CN113979996B

CN113979996B - Phthalimide fluorescent probe for detecting copper ions, preparation method and application thereof, and copper ion detection method

Info

Publication number: CN113979996B
Application number: CN202111256277.7A
Authority: CN
Inventors: 盛筱; 王守信; 孔令栋; 王军; 丁林
Original assignee: JINING MEDICAL UNIVERSITY
Current assignee: JINING MEDICAL UNIVERSITY
Priority date: 2021-10-27
Filing date: 2021-10-27
Publication date: 2022-08-23
Anticipated expiration: 2041-10-27
Also published as: CN113979996A

Abstract

The invention belongs to the technical field of organic synthesis and analytical chemistry, and particularly relates to a phthalimide fluorescent probe for detecting copper ions, a preparation method and application thereof, and a copper ion detection method. The fluorescent probe LXJ-3 provided by the invention is a phthalimide compound taking pyridine-2-formate as a recognition receptor, and is a phthalimide derivative. The preparation method comprises the steps of taking N-butyl-3-hydroxyphthalimide and pyridine-2-formyl chloride as raw materials, and carrying out acylation reaction under the catalysis of alkali and 4-dimethylaminopyridine to obtain the fluorescent probe. The method can prepare the fluorescent probe LXJ-3 through two steps, and the synthesis process is simple.

Description

Phthalimide fluorescent probe for detecting copper ions, preparation method and application thereof, and copper ion detection method

Technical Field

The invention belongs to the technical field of organic synthesis and analytical chemistry, and particularly relates to a phthalimide fluorescent probe for detecting copper ions, a preparation method and application thereof, and a copper ion detection method.

Background

Copper is the third most abundant transition metal element in the human body and widely distributed in the blood and tissues of the human body. Cu ²⁺ Plays an important role in physiological processes such as energy generation, signal transduction, oxygen transport and activation of cells. Cu in human body ²⁺ Abnormal levels can cause a series of diseases such as Mengkin's disease, Wilson's disease, Alzheimer's disease, Parkinson's disease, coronary heart disease, amyotrophic lateral sclerosis, myelopathy, and the like. Furthermore, Cu is widely used in agricultural and industrial production ²⁺ Has become an important environmental pollutant. The U.S. Environmental Protection Agency (EPA) has treated Cu in drinking water ²⁺ Is set to 20 μ M. Therefore, the development of a method for detecting Cu with high sensitivity and selectivity ²⁺ The method of (a) is very important for human health and environmental safety.

Currently detecting Cu ²⁺ The method of (3) includes atomic absorption spectrometry, atomic emission spectrometry, inductively coupled plasma mass spectrometry, electrochemical method, and the like. However, these methods are costlyTime, complicated sample preparation, expensive instrument, unsuitability for real-time analysis and the like. Compared with the method, the fluorescent probe has the advantages of low cost, simple and convenient operation, real-time analysis, nondestructive biological imaging and the like, and is paid more and more attention in the detection of heavy metal ions in recent years. To date, the literature reports a number of Cu ²⁺ Fluorescent probes, but many of them still have some limitations, such as poor selectivity, organic solvent requirement, complicated synthesis steps, fluorescence quenching, etc. Therefore, a Cu having good water solubility, simple synthesis and excellent performance has been developed ²⁺ Fluorescent probes have important practical significance.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a phthalimide fluorescent probe for detecting copper ions, a preparation method and application thereof, and a copper ion detection method. The fluorescent probe LXJ-3 provided by the invention has the advantages of simple synthesis steps, large Stokes displacement, good water solubility and the like, is an excellent fluorescence-enhanced probe, and has good application prospect.

The technical scheme provided by the invention is as follows:

a phthalimide-based fluorescent probe for detecting copper ions, the fluorescent probe having the following structure:

the fluorescent probe LXJ-3 provided by the technical scheme is a phthalimide compound taking pyridine-2-formate as a recognition receptor, and is a phthalimide derivative.

The invention also provides a preparation method of the phthalimide fluorescent probe for detecting the copper ions, which comprises the following steps:

1) dissolving N-butyl-3-hydroxyphthalimide (BHIO), an acid-binding agent and a catalyst 4-Dimethylaminopyridine (DMAP) in dichloromethane, and reacting at 0-30 ℃;

2) dissolving pyridine-2-formyl chloride hydrochloride in dichloromethane, slowly adding the dichloromethane dropwise into the reaction solution, and purifying after the reaction is finished to obtain the phthalimide fluorescent probe for detecting copper ions.

According to the technical scheme, N-butyl-3-hydroxyphthalimide and pyridine-2-formyl chloride are used as raw materials, and the fluorescent probe is prepared through acylation reaction under the catalysis of alkali and 4-dimethylaminopyridine. The method can prepare the fluorescent probe LXJ-3 through two steps, and the synthesis process is simple.

Specifically, a compound BHIO, an acid-binding agent and DMAP are dissolved in dichloromethane, pyridine-2-formyl chloride hydrochloride is dissolved in dichloromethane, the mixture is dripped into a reaction liquid through a constant-pressure dropping funnel under stirring, reaction is carried out at 0-30 ℃, and the phthalimide probe LXJ-3 is obtained through purification after the reaction is finished.

The compound BHIO of the invention is prepared by heating 3-hydroxyphthalic anhydride and n-butylamine in acetic acid by a disclosed method (analysis laboratory 2020, 39(12): 1430-1434).

The chemical name of the compound BHIO is N-butyl-3-hydroxyphthalimide, and the structural formula is as follows:

specifically, in the step 2), the reaction time is 4-10 h.

Specifically, in the step 1), the acid-binding agent is selected from one or more of N, N-Diisopropylethylamine (DIPEA), triethylamine or pyridine.

Specifically, in the step 2), the reaction solution obtained after the reaction is finished is subjected to reduced pressure distillation to remove the solvent, so as to obtain a crude product, and then the crude product is separated and purified by a silica gel column, wherein the mobile phase is a mixed solvent of ethyl acetate and petroleum ether in a volume ratio of 1: 2.

Specifically, the molar ratio of the compound N-butyl-3-hydroxyphthalimide to the pyridine-2-formyl chloride hydrochloride is 1 (1-2); the molar ratio of the compound N-butyl-3-hydroxyphthalimide to the acid-binding agent is 1 (2-4); the molar ratio of the compound N-butyl-3-hydroxyphthalimide to the catalyst is (5-20): 1.

The invention also provides application of the phthalimide fluorescent probe for detecting copper ions as Cu ²⁺ Fluorescent probe of (2), qualitative or quantitative detection of Cu ²⁺ The concentration of (c).

The technical proposal utilizes Cu ²⁺ And catalyzing the probe LXJ-3 in a buffer solution to perform hydrolysis reaction, removing pyridine-2-formyl and regenerating a fluorescence parent BHIO, so that the ESIPT process is recovered, and the fluorescence is obviously enhanced. Specifically, under the irradiation of 365nm ultraviolet lamp, the probe solution has weak fluorescence, and Cu is added ²⁺ Then strong green fluorescence is emitted, thereby realizing the aim of Cu ²⁺ Detection of (3). Probe LXJ-3 vs. Cu in PBS buffer (10mM, pH 7.4, physiological conditions pH 7.4 being chosen for ease of use in biology) ²⁺ Has specific selectivity, strong anti-interference capability and obvious fluorescence enhancement effect, and the detection limit is 31 nM.

Specifically, Cu ²⁺ The detection method comprises the following steps:

1) establishing a standard curve of the gradient change of the concentration of the copper ions and the change value of the fluorescence intensity of the fluorescent probe solution;

2) obtaining Cu in the solution to be detected from the standard curve according to the fluorescence intensity of the solution to be detected ²⁺ The concentration of the ions.

More specifically, the method comprises the following steps:

1) respectively mixing the copper ion solution with the gradient change of the concentration with the probe LXJ-3 solution with the specific concentration, measuring the corresponding fluorescence intensity, then drawing by taking the concentration of the copper ions as an abscissa and the fluorescence intensity of the corresponding mixed solution as an ordinate, and establishing a standard curve of the gradient change of the concentration of the copper ions and the change value of the fluorescence intensity of the probe LXJ-3 solution;

2) and obtaining the concentration of the copper ions in the solution to be detected from the standard curve according to the fluorescence intensity.

Specifically, the fluorescence detection conditions are as follows: the excitation wavelength is 344nm, the maximum fluorescence emission wavelength is 515nm, the slit width is 5nm and 5nm, and the fluorescence emission spectrum is detected at 420-650 nm.

Specifically, the fluorescent probe stock solution adopted by the fluorescence detection comprises a fluorescent probe, a solvent and a buffer solution, wherein the fluorescent probe is phthalimide derivative LXJ-3.

Specifically, the molar concentration of the fluorescent probe in the stock solution is 0.01-10 mM; the solvent is at least one of methanol, ethanol and dimethyl sulfoxide; the buffer solution is PBS buffer solution or Tris-HCl buffer solution, and the pH value of the buffer solution is 5-10.

Compared with the prior art, the invention has the following advantages:

the phthalimide fluorescent probe LXJ-3 provided by the invention has the advantages of simple structure, easy synthesis and higher yield. Probe LXJ-3 realizes selective recognition of copper ions based on ESIPT process of N-butyl-3-hydroxyphthalimide, has the advantages of large Stokes displacement, high fluorescence quantum yield, strong optical stability and the like, and can sensitively and specifically recognize and detect Cu in a pure water phase and a wider pH range ²⁺ The method is not interfered by other ions basically, the minimum detection limit of the method to the copper ions is 31nM, the quantitative detection of trace copper ions in complex environment or in organisms can be well met, and the method has important application value.

Drawings

FIG. 1 is a view of probe LXJ-3 prepared in example 1 ¹ H-NMR spectrum;

FIG. 2 is a diagram showing a probe LXJ-3 prepared in example 1 ¹³ A C-NMR spectrum;

FIG. 3 is a high resolution mass spectrum of probe LXJ-3 prepared in example 1;

FIG. 4 is a graph showing the change of fluorescence intensity with time of the probe LXJ-3 prepared in example 1 reacting with copper ions;

FIG. 5 is a graph showing the change in fluorescence spectrum of probe LXJ-3 prepared in example 1 when copper ions were added at different concentrations;

FIG. 6 is a graph showing the change in fluorescence intensity of probe LXJ-3 prepared in example 1 when added to copper ions of different concentrations and the linear graph showing the fluorescence intensity versus the concentration of copper ions;

FIG. 7 is a graph showing the change of fluorescence response with pH of probe LXJ-3 and probe LXJ-3 reacted with copper ions prepared in example 1;

FIG. 8 shows fluorescence spectra of probe LXJ-3 prepared in example 1 with different interfering ions added in PBS buffer system;

FIG. 9 shows the effect of other interfering ions on the fluorescence intensity of probe LXJ-3 plus copper ion system prepared in example 1;

FIG. 10 shows probes LXJ-3 vs. Cu prepared in example 1 ²⁺ A possible recognition mechanism;

FIG. 11 shows the presence and absence of Cu ²⁺ Fluorescence spectra of probe LXJ-3 (10. mu.M) prepared in example 1 and BHIO (10. mu.M) in PBS (10mM, pH 7.4) (20. mu.M);

FIG. 12 shows probes LXJ-3 and Cu prepared in example 1 ²⁺ High resolution mass spectrogram of the reacted solution.

Detailed Description

The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.

The following discloses many different embodiments or examples for implementing the subject technology described. The following description sets forth specific examples of one or more permutations of various features for the purpose of simplifying the disclosure, but the examples should not be construed as limiting the invention, and any combination of a first feature with a second feature set forth later in the specification can include embodiments that are directly connected, and can also include embodiments that form additional features, and further can include embodiments that use one or more additional intervening features to indirectly connect or combine the first and second features with each other, such that the first and second features may not be directly connected.

In embodiments disclosed herein are phthalimide derivatives having the formula:

the application also discloses a preparation method of the phthalimide derivative, and the synthetic route is as follows:

for ease of understanding, the present application provides several specific implementations:

example 1

The phthalimide derivative is prepared by the following steps, and specifically comprises the following steps:

BHIO (0.1g,0.46mmol), DIPEA (0.18g,1.38 mmol), DMAP (6mg,0.046mmol) and 15mL of dried dichloromethane are added into a 100mL round-bottom flask, stirring is carried out at room temperature, pyridine-2-formyl chloride hydrochloride (0.12g,0.69mmol) is dissolved in 5mL of dried dichloromethane and slowly dropped into the reaction liquid, after the reaction is completed for 8 hours by dropping, TLC monitors that the reaction is completed, and the solvent is removed by distillation under reduced pressure to obtain a crude product; passing through silica gel column (mobile phase is petroleum ether: ethyl acetate 2:1, V: V) to obtain white solid product 0.13g, yield 85%.

The nuclear magnetic analysis of the obtained product showed the following results:

¹ H NMR(400MHz,DMSO-d ₆ )δ:8.84-8.86(m,1H),8.28-8.30(m,1H), 8.10-8.15(m,1H),7.95(t,1H,J＝7.5Hz),7.84(d,1H,J＝7.4Hz),7.75-7.80(m, 2H),3.50(t,2H，J＝7.0Hz),1.48-1.55(m,2H),1.20-1.29(m,2H),0.85(t,3H, J＝7.4Hz)； ¹³ C NMR(100MHz，DMSO-d ₆ )δ:167.02,165.53,162.54,150.21, 146.02,145.96,137.87,136.56,133.27,128.73,128.31,126.03,122.34,121.13, 37.20,29.85,19.46,13.43.

calculated value of ESI-HRMS (m/z) C ₁₈ H ₁₆ N ₂ NaO ₄ [M+Na] ⁺ 347.10023, found 347.10040.

Example 2

Adding BHIO (0.1g,0.46mmol), DIPEA (0.24g, 1.84mmol), DMAP (12mg,0.092mmol) and 15mL of dried dichloromethane into a 100mL round-bottom flask, stirring at room temperature, dissolving pyridine-2-formyl chloride hydrochloride (0.16g,0.92mmol) in 5mL of dried dichloromethane, slowly dropping into the reaction solution, after 4 hours of reaction completion, monitoring by TLC that the reaction is complete, and distilling off the solvent under reduced pressure to obtain a crude product; passing through silica gel column (mobile phase is petroleum ether: ethyl acetate: 2:1, V: V) to obtain white solid product 0.13g, yield 88%.

Example 3

BHIO (0.1g,0.46mmol), triethylamine (0.14g,1.38 mmol), DMAP (6mg,0.046mmol) and 15mL of dried dichloromethane are added into a 100mL round-bottom flask, stirring is carried out at room temperature, pyridine-2-formyl chloride hydrochloride (0.12g,0.69mmol) is dissolved in 5mL of dried dichloromethane and slowly dropped into the reaction liquid, after the completion of the reaction for 8 hours, TLC monitors the completion of the reaction, and the solvent is removed by distillation under reduced pressure to obtain a crude product; passing through silica gel column (mobile phase is petroleum ether: ethyl acetate: 2:1, V: V) to obtain white solid product 0.12g, yield 80%.

Example 4

BHIO (0.1g,0.46mmol), pyridine (0.11g,1.38 mmol), DMAP (6mg,0.046mmol) and 15mL of dried dichloromethane are added into a 100mL round-bottom flask, stirring is carried out at room temperature, pyridine-2-formyl chloride hydrochloride (0.123g,0.69mmol) is dissolved in 5mL of dried dichloromethane and slowly dropped into the reaction liquid, after 8 hours of dropping reaction, TLC monitors that the reaction is complete, and the solvent is removed by distillation under reduced pressure to obtain a crude product; passing through silica gel column (mobile phase is petroleum ether: ethyl acetate 2:1, V: V) to obtain white solid product 0.11g, yield 76%.

The application also discloses several embodiments of the phthalimide derivative used as a fluorescent probe for detecting copper ions.

Example 5

Method for testing spectral properties

Dissolving a probe LXJ-3 in DMSO to prepare a 5mM stock solution, and storing at low temperature; metal cation (K) ⁺ 、Na ⁺ 、Ag ⁺ 、Ca ²⁺ 、Mg ²⁺ 、Cu ²⁺ 、Zn ²⁺ 、Al ³⁺ 、Fe ³⁺ 、Fe ²⁺ 、Cd ²⁺ 、Cr ³⁺ 、 Co ²⁺ 、Hg ²⁺ 、Ni ²⁺ 、Pb ²⁺ 、Mn ²⁺ ) Dissolving with secondary water to obtain a solution with the concentration of 10 mM. The measurement system comprises: 30 μ L of stock solution of probe LXJ-3 was mixed with 30 μ L of the test solutionAnd mixing the metal ion solutions, fixing the volume to 3mL by using PBS buffer solution, placing the mixture in a cuvette, reacting for 15min, and then determining. And (3) testing conditions are as follows: room temperature, measured by a fluorescence spectrophotometer, with a parameter of lambda _ex ＝344nm，λ _em 515nm, slit widths of 5nm and 5nm, voltage of 700V, wavelength range of 420 and 650 nm.

Example 6

Probe LXJ-3 and Cu ²⁺ Influence of reaction time on fluorescence intensity

In the PBS buffer solution (10mM, pH 7.4) system, the fluorescence of the probe LXJ-3(10 μ M) solution itself is very weak; 2 equivalents of Cu were added to the solution of probe LXJ-3 ²⁺ Then, the fluorescence intensity of the probe is recorded at regular intervals, and the result shows that the fluorescence intensity at 515nm is rapidly enhanced, the increase trend of the fluorescence intensity gradually becomes smaller along with the progress of the reaction, and the fluorescence intensity basically tends to be stable after 15 min. Therefore, the reaction time was controlled within 15min in all subsequent experiments (FIG. 4).

Example 7

Cu ²⁺ Influence of concentration of (2) on fluorescence intensity of Probe LXJ-3

As shown in FIG. 5, Cu was added to PBS buffer (10mM, pH 7.4) ²⁺ The fluorescence intensity of probe LXJ-3(10 μ M) at 515nm is dependent on Cu in the concentration range of 0-50 μ M ²⁺ The increase in concentration is markedly enhanced. As can be seen from FIG. 6, in Cu ²⁺ When the concentration was increased to 20. mu.M, the fluorescence intensity of probe LXJ-3 reached a maximum, and further increase of Cu was continued ²⁺ The fluorescence intensity remains substantially unchanged. The fluorescence intensity of the probe LXJ-3 and Cu in the concentration range of 0-10 μ M ²⁺ Has a good linear relation with the regression equation of y being 532.9x +909.7 (R) ² 0.9938). Calculating formula according to limit of detection (LOD): LOD is 3 sigma/K (wherein sigma is Cu is not added) ²⁺ Standard deviation of fluorescence spectrum change of the time probe LXJ-3, and K is the slope of a straight line), calculating to obtain the probe LXJ-3 for Cu ²⁺ The detection limit of (2) was 31nM, indicating that probe LXJ-3 is paired with Cu ²⁺ Has better detection sensitivity.

Example 8

Effect of pH on the detection Performance of Probe LXJ-3

As shown in FIG. 7, in the PBS buffer solution (10mM, pH 7.4), the fluorescence intensity of the probe LXJ-3(10 μ M) solution does not change much in the range of pH 3-11, indicating that the probe LXJ-3 is relatively stable under acidic and weakly alkaline conditions. When 2 equivalents of Cu are added ²⁺ Then, the fluorescence intensity of the probe LXJ-3 and the fluorescence intensity of the probe without Cu are within the pH range of 3-4 ²⁺ The time variation is not large; and in the range of pH 5-10, probes LXJ-3 and Cu ²⁺ The increase in fluorescence intensity after the action was very significant and reached a peak at pH 7.4. Indicating that probe LXJ-3 can detect copper ions over a wide pH range. To facilitate its use in biology, a PBS buffer system at physiological conditions pH 7.4 was chosen for testing.

Example 9

Selectivity and interference of probe LXJ-3 with other metal ions

Selection of cations (K) which may cause interference ⁺ 、Na ⁺ 、Ag ⁺ 、Ca ²⁺ 、Mg ²⁺ 、Zn ²⁺ 、Al ³⁺ 、 Fe ³⁺ 、Fe ²⁺ 、Cd ²⁺ 、Cr ³ ⁺ 、Co ²⁺ 、Hg ²⁺ 、Ni ²⁺ 、Pb ²⁺ 、Mn ²⁺ ) To examine probe LXJ-3 vs. Cu ²⁺ Selectivity of (2). The change in fluorescence spectrum of probe LXJ-3 was measured after reaction at room temperature for 15min by adding 2 equivalents of each of the above ions to 10. mu. mol/L of probe LXJ-3 in PBS buffer (10mM, pH 7.4). When interfering ions are added, the fluorescence of probe LXJ-3 is not significantly changed; while when 2 equivalents of Cu are added ²⁺ After that, the fluorescence intensity was significantly increased (fig. 8). Thus, probe LXJ-3 was aligned with Cu ²⁺ Has specific selectivity and can be used for Cu ²⁺ The analysis and detection of (2).

To further test probes LXJ-3 in Cu ²⁺ Detection of Cu in the Presence of other competitive ions ²⁺ For probe LXJ-3, an anti-interference experiment was performed. 10 μ M Probe LXJ-3 and 2 equivalents Cu in a PBS buffer (10mM, pH 7.4) system ²⁺ After mixing, 2 equivalents of each of the above ionic solutions was added, and the fluorescence intensity of the mixed solution was measuredAnd (4) changing. As shown in FIG. 9, the fluorescence intensity of probe LXJ-3 was compared with that of Cu alone ²⁺ When present, are substantially the same. The above results indicate that the presence of other competitive materials detects Cu for probe LXJ-3 ²⁺ No significant interference is generated. Thus, probe LXJ-3 can be used for Cu in complex environments ²⁺ The detection of (3).

Example 10

Probe LXJ-3 pairs of Cu ²⁺ Mechanism of recognition of

Identification of Cu by pyridine-2-formyl group was reported according to the literature (sensor. actuat. b-chem.,2017,252,134.) ²⁺ We propose LXJ-3 pairs of Cu ²⁺ Possible response mechanism (fig. 10). Because of the presence of pyridine-2-formyl, the ESIPT process based on keto-enol tautomer in BHIO was inhibited, and probe LXJ-3 itself exhibited weak fluorescence. When the probe and Cu are present ²⁺ When the probe is used, pyridine-2-formate is catalyzed and hydrolyzed, the probe LXJ-3 removes pyridine-2-formyl, the fluorescent parent BHIO is released to recover the ESIPT process, and strong green fluorescence is re-presented, so that Cu is realized ²⁺ Identification and detection. To verify our conjecture, LXJ-3 and Cu were paired ²⁺ The fluorescence spectrum and mass spectrum of the product after the reaction were analyzed. Fluorescence spectrum experiments show that under excitation at 344nm, the autofluorescence of the probe LXJ-3 is very weak, while when LXJ-3 and Cu are mixed ²⁺ After the reaction, a significantly enhanced fluorescence signal appeared at 515nm, which is consistent with the fluorescence spectrum of the parent fluorescent BHIO (see FIG. 11). LXJ-3 and Cu ²⁺ An ion peak at M/z 218.08215 appears in the high resolution mass spectrum after the reaction (see FIG. 12), which is related to the deprotonated molecular weight of the parent BHIO [ M-H ]] ^- 218.08227, in accordance.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.

Claims

1. A phthalimide fluorescent probe for detecting copper ions, wherein the fluorescent probe has the following structure:

2. the method for preparing the phthalimide-based fluorescent probe for detecting copper ions according to claim 1, comprising the steps of:

1) dissolving N-butyl-3-hydroxyphthalimide, an acid-binding agent and 4-dimethylaminopyridine serving as a catalyst in dichloromethane, and reacting at 0-30 ℃, wherein the acid-binding agent is selected from any one or more of N, N-diisopropylethylamine, triethylamine and pyridine;

2) dissolving pyridine-2-formyl chloride hydrochloride in dichloromethane, slowly dropwise adding the dichloromethane into a reaction solution, and purifying after the reaction is finished to obtain the phthalimide fluorescent probe for detecting copper ions.

3. The method for preparing a phthalimide-based fluorescent probe for detecting copper ions according to claim 2, wherein the method comprises the steps of: in the step 2), the reaction time is 4-10 h.

4. The method for preparing a phthalimide-based fluorescent probe for detecting copper ions according to claim 2, wherein the method comprises: in the step 2), the purification specifically comprises the steps of distilling the reaction liquid obtained after the reaction under reduced pressure to remove the solvent to obtain a crude product, and then separating and purifying the crude product through a silica gel column, wherein the mobile phase is a mixed solvent of ethyl acetate and petroleum ether in a volume ratio of 1: 2.

5. The method for producing a phthalimide-based fluorescent probe for detecting copper ions according to any one of claims 2 to 4, wherein the method comprises the steps of: the molar ratio of the compound N-butyl-3-hydroxyphthalimide to the pyridine-2-formyl chloride hydrochloride is 1 (1-2); the molar ratio of the compound N-butyl-3-hydroxyphthalimide to the acid-binding agent is 1 (2-4); the molar ratio of the compound N-butyl-3-hydroxyphthalimide to the catalyst is (5-20): 1.

6. Use of a phthalimide-based fluorescent probe for detecting copper ions according to claim 1, wherein: as Cu ²⁺ The fluorescent probe of (1).

7. A method for detecting copper ions is characterized by comprising the following steps:

1) establishing a standard curve of the change of the copper ion concentration gradient and the change value of the fluorescence intensity of the phthalimide fluorescent probe solution for detecting copper ions according to claim 1;

2) detecting the fluorescence intensity of the detected solution, and obtaining Cu in the solution to be detected from the standard curve according to the fluorescence intensity of the detected solution ²⁺ The concentration of the ions.

8. The method for detecting copper ions according to claim 7, wherein the fluorescence detection conditions in each step are as follows: the excitation wavelength is 344nm, the maximum fluorescence emission wavelength is 515nm, the slit width is 5nm and 5nm, and the fluorescence emission spectrum is detected at 420-650 nm.