Preparation of zinc (II) coordination type naphthalimide fluorescent probe and application of zinc (II) coordination type naphthalimide fluorescent probe in pasture detection

Abstract

The invention discloses preparation of a zinc (II) coordination type naphthalimide fluorescent probe and application of the zinc (II) coordination type naphthalimide fluorescent probe in pasture detection, and relates to the field of pasture detection and fluorescent probes, and the preparation method of the fluorescent probe comprises the following steps: (1) preparation of an intermediate a: preparing the intermediate a by using 2-bromo-1, 8-naphthalic anhydride; (2) preparation of an intermediate c: preparing the intermediate c by using the intermediate a; (3) preparation of an intermediate d: preparing the intermediate d by using the intermediate c; (4) preparing Nap3: preparing Nap3 by using the intermediate d; the raw materials for preparing the Nap3 are easy to obtain, the synthesis steps are simple, the operation is simple and convenient, and the Nap3 has high application value in the fields of rapid detection of zinc ions in pasture and quality identification of the pasture.

Description

Technical Field

[0001] This invention relates to the fields of forage detection and fluorescent probes, and particularly to the preparation of a zinc(II)-coordinated naphthalimide fluorescent probe and its application in forage detection. Background Technology

[0002] Zinc plays a vital role in the human body, significantly regulating various physiological responses such as gene expression, apoptosis, neurotransmission, enzyme regulation, and metabolic function. Studies have shown that zinc deficiency can lead to a variety of clinical diseases, including reproductive dysfunction, immune system dysfunction, growth retardation, anorexia, and diabetes. Excessive zinc can cause symptoms such as diarrhea, nausea, and poisoning, and may also trigger coronary heart disease and atherosclerosis. Furthermore, zinc imbalance may lead to neurological disorders such as Alzheimer's disease and Parkinson's disease. Therefore, zinc has a significant impact on human health. The human body primarily obtains zinc from food. Grains and meats are high in zinc, and a balanced diet is beneficial to health. Meat is an important source of zinc, containing 5-60 mg of zinc per 100g, making it an effective and rapid way to supplement zinc. Secondly, in many developing countries, rice is a staple food; increasing the zinc content of rice can help prevent zinc deficiency. Consuming zinc-rich rice and meat can effectively meet the body's zinc requirements, promoting overall health and normal bodily functions. Therefore, the detection of zinc content in rice and meat products is of great significance.

[0003] Traditional methods for zinc detection include atomic absorption spectrometry, inductively coupled plasma mass spectrometry, spectrophotometry, electrochemical analysis, and chromatographic analysis. While these methods offer good selectivity and high sensitivity for zinc ion detection, they suffer from high equipment costs, complex procedures, and long detection times, failing to meet the demands of real-time, in-situ online detection. In contrast, small-molecule fluorescent probes offer advantages such as structural diversity, good reproducibility, ease of modification, and well-defined significance. Therefore, developing a zinc ion fluorescent probe with high sensitivity, good selectivity, low cost, and simple operation is of great importance for food quality testing. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention discloses the preparation of a zinc(II)-coordinated naphthalimide fluorescent probe and its application in forage detection.

[0005] A zinc(II)-coordinated naphthaleneimide fluorescent probe, the chemical structure of which is as follows: ( Figure 1 ), denoted as Nap3.

[0006] Furthermore, the preparation method of the coordination-type naphthalimide fluorescent probe includes the following steps: (1) Preparation of intermediate a 2-Bromo-1,8-naphthalenedicarboxylic anhydride was dissolved in anhydrous ethanol (EtOH), and then n-butylamine was added to react. After 2 hours, a large amount of precipitate was produced. The solvent was removed by vacuum distillation, and then ice water was added to precipitate the product. The product was filtered to obtain a filter cake, and washed with ice ethanol to obtain intermediate a. The chemical structural formula of intermediate a is as follows: ; The chemical reaction formula for the preparation process of intermediate a is as follows: ; (2) Preparation of intermediate c Weigh intermediate a and dissolve it in a round-bottom flask using N,N-dimethylformamide (DMF) as solvent. Weigh an appropriate amount of sodium azide (NaN3) and dissolve it in ultrapure water, then add this solution to the flask. Place the reaction system in an oil bath and reflux at 105°C for 8 hours. After the reaction, remove some of the solvent by vacuum distillation, then add an appropriate amount of saturated sodium chloride solution to the reaction solution, precipitating a large amount of solid. Collect the insoluble matter by filtration, wash the filter cake with ice-cold methanol, and dry to obtain a solid crude product. Dissolve the crude product in anhydrous ethanol, and slowly add freshly prepared sodium sulfide (Na2S) solution dropwise while stirring. Observe the color change of the reaction solution; continue adding until the color no longer changes. Continue stirring the reaction mixture at room temperature for 2 hours. After the reaction, remove the solvent by vacuum distillation, and finally purify by silica gel column chromatography to obtain intermediate c. ; The chemical reaction formula for the preparation process of intermediate c is as follows: ; (3) Preparation of intermediate d Weigh intermediate c and place it in a round-bottom flask. Add dichloromethane to dissolve it, then add triethylamine and cool it to 0°C in an ice-water bath. Then slowly add chloroacetyl chloride dropwise using a constant-pressure separatory funnel. After that, move the mixture to room temperature and continue the reaction for 3 hours. Remove the solvent by vacuum distillation and purify by silica gel column chromatography to obtain a white solid intermediate d. ; The chemical reaction formula for the preparation process of intermediate d is as follows: ; in, Chloroacetyl chloride; CH2Cl2: Dichloromethane; Et3N: Triethylamine; (4) Preparation of the coordination-type naphthalimide fluorescent probe Nap3 Weigh intermediate d and place it in a round-bottom flask. Dissolve it in acetonitrile (MeCN), then add dimethylpyridinium (DPA) and N,N-diisopropylethylamine (DIPEA) and stir until homogeneous. Add potassium iodide (KI), and reflux in an oil bath at 60°C overnight. Remove the solvent by vacuum distillation. Purify the product by silica gel column chromatography to obtain a white solid product, which is the fluorescent probe Nap3. The chemical reaction formula is as follows: ; in, :DPA.

[0007] Moreover, in the preparation of intermediate a in step (1), the molar ratio of 2-bromo-1,8-naphthalenedicarboxylic anhydride and n-butylamine is 1:1.5 equivalents; the reaction in step (1) is carried out under the following conditions: refluxed at 80°C for 2 hours under magnetic stirring.

[0008] Furthermore, in the preparation of intermediate b in step (2), the ratio of intermediate a to sodium azide is preferably 1:1.5 equivalents; the reaction in step (2) is carried out under the following conditions: sodium azide is first dissolved in a small amount of pure water and slowly added to solution a under magnetic stirring, and refluxed at 105°C for 8 hours under nitrogen protection; after removing part of the solvent by vacuum distillation, a large amount of saturated sodium chloride solution is added, the solid is precipitated, the insoluble matter is filtered, and the filter cake is washed with ice-cold methanol and dried to obtain a solid crude product. In the preparation of intermediate c in step (2), the ratio of intermediate b to sodium sulfide is preferably 1:2 equivalents; the reaction in step (2) is carried out under the following conditions: the dissolved sodium sulfide is added to the intermediate b solution and reacted at room temperature for 2 hours under magnetic stirring.

[0009] Moreover, in the preparation of intermediate d in step (3), the ratio of intermediate c to chloroacetyl chloride is preferably 1:12 equivalents; the reaction in step (3) is carried out under the following conditions: under magnetic stirring, triethylamine is added and cooled to 0°C in an ice-water bath, then chloroacetyl chloride is slowly added dropwise using a constant pressure separatory funnel, and then the reaction is continued at room temperature for 3 hours.

[0010] Moreover, in step (4), when preparing the coordination-type naphthalimide fluorescent probe Nap3, the molar ratio of intermediate d to DPA is 1:4 equivalent; the reaction in step (4) is carried out under the following conditions: DPA is slowly added dropwise under magnetic stirring, and the reaction is refluxed at 60°C for 16 hours under nitrogen protection.

[0011] On the other hand, the present invention discloses the application of a zinc(II)-coordinated naphthalimide fluorescent probe for the qualitative or quantitative detection of zinc(II) in forage.

[0012] Furthermore, the forage grass is any one or a combination of alfalfa, oats, and clover; the forage grass sample is pretreated and then reacted with the fluorescent probe, and the fluorescence intensity of the reaction product is measured to quantitatively detect the zinc(II) concentration in the forage grass, as detailed below: Step 1: After crushing and dissolving the forage sample by centrifugation, take the supernatant, adjust the pH of the supernatant to 7, and filter it through a microporous membrane to obtain the forage sample solution. Step 2: Add the fluorescent probe to the forage sample solution. The reaction product can be initially observed to change color under ultraviolet light to qualitatively detect zinc (II) in the forage. Then, measure the fluorescence intensity of the mixture at an excitation wavelength of 365 nm and an emission wavelength of 501 nm, and calculate the content of free zinc (II) in the forage sample to be tested according to the pre-established standard curve.

[0013] Furthermore, the fluorescent probe was added to the forage sample solution to a final concentration of 10 μM, and the reaction temperature was 20-25℃.

[0014] The detection principle of this invention is that the DPA structure detection group of the coordination-type naphthimide fluorescent probe coordinates and binds to zinc(II), generating fluorescence activation, which can accurately detect the zinc(II) content in forage. Specifically, the naphthimide carbonyl group, the carbonyl group on the linker arm (2-amino-acetamido), and the DPA group in the probe form a co-coordination system with Zn. 2+ After specific coordination, the photoinduced electron transfer (PET) process from DPA to the naphthalimide fluorophore is suppressed. Simultaneously, the chelate-enhanced fluorescence (CHEF) effect increases molecular rigidity, thereby significantly enhancing fluorescence emission and enabling the collection of Zn from the analyte system. 2+ This method offers highly sensitive and selective fluorescence detection. It is fast, highly sensitive, selective, simple to operate, and low in cost, and can be used for the rapid detection of zinc(II) in forage.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The fluorescent probe Nap3 provided by this invention exhibits strong fluorescence enhancement and high affinity for zinc(II). Even in mixed solutions containing multiple metal ions, it preferentially binds to zinc(II). For ions that can cause fluorescence quenching (such as Fe), it does not bind to zinc(II). 2+ Co 2+ Ni 2+ Cu 2+ Cu + The fluorescence intensity ratio of the fluorescent probe Nap3 was significantly increased after the addition of zinc (II); while Cd 2+The fluorescence enhancement effect produced was consistent with that of zinc(II) alone after the addition of zinc(II), indicating that it does not interfere with the selective recognition of zinc(II) by Nap3 and can achieve specific binding of zinc(II) under the condition of coexistence of interfering ions.

[0016] 2. The fluorescent probe Nap3 provided by this invention exhibits a gradual increase in fluorescence intensity with increasing zinc (II) concentration. The fluorescence intensity at 501 nm shows a linear relationship with zinc (II) (0-10 μM), expressed by the equation y = 388.39x + 185550, R. 2 =0.9976. Combined with 20 blank measurements in PBS matrix, the detection limit of the fluorescent probe Nap3 for zinc(II) was calculated to be 1.28 nM according to the detection limit calculation formula, indicating that it has very high sensitivity for the detection of zinc(II).

[0017] 3. After adding EDTA to the complex formed by the fluorescent probe Nap3 and zinc(II) provided by this invention, the fluorescence of Nap3 is reversibly restored. This further confirms that the interaction between the probe and zinc(II) is reversible, which is beneficial for the repeated use of the probe and further research on its binding mechanism with zinc(II).

[0018] 4. The working curve was analyzed by fluorescence spectroscopy titration. When the molar fraction of the fluorescent probe Nap3 and zinc (II) in the solution reached 0.5, the concentration of zinc (II) in the solution was equal to the concentration of the probe, and the fluorescence intensity reached its maximum value. This indicates that the stoichiometric binding ratio of the probe and zinc (II) is 1:1, which provides a theoretical basis for the accurate detection of zinc (II) content.

[0019] 5. When the fluorescent probe Nap3 provided by this invention undergoes a coordination reaction with zinc (II), the carbonyl double bond of the linking group at the 2-position of naphthalimide migrates, and the original carbonyl group is transformed into a hydroxyl group. At this time, zinc (II) forms a co-coordinated complex with DPA, the nitrogen atom of the linking group and the carbonyl group of naphthalimide. Combined with the strong electron-donating effect of the hydroxyl group, the fluorescence is sharply enhanced, thus clarifying the response mechanism of the probe to zinc (II).

[0020] 6. Using the fluorescent probe Nap3 provided by this invention to detect zinc(II) in alfalfa, oats and clover, the error between the measured free zinc content in alfalfa, oats and clover and the ICP-OES determination results was less than 5%; the zinc(II) spiked recovery rate was 95%-102% and RSD<3%, indicating that Nap3 has the potential for quantitative detection of zinc(II) in forage.

[0021] 7. This invention provides a zinc (II)-coordinated naphthalimide fluorescent probe, its preparation method, and its application in forage detection. The raw materials are readily available, the synthesis steps are simple, and the operation is convenient. It has high application value in the field of rapid detection of zinc content in forage.

[0022] 8. The DPA structure of the coordination-type naphthalimide fluorescent probe provided by this invention coordinates with zinc (II) to generate fluorescence activation, enabling accurate detection of zinc (II) content in forage. Specifically, the carbonyl group of the naphthalimide in the probe, the carbonyl group on the linker arm (2-amino-acetamido), and the DPA group form a co-coordination system with Zn. 2+ After specific coordination, the photoinduced electron transfer (PET) process from DPA to the naphthalimide fluorophore is suppressed. Simultaneously, the chelate-enhanced fluorescence (CHEF) effect increases molecular rigidity, thereby significantly enhancing fluorescence emission and enabling the collection of Zn from the analyte system. 2+ This highly sensitive and selective fluorescence detection method is fast, sensitive, selective, simple to operate, and low in cost. It can be used for the rapid detection of zinc(II) in forage, overcoming the disadvantages of existing large-scale instruments that have high detection costs and limited detection scenarios for zinc(II). Attached Figure Description

[0023] Figure 1 The structural formula of the coordination-type naphthalimide fluorescent probe compound of this invention is shown below; Figure 2 This is a synthetic route diagram of the coordination-type naphthalimide fluorescent probe compound of the present invention; Figure 3 This is the 1H NMR spectrum of the coordination-type naphthalimide fluorescent probe compound of this invention; Figure 4 This is the carbon NMR spectrum of the coordination-type naphthalimide fluorescent probe compound of the present invention; Figure 5 This is the mass spectrum of the coordination-type naphthalimide fluorescent probe compound of the present invention; Figure 6 The excitation wavelengths of the coordination-type naphthalimide fluorescent probe compound (10 μM) of this invention were measured before and after binding with zinc(II) (20 μM) in PBS buffer solution at pH=7.4. Figure 7 The emission wavelengths of the coordination-type naphthalimide fluorescent probe compound (10 μM) of this invention were measured before and after binding with zinc(II) (20 μM) in PBS buffer solution at pH=7.4. Figure 8 The fluorescence spectra of the coordination-type naphthalimide fluorescent probe compound (10 μM) of this invention in PBS buffer solution at pH 7.4 with different interfering matrices are shown. Figure 9 The bar chart shows the addition of different metal ions and some interfering factors to the coordination-type naphthimide fluorescent probe compound (10 μM) of this invention in PBS buffer solution at pH=7.4. Figure 10 The fluorescence intensity spectrum of the coordination-type naphthalimide fluorescent probe compound (10 μM) of the present invention in PBS buffer solution at pH=7.4 is a spectrum showing the fluorescence intensity change with the zinc(II) concentration as the zinc(II) concentration gradually increases (0-50 μM). Figure 11 The linear relationship between fluorescence intensity and zinc(II) concentration was observed when different concentrations of zinc(II) (0-50 μM) were added to a PBS buffer solution at pH 7.4 for the coordination-type naphthimide fluorescent probe compound (10 μM) of the present invention. Figure 12 The diagram shows the detection specificity of the coordination-type naphthalimide fluorescent probe compound of the present invention in a mixed solution of PBS buffer solution at pH 7.4 when the probe is interfered with by different metal ions in the same system. Figure 13 The working curve of the coordination-type naphthalimide fluorescent probe compound of the present invention binding with zinc(II) in PBS buffer solution at pH=7.4 is shown. Figure 14 The reversibility of binding of the coordination-type naphthalimide fluorescent probe compound (10 μM) of this invention with zinc(II) in PBS buffer solution at pH=7.4 was tested. Figure 15 This diagram illustrates the binding mechanism between the coordination-type naphthalimide fluorescent probe compound of this invention and zinc(II). Detailed Implementation

[0024] The present invention will be further described below with reference to specific embodiments, and the features and advantages of the technical solution of the present invention will become clearer with the description. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention.

[0025] Example 1: Synthesis and Characterization of Coordination-Type Naphthalimide Fluorescent Probe Synthesis route diagram of fluorescent probe as follows Figure 2 As shown, the steps include: 2-Bromo-1,8-naphthalenedicarboxylic anhydride (4 g, 14.44 mmol, 1 eq) was placed in a 100 mL round-bottom flask and dissolved in 60 mL of anhydrous ethanol. Then, n-butylamine (2.1 mL, 21.6 mmol, 1.5 eq) was added. The reaction mixture was placed in an oil bath and refluxed at 80 °C for 2 hours. The reaction was monitored by thin-layer chromatography (TLC). After the reaction was complete, the reaction mixture was cooled and ice water was added. A large amount of solid precipitated. The solid was filtered through a Buchner funnel to obtain a gray, loose, crystalline crude product. The product was then washed with ice ethanol and water to obtain intermediate a, with a yield of 83.68%.

[0026] intermediate a 1 The H NMR data are as follows: 1 H NMR (500 MHz, DMSO- d 6) δ 8.42 (ddt, J =21.3, 8.4, 1.1 Hz, 2H), 8.20 (dd, J = 7.8, 1.0 Hz, 1H), 8.10 (dd, J = 7.8, 1.0 Hz, 1H), 7.89 (ddd, J = 8.4, 7.3, 1.0 Hz, 1H), 3.99 – 3.92 (m, 2H), 1.61 – 1.51 (m, 2H), 1.37 – 1.26 (m, 2H), 0.89 (td, J = 7.4, 1.0 Hz, 3H).

[0027] intermediate a 13 The C NMR data are as follows: 13 C NMR (126 MHz, DMSO- d 6) δ 163.15 (d, J =6.3 Hz), 132.90, 131.90, 131.69, 131.28, 129.12, 39.94, 29.98, 20.24, 14.13.

[0028] Accurately weigh intermediate a (6.62 g, 20 mmol, 1 eq) and place it in a 250 mL round-bottom flask, then dissolve it in 50 mL of DMF. Separately weigh NaN3 (1.95 g, 30 mmol, 1.5 eq) and dissolve it in 15 mL of ultrapure water, then add this solution to the reaction flask. After three vacuum-nitrogen purging cycles, reflux the reaction mixture in an oil bath at 105 °C for 8 hours under a nitrogen atmosphere. After the reaction is complete as monitored by TLC, concentrate under reduced pressure to remove some of the solvent, and add saturated sodium chloride solution to precipitate a solid. Filter, wash the filter cake 1-2 times with ice-cold methanol, and dry to obtain intermediate b. Dissolve intermediate b in anhydrous ethanol, and add freshly prepared Na2S solution dropwise with stirring until the system color no longer changes. Continue stirring at room temperature for 2 hours. TLC monitoring shows that the product has absorption at 256 nm and 365 nm UV and exhibits yellow-green fluorescence. After the reaction solution was concentrated under reduced pressure, it was dry-loaded and purified by silica gel column chromatography (eluent: PE / EA = 5:1, v / v). After drying, 3.10 g of intermediate c, a yellow solid, was obtained, with a yield of 57.84%.

[0029] intermediate c 1 The H NMR data are as follows: 1 H NMR (500 MHz, DMSO- d 6) δ 8.55 (dd, J = 8.4, 1.1 Hz, 1H), 8.35 (dd, J = 7.3, 1.0 Hz, 1H), 8.13 (d, J = 8.3 Hz, 1H), 7.58 (dd, J = 8.4, 7.2 Hz, 1H), 7.36 (s, 2H), 6.80 (d, J = 8.4 Hz, 1H), 3.95 (dd, J = 8.2, 6.6 Hz, 2H), 1.58–1.48 (m, 2H), 1.28 (h, J = 7.4 Hz, 2H), 0.86 (t, J = 7.4 Hz, 3H).

[0030] intermediate c 13 The C NMR data are as follows: 13 C NMR (126 MHz, DMSO- d6) δ 164.13, 163.28,153.04, 134.26, 131.30, 130.05, 129.61, 124.28, 122.18, 119.76, 108.54,39.31, 30.26, 20.28, 14.16.

[0031] MS(ESI)m / z: C 16 H 16 N₂O₂[M+H] + = 269.1293.

[0032] Intermediate c (0.54 g, 2 mmol, 1 eq) was accurately weighed and placed in a 100 mL round-bottom flask. 30 mL of dichloromethane was added to dissolve it, followed by the addition of triethylamine (1.67 mL, 12 mmol, 6 eq). The reaction mixture was cooled to 0°C in an ice bath, and chloroacetyl chloride (1.9 mL, 24 mmol, 12 eq) was slowly added dropwise. After the addition was complete, the mixture was allowed to return to room temperature and reacted for 3 hours. TLC monitoring showed absorption at 256 nm and 365 nm and blue fluorescence in the product. After the reaction was complete, the solvent was removed by concentration under reduced pressure. Due to severe tailing of impurities, the product was purified by silica gel column chromatography using pure EA as the eluent. The obtained product was then recrystallized from a CH2Cl2 / MeOH mixed solvent to give intermediate d as a white solid, 0.35 g, with a yield of 50.87%.

[0033] intermediate d 1 The H NMR data are as follows: 1 H NMR (500 MHz, DMSO- d 6) δ 10.61 (s, 1H), 8.55 (dd, J = 8.6, 1.1 Hz, 1H), 8.45–8.34 (m, 2H), 8.18 (d, J = 8.1 Hz, 1H), 7.80 (dd, J = 8.5, 7.3 Hz, 1H), 4.52 (s, 2H), 3.95 (dd, J = 8.4, 6.5 Hz, 2H),1.60–1.50 (m, 2H), 1.30 (h, J = 7.4 Hz, 2H), 0.88 (t, J = 7.4 Hz, 3H).

[0034] intermediate d 13 The C NMR data are as follows: 13C NMR (126 MHz, DMSO- d 6) δ 166.38, 163.68,163.13, 139.70, 131.75, 131.22, 129.30, 128.53, 126.94, 124.61, 122.64,120.28, 118.60, 44.01, 39.72, 30.05, 20.24, 14.12.

[0035] MS(ESI)m / z: C 18 H 17 ClN2O3[M+H] + = 345.0998.

[0036] Accurately weigh compound d (0.20 g, 0.6 mmol, 1 eq) and place it in a 50 mL round-bottom flask. Add 30 mL of MeCN to dissolve it, then add DPA (0.48 g, 2.4 mmol, 4 eq) and 2 mL of DIPEA, and stir until homogeneous. Then add KI (0.12 g, 0.72 mmol, 1.2 eq). After three vacuum nitrogen cycles, under nitrogen protection, place the reaction mixture in an oil bath, heat to 60 °C, and reflux overnight. Monitor the reaction progress by TLC. After the reaction is complete, remove the solvent by vacuum distillation. The obtained product has UV absorption at 256 nm and exhibits blue fluorescence under 365 nm UV light. Purify the crude product by column chromatography using silica gel, eluting with CH2Cl2:MeOH:NH3·H2O = 500 mL:2 mL:1 drop. After drying, 0.09 g of the fluorescent probe Nap3 white solid product was obtained, with a yield of 30.6%.

[0037] Coordination-type naphthalimide fluorescent probe compounds 1 The H NMR data are as follows: 1 H NMR (500 MHz, DMSO- d 6)δ 11.42 (s, 1H), 8.91 (dd, J = 8.7, 1.1 Hz, 1H), 8.50–8.42 (m, 2H), 8.41(ddd, J = 4.9, 1.8, 0.9 Hz, 2H), 8.33 (d, J = 8.2 Hz, 1H), 7.91 (dd, J = 8.5, 7.2 Hz, 1H), 7.72 (td, J= 7.6, 1.8 Hz, 2H), 7.43 (dd, J = 7.8, 1.1 Hz, 2H), 7.22 (ddd, J = 7.5, 4.8, 1.2 Hz, 2H), 4.02 (s, 4H), 4.00–3.93 (m, 2H), 3.62(s, 2H), 1.62–1.52 (m, 2H), 1.31 (q, J = 7.4 Hz, 2H), 0.89 (t, J = 7.4 Hz, 3H).

[0038] Coordination-type naphthalimide fluorescent probe compounds 13 The C NMR data are as follows: 13 C NMR (126 MHz, Chloroform- d ) δ 170.75, 163.77, 157.64, 149.55, 139.81, 136.71, 132.70, 131.03, 128.17, 126.11, 123.44 (d, J = 19.9 Hz), 123.08, 122.69, 117.70, 117.09, 60.63, 59.14, 40.15, 30.24, 29.68, 20.40, 13.86.

[0039] MS(ESI)m / z: C 30 H 29 N5O3[M+H] + = 508.2285.

[0040] Coordination-type naphthalimide fluorescent probe 1 H NMR image as follows Figure 3 As shown, 13 C NMR such as Figure 4 As shown, the mass spectrum is as follows Figure 5 As shown.

[0041] Experimental Example 2: Spectral Response of Probe Molecules to Zinc(II) The fluorescent coordination type naphthalimide fluorescent probe compound prepared in Example 1 was used to detect zinc(II) in forage under the condition of pH=7.4 with PBS as the dissolving matrix. The fluorescent probe was dissolved in dimethyl sulfoxide to prepare a probe stock solution with a concentration of 2 mM and a metal ion stock solution with a concentration of 2 mM.

[0042] Figure 6 The UV absorption wavelengths of the coordination-type naphthimide fluorescent probe compound (10 μM) of this invention before and after binding with zinc(II) (20 μM) in PBS buffer solution at pH 7.4 are shown; the interaction between the fluorescent probe Nap3 and Zn is also illustrated. 2+ Combined with the UV absorption measurements before and after the experiment, the excitation wavelength remained at 365 nm, showing no significant change. This wavelength was used as the probe excitation wavelength for subsequent spectroscopic experiments.

[0043] Figure 7 The emission wavelengths of the coordination-type naphthalimide fluorescent probe compound (10 μM) of this invention were measured before and after binding with zinc (II) (20 μM) in PBS buffer solution at pH=7.4. Fluorescence measurements showed that the emission wavelength slightly red-shifted from 489 nm to 501 nm, and the fluorescence intensity was significantly enhanced.

[0044] Figure 8 To prepare the coordination-type naphthimide fluorescent probe compound (10 μM) of this invention, different interfering matrices were added to a PBS buffer solution at pH 7.4: magnesium ions (Mg... 2+ ), aluminum ions (Al 3+ ), chromium ions (Cr 3+ ), manganese ions (Mn 2+ ), ferrous ions (Fe 2+ ), iron ions (Fe 3+ ), cobalt ions (Co) 2+ Nickel ions (Ni) 2+ ), copper ions (Cu) 2+ ), cuprous ion (Cu + ), zinc ions (Zn 2+ ), silver ions (Ag) + ), cadmium ions (Cd) 2+ ), mercury ions (Hg) 2+ ), lead ions (Pb 2+ ), glutathione (GSH), cysteine ​​(Cys), sulfide ions (S) 2- sulfate ions (SO4) 2- Vitamin C, hydrogen peroxide (H2O2), hypochlorite (ClO2) - ), chloride ions (Cl - ), nitrate (NO - The fluorescence spectrum after emission was obtained, with an excitation wavelength of 365 nm and an emission wavelength of 501 nm. The results show that this fluorescent probe (Nap3) is effective against zinc ions (Zn). 2+ It exhibits extremely high selectivity and can induce significant fluorescence enhancement; although it is sensitive to cadmium ions (Cd) 2+It also has some response, but its intensity is much lower than that of zinc ions; while many other common metal ions (such as Cu) also have a response. 2+ Co 2+ Ni 2+ Interfering substances such as α-monomers and biomolecules (e.g., GSH, Cys) have minimal impact on the fluorescence signal, demonstrating that the probe can detect Zn in complex environments. 2+ Its strong anti-interference capability.

[0045] like Figure 9 The bar chart shows the fluorescence response of the coordination-type naphthimide fluorescent probe compound (10 μM, pH=7.4 PBS buffer solution) described in this invention under the presence of different metal ions and interfering factors. The fluorescent probe Nap3 exhibits strong fluorescence enhancement and high affinity for zinc(II), while also showing strong fluorescence for Cd. 2+ There was also some response, and regarding Co 2+ Ni 2+ Cu 2+ and Cu + A slight fluorescence quenching was observed. The increase in carbon chain length in the linker arm and the introduction of a carbonyl group led to a slight decrease in specificity, but the PET effect caused by the carbonyl group resulted in low initial fluorescence. The fluorescence significantly recovered and increased after binding to zinc(II). This probe can achieve highly sensitive detection of zinc(II) in forage under specific conditions. This probe (Nap3) is sensitive to zinc ions (Zn... 2+ It exhibits excellent selectivity, with its fluorescence intensity response value far exceeding that of all other test ions. However, the introduction of the linker arm (2-amino-acetamido) keeps the DPA group away from the naphthimide fluorophore, making it difficult to use for copper ion detection. Simultaneously, the introduction of the carbonyl group on the linker arm to participate in the coordination system of DPA allows the system to be used for zinc ion detection. Although it is effective for cadmium ions (Cd... 2+ While exhibiting some cross-response, its signal intensity is significantly lower than that of zinc ions; and it shows a fluorescence quenching effect on transition metal ions such as cobalt and nickel, proving that the fluorescent probe Nap3 can achieve highly selective and sensitive specific recognition and detection of zinc ions in an environment with multiple coexisting interferences.

[0046] like Figure 10 and Figure 11 As shown, the fluorescence intensity of the fluorescent coordination-type naphthalimide fluorescent probe compound (10 μM) changed significantly after the addition of copper(II); when zinc(II) (0-50 μM) was added to the fluorescent probe Nap3 (10 μM), the fluorescence intensity gradually increased with the increase of zinc(II) concentration, and the fluorescence intensity showed a linear relationship with zinc(II) (0-10 μM) at 501 nm, as shown in the equation. y = 388.39 x +185550, R2 = 0.9976 ( y Fluorescence intensity x Zinc (II) concentration (nM) R 2 (Correlation coefficient). Based on 20 sets of blank measurements using the PBS matrix, and according to the limit of detection formula (detection limit is 3σ / S, where σ is the standard deviation of the blank measurement and S is the slope), the detection limit of the fluorescent probe Nap3 for zinc(II) was found to be 1.28 nM. The results indicate that the fluorescent probe Nap3 has very high sensitivity for the detection of zinc(II).

[0047] like Figure 12 As shown, in mixed solutions where multiple metal ions coexist, the coordination-type naphthalimide fluorescent probe compound of this invention can still preferentially bind to zinc(II). For ions that can cause fluorescence quenching (such as Fe), the binding remains constant. 2+ Co 2+ Ni 2+ Cu 2+ Cu + The fluorescence intensity ratio of the fluorescent probe Nap3 was significantly increased after the addition of zinc (II); while Cd² + The fluorescence enhancement effect produced after the addition of zinc(II) was consistent with that when zinc(II) was bound alone, indicating that it did not interfere with the selective recognition of zinc(II) by Nap3. Experiments showed that the probe could still achieve specific binding to zinc(II) even under the condition of coexistence of interfering ions.

[0048] like Figure 13 As shown, the working curve of the fluorescent coordination type naphthalimide fluorescent probe compound was analyzed by fluorescence spectral titration. When the molar fraction of fluorescent probe Nap3 and zinc (II) in the solution reached 0.5, the concentration of zinc (II) in the solution was equal to the concentration of the probe, and the fluorescence intensity reached its maximum value, indicating that the stoichiometric binding ratio of the probe to zinc (II) was 1:1.

[0049] like Figure 14 As shown, a reversibility test was performed to more clearly determine the response mechanism of the fluorescent probe Nap3 and zinc(II). After adding EDTA (diaminetetraacetic acid) to the complex formed by the fluorescent probe Nap3 and zinc(II), the fluorescence of Nap3 was reversibly restored, which further confirmed the interaction between the probe and zinc(II).

[0050] like Figure 15As shown, when the fluorescent probe Nap3 undergoes a coordination reaction with zinc(II), the carbonyl double bond of the linker group at the 2-position of the naphthalimide migrates, and the original carbonyl group is transformed into a hydroxyl group. At this point, zinc(II) forms a co-coordinated complex with DPA, the nitrogen atom of the linker group, and the carbonyl group of the naphthalimide. The strong electron-donating effect of the bound hydroxyl group causes a sharp increase in fluorescence.

[0051] Example 3: Application of Coordination-type Naphthalimide Fluorescent Probe Compounds The coordination-type naphthalimide fluorescent probe compound prepared in Example 1 was used to detect zinc(II) in alfalfa, oats, and clover.

[0052] Alfalfa, oats, and clover were dried and ground into powder. 2 grams of the powder were added to 50 ml of deionized water, stirred for 2 hours, and then centrifuged at 4000 rpm for 10 minutes. The supernatant was collected and the pH adjusted to 7 (to neutralize the interference of Cu ions). 2+ (Partial precipitation), then the filtrate was centrifuged three times until clear, filtered through a 0.22 μm microporous membrane and stored at 4℃ to obtain the test solution and measure the fluorescence.

[0053] Next, take the same amount of the three types of forage powder, and process the samples by wet digestion according to the pretreatment steps in the national standard GB 5009.268-2016 to prepare sample solutions for later use. The copper, zinc and cadmium contents in the three types of forage were determined by ICP-OES (Table 1).

[0054] The fluorescent coordination type naphthalimide fluorescent probe compound of the present invention was mixed with the above three forage test solutions to make the concentration of the fluorescent coordination type naphthalimide fluorescent probe compound 10 μM. The fluorescence intensity value was measured and substituted into the standard curve to calculate the zinc content (Table 2) and the spiked recovery rate (Table 3).

[0055] Table 1. Copper, zinc, and cadmium content in three types of forage as measured by ICP-OES.

[0056] Table 2. Zinc content in three types of forage measured by two methods.

[0057] Table 3 Spiked recovery rates of three forage substrates

[0058] like Figure 8 As shown, the fluorescent probe Nap3 is susceptible to Cu when detecting zinc(II). 2+ and Cd 2+ Interference. To suppress Cu 2+ Interference, in the preparation of fluorescent sample solutions, based on Cu 2+The difference in solubility product between zinc (II) and Cu effectively reduced the concentration of Cu by adjusting the solution pH. 2+ The effects of this on the forage were also observed. Meanwhile, ICP-OES measurements (Table 1) showed that the cadmium content in the three forages was only one percent of that of copper and one-thousandth of that of zinc; these extremely low concentrations resulted in very low cadmium levels. 2+ The impact is negligible in actual testing.

[0059] Table 2 shows that the free zinc content in alfalfa, oats, and clover measured by the fluorescent probe method was 8.1%, 7.8%, and 7.1% of the total zinc content measured by ICP-OES, respectively. The error was less than 5% when the test solutions were re-analyzed by the probe method using ICP-OES. Table 3 shows that the zinc (II) spiked recovery rate was 95%-102% in the above three matrices, with RSD < 3% (N = 3), indicating that Nap3 has the potential for quantitative detection of zinc (II) in forage.

[0060] In summary, this invention provides an application of a coordination-type naphthalimide fluorescent probe for detecting zinc(II) in forage and its preparation method. This method has good selectivity and high sensitivity, and can accurately detect zinc(II) qualitatively and quantitatively, which is of great significance for forage quality detection.

[0061] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent scheme adjustments, technical element substitutions or innovative improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A zinc(II)-coordinated naphthalimide fluorescent probe, characterized in that, The chemical structure of the coordination-type naphthimide fluorescent probe is as follows: , denoted as Nap3.

2. A method for preparing a zinc(II)-coordinated naphthalimide fluorescent probe as described in claim 1, characterized in that, The preparation method of the coordination-type naphthalimide fluorescent probe includes the following steps: (1) Preparation of intermediate c Intermediate a undergoes a nucleophilic substitution reaction with NaN3 in N,N-dimethylformamide to generate azide compound intermediate b; intermediate b is then reduced with Na2S in ethanol solvent to generate intermediate c. The chemical structural formula of intermediate a is as follows: ； The chemical structural formula of intermediate b is as follows: ； The chemical structural formula of intermediate c is as follows: ； (2) Preparation of intermediate d: Using triethylamine as an acid-binding agent, intermediate c is acylated with chloroacetyl chloride in dichloromethane solvent to generate N-chloroacetylated product intermediate d; the chemical structural formula of intermediate d is as follows: ； (3) Preparation of fluorescent probe Nap3: Using N,N-diisopropylethylamine as a base and potassium iodide as a catalyst, intermediate d undergoes a nucleophilic substitution reaction with dimethylpyridinium in acetonitrile solvent to synthesize fluorescent probe Nap3.

3. The method for preparing a zinc(II)-coordinated naphthalimide fluorescent probe as described in claim 2, characterized in that, In step (1) when preparing intermediate c, the molar ratio of intermediate a to NaN3 is 1:1.5 equivalents. NaN3 is dissolved in pure water and added to intermediate a under magnetic stirring. The mixture is then refluxed at 105°C for 8 hours under nitrogen protection. The molar ratio of intermediate b to Na2S is 1:2 equivalent. Na2S is added to intermediate b, and the reaction is carried out for 2 hours under magnetic stirring.

4. The preparation method according to claim 2, characterized in that, In step (2), during the preparation of intermediate d, the molar ratio of intermediate c to chloroacetyl chloride is 1:12 equivalent. Intermediate c is dissolved in dichloromethane. Triethylamine is cooled to 0°C in an ice-water bath under magnetic stirring, and then chloroacetyl chloride is added dropwise for 3 hours.

5. The preparation method according to claim 2, characterized in that, In step (3) of preparing the fluorescent probe Nap3, the molar ratio of intermediate d to dimethylpyridinium amine is 1:4 equivalent. Intermediate d is dissolved in acetonitrile, and dimethylpyridinium amine is added dropwise under magnetic stirring. The mixture is then refluxed at 60°C for 16 hours under nitrogen protection.

6. The application of the zinc(II)-coordinated naphthalimide fluorescent probe as described in claim 1, characterized in that, Used for qualitative or quantitative detection of zinc (II) in forage.

7. The application as described in claim 6, characterized in that, The forage grass is any one or a combination of alfalfa, oats, and clover.

8. The application as described in claim 7, characterized in that, The specific application methods are as follows: Step 1: After crushing and dissolving the forage sample by centrifugation, take the supernatant, adjust the pH of the supernatant to 7, and filter it through a microporous membrane to obtain the forage sample solution. Step 2: Add the fluorescent probe to the forage sample solution, measure the fluorescence intensity of the mixture at an excitation wavelength of 365 nm and an emission wavelength of 501 nm, and calculate the content of free zinc (II) in the forage sample to be tested according to the pre-established standard curve.

9. The application as described in claim 8, characterized in that, The fluorescent probe was added to the forage sample solution to a final concentration of 10 μM, and the reaction temperature was 20-25℃.

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