Preparation method of ratio type fluorescent probe for detecting mercury ions

Abstract

The invention belongs to the technical field of ion detection, and particularly relates to a preparation method of a ratio type fluorescent probe for detecting collected ions, which comprises the following steps: dissolving ethylenediamine and di-tert-butyl dicarbonate in perchloroform to obtain a product 1; dissolving the product 1 in absolute ethyl alcohol, and adding 4-Australio-1, 8-dianhydride to obtain a product 2; dissolving the product 2 in thousand dimethyl sulfoxide, and adding N-isothiazolyl phthaloyl to obtain a product 3; dissolving the product 3 in dichloromethane, and adding phenyl thiochloroformate to obtain the fluorescent probe NAP. The preparation method comprises the following steps: dissolving a fluorescent probe NAP in DMSO to prepare a mother liquor A, and dissolving methoxy chitosan CS-MeO in water to prepare a mother liquor B; and mixing the mother liquor A and the mother liquor B to prepare a Nano-NAP solution. The ratiometric fluorescent probe is used for detecting collected ions, has the advantages of high selectivity, small interference, low detection limit, good pH stability and the like, is high in use value, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of ion detection, and particularly relates to a preparation method of a ratio fluorescent probe for detecting mercury ions. Background Art

[0002] Mercury ion (Hg 2+ ) as a harmful heavy metal ion is extremely easy to migrate and diffuse into the ecological cycle, causing serious harm to biological systems and human health. At present, due to human activities such as coal combustion, gold mining, cement production, etc., more and more Hg 2+ is released into the environment, and the environmental pollution problem caused by Hg 2+ is becoming more and more serious. In addition, the continuous accumulation of Hg 2+ in the human body will have an adverse impact on the normal physiological functions of important organs such as the kidney, brain, heart, etc., and even lead to gene mutations. Therefore, it is crucial to develop efficient analytical methods to detect and identify Hg 2+ in biological and environmental systems. So far, the methods for detecting Hg 2+ mainly include mercury analyzers, atomic absorption spectrometry, inductively coupled plasma mass spectrometry, ion chromatography, etc. However, these detection technologies have some obvious disadvantages, such as relatively cumbersome sample preparation, generally inconvenient portability of instruments, non-intuitive signal transmission methods, etc., which greatly limit their practicality. In order to overcome these defects, currently more and more people are focusing on developing Hg 2+ fluorescent sensors with good availability, high sensitivity, convenient operation, in-situ detection and application in biology. Most of the currently reported probes for detecting Hg 2+ only rely on the change in single emission fluorescence intensity as the response signal, which is extremely vulnerable to interference from the surrounding environment, background fluorescence, probe concentration, excitation power and instrument performance, resulting in inaccurate analysis results or false positives. Considering that mercury ions are highly harmful and toxic to the environment and human health, developing a dual-functional ratio fluorescent probe that can specifically detect Hg 2+ in a real environment and can effectively adsorb Hg 2+ has important scientific significance and application prospects. Summary of the Invention

[0003] Aiming at the problems existing in the process of the existing Hg 2+ fluorescent probe detection, the present invention provides a preparation method of a ratio fluorescent probe for detecting mercury ions. This method has simple steps, fast response speed, good selectivity, high sensitivity, low detection limit and adsorption performance.

[0004] To solve the above technical problems, the present invention is realized as follows:

[0005] A preparation method of a ratiometric fluorescent probe for detecting mercury ions, comprising the following steps:

[0006] (1) Dissolve ethylenediamine in chloroform, stir, and then dropwise add a chloroform solution of di-tert-butyl dicarbonate. After the reaction is completed, extract with dichloromethane, dry over anhydrous sodium sulfate, and then dry under vacuum to obtain product 1;

[0007] (2) Take the product 1 obtained in step (1) and dissolve it in absolute ethanol, add 4-bromo-1,8-naphthalic anhydride, heat and stir. After the reaction is completed, cool to room temperature, filter by suction, wash with ice ethanol, and dry under vacuum to obtain product 2;

[0008] (3) Dissolve the product 2 obtained in step (2), N-hydroxyphthalimide and potassium carbonate in dimethyl sulfoxide, heat and stir. After the reaction is completed, cool to room temperature, add water and adjust the pH, filter by suction, and dry under vacuum to obtain product 3;

[0009] (4) Dissolve the product 3 obtained in step (3) in dry dichloromethane, add phenyl chloroformate and triethylamine, and stir the obtained mixture at room temperature. After the reaction is completed, remove dichloromethane, purify by column chromatography, and dry under vacuum to obtain the fluorescent probe NAP;

[0010] (5) Dissolve the fluorescent probe NAP obtained in step (4) in DMSO to prepare stock solution A; dissolve methoxy chitosan CS-MeO in water to prepare stock solution B; mix stock solution A and stock solution B to prepare a Nano-NAP solution; after ultrasonic treatment and dialysis, the target product, a ratiometric fluorescent probe for detecting mercury ions, is obtained.

[0011] Furthermore, in step (5), the preparation steps of the methoxy chitosan CS-MeO are as follows:

[0012] a. Dissolve chitosan and 4-bromo-1,8-naphthalic anhydride in dimethyl sulfoxide, heat and stir under nitrogen protection. After the reaction is completed, cool to room temperature, filter by suction, and wash successively with dimethyl sulfoxide, water, and ethanol, and then dry under vacuum to obtain product 4;

[0013] b. Dissolve the product 4 obtained in step a and potassium carbonate in methanol, heat and stir under nitrogen protection. After the reaction is completed, cool to room temperature, filter by suction, and wash successively with dimethyl sulfoxide, water, and ethanol, and then dry under vacuum to obtain methoxy chitosan CS-MeO.

[0014] Furthermore, in step a, the mass ratio of 4-bromo-1,8-naphthalic anhydride to chitosan is 1:9; in step b, the mass ratio of potassium carbonate to product 4 is 1:5.

[0015] Furthermore, in step (4), the structural formula of the fluorescent probe NAP is as follows:

[0016]

[0017] Further, in the step b, the structural formula of methoxy chitosan CS-MeO is as follows:

[0018]

[0019] Further, in the step (1), the molar ratio of di-tert-butyl dicarbonate to ethylenediamine is 1:10.

[0020] Further, in the step (2), the molar ratio of product 1 to 4-bromo-1,8-naphthalic anhydride is 1:2.

[0021] Further, in the step (3), the molar ratio of product 2 to N-hydroxyphthalimide is 1:1.2.

[0022] Further, in the step (4), the molar ratio of product 3 to phenyl chlorothionoformate is 1:1.2.

[0023] Further, in the step (5), the volume ratio of the fluorescent probe NAP in mother liquor A to methoxy chitosan CS-MeO in mother liquor B is 1:2.

[0024] The preparation method of the above ratio-type fluorescent probe for detecting mercury ions is as follows in specific reaction processes:

[0025]

[0026]

[0027] The present invention prepares the required fluorescent probe Nano-NAP by reacting ethylenediamine, di-tert-butyl dicarbonate, 4-bromo-1,8-naphthalic anhydride, N-hydroxyphthalimide, phenyl chlorothionoformate, triethylamine, chitosan and potassium carbonate. When the fluorescent probe is under the condition of DMF:H2O (2:8) in the presence of mercury ions, significant fluorescence changes will occur with the increase of mercury ion concentration; the fluorescent probe has high selectivity, high sensitivity and excellent adsorption ability for the detection of mercury ions. By using the ratio fluorescent probe to detect the fluorescence intensity ratio of the mercury ion solution at 550 nm and 460 nm, the concentration of the mercury ions to be measured can be obtained according to the linear equation between the fluorescence intensity ratio and the mercury ion concentration; compared with the conventional detection methods, the present invention uses the ratio fluorescent probe to detect mercury ions, which has the advantages of high selectivity, small interference, low detection limit and good pH stability, and has high use value and broad application prospects. Compared with some existing detection technologies, the chemical fluorescent probe in the present invention has less cost investment, a simple synthesis route, convenient post-treatment, and can directly achieve specific recognition of mercury ions in the DMF:H2O (2:8) system and also has an adsorption effect, especially has potential application value in environmental water samples and biological systems. Description of the Drawings

[0028] The present invention will be described in detail below through specific examples. These examples are provided to be able to more thoroughly understand the present invention and to be able to fully convey the scope of the present invention to those skilled in the art. As mentioned throughout the specification and claims, "comprising" or "including" is an open-ended term and is interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment for implementing the present invention, but the description is for the purpose of the general principle of the specification and is not intended to limit the scope of the present invention. The protection scope of the present invention shall be determined by the scope defined by the appended claims. Unless otherwise specified, various reagents and materials used in the present invention can be purchased from the market.

[0029] Figure 1 1H NMR spectrum of the prepared fluorescent probe NAP;

[0030] Figure 2 13C NMR spectrum of the prepared fluorescent probe NAP;

[0031] Figure 3 Fluorescence emission spectrum of the fluorescent probe Nano-NAP at different mercury ion concentrations;

[0032] Figure 4 Fitting curve of the fluorescence intensity corresponding to the change of the fluorescent probe Nano-NAP with the mercury ion concentration and the function graph corresponding to the curve;

[0033] Figure 5It is the time scan chart of the response of the fluorescent probe Nano-NAP to mercury ions;

[0034] Figure 6 It is the selectivity of the fluorescent probe Nano-NAP to other analytes.

[0035] The concentration of the added ions is 1.5×10 -5 mol / L, and the excitation wavelength is 380 nm. (Note: The concentration of this probe is 10 μM; Hg 2+ The response time is 100 seconds.) Detailed implementation methods

[0036] Example 1

[0037] (1) Preparation of Product 1

[0038] Add ethylenediamine (100 mmol) and di-tert-butyl dicarbonate (10 mmol) to a round-bottom flask containing 30 mL of chloroform, and stir at room temperature for 24 h. After the reaction is completed, add 30 mL of secondary water, extract three times with dichloromethane, and dry under vacuum to synthesize Product 1.

[0039] (2) Preparation of Product 2

[0040] Dissolve Product 1 (5 mmol) in 10 mL of absolute ethanol, and add 4-bromo-1,8-naphthalic anhydride (10 mmol). Heat to 75 °C and stir for 2 hours under nitrogen protection. Detect with a TLC plate. After the reaction is completed, cool to room temperature, filter by suction, wash with ice ethanol, and dry under vacuum to synthesize Product 2.

[0041] (3) Preparation of Product 3

[0042] Dissolve Product 2 (4 mmol) and N-hydroxyphthalimide (4.8 mmol) in 30 mL of dimethyl sulfoxide, add potassium carbonate, and heat to 100 °C and stir for 5 hours. Detect with a TLC plate. After cooling to room temperature, add secondary water, adjust the pH to 3, filter by suction to obtain the required product, and dry under vacuum to synthesize Product 3.

[0043] (4) Synthesis of the fluorescent probe using Product 3 and phenyl chloroformate

[0044] Dissolve Product 3 (1.5 mmol) in 10 mL of dichloromethane, and add triethylamine and phenyl chloroformate (1.8 mmol). Stir at room temperature for 2 h. After the reaction is completed, remove dichloromethane, and purify by column chromatography. The mobile phase for column chromatography separation is dichloromethane:methanol with a volume ratio of 240:1. Remove the solvent from the purified product by rotary evaporation, and dry under vacuum to obtain the fluorescent probe NAP.

[0045] (5) Preparation of the target product

[0046] Chitosan and 4-bromo-1,8-naphthalic anhydride were dissolved in dimethyl sulfoxide, and the temperature was raised to 100 °C under nitrogen protection and stirred for 5 hours; after the reaction was completed, it was cooled to room temperature, filtered by suction, washed successively with dimethyl sulfoxide, water, and ethanol, and dried under vacuum to obtain product 4; product 4 was dissolved in methanol, potassium carbonate was added, and the temperature was raised to 65 °C under nitrogen protection and stirred for 3 hours. After the reaction was completed, it was cooled to room temperature, filtered by suction, washed successively with dimethyl sulfoxide, water, and ethanol, and dried under vacuum to obtain methoxy chitosan CS-MeO;

[0047] Weigh NAP (1 mmol) and dissolve it in DMSO to prepare a 1 mM mother liquor. Dissolve CS-MeO (2.5 mg) in secondary water (pH = 7.4) to prepare a 1 mM mother liquor. Mix the two mother liquors of NAP and CS-MeO to prepare a Nano-NAP solution (NAP:CS-MeO = 1:2, v / v). After ultrasonic treatment for 30 minutes, place it in a MW1000 dialysis bag and dialyze for one day to obtain a probe Nano-NAP solution, which is a ratiometric fluorescence probe for detecting mercury ions.

[0048] Detection test of mercury ions:

[0049] Take 11 5-mL sample bottles, and add 20 μL of the solution Nano-NAP prepared from the fluorescent probe obtained in Example 1 (the concentration of this fluorescent probe is 10 μM) to each. Then, add mercury ion solutions with concentrations of [Hg 2+ = 0 (a), 2×10 - 6 mol / L (b), 4×10 -6 mol / L (c), 6×10 -6 mol / L (d), 8×10 -6 mol / L (e), 1×10 -5 mol / L (f), 1.1×10 -5 mol / L (g), 1.2×10 -5 mol / L (h), 1.3×10 -6 mol / L (i), 1.4×10 -5 mol / L (.j), 1.5×10 - 5 mol / L (k) to the 11 sample bottles respectively. After stirring at room temperature for 1 second, using 380 nm as the excitation wavelength, measure the fluorescence intensities of these samples respectively to obtain the fluorescence intensity emission spectrum change diagram of 11 samples, as shown in Figure 3 , and the measurement results show that: the fluorescence intensity at 550 nm of this fluorescence probe gradually increases with the gradual increase of mercury ion concentration, and the fluorescence intensity at 460 nm remains unchanged with the gradual increase of mercury ion concentration. According to Figure 3The fluorescence intensity change value can be used to create a corresponding fitted function curve and the function graph corresponding to this curve (y = ax + b, a = 0.07936, b = 0.16456, R 2 = 0.99845) as shown in Figure 4 . Add 20 μL of the probe solution (the concentration of this fluorescent probe is 10 μM) to the sample vial, and add a concentration of [Hg 2+ = 1×10 -3 mol / L (the ion concentration is 15 μM). Measure the response time of the probe to mercury ions at an emission wavelength of 550 nm. The response time of the probe to mercury ions is approximately 100 s, and the fluorescence intensity remains stable within 100 s, as shown in Figure 5 .

[0050] Comparative detection test for other analytes:

[0051] Take 30 5-mL sample vials, and add 20 μL of solution Nano-NAP prepared with the fluorescent probe obtained in Example 1 to each (the concentration of this fluorescent probe is 10 μM). Then, add 30 μL each of other analytes with a concentration of 1×10 -3 mol / L and Hg 2+ to the other 29 sample vials. The first sample is a blank sample. Then, measure the fluorescence emission intensity of the 30 samples at an excitation wavelength of 380 nm and an emission wavelength of 550 nm. The results are shown in Figure 6 . The measurement results show that the above-mentioned other analytes have no obvious effect on the intensity of the prepared fluorescent probe.

[0052] The above embodiments are used to explain the present invention, rather than to limit the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims of the present invention fall within the protection scope of the present invention.

Claims

1. A preparation method of a ratiometric fluorescence probe for detecting mercury ions, characterized in that It includes the following steps: (1) Dissolve ethylenediamine in chloroform, stir, and then dropwise add a chloroform solution of di-tert-butyl dicarbonate. After the reaction is completed, extract with dichloromethane, dry over anhydrous sodium sulfate, and then dry under vacuum to obtain Product 1; (2) Take the Product 1 obtained in step (1) and dissolve it in absolute ethanol. Add 4-bromo-1,8-naphthalic anhydride, heat and stir. After the reaction is completed, cool to room temperature, filter by suction, wash with ice ethanol, and dry under vacuum to obtain Product 2; (3) Dissolve the Product 2 obtained in step (2), N-hydroxyphthalimide and potassium carbonate in dimethyl sulfoxide, heat and stir. After the reaction is completed, cool to room temperature, add water and adjust the pH, filter by suction, and dry under vacuum to obtain Product 3; (4) Dissolve the Product 3 obtained in step (3) in dry dichloromethane, add phenyl chlorothionoformate and triethylamine, and stir the resulting mixture at room temperature. After the reaction is completed, remove dichloromethane, purify by column chromatography, and dry under vacuum to obtain the fluorescent probe NAP; (5) Dissolve the fluorescent probe NAP obtained in step (4) in DMSO to prepare mother liquor A; dissolve methoxy chitosan CS-MeO in water to prepare mother liquor B; mix mother liquor A and mother liquor B to prepare a Nano-NAP solution; after ultrasonic treatment and dialysis, the target product, a ratiometric fluorescent probe for detecting mercury ions, is obtained.

2. The preparation method of the ratiometric fluorescence probe for detecting mercury ions according to claim 1, characterized in that: In step (5), the preparation steps of the methoxy chitosan CS-MeO are as follows: a. Dissolve chitosan and 4-bromo-1,8-naphthalic anhydride in dimethyl sulfoxide, heat and stir under nitrogen protection. After the reaction is completed, cool to room temperature, filter by suction, and wash successively with dimethyl sulfoxide, water, and ethanol, and then dry under vacuum to obtain Product 4; b. Dissolve the Product 4 obtained in step a and potassium carbonate in methanol, heat and stir under nitrogen protection. After the reaction is completed, cool to room temperature, filter by suction, and wash successively with dimethyl sulfoxide, water, and ethanol, and then dry under vacuum to obtain methoxy chitosan CS-MeO.

3. The preparation method of the ratiometric fluorescence probe for detecting mercury ions according to claim 2, wherein: In step a, the mass ratio of 4-bromo-1,8-naphthalic anhydride to chitosan is 1:9; in step b, the mass ratio of potassium carbonate to Product 4 is 1:

5.

4. The preparation method of the ratiometric fluorescence probe for detecting mercury ions according to claim 3, characterized in that: In step (4), the structural formula of the fluorescent probe NAP is as follows:

5. The preparation method of the ratiometric fluorescence probe for detecting mercury ions according to claim 3, wherein: In step b, the structural formula of the methoxy chitosan CS-MeO is as follows:

6. The preparation method of the ratiometric fluorescence probe for detecting mercury ions according to claim 3, wherein: In step (1), the molar ratio of di-tert-butyl dicarbonate to ethylenediamine is 1:

10.

7. The preparation method of the ratiometric fluorescent probe for detecting mercury ions according to claim 6, wherein: In step (2), the molar ratio of Product 1 to 4-bromo-1,8-naphthalic anhydride is 1:

2.

8. The preparation method of the ratiometric fluorescent probe for detecting mercury ions according to claim 7, characterized in that: In step (3), the molar ratio of Product 2 to N-hydroxyphthalimide is 1:1.

2.

9. The preparation method of the ratiometric fluorescent probe for detecting mercury ions according to claim 8, characterized in that: In step (4), the molar ratio of Product 3 to phenyl chlorothionoformate is 1:1.

2.

10. The preparation method of the ratiometric fluorescent probe for detecting mercury ions according to claim 9, wherein: In step (5), the volume ratio of the fluorescent probe NAP in mother liquor A to the methoxy chitosan CS-MeO in mother liquor B is 1:2.

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