A preparation method of a ratio type fluorescent probe for detecting mercury ions

Abstract

The application belongs to the technical field of ion detection, and particularly relates to a preparation method of a ratio type fluorescent probe for detecting ions, which comprises the following steps: dissolving ethylenediamine and di-tert-butyl dicarbonate in chloroform to obtain product 1; dissolving the product 1 in anhydrous ethanol, adding 4-nitro-1,8-naphthalic anhydride to obtain product 2; dissolving the product 2 in dimethylformamide, and adding N-phthaloylglycine to obtain product 3; dissolving the product 3 in dichloromethane, and adding phenylthiochloroformate to obtain a fluorescent probe NAP; dissolving the fluorescent probe NAP in DMSO to prepare mother liquor A, dissolving methoxyl chitosan CS-MeO in water to prepare mother liquor B; and mixing the mother liquor A and the mother liquor B to prepare a Nano-NAP solution. The ratio fluorescent probe is used for detecting ions, and has the advantages of high selectivity, small interference, low detection limit, good pH stability and the like, and has high use value and wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of ion detection technology, specifically relating to a method for preparing a ratiometric fluorescent probe for detecting mercury ions. Background Technology

[0002] Mercury ions (Hg) 2+ As a harmful heavy metal ion, Hg easily migrates and diffuses into the ecological cycle, causing serious harm to biological systems and human health. Currently, due to human activities such as coal combustion, gold mining, and cement production, Hg is released into the environment... 2+ More and more, Hg 2+ The resulting environmental pollution problems are becoming increasingly serious. Furthermore, Hg... 2+ The continuous accumulation of Hg in the human body can adversely affect the normal physiological functions of vital organs such as the kidneys, brain, and heart, and may even lead to gene mutations. Therefore, developing efficient analytical methods to detect and identify Hg in biological and environmental systems is crucial. 2+ Crucial. To date, detecting Hg... 2+ The main methods include mercury analyzers, atomic absorption spectrometry, electron-coupled plasma mass spectrometry, and ion chromatography. However, these detection techniques have some significant drawbacks, such as relatively cumbersome sample preparation, generally inconvenient instruments, and unintuitive signal transmission methods, which greatly limit their practicality. To overcome these shortcomings, more and more researchers are focusing on developing mercury analyzers that are readily available, highly sensitive, easy to operate, and capable of in-situ detection and application in biology. 2+ Fluorescent sensors. Most reported methods for detecting Hg... 2+ The probe relies solely on changes in single-emission fluorescence intensity as its response signal, making it highly susceptible to interference from the surrounding environment, background fluorescence, probe concentration, excitation power, and instrument performance, leading to inaccurate analytical results or false positives. Considering the high environmental and human health hazards and toxicity of mercury ions, it is necessary to develop a probe that can specifically detect Hg in real-world environments. 2+ It can also effectively adsorb Hg 2+ This is a bifunctional ratiometric fluorescent probe with significant scientific value and promising application prospects. Summary of the Invention

[0003] For existing Hg detection 2+ To address the problems existing in the fluorescent probe process, this invention provides a method for preparing a ratiometric fluorescent probe for detecting mercury ions. This method is simple, has a fast response speed, good selectivity, high sensitivity, low detection limit, and adsorption properties.

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

[0005] A method for preparing a ratiometric fluorescent probe for detecting mercury ions includes the following steps:

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

[0007] (2) Dissolve product 1 obtained in step (1) in anhydrous ethanol, add 4-bromo-1,8-naphthalene anhydride, heat and stir, cool to room temperature after the reaction is complete, filter, 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, cool to room temperature after the reaction is completed, add water and adjust pH, filter and dry under vacuum to obtain product 3.

[0009] (4) Dissolve the product 3 obtained in step (3) in dry dichloromethane, add phenyl thiochloroformate and triethylamine, stir the mixture at room temperature, remove dichloromethane after the reaction is complete, 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 mother solution A; dissolve methoxy chitosan CS-MeO in water to prepare mother solution B; mix mother solution A and mother solution B to prepare Nano-NAP solution; after sonication and dialysis, the ratiometric fluorescent probe for detecting mercury ions is obtained.

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

[0012] a. Chitosan and 4-bromo-1,8-naphthalene anhydride were dissolved in dimethyl sulfoxide. The mixture was heated and stirred under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, filtered, and washed successively with dimethyl sulfoxide, water, and ethanol. The product was then dried under vacuum to obtain product 4.

[0013] b. Dissolve product 4 obtained in step a and potassium carbonate in methanol, heat and stir under nitrogen protection, cool to room temperature after the reaction is complete, filter, wash with dimethyl sulfoxide, water and ethanol in sequence, and dry under vacuum to obtain methoxychitosan CS-MeO.

[0014] Further, in step a, the mass ratio of 4-bromo-1,8-naphthalenediic 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 fluorescent probe NAP has the following structural formula:

[0016]

[0017] Furthermore, in step b, the methoxychitosan CS-MeO structure is as follows:

[0018]

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

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

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

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

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

[0024] The specific reaction process for preparing the ratiometric fluorescent probe for detecting mercury ions described above is as follows:

[0025]

[0026]

[0027] This invention utilizes a reaction of ethylenediamine, di-tert-butyl dicarbonate, 4-bromo-1,8-naphthalenediic anhydride, N-hydroxyphthalimide, phenyl thiochloroformate, triethylamine, chitosan, and potassium carbonate to prepare the desired fluorescent probe, Nano-NAP. Under DMF:H₂O (2:8) conditions, this fluorescent probe exhibits a significant fluorescence change with increasing mercury ion concentration in the presence of mercury ions. This fluorescent probe demonstrates high selectivity, high sensitivity, and excellent adsorption capacity for mercury ion detection. By detecting the fluorescence intensity ratio of the mercury ion solution at 550 nm and 460 nm using a ratiometric fluorescent probe, the concentration of the mercury ions can be obtained based on the linear equation between the fluorescence intensity ratio and the mercury ion concentration. Compared with conventional detection methods, this invention, using a ratiometric fluorescent probe to detect mercury ions, offers advantages such as high selectivity, low interference, low detection limit, and good pH stability, making it highly valuable and promising for broad applications. Compared to some existing detection technologies, the chemical fluorescent probe in this invention has lower cost, simpler synthesis route, and convenient post-processing. It can directly and specifically identify mercury ions in the DMF:H2O (2:8) system while also having an adsorption effect, and has potential application value, especially in environmental water samples and biological systems. Attached Figure Description

[0028] The present invention will now be described in detail through specific embodiments. These embodiments are provided to enable a more thorough understanding of the invention and to fully convey the scope of the invention to those skilled in the art. As used throughout the specification and claims, the terms "comprising" or "including" are open-ended and are interpreted as "comprising but not limited to". The following description is a preferred embodiment for carrying out the invention; however, this description is intended to illustrate the general principles of the specification and is not intended to limit the scope of the invention. The scope of protection of the invention is determined by the appended claims. Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

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

[0030] Figure 2 The NMR C-ray spectroscopy (NMR) spectrum of the prepared fluorescent probe NAP is shown below.

[0031] Figure 3 The fluorescence emission spectra of the fluorescent probe Nano-NAP are shown for different mercury ion concentrations.

[0032] Figure 4 The fitted curve of the fluorescence intensity of the fluorescent probe Nano-NAP as a function of mercury ion concentration and the corresponding function graph are shown.

[0033] Figure 5This is a time-scan graph showing the response of the fluorescent probe Nano-NAP to mercury ions;

[0034] Figure 6 This demonstrates the selectivity of the fluorescent probe Nano-NAP for other analytes.

[0035] The concentration of the added ions was 1.5 × 10⁻⁶. -5 mol / L, 380 nm is the excitation wavelength. (Note: The concentration of this probe is 10 μM; Hg) 2+ The response time is 100 seconds. Detailed Implementation

[0036] Example 1

[0037] (1) Preparation of product 1

[0038] Ethylenediamine (100 mmol) and di-tert-butyl dicarbonate (10 mmol) were added to a round-bottom flask containing 30 mL of chloroform and stirred at room temperature for 24 h. After the reaction was complete, 30 mL of deionized water was added, and the mixture was extracted three times with dichloromethane and dried under vacuum to obtain product 1.

[0039] (2) Preparation of product 2

[0040] Product 1 (5 mmol) was dissolved in 10 mL of anhydrous ethanol, and 4-bromo-1,8-naphthalenediic anhydride (10 mmol) was added. The mixture was heated to 75 °C and stirred for 2 hours under nitrogen protection. The reaction was monitored by TLC. After the reaction was completed, the mixture was cooled to room temperature, filtered, washed with ice-cold ethanol, and dried under vacuum to synthesize product 2.

[0041] (3) Preparation of product 3

[0042] Product 2 (4 mmol) and N-hydroxyphthalimide (4.8 mmol) were dissolved in 30 mL of dimethyl sulfoxide, potassium carbonate was added, and the mixture was heated to 100 °C and stirred for 5 hours. The mixture was then analyzed by TLC. After cooling to room temperature, deionized water was added to adjust the pH to 3, and the mixture was filtered to obtain the desired product. The product was then dried under vacuum to synthesize product 3.

[0043] (4) Synthesize fluorescent probes using product 3 and phenyl thiochloroformate.

[0044] Product 3 (1.5 mmol) was dissolved in 10 mL of dichloromethane, and triethylamine and phenyl thiochloroformate (1.8 mmol) were added. The mixture was stirred at room temperature for 2 h. After the reaction was complete, the dichloromethane was removed, and the product was purified by column chromatography using a mobile phase of dichloromethane:methanol at a volume ratio of 240:1. The purified product was then dried under vacuum to remove the solvent, yielding the fluorescent probe NAP.

[0045] (5) Preparation of target product

[0046] Chitosan and 4-bromo-1,8-naphthalene anhydride were hydrolyzed in dimethyl sulfoxide (DMSO). The mixture was heated to 100°C and stirred for 5 hours under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, filtered, and washed successively with DMSO, water, and ethanol. The product was then dried under vacuum to obtain product 4. Product 4 was dissolved in methanol, potassium carbonate was added, and the mixture was heated to 65°C and stirred for 3 hours under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, filtered, and washed successively with DMSO, water, and ethanol. The product was then dried under vacuum to obtain methoxychitosan CS-MeO.

[0047] 1 mmol of NAP was dissolved in DMSO to prepare a 1 mM stock solution. 2.5 mg of CS-MeO was dissolved in deionized water (pH 7.4) to prepare another 1 mM stock solution. The NAP and CS-MeO stock solutions were mixed to prepare a Nano-NAP solution (NAP:CS-MeO = 1:2, v / v). After sonication for 30 minutes, the solution was placed in a MW1000 dialysis bag and dialyzed for one day to obtain the Nano-NAP probe solution, which is a ratiometric fluorescent probe for detecting mercury ions.

[0048] Tests for detecting mercury ions:

[0049] Take 11 5mL sample vials and add 20μL of Nano-NAP solution (10μM concentration) prepared with the fluorescent probe obtained in Example 1 to each vial. Then, add [Hg] to each of the 11 sample vials. 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 A mercury ion solution of mol / L(k) was stirred at room temperature for 1 second, and the fluorescence intensity of these samples was measured at an excitation wavelength of 380 nm. The fluorescence intensity emission spectrum changes of the 11 samples are shown in the figure. Figure 3 The measurement results showed that the fluorescence intensity of the fluorescent probe at 550 nm gradually increased with the gradual increase of mercury ion concentration, while the fluorescence intensity at 460 nm remained unchanged with the gradual increase of mercury ion concentration. According to... Figure 3The fluorescence intensity change values ​​can be used to plot the corresponding fitted function curve and the corresponding function graph (y = ax + b, a = 0.07936, b = 0.16456, R). 2 =0.99845) See Figure 4 Add 20 μL of probe solution (the fluorescent probe concentration is 10 μM) to the sample vial, and add [Hg] solution. 2+ ] = 1 × 10 -3 The response time of the probe to mercury ions was measured at an emission wavelength of 550 nm using a concentration of mol / L (15 μM). The result showed that the response time of the probe to mercury ions was approximately 100 s, and the fluorescence intensity remained stable within 100 seconds. Figure 5 .

[0050] Other analyte comparison tests:

[0051] Take 30 5mL sample vials and add 20μL of Nano-NAP solution (10μM concentration) prepared with the fluorescent probe obtained in Example 1 to each vial. Then, add 1×10⁻⁶ solution to each vial. -3 Other analytes and Hg (mol / L) 2+ Take 30 μL of each sample and add it to the remaining 29 sample vials, with sample 1 serving as the blank sample. Then, measure the fluorescence emission intensity of each of the 30 samples at an excitation wavelength of 380 nm and an emission wavelength of 550 nm. The results are shown below. Figure 6 The results showed that the other analytes had no significant effect on the intensity of the prepared fluorescent probe.

[0052] The above embodiments are used to explain and illustrate the present invention, but not to limit the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims fall within the protection scope of the present invention.

Claims

1. A method for preparing a ratiometric fluorescent probe for detecting mercury ions, characterized by, Comprising the following steps: (1) Preparation of product 1 100 mmol of ethylenediamine and 10 mmol of di-tert-butyl dicarbonate were added to a round-bottom flask containing 30 mL of chloroform, stirred at room temperature for 24 h, after the reaction was completed, 30 mL of water was added, extracted with dichloromethane three times, dried in vacuum to synthesize product 1; (2) Preparation of product 2 5 mmol of product 1 was dissolved in 10 mL of anhydrous ethanol, 10 mmol of 4-bromo-1,8-naphthalene anhydride was added; under nitrogen protection, the temperature was raised to 75 ℃ and stirred for 2 hours; TLC plate detection, after the reaction was completed, it was cooled to room temperature, filtered, washed with ice ethanol, and dried in vacuum to synthesize product 2; (3) Preparation of product 3 4 mmol of product 2 and 4.8 mmol of N-hydroxyphthalimide were dissolved in 30 mL of dimethyl sulfoxide, potassium carbonate was added, the temperature was raised to 100 ℃ and stirred for 5 hours; TLC plate detection; after cooling to room temperature, water was added to adjust the pH to 3, and the desired product was obtained by filtration, and dried in vacuum to synthesize product 3; (4) Synthesis of fluorescent probe using product 3 and phenyl chlorothioformate 1.5 mmol of product 3 was dissolved in 10 mL of dichloromethane, triethylamine and 1.8 mmol of phenyl chlorothioformate were added; stirred at room temperature for 2 h; after the reaction was completed, dichloromethane was removed, and column chromatography was used for purification, the mobile phase of column chromatography separation was dichloromethane:methanol with a volume ratio of 240:1; the purified product was removed by rotary evaporation, and dried in vacuum to obtain fluorescent probe NAP; (5) Preparation of the target product Chitosan and 4-bromo-1,8-naphthalene anhydride were dissolved in dimethyl sulfoxide, stirred at 100 ℃ for 5 hours under nitrogen protection; after the reaction was completed, it was cooled to room temperature, filtered, and washed with dimethyl sulfoxide, water, and ethanol in sequence, and dried in vacuum to obtain product 4; product 4 was dissolved in methanol, potassium carbonate was added, and stirred at 65 ℃ for 3 hours under nitrogen protection; after the reaction was completed, it was cooled to room temperature, filtered, and washed with dimethyl sulfoxide, water, and ethanol in sequence, and dried in vacuum to obtain methoxy chitosan CS-MeO; the fluorescent probe NAP obtained in step (4) was dissolved in DMSO to prepare mother liquor A; methoxy chitosan CS-MeO was dissolved in water to prepare mother liquor B; mother liquor A and mother liquor B were mixed to prepare Nano-NAP solution; after ultrasonic treatment and dialysis, the target product, a ratiometric fluorescent probe for detecting mercury ions, was obtained.

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