Fluorescent probe and detection method for continuous detection of copper ions and hypochlorite

Abstract

The application discloses a fluorescent probe and a detection method for continuously detecting copper ions and hypochlorite. The fluorescent probe is a triphenylamine compound containing an acylhydrazone group and has a structure shown in formula (I). The complex of the application is used as a fluorescent probe for detecting hypochlorite in an aqueous solution, and shows a clear fluorescence enhancement response, has a fast response speed, and can realize real-time detection of hypochlorite. The detection sensitivity is high, and the detection limit is as low as 120 nM. By using a standard curve, the concentration of hypochlorite in a to-be-detected sample can be accurately quantitatively analyzed, especially the concentration of hypochlorite in a commercially available disinfectant sample. The hypochlorite has ultrahigh selectivity, and other common anions have almost no interference. The detection method provided by the application can realize rapid detection of harmful substances, copper ions and hypochlorite, is more efficient, and has a relatively low cost.

Description

Technical Field

[0001] This invention relates to the field of environmental monitoring, and in particular to a fluorescent probe and detection method for the continuous detection of copper ions and hypochlorite ions. Background Technology

[0002] Environmental pollution monitoring and control have become a global concern. Copper is an essential trace element for organisms, playing a crucial regulatory role in physiological processes such as cellular respiration, biosynthesis, and metabolism. However, excessive copper can disrupt the metabolic balance in the human body, leading to various neurodegenerative diseases such as Parkinson's and Alzheimer's. Therefore, efficient detection of copper ions in the environment is of great significance. Hypochlorous acid (HClO) is a reactive oxygen species that plays an important role in daily life and in defending against invading pathogens. It is a strong oxidant and can therefore be used as a bleaching agent, oxidant, deodorant, and disinfectant. However, excessive hypochlorous acid in the body can damage the host's tissues, causing a series of diseases such as kidney disease, arteriosclerosis, and arthritis. Therefore, efficient detection of hypochlorous acid ions in the environment is also of great significance.

[0003] In recent years, fluorescence technology, as an emerging detection method, has gained popularity due to its high sensitivity, fast response speed, and low detection limit, and has been widely applied in fields such as biochemistry, cell biology, and analytical chemistry. However, most reported fluorescent probes currently only detect a single target and are relatively expensive; fluorescent probes capable of continuously detecting multiple harmful substances are rarely reported. Moreover, current methods for directly detecting hypochlorite often require long reaction times, which is not conducive to real-time detection in actual samples. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned technical deficiencies and propose a fluorescent probe and detection method for continuous detection of copper ions and hypochlorite ions, thereby solving the technical problems of existing fluorescent probes having a single detection target and being unable to achieve rapid and real-time detection of hypochlorite ions.

[0005] In a first aspect, the present invention provides a fluorescent probe for the continuous detection of copper ions and hypochlorite ions, which is a triphenylamine compound containing an acylhydrazone group and has the structure shown in formula (I):

[0006]

[0007] Secondly, the present invention provides a method for preparing a fluorescent probe for continuous detection of copper ions and hypochlorite ions, comprising the following steps:

[0008] The aldehyde compound of triphenylamine and 2-pyridylhydrazone were dissolved in a first organic solvent, followed by the addition of a catalyst and reflux reaction. After the reaction was completed, the mixture was naturally cooled to room temperature, and a solid precipitated. The solid was filtered and recrystallized from anhydrous ethanol to obtain triphenylamine compounds containing hydrazone groups. The reaction formula is as follows:

[0009]

[0010] Thirdly, the present invention provides a method for the continuous detection of copper ions and hypochlorite ions, comprising the following steps:

[0011] S1. Copper ions are detected by triphenylamine compounds containing acylhydrazone groups, and complexes formed by triphenylamine compounds containing acylhydrazone groups and copper ions are obtained;

[0012] S2. Detection of hypochlorite ions using the above complex; wherein the above complex has the structure shown in formula (II):

[0013]

[0014] Compared with the prior art, the beneficial effects of the present invention include:

[0015] The fluorescent probe of this invention—a triphenylamine compound containing an acylhydrazone group—complexes with copper ions, resulting in a significant quenching of the solution fluorescence. Simultaneously, the complexation weakens the carbon-nitrogen double bond in the molecule, making it more favorable for hypochlorite attack. Therefore, after adding hypochlorite to the complex solution, the unique oxidizing property of hypochlorite oxidizes the acylhydrazone group in the molecule to an aldehyde group, causing the solution to emit a strong orange fluorescence. When used as a fluorescent probe for the detection of hypochlorite in aqueous solutions, the complex of this invention exhibits a significant fluorescence enhancement response, a fast response speed, and enables real-time detection of hypochlorite. It has high detection sensitivity, with a detection limit as low as 120 nM. Using a standard curve, it can accurately quantify the concentration of hypochlorite in the sample, especially in commercially available disinfectant samples. It exhibits extremely high selectivity for hypochlorite, with almost no interference from other common anions. The detection method provided by this invention can sequentially achieve rapid detection of harmful substances such as copper ions and hypochlorite, which is more efficient and lower in cost. Attached Figure Description

[0016] Figure 1 Fluorescence titration diagrams of copper ions at different concentrations for a solution of a triphenylamine compound containing an acylhydrazone group provided by the present invention;

[0017] Figure 2 A linear relationship between the fluorescence intensity change of the triphenylamine compound solution containing an acylhydrazone group at 490 nm and the concentration of copper ions, provided by the present invention.

[0018] Figure 3 The recognition response diagram of copper ions in a solution of triphenylamine compound containing an acylhydrazone group provided by the present invention when common interfering ions coexist;

[0019] Figure 4 The fluorescence titration diagrams of the complex solution provided by this invention on hypochlorite ions of different concentrations are shown.

[0020] Figure 5 The linear relationship between the fluorescence intensity change of the complex solution at 595 nm and the hypochlorite concentration provided by the present invention;

[0021] Figure 6 The fluorescence spectrum of the complex solution provided by this invention after adding commercially available disinfectant diluted 1000 times;

[0022] Figure 7 The diagram shows the recognition response of the complex solution provided by this invention to hypochlorite ions when common interfering ions coexist. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0024] In recent years, fluorescence technology, as an emerging detection method, has gained popularity due to its high sensitivity, fast response speed, and low detection limit. It has been widely applied in fields such as biochemistry, cell biology, and analytical chemistry. However, current fluorescent probes are limited to copper ion detection or hypochlorite detection, resulting in a single detection target, cumbersome operation, and high cost. Furthermore, some reported hypochlorite fluorescent probes exhibit slow response speeds to hypochlorite, hindering real-time detection.

[0025] Therefore, this invention is proposed.

[0026] In a first aspect, the present invention provides a fluorescent probe for the continuous detection of copper ions and hypochlorite ions, which is a triphenylamine compound containing an acylhydrazone group and has the structure shown in formula (I):

[0027]

[0028] Secondly, the present invention provides a method for preparing a fluorescent probe for continuous detection of copper ions and hypochlorite ions, comprising the following steps:

[0029] The aldehyde compound of triphenylamine and 2-pyridylhydrazone were dissolved in a first organic solvent, followed by the addition of a catalyst, and the mixture was heated under reflux. After the reaction was complete, the mixture was allowed to cool naturally to room temperature, and a solid precipitated. The solid was filtered and recrystallized from anhydrous ethanol to obtain a triphenylamine compound containing an acylhydrazone group. The reaction formula is as follows:

[0030]

[0031] The molar ratio of the aldehyde compound of triphenylamine to 2-pyridylhydrazone is 1:(1-2), and more specifically 1:(1.1-1.3).

[0032] The ratio of the amount of triphenylamine aldehyde compound to the first organic solvent is 1 g: (10-30) mL.

[0033] The first organic solvent is anhydrous ethanol.

[0034] The amount of catalyst added is 5-20% of the mass of the aldehyde compound of triphenylamine.

[0035] The catalyst is acetic acid.

[0036] The reflux reaction temperature is 80–90℃, and the reflux reaction time is 6–7 hours.

[0037] The reflux reaction is carried out under a nitrogen atmosphere.

[0038] Thirdly, the present invention provides a method for the continuous detection of copper ions and hypochlorite ions, comprising the following steps:

[0039] S1. Copper ions are detected by triphenylamine compounds containing acylhydrazone groups, and complexes formed by triphenylamine compounds containing acylhydrazone groups and copper ions are obtained;

[0040] S2. Detection of hypochlorite ions using the above complex; wherein the above complex has the structure shown in formula (II):

[0041]

[0042] The detection process is as follows: A solution of triphenylamine compound (I) containing an acylhydrazone group emits strong green fluorescence. When it reacts with copper ions (the analyte), a stable complex (II) is formed, and the fluorescence of the solution is greatly quenched. The resulting complex (II) solution is then used to detect hypochlorite. Utilizing the oxidizing property of hypochlorite, the C=N bond in complex (II) is broken, oxidizing it to an aldehyde compound, and the solution emits strong orange fluorescence. The specific process is as follows:

[0043]

[0044] The key technology of this invention lies in the fact that the complexation of copper ions with the acylhydrazone group in compound (I) weakens the C=N bond, which is more conducive to the subsequent oxidation reaction of hypochlorite. Therefore, the response speed to hypochlorite is faster, enabling real-time detection of hypochlorite. In contrast, directly adding hypochlorite to the solution of compound (I) results in a slower response speed and very low detection sensitivity. Even with a 10-fold increase in hypochlorite concentration and a reaction time extended to 10 minutes, the change in the fluorescence spectrum is still not significant. Therefore, the detection method provided by this invention can sequentially detect harmful substances such as copper ions and hypochlorite, which is more efficient and lower in cost.

[0045] In this embodiment, step S1 includes:

[0046] S11. Provide a solution of triphenylamine compounds containing acylhydrazone groups and a copper ion solution;

[0047] S12. Add copper ion solution to a triphenylamine compound solution containing acylhydrazone group, mix well, and use a fluorescence spectrophotometer to test its emission spectrum to determine the fluorescence intensity corresponding to different copper ion concentrations.

[0048] S13. Determine the fluorescence intensity change value corresponding to different copper ion concentrations based on the fluorescence intensity corresponding to different copper ion concentrations, and perform linear fitting with the fluorescence intensity change value corresponding to different copper ion concentrations as the ordinate and the copper ion concentration as the abscissa to establish a standard curve for copper ions.

[0049] S14. Use a fluorescence spectrophotometer to test the fluorescence intensity of the copper ion sample to be tested, and determine the copper ion concentration in the sample to be tested according to the standard curve of copper ions.

[0050] The water-soluble copper salt used in the copper ion solution can be CuCl2 or Cu(NO3)2, etc., and this invention does not impose any restrictions.

[0051] The solution of triphenylamine compounds containing acylhydrazone groups is obtained through the following steps:

[0052] The concentration range of 1×10⁻⁶ was prepared using a second organic solvent. -4 ~1×10 -1 A stock solution of triphenylamine compounds containing acylhydrazone groups was prepared at mol / L, and then diluted to 5 × 10⁻⁶ mol / L using a buffer solution and a second organic solvent. -7 ~1×10 -5 mol / L.

[0053] Furthermore, the second organic solvent can be dimethyl sulfoxide, acetonitrile, tetrahydrofuran (THF), N,N-dimethylformamide, acetone, etc., and the present invention does not limit it.

[0054] Furthermore, the buffer solution can be 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer solution, etc., and the present invention is not limited thereto.

[0055] Furthermore, the pH of the HEPES buffer is 6–10.

[0056] Furthermore, in the solution of triphenylamine compounds containing acylhydrazone groups, the volume ratio of the buffer solution to the second organic solvent is in the range of 1:(0.2 to 5), and even more specifically 1:1.

[0057] In the process of testing its emission spectrum using a fluorescence spectrophotometer, the excitation wavelength was 390 nm.

[0058] Linear fitting was performed using the fluorescence intensity change at 490 nm corresponding to different copper ion concentrations as the ordinate and the copper ion concentration as the abscissa.

[0059] In the process of testing the fluorescence intensity of the copper ion sample using a fluorescence spectrophotometer, if the copper ion concentration in the sample is too high, the sample must first be diluted to the copper ion concentration range applicable to the standard curve of copper ions.

[0060] In this embodiment, step S2 includes:

[0061] S21. Provide a solution of a triphenylamine compound containing an acylhydrazone group, a copper ion solution, and a sodium hypochlorite solution;

[0062] S22. Add copper ion solution to a solution of triphenylamine compound containing acylhydrazone group, mix well to obtain complex solution;

[0063] S23. Add sodium hypochlorite solution to the complex solution, mix well, and use a fluorescence spectrophotometer to test its emission spectrum to determine the fluorescence intensity corresponding to different hypochlorite concentrations.

[0064] S24. Determine the fluorescence intensity change value corresponding to different hypochlorite concentrations based on the fluorescence intensity corresponding to different hypochlorite concentrations, and perform linear fitting with the fluorescence intensity change value corresponding to different hypochlorite concentrations as the ordinate and the hypochlorite concentration as the abscissa to establish a standard curve for hypochlorite.

[0065] S25. Add the hypochlorite sample to be tested to the complex solution, mix well, test its fluorescence intensity using a fluorescence spectrophotometer, and determine the concentration of hypochlorite in the hypochlorite sample according to the standard curve of hypochlorite.

[0066] The methods for obtaining the triphenylamine compound solution containing acylhydrazone group and the copper ion solution are as described above, and will not be repeated here.

[0067] In the process of adding copper ion solution to a solution of triphenylamine compound containing hydrazone group, the molar ratio of triphenylamine compound containing hydrazone group to copper ion is 1:(1~3).

[0068] In the process of testing its emission spectrum using a fluorescence spectrophotometer, the excitation wavelength was 390 nm.

[0069] The fluorescence intensity change at 595 nm corresponding to different hypochlorite concentrations was used as the ordinate and the hypochlorite concentration as the abscissa for linear fitting.

[0070] In the process of testing the fluorescence intensity of the hypochlorite sample using a fluorescence spectrophotometer, if the hypochlorite concentration in the sample is too high, the sample must first be diluted to the hypochlorite concentration range applicable to the standard curve of hypochlorite.

[0071] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0072] Example 1: Preparation of triphenylamine compounds (I) containing acylhydrazone groups

[0073] (1) Under a nitrogen atmosphere, the aldehyde compound of triphenylamine (1.27 g, 2.0 mmol) and 2-pyridylhydrazone (0.34 g, 2.5 mmol) were dissolved in 30 mL of anhydrous ethanol. Then, 3 drops of acetic acid were added as a catalyst, and the mixture was heated under reflux at 85 °C for 7 hours. After the reaction was completed, the mixture was allowed to cool naturally to room temperature, and a solid precipitated. The solid was filtered and recrystallized from anhydrous ethanol to obtain a triphenylamine compound (I) containing an acylhydrazone group. The structural characterization results are as follows:

[0074] 1 H NMR (500MHz, CDCl3): δ = 11.02 (1H, s), 8.6 (1H, s), 8.52-8.53 (1H, d, J = 5.0Hz), 8.26-8.27 (1H, d, J = 5.0Hz), 7.87-7.90 (1H, t, J = 5.0Hz) ,7.45-7.47(1H,t,J=5.0Hz),7.29-7.30(6H,d,J=5.0Hz),7.17-7.19(4H,m),7.05-7.09(2H,m),6.89-6.95(5H,m),6.77-6.80(1H,m). 13C NMR(125MHz, CDCl3):159.7,148.9,147.8,146.6,146.1,143.9,143.3,141.0,138.3,137.4,135.3,132 .6,132.0,131.7,129.3,127.6,126.9,125.5,124.1,123.6,123.3,120.6,115.9ppm.MS(ESI),m / z[M+H] + :741.5,calcd,740.9.

[0075] Example 2: Detection of copper ions

[0076] The detection of copper ions by triphenylamine compounds (I) containing acylhydrazone groups mainly includes the following steps:

[0077] (1) Prepare a 10 mM HEPES buffer solution with a pH of 7.4; prepare a THF solution of compound (I) to obtain a concentration of 1 × 10⁻⁶ mM. -4 The first solution was prepared at a concentration of 1 mol / L. 1 mL of this first solution was placed in a 10 mL colorimetric tube and diluted to volume with 4 mL THF and 5 mL HEPES buffer solution to obtain a concentration of 1 × 10⁻⁶ mol / L. -5 A mol / L solution of compound (I) (THF to HEPES buffer solution volume ratio 1:1).

[0078] (2) Prepare a solution with a concentration of 3.0 × 10⁻⁶ using deionized water. -3 A mol / L aqueous solution of Cu(NO3)2.

[0079] (3) Take 3 mL of the compound (I) solution prepared in step (1) and place it in a quartz cuvette. Under room temperature conditions, excite it at a wavelength of 390 nm and test its emission spectrum using a fluorescence spectrophotometer. Then, add to the solution in sequence volumes (unit: μL) of Cu(NO3)2 aqueous solution prepared in step (2) (the effect on the total volume is negligible), and continuously test the fluorescence spectrum under the same conditions.

[0080] like Figure 1 As shown, the titration curves at 490 nm, from top to bottom, represent the copper ion concentrations in the system (unit: 10). -6 The fluorescence spectra of the system at concentrations of mol / L were obtained at 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0, respectively. From... Figure 1As can be seen, the fluorescence emission spectrum at 490 nm gradually weakens with increasing copper ion concentration. Furthermore, when the copper ion concentration in the system is only 5 × 10⁻⁶, the fluorescence emission spectrum at 490 nm gradually decreases. -7 At a concentration of mol / L, the fluorescence intensity decreased to 90% of its original value; when the copper ion concentration was 9 × 10⁻⁶ mol / L, the fluorescence intensity decreased to 90% of its original value. -6 At mol / L, the fluorescence emission peak was almost completely quenched.

[0081] like Figure 2 As shown, within the concentration range of 0–9 μM, the change in fluorescence spectrum exhibits a good linear relationship with copper ions. The detection limit of this complex is calculated to be as low as 90 nM. Therefore, the compound (I) described in this invention can achieve highly sensitive detection of copper ions.

[0082] Example 3: Selectivity experiment on copper ions

[0083] The selective experiments on copper ions by triphenylamine compounds (I) containing acylhydrazone groups mainly include the following steps:

[0084] (1) Prepare a 10 mM HEPES buffer solution with a pH of 7.4; prepare a tetrahydrofuran (THF) solution of compound (I) to obtain a concentration of 1×10⁻⁶. -4 The first solution was prepared at a concentration of 1 mol / L. 1 mL of this first solution was placed in a 10 mL colorimetric tube and diluted to volume with 4 mL of THF and 5 mL of HEPES buffer solution to obtain a concentration of 1 × 10⁻⁶ mol / L. -5 A mol / L solution of compound (I) (THF to HEPES buffer solution volume ratio 1:1).

[0085] (2) Prepare deionized water solutions of NaNO3, KNO3, LiCl, AgNO3, MgSO4, MnSO4·2H2O, Zn(NO3)2·6H2O, Ni(NO3)2·6H2O, Pb(NO3)2, Ba(NO3)2, Ca(NO3)2·4H2O, CoCl2·6H2O, CdSO4·8H2O, Fe(NO3)3·9H2O, Al(NO3)3·9H2O, and Cr(NO3)3·9H2O, with a concentration of 1×10⁻⁶ for each of the following: -2 mol / L.

[0086] (3) Take 3 mL of the compound (I) solution prepared in step (1) and place it in a quartz cuvette. Then, add 15 μL of the aqueous solutions of each interfering substance prepared in step (2) to the solution. Under room temperature conditions, excite at a wavelength of 390 nm and measure the emission spectrum using a fluorescence spectrophotometer. Then, continue to add copper ion solution with a concentration of 1 × 10⁻⁶. -5The concentration was mol / L, the excitation wavelength was 390 nm, and the emission spectrum was measured using a fluorescence spectrophotometer.

[0087] like Figure 3 As shown, the vertical axis represents the fluorescence intensity change at 490 nm, and the horizontal axis represents the concentration at 5 × 10⁻⁶. -5 Various interfering ions at mol / L, and the addition of copper ions to the interfering ions. Comparison. Figure 3 As can be seen from the height of the bar chart, the fluorescence spectral response of compound (I) of the present invention to other common metal ions is much smaller than that to copper ions. The technical solution described in the present invention can achieve highly selective detection of copper ions.

[0088] Example 4: Detection of hypochlorite by complex (II)

[0089] (1) Prepare a solution of compound (I) according to the above method;

[0090] (2) Take 10 mL of the above compound (I) solution and add 40 μL of a solution with a concentration of 3.0 × 10⁻⁶. -3 A solution of complex (II) was prepared by thoroughly mixing a copper ion aqueous solution at a concentration of mol / L.

[0091] (3) Prepare a solution with a concentration of 3.0 × 10⁻⁶ using deionized water. -3 A mol / L NaClO aqueous solution was prepared. 3 mL of the above complex (II) solution was placed in a quartz cuvette, and its emission spectrum was measured using a fluorescence spectrophotometer at room temperature and an excitation wavelength of 390 nm. Then, volumes (in μL) of the above-prepared NaClO aqueous solution were added sequentially (the effect on the total volume is negligible), and the fluorescence spectra were continuously measured under the same conditions. The results are as follows: Figure 4 As shown, the titration curves at 595 nm, from bottom to top, represent the NaClO concentrations in the system (unit: 10). -6 The fluorescence spectra of the system were obtained at concentrations (mol / L) of 0.0, 0.2, 0.6, 1.0, 1.5, 2.0, 3.0, 4.0, 7.0, 10.0, 15.0, 20.0, and 25.0. Notably, complex (II) responds very rapidly to hypochlorite; the fluorescence spectrum changes immediately upon the addition of NaClO, which is beneficial for real-time detection of hypochlorite. A standard curve was then plotted with the change in fluorescence intensity at 595 nm as the ordinate and the NaClO concentration as the abscissa. The results are shown below. Figure 5 As shown in the figure. Thus, the hypochlorite content in the test solution can be calculated based on the standard curve.

[0092] like Figure 4 As shown, the fluorescence emission spectrum at 595 nm gradually increases with increasing NaClO concentration. Furthermore, when the NaClO concentration is 2.5 × 10⁻⁶, the fluorescence emission spectrum at 595 nm gradually increases. -5 At mol / L, the fluorescence emission intensity increased by nearly 1600 times.

[0093] like Figure 5 As shown, within the concentration range of 0–25 μM, the change in fluorescence intensity exhibits a good linear relationship with the NaClO concentration (R0). 2 =0.9978), and the linear equation obtained from the simulation is y = 67.9236x - 2.8935 (y represents the fluorescence intensity change at 595nm, x represents the concentration of NaClO, in units of 10). -6 The concentration of mol / L indicates that the detection method provided by this invention can achieve accurate quantitative analysis of hypochlorite. According to the formula: Detection limit = 3σ / k, the detection limit of this complex is calculated to be as low as 120 nM. Where σ represents the standard deviation of the fluorescence intensity of the blank solution in ten measurements, and k represents... Figure 5 The slope of the fitted curve. It can be seen that the complex (II) described in this invention can achieve highly sensitive detection of hypochlorite.

[0094] Example 5: Application of Complex (II) in the Detection of Commercially Available Disinfectant Samples

[0095] (1) Prepare a solution of complex (II) according to the above method;

[0096] (2) Dilute the commercially available disinfectant with deionized water 1000 times to obtain a disinfectant sample. Then take 200 μL of the sample and add it to 15 mL of complex (II) solution. After shaking well, divide it into five equal parts.

[0097] like Figure 6 As shown, the fluorescence intensity of five equal portions of the above solution was tested, and the average value of the fluorescence intensity change (I / I0-1) was 1160, corresponding to a hypochlorite concentration of 1.73 × 10⁻¹ in the standard curve. -5 The concentration of chlorine in the sample is approximately mol / L, which is basically consistent with the available chlorine content of 14.4–17.6 g / L on the product label, indicating that the detection method of the present invention can be used for accurate quantitative detection of hypochlorite in actual disinfectant samples.

[0098] Example 6: Selectivity experiment of complex (II) for hypochlorite ions

[0099] (1) Prepare a solution of compound complex (II) according to the above method;

[0100] (2) Prepare deionized aqueous solutions of NaOAc·3H2O, NaNO2, NaNO3, KClO3, NaF, NaClO4, NaHCO3, K3PO4·3H2O, NaCl, NaIO3, K2HPO4·3H2O, KBr, NaHSO3, NaHSO4, KI, Na2S2O3·5H2O, KSCN, and H2O2 respectively, i.e., aqueous solutions of interfering substances, with a concentration of 1×10⁻⁶. -2 mol / L;

[0101] (3) Take 3 mL of the solution prepared in step (1) above and place it in a quartz cuvette. Under room temperature conditions, excite it at a wavelength of 390 nm and test its emission spectrum using a fluorescence spectrophotometer. Then add 15 μL of the above-mentioned interfering aqueous solution (the effect on the total volume is negligible) to the resulting solution and test its fluorescence spectrum under the same conditions. Then continue to add sodium hypochlorite solution with a concentration of 2.2 × 10⁻⁶. -5 The concentration was mol / L, and its emission spectrum was measured using a fluorescence spectrophotometer. The results are as follows: Figure 7 As shown, the vertical axis represents the fluorescence intensity change at 595 nm, and the horizontal axis represents the concentration at 5 × 10⁻⁶. -5 Various interfering ions at mol / L, and interfering ions plus hypochlorite.

[0102] like Figure 7 As shown, in the detection method provided by this invention, the fluorescence spectral response to other common interfering substances is much smaller than that to ClO. - Therefore, the continuous detection method for copper ions and hypochlorite provided by the present invention has high selectivity and high specificity for hypochlorite.

[0103] In summary, the fluorescent probe and detection method for continuous detection of copper ions and hypochlorite provided by this invention can accurately detect copper ions and hypochlorite ions qualitatively and quantitatively in sequence, saving costs, and has high sensitivity and selectivity, with high specificity for copper ions and hypochlorite ions, making it highly practical.

[0104] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A fluorescent probe for the continuous detection of copper ions and hypochlorite ions, characterized in that, The fluorescent probe used for continuous detection of copper ions and hypochlorite ions is a triphenylamine compound containing an acylhydrazone group, having the structure shown in formula (I): 。 2. A method for preparing a fluorescent probe for continuous detection of copper ions and hypochlorite ions as described in claim 1, characterized in that, Includes the following steps: The aldehyde compound of triphenylamine and 2-pyridylhydrazone were dissolved in a first organic solvent, followed by the addition of a catalyst, and the mixture was heated under reflux. After the reaction was completed, the mixture was allowed to cool naturally to room temperature, and a solid precipitated. The solid was filtered and recrystallized from anhydrous ethanol to obtain a triphenylamine compound containing an acylhydrazone group. The catalyst was acetic acid. The reflux reaction was carried out under a nitrogen atmosphere. The reaction formula is as follows: 。 3. The method for preparing the fluorescent probe for continuous detection of copper ions and hypochlorite ions according to claim 2, characterized in that, The molar ratio of the aldehyde compound of triphenylamine to 2-pyridylhydrazone is 1:(1~2); the molar ratio of the aldehyde compound of triphenylamine to the first organic solvent is 1g:(10~30)mL; the first organic solvent is anhydrous ethanol; the amount of catalyst added is 5~20% of the mass of the aldehyde compound of triphenylamine; the reflux reaction temperature is 80~90°C. o C, the reflux reaction time is 6~7h.

4. A method for continuous detection of copper ions and hypochlorite ions, characterized in that, Includes the following steps: S1. Detect copper ions using the triphenylamine compound containing an acylhydrazone group as described in claim 1, and obtain a complex formed by the triphenylamine compound containing an acylhydrazone group and copper ions; S2. Detection of hypochlorite ions using the complex; wherein... The complex has the structure shown in formula (II): ； The detection method for continuous detection of copper ions and hypochlorite ions is intended for non-disease diagnosis or treatment.

5. The detection method for continuous detection of copper ions and hypochlorite ions according to claim 4, characterized in that, Step S1 includes: S11. Provide a solution of triphenylamine compounds containing acylhydrazone groups and a copper ion solution; S12. Add the copper ion solution to the triphenylamine compound solution containing the acylhydrazone group, mix well, and use a fluorescence spectrophotometer to test its emission spectrum to determine the fluorescence intensity corresponding to different copper ion concentrations. S13. Determine the fluorescence intensity change value corresponding to different copper ion concentrations based on the fluorescence intensity corresponding to different copper ion concentrations, and perform linear fitting with the fluorescence intensity change value corresponding to different copper ion concentrations as the ordinate and the copper ion concentration as the abscissa to establish a standard curve for copper ions. S14. Use a fluorescence spectrophotometer to test the fluorescence intensity of the copper ion sample to be tested, and determine the copper ion concentration in the copper ion sample to be tested according to the standard curve of copper ions.

6. The detection method for continuous detection of copper ions and hypochlorite ions according to claim 5, characterized in that, The solution of the triphenylamine compound containing the acylhydrazone group is obtained through the following steps: The concentration range of 1×10⁻⁶ was prepared using a second organic solvent. -4 ~ 1×10 -1 A stock solution of triphenylamine compounds containing acylhydrazone groups was prepared at mol / L, and then diluted to 5 × 10⁻⁶ mol / L using a buffer solution and a second organic solvent. -7 ~ 1×10 -5 mol / L; where, The second organic solvent is at least one of dimethyl sulfoxide, acetonitrile, tetrahydrofuran, N,N-dimethylformamide, and acetone; The buffer solution is a HEPES buffer solution, and the pH value of the HEPES buffer solution is 6~10; In the solution of the triphenylamine compound containing an acylhydrazone group, the volume ratio of the buffer solution to the second organic solvent is in the range of 1:(0.2~5).

7. The detection method for continuous detection of copper ions and hypochlorite ions according to claim 5, characterized in that, During the process of testing its emission spectrum using a fluorescence spectrophotometer, the excitation wavelength was 390 nm; linear fitting was performed with the fluorescence intensity change corresponding to different copper ion concentrations at 490 nm as the ordinate and the copper ion concentration as the abscissa.

8. The detection method for continuous detection of copper ions and hypochlorite ions according to claim 4, characterized in that, Step S2 includes: S21. Provide a solution of a triphenylamine compound containing an acylhydrazone group, a copper ion solution, and a sodium hypochlorite solution; S22. Add the copper ion solution to the triphenylamine compound solution containing the acylhydrazone group, mix well, and obtain a complex solution; S23. Add sodium hypochlorite solution to the complex solution, mix well, and use a fluorescence spectrophotometer to test its emission spectrum to determine the fluorescence intensity corresponding to different hypochlorite concentrations. S24. Based on the fluorescence intensity corresponding to different hypochlorite concentrations, determine the fluorescence intensity change value corresponding to different hypochlorite concentrations, and perform linear fitting with the fluorescence intensity change value corresponding to different hypochlorite concentrations as the ordinate and the hypochlorite concentration as the abscissa to establish a standard curve for hypochlorite. S25. Add the hypochlorite sample to be tested to the complex solution, mix well, test its fluorescence intensity using a fluorescence spectrophotometer, and determine the concentration of hypochlorite in the hypochlorite sample according to the standard curve of hypochlorite.

9. The detection method for continuous detection of copper ions and hypochlorite ions according to claim 8, characterized in that, During the process of adding copper ion solution to the solution of triphenylamine compound containing hydrazone group, the molar ratio of the triphenylamine compound containing hydrazone group to the copper ions is 1:(1~3).

10. The detection method for continuous detection of copper ions and hypochlorite ions according to claim 8, characterized in that, During the process of testing its emission spectrum using a fluorescence spectrophotometer, the excitation wavelength was 390 nm; linear fitting was performed with the fluorescence intensity change corresponding to different hypochlorite concentrations at 595 nm as the ordinate and the hypochlorite concentration as the abscissa.

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