KR102270656B1 - Tetraphenylene-based mercury ion detection sensor and mercury ion detection method using the same - Google Patents

Abstract

Disclosed herein are a tetraphenylene-based mercury ion detection sensor and a mercury ion detection method using the same.

Description

Tetraphenylene-based mercury ion detection sensor and mercury ion detection method using the same {TETRAPHENYLENE-BASED MERCURY ION DETECTION SENSOR AND MERCURY ION DETECTION METHOD USING THE SAME}

The present specification relates to a tetraphenylene-based mercury ion detection sensor and a mercury ion detection method using the same.

More specifically, it relates to a mercury ion detection sensor with high sensitivity and high selectivity for detecting mercury ions using a tetraphenylene-based compound, and a method for detecting mercury ions using the same.

The detection of metal ions is of considerable significance because of its high toxicity to organisms and serious risks to the environment and human health. In particular, mercury in heavy metal ions (Hg ²⁺ ) The ion is one of the toxic, non-radioactive metals present in the environment, and traces of mercury (Hg ²⁺ ) Even ions are very harmful to humans.

Mercury for industrial applications and waste (Hg ^{2 +)} devoting a lot of effort into reducing the ion, but remains of mercury (Hg ^{2 +)} pollution of ions variety of natural and anthropogenic causes (hydropower, mining, pulp and paper industries, etc.) It is approaching as a constant threat to humans through

According to the US Environmental Protection Agency, the acceptable amount of mercury ions in drinking water is 2 μg/L.

Also mercury (Hg ^{2 +} ) The strong affinity between ions and sulfur-containing ligands leads to dysfunction of enzymes and proteins, leading to various diseases related to neurotoxicity, hepatotoxicity and nephrotoxicity.

Therefore, while rapid, efficient mercury (Hg ^{+ 2)} Method of measuring ion concentration of a low cost is essential for biological, environmental, and real-time monitoring of industrial samples.

According to previous reports, mercury (Hg ^{2 +} ) Ion detection methods include atomic absorption spectroscopy (AAS), inductively coupled plasma mass spectrometry (ICP-MS), flame atomic absorption spectroscopy (FAAS), reversed-phase high-performance liquid chromatography (RP-HPLC), and cyclic voltammetry (CV). . However, these techniques have various drawbacks such as the construction of expensive measuring equipment, the long time required for sample preparation, and limitations in application in field and real-time monitoring.

Recently, fluorescence probes have attracted attention due to their advantages such as high sensitivity, good selectivity, cost-effectiveness, cell image measurement, and easy manipulation.

Recently fluorescence resonance energy transfer for decades (Fluorescence Resonance Energy Transfer, FRET) , luminescence induced aggregation (Aggregation Induced Emission, AIE), such as mercury fluorescent ?? Ching (Fluorescence quenching) and ratiometric fluorescence (Ratiometric fluorescence) (Hg ^{2 +} ) Several types of fluorescent probes using various signal output methods for ion detection have been developed.

Among the fluorescence-based technologies listed above, AIE emitters have recently attracted attention, and some of them have the disadvantages of low solubility, lack of biocompatibility and selectivity for other ions, and insensitivity to pH values.

Therefore, under neutral pH, mercury (Hg ^{2 +} ) There is a need to develop AIE illuminators for rapid and selective detection of ions.

Korean Patent Publication No. 10-2014-0098436 Korean Patent Registration No. 10-1642406

New J. Chem., 2018, 42, 13836 Sensors & Actuators: B. Chemical 296 (2019) 126670 Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 223 (2019) 117315

An object of the present specification is to provide a tetraphenylene-based mercury ion detection sensor and a mercury ion detection method using the same.

In one embodiment of the present invention, there is provided a mercury ion detection sensor including a compound represented by the following formula (1).

[Formula 1]

In another embodiment of the present invention, a mercury ion detection kit comprising: a concentration measuring unit in a biological sample separated from the body of the human body, wherein the concentration measuring unit in the biological sample includes the above-described mercury ion detection sensor to provide.

In another embodiment of the present invention, preparing a mercury ion detection sensor; reacting the mercury ion detection sensor with the analyte sample; and detecting mercury ions by measuring a change in fluorescence of the mercury ion detection sensor after the reaction between the mercury ion detection sensor and the analyte sample.

Mercury ion detection sensor according to an embodiment of the present invention is mercury (Hg ^{2 +} ) The detection limit of the ion is 0.9 nM, and it shows clear selectivity even in the presence of other metal ions in concentrations greater than 1000 times. It maintains optimal conditions in physiologically active conditions, and can be successfully achieved in cell image measurement and environmental sample analysis.

1 is a schematic diagram showing a mechanism for synthesizing a compound represented by Formula 1 (hereinafter, TPE-BTA) according to an embodiment of the present invention.
^{2 is a 1} H NMR spectrum of TPE-BTA and an expanded image thereof.
3 is a ¹³ C NMR spectrum of TPE-BTA.
Figures 4a and 4b is a HR-LC-MS spectrum of (4a) TPE-BTA and (4b) TPE-BTA-Hg 2 + respectively.
5 shows (a) fluorescence excitation spectrum, (b) of 0.1 μM TPE-BTA in 0.2 M Hepes buffer containing 2 vol% of THF/H ₂ O (f _{w = 70%, pH 7.4);} ) Fluorescence emission spectrum (Emission spectrum) and (c) Graph showing fluorescence emission spectrum of TPE-BTA after reaction with ^{5 μM Hg 2 +} and color ^{of TPE-BTA and TPE-BTA-Hg 2 + solution under UV lamp} It is a photographic image.
6a-6c are respectively (6a) _{a graph showing a fluorescence spectrum obtained from TPE-BTA in a THF/H 2} O solution, (6b) a graph showing a change in quantum yield according to a change in water fraction, and (6c) a corresponding graph; It is a color photographic image.
Figure 7 is a particle size of 2 vol% of _{THF / H 2 O (f w} = 70%, pH 7.4) 0.2 M Hepes TPE-BTA-Hg of 0.1 μm of the ^second buffer containing ^{+.
Figure 8 shows 0.5 μM Hg 2 +} (a ~ k; 0 ~) in 0.1 μM TPE-BTA in 0.2 M Hepes buffer solution containing 2 vol % THF/H ₂ O (f _{w = 70%, pH 7.4).} 5 μM) is a graph showing the fluorescence spectra to which 0 to 5 μM of Hg ^{2 + was added while increasing by increments.
9 is a 1} H NMR spectrum of a solution in which ^{Hg 2 +} is added to TPE-BTA, respectively (a) 0 equivalents (equiv.) Hg ^{2 +} , (b) 1 equivalent (equiv.) Hg ^{2 +} , (c) ) 5 equivalents (equiv.) Hg ^{+ 2,} (d). 10 eq (equiv), Hg ^2+, and (e) 15 equivalents (equiv.) Hg is a graph showing the spectrum of: ^{2 +} added.
10A-10F are graphs showing XPS spectra. XPS wide scan (10a), S2p (10b), and N1s (10c) are XPS spectra for TPE-BTA, respectively, XPS wide scan (10d), S2p (10e) , N1s (10f) is an XPS spectrum for the TPE-BTA-Hg ^{2 +.}
11a-11c are the results of analyzing the selectivity and interference effects of TPE-BTA. 11a and 11b are _{0.2 M Hepes buffer solutions containing 2 vol % of THF/H 2} O (f _w = 70%, pH 7.4). in 0.1 μM TPE-BTA and (1) 0 μM Hg ^{2 +} (2) 5 μM Hg ^{2 +} , (3-18) 5 mM Ag ⁺ , Na ⁺ , K ⁺ , Co ^{2 +} , Mg ²⁺ , Cu ^{2 + , Ca 2 +, Zn 2 +} , Sr 2 +, Ba 2 +, Pb 2 +, Ni 2 +, Hg 2 +, Fe 2 +, Cd 2 + and Fe ^{3 +} selectivity through a fluorescence intensity change of an ion addition ( 11a) and its 5 μM Hg ^{+ 2} will showing the effect of interference (11b) at the time of addition, 11c is above the analyte (1-18) and Hg ^{2 +} selectivity (brown) and interference (blue) bar illustration of the effect it's a chart
Figure 12a-12c are each under (12a) member and the presence of Hg ^{2 +} Fluorescence intensity change according to pH of TPE-BTA and TPE-BTA-Hg ^{2 +} , (12b) Time course for the reaction of TPE-BTA (0.1 μM) with Hg ^{2 +} (ad: 0, 1, 3, 5 μM ) shows a fluorescence image in accordance with various pH values, under a TPE-BTA (0.1 μM) change with time of the fluorescence intensity, (12c), the presence and absence of the Hg ^{+ 2} in accordance with the concentration.
13 is a cytotoxicity test as a result of MTT analysis for TPE-BTA, and is a graph showing the results measured as a percentage of cell viability (number of measurements, repeated 5 times). (1) control, (2) 0.01, (3) 0.1, (4) 0.5, (5) 1.0, (6) 10.0, (7) 25 and (8) 50 μM TPE-BTA.
14a-14c are dark-field images of a confocal fluorescence microscope, respectively, (14a) cells incubated for 30 minutes after addition of 10 μM TPE-BTA fluorescent probe to Hela cells, (14b) 10 μM TPE to Hela cells, respectively. -BTA fluorescent probe and 1 μm Hg ^{2 +} ions by the addition of 30 minute incubation in which cell images, and (14c) 10 μM TPE-BTA fluorescent probe and the 2 μm Hg ²⁺ ion was added to the cells, incubation 30 minutes image on Hela cells to be.

In the present specification, when a part "includes" a certain component, this means that other components may be further included, rather than excluding other components, unless otherwise stated.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. Embodiments of the present invention have been described with reference to the accompanying drawings, which are described for purposes of illustration, and the technical spirit of the present invention and its configuration and application are not limited thereby.

As used herein, the term “mercury ion detection sensor” includes not only a sensor detecting the presence of mercury ions in a sample, but also a sensor measuring the concentration of mercury ions in the sample and an image sensor capable of measuring a mercury detection image in cells. covers all

Mercury ion detection sensor

In exemplary embodiments of the present invention, there is provided a mercury ion detection sensor including a compound represented by Formula 1 below.

[Formula 1]

The compound represented by Formula 1 is tetraphenylethylene (TPE) bonded to bis(thiophen-2-ylmethyl)amine (BTA) that reacts with mercury ions.

When the mercury ion detection sensor including the compound represented by Formula 1 reacts with mercury ions, strong fluorescence may be expressed, and specifically green fluorescence may be expressed.

Tetraphenylethene (TPE) is one of the most common AIE emitters, and research is still ongoing. Molecules with propeller-like structures such as TPE and TPE derivatives show weak emission in solution, but high fluorescence emission in the aggregated state. HSAB (hard soft acid base) theory has shown a strong affinity between the Hg ^{+ 2} and the organic molecule containing sulfur.

According to all the above descriptions, TPE-based bis(thiophen-2-ylmethyl)amine (BTA) according to an embodiment of the present invention is a group containing sulfur, and can be synthesized by a simple route, and mercury (Hg ^{2 ) +} ) The ion selectivity is high and the sensitivity can be increased (see FIG. 1 ).

Specifically, referring to FIG. 1 , the compound represented by Formula 1 may be prepared by first synthesizing TPE-Br, and then bonding the BTA functional group thereto.

In an exemplary embodiment, the compound represented by Formula 1 may react with a mercury ion to express a green fluorescence color.

In an exemplary embodiment, when the compound represented by Formula 1 reacts with mercury ions, the fluorescence intensity may increase at a wavelength of 450 to 700 nm, for example, 450 to 650 nm, 450 to 600 nm, or 500 to 600 nm It may increase at a wavelength of , preferably at a wavelength of 550 to 570 nm.

Therefore, mercury ions can be detected by observing changes in fluorescence color and intensity with the naked eye or with a fluorescence intensity meter (or fluorescence image measuring equipment such as confocal).

In an exemplary embodiment, the mercury ion detection sensor may further include an aqueous solution of tetrahydrofuran (THF), a HEPES buffer, and water (distilled water), so that the mercury ion detection sensor according to an embodiment of the present invention is in a solution state. , the sensor solution can be used in the form of a liquid for the sample, or it can be used as a paper strip-type sensor supported on paper, so that various applications are possible.

In an exemplary embodiment, the water ratio (vol%) in the mercury ion detection sensor may be 40 to 90 vol% based on the total volume of the mercury ion detection sensor, such as 50 vol% or more, 60 vol% or more, 65 It may be vol% or more or 70 vol% or more, 85 vol% or less, 80 vol% or less, or 75 vol% or less, and preferably 70 vol%.

In an exemplary embodiment, the pH of the mercury ion detection sensor may be adjusted to 6 to 8, and preferably, the pH may be 7. When the pH value is out of the above range, the ratio of fluorescence intensity is lowered, so that fluorescence expression may be insufficient.

In an exemplary embodiment, the mercury ion detection sensor can detect mercury ions at room temperature to 37° C., and thus can be widely used because mercury innoy can be detected even at room temperature.

In an exemplary embodiment, the mercury ion detection sensor can display the mercury ion detection result within 15 seconds, so there is an advantage that the mercury ion can be detected very quickly.

In an exemplary embodiment, the detection limit of the mercury ion detection sensor may be 0.9 nM, so there is an advantage in that a small amount of mercury ions can be detected.

In an exemplary embodiment, the mercury ion detection sensor may be for detecting mercury ions in a cell.

In an exemplary embodiment, since the mercury ion detection sensor may be a paper-strip type sensor in which the compound represented by Formula 1 is supported on paper, it is very easily carried and used in environmental samples or cell samples. Mercury ions can be detected.

In an exemplary embodiment, the mercury ion detection sensor may be an imaging sensor, and may be utilized as a cell image sensor by reacting with mercury ions in a cell to express a fluorescence image unlike cells that do not contain mercury ions.

As such, the mercury ion detection sensor according to an embodiment of the present invention can be used to detect the presence and/or concentration of mercury ions with high sensitivity and selectively, and thus can be widely used in various fields.

Mercury ion detection kit

In another embodiment of the present invention, a mercury ion detection kit comprising: a concentration measuring unit in a biological sample separated from the body of the human body, wherein the concentration measuring unit in the biological sample includes the above-described mercury ion detection sensor to provide.

The presence and/or concentration of mercury ions in a body fluid and/or blood separated from the human body can be measured by using the mercury ion detection kit according to an embodiment of the present invention.

Mercury ion detection Way

In another embodiment of the present invention, preparing the above-described mercury ion detection sensor; reacting the mercury ion detection sensor with an analysis sample; and detecting mercury ions by measuring a change in fluorescence of the mercury ion detection sensor after the reaction between the mercury ion detection sensor and the analyte sample.

In an exemplary embodiment, the reacting the sensor and the analyte sample may include: introducing the sensor and the analyte sample into a reaction chamber; adjusting the pH in the reaction chamber; and reacting the sensor and the analyte sample in the reaction chamber. may include.

In an exemplary embodiment, the pH in the reaction chamber may be adjusted to 6 to 8.

In an exemplary embodiment, the analysis sample may include at least one of a human body fluid and blood.

As described above, the detection of mercury ions using the mercury ion detection sensor can be performed through a very simple method, and the compound represented by Formula 1 has very high selectivity and sensitivity to mercury ions, so that the naked eye and/or fluorescence Mercury ions can be easily detected with only a sensitivity meter. Accordingly, the mercury ion detection sensor can be widely used in various fields.

Hereinafter, the present invention will be described in detail with reference to preferred embodiments so that those skilled in the art can easily carry out the present invention. However, the present invention may be embodied in various different forms, and is not limited to the embodiments described herein.

production example One: TPE -Br synthesis

4-bromobenzophenone (2.00 g, 0.66 mmol) and zinc powder (1.25 g, 19.2 mmol) were added to 250 mL of a round bottom flask equipped with a reflux condenser. The flakes were placed under vacuum and pumped three times under _{nitrogen (N 2 ) conditions.}

50 ml of anhydrous THF were added, the mixture was cooled to -78 °C, and TiCl ₄ (0.84 mL, 7.66 mmol) was added slowly by syringe. The mixture was stirred for 30 min while slowly warming to room temperature and refluxed for 24 h. The reaction was quenched with 10% aqueous K ₂ CO ₃ solution and then filtered. The organic layer was extracted 3 times with Dichloromethane, washed 2 times with brine and dried over Na ₂ SO ₄ .

The solvent was evaporated under reduced pressure, and the initial product was purified by silica gel column chromatography to give a white solid (2.70 g, 74%). ¹ H NMR analysis results are as follows.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.27-7.16 (m, 5H), 7.17-7.06 ppm (m, 5H), 7.03-6.95 (m, 4H), 6.89-6.82 (m, 4H). ¹³ C NMR (100 MHz, CDCl ₃ ) δ 143.3, 142.5, 142.2, 142.0, 140.5, 132.9, 131.6, 131.4, 130.7, 128.4, 127.5, 127.3, 126.7, 120.5, 120.6 ppm.

production example 2: TPE - BTA's synthesis

BTA (8.16 mg, 4.82 mM) and Potassium tert-butoxide (t-BuOK) (541 mg, 4.82 mM) were placed in a 250 ml two-neck round bottom flask equipped with a reflux condenser. The flask was evacuated under vacuum and pumped three times under nitrogen conditions.

Then, 100 mL of anhydrous Dimethylformamide (DMF) was added, and the mixture was stirred for 20 minutes and TPE-Br (1 g, 30 mmol) was added. The mixture was stirred at 110 °C for 24 h, and the reaction was quenched with 10% aqueous HCl solution.

Then the mixture was extracted three times with dichloromethane, the organic layers were combined and washed twice with brine. The solvent was evaporated under reduced pressure and the crude product was purified on a silica gel column using dichloromethane/petroleum ether mixture (1:10) as eluent.

A yellow solid TPE-BTA (2.16 g, 86%) was obtained. The synthesis process of the compound TPE-BTA is shown in FIG. 1 . ¹ H NMR results are as follows, and ^{analysis results of compounds such as 1} H NMR, ¹³ C NMR and HR-MS are shown in FIGS. 2 to 4A and 4B.

¹ H NMR: δ 7.47-7.40 ppm (8H, dddd, J = 7.7, 6.9, 1.5, 0.5 Hz), 7.40-7.38 (4H, dd, J = 4.9, 1.3 Hz), 7.20-7.13 (10H, ddd, J = 8.2, 1.9, 0.4 Hz), 7.01-6.93 (8 H, ddd, J = 8.2, 1.3, 0.5 Hz), 5.01 (8 H, s). ¹³ C NMR: δ 149.1, 143.8, 141.0, 139.2, 138.8, 136.3, 131.3, 128.9, 128.6, 126.4, 125.7, 125.3, 111.6, 38.6 ppm. HR-LC-Mass: 748.1 m/z.

Example 1: Spectroscopic measurement

A solution of TPE-BTA was prepared in 10 mM Hepes buffer at pH 7.4 as a stock _{solution of 2 vol% THF/H 2} O (f _{w = 70 %).} For UV-vis and fluorescence measurements, the final concentration of the probe solution was 0.1 μM TPE in 10 mM Hepes buffer at pH 7.4 with 2 vol% of the stock solution (f _{w = 70%) of THF/H 2 O.} -Measured as BTA.

5 mM of metal ions were prepared for selectivity experiments.

The probe solution of the present invention was completely mixed with 0.1 μM TPE-BTA, and the mixture of the probe and metal ion was mixed with 5 μM Hg ^{2 + ,} and the fluorescence test was performed again.

TPE-BTA showed an emission peak at 550 nm and corresponding excitation at 372 nm.

Example 2: cell culture, cytotoxicity test and confocal microscope image

_{HeLa cell lines were grown at 37° C. and 5% CO 2} in Dulbeco's Modified Eagle's Media (DMEM) supplemented with 10% Fetal Bovone Serum (FBS). Cells were grown in a 96-well plate in culture medium for 24 hours to perform a cytotoxicity test.

Cells were incubated with different concentrations of TPE-BTA (0-50 μM) for 24 hours. To determine the cytotoxicity of TPE-BTA, 0.5 mg/ml of 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) medium was added and the cells were cultured for about 4 hours. .

The resulting Formazan (formazan) crystals were dissolved in 0.1 ml DMSO, read with a SpectraMax M2 micro well plate reader, and cell imaging was performed with a fluorescence microscope with blue and green channels.

The cytotoxicity of TPE-BTA was measured according to the following formula (1).

Equation (1): Cell viability (%) = absorbance of sample / absorbance of control × 100

First, the results of the MTT analysis of the probe biotoxicity test (percentage of survival rate (number of measurements, repeated 5 times)) are shown in FIG. 13 . 13, TPE-BTA is (1) control, (2) 0.01, (3) 0.1, (4) 0.5, (5) 1.0, (6) 10.0, (7) 25 and (8) 50 μM showed low cytotoxicity. Thus, this probe can be applied to and applied to the superior in vivo Hg ^{2 +} fluorescence detection probe.

Furthermore, washed and incubated with ^{0, 1, 2 μM Hg 2} + ion for 30 minutes, and the cultured HeLa cells by incubation with 10 μM of the probe and, 2,4-Dibenzylidene-D-sorbitol (DBS) for 30 minutes .

Fluorescence images were observed with a Zeiss LSM 700 confocal microscope.

As a result of observation, the probe TPE-BTA of the present invention exhibited excellent sensitivity at the nanomolar level under physiological conditions with a short time response. Therefore, it is considered to be suitable for tracking the Hg ^{+ 2} in the cell lines.

Specifically, Hela cells stained in the absence of a probe Hg ^{+ 2,} as shown in Figure 14a is shown a primary green fluorescence. The signal intensity as the probe staining the cells with a 1 μM Hg ^{2 +} 2 minutes and also cultured 14b appears more strongly. Further, it becomes brighter fluorescence signal as shown in Figure 14c, increasing the amount of Hg ^{2 +} to 2 μM. Cell confocal images of our current probe showed the interest to be aware of changes in the concentrations of Hg ^{2 +.} This showed that the TPE-BTA can be used as bio-imaging of Hg ^{2 +.}

Example 3: TPE - BTA's photophysical properties and Hg ^{2 +} sensing behavior

At an early stage in the presence and absence of Hg ^{2 +} ions and irradiating the optical properties of the TPE-BTA, it is shown in Fig. The aggregated stimulated release (AIE) properties of TPE-BTA were studied in _{THF/H 2 O solution.}

Specifically, the fluorescence spectrum of TPE-BTA in 0.2 M Hepes buffer containing 2 vol% THF/H ₂ O (f _w = 70%, pH 7.4) was excitation at 372 nm with a stoke shift of 178 nm ( A) while showing the emission fluorescence spectrum (b) at 550 nm.

In addition, Hg ^{+ 2} showed a bright green to a strong green (c) increased significantly the probe emission peak intensity under UV lamp in accordance with the addition (5 μM). And, the image inserted in the upper right of FIG. 5 is a corresponding photographic image of ^{TPE-BTA and TPE-BTA-Hg 2 + under a UV lamp.}

This is due mainly to form a weak fluorescence TPE-BTA the Hg ²⁺ ion and, BTA moieties electron deficient group with the high-fluorescence TPE BTA-Hg, while the combined ^{2 +.}

Due to the soft flexible interaction specific fluorescent markers generated for Hg ^{2 +} detection, it is due to the soft metal that can interact with a sulfur-containing organic molecules.

The enhanced fluorescent strength and the formation of a strong green fluorescence TPE-BTA-Hg ^{2 +} complex belongs to the aggregation induced emission (Aggregation Induced Emission, AIE) mechanistic way the scope of the.

In addition, the fluorescence intensity of TPE-BTA at 550 nm according to the ratio of water in the solvent mixture was monitored and shown in FIGS. 6A-6C . A gradual increase in the ratio of water to TPE-BTA solution was monitored under a UV lamp.

As shown in Figures 6a and 6b, TPE-BTA shows little emission in pure THF solution with a fluorescence quantum yield of 0.5%. When the moisture is less than 40%, the fluorescence measurement curve has very low meaning for the horizontal coordinate, indicating that the emission is small in pure THF solution.

The fluorescence intensity at 550 nm increased significantly as the water ratio increased from 50 vol% to 70 vol%. In particular, it was confirmed that the fluorescence of TPE-BTA induces a maximum increase in fluorescence intensity with a quantum yield of 7.2% when the water fraction is 70 vol %, compared to the case where the water ratio is less than 70 vol %.

The cause of the increased fluorescence intensity was attributed to the formation of molecular aggregation with intramolecular free rotation, which was captured via the AIE kinetic pathway and was previously reported [NewJ.Chem., 2018, 42, 13836, Sensors & Actuators: B. Chemical 296 (2019) 126670, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 223 (2019) 117315].

Finally, the presence of AIE was investigated by monitoring the aggregates of TPE-BTA using dynamic light scattering (DLS) and shown in FIG. 7 .

From Figure 7, it has been found that the TPE-BTA form aggregates of an average size of 224nm to react with Hg ^{2 +.}

Example 4: Hg ^{2 +} High sensitivity detection for ions

The detectability of the TPE-BTA for different concentrations of Hg ^{2 +} initially were tested using the UV-Vis absorption spectroscopy, investigated the absorption spectra obtained for the TPE-BTA and TPE-BTA-Hg ^{2 +} complexes and Figure 8 shown in

Specifically, in order to investigate the sensitivity of TPE-BTA for the detection of Hg ^{2 +,} of different concentrations in 0.2 M Hepes buffer solution containing _{2 vol% THF / H 2 O} (fw = 70%, pH 7.4) Hg ^{After addition of 2+} , the fluorescence emission intensity was monitored for TPE-BTA.

Initially, 0.1 μM of TPE-BTA showed an emission peak at 550 nm during excitation at 372 nm (curve 'a' in FIG. 8). The emission intensity at 550 nm was increased with the addition of ^{0.5 μM Hg 2+ .}

Referring to Figure 8, the right side of the graph of the above, a result of further increasing the concentration of Hg ^{2 +,} fluorescence increased linearly (in a curve 'a''k') 0.9918 in correlation to the detection limit of 0.9 nM.

In addition, referring to the shape of the left arrow in FIG. 8 , as a result of specifying the fluorescence intensity with a paper-strip-based sensor, when the TPE-BTA paper strip sensor ^{is immersed in Hg 2 +} , the light green piece of paper changes to light green. can be confirmed (Hg ^{2 +} 1, 2, 3 μM).

Example 5: Binding form and experimental basis

^In order to confirm the basis for the binding ^{of Hg 2 +} and TPE-BTA by 1 H NMR titration, ^{the binding position of Hg 2 +} and TPE was confirmed using NMR spectroscopy, and is shown in FIG. 9 .

Specifically, FIG. 9 shows ¹ H NMR for TPE-BTA upon addition of 0(a), 1(b), 5(c), 10(d) and 15(e) equivalents of Hg ²⁺ _{in DMSO-d 6 .} spectrum is shown.

^{During the analysis of the 1} H NMR spectrum of the probe TPE-BTA, the protons of the methylene thiophene group, Ha, Hb, Hc and Hd, at 7.39, 7.19, 6.97 and 5.01 ppm can be significantly affected by Hg2+ ions.

Referring to FIG. 9 , according to ^{the addition of Hg 2 +} ions (a->e), protons corresponding to protons of the methylene thiophene group (δHa = 0.17 ppm , δHb = 0.10 ppm, δHc = 0.05 ppm and δHd = 0.04) to demonstrate that the ppm) can be confirmed a marked-up field moves (upfield shift) (a to e in Fig. 9), Hg ^{2 +} is coupled to a sulfur and nitrogen atom of the compound of TPE-BTA.

In other words, a similar shift of the methylene thiophene moiety during binding with the heavy metal atom was confirmed.

XPS analysis was additionally performed on TPE-BTA and TPE-BTA-Hg ^{2 +} , and the results are shown in FIGS. 10a-10f . From XPS wide scan spectrum of Figure 10a (TPE-BTA) and ^{10d (TPE-BTA-Hg 2+} ), TPE-BTA-Hg 2 + is confirmed that represents an additional Hg ^{2 +} signal in contrast to the TPE-BTA can

Sulfur (S) regions obtained by both TPE-BTA and TPE-BTA-Hg ^{2 +} are shown in Figs. 10b (S2p) and 10e (S2p). In TPE-BTA, S2p bound to a thiol group appeared at 163.2 eV, and unbound thiol was less abundant at 163.9 eV. Interestingly, referring to Figure ^{10e, TPE-BTA-Hg 2} + showed a new peaks at 161.7 eV for the S-Hg ^{2 +} binding. Similarly, in the nitrogen region (Figs. 10c and 10f), TPE exhibits 399.5 and 399.0 eV of NC and neutral NH, respectively. After combining with the Hg ^{+ 2,} Hg ^{2 +} N- showed a peak at 400.2 eV. The NMR and XPS results are shown clearly involved in the coordination with the Hg ^{+ 2.}

In addition, 2vol% of THF/H ₂ O control solution (0), 5 μM Hg ^{2 +} ions (1) and 5 mM concentration of Ag ⁺ , Na ⁺ , K ⁺ , Co ^{2 +} , Mg ^{2 +} , Cu ^{2 +} , Ca ^{+ 2, + 2} for Zn, Sr ^2+, Ba ^2+, Pb ^2+, Ni ^2+, Hg ^2+, Fe ^2+, Cd ^2+, and various metal ions, (3 to 18) such as Fe ³⁺ The selectivity and interference effects of 0.1 μM TPE-BTA in 0.2 M Hepes buffer solution containing 2 vol% THF/H ₂ O (f _{w = 70%, pH 7.4) were evaluated using fluorescence spectroscopy, Fig. 11a} -11c.

Hg ^{2 +} fluorescent response of the TPE-BTA-free showed a very weak fluorescence emission at 550 nm. When the addition of various metal ions than the Hg ^{+ 2} in the probe solution (5 mM, about 1000 times), the fluorescence intensity was not changed. However, ^{after adding Hg 2 +} (0.5 μM) to the probe solution, the fluorescence intensity at 550 nm dramatically increased as shown in FIG. 11A .

More specifically, referring to Figure 11a on the selectivity, Hg ^{2 +} fluorescent response of the TPE-BTA-free indicates a very weak fluorescence emission at 550 nm. It was confirmed that the fluorescence intensity did not change when various metal ions (5 mM) were added to the probe solution. On the other hand, when Hg ^{2 +} (0.5 μM) was added to the probe solution, the fluorescence intensity at 550 nm was dramatically increased as shown in FIG. 11A .

Referring to Figure 11b, the detection of the interference study (interference test) Hg ^{+ 2} can be found in the unaffected in the presence of other metal ions in excess of 1000 times. Corresponding photographic images of the selectivity and interference studies are inset in FIGS. 11A and 11B .

As Referring to Figure 11c, showing a selectivity and a bar chart representation of the interference result, with reference purpose, there under the different metal ions in the 1000-fold greater than without the TPE-BTA probe of the present invention, interference is also an optional start Hg ^{2 +} detection can be confirmed.

Example 6: Hg ^{2 +} For measuring the concentration of ions TPE - BTA's optimum conditions

It is shown in Figure 12a to assess the ability to detect and fluorescence behavior of the TPE-BTA probe as a function of pH for the Hg ^{+ 2} is detected. Referring to Figure 12a, emission intensity at 550 nm for the TPE-BTA probe in the range pH 3 to 13 showed no significant change in the absence of Hg ^{+ 2.} However, the Hg ^{+ 2} of 5 μM was added, at about pH 7 was achieved the maximum value of the emission intensity at 550 nm.

In addition, the exhibited concentration-dependent fluorescence kinetics of TPE-BTA with the Hg ^{+ 2} in Fig. 12b by measuring the fluorescence intensity at 550 nm as a function of time. Referring to Figure 12b, if there is no early Hg ^{2 +} 0.1 μM TPE-BTA showed a stable reaction between the 10th seconds 15 seconds (Fig. 12b: curve a). Similarly, the 1, 3, and 5 μM of after adding different concentrations, such as Hg ^{+ 2,} the emission intensity was increased and reached a steady state within 10 to 15 seconds (Fig. 12b: curve bd).

Thus, the incubation time of 15 seconds of the Hg ^{+ 2} and the probe was sufficient to detect the Hg ^{+ 2.} The short time response of the probe TPE-BTA to physiological conditions can be used for further applications in real samples and cell imaging.

Example 7: Hg ^{2 +} Validation of Ion Detection Sensors

Furthermore, river water (River water), it was using tap water (Tap water) and ponds can (Pond water) assess the applicability of the method of measuring the Hg ^{+ 2} in the environmental sample. Actual water quality samples were collected at the Korea Institute of Science and Technology, a nearby location.

The recovery rate had a value in the range of 99.2% to 99.8% (Table 1), additionally verified by the ICP-AES method in an actual water sample, and the Hg ^{2 +} measurement results are shown in Table 2, based on the T-test , it can be seen that there is good agreement between the ICP-AES method and the current decision method.

Validation of detection of Hg ^{2 +} ions in water samples (n=5) Samples Hg ^{2 +} added
(ng L ^-1 ) Hg ^{2 +} found
(ng L ^-1 ) R.S.D. Recovery (%) river water 10 9.94±0.09 0.91 99.4 20 19.90±0.06 0.30 99.5 tap water 10 9.98±0.05 0.50 99.8 20 19.92±0.11 0.55 99.6 Pond water 10 9.92±0.13 0.85 99.2 20 19.91±0.15 0.75 99.6

Hg ^{2 +} ion detection compared using the present invention detects and ICP-AES Hg ^{2 +} added
(ng L ^-1 ) Hg ^{2 +} found (ng L ^-1 ) R.S.D. T-test Fluorimetry method
(this work) ICP-AES
method 5 4.98±0.02 4.98±0.03 0.37 0.3153 8 7.99±0.03 7.98±0.01 0.26 0.6130

The embodiments of the present invention described above should not be construed as limiting the technical spirit of the present invention. The protection scope of the present invention is limited only by the matters described in the claims, and those skilled in the art can improve and change the technical idea of the present invention in various forms. Accordingly, such improvements and modifications will fall within the protection scope of the present invention as long as it is apparent to those of ordinary skill in the art.

Claims (16)

A mercury ion detection sensor comprising a compound represented by Formula 1 below:
[Formula 1]

According to claim 1,
The compound represented by Formula 1 reacts with mercury ions, and a green fluorescent color is expressed, a mercury ion detection sensor. According to claim 1,
When the compound represented by Formula 1 reacts with mercury ions, the fluorescence intensity increases at a wavelength of 450 to 700 nm, a mercury ion detection sensor. According to claim 1,
The mercury ion detection sensor, wherein the water ratio (vol%) in the mercury ion detection sensor is 40 to 90 vol% based on the total volume of the mercury ion detection sensor. According to claim 1,
A pH of the mercury ion detection sensor is adjusted to 6 to 8, a mercury ion detection sensor. According to claim 1,
The mercury ion detection sensor detects mercury ions at room temperature to 37°C. According to claim 1,
The mercury ion detection sensor displays a mercury ion detection result within 15 seconds. According to claim 1,
The detection limit of the mercury ion detection sensor is 0.9 nM, the mercury ion detection sensor. According to claim 1,
The mercury ion detection sensor is for detecting mercury ions in a cell. According to claim 1,
The mercury ion detection sensor is a paper-strip type sensor in which the compound represented by Formula 1 is supported on paper. According to claim 1,
wherein the mercury ion detection sensor is an imaging sensor. Containing a; concentration measuring unit in the biological sample separated from the body of the human body;
The mercury ion detection kit comprising the mercury ion detection sensor of any one of claims 1 to 11, wherein the concentration measuring unit in the biological sample. 12. A method comprising: preparing a mercury ion detection sensor according to any one of claims 1 to 11;
reacting the mercury ion detection sensor with an analysis sample; and
and detecting mercury ions by measuring a change in fluorescence of the mercury ion detection sensor after the reaction between the mercury ion detection sensor and the analyte sample. 14. The method of claim 13,
The step of reacting the sensor and the analyte sample,
introducing the sensor and the analyte sample into a reaction chamber;
adjusting the pH in the reaction chamber; and
reacting the sensor and the analyte sample in the reaction chamber; A method for detecting mercury ions, comprising: 15. The method of claim 14,
The pH in the reaction chamber is adjusted to 6 to 8, mercury ion detection method. 14. The method of claim 13,
The method for detecting mercury ions, wherein the analysis sample includes at least one of a human body fluid and blood.

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