.25, 128.44, 127.70, 118.34 ppm

MS: 351 (M+1) m/z. Elemental analysis calcd (%) for C ₁₈ H ₁₄ N ₄ O ₂ S: C 61.70; H 4.03; N 15.99; O 9.13; S 9.15; found: C 61.68; H 4.04; N 15.99; O 9.14; S 9.15.

The present invention provides a composition for detecting heavy metal ions selected from the group consisting of Ni ²⁺ , Cu ²⁺ , Cd ²⁺ and Co ²⁺ , including the compound represented by Formula 1 above. Since the four heavy metal ions induce fluorescent quenching upon reaction with SB, they can be detected using SB.

The composition may have a pH of 6 to 8, preferably pH 7. This is because it shows the maximum fluorescence intensity at the above pH.

The present invention provides a method for detecting heavy metal ions selected from the group consisting of Ni ²⁺ , Cu ²⁺ , Cd ²⁺ and Co ²⁺ , comprising reacting the compound represented by Formula 1 with a sample. As the detection method, a method of detecting colorimetric with the naked eye, a method of measuring a change in absorbance, and a method of observing fluorescence extinction may be used, but the present invention is not limited thereto.

For example, when Ni ²⁺ , Cd ²⁺ , Cu ²⁺ or Co ²⁺ is added to the chemical sensor SB, the absorption spectrum of the SB shifts, and the degree of spectrum shift varies depending on the type of heavy metal ion added. Ions can be detected. In addition, when the heavy metal ion is added to the chemical sensor SB, quenching is induced from the original fluorescence intensity of the chemical sensor SB, and the degree of quenching is different depending on the type of heavy metal ion, so that a specific heavy metal ion can be detected.

Meanwhile, observing the absorbance and fluorescence quenching may be performed by a conventional method in the art.

The present invention comprises the steps of (a) reacting a compound represented by the following formula (2) and a compound represented by the following formula (3) to prepare a compound represented by the following formula (4);

(b) preparing a compound represented by Formula 1 by reacting the compound represented by Formula 4 obtained in step (a) with a compound represented by Formula 5 below to prepare a compound represented by Formula 1 provides

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

Hereinafter, the present invention will be described in more detail through Examples and Test Examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto.

<Example 1: Materials and Preparation>

Example 1-1. material

Precursors for SB synthesis were purchased from commercial suppliers, and Thiosemicarbazide, quinoline 2-carboxaldehyde and 2,4-dihydroxy benzaldehyde were purchased from Sigma-Aldrich. Solvents such as ethanol and methanol were purchased from Samchun Chemical, and secondary distilled water was used in all experiments.

Example 1-2. Experimental method

Spectral characterization of the synthesized compound SB was performed using analytical techniques such as FT-IR (Frontier IR, Perkin Elmer), NMR (Bruker Avance 400 MHz spectrometer, Germany and DMSO-d ₆ solvent) and GC-MS (2795/ZQ2000, waters). was performed using The optical properties of SBs were investigated in the presence of specific metal ions and in the presence of specific metal ions using a UV-Vis spectrophotometer (UV-2600, Shimadzu, Japan) and a fluorescence spectrophotometer (FS-2, Scinco, Korea). Fluorescence lifetime values were obtained using a time-correlated single photon counting (TCSPC) spectrophotometer (HORIBA-iHR320, Japan) using a nanosecond diode laser operating at the excitation wavelength. The structure of SB and its complexes with respective metal ions, namely Ni ²⁺ , Cu ²⁺ , Cd ²⁺ and Co ²⁺ were optimized by TD-DFT using Gaussian 09 software.

Examples 1-3. (2E,NE)-N-(2,4-dihydroxybenzylidene)-2-

Synthesis and spectral characterization of (quinolin-2-ylmethylene)hydrazinecarbothioamide (SB)

Compound SB was synthesized using the condensation route. Scheme 1 below shows a two-step synthetic approach for the synthesis of compound SB.

[Scheme 1]

The first step involves a reaction between quinoline 2-carboxaldehyde (6.3 mmol, 1 g) and thiosemicarbazide (6.3 mmol, 0.58 g) (reflux conditions in ethanol for 4 h). The reaction progress was monitored using TLC technique, and after reaction completion the mixture was cooled to ambient temperature. Distilled water was added thereto and quinoline thiosemicarbazone (QTS) was filtered, and it was confirmed that the actual yield of the first-stage compound was 88.12%.

For the synthesis of the target compound, SB, the second step was performed by reacting quinoline thiosemicarbazone (3.03 mmol, 0.7 g) and 2,4-dihydroxybenzaldehyde (3.03 mmol, 0.42 g) with ethanol reflux for 5 hours. TLC was then monitored until the reaction was complete. The obtained hot solution was cooled and poured into cold water with vigorous stirring to obtain the desired SB, a yellowish solid in 79.68 % yield.

Examples 1-4. Absorption and Fluorescence Measurements

To investigate the characteristic colorimetric and fluorescence measurements of each metal ion using SB, solvent mixtures using a 7:3 ratio of methanol and water were prepared. Specifically, a solution containing SB (5 x 10 ^-5 M) in methanol:water (7:3 v/v) and secondary distilled water containing various metal ions (100 nM) were prepared. 1 mL of the SB stock solution was added to a 10 mL volumetric flask, followed by 1 mL of each metal ion solution and 1 mL of a pH 7 solution. The series of solutions was then diluted to 10 mL to an effective concentration of metal ions of 10 nM. Then, these solutions were shaken thoroughly, mixed and kept at room temperature for 10 minutes. The absorption and fluorescence emission spectra of all these solutions were recorded and compared with the spectra of SB.

As a result, an immediate macroscopic colorimetric change was observed by adding each metal ion. That is, Ni ²⁺ (yellow), Cu ²⁺ (yellow), Cd ²⁺ (light yellow) and Co ²⁺ (dark yellow) were observed within a very short time.

<Example 2: Observation of effect according to pH>

The pH of a solution plays a pivotal role in all fluorescence chemical measurements used for the detection of analytes in aqueous solutions. To optimize the pH for the fluorescence-based assay of the present invention, experiments were performed in different pH solutions ranging from 1-12. Chemistry sensor SB was treated with each pH solution and the fluorescence emission intensity output was scanned. As a result, as shown in FIG. 1 , the fluorescence intensity response of the chemical sensor SB was confirmed in different pH solutions, and the maximum fluorescence intensity was observed at pH 6 to 8. For consistency, the experiment was repeated 3 times, and it was confirmed that pH 7 was an ideal condition for performing the experiment.

Very low and high pH values can interfere with the stability of the chemosensor in methanol:water systems, thereby reducing the fluorescence intensity. The effect of the pH solution on the fluorescence intensity of the chemical sensor SB in the presence of metal ions such as Cd ²⁺ , Co ²⁺ , Ni ²⁺ and Cu ²⁺ at a concentration of 10 nM was investigated, and the results are shown in FIG. 19 .

Through the above experiment, it was found that the complexation-related fluorescence intensity between the chemical sensor SB and each metal ion was maximum in the range of pH 6-8, and that the complex between the metal ion and the chemical sensor SB at neutral pH was stable and most biocompatible. Confirmed.

<Example 3: Visual colorimetric detection of each metal ion>

In methanol: water (7: 3, v/v), the chemical sensor SB is colorless.

1 mL (100 nM) solution of various metal ions, ie. 10 nM Ag ⁺ , Na ⁺ , Fe ²⁺ , Ni ²⁺ , Pb ²⁺ , Al ³⁺ , Hg ²⁺ , Zn ²⁺ , Ba ²⁺ , Mn ²⁺ , Cu ²⁺ , NH ⁴⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , Cd ²⁺ , Cr ³⁺ , Co ²⁺ and Cr ⁶⁺ were added to the chemical sensor SB (5 × 10 ^-5 M, 1 mL) and the solution was kept at ambient temperature. (effective concentration of metal ions is 10 nM). As a result, the color change of the solution was easily discernable with the naked eye, with a short reaction time (3) from colorless to yellow for Ni ²⁺ and Cu ²⁺ , pale yellow for Cd ²⁺ , and dark yellow for Co ²⁺ -5 min) showed a color change. 2 shows digital photographs of color change of solutions with chemosensor SB in the presence and absence of various metal ions. Accordingly, the present invention provides a sensitive and selective color change for the detection of multiple ions in aqueous solution, which is further supported by the following examples.

<Example 4: Absorption Titration Analysis>

To investigate the detection of specific metal ions in aqueous solution, the absorption spectrum of a chemical sensor SB (5 x 10 ^-6 M) was measured in the presence of each metal solution at a concentration of 10 nM. Different metal ion solutions, i.e. Ag ⁺ , Na ⁺ , Fe ²⁺ , Ni ²⁺ , Pb ²⁺ , Al ³⁺ , Hg ²⁺ , Zn ²⁺ , Ba ²⁺ , Mn ²⁺ , Cu ²⁺ , NH ⁴ The absorption characteristics of the chemical sensor SB were analyzed using ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , Cd ²⁺ , Cr ³⁺ , Co ²⁺ and Cr ⁶⁺ . Among the metal ions, except for Ni ²⁺ , Cd ²⁺ , Cu ²⁺ and Co ²⁺ , there was no significant change in absorbance and spectral shift for the chemical sensor SB. 3 shows absorption spectra of the chemical sensor SB in the presence or absence of various metal ion solutions.

The absorption characteristics of the chemical sensor SB were significantly changed in terms of absorption intensity and spectral shift. The chemical sensor SB initially exhibits an absorption spectrum at 338 nm, which may be due to the conjugation π binding system present in the chemical sensor structure.

When Ni ²⁺ , Cd ²⁺ , Cu ²⁺ and Co ²⁺ are added, the absorption spectrum of the chemical sensor SB changes with bathochromic spectrum shifts of 84, 61, 86 and 58 nm, respectively. Surprisingly, the bathochromic spectral shifts of the four metal ions in the absorption spectrum were observed to be distinct from each other and also from other metal ions. The spectrophotometric titration of the chemical sensor SB was performed independently of the corresponding metal ions (Ni ²⁺ , Cd ²⁺ , Cu ²⁺ , and Co ²⁺ ) to investigate the effect of metal ions on the absorption properties of the chemical sensor SB. .

As a result, absorption spectra of the chemical sensor SB in the presence of Ni ²⁺ , Cd ²⁺ , Cu ²⁺ and Co ²⁺ were shown as shown in FIGS. 4 a to 4 , respectively. The results indicate that the absorption spectrum of the chemical sensor SB at 338 nm is reduced with the appearance of a new bathochromic shift absorption band by the metal ions, ie, Ni ²⁺ , Cd ²⁺ , Cu ²⁺ and Co ²⁺ . When the amounts of metal ions Ni ²⁺ , Cd ²⁺ , Cu ²⁺ and Co ²⁺ are increased, the absorption bands of the chemical sensor SB shift from 338 to 422 nm, 399 nm, 424 nm and 396 nm, respectively.

The change in the absorption value with the further addition of the metal ion solution was also significant. These observations suggest strong interactions between individual metal ions (Ni ²⁺ , Cd ²⁺ , Cu ²⁺ and Co ²⁺ ) and functional groups such as sulfur (-S) and hydroxyl (-OH) groups in the chemical sensor SB. This means that it may exist.

The results of individually adding various amounts of metal ion Ni ²⁺ , Cd ²⁺ , Cu ²⁺ and Co ²⁺ solutions to the chemical sensor SB are shown in FIGS. 5A to 5D . All these colorimetric and spectrophotometric results show distinct properties of Ni ²⁺ , Cd ²⁺ , Cu ²⁺ and Co ²⁺ when in contact with chemical sensor SB compared to other metal ion solutions. Accordingly, these properties can be usefully used to distinguish the four metal ions from other metal ion solutions at a specific concentration level.

<Example 5: Fluorescence titration analysis>

The effect of each metal ion on the fluorescence response was studied through spectrofluorescence titration. Ag ⁺ , Na ⁺ , Fe ²⁺ , Ni ²⁺ , Pb ²⁺ , Al ³⁺ , Hg ²⁺ , Zn ²⁺ , Ba ²⁺ , Mn ²⁺ , Cu ²⁺ , NH ⁴⁺ , K+, Ca ² Changes in fluorescence properties of a series of solutions containing chemosensor SB with and without addition of metal ions (10 nM) such as ⁺ , Mg ²⁺ , Cd ²⁺ , Cr ³⁺ , Co ²⁺ and Cr ⁶⁺ investigated.

6 shows the fluorescence emission spectra of chemical sensor SB measured in various metal ion solutions having an excitation wavelength of 340 nm and 10 nM concentration, and only Ni ²⁺ , Cd ²⁺ , Cu ²⁺ and Co ²⁺ are chemical sensor SBs. It was confirmed that the fluorescence intensity was quenched, and other metal ions did not cause a significant change. Cu ²⁺ induced a 10.5 fold quenching with respect to the initial fluorescence intensity of the chemical sensor SB, whereas the other metal ions, Ni ²⁺ , Co ²⁺ and Cd ²⁺ , respectively, 9.2, 8.6 and 8.1 times the original value of the chemical sensor SB. Fluorescence quenching was induced. These results imply that strong complexation between the chemical sensor SB and -S (sulfur) and -O (oxygen) and metal ions is responsible for the fluorescence quenching process. In addition, it was confirmed that the strong complex formation ability through the bonding of the chemical sensor and metal ions was in the order of SB-Cu ²⁺ > SB-Ni ²⁺ > SB-Co ²⁺ > SB-Cd ²⁺ .

<Example 6: Confirmation of influence of foreign material ions>

In order to successfully use fluorescent chemical sensors, the influence of external metal ions present in the aqua system must be minimized or eliminated. Therefore, to investigate the influence of foreign metal ions on binding capacity and fluorescence reaction, a simple spectrofluorescence titration was performed, and then the emission spectra of each band solution were recorded. The fluorescence emission spectra of the chemical sensor SB were recorded separately in the presence of Ni ²⁺ , Cd ²⁺ , Cu ²⁺ and Co ²⁺ (10 nM). Ag ⁺ , Na ⁺ , Fe ²⁺ , Pb ²⁺ , Al ³⁺ , Hg ²⁺ , Zn ²⁺ , Ba ²⁺ , Mn ²⁺ , NH ⁴⁺ , K ⁺ at equimolar concentrations (10 nM) , Ca ²⁺ , Mg ²⁺ , Cr ³⁺ , and Cr ⁶⁺ were treated with a compound including a chemical sensor SB, and then the change in fluorescence response by each metal solution was measured.

Fig. 7a shows that the fluorescence quenching by the addition of Cd ²⁺ was affected by the addition of Co ²⁺ , Ni ²⁺ and Cu ²⁺ , while the remaining metal ions did not affect the results. The most prominent effects on fluorescence intensity were Co ²⁺ about 8.5 times, Ni ²⁺ about 9.1 times and Cu ²⁺ about 10.4 times, which were important features of each metal ion when exposed to independent fluorescence titration. In addition, Fig. 7b shows that only Ni ²⁺ and Cu ²⁺ ions affect the fluorescence intensity of the chemical sensor SB quenched by Co ²⁺ . When Ni ²⁺ and Cu ²⁺ ions were added to a solution containing chemical sensors SB and Co ²⁺ , 9.2-fold and 10.4-fold quenching was observed. Nevertheless, almost all metal ions except Cu ²⁺ had little effect on the fluorescence intensity of the chemical sensor SB quenched by Ni ²⁺ as shown in Fig. 7c. The equimolar addition of Cu ²⁺ to the same solution containing the chemical sensor SB and Ni ²⁺ induced fluorescence quenching 10.5 times the initial value, a characteristic induced by Cu ²⁺ ions seen during fluorescence titration experiments. In contrast, Fig. 7d shows that all metal ion solutions did not affect the fluorescence intensity induced by the addition of Cu ²⁺ to the chemical sensor SB. These results show that chemical sensor SB has strong binding affinity for metal ions in ascending order, such as Cd ²⁺ < Co ²⁺ < Ni ²⁺ < Cu ²⁺ . This means that Cu ²⁺ can release other metal ions from the chemosensor SB complex. Also, Ni ²⁺ can remove Co ²⁺ and Cd ²⁺ from the complex with SB. In addition, Co ²⁺ can remove only Cd ²⁺ metal ions from the complex including SB. This selective and sensitive reaction can be used for the differentiation and quantitative analysis of these four metal ions in aqueous solution.

The above results indicate that the stability order of each metal ion with the chemical sensor SB is SB-Cu ²⁺ > SB-Ni ²⁺ > SB-Co ²⁺ > SB-Cd ²⁺ , which supports the density functional theory over time. It is further supported by computational analysis and evaluation of binding parameters via time-dependent density functional theory (TD-DFT).

Scheme 2 below shows a schematic of the competitive sensing reaction of a chemical sensor SB designed for Ni ²⁺ , Cd ²⁺ , Cu ²⁺ and Co ²⁺ ion targets.

[Scheme 2]

Moreover, all these experimental observations imply maximum stability for the composite with chemical sensor SB by the Irving William stability sequence dealing with divalent first-row transition metal ions. According to the Irving William stability sequence, the stability of complexes formed by divalent first-row transition metal ions generally increases over the cycle, with the exception of SB-Cd ²⁺ SB-Cu ²⁺ > SB-Ni ²⁺ > SB -Co ²⁺ It was confirmed that it is similar to the previous experiment.

<Example 7: Evaluation of binding parameters>

Job's method was used to investigate the binding stoichiometry between the chemical sensor SB and a representative metal ion. The sum of the concentrations of the specific metal ion and the chemical sensor SB was kept constant, and the mole fraction of the metal ion was varied from 0.1 to 0.9. Job's plots derived from fluorescence and absorption outputs for each metal ion are shown in FIGS. 8 a to d. From this, it was confirmed that the absorbance and fluorescence emission intensity reached the maximum when the mole fraction of each metal ion was 0.5. The above result means that specific metal ions, ie, chemical sensor SB and Ni ²⁺ , Cd ²⁺ , Cu ²⁺ and Co ²⁺ are combined in a stoichiometric ratio 1:1.

In addition, the binding constant was evaluated through a modified Benesi-Hilderbrand (BH) equation based on the fluorescence output. Equation (1) given below is known as a modified Benesi-Hildebrand (BH) equation used to estimate the binding constant (K _B ) between the chemical sensor SB and a specific metal ion.

[Equation 1]

Here, F _m , F ₀ and F mean the minimum fluorescence intensity, initial fluorescence intensity, and fluorescence intensity at each metal concentration of the chemical sensor SB for metal ions, respectively. The number of bound metal ions per chemical sensor SB unit is denoted by 'n'. Modified Benesi-Hildebrand (BH) plots are shown for representative metal ions as shown in FIGS. 9 a to 9 , and the figures show straight lines with slope values, respectively.

Estimated values of binding constants for the chemical sensor SB and each metal ion Ni ²⁺ , Cd ²⁺ , Cu ²⁺ and Co ²⁺ were calculated and shown in Table 1 below.

A high binding constant value indicates a strong and efficient binding between the target metal ion and the chemical sensor SB. From these calculations, it was confirmed that the chemical sensor SB forms the most stable complex with Cu ²⁺ .

Through IR and ¹ H NMR analysis, each metal ion and bond between functional groups having -S and -O atoms of the chemical sensor SB was investigated. 10 and 11 show the IR and ¹ H NMR titration results between the chemical sensor SB and the metal ion (1 : 1 equivalent), respectively. Experiments on IR titration (Fig. 10) clearly show the disappearance of the stretching frequencies of -OH (3750 cm ^-1 ) and -C = S (1225 cm ^-1 ) functional groups when each metal ion is in contact with the chemical sensor SB. indicates. The above result means that there is an interaction between -S and -O atoms of the chemical sensor SB and metal ions.

In addition, ¹ H NMR titration also supports the interaction pattern at -S and -O of the chemosensor SB. 11 shows the partially expanded ¹ H NMR spectrum of the chemical sensor SB with the addition of each metal ion. In the ¹ H NMR spectrum of the chemical sensor SB, two singlets at δ 8.44 and 8.47 (Fig. 11 a) correspond to protons belonging to -OH at the ortho position (*) of the phenyl ring and -OH at the para position (**) . Addition of metal ions Cu ²⁺ , Ni ²⁺ , Co ²⁺ and Cd ²⁺ (Fig. 11 b to e) to the chemical sensor SB in equal amounts resulted in a change in δ values for aromatics and other protons at δ 8.47. It is clearly seen that one -OH peak is missing. Full and partial extended spectra for ¹ H NMR titrations between the chemical sensor SB and metal ions are shown in FIGS. 20 to 23 .

<Example 8: Stern-Volmer plot and detection limit confirmation>

Fluorometric titration was performed to investigate the change in fluorescence intensity of the chemical sensor SB upon further addition of a specific metal ion solution within the range of 0-10 nM. 12A to 12D show fluorescence quenching of the chemical sensor SB to which Ni ²⁺ , Cu ²⁺ , Co ²⁺ and Cd ²⁺ concentrations of 0-10 nM are added, respectively. This indicates that the fluorescence intensity of the chemical sensor SB decreases regularly without spectral shift when in contact with the target metal ion. Fluorescence quenching efficiency was evaluated through the conventional Stern-Volmer equation for fluorescence quenching data, and Equation 2 below was used.

[Equation 2]

Here, F ₀ , F and K _SV denote the initial fluorescence intensity of the chemical sensor SB, the fluorescence intensity of the chemical sensor SB when a specific concentration of a quencher [Q] is added, and the Stern-Volmer constant. 13A to 13D show Stern-Volmer plots for each metal ion having a chemical sensor SB. The Stern-Volmer constant values for metal ions such as Ni ²⁺ , Cd ²⁺ , Cu ²⁺ and Co ²⁺ with the chemical sensor SB are shown in Table 1 above.

In addition, absorption titration was performed with or without increasing (0-10 nM) metal ions in the solution containing the chemical sensor SB. 4a to 4d show that the absorption values of the chemical sensor SB to which 0-10 nM of Ni ²⁺ , Cd ²⁺ , Cu ²⁺ and Co ²⁺ are added consistently increase.

Absorption titration profiles were used to plot the change in absorbance values for specific concentrations of metal ions. 24 was used as a calibration plot based on absorption titration for the chemical sensor SB to which Ni ²⁺ , Cd ²⁺ , Cu ²⁺ and Co ²⁺ were added, respectively. 13 and 24 were used as calibration plots for estimation of the limit of detection (LOD) for metal ions based on fluorescence and absorption methods, respectively. The detection limits for the metal ions were calculated using Equation 3 below and are listed in Table 1 above.

[Equation 3]

Here, the standard deviation and slope of the calibration curve are denoted by σ and K, respectively.

Relatively similar LOD values for Ni ²⁺ , Cd ²⁺ , Cu ²⁺ and Co ²⁺ metal ions via fluorescence and absorption indicate selectivity and sensitivity for the detection scheme. The LOD values obtained were extremely lower than the LOD values studied by other methods for this metal ion. LOD values obtained through the present invention and Cd ²⁺ , Co ²⁺ , Ni ²⁺ and Cu ²⁺ LOD values known through prior studies were compared in Tables 2 to 5 below (Table 2 is Cd ²⁺ , Table 3 is for Co ²⁺ , Table 4 is for Ni ²⁺ , and Table 5 is for Cu ²⁺ ).

(In Table 2 above, 8 and 9 are cases using SB)

(In Table 3 above, 9 and 10 are cases using SB)

(In Table 4 above, 9 and 10 are cases using SB)

(In Table 5, 9 and 10 are the case of using SB)

<Example 9: Confirmation of fluorescence quenching mechanism of SB by each metal ion>

A functional group having heteroatoms -S and -O interacts with respective metal ions, resulting in changes in absorption and fluorescence properties of the chemical sensor SB. According to the previous study on the effect of adding foreign substances to the chemical sensor SB and a solution containing specific metal ions, it was confirmed that Cu ²⁺ exhibits a strong interaction with the chemical sensor SB. The interaction of the metal ion with the chemical sensor SB reduces the radiation path in the excited state and further quenches the fluorescence. Scheme 2 illustrates the binding properties of the target metal ion responsible for the fluorescence quenching process and the chemical sensor SB.

The fluorescence quenching process was investigated by absorption titration in the presence and absence of metal ions in solution of chemosensor SB, Stern-Volmer plots and experimental observations for estimation of fluorescence lifetime values. The absorption titration profile (FIG. 4) shows that the maximum absorption wavelength was changed to a longer wavelength by adding various concentrations of metal ions. Furthermore, observing the linear Stern-Volmer plot (Fig. 13) for each metal ion study, the nature of the fluorescence quenching process could be static rather than dynamic. The fluorescence quenching process is static when the interaction between the chemosensor and the analyte must occur in the ground state. To support the results of the static type fluorescence quenching process, fluorescence lifetime estimation was used. If the interaction between the chemical sensor and the analyte is static, the fluorescence lifetime remains constant even if the concentration change of the analyte molecule is dynamic. This is because only unassociated fluorescent probes in contact with analyte molecules contribute to lifetime. The average fluorescence lifetime values of the chemical sensor SB with or without the addition of a specific metal ion solution having a specific concentration are given in Table 6 below.

In this study, the fluorescence lifetime values of the chemical sensor SB with and without the addition of target molecules remain unchanged, meaning that the nature of the fluorescence quenching process is purely static.

The formation of a non-fluorescent complex between the chemosensor SB and the metal ion in the ground state follows a static quenching process. In this process, the metal ion and the chemosensor SB complex absorb energy from the light and then immediately return to the ground state without emission of a photon, resulting in fluorescence quenching. Scheme 3 below illustrates the static fluorescence quenching of a chemical sensor SB with specific metal ions.

[Scheme 3]

<Example 10: Computational analysis through TD-DFT (Time-dependent density functional theory)>

The study of theoretical calculations was carried out through a calculation method based on time-dependent density functional theory (TD-DFT) using the Gaussian 09 program. This study was carried out to determine the cooperative behavior, investigate the theoretical properties of the chemical sensor SB, and investigate its complexation with metal ions such as Ni ²⁺ , Cd ²⁺ , Cu ²⁺ and Co ²⁺ . Becke-3-Lee-Yang-Parr (B3LYP) functionality and the lanl2dz base set were used to optimize the structure of the target complex of compound SB with a metal ion.

The calculated energetically optimized stable structure together with the binding interaction mode between the chemical sensor SB and each metal ion is shown in FIG. 14, and the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (HOMO) of the chemical sensor SB are shown in LUMO) characterized by electron distribution of energy levels and complexation with each metal ion. 14 shows a HOMO-LUMO diagram of a chemical sensor SB in which the electron distribution of HOMO mainly spans the dihydroxybenzylthiosemicarbazone backbone, and LUMO is predominantly in the quinoline moiety.

The HOMO side of the chemical sensor SB has the greatest influence on metal ions. The change in HOMO-LUMO energy was caused by complexation between the chemical sensor SB and each metal ion. The strong electrostatic interaction through functional groups having heteroatoms such as -S and -O of the chemical sensor SB has been demonstrated to form complexes with metal ions. The energy values of HOMO and LUMO for the corresponding composite and free chemical sensor SB are provided in Table 7 below.

It was found that the energy values of HOMO and LUMO for the composite were lower than those of the chemical sensor SB. The high stability of the composite chemical sensor SB with metal ions was confirmed through a lower value of the total energy of the composite than that of the glass SB. Therefore, the TD-DFT calculation implies that the composite with lower stabilization energy has higher stability. In this case, it was found that the decreasing order of stabilization energy for the complex of the chemical sensor SB and the metal ion was SB-Cd ²⁺ > SB-Co ²⁺ > SB-Ni ²⁺ > SB-Cu ²⁺ . On the other hand, the order of increased stability of the complex was found to be that of SBCd ²⁺ < SB-Co ²⁺ < SB-Ni ²⁺ < SB-Cu ²⁺ . Therefore, these findings support experimental observations during absorption and fluorescence studies.

<Example 11: Application of fluorescence quenching method through river sample analysis>

Water samples were collected from a river near the university to apply the heavy metal detection method of the present invention. The collected sample was filtered and boiled to remove solid impurities and dissolved gases. The target solution was prepared by spiking each of the standard metal ions (Ni ²⁺ , Cu ²⁺ , Cd ²⁺ and Co ²⁺ ) at specific concentrations into the solution of the chemical sensor SB and diluting it in the collected water sample. 13A to 13D were used as calibration curves for estimating the spiked amount of each metal ion. The results investigated for each metal ion are presented in Tables 8 and 9 below.

Through the above results, it was confirmed that the observed experimental value was very close to the expected value.

As described above, when the chemical sensor SB of the present invention is used, it is possible to quickly and accurately measure four heavy metal ions (Ni ²⁺ , Cu ²⁺ , Cd ²⁺ and Co ²⁺ ) in aqueous solution.

Claims (5)

As a compound represented by Formula 1 below, Ni ²⁺ , Cu ²⁺ , Cd ²⁺ and Co ²⁺ A compound in which fluorescence quenching is induced when a heavy metal ion selected from the group consisting of the compound reacts with the compound.
[Formula 1]

A composition for detecting heavy metal ions selected from the group consisting of Ni ²⁺ , Cu ²⁺ , Cd ²⁺ and Co ²⁺ , comprising the compound represented by Formula 1 of claim 1 . The composition for detecting heavy metals according to claim 2, wherein the composition has a pH of 6 to 8. A method for detecting heavy metal ions selected from the group consisting of Ni ²⁺ , Cu ²⁺ , Cd ²⁺ and Co ²⁺ comprising reacting the compound represented by Formula 1 of claim 1 with a sample. (a) reacting a compound represented by the following formula (2) and a compound represented by the following formula (3) to prepare a compound represented by the following formula (4);
(b) reacting the compound represented by Formula 4 obtained in step (a) and the compound represented by Formula 5 below to prepare a compound represented by Formula 1 below.
[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

Images