Method for detecting hydrogen peroxide based on naphthalimide fluorescent probe

Abstract

The invention provides a method for detecting hydrogen peroxide based on a naphthalimide fluorescent probe, the chemical name of the probe is (E)-(5-(((2-(2-mercaptoethyl)-1, 3-dioxo-2, 3-dihydro-1H-benzo [de] isoquinoline-6-yl) imino) methyl) thiophene-2-yl) boric acid, and the structural formula of the probe is shown in the description. Thiophene plays an important role in constructing a fluorescent probe based on naphthalimide. The fluorescence of the probe is changed into yellow green fluorescence from blue before and after reaction, and the probe has excellent sensing performance on hydrogen peroxide, including quite excellent selectivity and detection limit as low as 38.5 nM. Besides, on the basis of a reaction product aggregation phenomenon induced by cellulose, an (E)-(5-(((2-(2-mercaptoethyl)-1, 3-dioxo-2, 3-dihydro-1H-benzo [de] isoquinoline-6-yl) imino) methyl) thiophene-2-yl) boric acid / cellulose probe is further developed, and it is proved that the probe is higher in hydrogen peroxide detection speed, higher in sensitivity and capable of being used for detecting hydrogen peroxide. And the detection limit is as low as 4.0 nM.

Description

Technical Field

[0001] The present invention belongs to the field of analytical detection, and provides a method for detecting hydrogen peroxide based on a naphthalimide-based fluorescent probe. Background Art

[0002] Hydrogen peroxide (H2O2), as a representative and extremely important precursor in improvised explosives, is not only a raw material for synthesizing peroxide explosives, but also a decomposition product of peroxide explosives. For example, highly dangerous improvised explosives such as triacetone triperoxide (TATP), diacetone diperoxide (DADP), and hexamethylene triperoxide diamine (HMTD) are indispensable key raw materials during the synthesis process. Therefore, in order to effectively protect human health and maintain public safety, it is urgent to develop a method that can quickly and accurately detect hydrogen peroxide on-site. Among them, on-site detection has strict requirements for detection means. It not only needs to give results in a short time so as to take timely measures to effectively prevent the further synthesis and use of explosives, but also requires high sensitivity to ensure that extremely trace amounts of hydrogen peroxide can be detected and potential dangers can be avoided due to too high detection limits.

[0003] So far, researchers have developed a large number of sensing methods for hydrogen peroxide detection, including mass spectrometry, chromatography, electrochemistry, chemiluminescence, etc. However, these methods are costly, require professional personnel to operate, and are not convenient for real-time detection and monitoring. Fluorescence analysis has made certain progress in the field of hydrogen peroxide detection due to its low cost, simple operation, high sensitivity, and strong selectivity. However, the performance of the reported fluorescence sensing materials still needs to be improved. For example, it is difficult to achieve highly sensitive and highly selective detection of hydrogen peroxide at low concentrations, and there are generally problems such as long response time, complex detection methods, harsh detection conditions, and difficulty in gas-phase detection. In summary, there are still great challenges in the practical application of current fluorescence sensing materials for hydrogen peroxide detection. Therefore, constructing a material with advantages such as naked-eye observation, fast detection speed, high sensitivity, and strong anti-interference ability is of great significance for the detection of hydrogen peroxide. Summary of the Invention

[0004] The object of the present invention is to propose a method for detecting hydrogen peroxide based on a naphthalimide-based fluorescent probe in order to achieve highly sensitive detection of hydrogen peroxide. The chemical name of this probe is (E)-(5-(((2-(2-mercaptoethyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)imino)methyl)thiophen-2-yl)boronic acid. During the detection of hydrogen peroxide, the boronic acid it contains will be oxidized, and the carbon-nitrogen double bond in the π-conjugated bridge will break, resulting in the ratio fluorescence changing from light blue to yellow-green. The (E)-(5-(((2-(2-mercaptoethyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)imino)methyl)thiophen-2-yl)boronic acid probe exhibits excellent sensing performance towards hydrogen peroxide, with a detection limit as low as 38.5 nM and quite good selectivity even in the presence of 18 interfering substances. In addition, due to the advantages of cellulose such as chemically modifiable structure, biodegradability, high strength, and high specific surface area, and cellulose contains abundant hydroxyl and carboxyl groups, which can provide more active sites for binding with probe molecules. Therefore, in order to further improve the sensing performance of this probe, cellulose rich in hydroxyl groups was introduced, and an (E)-(5-(((2-(2-mercaptoethyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)imino)methyl)thiophen-2-yl)boronic acid / cellulose colloidal probe was designed to achieve signal enrichment and enhance the visualization effect. During the detection process, the fluorescence color of the probe changes from blue to yellow-green, and the detection limit is further reduced to 4.0 nM, showing more excellent detection performance.

[0005] A method for detecting hydrogen peroxide based on a naphthalimide-based fluorescent probe according to the present invention, the chemical name of this probe is (E)-(5-(((2-(2-mercaptoethyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)imino)methyl)thiophen-2-yl)boronic acid, and the specific operation is carried out according to the following steps:

[0006] Preparation of naphthalimide-based fluorescent probe:

[0007] a. Dissolve 4-amino-1,8-naphthalic anhydride and 2-aminoethanethiol in 20 mL of ethanol according to a molar ratio of 1:8, stir overnight at a temperature of 80 °C, then cool the solution to room temperature to form a yellow precipitate, filter to obtain a solid product, wash it 3 times with pure water, and then dry the product in a vacuum oven to obtain the intermediate 6-amino-2-(2-mercaptoethyl)-1H-benzoisoquinoline-1,3(2H)-dione, and its structural formula (Ⅰ) is:

[0008]

[0009] b. Mix the intermediate obtained in step a and 5-formyl-2-thiopheneboronic acid in a molar ratio of 1:1 and dissolve them in 20 mL of ethanol. Stir overnight at 70 °C, evaporate the solvent to obtain a crude product, and further recrystallize it with ethanol. Filter to obtain a solid product. Wash the solid product three times with ethanol, and then dry the product in a vacuum oven to obtain the probe (E)-(5-(((2-(2-mercaptoethyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)imino)methyl)thiophen-2-yl)boronic acid, and its structural formula (II) is:

[0010]

[0011]

[0012] Detection of hydrogen peroxide:

[0013] c. At room temperature, take a solution of the probe (E)-(5-(((2-(2-mercaptoethyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)imino)methyl)thiophen-2-yl)boronic acid with a concentration of 0.1 mM. The organic solvent is ethanol, and a fluorescent probe solution for detecting hydrogen peroxide is obtained.

[0014] d. Pipette the fluorescent probe solution obtained in step c and tetrabutylammonium hydroxide into a small centrifuge tube, add the analyte, and when the excitation light wavelength is 365 nm, the fluorescence changes from blue to yellow-green fluorescence.

[0015] e. Add 100 μL of hydrogen peroxide solutions with concentrations of 0 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, 1 mM, 2 mM, and 3 mM respectively to a cuvette containing 200 μL of tetrabutylammonium hydroxide solution and 0.5 mL of the fluorescent probe solution. After the reaction is completed, use an Edinburgh FLS1000 fluorescence spectrophotometer to measure and record the fluorescence spectrum and image. By comparing the fluorescence intensity at 531 nm in the fluorescence spectrum, it shows a linear relationship with the hydrogen peroxide concentration from 0 μM to 375 μM, and the detection limit is 38.5 nM.

[0016] Introduce the obtained probe into cellulose rich in hydroxyl groups to obtain (E)-(5-(((2-(2-mercaptoethyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)imino)methyl)thiophen-2-yl)boronic acid / cellulose colloidal probe, and use the obtained probe to detect hydrogen peroxide. The specific operation is carried out according to the following steps:

[0017] a. At room temperature, take 10 mL of a 0.1 mM solution of the probe (E)-(5-(((2-(2-mercaptoethyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)imino)methyl)thiophen-2-yl)boronic acid, add 2 mg of cellulose, and stir until completely dissolved to obtain a fluorescent probe / cellulose colloidal solution for detecting hydrogen peroxide;

[0018] b. Pipette the fluorescent probe / cellulose colloidal solution obtained in step a and tetrabutylammonium hydroxide into a small centrifuge tube, add the analyte, and when the excitation light wavelength is 365 nm, the fluorescence changes from blue to yellow-green fluorescence;

[0019] c. Add 100 μL of hydrogen peroxide solutions with concentrations of 0 mM, 0.08 mM, 0.16 mM, 0.32 mM, 0.48 mM, and 0.56 mM respectively to a cuvette containing 200 μL of tetrabutylammonium hydroxide solution and 0.5 mL of (E)-(5-(((2-(2-mercaptoethyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)imino)methyl)thiophen-2-yl)boronic acid / cellulose probe mixed solution. After the reaction is completed, use an Edinburgh FLS1000 fluorescence spectrophotometer to measure and record the fluorescence spectrum and image. By comparing the fluorescence spectra, it can be seen that the fluorescence intensity at 531 nm has a linear relationship with the hydrogen peroxide concentration of 0 μM - 70 μM, and the detection limit is 4.0 nM.

[0020] The preparation method of a solution for detecting hydrogen peroxide based on a naphthalimide-based fluorescent probe according to the present invention is as follows:

[0021] Dilute H2O2 (30%) with deionized water to obtain H2O2 solutions with concentrations of 0 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, 1 mM, 2 mM, 3 mM, 5 mM, 10 mM, 12 mM, 20 mM, and 40 mM respectively;

[0022] Dilute 0.13 mL of 40% aqueous solution of tetrabutylammonium hydroxide with 10 mL of deionized water to obtain a 20 mM tetrabutylammonium hydroxide stock solution

[0023] Dissolve 4.11 mg of the probe (E)-(5-(((2-(2-mercaptoethyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)imino)methyl)thiophen-2-yl)boronic acid in 100 mL of ethanol solution to obtain a 0.1 mM solution of the probe (E)-(5-(((2-(2-mercaptoethyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)imino)methyl)thiophen-2-yl)boronic acid.

[0024] A method for detecting hydrogen peroxide based on a naphthalimide-based fluorescent probe according to the present invention mainly targets explosive hydrogen peroxide and solves the problem of highly sensitive and visual detection of hydrogen peroxide in current on-site detection.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] 1. During the detection of hydrogen peroxide by this probe, the boric acid contained therein will be oxidized, and the carbon-nitrogen double bond in the π-conjugated bridge will break, resulting in the ratio fluorescence changing from light blue to yellow-green.

[0027] 2. The naphthalimide-based probe described in the present invention exhibits excellent sensing performance for hydrogen peroxide, including a detection limit of 38.5 nM, and has quite good selectivity even in the presence of 18 interfering substances. To further improve the sensing performance of this probe, a MOHB-IMTP / cellulose colloid probe was designed to achieve signal enrichment, thereby further reducing the detection limit to 4.0 nM. Description of the Drawings

[0028] Figure 1 For the probe of the present invention, the concentration in ethanol solvent is 0.1 mM, and the concentration of tetrabutylammonium hydroxide is 1 mM. Among them, a is the fluorescence emission spectrum obtained after reacting with 0 μM - 375 μM hydrogen peroxide, and b is the fitting equation with the hydrogen peroxide concentration as the abscissa and the fluorescence intensity value at 531 nm as the ordinate;

[0029] Figure 2 For the cellulose probe of the present invention, the concentration in ethanol solvent is 0.1 mM, and the cellulose content is 0.2 mg / mL. Among them, a is the fluorescence emission spectrum obtained after reacting with 0 μM - 70 μM hydrogen peroxide, and b is the fitting equation with the hydrogen peroxide concentration as the abscissa and the fluorescence intensity at 531 nm as the ordinate;

[0030] Figure 3 For the cellulose probe of the present invention, the concentration in ethanol solvent is 0.1 mM, and the cellulose content is 0.2 mg / mL, Figure 3 is the fluorescence image obtained after reacting with 18 interfering substances. Detailed Embodiments

[0031] The present invention will be further described below through specific examples.

[0032] Example 1

[0033] Prepare a naphthalimide-based fluorescent probe:

[0034] a. In a 100 mL round-bottom flask, 0.5 mmol of 4-amino-1,8-naphthalic anhydride and 4 mmol of 2-aminoethanethiol were dissolved and mixed in 20 mL of ethanol. The mixture was stirred overnight at 80 °C. After that, the solution was cooled to room temperature to form a yellow precipitate. The solid product was filtered, washed three times with pure water, and then dried in a vacuum oven to obtain the desired intermediate 6-amino-2-(2-mercaptoethyl)-1H-benzoisoquinoline-1,3(2H)-dione (MHB-D) with a yield of 85%.

[0035] 1 H NMR (400 MHz, DMSO-d6) δ 8.62 (d, J = 8.6 Hz, 1H), 8.43 (d, J = 7.2 Hz, 1H), 8.20 (d, J = 8.4 Hz, 1H), 7.69–7.62 (m, 1H), 7.47 (s, 2H), 6.84 (d, J = 8.4 Hz, 1H), 4.37–4.26 (m, 2H), 3.00–2.94 (m, 2H), 1.23 (s, 1H). 13 C NMR (101 MHz, DMSO-d6) δ 163.59, 162.62, 152.70, 133.91, 130.96, 129.62, 129.29, 123.84, 121.52, 119.21, 108.05, 107.19, 41.90, 35.21. HRMS: [M] + Calcd. for 272.06; Found 272.06.

[0036] b. In a 100 mL round-bottom flask, 0.1 mmol of the intermediate obtained in step a and 0.10 mmol of 5-formyl-2-thiopheneboronic acid were dissolved and mixed in 20 mL of ethanol. The mixture was stirred overnight at 70 °C. After evaporating the solvent, a crude product was obtained, which was further recrystallized with ethanol, filtered to obtain a solid product. The solid product was washed three times with ethanol and then dried in a vacuum oven to obtain the desired probe (E)-(5-(((2-(2-mercaptoethyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)imino)methyl)thiophen-2-yl)boronic acid (MOHB-IMTP) with a yield of approximately 45%.

[0037] 11H NMR (400 MHz, DMSO-d6) δ 8.61 (d, J = 8.4 Hz, 1H), 8.47 (s, 1H), 8.43 (d, J = 7.4 Hz, 1H), 8.20 (d, J = 8.4 Hz, 1H), 7.64 (dd, J = 14.5, 6.2 Hz, 2H), 7.49–7.45 (m, 1H), 6.84 (d, J = 8.4 Hz, 1H), 4.33 (q, J = 7.2 Hz, 2H), 3.86–3.76 (m, 2H), 1.23 (s, 1H). 13 13C NMR (151 MHz, DMSO-d6) δ 188.00, 187.64, 177.83, 164.00, 163.04, 153.22, 152.92, 134.32, 131.38, 130.05, 129.68, 124.23, 121.95, 119.61, 119.10, 108.40, 107.68, 35.71, 14.08. HRMS: [M] + Calcd. for 410.06; Found 410.98。

[0038] Detection of hydrogen peroxide:

[0039] c. At room temperature, take a solution of the probe (E)-(5-(((2-(2-mercaptoethyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)imino)methyl)thiophen-2-yl)boronic acid with a concentration of 0.1 mM, and the organic solvent is ethanol, thus obtaining a fluorescent probe solution for detecting hydrogen peroxide;

[0040] d. Pipette the fluorescent probe solution obtained in step c and tetrabutylammonium hydroxide into a small centrifuge tube, add the analyte, and when the excitation light wavelength is 365 nm, the fluorescence changes from blue to yellow-green fluorescence;

[0041] e. Add 100 μL of hydrogen peroxide solutions with concentrations of 0 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, 1 mM, 2 mM, and 3 mM respectively to a cuvette containing 200 μL of tetrabutylammonium hydroxide solution and 0.5 mL of the fluorescent probe solution. After the reaction is completed, use an Edinburgh FLS1000 fluorescence spectrophotometer to measure and record the fluorescence spectra and images. By comparing the fluorescence intensity at 531 nm in the fluorescence spectra, it shows a linear relationship with the hydrogen peroxide concentration of 0 μM - 375 μM, and the detection limit is 38.5 nM.

[0042] Example 2

[0043] The probe obtained in Example 1 was introduced into cellulose rich in hydroxyl groups to obtain (E)-(5-(((2-(2-mercaptoethyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)imino)methyl)thiophen-2-yl)boric acid / cellulose colloidal probe, and the obtained probe was used to detect hydrogen peroxide. The specific operation was carried out according to the following steps:

[0044] a. At room temperature, take 10 mL of 0.1 mM probe (E)-(5-(((2-(2-mercaptoethyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)imino)methyl)thiophen-2-yl)boric acid solution, add 2 mg of cellulose and stir until completely dissolved to obtain a fluorescent probe / cellulose colloidal solution for detecting hydrogen peroxide;

[0045] b. Aspirate the fluorescent probe / cellulose colloidal solution obtained in step a and tetrabutylammonium hydroxide into a small centrifuge tube, add the analyte, and when the excitation light wavelength is 365 nm, the fluorescence changes from blue to yellow-green fluorescence;

[0046] c. Add 100 μL of hydrogen peroxide solutions with concentrations of 0 mM, 0.08 mM, 0.16 mM, 0.32 mM, 0.48 mM and 0.56 mM respectively to a cuvette containing 200 μL of tetrabutylammonium hydroxide solution and 0.5 mL of (E)-(5-(((2-(2-mercaptoethyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)imino)methyl)thiophen-2-yl)boric acid / cellulose probe mixed solution. After the reaction, use an Edinburgh FLS1000 fluorescence spectrophotometer to measure and record the fluorescence spectrum and image. It can be seen from the comparison of the fluorescence spectra that the fluorescence intensity at 531 nm has a linear relationship with the hydrogen peroxide concentration of 0 μM - 70 μM, and the detection limit is 4.0 nM.

[0047] Example 3

[0048] Add 200 μL of hydrogen peroxide solution and 18 kinds of interfering substances such as sodium acetate, potassium acetate, zinc acetate, sodium chloride, calcium chloride, magnesium chloride, potassium chloride, ammonium chloride, sodium chlorite, potassium fluoride, sodium bromate, potassium bromate, sodium perchlorate, sodium iodide, urea, boric acid, sodium iodate, potassium chlorate to 0.3 mL of probe (E)-(5-(((2-(2-mercaptoethyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)imino)methyl)thiophen-2-yl)boric acid (MOHB-IMTP) / cellulose colloidal probe solution respectively, and take optical images using a smartphone under 365 nm light excitation.

Claims

1. A method for detecting hydrogen peroxide based on a naphthylimide-based fluorescent probe, characterized in that: The chemical name of the probe is (E)-(5-(((2-(2-mercaptoethyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)imino)methyl)thiophen-2-yl)boronic acid. The specific operation is carried out according to the following steps: Preparation of naphthaleneimide-based fluorescent probes: a. Dissolve the compound 4-amino-1,8-naphthalene dicarboxylic anhydride and 2-aminoethanethiol in 20 mL of ethanol at a molar ratio of 1:8, stir overnight at 80°C, then cool the solution to room temperature to form a yellow precipitate, filter to obtain a solid product, wash it with pure water 3 times, and then dry it in a vacuum oven to obtain an intermediate 6-amino-2-(2-mercaptoethyl)-1H-benzisinoquinoline-1,3(2H)-dione, whose structural formula (I) is: b. The intermediate obtained in step a and 5-formyl-2-thiopheneboronic acid were dissolved in 20 mL of ethanol in a molar ratio of 1:1, and the mixture was stirred overnight at 70° C. The solvent was evaporated to obtain a crude product, which was further recrystallized with ethanol and filtered to obtain a solid product. The solid product was washed with ethanol three times, and the product was dried in a vacuum oven to obtain a probe (E)-(5-(((2-(2-mercaptoethyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)imino)methyl)thiophene-2-yl)boric acid, whose structural formula (II) is: Detection of Hydrogen Peroxide: c. At room temperature, a probe (E)-(5-(((2-(2-mercaptoethyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)imino)methyl)thiophen-2-yl)boric acid solution with a concentration of 0.1 mM is taken, and the organic solvent is ethanol, to obtain a fluorescent probe solution for detecting hydrogen peroxide; d. Pipette the fluorescent probe solution and tetrabutylammonium hydroxide obtained in step c into a small centrifuge tube, add the analyte, and when the excitation light wavelength is 365 nm, the fluorescence changes from blue to yellow-green fluorescence; e. Add 100 μL of hydrogen peroxide solution with concentrations of 0 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, 1 mM, 2 mM and 3 mM respectively into cuvettes containing 200 μL of tetrabutylammonium hydroxide solution and 0.5 mL of fluorescent probe solution. After the reaction, use Edinburgh FLS1000 fluorescence spectrophotometer to measure and record the fluorescence spectrum and image. By comparing the fluorescence intensity at 531 nm of the fluorescence spectrum, it is linearly related to the hydrogen peroxide concentration of 0 μM-375 μM, and the detection limit is 38.5 nM.

2. A method for detecting hydrogen peroxide based on a naphthylimide-based fluorescent probe as claimed in claim 1, characterized in that: The obtained probe is introduced into hydroxyl-rich cellulose to obtain a (E)-(5-(((2-(2-mercaptoethyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)imino)methyl)thiophen-2-yl)boric acid / cellulose colloid probe, and the obtained probe is used to detect hydrogen peroxide. The specific operation is carried out according to the following steps: a. At room temperature, take 10 mL of 0.1 mM probe (E)-(5-(((2-(2-mercaptoethyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)imino)methyl)thiophen-2-yl)boric acid solution, add 2 mg of cellulose and stir until completely dissolved to obtain a fluorescent probe / cellulose colloid solution for detecting hydrogen peroxide; b. Pipette the fluorescent probe / cellulose colloid solution and tetrabutylammonium hydroxide obtained in step a into a small centrifuge tube, add the analyte, and when the excitation light wavelength is 365 nm, the fluorescence changes from blue to yellow-green fluorescence; c. 100 μL of hydrogen peroxide solution with concentrations of 0 mM, 0.08 mM, 0.16 mM, 0.32 mM, 0.48 mM and 0.56 mM were added into a cuvette containing 200 μL of tetrabutylammonium hydroxide solution and 0.5 mL of (E)-(5-(((2-(2-mercaptoethyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)imino)methyl)thiophen-2-yl)boric acid / cellulose probe mixed solution. After the reaction, an Edinburgh FLS1000 fluorescence spectrophotometer was used to measure and record the fluorescence spectrum and image. By comparing the fluorescence spectrum, it can be seen that the fluorescence intensity at 531 nm is linearly related to the hydrogen peroxide concentration of 0 μM-70 μM, and the detection limit is 4.0 nM.

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