Fluorescent probe for detecting copper (I) in pasture as well as preparation method and application of fluorescent probe

Abstract

The invention discloses a fluorescent probe for detecting copper (I) in forage grass and a preparation method and application thereof, and relates to the field of forage grass detection and 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 a probe Nap1: preparing the Nap1 by using the intermediate a; according to the probe Nap1 provided by the invention, a DPA group is introduced to the site 2 of naphthalimide, the specificity of the spatial position enables the probe Nap1 to have very high specificity to copper (I), almost no reaction is generated to other metal ions, the response to copper (I) is obviously higher than that of copper (II), the probe Nap1 is not easily interfered by other substances in the detection process, and copper (I) in pasture can be selectively detected.

Description

Technical Field

[0001] This invention relates to the field of forage detection and fluorescent probes, and particularly to a fluorescent probe for detecting copper (I) in forage, its preparation method, and its application. Background Technology

[0002] Copper is an essential trace element for living organisms. As a cofactor for the redox activity of many enzymes, copper participates in important physiological functions such as respiration, neurotransmitter synthesis and metabolism, gene expression, and antioxidant defense. In forage grasses, copper exists mainly 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 redox reactions within plants. In contrast, bound copper is tightly bound to proteins, enzymes, or other biomolecules, forming stable complexes. Its chemical activity is relatively low, and it can only be released or activated under specific physiological conditions to exert its biological functions. In practical applications, free copper can usually be obtained from forage grasses through simple and rapid physical extraction processes. However, excessive free copper may generate hydroxyl radicals through the Fenton reaction, thereby causing oxidative damage to proteins, lipids, and nucleic acids, thus harming the organism. Secondly, in mitochondria, there are a large number of reducing agents (such as glutathione), making intracellular copper mainly exist as Cu. + It exists in form. Based on this characteristic, on the one hand, Cu + Probes can be used for in vitro Cu + Concentration determination provides fundamental data for research; on the other hand, it also allows for the determination of Cu concentration in subcellular structures. + To conduct spatial imaging and then conduct in-depth research on the physiological processes of cells.

[0003] Traditional methods for copper detection, such as atomic absorption spectroscopy and ICP-MS, offer high sensitivity, but the equipment is expensive, the operation is complex, and real-time in-situ detection is difficult. Small molecule fluorescent probes offer advantages such as tunable structure, good selectivity, ease of operation, and easy modification. Therefore, developing high-sensitivity, low-cost copper (I) fluorescent probes suitable for forage grasses is of great significance. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention discloses a fluorescent probe for detecting copper (I) in forage, its preparation method, and its application, as detailed below:

[0005] A copper (I)-coordinated naphthaleneimide fluorescent probe, the chemical structure of which is as follows: ( Figure 1 ), denoted as Nap1.

[0006] Furthermore, the preparation method of the coordination-type naphthalimide fluorescent probe includes the following steps: (1) Preparation of intermediate a Commercially available 2-bromo-1,8-naphthalenedicarboxylic anhydride was dissolved in anhydrous ethanol, and then n-butylamine was added to react. After 2 hours, a large amount of precipitate was produced. The solvent was removed by vacuum distillation, and then ice water was added to precipitate the product. The product was filtered to obtain a filter cake, and washed with ice ethanol (EtOH) to obtain intermediate a. The chemical structural formula of intermediate a is as follows: ; The chemical reaction formula for the preparation process of intermediate a is as follows: ; n-Butylamine; 2-Bromo-1,8-naphthoic anhydride; (2) Preparation of coordination-type naphthimide fluorescent probe Nap1 Compound a was weighed and placed in a round-bottom flask. Ethylene glycol methyl ether (MOE) was added, and the mixture was heated to dissolve. Then, dimethylpyridinium chloride (DPA) was added. Under nitrogen protection, the reaction system was placed in an oil bath and heated to 110°C, and refluxed for 48 hours. After the reaction was completed, the solvent was removed by vacuum distillation. The crude product was purified by column chromatography with silica gel to obtain a yellow solid product, namely probe Nap1. The chemical reaction formula is as follows: ; : Dimethylpyridinium.

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

[0008] Moreover, in the preparation of intermediate b in step (2), the ratio of intermediate a to DPA is preferably 1:4 equivalent; the reaction in step (2) is carried out under the following conditions: DPA is slowly added dropwise under magnetic stirring, and the reaction is refluxed at 110°C for 48 hours under nitrogen protection.

[0009] On the other hand, the present invention discloses the application of a copper(I)-coordinated naphthalimide fluorescent probe for the quantitative detection of copper(I) in forage.

[0010] Furthermore, the forage grass is any one or a combination of alfalfa, oats, and clover; after pretreatment, the forage grass sample is reacted with the fluorescent probe, and the fluorescence intensity of the reaction product is measured to quantitatively detect the copper (I) concentration in the forage grass, as detailed below: (1) Preparation of forage test solution: After drying and pulverizing the forage sample, mix it with water, centrifuge, and take the supernatant; add vitamin C to the supernatant, adjust the pH to 5.5-6.5, centrifuge and filter to obtain the forage test solution; the amount of vitamin C added is sufficient to remove Cu in the test solution. 2 ⁺ is reduced to Cu⁺, and excess vitamin C does not interfere with subsequent fluorescence detection; (2) Qualitative detection: The copper (I) coordination type naphthalimide fluorescent probe is added to the forage test solution and mixed evenly. The reaction product can be preliminarily observed under ultraviolet light to qualitatively detect the copper (I) in the forage. (3) Quantitative detection: The fluorescence intensity is measured, and the concentration of total free copper in the test solution of the forage is calculated using a standard curve based on the fluorescence intensity.

[0011] Furthermore, the pH was adjusted to 6, and the detection temperature was 20-25℃.

[0012] Moreover, in step (2), the wavelength for measuring the fluorescence intensity is 527 nm.

[0013] Furthermore, the final concentration of the fluorescent probe in the detection system is 18-22 μM, preferably 20 μM.

[0014] The detection principle of this invention is that the DPA structure detection group of the coordination-type naphthalimide fluorescent probe coordinates and binds to copper (I), generating fluorescence activation, which can accurately detect the copper (I) content in forage. This method is fast, sensitive, selective, simple to operate, and low in cost, and can be used for the rapid detection of copper (I) in forage.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The probe Nap1 provided by this invention introduces a DPA group at the 2 position of naphthalimide. The special spatial position makes it highly specific for copper (I) and almost unresponsive to other metal ions. Moreover, its response to copper (I) is significantly higher than that to copper (II). It is not easily interfered with by other substances during detection and can selectively detect copper (I) in forage.

[0016] 2. The probe Nap1 provided by this invention has a fluorescence intensity at 527 nm that is similar to that of Cu. + The concentration (0-20 μM) showed a good linear relationship, with a correlation coefficient R0. 2 = 0.9809, the detection limit for copper (I) is 10.19 nM, which can accurately detect low concentrations of copper (I).

[0017] 3. The fluorescence of the probe Nap1 provided by this invention is reversibly restored after adding BCP (bath copper chelating agent) which has a strong chelating effect on copper (I) to the complex formed with copper (I), which further confirms the interaction between the probe and copper (I) and provides the possibility for the repeated use of the probe and in-depth research.

[0018] 4. In this invention, adding vitamin C to the forage solution effectively eliminates Cu. 2+ Interference, while Cu 2+ Reduced to Cu + And by measuring Cu + Concentration allows for the quantification of total free copper, and excessive vitamin C does not affect the detection system.

[0019] 5. The total free copper content in alfalfa, oats, and clover measured by the probe Nap1 provided by this invention is consistent with the results determined by ICP-OES, and the Cu content in the three forage substrates is relatively low. + The spiked recoveries reached 95%–105%, with RSD <5% (N = 3), indicating that the fluorescent probe Nap1 is suitable for the quantitative detection of total free copper in forage, providing an accurate and reliable method for forage quality testing.

[0020] 6. This invention provides a copper (I)-coordinated naphthalimide fluorescent probe, its preparation method, and its application in forage detection. The raw materials are readily available, the synthesis steps are simple, and the operation is convenient. It has high application value in the field of rapid detection of zinc pollution in forage.

[0021] 7. The present invention mainly overcomes the disadvantages of existing large-scale instruments for detecting copper (I) being costly and limited in detection scenarios; the coordination-type naphthalimide fluorescent probe can rapidly detect copper (I) in forage grass, and the method has the advantages of high detection sensitivity, low cost and simple operation. Attached Figure Description

[0022] Figure 1 The structural formula of the coordination-type naphthalimide fluorescent probe compound of this invention is shown below; Figure 2 This is a synthetic route diagram of the coordination-type naphthalimide fluorescent probe compound of the present invention; Figure 3 This is the 1H NMR spectrum of the coordination-type naphthalimide fluorescent probe compound of this invention; Figure 4 This is the carbon NMR spectrum of the coordination-type naphthalimide fluorescent probe compound of the present invention; Figure 5 This is the mass spectrum of the coordination-type naphthalimide fluorescent probe compound of the present invention; Figure 6The excitation wavelengths of the coordination-type naphthalimide fluorescent probe compound (20 μM) of this invention were measured before and after binding with copper (I) (40 μM) in PBS buffer solution at pH=7.4. Figure 7 The emission wavelengths of the coordination-type naphthalimide fluorescent probe compound (20 μM) of this invention were measured before and after binding with copper (I) (40 μM) in PBS buffer solution at pH=7.4. Figure 8 The fluorescence spectra of the coordination-type naphthalimide fluorescent probe compound (20 μM) of this invention in PBS buffer solution at pH 7.4 with different interfering matrices are shown. Figure 9 The bar chart shows the addition of different metal ions and some interfering factors to the coordination-type naphthalimide fluorescent probe compound (20 μM) of this invention in PBS buffer solution at pH=7.4. Figure 10 The fluorescence intensity spectrum of the coordination-type naphthalimide fluorescent probe compound (20 μM) of the present invention in PBS buffer solution at pH=7.4 is a spectrum showing the fluorescence intensity change with the concentration of copper (I) as the concentration of copper (I) is gradually increased (0-50 μM). Figure 11 The linear relationship between fluorescence intensity and copper(I) concentration was observed when different concentrations of copper(I) (0-50 μM) were added to PBS buffer solution at pH=7.4 for the coordination-type naphthalimide fluorescent probe compound (20 μM) of this invention. Figure 12 The working curve of the coordination-type naphthalimide fluorescent probe compound of the present invention binding to copper (I) in PBS buffer solution at pH=7.4 is shown. Figure 13 The reversibility of binding of the coordination-type naphthalimide fluorescent probe compound (20 μM) of this invention with copper (I) in PBS buffer solution at pH=7.4 was tested. Figure 14 This is a diagram illustrating the binding mechanism between the coordination-type naphthalimide fluorescent probe compound of this invention and copper (I); Figure 15 Fluorescence spectra of the fluorescent probe Nap2 in PBS buffer solution at pH 7.4 with different interfering matrices added; Figure 16 A bar chart showing the addition of different metal ions and some interfering factors to the fluorescent probe Nap2 in PBS buffer solution at pH 7.4. Detailed Implementation

[0023] The present invention will be further described below with reference to specific embodiments, and the features and advantages of the technical solution of the present invention will become clearer with the description. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention.

[0024] Example 1: Synthesis and Characterization of Coordination-Type Naphthalimide Fluorescent Probe Synthesis of a coordination-type naphthalimide fluorescent probe, the synthetic route is as follows: Figure 2 As shown, the steps include: 2-Bromo-1,8-naphthalenedicarboxylic anhydride (4 g, 14.44 mmol, 1 eq) was placed in a 100 mL round-bottom flask and dissolved in 60 mL of anhydrous ethanol. Then, n-butylamine (2.1 mL, 21.6 mmol, 1.5 eq) was added. The reaction mixture was placed in an oil bath and refluxed at 80 °C for 2 hours. The reaction was monitored by thin-layer chromatography (TLC). After the reaction was complete, the reaction mixture was cooled and ice water was added. A large amount of solid precipitated. The solid was filtered through a Buchner funnel to obtain a gray, loose, crystalline crude product. The product was then washed with ice ethanol and water to obtain intermediate a, with a yield of 83.68%.

[0025] 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).

[0026] intermediate a 13 The 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.

[0027] Accurately weigh compound a (0.66 g, 2 mmol, 1 eq) and place it in a 100 mL round-bottom flask. Add 60 mL of MOE (ethylene glycol methyl ether) to the flask and heat to 60 °C. Under stirring, slowly add DPA (1.45 mL, 8 mmol, 4 eq). After three vacuum nitrogen cycles, place the reaction mixture in an oil bath under nitrogen protection, heat to 110 °C, and reflux for 48 h. Monitor the reaction progress by TLC. After the reaction is complete, remove the solvent by vacuum distillation. The obtained product has UV absorption at 256 nm and green fluorescence at 365 nm. Purify the crude product by column chromatography using silica gel, eluting with a gradient of PE:EA = 1:1 (v / v) to PE:EA = 1:2 (v / v). After drying, 0.11 g of a yellow solid product was obtained, which was a coordination-type naphthalimide fluorescent probe with a yield of 12.2%. The binding mechanism diagram is shown below. Figure 14 .

[0028] Coordination-type naphthalimide fluorescent probe compounds 1 The 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).

[0029] Coordination-type naphthalimide fluorescent probe compounds 13 The 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.

[0030] MS(ESI) m / z: C 28 H 26 N4O2[M+H] + = 451.2127.

[0031] Coordination-type naphthalimide fluorescent probe 1 H NMR image as follows Figure 3 As shown, 13 C NMR such as Figure 4 As shown, the mass spectrum is as follows Figure 5 As shown.

[0032] Experimental Example 2: Spectral Response of Probe Molecules with Copper (I) The fluorescent coordination type naphthalimide fluorescent probe compound prepared in Example 1 was used to detect copper (I) in forage under the condition of pH=7.4 with PBS as the dissolving matrix. The fluorescent probe was dissolved in dimethyl sulfoxide to prepare a probe stock solution with a concentration of 2 mM and a metal ion stock solution with a concentration of 2 mM.

[0033] Figure 6 The UV absorption wavelengths of the coordination-type naphthalimide fluorescent probe compound (20 μM) of this invention were measured before and after binding with copper (I) (40 μM) in PBS buffer solution at pH=7.4. These wavelengths were used as the probe excitation wavelengths for subsequent spectroscopic experiments. Figure 7 The emission wavelengths of the coordination-type naphthalimide fluorescent probe compound (20 μM) of this invention were measured in PBS buffer solution at pH=7.4 before and after binding with copper (I) (40 μM). After binding with copper (I), the UV absorbance of the probe decreased slightly, while the fluorescence intensity was significantly weakened, indicating that the probe's solubility was enhanced and the fluorescence quenching effect was significant after complexing with copper (I).

[0034] Figure 8To prepare the coordination-type naphthimide fluorescent probe compound (20 μM) of this invention, different interfering matrices were added to a PBS buffer solution at pH 7.4: magnesium ions (Mg... 2+ ), aluminum ions (Al 3+ ), chromium ions (Cr 3+ ), manganese ions (Mn 2+ ), ferrous ions (Fe 2+ ), iron ions (Fe 3+ ), cobalt ions (Co) 2+ Nickel ions (Ni) 2+ ), copper ions (Cu) 2+ ), cuprous ion (Cu + ), zinc ions (Zn 2+ ), silver ions (Ag) + ), cadmium ions (Cd) 2+ ), mercury ions (Hg) 2+ ), lead ions (Pb 2+ ), glutathione (GSH), cysteine ​​(Cys), sulfide ions (S) 2- ), sulfate ions (SO4) 2- Vitamin C, hydrogen peroxide (H2O2), hypochlorite (ClO2) - ), chloride ions (Cl - ), nitrate (NO - The fluorescence spectrum after emission was obtained, with an excitation wavelength of 432 nm and an emission wavelength of 527 nm. In the presence of numerous interfering substances, only Cu... + This causes a significant quenching of the probe's fluorescence intensity, while other ions or molecules (such as Cu) cause this. 2 + Zn 2+ Fe 2+ Fe 2+ The presence of substances such as GSH, Cys, H2O2, and Vitamin C has little or no effect on the fluorescence signal of the probe. Even under conditions where high concentrations of interfering substances coexist, the probe can still specifically recognize Cu. + This indicates that it has good anti-interference performance and is suitable for the specific detection of Cu⁺ in complex matrices; the close proximity of the naphthalimide carbonyl group and the DPA (dimethylpyridinium amine) group in the probe molecule forms a co-coordination system for Cu. + It exhibits strong coordination ability; the introduction of a carbonyl group into the coordination system enhances its ability to coordinate with the more electronegative Lewis weak acid Cu. + Its coordination ability and spatial structure effectively avoid the non-specific binding of other metal ions, thus achieving the coordination of Cu + Highly selective identification.

[0035] Figure 9This is a bar chart showing the addition of different metal ions and some interfering factors to the coordination-type naphthimide fluorescent probe compound (20 μM) of this invention in PBS buffer solution at pH 7.4. As can be seen from the figure, the addition of common metal ions, such as magnesium ions (Mg...), to the fluorescent coordination-type naphthimide fluorescent probe compound... 2+ ), aluminum ions (Al 3+ ), chromium ions (Cr 3+ ), manganese ions (Mn 2+ ), ferrous ions (Fe 2+ ), iron ions (Fe 3+ ), cobalt ions (Co) 2+ Nickel ions (Ni) 2+ ), copper ions (Cu 2+ ), cuprous ion (Cu + ), zinc ions (Zn 2+ ), silver ions (Ag) + ), cadmium ions (Cd) 2+ ), mercury ions (Hg) 2+ ), lead ions (Pb 2+ ), as well as redox disruptors and anions: including glutathione (GSH), cysteine ​​(Cys), sulfide ions (S... 2- ), sulfate ions (SO4) 2- Vitamin C, hydrogen peroxide (H2O2), hypochlorite (ClO2) - ), chloride ions (Cl - ), nitrate (NO - After the reaction, the fluorescence spectrum shows that probe Nap1 is effective against Cu. + A significant fluorescence quenching effect is observed. The probe Nap1 incorporates a DPA group at position 2 of the naphthalimide, a spatially specific feature that renders Nap1 almost unresponsive to other metal ions. Furthermore, Nap1 exhibits a significantly higher response to copper (I) than copper (II), demonstrating high specificity for copper (I) and making it less susceptible to interference from other substances during detection. Therefore, the coordination-type fluorescent naphthalimide fluorescent probe compound described in this invention can selectively detect copper (I) in forage under specific conditions.

[0036] like Figure 10 and Figure 11 As shown, the fluorescence intensity of the fluorescent coordination-type naphthalimide fluorescent probe compound (20 μM) changed significantly after the addition of copper (I). The fluorescence intensity of probe Nap1 gradually decreased with increasing copper (I) concentration. The fluorescence intensity of probe Nap1 at 527 nm was similar to that of Cu. + The linear equation for concentration (0-20 μM) is: y = -23.291 x+ 541270, R 2 =0.9809 ( y Fluorescence intensity x Cu + Concentration (nM) R 2 (Correlation coefficient). Based on 20 sets of blank measurements in PBS matrix, and according to the limit of detection formula (the limit of detection is 3σ / S, where σ is the standard deviation of the blank measurement and S is the slope), the limit of detection for copper (I) by probe Nap1 is 10.19 nM.

[0037] like Figure 12 As shown, the working curve of the fluorescent coordination type naphthalimide fluorescent probe compound was analyzed by fluorescence spectral titration. When the molar fraction of probe Nap1 and copper (I) in the solution reached 0.5, the concentration of copper (I) was equal to the concentration of the probe, and the fluorescence intensity change (F0-F) reached its maximum value, indicating that the stoichiometric binding ratio of the probe to copper (I) was 1:1.

[0038] like Figure 13 As shown, a reversibility test was performed to more clearly determine the response mechanism of probe Nap1 to copper(I). The fluorescence of Nap1 was reversibly restored after adding BCP (copper bath chelate), a chelating agent with a strong chelating effect on copper(I), to the complex formed by the probe and copper(I), further confirming the interaction between the probe and copper(I).

[0039] Example 3: Application of Coordination-type Naphthalimide Fluorescent Probe Compounds The total free copper in alfalfa, oats, and clover was detected using the coordination-type naphthalimide fluorescent probe compound prepared in Example 1.

[0040] After drying and crushing the three types of forage into powder, 2 grams of powder were weighed and added to 50 ml of deionized water. The mixture was stirred for 2 hours and then centrifuged at 4000 rpm for 10 minutes. An appropriate amount of vitamin C was added, and the pH of the supernatant was adjusted to 6. The filtrate was then centrifuged three times repeatedly until the solution was clear and transparent. The solution was then filtered through a 0.22 μm microporous membrane and stored at 4°C for later use, yielding the test solutions for the three types of forage.

[0041] Next, take the same amount of three types of forage powder, and process the samples using the wet digestion method according to the pretreatment steps in the national standard GB 5009.268-2016 to prepare sample solutions for later use. The total copper content was determined by ICP-OES (Table 1).

[0042] The fluorescent coordination type naphthalimide fluorescent probe compound of the present invention was mixed with the above three forage test solutions to make the concentration of the fluorescent coordination type naphthalimide fluorescent probe compound 20 μM. 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).

[0043] Table 1. Copper content in three types of forage determined by two methods.

[0044] Table 2 Spiked recovery rates of three forage substrates

[0045] To verify the practicality of the fluorescent probe, a series of comparative experiments with the ICP-OES method were conducted. Adding vitamin C to the forage solution effectively eliminated Cu. 2+ Interference, while Cu 2+ Reduced to Cu + And by measuring Cu + Concentration allows for the quantification of total free copper, and excessive vitamin C does not affect the detection system. Table 1 shows that the total free copper content in alfalfa, oats, and clover measured by the fluorescent probe method was 6.4%, 7.5%, and 17.6% of the total copper content measured by the ICP-OES method, respectively. The difference stems from the fact that ICP-OES measures the total copper after wet digestion, while the probe method detects bioavailable free copper after pretreatment and reduction. Although the free copper content is low, it is a key indicator affecting plant physiological processes. Furthermore, the probe method solution showed high consistency with ICP-OES retesting results. Table 2 shows the Cu content in the three forage substrates. + The spiked recoveries reached 95%–105%, with RSD <5% (N = 3), indicating that the fluorescent probe Nap1 is suitable for the quantitative detection of total free copper in forage.

[0046] Example 4: Fluorescent probes Nap1 and Nap2 for Cu + and Cu 2+ Detection The total free copper (as Cu) in alfalfa, oats, and clover was detected using Nap1, a coordination-type naphthimide fluorescent probe compound prepared in Example 1. + (Form exists).

[0047] The coordination-type naphthimide fluorescent probe compound Nap2, prepared using the comparative invention patent (ZL202510227820.2), was used to detect copper(II) in alfalfa, oats, and clover. 2+ (Form exists).

[0048] The forage sample also contained Cu + and Cu 2+The content varies, and some Cu in the forage grass is reduced after pretreatment. + It may be oxidized to Cu 2+ This experiment aims to verify the effectiveness of fluorescent probes Nap1 and Nap2 in detecting Cu. + and Cu 2+ The differences in time were investigated, and the contents of these two copper ions in alfalfa, oats, and clover were determined respectively.

[0049] Using the treatment method in Example 3, test solutions of alfalfa, oats, and clover were obtained.

[0050] Fluorescent probes Nap1 and Nap2 were mixed with the three forage samples mentioned above, respectively, to a concentration of 20 μM for both Nap1 and Nap2. Fluorescence intensity values ​​were measured at different emission wavelengths (527 nm for Nap1), and substituted into standard curves to calculate the effects of Nap1 and Nap2 on Cu in alfalfa, oats, and clover. + and Cu 2+ The content of [the substance] was determined, and the experimental results are shown in Table 3.

[0051] Table 3. Cu content in three forages measured using Nap1 and Nap2, respectively. + and Cu 2+ content

[0052] Since Nap1 and Nap2 are respectively for Cu + and Cu 2+ The detection conditions and procedures differ, therefore Nap1 is used for detecting Cu. + Fluorescent probes cannot be used to detect Cu. 2+ Nap2 is used for detecting Cu. 2+ Fluorescent probes cannot be used to detect Cu. + ( Figure 15 and Figure 16 ), fluorescent probe Nap2 for Cu 2+ It exhibits a stronger response to Cu and other ions; while the probe Nap1 of this invention shows a stronger response to Cu. + It has higher specificity ( Figure 8 , Figure 9 Furthermore, the fluorescence intensity of the probe Nap1 of the present invention is related to Cu. + The concentration showed a good linear relationship at 527 nm. Figure 11 The detection limit is lower (10.19 nM), while the fluorescent probe Nap2 does not have this function.

[0053] In summary, this invention provides an application of a coordination-type naphthimide fluorescent probe for detecting copper (I) in forage and its preparation method. This method exhibits good selectivity and can accurately perform qualitative and quantitative detection of copper (I). Furthermore, the addition of vitamin C to the detection solution helps to neutralize the Cu(I) content. 2+ Reduced to Cu + It is used for the detection of total free copper in forage, which is of great significance for the quality testing of forage.

[0054] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent scheme adjustments, technical element substitutions or innovative improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A copper(I)-coordinated naphthalimide fluorescent probe, characterized in that, The chemical structure of the coordination-type naphthimide fluorescent probe is as follows: , denoted as Nap1.

2. The copper(I)-coordinated naphthalimide fluorescent probe as described in claim 1, characterized in that, The preparation method of the copper (I) coordinated naphthalimide fluorescent probe is as follows: intermediate a is reacted with dimethylpyridinium in ethylene glycol methyl ether solvent under reflux conditions under inert gas protection to obtain the coordinated naphthalimide fluorescent probe, denoted as Nap1; The chemical structural formula of intermediate a is as follows: 。 3. The copper(I)-coordinated naphthalimide fluorescent probe as described in claim 2, characterized in that, The molar ratio of intermediate a to dimethylpyridinium amine is 1:4 equivalent. Dimethylpyridinium amine is added dropwise under magnetic stirring, and the mixture is refluxed at 110°C for 48 hours under nitrogen protection.

4. The application of the copper(I)-coordinated naphthalimide fluorescent probe as described in any one of claims 1-3, characterized in that, Used for qualitative or quantitative detection of copper (I) in forage.

5. The application of the copper(I)-coordinated naphthalimide fluorescent probe as described in claim 4, characterized in that, The forage grass is any one or a combination of alfalfa, oats, and clover.

6. The application of the copper(I)-coordinated naphthalimide fluorescent probe as described in any one of claims 1-3, characterized in that, The specific application method for detecting the free copper content in pasture is as follows: (1) Preparation of forage test solution: After drying and crushing the forage sample, mix it with water, centrifuge, take the supernatant, add vitamin C to the supernatant, adjust the pH to 5.5-6.5, and obtain the forage test solution after centrifugation and filtration; (2) Detection: The copper (I)-coordinated naphthalimide fluorescent probe was added to the forage test solution, mixed evenly, and its fluorescence intensity was measured. (3) Quantitative analysis: Based on the fluorescence intensity, the content of free copper in the test solution of the forage is calculated using a standard curve.

7. The application of the copper(I)-coordinated naphthalimide fluorescent probe as described in claim 6, characterized in that, Add vitamin C to the supernatant to adjust the pH to 6, and test the temperature at 20-25℃.

8. The application of the copper(I)-coordinated naphthalimide fluorescent probe as described in claim 6, characterized in that, In step (2), the wavelength for measuring the fluorescence intensity is 527 nm.

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

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