ESIPT-based "off-on" fluorescent probe NS and its synthesis method and application

Abstract

The present invention relates to the field of fluorescent probe analysis, and provides an "off-on" fluorescent probe N-S based on ESIPT and its synthesis method and application. A fluorescent probe specifically detecting Hg is designed and synthesized using 3-benzothiazolenaphthalene-2-ol as a fluorophore and phenylthiochloroformate as a recognition group. 2+ Fluorescent probes. 1 H‑NMR and 13 C-NMR confirmed its structure. The probe was able to rapidly detect Hg in DMF / H2O (1:1, v / v) solution. 2+ , for Hg 2+ It has good selectivity and high sensitivity, and is not interfered by other metal ions. Based on the principle of ESIPT, Hg 2+ The detection process produces a large Stokes shift (236 nm). The fluorescent probe synthesis method of the present invention is simple, has stable performance, fast response speed, low detection limit and high sensitivity, and can be used for qualitative and quantitative detection of mercury ions in actual water samples.

Description

Technical Field

[0001] The invention belongs to the field of fluorescent probe analysis, and relates to an "off-on" type fluorescent probe NS based on ESIPT and a synthesis method and application thereof. Background Art

[0002] With the development of industrial activities, heavy metals accumulate in the human living environment through the atmosphere, water, soil and other channels. Because of its characteristics of non-biodegradability, accumulation and irreversibility, when it accumulates to a certain extent in the human body, it causes chronic poisoning and endangers human health. Large intake of mercury can damage multiple physiological systems or organs such as the brain, kidneys, central nervous system, respiratory, immune, endocrine system, etc., and can cause mercury poisoning, hepatitis, uremia, nephritis, dementia and other diseases. Therefore, it is necessary to establish a rapid and accurate detection method for Hg 2+ method becomes particularly important.

[0003] Traditional detection of Hg 2+ There are many methods such as atomic absorption spectrophotometry (AAS) and inductively coupled plasma mass spectrometry (ICP-MS), which have the disadvantages of expensive instruments, high operating costs, portability, cumbersome procedures and long time consumption. In recent years, fluorescent probe molecules have been rapidly developed in the field of heavy metal detection due to their low cost, ability to achieve rapid real-time detection, visualization, high sensitivity and high accuracy.

[0004] Fluorescent probes are mainly composed of three parts: fluorophore, recognition group and bridge. 2+ Fluorophores include coumarin, benzothiazole, naphthylimide, rhodamine, etc. Among them, benzothiazoles and their derivatives have rigid conjugated structures, good optical properties, high quantum yields and other characteristics, and are often used to make fluorescent materials, luminescent materials, etc. In particular, 2-hydroxyphenyl-benzothiazole (HBT) is a typical luminophore with ESIPT characteristics, which exhibits excited state intramolecular proton transfer (ESIPT) through keto-enol tautomers, resulting in a larger stokes shift, thereby increasing the fluorescence intensity of the fluorescent dye. The present invention increases its conjugated system through structural modification, and connects phenylthiochloroformate as a recognition group to obtain a fluorescent probe NS for Hg 2+ Testing has potential implications. Summary of the invention

[0005] The object of the present invention is to provide an "off-on" type fluorescent probe NS based on ESIPT.

[0006] Another object of the present invention is to provide a method for synthesizing an "off-on" type fluorescent probe NS based on ESIPT.

[0007] Another object of the present invention is to provide an "off-on" type fluorescent probe NS based on ESIPT for detecting Hg 2+ Application in.

[0008] In order to achieve the purpose of the present invention, the present invention adopts the following technical solutions and steps:

[0009] The "off-on" fluorescent probe NS described in the present invention has a Chinese name: O-(3-(benzo[d]thiazol-2-yl)naphtha len-2-yl)oxy-phenyl thiocarboxylate, an English name: O-(3-(benzo[d]thiazol-2-yl)naphtha len-2-yl)O-phenyl carbonothioate, and a structural formula of:

[0010]

[0011] The synthetic route of the oxygen-(3-(benzothiazol-2-yl)naphthalene-2-yl)oxy-phenylthioformate of the present invention is as follows:

[0012]

[0013] The synthesis method of the ESIPT-based "off-on" fluorescent probe NS of the present invention comprises the following steps:

[0014] (1) 3-Hydroxy-2-naphthoic acid and 2-aminobenzenethiol are dissolved in a toluene solution, and when the solution is refluxed to 90°C, a toluene solution of PBr3 is slowly added dropwise, and the solution is heated under reflux for 2 hours under nitrogen protection. When the reaction is complete, the solution is cooled to 60°C, a methanol solution is added, and the solution is cooled to room temperature and filtered, and washed with a methanol solution to obtain a yellow solid, which is compound A;

[0015] (2) Phenyl thiochloroformate was dissolved in anhydrous dichloromethane, and a solution of compound A (3-benzothiazonaphthalene-2-ol) and DIPEA (N,N-diisopropylethylamine) in anhydrous dichloromethane was slowly added dropwise under stirring at room temperature. After the reaction was completed as monitored by TLC, the solvent was dried using a rotary evaporator, and the crude product was separated and purified by silica gel column chromatography, and recrystallized from a n-hexane solution to obtain a white solid, which was the fluorescent probe NS.

[0016] In step (1) of the present invention, the molar ratio of 3-hydroxy-2-naphthoic acid, 2-aminothiophenol and PBr3 is 1:1.1:1.

[0017] In step (2) of the present invention, the molar ratio of phenyl chlorothioformate, 3-benzothiazolylnaphthalene-2-ol and DIPEA is 1.5:1:1.5.

[0018] The eluent used in the silica gel column chromatography in step (2) of the present invention is n-hexane and ethyl acetate in a volume ratio of 10:1.

[0019] The fluorescent probe NS of the present invention or the fluorescent probe NS synthesized by the synthesis method is effective in identifying Hg in the detection environment. 2+ application.

[0020] The application of the present invention includes the detection of Hg by fluorescent probe NS in the actual environment of mineral water, tap water and lake water. 2+ .

[0021] The present invention is to detect Hg 2+ Fluorescence spectroscopy was used to quickly detect Hg in a DMF / H2O (1:1, v / v) solution system. 2+ .

[0022] The present invention is to detect Hg 2+ The specific steps are as follows: take 40 ml of water sample, treat with ultrasound for 10 minutes, filter into a 100 ml volumetric flask with a 0.22 μm microporous filter membrane, add 10 ml of Hg 2+ (50μM and 100μM) stock solutions were added to DMF to make up to the mark to prepare the test solution; in a 10ml volumetric flask, 1ml of the test solution was added, and then 1ml of the 10μM stock solution of the probe NS in DMF / H2O (1:1, v / v) was added, and the volume was made up to the mark with DMF / H2O (1:1, v / v), and the fluorescence emission spectrum was measured by fluorescence spectrometer.

[0023] The working principle of the fluorescent probe of the present invention is as follows: 3-benzothiazolenaphthalene-2-ol is used as a fluorophore, and when phenylthiochloroformate is connected as a recognition group, the hydroxyl group of 3-benzothiazolenaphthalene-2-ol is protected, resulting in the disappearance of the ESIPT process; when mercuric ions are added, the mercuric ions promote hydrolysis, remove the thiocarbonate part and generate free 3-benzothiazolenaphthalene-2-ol, and restore the ESIPT process. In other words, the selective detection of mercuric ions is achieved through the "protection-deprotection" process of phenylthiochloroformate.

[0024] Beneficial effects of the present invention:

[0025] 1. The fluorescent probe NS of the present invention has a simple synthesis route, high yield, and is easy to obtain crystals. It can detect Hg in actual water environments such as mineral water, tap water, and lake water. 2+ Qualitative and quantitative detection.

[0026] 2. Rapid detection of Hg by the fluorescent probe NS of the present invention in DMF / H2O (1:1, v / v) solution 2+ , for Hg 2+It has good selectivity and high sensitivity, and can quickly identify Hg within 15 minutes. 2+ And it is not interfered by other metal ions. In the fluorescence emission spectrum, the fluorescent probe recognizes mercury ions without interference from other metal ions; the fluorescence intensity of the fluorescent probe recognizes mercury ions gradually increases and tends to equilibrium within 15 minutes, with fast response speed and stable performance; with 335nm as the excitation wavelength and a strong emission peak at 567nm, the fluorescent probe detects Hg 2+ The concentration range was linear (R 2 =0.9859), and the detection limit was as low as 0.06 μM, indicating that the probe has extremely high sensitivity in detecting mercury ions.

[0027] 3. The fluorescent probe NS of the present invention is based on the ESIPT principle, produces a large Stokes shift, and realizes the "off-on" detection of mercury ions, and has potential application value in the fields of environment, food, biochemistry, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the H NMR spectrum of probe NS.

[0029] Figure 2 This is the carbon NMR spectrum of probe NS.

[0030] Figure 3 The water content in DMF / H2O is probed by N-S+Hg 2+ (1 μM) Effect of fluorescence intensity.

[0031] Figure 4 Probe N-S+Hg in DMF / H2O (1:1, v / v) 2+ Time kinetics of the spectral intensity of the (1 μM) system, and the inset is the time kinetic scatter plot.

[0032] Figure 5 The fluorescence emission spectra of the probe NS (1 μM) in DMF / H2O (1:1, v / v) solution interacting with equal amounts of different metal ions.

[0033] Figure 6 Recognition of Hg by probe NS 2+ Anti-interference experiment.

[0034] Figure 7 The probe NS (1 μM, DMF / H2O, 1 / 1, v / v) and Hg 2+ Fluorescence spectra titration diagram of the effect, and the inset is the nonlinear fitting curve.

[0035] Figure 8The probe NS (1 μM, DMF / H2O, 1 / 1, v / v) and Hg 2+ Linear fitting curve of the effect. DETAILED DESCRIPTION

[0036] Example 1: Preparation method of fluorescent probe NS

[0037] (1) In a 100 ml round-bottom flask, add 3-hydroxy-2-naphthoic acid (1.88 g, 10 mmol), 2-aminothiophenol (1.38 g, 11 mmol), and 20 ml of toluene. Reflux and stir until it is completely dissolved, then lower the temperature to 90°C, and add 5 ml of toluene solution of PBr3 (2.7 g, 10 mmol) dropwise with a constant pressure dropping funnel. After the addition is complete, continue to reflux for 2 h, cool to 60°C, add 40 ml of methanol, cool to room temperature, filter, and wash the precipitate with methanol solution. The precipitate is intermediate A with a yield of 72%. 1 H NMR (400MHz, DMSO-d6) δ11.55 (s, 1H), 8.90 (s, 1H), 8.19 (d, J = 7.6Hz, 1H), 8.13 (d, J = 8.0Hz, 1H), 8.05 (d, J = 8.1Hz, 1H), 7 .79 (d, J=8.2Hz, 1H), 7.58 (td, J=8.3, 7.7, 1.2Hz, 1H), 7.55-7.45 (m, 2H), 7.43 (s, 1H), 7.37 (td, J=7.4, 6.9, 1.0Hz, 1H).

[0038] (2) In a 25 ml round-bottom flask, add phenyl thiochloroformate (374 mg, 2.17 mmol) dissolved in 10 ml of anhydrous dichloromethane. While stirring, slowly add 3-benzothiazole naphthalene-2-ol (400 mg, 1.44 mmol) and N,N-diisopropylethylamine DIPEA (0.4 ml, 2.17 mmol), stir at room temperature, monitor the reaction progress by TLC, spin dry after the reaction is complete, separate and purify by column chromatography (n-hexane / ethyl acetate = 10:1, v / v), and recrystallize from the n-hexane solution to obtain a white solid, which can be used to obtain probe NS, with a yield of 72%. 1 HNMR (400MHz, CDCl3) δ8.81 (s, 1H), 8.19 (d, J=7.9Hz, 1H), 8.02 (dd, J=12.9, 7.8Hz, 2H), 7.90 (d, J=7. 6Hz, 1H), 7.81 (s, 1H), 7.64-7.53 (m, 3H), 7.49-7.38 (m, 3H), 7.30 (t, J=7.4Hz, 1H), 7.23-7.16 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 194.33, 162.80, 153.76, 153.45, 147.83, 135.68, 134.30, 131.73, 131.40, 129.69, 128.90, 128.35, 127.65, 127.19, 126.87, 126.54, 125.56, 125.37, 123.53, 121.82, 121.56, 121.35. H NMR spectrum Figure 1 As shown, the carbon NMR spectrum is Figure 2 shown.

[0039] The obtained fluorescent probe NS is: oxygen-(3-(benzothiazol-2-yl)naphthalene-2-yl)oxy-phenylthiocarboxylate, and its structural formula is:

[0040]

[0041] Example 2: Fluorescent probe NS for Hg in mineral water samples 2+ Detection

[0042] Take 40 ml of mineral water sample, treat it with ultrasound for 10 minutes, filter it into a 100 ml volumetric flask with a 0.22 μm microporous filter membrane, and add 10 ml of Hg 2+ (50μM) stock solution, add DMF to the mark, and prepare the test solution. In a 10ml volumetric flask, add 1ml of the test solution, then add 1ml of the 10μM stock solution of the probe NS in DMF / H2O (1:1, v / v), and make up to the mark with DMF / H2O (1:1, v / v), and measure the fluorescence emission spectrum using a fluorescence spectrometer.

[0043] Example 3: Fluorescent probe NS for Hg in mineral water samples 2+ Detection

[0044] Take 40 ml of mineral water sample, treat it with ultrasound for 10 minutes, filter it into a 100 ml volumetric flask with a 0.22 μm microporous filter membrane, and add 10 ml of Hg 2+ (100μM) stock solution, add DMF to the mark, and prepare the test solution. In a 10ml volumetric flask, add 1ml of the test solution, then add 1ml of the 10μM stock solution of the probe NS in DMF / H2O (1:1, v / v), and make up to the mark with DMF / H2O (1:1, v / v), and measure the fluorescence emission spectrum using a fluorescence spectrometer.

[0045] Example 4: Fluorescent probe NS for Hg in tap water samples 2+ Detection

[0046] Take 40 ml of tap water sample, treat it with ultrasound for 10 minutes, filter it into a 100 ml volumetric flask with a 0.22 μm microporous filter membrane, and add 10 ml of Hg 2+ (50μM) stock solution, add DMF to the mark, and prepare the test solution. In a 10ml volumetric flask, add 1ml of the test solution, then add 1ml of the 10μM stock solution of the probe NS in DMF / H2O (1:1, v / v), and make up to the mark with DMF / H2O (1:1, v / v), and measure the fluorescence emission spectrum using a fluorescence spectrometer.

[0047] Example 5: Fluorescent probe NS for Hg in tap water samples 2+ Detection

[0048] Take 40 ml of tap water sample, treat it with ultrasound for 10 minutes, filter it into a 100 ml volumetric flask with a 0.22 μm microporous filter membrane, and add 10 ml of Hg 2+ (100μM) stock solution, add DMF to the mark, and prepare the test solution. In a 10ml volumetric flask, add 1ml of the test solution, then add 1ml of the 10μM stock solution of the probe NS in DMF / H2O (1:1, v / v), and make up to the mark with DMF / H2O (1:1, v / v), and measure the fluorescence emission spectrum using a fluorescence spectrometer.

[0049] Example 6: Fluorescent probe NS for Hg in lake water samples 2+ Detection

[0050] Take 40 ml of lake water sample, treat it with ultrasound for 10 minutes, filter it into a 100 ml volumetric flask with a 0.22 μm microporous filter membrane, and add 10 ml of Hg 2+ (50μM) stock solution, add DMF to the mark, and prepare the test solution. In a 10ml volumetric flask, add 1ml of the test solution, then add 1ml of the 10μM stock solution of the probe NS in DMF / H2O (1:1, v / v), and make up to the mark with DMF / H2O (1:1, v / v), and measure the fluorescence emission spectrum using a fluorescence spectrometer.

[0051] Example 7: Fluorescent probe NS for Hg in lake water samples 2+ Detection

[0052] Take 40 ml of lake water sample, treat it with ultrasound for 10 minutes, filter it into a 100 ml volumetric flask with a 0.22 μm microporous filter membrane, and add 10 ml of Hg 2+(100μM) stock solution, add DMF to the mark, and prepare the test solution. In a 10ml volumetric flask, add 1ml of the test solution, then add 1ml of the 10μM stock solution of the probe NS in DMF / H2O (1:1, v / v), and make up to the mark with DMF / H2O (1:1, v / v), and measure the fluorescence emission spectrum using a fluorescence spectrometer.

[0053] Test Example 1: The test conditions of the fluorescent probe of the present invention are as follows:

[0054] (1) Selection of test solvent medium

[0055] DMF solution and water in different proportions were added to a series of cuvettes to maintain the total volume at 3 ml. Then the probe NS (10 μL, 1 μM) stock solution and Hg 2+ (10 μL, 10 μM) stock solution. The determination was performed with 335 nm as the excitation wavelength. Figure 3 As shown in the figure, as the water content increases to 50%, the fluorescence intensity reaches the maximum value. As the water content continues to increase, the fluorescence gradually decreases until it is quenched in pure water solution. Therefore, DMF / H2O (1:1, v / v) was selected as the test condition.

[0056] (2) Binding kinetics test

[0057] In a cuvette, add 3 ml of DMF / H2O (1:1, v / v) solution, probe NS stock solution (10 μL), Hg 2+ The stock solution (10 μL) was shaken and then immediately subjected to fluorescence measurement, once every 30 seconds. Figure 4 As shown in the figure, after adding mercury ions, the fluorescence intensity gradually increased within 15 minutes until it reached stability. Therefore, the reaction time is 15 minutes. In subsequent experiments, the prepared solution system can be left for 15 minutes before measurement. It also shows that the probe NS is effective for detecting Hg 2+ Has higher sensitivity.

[0058] Test Example 2: The selectivity test of the fluorescent probe of the present invention for metal ions, the steps are as follows:

[0059] In a series of cuvettes, 3 ml of DMF / H2O (1:1, v / v) solution and probe NS (10 μL, 1 μM) were added, and then equal amounts of different metal ions such as Li + , Na + , K + , Cs + ,Mg 2+ , Ca 2+ , Sr. 2+ , Ba 2+ ,Cr2+ , Fe 3+ , Fe 2+ ,Co 2+ , Ni 2 + , Cu 2+ , Zn 2+ , Cd 2+ , Al 3+ , Pb 2+ , Hg 2+ After shaking, let it stand for 15 minutes and perform fluorescence measurement with 335nm as the excitation wavelength. Figure 5 As shown in the figure, under the same conditions, when mercury ions were added, there was a strong fluorescence emission peak at 567nm. However, the probe NS had no selectivity for the other 18 metal ions. The results showed that the probe NS- 2+ Has higher selectivity.

[0060] Experimental Example 3: Recognition of Hg by the fluorescent probe of the present invention 2+ The anti-interference test steps are as follows:

[0061] In a series of quartz cuvettes, 3 ml of DMF / H2O (1:1, v / v) solution, probe NS (10 μL, 1 μM), Hg 2 + (10 μL, 10 μM), and then add 5 times the amount of different metal ions such as Li + , Na + , K + , Cs + ,Mg 2+ , Ca 2+ , Sr. 2+ , Ba 2+ ,Cr 2+ , Fe 3+ , Fe 2+ ,Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ , Cd 2+ , Al 3+ , Pb 2+ After 15 minutes of standing, the fluorescence was measured with 335 nm as the excitation wavelength. The fluorescence intensity of different metal ions at 567 nm was plotted as a bar graph. Figure 6 As shown in Figure 2, in the presence of other metal ions, the probe NS detects Hg 2+ The fluorescence intensity of the probe NS was almost unaffected. The results showed that the coexistence of other metal ions did not affect the fluorescence intensity of Hg 2+ Detection.

[0062] Experimental Example 4: Detection of Hg by the fluorescent probe of the present invention 2+ The standard curve is as follows:

[0063] Different concentrations of Hg were added to the probe NS (1 μM, DMF / H2O, 1 / 1, v / v). 2+ , and the fluorescence spectrum titration experiment was carried out with 335nm as the excitation wavelength. Figure 7 As shown, with the increase of Hg 2+ As the concentration increases, the fluorescence intensity increases until the mercury ion concentration reaches 10 μM and gradually reaches equilibrium. The fluorescence intensity of the probe NS at 567 nm is plotted against the concentration of mercury ions, as shown in Figure 2. Figure 8 As shown in Figure 2, the probe detected mercury ion concentration in the range of 0-6 μM and showed a good linear relationship (R 2 =0.9859). The detection limit LOD is 0.06 μM. The results show that the probe has a very high sensitivity in detecting mercury ions and can be applied to Hg 2+ Detection.

[0064] Experimental Example 5: The fluorescence probe of the present invention is used to detect Hg in actual water samples 2+ The detection steps are as follows:

[0065] 40 ml of each of three water samples, namely mineral water, tap water and lake water, were selected and ultrasonically treated for 10 minutes. The samples were filtered into 100 ml volumetric flasks using a 0.22 μm microporous filter membrane and 10 ml of Hg 2+ (50μM and 100μM) stock solutions were added to DMF to make up to the mark to prepare the test solution. In a 10ml volumetric flask, 1ml of the test solution was added, and then 1ml of the 10μM stock solution of the probe NS in DMF / H2O (1:1, v / v) was added, and the volume was made up to the mark with DMF / H2O (1:1, v / v), and the fluorescence emission spectrum was measured by fluorescence spectrometer. As shown in Table 1, Hg was detected in different actual water samples. 2+ The recovery rate was over 95%, and the RSD% was in the range of 0.4%-3.4%, indicating that the detection accuracy and instrument precision were high. It also showed that the probe could detect Hg in a complex environment. 2+ Qualitative detection.

[0066] Table 1 Recovery values ​​determined when detecting Hg2+ in different actual water samples

[0067]

[0068] Although the present invention has been described in detail above by means of general description, specific implementation methods and experiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. An "off-on" fluorescent probe NS based on ESIPT, characterized in that: The fluorescent probe NS is oxygen-(3-(benzothiazol-2-yl)naphthalene-2-yl)oxy-phenylthiocarboxylate, and its structural formula is:

2. A method for synthesizing an "off-on" fluorescent probe NS based on ESIPT as claimed in claim 1, characterized in that: The steps include: (1) 3-Hydroxy-2-naphthoic acid and 2-aminobenzenethiol are dissolved in a toluene solution, and when the solution is refluxed to 90°C, a toluene solution of PBr3 is slowly added dropwise, and the solution is heated under reflux for 2 hours under nitrogen protection. When the reaction is complete, the solution is cooled to 60°C, a methanol solution is added, and the solution is cooled to room temperature and filtered, and washed with a methanol solution to obtain a yellow solid, which is compound A; (2) dissolving phenyl thiochloroformate in anhydrous dichloromethane, slowly adding a solution of compound A and DIPEA in anhydrous dichloromethane dropwise under stirring at room temperature, monitoring the reaction to be complete by TLC, drying the solvent using a rotary evaporator, separating and purifying the crude product by silica gel column chromatography, and recrystallizing from a n-hexane solution to obtain a white solid, which is the fluorescent probe NS; The compound A is 3-benzothiazolylnaphthalene-2-ol; the DIPEA is N,N-diisopropylethylamine; The synthetic route of the fluorescent probe NS is as follows:

3. The method for synthesizing the ESIPT-based "off-on" fluorescent probe NS according to claim 2, characterized in that: In the step (1), the molar ratio of 3-hydroxy-2-naphthoic acid, 2-aminothiophenol and PBr3 is 1:1.1:

1.

4. The method for synthesizing the ESIPT-based "off-on" fluorescent probe NS according to claim 2, characterized in that: In the step (2), the molar ratio of phenyl chlorothioformate, 3-benzothiazolylnaphthalene-2-ol and DIPEA is 1.5:1:1.

5.

5. The method for synthesizing the ESIPT-based "off-on" fluorescent probe NS according to claim 2, characterized in that: The eluent used for silica gel column chromatography in step (2) is n-hexane and ethyl acetate in a volume ratio of 10:

1.

6. The fluorescent probe NS according to claim 1 or the fluorescent probe NS synthesized by the synthesis method according to any one of claims 2 to 5 is effective in identifying Hg in the detection environment. 2+ application.

7. The use according to claim 6, characterized in that: The detection is the fluorescent probe NS to detect Hg in the actual environment of mineral water, tap water and lake water 2+ .

8. The use according to claim 7, characterized in that: The detection of Hg 2+ Rapid detection of Hg by fluorescence spectroscopy in a DMF / H2O, v / v 1:1 solution system 2+ .

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