A fluorescence probe for detecting copper (I) in forage grasses, and a preparation method and application thereof

Abstract

The application discloses a fluorescence probe for detecting copper (I) in pasture, and a preparation method and application thereof, relates to the field of pasture detection and fluorescence probes, and the preparation method of the fluorescence probe is as follows: (1) preparation of an intermediate a: the intermediate a is prepared by using 2-bromo-1,8 naphthalene dicarboxylic anhydride; (2) preparation of a probe Nap1: the Nap1 is prepared by using the intermediate a; the probe Nap1 provided by the application introduces a DPA group at the 2nd position of naphthalimide, the particularity of the spatial position makes the probe have high specificity for copper (I), almost no reaction is generated for other metal ions, the response of the probe to copper (I) is significantly higher than that to copper (II), the probe is not easily disturbed by other substances in detection, and the probe can be used for selectively detecting copper (I) in pasture.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of forage detection and fluorescent probes, and particularly relates to a fluorescent probe for detecting copper (I) in forage and a preparation method and application thereof. BACKGROUND

[0002] Copper is one of the essential trace elements for life. As the redox active cofactor of a variety of enzymes, copper is involved in important physiological functions such as respiration, synthesis and metabolism of neurotransmitters, gene expression, and antioxidant defense. In forage, copper mainly exists in two forms: free copper and bound copper. Free copper is in the form of free ions, mainly distributed in the cytoplasm and cell sap. This form of copper has high chemical activity and availability, and can directly participate in the redox reaction in the body. In contrast, bound copper is tightly bound to proteins, enzymes or other biological macromolecules to form stable complexes. Its chemical activity is relatively low, and it can only be released or activated under specific physiological conditions to exert its biological function. In practical applications, free copper can be obtained more from forage through a simple and rapid physical extraction process. However, excessive free copper can generate hydroxyl radicals through the Fenton reaction, which in turn can cause oxidative damage to proteins, lipids and nucleic acids, thereby causing harm to the organism. Secondly, in mitochondria, there are a large number of reducing agents (glutathione, etc.), so that copper mainly exists in the form of Cu + in cells. Based on this characteristic, on the one hand, the Cu + probe can be used for the determination of Cu + concentration in vitro, providing basic data for research; on the other hand, it can also be used for spatial imaging of Cu + in subcellular structures, and further study the physiological processes of cells.

[0003] Traditional copper detection methods such as atomic absorption spectrometry and ICP-MS have high sensitivity, but are expensive, complex to operate and difficult to detect in real time in situ. Small molecule fluorescent probes have the advantages of adjustable structure, good selectivity, simple operation and easy modification, so it is of great significance to develop a fluorescent probe for copper (I) with high sensitivity, low cost and suitable for forage. SUMMARY

[0004] To solve the above technical problems, the present application discloses a fluorescent probe for detecting copper (I) in forage and a preparation method and application thereof, which are as follows:

[0005] A copper (I) coordination naphthalimide fluorescent probe, the chemical structural formula of the coordination naphthalimide fluorescent probe is as follows:

[0006] Figure 1 , denoted as Nap1. ​

[0007] Further, the preparation method of the coordination type naphthalimide fluorescent probe comprises the following steps:

[0008] (1) Preparation of intermediate a

[0009] Weigh the commercially available 2-bromo-1,8 naphthalene dianhydride and dissolve it in anhydrous ethanol, then add n-butylamine for reaction. After 2 hours, a large amount of precipitate is produced. Remove part of the solvent by distillation under reduced pressure, then add ice water to precipitate the product. After suction filtration, the filter cake is obtained and washed with ice ethanol (EtOH) to obtain intermediate a, and the chemical structural formula of the intermediate a is as follows:

[0010] ;

[0011] The chemical reaction formula of the preparation process of the intermediate a is as follows:

[0012] ;

[0013] : n-butylamine;

[0014] : 2-bromo-1,8 naphthalene dianhydride;

[0015] (2) Preparation of coordination type naphthalimide fluorescent probe Nap1

[0016] Weigh compound a and place it in a round-bottom flask, then add ethylene glycol methyl ether (MOE). After heating and dissolving, add dimethylpyridine amine (DPA). Under nitrogen protection, the reaction system is heated to 110°C in an oil bath and refluxed for 48 hours. After the reaction is completed, the solvent is removed by distillation under reduced pressure. The obtained crude product is purified by column chromatography on silica gel to obtain yellow solid product, i.e. probe Nap1, and the chemical reaction formula is as follows:

[0017] ;

[0018] : dimethylpyridine amine.

[0019] Further, in the preparation of the intermediate a in step (1), the molar ratio of 2-bromo-1,8 naphthalene dianhydride to n-butylamine is 1:1.5 equivalent; and the reaction in step (1) is carried out under the following conditions: under magnetic stirring, refluxing at 80°C for 2 hours.

[0020] Further, in the preparation of the intermediate b in step (2), the ratio of intermediate a to DPA is preferably 1:4 equivalent; and the reaction in step (2) is carried out under the following conditions: under magnetic stirring, slowly adding DPA, refluxing at 110°C under nitrogen protection for 48 hours.

[0021] In another aspect, the application discloses application of a copper (I) coordination type naphthalimide fluorescent probe to quantitative detection of copper (I) in forage grasses.

[0022] Moreover, the forage grass is any one or a combination of alfalfa, oat and clover; the forage grass sample is reacted with the fluorescent probe after pretreatment, and fluorescence intensity of the reaction product is determined to quantitatively detect the copper (I) concentration in the forage grass, specifically as follows:

[0023] (1) Preparation of forage grass sample to be detected: the forage grass sample is dried and crushed, mixed with water, and centrifuged to obtain supernatant; vitamin C is added to the supernatant, and the pH is adjusted to 5.5-6.5; after centrifugation and filtration, the forage grass sample to be detected is obtained; the amount of the added vitamin C is sufficient to reduce Cu 2 ⁺ in the sample to be detected to Cu⁺, and the excessive vitamin C does not interfere with subsequent fluorescence detection;

[0024] (2) Qualitative detection: the copper (I) coordination type naphthalimide fluorescent probe is added to the forage grass sample to be detected, and the reaction product can be preliminarily detected for color change under ultraviolet light to qualitatively detect copper (I) in the forage grass.

[0025] (3) Quantitative detection: the fluorescence intensity is determined, and the concentration of total free copper in the forage grass sample to be detected is calculated according to the fluorescence intensity and a standard curve.

[0026] Moreover, the pH is adjusted to 6, and the detection temperature is 20-25℃.

[0027] Moreover, in step (2), the wavelength for measuring the fluorescence intensity is 527 nm.

[0028] Moreover, the final concentration of the fluorescent probe in the detection system is 18-22 μM, preferably 20 μM.

[0029] The detection principle of the application is that the DPA structure detection group of the coordination type naphthalimide fluorescent probe is combined with copper (I) to produce fluorescence opening, so that the copper (I) content in the forage grass can be accurately detected. The method has the advantages of fast detection speed, high sensitivity, good selectivity, simple operation, low cost and can be used for rapid detection of copper (I) in forage grass.

[0030] Compared with the prior art, the application has the following beneficial effects:

[0031] 1. The probe Nap1 provided by the application introduces a DPA group at the 2nd position of naphthalimide, and the particularity of the spatial position makes it have high specificity for copper (I) and almost no reaction with other metal ions, and the response to copper (I) is significantly higher than that to copper (II), so that the probe is not easily disturbed by other substances in detection and can selectively detect copper (I) in forage grass.

[0032] 2. The probe Nap1 provided by the application has a good linear relationship with Cu + at 527 nm, and the correlation coefficient R 2 = 0.9809, and the detection limit of copper (I) is 10.19 nM, and the copper (I) with low concentration can be accurately detected.

[0033] 3. After adding the chelating agent BCP (bathocuproin) with strong chelation effect on copper (I) to the complex formed by the probe Nap1 and copper (I), the fluorescence reversibility of Nap1 is restored, which further proves the interaction between the probe and copper (I), and provides the possibility for repeated use and in-depth research of the probe.

[0034] 4. In the application, by adding vitamin C to the forage solution, the Cu 2+ interference is effectively eliminated, and the Cu 2+ is reduced to Cu + , and the total free copper is quantified by measuring the Cu + concentration, and the excess vitamin C does not affect the detection system.

[0035] 5. The total free copper content of alfalfa, oat and clover measured by the probe Nap1 provided by the application is consistent with the results measured by the ICP-OES method, and the Cu + spiked recovery rate reaches 95% to 105%, and the RSD is less than 5% (N=3), which indicates that the fluorescent probe Nap1 is suitable for the quantitative detection of total free copper in forage, and provides an accurate and reliable method for forage quality detection.

[0036] 6. The application provides a copper (I) coordination type naphthalimide fluorescent probe, a preparation method thereof and application in forage detection, raw materials are easy to obtain, synthesis steps are simple, and operation is simple, and the copper (I) coordination type naphthalimide fluorescent probe has high application value in the field of rapid detection of forage zinc pollution.

[0037] 7. The application mainly overcomes the shortcomings that the detection cost of copper (I) is high and the detection scene is limited by using a large instrument; the copper (I) in forage can be rapidly qualitatively or quantitatively detected by using the coordination type naphthalimide fluorescent probe, and the method has the advantages of high detection sensitivity, low cost and simple operation. DETAILED DESCRIPTION

[0038] Figure 1 The structural formula of the coordination type naphthalimide fluorescent probe compound in the application is shown in the figure;

[0039] Figure 2 The synthesis route map of the coordination type naphthalimide fluorescent probe compound in the application is shown in the figure;

[0040] Figure 3 The nuclear magnetic resonance hydrogen spectrum of the coordination type naphthalimide fluorescent probe compound of the application;

[0041] Figure 4 The nuclear magnetic resonance carbon spectrum of the coordination type naphthalimide fluorescent probe compound of the application;

[0042] Figure 5 The mass spectrum of the coordination type naphthalimide fluorescent probe compound of the application;

[0043] Figure 6 The excitation wavelength of the coordination type naphthalimide fluorescent probe compound (20 μM) of the application before and after being combined with copper (I) (40 μM) in a PBS buffer solution with pH = 7.4;

[0044] Figure 7 The emission wavelength of the coordination type naphthalimide fluorescent probe compound (20 μM) of the application before and after being combined with copper (I) (40 μM) in a PBS buffer solution with pH = 7.4;

[0045] Figure 8 The fluorescence spectrum of the coordination type naphthalimide fluorescent probe compound (20 μM) of the application in a PBS buffer solution with pH = 7.4 under the addition of different interference substrates;

[0046] Figure 9 The columnar chart of the coordination type naphthalimide fluorescent probe compound (20 μM) of the application in a PBS buffer solution with pH = 7.4 under the addition of different metal ions and part of interference factors;

[0047] Figure 10 The fluorescence intensity change spectrum of the coordination type naphthalimide fluorescent probe compound (20 μM) of the application in a PBS buffer solution with pH = 7.4 when the concentration of copper (I) gradually increases (0-50 μM), and the fluorescence intensity and the concentration of copper (I);

[0048] Figure 11 The linear relationship of the fluorescence intensity and the concentration of copper (I) when the coordination type naphthalimide fluorescent probe compound (20 μM) of the application in a PBS buffer solution with pH = 7.4 is added with different concentrations of copper (I) (0-50 μM);

[0049] Figure 12 The working curve of the coordination type naphthalimide fluorescent probe compound of the application combined with copper (I) in a PBS buffer solution with pH = 7.4;

[0050] Figure 13 The reversibility test of the coordination type naphthalimide fluorescent probe compound (20 μM) of the application combined with copper (I) in a PBS buffer solution with pH = 7.4;

[0051] Figure 14 The binding mechanism diagram of the coordination type naphthalimide fluorescent probe compound of the application and copper (I);

[0052] Figure 15 The fluorescence spectrum diagram of the fluorescent probe Nap2 in the PBS buffer solution with pH = 7.4 under the addition of different interference substrates;

[0053] Figure 16 The column chart of the fluorescent probe Nap2 in the PBS buffer solution with pH = 7.4 under the addition of different metal ions and part of interference factors. DETAILED DESCRIPTION

[0054] The technical scheme of the application will be further described below in combination with specific embodiments, and the features and advantages of the application will be clearer with the description. It should be understood that the specific embodiments described herein are only used to explain the application, and are not used to limit the application.

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

[0056] The synthesis of the coordination type naphthalimide fluorescent probe is shown in the synthesis route diagram as Figure 2 The steps include:

[0057] 2-bromo-1,8 naphthalic 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 for dissolution, then n-butylamine (2.1 mL, 21.6 mmol, 1.5 eq) was added, and the reaction liquid was placed in an oil bath pot and warmed to 80 ℃ for reflux reaction for 2 hours; TLC thin layer chromatography was used for monitoring, after the reaction was completed, the reaction liquid was cooled, a large amount of solid was precipitated after ice water was added, and the crude product in the form of gray loose crystals was obtained by using a Buchner funnel for suction filtration; then the obtained product was washed with ice ethanol and water to obtain intermediate a, and the yield was 83.68%.

[0058] The H NMR data of intermediate a are as follows 1 The H NMR data of intermediate a 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)。

[0059] Intermediate a of 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。

[0060] Compound a (0.66 g, 2 mmol, 1 eq) was accurately weighed and placed in a 100 mL round bottom flask. To the flask was added 60 mL MOE (methyl glycol ether) and heated to 60 °C. Under stirring condition, DPA (1.45 mL, 8 mmol, 4 eq) was added slowly. After three vacuum nitrogen cycles, the reaction mixture was placed in an oil bath and heated to 110 °C under nitrogen protection and refluxed for 48 h. The reaction progress was monitored by TLC thin layer chromatography, after the reaction was completed, the solvent was removed by distillation under reduced pressure, the resulting product had UV absorption at 256 nm and green fluorescence at 365 nm. The crude product was purified by column chromatography on silica gel, eluted with a gradient of PE:EA = 1:1 (v / v) to PE:EA = 1:2 (v / v). After the product was dried, a yellow solid product 0.11 g was obtained, which was a coordination naphthalimide fluorescent probe, with a yield of 12.2%, and the binding mechanism diagram is shown in Figure 14 .

[0061] Preparation of coordination naphthalimide fluorescent probe compounds 1 H NMR data are as follows: 1 H NMR (500 MHz, DMSO- d 6)δ 8.88 (d, J = 8.5 Hz, 4H), 8.56 – 8.47 (m, 8H), 8.37 (d, J = 7.2 Hz, 4H),8.16 (dd, J= 8.2, 1.6 Hz, 4H), 7.78–7.64 (m, 13H), 7.52–7.48 (m, 1H), 7.42(d, J = 7.8 Hz, 8H), 7.21 (td, J = 5.4, 2.4 Hz, 12H), 3.93 (t, J = 7.5 Hz,8H), 3.43 (s, 6H), 1.51 (dt, J = 14.5, 7.1 Hz, 9H), 1.33 – 1.20 (m, 9H), 0.85(td, J = 7.4, 1.6 Hz, 13H)。

[0062] Coordination-type naphthalimide fluorescent probe compounds 13 C NMR data are as follows: 13 C NMR (126 MHz, DMSO- d 6) δ 163.85, 163.24, 157.69, 154.17, 149.65, 137.20, 131.85, 130.97 (d, J =8.6 Hz), 129.76, 126.29 (d, J = 7.2 Hz), 122.93, 122.75, 117.44, 115.55,59.51, 39.52, 30.10, 20.22, 14.11.

[0063] MS (ESI) m / z: C 28 H 26 N4O2[M+H] + = 451.2127.

[0064] Coordination-type naphthalimide fluorescent probe 1 H NMR chart as Figure 3 shown, 13 C NMR as Figure 4 shown, mass spectrum chart as Figure 5 shown.

[0065] Experimental Example 2 Spectral response of probe molecule to copper (I)

[0066] The fluorescent coordination naphthalimide fluorescent probe compound prepared in Example 1 was used to detect copper (I) in grass under the condition that PBS was used as a dissolving base with pH = 7.4, the fluorescent probe was dissolved in dimethyl sulfoxide to prepare a probe mother liquor with a concentration of 2 mM, and a metal ion mother liquor with a concentration of 2 mM.

[0067] Figure 6 The ultraviolet absorption wavelength of the coordination naphthalimide fluorescent probe compound (20 μM) of the application before and after being combined with copper (I) (40 μM) in a PBS buffer solution with pH = 7.4 was measured, and the wavelength was used as the probe excitation wavelength in subsequent spectral experiments;

[0068] Figure 7 The emission wavelength of the coordination naphthalimide fluorescent probe compound (20 μM) of the application before and after being combined with copper (I) (40 μM) in a PBS buffer solution with pH = 7.4 was measured; after being combined with copper (I), the ultraviolet absorption value of the probe slightly decreased, and the fluorescence intensity significantly weakened, which indicated that the solubility of the probe was enhanced after being combined with copper (I) and the fluorescence quenching effect was significant.

[0069] Figure 8 The fluorescent spectrum of the coordination naphthalimide fluorescent probe compound (20 μM) of the application in a PBS buffer solution with pH = 7.4 after being added with different interference substrates: magnesium ion (Mg 2+ ), aluminum ion (Al 3+ ), chromium ion (Cr 3+ ), manganese ion (Mn 2+ ), ferrous ion (Fe 2+ ), ferric ion (Fe 3+ ), cobalt ion (Co 2+ ), nickel ion (Ni 2+ ), 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), sulfur ion (S 2- ), sulfate ion (SO4 2- ), vitamin C (Vitamin C), hydrogen peroxide (H2O2), hypochlorite (ClO - ), chloride ion (Cl - ), nitrate (NO - ) was measured, the excitation wavelength was 432 nm, and the emission wavelength was 527 nm. In the presence of many interference substances, only Cu+ cause significant quenching of the probe fluorescence intensity, while other ions or molecules (such as Cu 2 + , Zn 2+ , Fe 2+ , Fe 2+ , GSH, Cys, H2O2, Vitamin C, etc.) have little or no effect on the fluorescence signal of the probe, even under high concentration of interference coexistence conditions, the probe can specifically recognize Cu + , indicating that it has good anti-interference performance and is suitable for specific detection of Cu⁺ in complex matrix; the naphthalimide carbonyl group and the DPA (dimethylpyridylamine) group of the probe molecule are close to each other, and the coordination system formed has strong coordination ability for Cu + , and because the carbonyl group is introduced into the coordination system, the coordination ability for the more electronegative Lewis acid Cu + is enhanced, and its spatial structure effectively avoids non-specific binding of other metal ions, thereby achieving high selective recognition of Cu + .

[0070] Figure 9 The column chart of the coordination type naphthalimide fluorescent probe compound (20 μM) of the application in a pH=7.4 PBS buffer solution adding different metal ions and some interference factors. As can be seen from the figure, the common metal ions of the fluorescent coordination type naphthalimide fluorescent probe compound, magnesium ion (Mg 2+ ), aluminum ion (Al 3+ ), chromium ion (Cr 3+ ), manganese ion (Mn 2+ ), ferrous ion (Fe 2+ ), ferric ion (Fe 3+ ), cobalt ion (Co 2+ ), nickel ion (Ni 2+ ), 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 redox interference substances and anions: including glutathione (GSH), cysteine (Cys), sulfur ion (S 2- ), sulfate ion (SO4 2- ), Vitamin C, hydrogen peroxide (H2O2), hypochlorite (ClO - ), chloride ion (Cl - ), nitrate (NO- After the reaction, it can be seen from the fluorescence spectrum that the probe Nap1 produces obvious fluorescence quenching effect on Cu + ; the probe Nap1 introduces DPA group at the 2nd position of naphthalimide, and the particularity of the spatial position makes Nap1 hardly produce reaction on other metal ions. In addition, the response of Nap1 to copper (I) is significantly higher than that to copper (II), and the probe has high specificity to copper (I), and is not easily interfered by other substances in detection. Therefore, the coordination type fluorescent naphthalimide fluorescent probe compound can selectively detect copper (I) in forage grass under specific conditions.

[0071] As shown in Figure 10 and Figure 11 , the fluorescence intensity of the fluorescent naphthalimide fluorescent probe compound (20 μM) obviously changes after the addition of copper (I), and the fluorescence intensity of the probe Nap1 gradually decreases with the increase of the concentration of copper (I); the linear equation of the fluorescence intensity of the probe Nap1 at 527 nm and the concentration (0-20 μM) of Cu + is y = -23.291 x + 541270, R 2 =0.9809( y : fluorescence intensity, x : Cu + concentration (nM), R 2 : correlation coefficient). According to the detection limit calculation formula (detection limit is 3σ / S, σ: standard deviation of blank measurement value, S: slope), the detection limit of the probe Nap1 to copper (I) is 10.19 nM, combined with 20 groups of blank measurement values of PBS matrix.

[0072] As shown in Figure 12 , the working curve analysis of the fluorescent naphthalimide fluorescent probe compound is carried out by fluorescence spectrum titration test, when the molar fraction of the probe Nap1 and copper (I) in the solution reaches 0.5, the concentration of copper (I) is equal to the probe concentration, and the fluorescence intensity change (F0-F) reaches the maximum value, indicating that the stoichiometric combination ratio of the probe and copper (I) is 1:1.

[0073] As shown in Figure 13 , in order to more clearly determine the response mechanism of the probe Nap1 and copper (I), reversibility test is carried out. After adding chelating agent BCP (bathocuproine) which has strong chelating effect on copper (I) in the complex formed by the probe and copper (I), the fluorescence reversibility of Nap1 is restored, which further confirms the interaction between the probe and copper (I).

[0074] Example 3 Application of the coordination naphthalimide fluorescent probe compound

[0075] The coordination naphthalimide fluorescent probe compound prepared in Example 1 was used to detect total free copper in alfalfa, oat and clover.

[0076] After the three kinds of forage were dried and crushed into powder, 2 grams of powder were weighed and added into 50 milliliters of deionized water to mix, stirred for 2 hours, and centrifuged at 4000 revolutions for 10 minutes. An appropriate amount of vitamin C was added, the supernatant was adjusted to pH 6, and the filtrate was repeatedly centrifuged for 3 times until the solution was clear and transparent. The solution was filtered with a 0.22 μm microporous filter membrane and stored at 4°C for standby, to obtain three kinds of forage test solutions.

[0077] Secondly, the same amount of three kinds of forage powder was taken, and the sample was treated by wet digestion method according to the pretreatment steps in the national standard GB 5009.268-2016, to prepare a sample solution for standby. The total copper content was determined by ICP-OES method (Table 1).

[0078] The fluorescent coordination naphthalimide fluorescent probe compound of the application was mixed with the above three kinds of forage test solutions, so that the concentration of the fluorescent coordination naphthalimide fluorescent probe compound therein was 20 μM. The fluorescence intensity value was measured, and the copper content (Table 1) and the standard addition recovery rate (Table 2) were calculated by substituting the standard curve.

[0079] Table 1 Copper content in three kinds of forage determined by two methods

[0080]

[0081] Table 2 Standard addition recovery rate of three kinds of forage substrates

[0082]

[0083] To verify the practicability of the fluorescent probe, a series of comparative tests with the ICP-OES method were carried out. By adding vitamin C to the forage solution, Cu 2+ interference was effectively eliminated, and Cu 2+ was reduced to Cu + , and the total free copper was quantified by determining the Cu + concentration, and the excess vitamin C did not affect the detection system. The data in Table 1 show that the total free copper content in alfalfa, oat and clover determined by the fluorescent probe method is 6.4%, 7.5% and 17.6% of the total copper determined by the ICP-OES method, respectively. The difference is due to the fact that the ICP-OES method determines the total copper after wet digestion, while the probe method detects the bioavailable free copper after pretreatment and reduction. Although the free copper content is low, it is a key indicator affecting plant physiological processes. In addition, the consistency of the ICP-OES retest results of the probe solution is high, and Table 2 shows that the Cu+ The recovery rate reached 95%-105%, RSD <5% (N = 3), indicating that the fluorescent probe Nap1 was suitable for the quantitative detection of total free copper in forage grass.

[0084] Example 4 Detection of Cu + and Cu 2+ by fluorescent probe Nap1 and fluorescent probe Nap2

[0085] The coordination type naphthalimide fluorescent probe compound Nap1 prepared in Example 1 was used to detect total free copper (in the form of Cu + ) in alfalfa, oat, and clover.

[0086] The coordination type naphthalimide fluorescent probe compound Nap2 prepared in the comparative invention patent (ZL202510227820.2) was used to detect copper (II) (in the form of Cu 2+ ) in alfalfa, oat, and clover.

[0087] The forage grass samples contain Cu + and Cu 2+ at different contents, and after pretreatment of the forage grass, part of the Cu + may be oxidized to Cu 2+ . This experiment aims to verify the difference between fluorescent probe Nap1 and Nap2 in detecting Cu + and Cu 2+ , and to determine the contents of these two copper ions in alfalfa, oat, and clover, respectively.

[0088] The treatment method in Example 3 was used to obtain the alfalfa, oat, and clover test solutions.

[0089] The fluorescent probe Nap1 and the fluorescent probe Nap2 were mixed with the above three forage grass test solutions, respectively, so that the fluorescent probe Nap1 and the fluorescent probe Nap2 in them were both 20 μM. According to the different emission wavelengths (for Nap1, the emission wavelength was 527 nm), the fluorescence intensity values were measured and substituted into the standard curve to calculate the contents of Cu + and Cu 2+ in alfalfa, oat, and clover for fluorescent probe Nap1 and fluorescent probe Nap2, respectively. The experimental results are shown in Table 3.

[0090] Table 3 Contents of Cu + and Cu 2+ in three forage grasses measured by Nap1 and Nap2, respectively

[0091]

[0092] Since Nap1 and Nap2 are respectively specific for Cu+ and Cu 2+ , so Nap1 is a fluorescent probe for detecting Cu + , and cannot be used for detecting Cu 2+ ; while Nap2 is a fluorescent probe for detecting Cu 2+ , and cannot be used for detecting Cu + Figure 15 and Figure 16 , the fluorescent probe Nap2 has a stronger response to Cu 2+ and other ions; while the probe Nap1 of the present application has higher specificity to Cu + Figure 8 , Figure 9 , and the fluorescence intensity of the probe Nap1 of the present application has a good linear relationship with the concentration of Cu + Figure 11 , and the detection limit is lower (10.19 nM), while the fluorescent probe Nap2 does not have this function.

[0093] In summary, the present application provides a kind of coordination type naphthalimide fluorescent probe for detecting copper (I) in the application and preparation method thereof in pasture, the method has good selectivity, can accurately qualitatively and quantitatively detect copper (I), and by adding vitamin C in the detection solution, Cu 2+ is reduced to Cu + , which is used for detecting total free copper in pasture, and has important significance for detecting the quality of pasture.

[0094] Finally, it should be noted that the above-mentioned is only the preferred embodiment of the present application, and does not limit the present application, any modification, equivalent solution adjustment, technical element replacement or innovative improvement within the spirit and principles of the present application should be included in the protection scope of the present application.​​​

Claims

1. A copper (I) coordination naphthoylimine fluorescent probe, characterized in that, The chemical structural formula of the copper (I) coordination naphthalimide fluorescent probe is as follows: , denoted Nap1.

2. A method for preparing the copper (I) coordination naphthoylimine fluorescent probe according to claim 1, characterized in that, The intermediate a is reacted with dimethylpyridine amine in ethylene glycol methyl ether solvent under inert gas protection at reflux condition to obtain the coordination naphthalimide fluorescent probe, denoted as Nap1. The chemical structural formula of the intermediate a is as follows: 。 3. The method for preparing copper (I) coordination naphthalimide fluorescent probe according to claim 2, characterized in that, The molar ratio of the intermediate a and dimethylpyridine amine is 1:4 equivalent, dimethylpyridine amine is added dropwise under magnetic stirring, and the reaction is carried out under nitrogen protection at 110℃ reflux for 48 hours.

4. The use of a copper (I) coordination naphthoylimine fluorescent probe according to claim 1, characterized in that, The copper (I) coordination naphthalimide fluorescent probe is used for qualitatively or quantitatively detecting copper (I) in forage grass.

5. The use of a copper (I) coordination naphthoylimine fluorescent probe according to claim 4, characterized in that, The forage grass is any one or combination of alfalfa, oat and clover.

6. The use of a copper (I) coordination naphthoylimine fluorescent probe according to claim 1, characterized in that, The copper (I) coordination naphthalimide fluorescent probe is used for detecting the content of free copper in forage grass, and the specific application method is as follows: (1) Preparation of forage grass test solution: the forage grass sample is dried and crushed, mixed with water, centrifuged, and the supernatant is taken, vitamin C is added to the supernatant, the pH is adjusted to 5.5-6.5, and after centrifugation and filtration, the forage grass test solution is obtained; (2) Detection: the copper (I) coordination naphthalimide fluorescent probe is added to the forage grass test solution, mixed uniformly, and then the fluorescence intensity is measured; (3) Quantification: according to the fluorescence intensity, the content of free copper in the forage grass test solution is calculated by using the standard curve.

7. The use of a copper (I) coordination naphthoylimine fluorescent probe according to claim 6, characterized in that, Vitamin C is added to the supernatant to adjust the pH to 6, and the detection temperature is 20-25℃.

8. The use of a copper (I) coordination naphthoylimine fluorescent probe according to claim 6, characterized in that, In step (2), the wavelength for measuring the fluorescence intensity is 527 nm.

9. The use of a copper (I) coordination naphthoylimine fluorescent probe according to claim 6, characterized in that, The final concentration of the copper (I) coordination naphthalimide fluorescent probe in the detection system is 18-22 μM.

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