Copper (II) coordination type naphthalimide fluorescent probe, preparation method thereof and application of copper (II) coordination type naphthalimide fluorescent probe in pasture detection

Abstract

The invention discloses a copper (II) coordination type naphthalimide fluorescent probe and a preparation method and application thereof in pasture detection, and relates to the field of fluorescent probes, 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) preparing an intermediate b: preparing the intermediate b by using the intermediate a; (3) preparing an intermediate c: preparing the intermediate c by using the intermediate b; (4) preparing Nap2: preparing Nap2 by using the intermediate c; the raw materials for preparing the Nap2 are easy to obtain, the synthesis steps are simple, the operation is simple and convenient, and the Nap2 has high application value in the field of rapid detection of pasture copper pollution.

Description

Technical Field

[0001] The invention relates to the field of fluorescent probes, and in particular to a copper (II) coordinated naphthalene imide fluorescent probe, a preparation method thereof, and an application thereof in forage grass detection. Background Art

[0002] Copper (II), as an essential trace element for life, plays multiple roles in plant physiological processes. For example, it participates in key metabolic pathways such as photosynthesis, electron transfer in the respiratory chain, oxidative stress defense, and enzyme activity regulation. In forage grasses (such as alfalfa, oats, and clover), appropriate amounts of copper (II) can promote chlorophyll synthesis, enhance stress resistance, and increase yield. However, excessive intake can inhibit forage growth and affect animal health. For example, excessive accumulation of copper in the soil (such as industrial pollution or excessive fertilization) can lead to plant copper poisoning, which manifests as inhibition of root development, leaf yellowing, and even death; the enrichment of copper by forage grasses such as alfalfa, oats, and clover may lead to copper poisoning in ruminants, causing metabolic disorders and even death.

[0003] Traditional methods for detecting copper include atomic absorption spectroscopy, inductively coupled plasma mass spectrometry, spectrophotometry, electrochemical analysis, and chromatography. Although these methods have good selectivity and high sensitivity for copper (II) detection, they have high equipment costs, complex operating procedures, and long detection times, and cannot meet the needs of real-time in-situ online detection. Therefore, the development of a copper (II) fluorescent probe with high detection sensitivity, good selectivity, low cost, and simple operation provides theoretical support for the development of portable forage copper (II) rapid detection equipment. Summary of the invention

[0004] In order to solve the above technical problems, the present invention discloses a copper (II) coordinated naphthalene imide fluorescent probe and a preparation method thereof and application thereof in forage detection.

[0005] A copper (II) coordinated naphthalene imide fluorescent probe, the chemical structure of the coordinated naphthalene imide fluorescent probe is as follows: ( Figure 1 ), denoted as Nap2.

[0006] Moreover, the preparation method of the coordination type naphthalimide fluorescent probe comprises the following steps: (1) Preparation of intermediate a Weigh the conventional commercially available drug 2-bromo-1,8-naphthalene dicarboxylic anhydride (provided by Anhui Zesheng Technology Co., Ltd., CAS No.: 21563-29-1) and dissolve it in anhydrous ethanol, then add n-butylamine to react. After 2 hours, a large amount of precipitation is generated. The solvent is partially distilled under reduced pressure, and then ice water is added to precipitate the product. The filter cake is filtered and washed with ice ethanol to obtain intermediate a. The chemical structure of intermediate a is as follows: ; The chemical reaction formula of the preparation process of the intermediate a is as follows: ; (2) Preparation of intermediate b Weigh the intermediate a and dissolve it in a round-bottom flask using ethylene glycol methyl ether (MOE) as a solvent; then slowly dropwise add an appropriate amount of ethanolamine, heat to 120°C in an oil bath and reflux for 16 hours, remove the solvent by vacuum distillation, and purify by silica gel column chromatography to obtain a yellow solid product intermediate b; the chemical structure of the intermediate b is as follows: ; The chemical reaction formula of the preparation process of the intermediate b is as follows: ; (3) Preparation of intermediate c Weigh the intermediate b and tetrabutylammonium bromide (TBAB) and dissolve them in dichloromethane to obtain a mixed solution A; then weigh 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) and triphenylphosphine (PPh3) and dissolve them in dichloromethane to obtain a mixed solution B. Add the mixed solution A to the mixed solution B under stirring and react at room temperature for 6 hours. Remove the solvent by vacuum distillation and purify by silica gel column chromatography to obtain a yellow solid product intermediate c; the chemical structure of the intermediate c is as follows: ; The chemical reaction formula of the preparation process of the intermediate c is as follows: ; (4) Preparation of the coordinated naphthaleneimide fluorescent probe Nap2 Weigh compound c and place it in a round-bottom flask, add toluene to dissolve it, then add dimethylpyridiniumamine DPA and stir evenly, heat to 80 °C in an oil bath and reflux for 48 hours, remove the solvent by vacuum distillation, and purify the product by column chromatography on silica gel to obtain a yellow solid product, which is the probe Nap2. The chemical reaction formula is as follows: .

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

[0008] Moreover, in the preparation of intermediate b in step (2), the molar ratio of intermediate a to ethanolamine is 1:4 equivalents; the reaction in step (2) is carried out under the following conditions: firstly, intermediate a is dissolved in MOE, ethanolamine is slowly added dropwise under magnetic stirring, and the mixture is refluxed at 120° C. for 16 hours under nitrogen protection.

[0009] Moreover, in the preparation of intermediate c in step (3), the molar ratio of intermediate b to TBAB, DDQ, and PPh3 is 1:1.3:1.3:1.3 equivalents; the reaction in step (3) is carried out under the following conditions: firstly, intermediate b and TBAB are dissolved in dichloromethane to obtain liquid A; DDQ and PPh3 are dissolved in dichloromethane to obtain liquid B; and then the two are slowly mixed. The reaction is stirred at room temperature under nitrogen protection for 6 hours.

[0010] Moreover, in the preparation of the coordination naphthalene imide fluorescent probe Nap2 in step (4), the molar ratio of intermediate c to DPA is 1:4 equivalents; the reaction in step (4) is carried out under the following conditions: under magnetic stirring, DPA is slowly added dropwise, and the reaction is refluxed at 80° C. under nitrogen protection for 48 hours.

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

[0012] Moreover, the forage grass is any one or a combination of alfalfa, oats, and clover; the forage grass sample is reacted with the fluorescent probe after pretreatment, the fluorescence intensity of the reaction product is measured, and the copper (II) concentration in the forage grass is quantitatively detected as follows: Step 1, crushing the grass sample, dissolving it, and centrifuging it, taking the supernatant, and filtering it through a microporous filter to obtain a grass sample solution with a certain concentration of copper (II); Step 2: reacting the grass sample solution with the coordinated naphthalene imide fluorescent probe, and the reaction product can be preliminarily seen to change color under ultraviolet light to qualitatively detect copper (II) in the grass, and the fluorescence intensity of the reaction product can be measured to quantitatively detect copper (II) in the grass.

[0013] Moreover, the reaction is carried out under the condition of pH=7.4, and the reaction temperature is room temperature 20-25°C.

[0014] Moreover, the reaction is carried out at pH = 7.4 and at a reaction temperature of 20-25 °C.

[0015] The detection principle of the present invention is that the dimethylpyridylamine (DPA) structure detection group of the coordinated naphthalimide fluorescent probe is coordinated with copper (II) to produce fluorescence quenching, which can accurately detect the copper (II) content in forage. The method has fast detection speed, high sensitivity, good selectivity, simple operation, low cost, and can be used for the rapid detection of copper (II) in forage.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides a copper (II) coordinated naphthaleneimide fluorescent probe and a preparation method thereof and application in forage detection. The raw materials are readily available, the synthesis steps are simple, the operation is easy, and it has high application value in the field of rapid detection of copper contamination in forage.

[0017] 2. The present invention mainly overcomes the shortcomings of existing large-scale instruments for copper (II) detection, such as high cost and limited detection scenarios; the coordinated naphthylimide fluorescent probe can be used for rapid qualitative or quantitative detection of copper (II) in forage, and this method has the advantages of high detection sensitivity, good selectivity, low cost and simple operation.

[0018] 3. During the experiment, it was found that as the concentration of copper (II) increased, the fluorescence intensity gradually weakened and showed a linear relationship in the range of 0-20μM. Through calculation, it was found that the detection limit of the coordinated naphthylimide fluorescent probe was 7.56nM, which can very sensitively detect trace amounts of copper (II).

[0019] 4. During the experiment, it was found that even in a mixed solution with interference from multiple metal ions, the coordinated naphthylimide fluorescent probe can preferentially bind to copper (II). Moreover, even if the probe binds to zinc ions first, low concentrations of copper (II) can displace the zinc ions in the probe, proving that the probe has a strong specificity for copper (II). BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the structural formula of the coordination-type naphthalimide fluorescent probe compound of the present invention.

[0021] Figure 2 The synthetic route of the coordination-type naphthalimide fluorescent probe compound of the present invention is shown in FIG.

[0022] Figure 3 It is the nuclear magnetic resonance hydrogen spectrum of the coordination type naphthalimide fluorescent probe compound of the present invention.

[0023] Figure 4 It is the carbon nuclear magnetic resonance spectrum of the coordination type naphthalimide fluorescent probe compound of the present invention.

[0024] Figure 5 It is the mass spectrum of the coordination-type naphthalimide fluorescent probe compound of the present invention.

[0025] Figure 6 It is the excitation wavelength measured before and after the coordination-type naphthalimide fluorescent probe compound (20 μM) of the present invention is combined with copper (II) (40 μM) in a PBS buffer solution with pH=7.4.

[0026] Figure 7 It is the emission wavelength measured before and after the coordination-type naphthalimide fluorescent probe compound (10 μM) of the present invention is combined with copper (II) (20 μM) in a PBS buffer solution with a pH of 7.4.

[0027] Figure 8 The fluorescence spectra of the coordination-type naphthalimide fluorescent probe compound (10 μM) of the present invention are obtained by adding different interfering matrices into a PBS buffer solution at pH=7.4.

[0028] Fig. 9 This is a bar graph showing the addition of different metal ions and some interference factors to the coordination-type naphthalimide fluorescent probe compound (10 μM) of the present invention in a PBS buffer solution at pH=7.4.

[0029] Fig.10 The fluorescence intensity of the coordination naphthylimide fluorescent probe compound (20 μM) of the present invention is in a PBS buffer solution with a pH of 7.4, and when the copper (II) concentration gradually increases (0-50 μM), the fluorescence intensity change spectrum of the copper (II) concentration is plotted.

[0030] Fig.11 The linear relationship between the fluorescence intensity and the copper (II) concentration is shown when different concentrations of copper (II) (0-50 μM) are added to the coordination-type naphthalimide fluorescent probe compound (20 μM) of the present invention in a PBS buffer solution at pH=7.4.

[0031] Fig.12 The coordination-type naphthalimide fluorescent probe compound of the present invention is a detection specificity diagram of copper (II) in a mixed solution in a PBS buffer solution of pH=7.4 when the probe is interfered by different metal ions in the same system.

[0032] Fig.13 The competitive binding diagram of the coordination naphthalimide fluorescent probe compound (10 μM) of the present invention to the probe by the interfering ion zinc ion and the target ion copper (II) in a PBS buffer solution at pH=7.4. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with specific embodiments, and the features and advantages of the technical solution of the present invention will become clearer as the description proceeds. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0034] Example 1 Synthesis and characterization of coordinated naphthalene imide fluorescent probe The synthesis and characterization of the coordination type naphthalene imide fluorescent probe compound prepared in Example 1, the synthetic route of the fluorescent probe is as follows Figure 2 As shown, the steps include: 2-Bromo-1,8-naphthalene dicarboxylic anhydride (4 g, 14.44 mmol, 1 eq) was placed in a 100 mL round-bottom flask, 60 mL of anhydrous ethanol was added to the round-bottom flask to dissolve, and then n-butylamine (14 mL, 144 mmol, 10 eq) was added. The reaction solution was placed in an oil bath and heated to 80 °C for reflux reaction for 2 hours; TLC thin-layer chromatography monitoring, after the reaction was complete, the reaction solution was cooled, and ice water was added thereto to precipitate a large amount of solid, which was filtered using a Buchner funnel to obtain a gray loose crystalline crude product; the obtained product was then washed with ice ethanol and water to obtain intermediate a, and the yield was 83.68%.

[0035] 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).

[0036] Intermediate a 13 C NMR data are as follows 13 C NMR (126 MHz, DMSO- d 6) δ 163.15 (d, J = 6.3Hz), 132.90, 131.90, 131.69, 131.28, 129.12, 39.94, 29.98, 20.24, 14.13.

[0037] Intermediate a (2 g, 6 mmol, 1 eq) was placed in a 100 mL round-bottom flask, 60 mL MOE was added, heated to 60°C, and ethanolamine (1.44 mL, 24 mmol, 4 eq) was added dropwise under stirring. Vacuum nitrogen was circulated 3 times, and the temperature was raised to 120°C in an oil bath under nitrogen protection for reflux reaction for 16 h. After the addition of ethanolamine, the solution changed from turbid to dark brown and clear. After TLC thin-layer chromatography monitored the reaction to be complete, the solvent was removed by vacuum distillation, and the product showed bright yellow fluorescence under 365 nm ultraviolet light. The crude product was purified by column chromatography on silica gel, using a gradient elution of petroleum ether (PE): ethyl acetate (EA) = 1:1 (v / v). After the product was dried, 1.5 g of a yellow solid product was obtained, with a yield of 83.3%.

[0038] Intermediate b 1 The H NMR data are as follows: 1 H NMR (500 MHz, Chloroform- d ) δ 8.57 (dd, J =7.3, 1.6 Hz, 3H), 8.44 (dd, J = 8.3, 1.7 Hz, 3H), 8.14 (d, J = 8.4 Hz, 3H), 7.67 – 7.59 (m, 3H), 6.71 (dd, J = 8.4, 1.7 Hz, 3H), 5.74 (s, 1H), 4.15 (td, J = 7.5, 1.7 Hz, 5H), 3.86 (td, J = 6.0, 1.7 Hz, 6H), 3.75 (td, J = 6.0, 1.7 Hz, 6H), 1.75 – 1.65 (m, 6H), 1.43 (hd, J = 7.4, 1.7 Hz, 6H), 1.25 (s, 2H), 0.96 (td, J = 7.4, 1.7 Hz, 8H).

[0039] Intermediate b 13 C NMR data are as follows 13 C NMR (126 MHz, DMSO- d 6) δ 134.55, 130.99, 128.91, 124.57, 104.22, 59.27, 46.01, 39.36, 30.28, 20.31, 14.20.

[0040] MS (ESI) m / z: C 18 H 20 N2O3[M+H] + =313.1555.

[0041] The intermediate b (300 mg, 1 mmol, 1 eq) and tetrabutylammonium bromide (420 mg, 1.3 mmol, 1.3 eq) were dissolved in 15 mL CH2Cl2 to obtain solution A; DDQ (295 mg, 1.3 mmol, 1.3 eq) and PPh3 (341 mg, 1.3mmol, 1.3 eq) were weighed and dissolved in a 100 mL round-bottom flask with 15 mL CH2Cl2, and stirred to obtain solution B; solution A was slowly added to solution B under stirring, and the solution gradually turned red-brown. The reaction was carried out at room temperature for 6 h. The product showed green fluorescence under 365 nm ultraviolet light under TLC thin-layer chromatography monitoring. After the reaction was complete, the solvent was distilled off under reduced pressure. The product was purified by column chromatography on silica gel, and gradient eluted with an eluent of PE:EA=5:1 (v / v). After the product was dried, 328 mg of a yellow solid product was obtained, and the yield was 90.6%.

[0042] Intermediate c 1 The H NMR data are as follows: 1 H NMR (500 MHz, Chloroform- d ) δ 8.57 (dd, J =7.3, 1.6 Hz, 3H), 8.44 (dd, J = 8.3, 1.7 Hz, 3H), 8.14 (d, J = 8.4 Hz, 3H), 7.67 – 7.59 (m, 3H), 6.71 (dd, J = 8.4, 1.7 Hz, 3H), 5.74 (s, 1H), 4.15 (td, J = 7.5, 1.7 Hz, 5H), 3.86 (td, J = 6.0, 1.7 Hz, 6H), 3.75 (td, J = 6.0, 1.7 Hz, 6H), 1.75 – 1.65 (m, 6H), 1.43 (hd, J = 7.4, 1.7 Hz, 6H), 1.25 (s, 2H), 0.96 (td, J = 7.4, 1.7 Hz, 8H).

[0043] Intermediate c 13 C NMR data are as follows13 C NMR (126 MHz, Chloroform- d ) δ 164.55, 164.04, 148.28, 134.07, 131.23, 125.89, 125.13, 123.18, 120.47, 111.49, 104.48, 44.62, 40.05, 30.89, 30.30, 20.45, 13.94.

[0044] MS (ESI) m / z: C 18 H 19 BrN2O2[M+H] + =375.0708.

[0045] Weigh the compound. Weigh compound c (270 mg, 0.6 mmol, 1 eq) and place it in a 100 mL round-bottom flask. Add 60 mL PhCH3 to dissolve it and then drop DPA (478 mg, 2.4 mmol, 4 eq) under stirring. Vacuum nitrogen is circulated 3 times. Heat to 80°C in an oil bath under nitrogen protection and reflux for 48 h. After the reaction is complete, the solvent is removed by vacuum distillation by TLC thin-layer chromatography. The product exhibits green fluorescence under 365 nm ultraviolet light. The crude product is purified by column chromatography on silica gel, eluted with an eluent of CH2Cl2: MeOH = 500: 6 (v / v). After the product is dried, 105 mg of a yellow solid product is obtained with a yield of 35.5%.

[0046] Coordination-type naphthalene imide fluorescent probe compounds 1 The H NMR data are as follows: 1 H NMR (500 MHz, CDCl3) δ8.80 (dt, J = 8.4, 1.4 Hz, 1H), 8.64 – 8.54 (m, 3H), 8.40 (dd, J = 8.4, 1.6 Hz, 1H), 7.84 (s, 1H), 7.68 (ddd, J = 8.4, 7.3, 1.6 Hz, 1H), 7.56 (tt, J = 7.7, 1.8 Hz, 2H), 7.40– 7.34 (m, 2H), 7.27 (s, 0H), 7.14 (ddt, J = 7.5, 4.9, 1.4 Hz, 2H), 6.53 (dd, J = 8.5, 1.6 Hz, 1H), 4.17 (td, J = 7.4, 1.6 Hz, 2H), 4.01 (d,J = 1.6 Hz, 4H), 3.39 (q, J =5.2, 4.7 Hz, 2H), 3.09 – 3.03 (m, 2H), 1.71 (dddd, J = 11.3, 9.4, 5.5, 1.7 Hz, 2H), 1.44(dqd, J = 14.8, 7.3, 1.6 Hz, 2H), 1.25 (s, 1H), 0.97 (td, J = 7.4, 1.6 Hz, 3H).

[0047] Coordination-type naphthalene imide fluorescent probe compounds 13 C NMR data are as follows: 13 C NMR (126 MHz, CDCl3) δ158.67, 150.35, 149.21, 136.64, 134.73, 130.96, 130.01, 127.51, 124.28, 123.31, 122 .93, 122.36, 120.77, 109.25, 103.92, 59.69, 51.00, 40.92, 39.91, 30.35, 20.45, 13.91.

[0048] MS (ESI) m / z: C 30 H 31 N5O2[M+H] + =494.2559.

[0049] Coordination-type naphthalene imide fluorescent probe 1 H NMR images Figure 3 As shown, 13 C NMR Figure 4 As shown in the mass spectrum (MS), Figure 5 shown.

[0050] Example 2 Spectral response of probe molecules to copper (II) The fluorescent coordination type naphthalene imide fluorescent probe compound prepared in Example 1 was used to detect copper (II) in forage grass at pH = 7.4 with PBS as the dissolution matrix. The fluorescent probe was dissolved in dimethyl sulfoxide to prepare a probe mother solution with a concentration of 2 mM and a metal ion mother solution with a concentration of 2 mM.

[0051] Figure 6 is the excitation wavelength measured before and after the coordination-type naphthalimide fluorescent probe compound (20 μM) of the present invention is combined with copper (II) (40 μM) in a PBS buffer solution at pH=7.4; Figure 7The emission wavelength of the coordination-type naphthalimide fluorescent probe compound (10 μM) of the present invention before and after binding with copper (II) (20 μM) in a PBS buffer solution at pH=7.4; Figure 6 It can be seen that the excitation wavelength of the coordinated naphthalene imide fluorescent probe compound undergoes a blue shift before and after binding to copper (II), accompanied by an obvious quenching effect of the fluorescence intensity. Figure 7 It can be seen from the excitation wavelength that the wavelength of the coordinated naphthalimide fluorescent probe compound has almost no change before and after binding to copper (II). However, the PET effect produced by the DPA group attached to the naphthalimide fluorophore in the coordinated naphthalimide fluorescent probe compound binding to copper (II) leads to a significant fluorescence quenching effect.

[0052] Figure 8 Different interfering matrices were added to the coordination-type naphthalimide fluorescent probe compound (10 μM) in a PBS buffer solution at pH = 7.4: magnesium ions (Mg 2+ ), aluminum ions (Al 3+ ), chromium ion (Cr 3+ ), manganese ion (Mn 2+ ), ferrous ions (Fe 2+ ), iron ions (Fe 3+ ), copper ions (Cu 2+ ), cuprous ions (Cu + ), zinc ion (Zn 2+ ), silver ions (Ag + ), cadmium ions (Cd 2+ ), mercury ion (Hg 2+ ), lead ion (Pb 2+ ), glutathione (GSH), cysteine ​​(Cys), sulfide ion (S2-), sulfate ion (SO4 2- ), Vitamin C, Hydrogen peroxide (H2O2), Hypochlorite (ClO - ), chloride ion (Cl - ), nitrate (NO - ), the excitation wavelength is 458 nm and the emission wavelength is 536 nm.

[0053] Fig. 9 The bar graph of the coordination type naphthalimide fluorescent probe compound (10 μM) of the present invention added different metal ions and some interference factors in the PBS buffer solution of pH=7.4. As can be seen from the figure, the fluorescent coordination type naphthalimide fluorescent probe compound was added with 13 common metal ions magnesium ion (Mg 2+ ), aluminum ions (Al 3+ ), chromium ion (Cr 3+ ), manganese ion (Mn2+ ), ferrous ions (Fe 2+ ), iron ions (Fe 3+ ), copper ions (Cu 2+ ), cuprous ions (Cu + ), zinc ion (Zn 2+ ), silver ions (Ag + ), cadmium ions (Cd 2+ ), mercury ion (Hg 2+ ), lead ion (Pb 2+ ), and a variety of redox interfering substances and anions, including glutathione (GSH), cysteine ​​(Cys), sulfide ions (S 2- ), sulfate ion (SO4 2- ), Vitamin C, Hydrogen peroxide (H2O2), Hypochlorite (ClO - ), chloride ion (Cl - ), nitrate (NO - ) after the reaction, we can see from the fluorescence spectrum that the DPA ligand has different degrees of response to copper, zinc and cadmium due to its own properties; it can be seen in the figure that the probe has a certain fluorescence enhancement effect with zinc and cadmium, but the quenching response to copper (II) is the strongest; subsequent experiments have proved that the probe has a very high affinity for copper, and preferentially combines with copper (II) in the mixed solution system. Therefore, the coordination-type fluorescent coordination-type naphthaleneimide fluorescent probe compound described in the present invention can selectively detect copper (II) in forage under specific conditions.

[0054] like Fig.10 and Fig.11 As shown in the figure, the fluorescence intensity of the fluorescent coordination type naphthylimide fluorescent probe compound (20 μM) changed significantly after the addition of copper (II); the fluorescence intensity gradually weakened with the increase of copper (II) concentration, and the fluorescence intensity was linearly related to the copper (II) concentration in the range of 0-20 μM, and the equation was y = -27.584x + 890289, R² = 0.9876 (y: fluorescence intensity, X: copper (II) concentration (nM), R 2 : correlation coefficient), combined with the PBS matrix blank measurement value, the detection limit (detection limit is 3σ / S, σ: standard deviation of blank measurement value, S: slope) was calculated to be 7.56nM.

[0055] like Fig.12 As shown, the fluorescent coordination type naphthylimide fluorescent probe compound preferentially binds to copper (II) in a mixed solution interfered by multiple metal ions. Except for the zinc-copper mixed solution system where the fluorescence is slightly enhanced, other ions do not affect the fluorescence quenching effect of copper (II).

[0056] Fig.13 This is a competitive experiment between copper (II) and zinc ions. In order to eliminate the influence of zinc ions in the actual sample mixture system, the probe is first combined with zinc ions, and then copper (II) is added thereto in sequence. The results show that the affinity of copper (II) for the probe is much greater than that for zinc ions. Low concentrations of copper (II) can displace the zinc ions in the probe. Therefore, in actual detection, the probe has a strong specificity for copper (II).

[0057] Example 3 Application of coordination-type naphthaleneimide fluorescent probe compound The coordinated naphthalene imide fluorescent probe compound prepared in Example 1 was used to detect copper (II) in alfalfa, oats and clover.

[0058] After the three kinds of forage grasses were dried and crushed into powder, 2 grams of powder were weighed and added into 50 milliliters of deionized water to mix well, stirred for 2 hours, centrifuged at 4000 rpm for 10 minutes, the supernatant was taken to adjust the pH to 5, activated carbon was added to adsorb impurities and filtered, and the filtrate was taken and centrifuged repeatedly for 3 times until the solution was clear and transparent, and the solution was filtered through a 0.45 μm microporous filter membrane and stored at 4°C for later use to obtain the three kinds of forage grass test solutions.

[0059] Secondly, take the same amount of the three forage powders, treat the samples by wet digestion method according to the pre-treatment steps in the national standard method GB 5009.268-2016, prepare the sample solution for use, and use ICP-OES to determine the copper content in the three forages (Table 1).

[0060] The fluorescent coordination type naphthalimide fluorescent probe compound of the present invention was mixed with the above three forage test solutions so that the concentration of the fluorescent coordination type naphthalimide fluorescent probe compound was 20 μM, and the fluorescence intensity value was measured and substituted into the standard curve to calculate the copper content (Table 1) and the spiked recovery rate (Table 2).

[0061] Table 1: Copper content measured by two methods

[0062] Table 2: Spiked recoveries of three forage matrices

[0063] As shown in Table 1, the value measured by the fluorescent probe method is much lower than the copper content measured by the ICP-OES method. One of the reasons is that after wet digestion, the ICP-OES solution contains the total copper content of the sample, while the fluorescent probe method pretreatment step mainly measures the free copper (II) content in the sample, and excessive free copper content in plants directly leads to copper toxicity.

[0064] As shown in Table 2, in the three forage plant extract matrices of alfalfa, oats and clover, when the concentration of 5 μM was spiked for the first time in each group of experiments, the recovery value was too large, which may be due to the enhanced probe response caused by some residual organic matrix in the plant matrix; but the spiked recoveries were all between 95% and 105%, which met the detection requirements, indicating that the probe has stable detection performance for copper ions and is sensitive.

[0065] In summary, the present invention provides an application of a coordination naphthylimide fluorescent probe for detecting copper (II) in forage and a preparation method thereof. The method has good selectivity and high sensitivity, can accurately perform qualitative and quantitative detection of copper (II), and is of great significance for forage quality detection.

[0066] Finally, it should be noted that the above 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 replacements or innovative improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A copper (II) coordination type naphthalimide fluorescent probe, characterized in that: The chemical structure of the coordination-type naphthalene imide fluorescent probe is as follows: , denoted as Nap2.

2. A copper (II) coordinated naphthalimide fluorescent probe as claimed in claim 1, characterized in that: The preparation method of the coordination type naphthalimide fluorescent probe comprises the following steps: (1) Preparation of intermediate a Weigh 2-bromo-1,8-naphthalene dicarboxylic anhydride and dissolve it in anhydrous ethanol, then add n-butylamine, react for 2 hours, distill the solvent under reduced pressure, add ice water to precipitate the product, filter to obtain a filter cake, wash with ice ethanol to obtain intermediate a, the chemical structure of intermediate a is as follows: ； The chemical reaction formula of the preparation process of the intermediate a is as follows: ； (2) Preparation of intermediate b Weigh the intermediate a and dissolve it in ethylene glycol methyl ether as a solvent, then drop ethanolamine, heat to 120°C and reflux for 16 hours, remove the solvent by distillation under reduced pressure, and purify by silica gel column chromatography to obtain a yellow solid product intermediate b; the chemical structure of the intermediate b is as follows: ； The chemical reaction formula of the preparation process of the intermediate b is as follows: ； (3) Preparation of intermediate c Weigh the intermediate b and tetrabutylammonium bromide and dissolve them in dichloromethane to obtain a mixed solution A; then weigh 2,3-dichloro-5,6-dicyano-p-benzoquinone and triphenylphosphine and dissolve them in dichloromethane to obtain a mixed solution B. Add the mixed solution A to the mixed solution B under stirring, react at 20-25°C for 6 hours, remove the solvent by reduced pressure distillation, and purify by silica gel column chromatography to obtain a yellow solid product intermediate c. The chemical structure of the intermediate c is as follows: ； The chemical reaction formula of the preparation process of the intermediate c is as follows: ； (4) Preparation of the coordinated naphthaleneimide fluorescent probe Nap2 Weigh the intermediate c, add toluene to dissolve it, then add dimethylpyridinium amine and stir evenly, heat to 80°C and reflux for 48 hours, remove the solvent by vacuum distillation, and purify the product by column chromatography on silica gel to obtain a yellow solid product, which is the coordinated naphthalene imide fluorescent probe Nap2. The chemical reaction formula is as follows: 。 3. A copper (II) coordinated naphthalene imide fluorescent probe as claimed in claim 2, characterized in that: In the preparation of intermediate a in step (1), the molar ratio of 2-bromo-1,8-naphthalene dicarboxylic anhydride to n-butylamine is 1:1.5 equivalents; n-butylamine is then added and the mixture is refluxed at 80° C. for 2 hours.

4. A copper (II) coordinated naphthalene imide fluorescent probe as claimed in claim 2, characterized in that: In the preparation of intermediate b in step (2), the molar ratio of intermediate a to ethanolamine is 1:4 equivalents; after dropwise addition of ethanolamine, the reaction is refluxed at 120° C. for 16 hours under nitrogen protection.

5. A copper (II) coordinated naphthalimide fluorescent probe as claimed in claim 2, characterized in that: In the preparation of the intermediate c in step (3), the molar ratio of the intermediate b to tetrabutylammonium bromide, 2,3-dichloro-5,6-dicyanobenzoquinone, and triphenylphosphine (PPh3) is 1:1.3:1.3:1.3 equivalents.

6. A copper (II) coordinated naphthalimide fluorescent probe as claimed in claim 2, characterized in that: In the preparation of the coordination-type naphthalene imide fluorescent probe Nap2 in step (4), the molar ratio of the intermediate c to dimethylpyridinium amine is 1:4 equivalents, DPA is added dropwise under magnetic stirring, and the mixture is refluxed at 80° C. under nitrogen protection for 48 hours.

7. Use of a copper (II) coordination type naphthalene imide fluorescent probe as claimed in any one of claims 1 to 6, characterized in that: For the qualitative or quantitative determination of copper (II) in forage grasses.

8. The use of a copper (II) coordinated naphthalene imide fluorescent probe as claimed in claim 7, characterized in that: The forage grass is any one or a combination of alfalfa, oats and clover.

9. The use of a copper (II) coordinated naphthalene imide fluorescent probe as claimed in claim 8, characterized in that: The specific application methods are as follows: Step 1, crushing the grass sample, dissolving it, and centrifuging it, taking the supernatant, and filtering it through a microporous filter to obtain a grass sample solution with a certain concentration of copper (II); Step 2: reacting the grass sample solution with the coordinated naphthalene imide fluorescent probe, qualitatively detecting copper (II) in the grass according to the color change of the reaction product under ultraviolet light, and quantitatively detecting copper (II) in the grass by measuring the fluorescence intensity of the reaction product.

10. The use of a copper (II) coordinated naphthalimide fluorescent probe as claimed in claim 9, characterized in that: The reaction is carried out at pH = 7.4 and the reaction temperature is 20-25°C.

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