A Copper(II) Coordination-Type Naphthalimide Fluorescent Probe, Its Preparation Method and Application in Forage Detection

Abstract

The present invention discloses a copper (II) coordination type naphthalimide fluorescent probe, a preparation method thereof, and an application thereof in forage detection, relating to the field of fluorescent probes. The preparation method of the fluorescent probe is as follows: (1) Preparation of intermediate a: Intermediate a is prepared using 2-bromo-1,8-naphthalic anhydride; (2) Preparation of intermediate b: Intermediate b is prepared using intermediate a; (3) Preparation of intermediate c: Intermediate c is prepared using intermediate b; (4) Preparation of Nap2: Nap2 is prepared using intermediate c. The raw materials for preparing Nap2 in the present invention are easily available, the synthesis steps are simple, the operation is convenient, and it has high application value in the field of rapid detection of copper pollution in forage.

Description

Technical Field

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

[0002] As an essential trace element in living organisms, copper (II) plays multiple roles in plant physiological processes. For example, it participates in key metabolic pathways such as photosynthesis, respiratory chain electron transfer, oxidative stress defense, and enzyme activity regulation. In forages (such as alfalfa, oats, and clover), an appropriate amount of copper (II) can promote chlorophyll synthesis, enhance stress resistance, and increase yield. However, excessive intake will inhibit the growth of forages and affect animal health. For example, excessive accumulation of copper in the soil (such as industrial pollution or excessive fertilization) will cause plant copper poisoning, manifested as inhibited root development, leaf yellowing, and even death; the enrichment of copper in forages such as alfalfa, oats, and clover may cause copper poisoning in ruminants, leading to metabolic disorders and even death.

[0003] Traditional methods for detecting copper include atomic absorption spectrometry, inductively coupled plasma mass spectrometry, spectrophotometry, electrochemistry analysis, and chromatography analysis. Although these methods have good selectivity and high sensitivity for copper (II) detection, they have high equipment costs, complex operation processes, and long detection times, and cannot meet the requirements 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 rapid detection equipment for copper (II) in forages. Summary of the Invention

[0004] To solve the above technical problems, the present invention discloses a copper (II)-coordinated naphthalimide fluorescent probe, a preparation method thereof, and an application thereof in forage detection.

[0005] A copper (II)-coordinated naphthalimide fluorescent probe, the chemical structural formula of the coordinated naphthalimide fluorescent probe is as follows: ( Figure 1 ), denoted as Nap2.

[0006] Moreover, the preparation method of the coordinated naphthalimide fluorescent probe includes the following steps:

[0007] (1) Preparation of intermediate a

[0008] Weigh the conventional commercially available drug 2-bromo-1,8-naphthalic anhydride (provided by Anhui Zesheng Technology Co., Ltd., CAS No.: 21563-29-1), dissolve it in absolute ethanol, then add n-butylamine for reaction. After 2 hours, a large amount of precipitate is produced. Distill off part of the solvent under reduced pressure, then add ice water to precipitate the product. Filter by suction to obtain the filter cake, and wash it with ice ethanol to obtain intermediate a. The chemical structural formula of the intermediate a is as follows:

[0009] ;

[0010] The chemical reaction formula for the preparation process of the intermediate a is as follows:

[0011] ;

[0012] (2) Preparation of intermediate b

[0013] Weigh intermediate a, dissolve it in a round-bottom flask using ethylene glycol methyl ether (MOE) as the solvent; then slowly add an appropriate amount of ethanolamine dropwise, heat it to 120 °C in an oil bath and reflux for 16 hours. Distill off the solvent under reduced pressure, and purify it by silica gel column chromatography to obtain the yellow solid product intermediate b. The chemical structural formula of the intermediate b is as follows:

[0014] ;

[0015] The chemical reaction formula for the preparation process of the intermediate b is as follows:

[0016] ;

[0017] (3) Preparation of intermediate c

[0018] Weigh intermediate b and tetrabutylammonium bromide (TBAB), dissolve them in dichloromethane to obtain mixture A; then weigh 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) and triphenylphosphine (PPh3), dissolve them in dichloromethane to obtain mixture B. Add mixture A to mixture B under stirring, and react at room temperature for 6 hours. Distill off the solvent under reduced pressure, and purify it by silica gel column chromatography to obtain the yellow solid product intermediate c. The chemical structural formula of the intermediate c is as follows:

[0019] ;

[0020] The chemical reaction formula for the preparation process of the intermediate c is as follows:

[0021] ;

[0022] (4) Preparation of the coordination-type naphthalimide fluorescent probe Nap2

[0023] Weigh compound c and place it in a round-bottom flask. Add toluene to dissolve it, then add dimethylpyridineamine (DPA) and stir evenly. Heat it to 80 °C in an oil bath and reflux for 48 hours. Distill off the solvent under reduced pressure, and purify the product by column chromatography silica gel to obtain a yellow solid product, which is the probe Nap2. The chemical reaction formula is as follows:

[0024] .

[0025] Moreover, when preparing intermediate a in step (1), the molar ratio of 2-bromo-1,8-naphthalic 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 at 80 °C for 2 hours.

[0026] Moreover, when preparing 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: first dissolve intermediate a in MOE, slowly add ethanolamine dropwise under magnetic stirring, and reflux at 120 °C for 16 hours under nitrogen protection.

[0027] Moreover, when preparing 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: first dissolve intermediate b and TBAB in dichloromethane to obtain solution A; dissolve DDQ and PPh3 in dichloromethane to obtain solution B; then slowly mix the two. Stir at room temperature for 6 hours under nitrogen protection.

[0028] Moreover, when preparing the coordination-type naphthalimide 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, slowly add DPA dropwise, and reflux at 80 °C for 48 hours under nitrogen protection.

[0029] On the other hand, the present invention discloses an application of a copper (II) coordination-type naphthalimide fluorescent probe for qualitatively or quantitatively detecting copper (II) in forage.

[0030] Moreover, the forage is any one or a combination of alfalfa, oats, and clover; the forage sample is pretreated and then reacted with the fluorescent probe, and the fluorescence intensity of the reaction product is measured to quantitatively detect the copper (II) concentration in the forage, specifically as follows:

[0031] Step 1: Crush the forage sample, dissolve it, centrifuge it, take the supernatant, and obtain a forage sample solution with a certain concentration of copper (II) after passing through a microporous filter membrane;

[0032] Step 2: React the forage sample solution with the coordination-type naphthalimide fluorescent probe. The color change of the reaction product can be preliminarily observed under ultraviolet light for qualitative detection of copper (II) in forage, and the fluorescence intensity of the reaction product can be measured for quantitative detection of copper (II) in forage.

[0033] Moreover, the reaction is carried out under the condition of pH = 7.4, and the reaction temperature is room temperature of 20 - 25 °C.

[0034] Moreover, the reaction is carried out under the condition of pH = 7.4, and the reaction temperature is 20 - 25 °C.

[0035] The detection principle of the present invention is that the dimethylpyridineamine (DPA) structural detection group of the coordination-type naphthalimide fluorescent probe coordinates with copper (II), resulting in fluorescence quenching, and the content of copper (II) in forage can be accurately detected. This method has the advantages of fast detection speed, high sensitivity, good selectivity, simple operation, low cost, and can be used for the rapid detection of copper (II) in forage.

[0036] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0037] 1. The present invention provides a copper (II) coordination-type naphthalimide fluorescent probe, its preparation method and application in forage detection. The raw materials are easily available, the synthesis steps are simple, and the operation is convenient, which has high application value in the field of rapid detection of copper pollution in forage.

[0038] 2. The present invention mainly overcomes the disadvantages of high detection cost and limited detection scenarios of existing large-scale instruments for copper (II); the coordination-type naphthalimide fluorescent probe can rapidly perform 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.

[0039] 3. It is found in the experiment that as the concentration of copper (II) increases, the fluorescence intensity gradually weakens, and there is a linear relationship in the range of 0 - 20 μM. Through calculation, the detection limit of the coordination-type naphthalimide fluorescent probe is 7.56 nM, which can very sensitively detect trace amounts of copper (II).

[0040] 4. It is found in the experiment that even in a mixed solution with interference of multiple metal ions, the coordination-type naphthalimide fluorescent probe can preferentially bind to copper (II). Moreover, even if the probe binds to zinc ions first, low-concentration copper (II) can displace zinc ions in the probe, proving that the probe has strong specificity for copper (II). Description of the Drawings

[0041] Figure 1 It is the structural formula of the coordination-type naphthalimide fluorescent probe compound of the present invention.

[0042] Figure 2 This is the synthetic route diagram of the coordination-type naphthalimide fluorescent probe compound of the present invention.

[0043] Figure 3 This is the 1H NMR spectrum of the coordination-type naphthalimide fluorescent probe compound of the present invention.

[0044] Figure 4 This is the 13C NMR spectrum of the coordination-type naphthalimide fluorescent probe compound of the present invention.

[0045] Figure 5 This is the mass spectrum of the coordination-type naphthalimide fluorescent probe compound of the present invention.

[0046] Figure 6 This is the excitation wavelength measured before and after the binding of the coordination-type naphthalimide fluorescent probe compound (20 μM) of the present invention with copper (II) (40 μM) in a PBS buffer solution at pH = 7.4.

[0047] Figure 7 This is the emission wavelength measured before and after the binding of the coordination-type naphthalimide fluorescent probe compound (10 μM) of the present invention with copper (II) (20 μM) in a PBS buffer solution at pH = 7.4.

[0048] Figure 8 This is the fluorescence spectrum of the coordination-type naphthalimide fluorescent probe compound (10 μM) of the present invention in a PBS buffer solution at pH = 7.4 with different interfering matrices added.

[0049] Figure 9 This is the bar chart of different metal ions and some interfering factors added to the coordination-type naphthalimide fluorescent probe compound (10 μM) of the present invention in a PBS buffer solution at pH = 7.4.

[0050] Figure 10 This is the fluorescence intensity change spectrum diagram of the fluorescence intensity and copper (II) concentration when the concentration of copper (II) gradually increases (0 - 50 μM) for the coordination-type naphthalimide fluorescent probe compound (20 μM) of the present invention in a PBS buffer solution at pH = 7.4.

[0051] Figure 11 This is the linear relationship between the fluorescence intensity and the copper (II) concentration 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.

[0052] Figure 12This is the detection specificity diagram of copper (II) in the mixed solution when the coordination-type naphthalimide fluorescent probe compound of the present invention is interfered by different metal ions in the same system in a PBS buffer solution with pH = 7.4.

[0053] Figure 13 This is the competitive binding diagram of the interfering ion zinc ion and the target ion copper (II) to the probe of the coordination-type naphthalimide fluorescent probe compound (10 μM) of the present invention in a PBS buffer solution with pH = 7.4. Detailed implementation manners

[0054] The present invention will be further described below in conjunction with specific embodiments, and the features and advantages of the technical solutions of the present invention will become clearer with the description. 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.

[0055] Example 1 Synthesis and characterization of a coordination-type naphthalimide fluorescent probe

[0056] For the synthesis and characterization of the coordination-type naphthalimide fluorescent probe compound prepared in Example 1, the synthesis route diagram of the fluorescent probe is as Figure 2 shown, and the steps include:

[0057] Take 2-bromo-1,8-naphthalic anhydride (4 g, 14.44 mmol, 1 eq) and place it in a 100 mL round-bottom flask. Add 60 mL of absolute ethanol to the round-bottom flask to dissolve it, and then add n-butylamine (14 mL, 144 mmol, 10 eq). Place the reaction solution in an oil bath and heat it to 80 °C for reflux reaction for 2 hours; monitor by TLC thin-layer chromatography. After the reaction is complete, cool the reaction solution, add ice water to it to precipitate a large amount of solid, and obtain a gray, loose, crystalline crude product by suction filtration using a Buchner funnel; then wash the obtained product with ice ethanol and water to obtain intermediate a, and the yield is 83.68%.

[0058] The 1 1H NMR data of intermediate a is as follows 1 1H NMR (500 MHz, DMSO- d 6) δ 8.42 (ddt, J J = 21.3, 8.4, 1.1 Hz, 2H), 8.20 (dd, J J = 7.8, 1.0 Hz, 1H), 8.10 (dd, J 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).

[0059] For intermediate a 13 The \(^{13}\)C NMR data are as follows 13 \(^{13}\)C NMR (126 MHz, DMSO- d 6) δ 163.15 (d, J \(J\) = 6.3Hz), 132.90, 131.90, 131.69, 131.28, 129.12, 39.94, 29.98, 20.24, 14.13.

[0060] Intermediate a (2 g, 6 mmol, 1 eq) was placed in a 100 mL round-bottom flask. 60 mL of MOE was added thereto, and the mixture was heated to 60 °C. Then, ethanolamine (1.44 mL, 24 mmol, 4 eq) was added dropwise under stirring. The vacuum was evacuated and refilled with nitrogen three times. Under nitrogen protection, the temperature was raised to 120 °C in an oil bath and refluxed for 16 h. After adding ethanolamine, the solution changed from turbid to dark brown and clear. After monitoring the completion of the reaction by TLC thin-layer chromatography, the solvent was removed by distillation under reduced pressure. The product showed bright yellow fluorescence under a 365 nm ultraviolet lamp. The crude product was purified by column chromatography silica gel, eluted with a gradient of eluent of petroleum ether (PE):ethyl acetate (EA) = 1:1 (v / v). After drying the product, 1.5 g of yellow solid product was obtained, and the yield was 83.3%.

[0061] For intermediate b 1 The \(^1\)H NMR data are as follows: 1 \(^1\)H NMR (500 MHz, Chloroform- d ) δ 8.57 (dd, J \(J\) =7.3, 1.6 Hz, 3H), 8.44 (dd, J \(J\) = 8.3, 1.7 Hz, 3H), 8.14 (d, J \(J\) = 8.4 Hz, 3H), 7.67 – 7.59 (m, 3H), 6.71 (dd, J \(J\) = 8.4, 1.7 Hz, 3H), 5.74 (s, 1H), 4.15 (td, J \(J\) = 7.5, 1.7 Hz, 5H), 3.86 (td, J \(J\) = 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).

[0062] For intermediate b 13 The \(^{13}\)C NMR data are as follows 13 \(^{13}\)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.

[0063] MS (ESI) m / z: C 18 H 20 N₂O₃ [M + H] + = 313.1555.

[0064] Dissolve intermediate b (300 mg, 1 mmol, 1 eq) and tetrabutylammonium bromide (420 mg, 1.3 mmol, 1.3 eq) in 15 mL of CH₂Cl₂ to obtain solution A; then weigh DDQ (295 mg, 1.3 mmol, 1.3 eq) and PPh₃ (341 mg, 1.3 mmol, 1.3 eq) and dissolve them in 15 mL of CH₂Cl₂ in a 100 mL round-bottom flask, stir well to obtain solution B; slowly add solution A to solution B under stirring, the solution gradually turns reddish-brown, react at room temperature for 6 h, monitor by TLC thin-layer chromatography, the product shows green fluorescence under a 365 nm ultraviolet lamp, after the reaction is complete, distill off the solvent under reduced pressure, purify the product by column chromatography silica gel, elute with an eluent gradient of PE:EA = 5:1 (v / v), and obtain 328 mg of a yellow solid product after drying the product, with a yield of 90.6%.

[0065] For intermediate c 1 The \(^1\)H NMR data are as follows: 1 \(^1\)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).

[0066] The 13 C NMR data of intermediate c are as follows 13 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.

[0067] MS (ESI) m / z: C 18 H 19 BrN2O2 [M + H] + = 375.0708.

[0068] Weigh compound c (270 mg, 0.6 mmol, 1 eq) and place it in a 100 mL round-bottom flask. Add 60 mL of PhCH3 to dissolve it, and then dropwise add DPA (478 mg, 2.4 mmol, 4 eq) under stirring. Conduct three cycles of vacuum nitrogen circulation, and heat it to 80 °C in an oil bath under nitrogen protection for reflux reaction for 48 h. After monitoring the completion of the reaction by TLC thin-layer chromatography, distill off the solvent under reduced pressure. The product shows green fluorescence under a 365 nm ultraviolet lamp. The crude product is purified by column chromatography silica gel, eluted with an eluent of CH2Cl2:MeOH = 500:6 (v / v), and 105 mg of yellow solid product is obtained after drying the product, with a yield of 35.5%.

[0069] The 1 H NMR data of the coordination-type naphthalimide fluorescent probe compound are as follows: 1 H NMR (500 MHz, CDCl3) δ 8.80 (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).

[0070] The 13 C NMR data of the coordination-type naphthalimide fluorescent probe compound 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.

[0071] MS (ESI) m / z: C 30 H 31 N5O2 [M + H] + = 494.2559.

[0072] The 1 1H NMR spectrum of the coordination-type naphthalimide fluorescent probe is as Figure 3 shown 1313C NMR is as follows Figure 4 As shown, the mass spectrometry (MS) is as follows Figure 5 As shown

[0073] Example 2 Spectral Response of Probe Molecule to Copper (II)

[0074] The fluorescent coordination naphthalimide fluorescent probe compound prepared in Example 1 was used to detect copper (II) in forage under the condition that PBS was used as the dissolution matrix at pH = 7.4. The fluorescent probe was dissolved in dimethyl sulfoxide to prepare a mother liquor concentration of the probe of 2 mM and a mother liquor concentration of metal ions of 2 mM

[0075] Figure 6 This is the excitation wavelength measured before and after the binding of the coordination naphthalimide fluorescent probe compound (20 μM) of the present invention to copper (II) (40 μM) in a PBS buffer solution at pH = 7.4 Figure 7 This is the emission wavelength measured before and after the binding of the coordination naphthalimide fluorescent probe compound (10 μM) of the present invention to 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 coordination naphthalimide fluorescent probe compound shows a blue shift before and after binding to copper (II), accompanied by an obvious fluorescence quenching effect Figure 7 It can be seen from the excitation wavelength that the wavelength of the coordination naphthalimide fluorescent probe compound hardly changes before and after binding to copper (II). However, the PET effect generated after the binding of the DPA group connected to the naphthalimide fluorophore in the coordination naphthalimide fluorescent probe compound to copper (II) leads to an obvious fluorescence quenching effect

[0076] Figure 8 This is the coordination naphthalimide fluorescent probe compound (10 μM) of the present invention adding different interfering matrices in a PBS buffer solution at pH = 7.4: magnesium ion (Mg 2+ ), aluminum ion (Al 3+ ), chromium ion (Cr 3+ ), manganese ion (Mn 2+ ), ferrous ion (Fe 2+ ), ferric ion (Fe 3+ ), copper ion (Cu 2+ ), cuprous ion (Cu + ), zinc ion (Zn 2+ ), silver ion (Ag + ), cadmium ion (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 ion (NO - ). The excitation wavelength is 458 nm and the emission wavelength is 536 nm for the fluorescence spectra after these substances.

[0077] Figure 9 This is a bar chart of the coordination-type naphthalimide fluorescent probe compound of the present invention (10 μM) in PBS buffer solution at pH = 7.4 after adding different metal ions and some interfering factors. As can be seen from the figure, when the fluorescent coordination-type naphthalimide fluorescent probe compound reacts with 13 common metal ions, including magnesium ion (Mg 2+ ), aluminum ion (Al 3+ ), chromium ion (Cr 3+ ), manganese ion (Mn 2+ ), ferrous ion (Fe 2+ ), ferric ion (Fe 3+ ), copper ion (Cu 2+ ), cuprous ion (Cu + ), zinc ion (Zn 2+ ), silver ion (Ag + ), cadmium ion (Cd 2+ ), mercury ion (Hg 2+ ), lead ion (Pb 2+ ), and various redox interfering substances and anions, including glutathione (GSH), cysteine (Cys), sulfide ion (S 2- ), sulfate ion (SO4 2- ), Vitamin C, hydrogen peroxide (H2O2), hypochlorite (ClO - ), chloride ion (Cl - ), nitrate ion (NO - ), it can be seen from the fluorescence spectra that the DPA ligand has different degrees of response to copper, zinc, and cadmium due to its own properties; it can be seen from the figure that the probe shows a certain fluorescence enhancement effect with zinc and cadmium, but the quenching response to copper (II) is the most intense; subsequent experiments prove 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 naphthalimide fluorescent probe compound described in the present invention can selectively detect copper (II) in forage under specific conditions.

[0078] Such as Figure 10 And Figure 11As shown, the fluorescence intensity of the fluorescent coordination-type naphthalimide fluorescent probe compound (20 μM) changes significantly after the addition of copper (II); as the concentration of copper (II) increases, the fluorescence intensity gradually weakens. The fluorescence intensity shows a linear relationship with the copper (II) concentration in the range of 0 - 20 μM, and the equation is y = -27.584x + 890289, R² = 0.9876 (y: fluorescence intensity, X: copper (II) concentration (nM), R 2 : correlation coefficient). The detection limit (the detection limit is 3σ / S, σ: standard deviation of the blank measurement value, S: slope) was calculated to be 7.56 nM by combining with the measured value of the PBS matrix blank.

[0079] As Figure 12 shown, the fluorescent coordination-type naphthalimide fluorescent probe compound preferentially binds to copper (II) in a mixed solution interfered by various metal ions. Except for a slight increase in fluorescence in the zinc-copper mixed solution system, other ions do not affect the fluorescence quenching effect of copper (II).

[0080] Figure 13 This is a competitive experiment between copper (II) and zinc ions. To exclude the influence of zinc ions in the actual sample mixing system, the probe was first combined with zinc ions, and then copper (II) was added to it successively; the results showed that the affinity of copper (II) for the probe is much greater than that of zinc ions, and low-concentration copper (II) can displace zinc ions in the probe. Therefore, this probe has strong specificity for copper (II) in actual detection.

[0081] Example 3 Application of the coordination-type naphthalimide fluorescent probe compound

[0082] The coordination-type naphthalimide fluorescent probe compound prepared in Example 1 was used to detect copper (II) in alfalfa, oats, and clover.

[0083] After drying and crushing the three kinds of forages into powder, 2 grams of the powder was weighed, added to 50 ml of deionized water and mixed evenly, stirred for 2 hours, centrifuged at 4000 rpm for 10 minutes in a centrifuge, the supernatant was taken and the pH was adjusted to 5, activated carbon was added to adsorb impurities and filtered, and then the filtrate was centrifuged repeatedly 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 standby to obtain the test solutions of the three kinds of forages.

[0084] Secondly, the same amount of the three kinds of forage powders was taken, and the samples were processed by the wet digestion method according to the pretreatment steps in the national standard method GB 5009.268-2016 to prepare the sample solutions for standby, and the copper contents in the three kinds of forages were measured by ICP-OES (Table 1).

[0085] Mix the fluorescent coordination-type naphthalimide fluorescent probe compound of the present invention with the above three forage grass test solutions so that the concentration of the fluorescent coordination-type naphthalimide fluorescent probe compound therein is 20 μM, measure the fluorescence intensity value, substitute it into the standard curve, and calculate the copper content (Table 1) and the spike recovery rate (Table 2).

[0086] Table 1: Copper contents measured by two methods

[0087]

[0088] Table 2: Spike recovery rates of three forage grass matrices

[0089]

[0090] As can be seen from Table 1, the measured value 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 by ICP-OES, the total copper content of the sample is in the solution, while the pretreatment step of the fluorescent probe method mainly measures the free copper (II) content in the sample, and the excessive free copper content in plants directly causes copper toxicity.

[0091] As can be seen from Table 2, in the matrix of the extraction solutions of three forage grass plants, alfalfa, oats, and clover, when spiked with a concentration of 5 μM for the first time in each group of experiments, the relatively large recovery rate may be due to the enhancement of the probe response caused by some residual organic matrices in the plant matrix; however, the spike recovery rates are all between 95% and 105%, meeting the detection requirements, indicating that the probe has stable detection performance for copper ions and sensitive reaction.

[0092] In summary, the present invention provides an application of a coordination-type naphthalimide fluorescent probe for detecting copper (II) in forage grass and its preparation method. This method has good selectivity and high sensitivity, can accurately qualitatively and quantitatively detect copper (II), and is of great significance for the detection of forage grass quality.

[0093] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modification, equivalent scheme adjustment, technical element replacement, or innovative improvement made within the spirit and principle of the present invention shall be included within 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. The method for preparing a copper (II) coordinated naphthalimide fluorescent probe according to claim 1, characterized in that: The following steps are involved: (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. The method for preparing a copper (II) coordinated naphthalene imide fluorescent probe according to 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. The method for preparing a copper (II) coordinated naphthalene imide fluorescent probe according to 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. The method for preparing a copper (II) coordinated naphthalene imide fluorescent probe according to 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. The method for preparing a copper (II) coordinated naphthalene imide fluorescent probe according to 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. The use of a copper (II) coordinated naphthalene imide fluorescent probe as claimed in claim 1, 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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