Method for distinguishing and detecting organic mercury or inorganic mercury in actual sample by using eutectic solvent

Abstract

The invention discloses a method for distinguishing and detecting organic mercury or inorganic mercury in an actual sample by using a deep eutectic solvent, the distinguishing and detection of Hg < 2 + > and CH3Hg < + > are realized by using DES as a solvent and PBA as a fluorescent probe, and the method is simple in steps and obvious in phenomenon; the used DES is good in stability, not easy to volatilize and small in environmental pollution. The method disclosed by the invention has good specificity, can be used for carrying out qualitative analysis and quantitative detection on Hg < 2 + > or CH3Hg < + > in an actual sample after carrying out simple pretreatment on the actual sample, and provides a new thought for distinguishing and detecting Hg < 2 + > or CH3Hg < + > in a complex system.

Description

(1) Technical field

[0001] The present invention relates to a method for distinguishing and detecting organic mercury or inorganic mercury in an actual sample by utilizing a deep eutectic solvent. (2) Background technology

[0002] Heavy metal mercury (Hg) in the environment has neurotoxic, immunotoxic, nephrotoxic and pulmonary toxicity to organisms, and has serious harm to organisms and the environment. Mercury usually exists in three chemical states, namely: elemental mercury (Hg 0 ), inorganic mercury (Hg 2+ ) and organic mercury (mainly CH3Hg + ). Since organic mercury is more toxic than inorganic mercury and their limit standards are different, it is very important to distinguish the forms of mercury based on the test. 2+ and CH3Hg + The methods for detecting Hg include high performance liquid chromatography, gas chromatography, atomic absorption spectrometry, inductively coupled plasma mass spectrometry, etc. These methods are limited by the need for expensive and complex instruments, long data acquisition time, and the participation of trained personnel. Therefore, it is necessary to develop a rapid, simple, and sensitive detection method for the detection of Hg 2+ and CH3Hg + .

[0003] Compared with the above methods, colorimetry has attracted much attention due to its advantages such as short response time, simple operation, high selectivity and sensitivity, and better suitability for analyzing metal ions in complex matrices. 2+ and CH3Hg + The research on Hg 2+ and CH3Hg + Similar properties are difficult to distinguish, and it is necessary to use colorimetry to determine Hg 2+ and CH3Hg + There are still difficulties in distinguishing detection. 2+ and CH3Hg + It is very easy to undergo a replacement reaction with arylboronic acid. Chatterjee et al. synthesized the aggregation-induced emission (AIE) probe tetraphenylethylene (TPE)-boronic acid to achieve Hg 2+ and CH3Hg + However, Hg 2+ and CH3Hg +Differentiation detection is performed. The principle of AIE is that luminescence is primarily caused by restricted intramolecular motion, and solvents play a significant role in influencing the aggregation state of AIE molecules. Some solvents, such as good solvents, low-polarity solvents, and low-viscosity solvents, promote the dispersion of AIE molecules, allowing them to move freely, resulting in weak or even no luminescence in dilute solutions. Other solvents, such as poor solvents, high-polarity solvents, and high-viscosity solvents, induce the aggregation of AIE molecules, restricting their motion and thus achieving efficient luminescence.

[0004] Therefore, it is necessary to explore the 2+ and CH3Hg + Differentiate detection methods. (3) Summary of the invention

[0005] The present invention aims to provide a method for distinguishing and detecting organic mercury or inorganic mercury in actual samples by using a deep eutectic solvent, so as to achieve the detection of Hg 2+ and CH3Hg + Differentiation detection, while deep eutectic solvents (DES) have the advantages of simple synthesis, environmental friendliness, easy biodegradation, low energy consumption and low toxicity, and have great potential in many fields, solving the problem that existing methods cannot distinguish and detect Hg 2+ and CH3Hg + problem.

[0006] The technical solution adopted in the present invention is:

[0007] The present invention provides a method for distinguishing and detecting organic mercury or inorganic mercury in an actual sample using a deep eutectic solvent, the method comprising the following steps:

[0008] Adding 1-pyreneboronic acid (PBA) probe and deep eutectic solvent (DES) to the actual sample pretreatment solution, detecting the fluorescence intensity at 380 nm (preferably at an excitation wavelength of 335 nm and with excitation and emission slit widths of 5 nm), using deionized water instead of the actual sample pretreatment solution as a control, and if the fluorescence intensity increases, it indicates that the detection sample contains inorganic mercury, and if the fluorescence intensity decreases, it indicates that the detection sample contains organic mercury, and then obtaining the concentration of organic mercury or inorganic mercury in the actual sample based on a standard curve of the concentration of inorganic mercury and organic mercury standards and the change in fluorescence intensity;

[0009] The deep eutectic solvent is prepared by mixing a hydrogen bond acceptor (HBA) and a hydrogen bond donor (HBD) in different molar ratios.

[0010] Furthermore, the actual sample pretreatment liquid is prepared by squeezing the fruit and vegetable samples, ultrasonically mixing them (preferably using an ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd., China) for 5 minutes), and diluting the resulting supernatant with deionized water to within the fluorescence detection range; diluting the liquid sample with deionized water to within the fluorescence detection range; and soaking tea leaves in deionized water overnight, taking the supernatant, and diluting it with deionized water to within the fluorescence detection range.

[0011] Furthermore, the hydrogen bond acceptor is choline chloride (ChCl); the hydrogen bond donor is one of glycerol (GLY), ascorbic acid (AA), ethylene glycol (EG) or urea (U).

[0012] Furthermore, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:1-8.

[0013] Furthermore, the deep eutectic solvent is prepared by mixing two components, a hydrogen bond acceptor (HBA) and a hydrogen bond donor (HBD), and heating the mixture at 60-100° C. and continuously stirring for 1-2 hours until the mixture becomes a colorless, uniform, viscous liquid.

[0014] Furthermore, the deep eutectic solvent is made of choline chloride and ethylene glycol (ChCl:EG) in a molar ratio of 1:6, and has the following structural formula:

[0015]

[0016] Furthermore, the probe is added to a final concentration of 2.0-7.0 μM, preferably 4.0 μM.

[0017] Furthermore, the volume ratio of the actual sample pretreatment solution to the deep eutectic solvent is 1:9-9:1, preferably 1:9.

[0018] Furthermore, the actual samples include fruit samples (such as apples and grapes), vegetable samples (such as tomatoes and corn), liquid samples (including wine samples such as Moutai and Wuliangye) and tea samples (such as Maojian and Biluochun).

[0019] Furthermore, the standard curve of the concentration of the inorganic mercury standard and the change in fluorescence intensity was prepared as follows:

[0020] After PBA was dissolved by DES, Hg 2+ The aqueous solution was added to the centrifuge tube and the volume was fixed with DES so that the final concentration of PBA was 4.0×10 -6 M, Hg 2+ Add the final concentration of 0-60nM (preferably 0, 20nM, 25nM, 30nM, 35nM, 40nM, 45nM, 50nM, 55nM, 60nM), mix well; add the mixture to the cuvette of the fluorescence spectrophotometer, and record the concentration of different Hg 2+The fluorescence intensity of the concentration mixture at 380nm, of which the fluorescence intensity of 0nM is recorded as F0, and the other concentrations are recorded as F1, 2+ The standard curve was drawn with the concentration as the horizontal axis and the change value of the fluorescence intensity F1-F0 at 380 nm as the vertical axis.

[0021] Furthermore, the standard curve of the concentration of the organic mercury standard and the change value of fluorescence intensity was prepared as follows:

[0022] PBA was dissolved in DES and mixed with CH3Hg + The aqueous solution was added to the centrifuge tube and the volume was fixed with DES so that the final concentration of PBA was 4.0×10 -6 M, CH3Hg + Add the final concentration of 0-100nM (preferably 0, 20nM, 30nM, 50nM, 60nM, 70nM, 80nM, 90nM, 100nM), mix well; add the mixture to the cuvette of the fluorescence spectrophotometer, and record the concentration of different CH3Hg + The fluorescence intensity of the concentration mixture at 380nm, of which the fluorescence intensity of 0nM is recorded as F0, and the other concentrations are recorded as F1, with CH3Hg + The standard curve was drawn with the concentration as the horizontal axis and the change value of the fluorescence intensity F0-F1 at 380 nm as the vertical axis.

[0023] Furthermore, the actual sample pretreatment solution is prepared by one of the following methods:

[0024] (1) Chop fruit and vegetable samples (preferably apples, grapes, tomatoes, and corn kernels) and add them to a juicer to extract juice. The obtained fruit and vegetable juice is ultrasonically treated in an ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd., China) for 5 min. The supernatant obtained after centrifugation is diluted 10 times with deionized water to serve as the actual sample pretreatment solution.

[0025] (2) Liquid samples (preferably Moutai and Wuliangye) were simply diluted 10 times with deionized water and used as the actual sample pretreatment solution;

[0026] (3) Tea samples (preferably Maojian and Biluochun) were soaked in deionized water overnight, and the supernatant was taken and diluted 10 times with deionized water as the actual sample pretreatment solution.

[0027] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0028] (1) The method of the present invention has simple steps and obvious phenomena.

[0029] The present invention provides a method for detecting Hg by using DES as solvent and PBA as fluorescent probe. 2+ and CH3Hg+ Since the probe PBA itself is fluorescent, the presence of mercury ions in DES will cause the fluorescence of the probe to be enhanced, while the presence of methylmercury will quench the fluorescence of the probe. The present invention achieves the detection of Hg by a simple method using DES as solvent and PBA as fluorescent probe. 2+ and CH3Hg + The method is simple in steps and has obvious results; the DES used has good stability, is not easy to volatilize, and has little pollution to the environment.

[0030] (2) The method of the present invention has good specificity.

[0031] PBA for Hg in DES 2+ and CH3Hg + The linear ranges of the two methods are 0-60nM and 0-100nM, respectively, and the detection limits are 3.14nM and 10.01nM, respectively. In addition, the method of the present invention can selectively identify Hg from 17 common metal ions. 2+ or CH3Hg + , with good specificity and anti-interference properties.

[0032] (3) The method of the present invention can be used to detect Hg in actual samples. 2+ or CH3Hg + Qualitative analysis and quantitative detection.

[0033] The method of the present invention can detect Hg in the actual sample after simple pre-treatment. 2+ or CH3Hg + Conduct qualitative analysis and quantitative detection to determine Hg in complex systems 2+ or CH3Hg + This provides a new approach for distinguishing detection. (IV) Description of the accompanying drawings

[0034] Figure 1 The probes in Example 1DES1 are respectively 2+ and CH3Hg + Fluorescence spectrum after reaction.

[0035] Figure 2 The probes in Example 1DES3 are respectively 2+ and CH3Hg + Fluorescence spectrum after reaction.

[0036] Figure 3 The probes in different molar ratios of DES in Example 2 were respectively mixed with Hg 2+ and CH3Hg + Fluorescence change rate curve after the reaction.

[0037] Figure 4 For Example 3 different Hg 2+ Fluorescence spectra at different concentrations (0-60 nM).

[0038] Figure 5 For Example 3 different Hg 2+ The linear calibration curve was obtained at different concentrations (0-60 nM).

[0039] Figure 6 For Example 3 different Hg 2+ Fluorescence visualization photos taken at concentrations (0-5 μM) under 365 nm UV light.

[0040] Figure 7 For Example 3 different CH3Hg + Fluorescence spectra at different concentrations (0-100 nM).

[0041] Figure 8 For Example 3 different CH3Hg + The linear calibration curve was obtained at different concentrations (0-100 nM).

[0042] Figure 9 For Example 3 different CH3Hg + Fluorescence visualization photos taken at concentrations (0-15 μM) under 365 nm UV light.

[0043] Figure 10 This is a histogram of the fluorescence intensity of different metal ions at 380 nm in Example 4. (V) Specific implementation methods

[0044] The present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto:

[0045] The chemical reagents and solvents used in the examples were of analytical grade.

[0046] Fluorescence spectra and fluorescence intensity were measured using a fluorescence spectrophotometer (F-7000, HITACHA) with an excitation wavelength of 335 nm, an emission wavelength of 355-460 nm, and excitation and emission slit widths of 5 nm.

[0047] Example 1: Synthesis and performance testing of different types of DES

[0048] 1. Synthesis of different types of DES

[0049] Different types of DES are prepared by mixing choline chloride (ChCl) as a hydrogen bond acceptor (HBA) and one of glycerol (GLY), ascorbic acid (AA), ethylene glycol (EG) or urea (U) as a hydrogen bond partner (HBD) in the same molar ratio. The mixture is heated at 100°C and stirred for 2 hours until it becomes a colorless, uniform viscous liquid. The obtained DES is stored in a dry place for future use. The specific steps are as follows:

[0050] (1) 28.49 g (0.2 mol) of choline chloride was added to 110.51 g (1.2 mol) of glycerol, and the mixture was heated at 100°C and stirred for 2 hours until it became a colorless, uniform, viscous liquid, to obtain 139.00 g of a deep eutectic solvent, which was designated as DES1.

[0051] (2) 28.49 g (0.2 mol) of choline chloride was added to 211.36 g (1.2 mol) of ascorbic acid, and the mixture was heated at 100°C and stirred for 2 hours until it became a colorless, uniform, viscous liquid, to obtain 239.85 g of a deep eutectic solvent, which was designated as DES2.

[0052] (3) 28.49 g (0.2 mol) of choline chloride was added to 76.00 g (1.2 mol) of ethylene glycol, and the mixture was heated at 100°C and stirred for 2 hours until it became a colorless, uniform, viscous liquid, to obtain 104.49 g of a deep eutectic solvent, which was designated as DES3.

[0053] (4) 28.49 g (0.2 mol) of choline chloride was added to 72.07 g (1.2 mol) of urea, and the mixture was heated at 100 °C and stirred for 2 h until it became a colorless, homogeneous, viscous liquid, to obtain 100.56 g of a deep eutectic solvent, which was designated as DES4.

[0054] 2. DES viscosity test

[0055] The viscosity of the four DES prepared above was tested using a rotational viscometer (DV-2pro, Brookfield). The results are shown in Table 1. The results show that ChCl:AA and ChCl:U are too thick to be accurately measured during the test and tend to solidify after cooling, making them unsuitable as solvents for the experiment.

[0056] Table 1. Viscosity of four DES

[0057]

[0058] 3. DES for Hg detection using PBA probe 2+ and CH3Hg + Impact

[0059] (1) Reagent preparation

[0060] PBA solution: PBA was accurately weighed and dissolved in DES1 or DES3 prepared in step 1 to prepare 1.0×10 -3 M and diluted with DES1 or DES3 to 1.0 × 10 -5 M for later use.

[0061] Hg 2+ Sample aqueous solution: prepare the mercuric chloride sample with deionized water to 1.0×10 -3 M and diluted with deionized water to 1.0 × 10 -5 M for later use.

[0062] CH3Hg + Sample aqueous solution: commercially available 1000 μg / mL (i.e. 3.98×10 -3 M) Methylmercuric chloride standard was diluted with deionized water to 1.0 × 10 -5 M for later use.

[0063] (2)DES1

[0064] Add 400 μL of 1.0 × 10 -5 M PBA solution and 400 μL of 1.0 × 10 -5 M of Hg 2+ Aqueous solution or CH3Hg + The aqueous solution was added to the centrifuge tube, and the total volume was adjusted to 1 mL with DES1 prepared in step 1 so that PBA, Hg 2+ or CH3Hg + The final concentration was 4.0×10 -6 M, mix well. Add the mixture to the cuvette of the fluorescence spectrophotometer and record the changes in the fluorescence spectrum. Under the same conditions, replace Hg with deionized water. 2+ Aqueous solution or CH3Hg + The aqueous solution was used as a control. Figure 1 As shown, in DES1 (ChCl: GLY), with the Hg 2+ or CH3Hg + There was no significant change in the fluorescence intensity of the probe.

[0065] (2)DES3

[0066] Add 400 μL of 1.0 × 10 -5 M PBA solution and 400 μL of 1.0 × 10 -5 M of Hg 2+ Aqueous solution or CH3Hg +The aqueous solution was added to the centrifuge tube, and the total volume was adjusted to 1 mL with DES3 prepared in step 1 so that PBA, Hg 2+ or CH3Hg + The final concentration was 4.0×10 -6 M, mix well. Add the mixture to the cuvette of the fluorescence spectrophotometer and record the changes in the fluorescence spectrum. Under the same conditions, replace Hg with deionized water. 2+ Aqueous solution or CH3Hg + The aqueous solution was used as a control. Figure 2 As shown, in DES3 (ChCl:EG), Hg 2+ The addition of CH3Hg + The reaction of the probe quenches the fluorescence, thus achieving the detection of Hg 2+ and CH3Hg + Therefore, ChCl:EG(DES3) was selected as the solvent for subsequent studies.

[0067] Example 2: Synthesis of DES3 with different molar ratios and detection of Hg by PBA probe 2+ and CH3Hg + Impact

[0068] 1. Synthesis of DES3 with different molar ratios

[0069] Different molar ratios of DES3 are prepared by mixing the hydrogen bond acceptor choline chloride and the hydrogen bond donor ethylene glycol in different molar ratios. The mixture is heated at 100°C and stirred for 2 hours until it becomes a colorless, uniform, viscous liquid. The obtained DES is stored in a dry place for future use. The specific steps are as follows:

[0070] (1)DES3-1

[0071] 28.49 g (0.2 mol) of choline chloride was added to 12.41 g (0.2 mol) of ethylene glycol, heated to 100° C. and stirred for 2 hours until it became a colorless, uniform viscous liquid, to obtain 40.90 g of a deep eutectic solvent, which was designated as DES3-1.

[0072] (2)DES3-2

[0073] 28.49 g (0.2 mol) of choline chloride was added to 24.83 g (0.4 mol) of ethylene glycol, heated to 100° C. and stirred for 2 hours until it became a colorless, uniform viscous liquid, to obtain 53.32 g of a deep eutectic solvent, which was designated as DES3-2.

[0074] (3)DES3-3

[0075] 28.49 g (0.2 mol) of choline chloride was added to 37.24 g (0.6 mol) of ethylene glycol, heated to 100° C. and stirred for 2 hours until it became a colorless, uniform viscous liquid, to obtain 65.73 g of a deep eutectic solvent, which was designated as DES3-3.

[0076] (4)DES3-4

[0077] 28.49 g (0.2 mol) of choline chloride was added to 49.66 g (0.8 mol) of ethylene glycol, heated to 100° C. and stirred for 2 hours until it became a colorless, uniform viscous liquid, to obtain 78.15 g of a deep eutectic solvent, which was designated as DES3-4.

[0078] (5)DES3-5

[0079] 28.49 g (0.2 mol) of choline chloride was added to 62.07 g (1.0 mol) of ethylene glycol, heated to 100° C. and stirred for 2 hours until it became a colorless, uniform viscous liquid, to obtain 90.56 g of a deep eutectic solvent, which was designated as DES3-5.

[0080] (6)DES3-6

[0081] 28.49 g (0.2 mol) of choline chloride was added to 74.48 g (1.2 mol) of ethylene glycol, heated to 100° C. and stirred for 2 hours until it became a colorless, uniform viscous liquid, to obtain 102.97 g of a deep eutectic solvent, which was designated as DES3-6.

[0082] (7)DES3-7

[0083] 28.49 g (0.2 mol) of choline chloride was added to 86.90 g (1.4 mol) of ethylene glycol, heated to 100° C. and stirred for 2 hours until it became a colorless, uniform viscous liquid, to obtain 115.39 g of a deep eutectic solvent, which was designated as DES3-7.

[0084] (8)DES3-8

[0085] 28.49 g (0.2 mol) of choline chloride was added to 99.31 g (1.6 mol) of ethylene glycol, heated to 100° C. and stirred for 2 hours until it became a colorless, uniform viscous liquid, to obtain 127.80 g of a deep eutectic solvent, which was designated as DES3-8.

[0086] 2. Detection of Hg by PBA probe using DES at different molar ratios 2+ and CH3Hg + Impact

[0087] (1) Preparation of reagents

[0088] PBA solution: PBA was accurately weighed and dissolved in the eight DES prepared in step 1 to prepare 1.0×10 -3 M and diluted with the corresponding DES to 1.0×10 -5 M for later use.

[0089] Hg 2+ Sample aqueous solution: prepare 1.0×10 -3 M and diluted with deionized water to 1.0 × 10 -5 M for later use.

[0090] CH3Hg + Sample aqueous solution: commercially available 1000 μg / mL (i.e. 3.98×10 -3 M) Methylmercuric chloride standard was diluted with deionized water to 1.0 × 10 -5 M for later use.

[0091] Add 400 μL of 1.0 × 10 -5 M PBA solution and 400 μL of 1.0 × 10 -5 M of Hg 2+ Aqueous solution or CH3Hg + The aqueous solution was added to the centrifuge tube, and the total volume was adjusted to 1 mL using the 8 DES prepared in step 2, so that PBA, Hg 2+ or CH3Hg + The final concentration was 4.0×10 -6 M, mix well. Add the mixture into the cuvette of the fluorescence spectrophotometer and record the fluorescence intensity of the mixture containing different molar ratios of DES at 380nm as F1; under the same conditions, 2+ or CH3Hg + The deionized water was used as the control, the fluorescence intensity was recorded as F0, the molar ratio of ChCl:EG was used as the horizontal axis, and the fluorescence intensity change rate ((F1-F0) / F0) was used as the vertical axis to draw a curve. The results are shown in Figure 2. Figure 3 The results show that when the molar ratio of ChCl and EG is 1:6, CH3Hg is added to PBA. + The fluorescence intensity change rate reaches the maximum value when the molar ratio of ChCl and EG is 1:7 and Hg 2+ After that, the fluorescence intensity change rate of PBA reached the maximum value. After comprehensive consideration, ChCl:EG (1:6) (DES3-6) was finally selected as the optimal solvent.

[0092] Example 3: DES detection of Hg using probe PBA 2+ and CH3Hg + Impact

[0093] The DES3-6 prepared in Example 2 was used as solvent and PBA was used as probe to detect Hg 2+ and CH3Hg + The specific steps of differentiation detection include:

[0094] (1) Probe PBA for different concentrations of Hg 2+ Fluorescence spectrum detection

[0095] PBA solution: PBA was accurately weighed and dissolved in DES3-6 to prepare 1.0×10 -3 M and diluted to 1.0 × 10 -5 M for later use.

[0096] Hg 2+ Sample aqueous solution: prepare the mercuric chloride sample with deionized water to 1.0×10 -3 M and diluted with deionized water to 1.0 × 10 -5 M for later use.

[0097] Add 400 μL of 1.0 × 10 -5 M PBA solution and different volumes of Hg 2+ The sample aqueous solution was added to the centrifuge tube and the total volume was adjusted to 1 mL with DES3-6 so that Hg 2+ The final concentrations of the mixtures were 0, 20nM, 25nM, 30nM, 35nM, 40nM, 45nM, 50nM, 55nM, and 60nM, and the final concentration of PBA was 4μM. The mixtures were added to the cuvette of the fluorescence spectrophotometer and the concentrations of Hg in the mixtures were recorded. 2+ The fluorescence spectrum of the concentration mixture is as follows Figure 4 As shown, the results show that with the increase of Hg 2+ As the concentration increases in the range of 0-60 nM, the fluorescence intensity at 380 nm gradually increases, so 380 nm is selected as the characteristic peak.

[0098] (2) Probe PBA for different concentrations of Hg 2+ Fluorescence intensity detection

[0099] Same as step (1), 400 μL of 1.0×10 -5 M PBA solution and different volumes of Hg 2+ The sample aqueous solution was added to the centrifuge tube and the total volume was adjusted to 1 mL with DES3-6 so that Hg 2+The final concentrations of the mixtures were 0, 20nM, 25nM, 30nM, 35nM, 40nM, 45nM, 50nM, 55nM, and 60nM, and the final concentration of PBA was 4μM. The mixtures were added to the cuvette of the fluorescence spectrophotometer and the concentrations of Hg in the mixtures were recorded. 2+ The fluorescence intensity of the concentration mixture at 380nm (the fluorescence intensity of 0nM is recorded as F0, and the other concentrations are recorded as F1) is expressed as Hg 2+ The concentration is the horizontal axis, and the change value of fluorescence intensity at 380nm (F1-F0) is the vertical axis to draw a standard curve. The results are shown in Figure 5 As shown in the figure, the results show that the fluorescence intensity change value of the emission peak at 380nm is consistent with that of Hg 2+ The concentration showed a linear relationship in the range of 0-60 nM, and the linear correlation equation was fitted as ΔF = 29.66x–285.49 (R 2 =0.992), with a detection limit of 3.14 nM.

[0100] (3) Probe PBA for different concentrations of Hg 2+ Fluorescence visualization detection

[0101] Same as step (1), 400 μL of 1.0×10 -5 M PBA solution and different volumes of Hg 2+ The sample aqueous solution was added to the centrifuge tube and the total volume was adjusted to 1 mL with DES3-6 so that Hg 2+ The final concentrations of the added products were 0, 0.1, 0.5, 1, 2, and 5 μM, and the final concentration of PBA was 4 μM. The products were mixed evenly. The fluorescence visualization photos taken under 365 nm UV light are shown in the figure. Figure 6 As shown, the blue fluorescence of PBA can be clearly observed by naked eyes as Hg 2+ Increased concentration enhances.

[0102] (4) Probe PBA to different concentrations of CH3Hg + Fluorescence spectrum detection

[0103] PBA solution: PBA was accurately weighed and dissolved in DES3-6 prepared in Example 2 to prepare 1.0×10 -3 M and diluted with DES3-6 to 1.0 × 10 -5 M for later use.

[0104] CH3Hg + Sample aqueous solution: commercially available 1000 μg / mL (i.e. 3.98×10 -3 M) methylmercuric chloride standards were diluted to 1.0 × 10 -4 M and 1.0×10 -5 M for later use.

[0105] Add 400 μL of 1.0 × 10 -5 M PBA solution and different volumes of 1.0 × 10 -5 M CH3Hg + The sample aqueous solution was added to the centrifuge tube and the total volume was adjusted to 1 mL with DES3-6 so that CH3Hg + The final concentrations of the samples were 0, 20nM, 30nM, 50nM, 60nM, 70nM, 80nM, 90nM, and 100nM, and the final concentration of PBA was 4μM. The mixture was added to the cuvette of the fluorescence spectrophotometer and the concentrations of CH3Hg in the sample were recorded. + The fluorescence spectrum of the concentration mixture is as follows Figure 7 As shown, the results show that with the increase of CH3Hg + As the concentration increases in the range of 0-100 nM, the fluorescence intensity at 380 nm gradually decreases, with 380 nm as the characteristic peak.

[0106] (5) Probe PBA for different concentrations of CH3Hg + Fluorescence intensity detection

[0107] Same as step (4), 400 μL of 1.0×10 -5 M PBA solution and different volumes of CH3Hg + The sample aqueous solution was added to the centrifuge tube and the total volume was adjusted to 1 mL with DES3-6 so that CH3Hg + The final concentrations of the samples were 0, 20nM, 30nM, 50nM, 60nM, 70nM, 80nM, 90nM, and 100nM, and the final concentration of PBA was 4μM. The mixture was added to the cuvette of the fluorescence spectrophotometer and the concentrations of CH3Hg in the sample were recorded. + The fluorescence intensity of the concentration mixture at 380nm (the fluorescence intensity of 0nM is recorded as F0, and the other concentrations are recorded as F1) is expressed as CH3Hg + The standard curve is drawn with the concentration as the horizontal axis and the fluorescence intensity change value (F0-F1) as the vertical axis. The results are as follows Figure 8 As shown in the figure, the fluorescence intensity change value of the 380nm emission peak is consistent with that of CH3Hg + The concentration showed a linear relationship in the range of 0-100 nM, and the linear correlation equation was fitted as ΔF = 9.31x-50.76 (R 2 =0.995), and the detection limit was 10.01 nM.

[0108] (6) Probe PBA to different concentrations of CH3Hg + Fluorescence visualization detection

[0109] Same as step (4), 400 μL of 1.0×10 -5 M PBA solution and different volumes of 1.0 × 10 -5 M or 1.0×10 -4 M CH3Hg + The sample aqueous solution was added to the centrifuge tube and the total volume was adjusted to 1 mL with DES3-6 so that CH3Hg + The final concentrations of the samples were 0, 0.1, 1, 5, 10, and 15 μM (10 and 15 μM were added at 1.0 × 10 -4 M CH3Hg + The sample solution was prepared by adding PBA to a final concentration of 4 μM and mixing well. The fluorescence visualization photos taken under 365 nm UV light are shown in the figure. Figure 9 As shown, the blue fluorescence of PBA can be clearly observed by naked eyes along with the + weakened with increasing concentration.

[0110] Example 4: DES detection of Hg using probe PBA 2+ and CH3Hg + Selective research

[0111] PBA solution: PBA was accurately weighed and dissolved in DES3-6 prepared in Example 2 to prepare 1.0×10 -3 M and diluted with DES3-6 to 1.0 × 10 -5 M for later use.

[0112] Metal ion aqueous solution: HgCl2, CH3HgCl, AlCl3, MgCl2, CaCl2, CdCl2, CuCl2, FeCl2, FeCl3, KCl, NaCl, NiCl2, PbCl2, ZnCl2, AgCl, BaCl2, MnCl2, MnCl4, CrCl3 samples were prepared with deionized water to 1.0×10 -3 M and diluted with deionized water to 1.0 × 10 -5 M for later use.

[0113] Add 400 μL of 1.0 × 10 -5 M PBA solution and 400 μL of 1.0 × 10 -5 Add the M metal ion aqueous solution to the centrifuge tube, and then adjust the total volume to 1 mL with DES3-6 so that the final concentration of PBA and metal ions is 4.0×10 -6 M, after mixing evenly. Add the mixture into the cuvette of the fluorescence spectrophotometer and measure the fluorescence intensity at 380nm. Figure 10 As shown, the PBA probe is effective for detecting Hg in DES3-6 solvent. 2+ and CH3Hg+ Has excellent selectivity.

[0114] Example 5: Hg in actual samples 2+ and CH3Hg + Detection

[0115] Eight real samples including apple, grape, tomato, corn, Moutai, Wuliangye, Maojian and Biluochun were selected, and the recovery rate was determined by spike recovery experiment.

[0116] 1. Sample pretreatment

[0117] 1.0 g each of apple, grape, tomato, and corn kernels were chopped and added to a juicer to extract the juice. The obtained fruit and vegetable juice was ultrasonically treated in an ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd., China) for 5 min. The supernatant obtained after centrifugation was diluted 10 times its volume with deionized water and used as the sample solution for later use.

[0118] Moutai and Wuliangye were simply diluted 10 times their volume with deionized water and used as sample test solutions.

[0119] After soaking Maojian and Biluochun in deionized water overnight, the supernatant was taken and diluted 10 times the volume with deionized water as the sample solution for later use.

[0120] 2. Detection

[0121] PBA solution: PBA was accurately weighed and dissolved in DES3-6 prepared in Example 2 to prepare 1.0×10 -3 M and diluted with DES3-6 to 1.0 × 10 -5 M for later use.

[0122] Hg 2+ Sample aqueous solution: prepare 1.0×10 -3 M and diluted with deionized water to 1.0 × 10 -6 M for later use.

[0123] CH3Hg + Sample aqueous solution: commercially available 1000 μg / mL (3.98×10 -3 M) Methylmercuric chloride standard was diluted with deionized water to 1.0 × 10 -6 M for later use.

[0124] In 20 μL or 50 μL of the above Hg 2+ Sample aqueous solution or CH3Hg + The sample aqueous solution and 80 μL or 50 μL of each sample solution were added to the centrifuge tube (sample aqueous solution or CH3Hg +The sample aqueous solution and each sample solution were added to a total volume of 100 μL, and then 400 μL of 1.0×10 -5 M PBA solution, and finally DES3-6 to 1mL, so that Hg 2+ and CH3Hg + The final concentrations were 20nM and 50nM respectively, and the final concentration of PBA was 4μM. The mixture was added to the cuvette of the fluorescence spectrophotometer and the fluorescence intensity at 380nm was measured (recorded as F1). 2+ or CH3Hg + The fluorescence intensity of deionized water as the test solution is recorded as F0. Figure 5 or Figure 8 The standard curve of Hg in the sample solution was obtained 2+ or CH3Hg + The recovery rate was calculated based on the concentration of Hg. The results are shown in Table 2 and Table 3. 2+ and CH3Hg + The recoveries were 82.96–108.14% and 84.59–110.30%, respectively, and the corresponding relative standard deviations (RSDs) were less than 0.97% and 0.91%, respectively, demonstrating that DES can be used for the selective detection of Hg in real samples. 2+ and CH3Hg + .

[0125] Table 2. Hg in actual samples 2+ The spike recovery results

[0126]

[0127] Table 3. CH3Hg in actual samples + The spike recovery results

[0128]

Claims

1. A method for distinguishing and detecting organic mercury or inorganic mercury in actual samples using a deep eutectic solvent, characterized in that: The method comprises the following steps: Add 1-pyreneboronic acid probe and deep eutectic solvent to the actual sample pretreatment solution, detect the fluorescence intensity at 380 nm, and use deionized water instead of the actual sample pretreatment solution as a control. If the fluorescence intensity increases, it means that the detection sample contains inorganic mercury, and if the fluorescence intensity decreases, it means that the detection sample contains organic mercury. Then, the concentration of organic mercury or inorganic mercury in the actual sample is obtained based on the standard curve of the concentration of inorganic mercury and organic mercury standards and the change in fluorescence intensity; The deep eutectic solvent is prepared by mixing a hydrogen bond acceptor and a hydrogen bond donor in different molar ratios.

2. The method according to claim 1, wherein The actual sample pretreatment solution is prepared by squeezing the juice of fruit or vegetable samples, ultrasonically mixing them, and diluting the resulting supernatant with deionized water to within the fluorescence detection range; diluting liquid samples with deionized water to within the fluorescence detection range; and diluting tea samples with deionized water overnight and then taking the supernatant with deionized water to within the fluorescence detection range.

3. The method according to claim 1, wherein The hydrogen bond acceptor is choline chloride; the hydrogen bond donor is one of glycerol, ascorbic acid, ethylene glycol or urea.

4. The method according to claim 1 or 3, wherein: The molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:1-8.

5. The method according to claim 4, wherein The deep eutectic solvent is prepared by mixing two components, a hydrogen bond acceptor and a hydrogen bond donor. The mixture is heated at 60-100° C. and continuously stirred for 1-2 hours until it becomes a colorless, uniform, viscous liquid.

6. The method according to claim 5, wherein The deep eutectic solvent is prepared from choline chloride and ethylene glycol in a molar ratio of 1:

6.

7. The method according to claim 1, wherein The probe is added at a final concentration of 2.0-7.0 μM; the volume ratio of the actual sample pretreatment solution to the low eutectic solvent is 1:9-9:

1.

8. The method according to claim 1, wherein The standard curve of inorganic mercury standard concentration and fluorescence intensity change was prepared as follows: The probe was dissolved in a low eutectic solvent and added to a centrifuge tube with an inorganic mercury aqueous solution. The volume was fixed with a low eutectic solvent so that the final concentration of the probe was 4.0×10 -6 M, inorganic mercury was added to a final concentration of 0-60 nM and mixed evenly; the mixture was added to the cuvette of a fluorescence spectrophotometer, and the fluorescence intensity at 380 nm was recorded respectively, where the fluorescence intensity of 0 nM was recorded as F0, and the remaining concentrations were recorded as F1. A standard curve was drawn with the inorganic mercury concentration as the horizontal axis and the fluorescence intensity change value F1-F0 at 380 nm as the vertical axis.

9. The method according to claim 1, wherein The standard curve of the concentration of the organic mercury standard and the change in fluorescence intensity was prepared as follows: The probe was dissolved in a low eutectic solvent and added to a centrifuge tube with an organic mercury aqueous solution. The volume was fixed with a low eutectic solvent so that the final concentration of the probe was 4.0×10 -6 M, organic mercury is added to a final concentration of 0-100 nM and mixed evenly; the mixture is added to the cuvette of a fluorescence spectrophotometer, and the fluorescence intensity at 380 nm is recorded respectively, where the fluorescence intensity of 0 nM is recorded as F0, and the remaining concentrations are recorded as F1. A standard curve is drawn with the organic mercury concentration as the horizontal axis and the fluorescence intensity change value F0-F1 at 380 nm as the vertical axis.

10. The method according to claim 1, wherein The actual sample pretreatment solution is prepared by one of the following methods: (1) Chop the fruit or vegetable samples into small pieces and add them to a juicer to extract the juice. The obtained fruit and vegetable juice is ultrasonically treated in an ultrasonic cleaner for 5 minutes. The supernatant obtained after centrifugation is diluted 10 times with deionized water to serve as the actual sample pretreatment solution. (2) Liquid samples were simply diluted 10 times with deionized water and used as the actual sample pretreatment solution; (3) After soaking the tea samples in deionized water overnight, the supernatant was taken and diluted 10 times with deionized water to serve as the actual sample pretreatment solution.

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