Near-infrared two-region fluorescent probe for detecting hydrogen peroxide as well as preparation method and application of near-infrared two-region fluorescent probe

Abstract

The invention discloses a near-infrared two-region fluorescent probe for detecting hydrogen peroxide as well as a preparation method and application of the near-infrared two-region fluorescent probe. The molecular formula of the probe is C71H94BN2O13 < + >. According to the design of the probe, 4-hydroxyphenylboronic acid pinacol ester is adopted as a functional group responding to hydrogen peroxide, and a heptamethine cyanine dye containing two pentaethylene glycol monomethyl ether benzoindole units is combined as a fluorophore. When the probe is subjected to the action of hydrogen peroxide, the probe can emit light in a near-infrared second region of 900-1200 nm, and shows excellent selectivity to hydrogen peroxide. Besides, the two pentaethylene glycol monomethyl ether parts in the probe not only improve the solubility of the probe in water, but also enhance the biocompatibility of the probe, so that the probe is suitable for detecting hydrogen peroxide generated in a plant body in an oxidative stress process induced by metal. The probe provides a powerful tool for researching and monitoring oxidative stress in plants.

Description

Technical Field

[0001] The present invention belongs to the field of analysis and detection of hydrogen peroxide generated in plants due to oxidative stress, and particularly relates to a near-infrared second-region fluorescent probe for detecting hydrogen peroxide, a preparation method thereof, and an application thereof. Background Art

[0002] Research shows that the generation and scavenging of reactive oxygen species (ROS) in plants maintain a dynamic balance. However, when plants are subjected to stress, this balance is broken, leading to a large accumulation of ROS. Hydrogen peroxide (H2O2), as a core ROS molecule mediating rapid systemic signal transduction in plants, is regarded as an acute stress indicator. Environmental stress factors (including high temperature, mechanical damage, salinity, low temperature, or pathogen infection, etc.) can induce plants to produce a large amount of H2O2, thereby activating the regulatory mechanisms of plants to respond to biotic / abiotic stress responses. In summary, H2O2 plays a key regulatory role in the process of plants responding to various stress conditions, and it is closely related to plant health. By monitoring the spatio-temporal distribution levels of H2O2 in plant organs (such as leaves, roots, petioles), the stress mechanism of plants can be deeply analyzed, which has important value for improving agricultural biosafety.

[0003] Currently, the main technical means for detecting the level of H2O2 in plants include fluorescent dyes, histochemical reagents, electrochemical sensing, and nanosensors (such as carbon nanotubes). However, traditional fluorescence detection methods are easily interfered by the autofluorescence of chlorophyll and are difficult to be directly applied to the in-situ detection of plant organs. The histochemical staining method requires complex and time-consuming extraction and staining processes and cannot achieve real-time dynamic monitoring. Therefore, there are still major technical challenges in the non-destructive analysis of the level of H2O2 and its transport process in plants. In recent years, the fluorescent probe technology has attracted much attention in the field of animal and plant disease detection due to its advantages of low cost, simple operation, high sensitivity, non-invasiveness, and real-time imaging. Among them, fluorescent probes with emission wavelengths in the near-infrared second region (900 - 1700 nm) have unique advantages such as significantly reducing the interference of autofluorescence of biological tissues and improving the imaging signal-to-noise ratio, showing stronger application potential and becoming an effective tool in the field of plant disease monitoring.

[0004] Currently, a variety of fluorescent probes for hydrogen peroxide detection have been developed. For example, Patent CN 116143814 A, "A Curcumin-based Fluorescent Probe for Detecting Hydrogen Peroxide, Its Preparation Method and Application", discloses a colorimetric and quenching-type fluorescent probe constructed with DFC formed by complexing natural curcumin with boron trifluoride diethyl etherate as the fluorophore and 4-bromomethylphenylboronic acid pinacol ester as the response group. Experiments show that as the concentration of hydrogen peroxide increases, the fluorescence emission intensity of the probe at 601 nm gradually decreases. However, this probe has significant defects: firstly, the emission wavelength is in the visible light band (around 600 nm), which is easily interfered by the fluorescence of other substances in plant tissues; secondly, the molecular structure contains a large conjugated backbone and lacks hydrophilic groups, resulting in poor water solubility, which significantly limits its practical application in plants. Another patent, CN 114230560 A, "A Water-soluble Fluorescent Probe for Visual Detection of Hydrogen Peroxide", proposes a fluorescent probe for hydrogen peroxide detection with a D-A structure constructed by the condensation reaction of xanthene and indole salt. This probe uses pentafluorobenzenesulfonyl as the hydrogen peroxide recognition unit. The results show that as the concentration of hydrogen peroxide increases, the fluorescence emission intensity of the probe at 715 nm gradually increases. Although this design solves the problem of poor water solubility of traditional probes, its emission wavelength is still within the range of the fluorescence emission wavelength of plant chlorophyll (680 - 730 nm), which is prone to serious background signal interference in in vivo plant imaging. Therefore, developing a fluorescent probe with both a second near-infrared emission wavelength and good water solubility is of great significance for achieving simple, rapid, and accurate detection of hydrogen peroxide in plants. Such probes can not only effectively avoid background signal interference but also provide higher detection sensitivity and specificity, thus meeting the requirements of practical applications. Summary of the Invention

[0005] Aiming at the deficiencies in the existing invention technology, the present invention aims to provide a second near-infrared fluorescent probe for detecting hydrogen peroxide in plants, its preparation method, and application. Specifically, the present invention relates to a second near-infrared fluorescent probe capable of detecting hydrogen peroxide, including the preparation method of the probe and its application in the detection of hydrogen peroxide in plants. The fluorescent probe of the present invention uses a heptamethine cyanine dye containing two pentaethylene glycol monomethyl ether benzindoles as the fluorophore. This structural design with a long conjugation degree enables the probe to emit fluorescence in the second near-infrared region of 900 - 1200 nm after reacting with hydrogen peroxide, thus effectively avoiding the interference problem caused by the autofluorescence of plant pigments by short-wavelength probes. In addition, the pentaethylene glycol monomethyl ether side chains on both sides of the probe significantly improve its water solubility, solving the problem that some other probes are difficult to achieve real-time and in-situ detection of hydrogen peroxide in plants due to poor water solubility.

[0006] The object of the present invention is achieved through the following technical solutions.

[0007] A near-infrared second near-infrared fluorescence probe for detecting hydrogen peroxide, wherein the fluorescence probe is (E)-2-[(E)-2-{(E)-3-[(E)-2-(3-(2,5,8,11,14-pentaoxahexadec-16-yl)-1,1-dimethyl-1H-benzo[e]indol-2-ylidene)ethylidene]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy]cyclopent-1-en-1-yl}vinyl]-3-(2,5,8,11,14-pentaoxahexadec-16-yl)-1,1-dimethyl-1H-benzo[e]indol-3-ium salt, and the molecular formula of the probe is C 71 H 94 BN2O 13 + , and the specific structure is as follows: .

[0008] The present invention provides a preparation method of a near-infrared second near-infrared fluorescence probe for detecting hydrogen peroxide, comprising the following steps: Dissolve the compound Cy-Cl p-toluenesulfonate shown in the following formula in N,N-dimethylformamide, add cesium carbonate, introduce an inert gas to make the reaction system in an inert atmosphere, stir in an ice-water bath, and then add 4-hydroxyphenylboronic acid pinacol ester for reaction; after the reaction is completed, separate and purify to obtain the near-infrared second near-infrared fluorescence probe.

[0009] .

[0010] Preferably, the molar ratio of the compound Cy-Cl to cesium carbonate is 1: (1.5-2).

[0011] Preferably, the molar ratio of the compound Cy-Cl to 4-hydroxyphenylboronic acid pinacol ester is 1: (1-1.5).

[0012] Preferably, the amount of N,N-dimethylformamide added per mmol of the compound Cy-Cl is 15-20 mL.

[0013] Preferably, the inert atmosphere is to evacuate the system and fill it with nitrogen, and repeat at least three times, and the inert gas is nitrogen.

[0014] Preferably, the stirring reaction time in the ice-water bath is 20-30 minutes.

[0015] Preferably, the stirring reaction time at room temperature is 8-12 h.

[0016] Preferably, the purification method is silica gel chromatography.

[0017] Preferably, the eluent used in the silica gel chromatography is dichloromethane / methanol.

[0018] The present invention also provides the application of the near-infrared second near-infrared fluorescence probe for detecting hydrogen peroxide in detecting hydrogen peroxide generated by metal-induced oxidative stress in plants.

[0019] Furthermore, the application of the near-infrared second near-infrared fluorescence probe for detecting hydrogen peroxide in detecting hydrogen peroxide generated by metal-induced oxidative stress in plants.

[0020] Compared with the prior art, the outstanding advantages of the fluorescence probe provided by the present invention include: (1) Optimization of optical properties: The fluorescence probe of the present invention uses 4-hydroxyphenylboronic acid pinacol ester as the response group and can specifically recognize hydrogen peroxide. In the heptamethine cyanine dye skeleton, a five-membered ring replaces the original six-membered ring at the middle position. Due to the smaller angle tension and lower ring tension of the five-membered ring, the molecule is more stable. At the same time, the smaller size of the five-membered ring is conducive to the effective overlap of π orbitals in the conjugated chain, enhancing the conjugation degree of the whole molecule, and further broadening the absorption spectrum or red-shifting the emission wavelength, so that it can emit near-infrared second near-infrared fluorescence of 900-1200 nm after reacting with hydrogen peroxide. Compared with other short-wavelength probes, the probe of the present invention can effectively reduce the interference of the autofluorescence of pigments in plants. In addition, since the fluorescence emission of the probe is located in the near-infrared second near-infrared region, the detection depth is significantly increased, which is beneficial to the detection of hydrogen peroxide in plants.

[0021] (2) Improvement of water solubility: The fluorescence probe of the present invention is modified with pentaethylene glycol monomethyl ether on the indole salt. Compared with the probe without a hydrophilic group or with triethylene glycol monomethyl ether, the water solubility of this probe is greatly improved, and when it is applied to the detection of hydrogen peroxide in plants, it can achieve a single-molecule dispersion state, avoiding the fluorescence quenching phenomenon caused by the aggregation of the probe. Therefore, the fluorescence probe of the present invention is particularly suitable for the detection of hydrogen peroxide in plants.

[0022] (3) High selectivity and anti-interference ability: The fluorescence probe of the present invention does not respond to some ions, amino acids, etc. that may exist in plants and has good selectivity for hydrogen peroxide. Therefore, the probe is simple to operate, fast, accurate and highly reliable in the application of detecting hydrogen peroxide in plants.

[0023] (4) Quantitative detection ability: When the fluorescence probe of the present invention reacts with hydrogen peroxide, there is a good linear relationship between the fluorescence intensity at the peak of 932 nm and the concentration of hydrogen peroxide. The linear regression equation is Y = 14.00441*X + 133.56223, R 2 = 0.995. Therefore, the probe can quantitatively detect hydrogen peroxide in plants. In addition, the synthesis route of the near-infrared second near-infrared fluorescence probe of the present invention is simple, efficient and low-cost, and has broad application prospects. Brief Description of the Drawings

[0024] Figure 1 is the synthetic route diagram of the fluorescent probe of the present invention.

[0025] Figure 2 is the 1H NMR spectrum of the fluorescent probe in Example 1.

[0026] Figure 3 is the high-resolution mass spectrum of the fluorescent probe in Example 1.

[0027] Figure 4 is the fluorescence emission spectrum measured by adding hydrogen peroxide with different concentrations to the fluorescent probe in Application Example 1.

[0028] Figure 5 is the linear relationship between the fluorescence intensity of the fluorescent probe in Application Example 1 at 932 nm and the hydrogen peroxide concentration.

[0029] Figure 6 is the spectrum of the change of fluorescence intensity with time measured by adding hydrogen peroxide to the fluorescent probe in Application Example 2.

[0030] Figure 7 is the relationship diagram between the fluorescence intensity at 932 nm and time measured by adding hydrogen peroxide to the fluorescent probe in Application Example 2.

[0031] Figure 8 is the selectivity test diagram of the fluorescent probe in Application Example 3.

[0032] Figure 9 is the plant imaging diagram of the fluorescent probe in Application Example 4. Detailed Embodiments

[0033] The following further illustrates the specific implementation of the present invention in conjunction with the drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that for the processes not specifically described in detail below, those skilled in the art can implement or understand them with reference to the prior art. The reagents or instruments used without indicating the manufacturer are regarded as conventional products that can be obtained through commercial purchase.

[0034] The synthetic route of the near-infrared second-region fluorescent probe for detecting hydrogen peroxide in the present invention is as Figure 1 shown.

[0035] Example 1 118.06 mg (0.10 mmol) of compound Cy-Cl p-toluenesulfonate was placed in a 25 mL two-necked flask, and 1.5 mL of N,N-dimethylformamide was added to dissolve it. Subsequently, 48.87 mg (0.15 mmol) of cesium carbonate was added, and nitrogen gas was introduced to protect the reaction system under nitrogen. After the reaction system was stirred in an ice-water bath for 20 minutes, 22.01 mg (0.10 mmol) of 4-hydroxyphenylboronic acid pinacol ester was added, and then the reaction was restored to room temperature for 8 h. After the reaction was completed, 5 mL of water was added to the reaction system, and the reaction system was extracted with 5 mL of dichloromethane (DCM). The organic phase was washed with water 2-3 times, the organic phase was collected, dried over anhydrous magnesium sulfate, and DCM was removed by rotary evaporation. The obtained oily liquid was purified by silica gel column chromatography (the eluent used was dichloromethane / methanol, V / V = 20:1), and 65.04 mg of the fluorescent probe was obtained (yield: 47.66%).

[0036] It was characterized by 1H NMR spectroscopy: 1 H NMR (400 MHz, DMSO) δ 8.10 (d, J J = 8.4 Hz, 2H), 8.02 (d, J J = 8.7 Hz, 4H), 7.86 (d, J J = 8.3 Hz, 2H), 7.69 (d, J J = 8.9 Hz,2H), 7.63 – 7.51 (m, 4H), 7.36 (d, J J = 8.4 Hz, 2H), 7.11 (d, J J = 8.1 Hz, 2H),6.09 (d, J J = 13.9 Hz, 2H), 4.44 (t, J J = 5.6 Hz, 4H), 3.81 (t, J J = 5.3 Hz, 4H), 3.53– 3.29 (m, 32H), 3.19 (s, 6H), 2.93 (s, 4H), 1.61 (s, 12H), 1.29 (s, 12H). The 1H NMR spectrum of the probe is as shown in Figure 2 Figure.

[0037] In addition, it was further verified by high-resolution mass spectrometry: HR-MS (ESI, m / z): The theoretically calculated molecular mass-to-charge ratio C 71 H 94 BN2O 13 + [M] +:1193.6844, the actual molecular mass-to-charge ratio is 1193.6802:. The high-resolution mass spectrum of this probe is as Figure 3 shown.

[0038] Example 2 Take 590.27 mg (0.50 mmol) of compound Cy-Cl p-toluenesulfonate and place it in a 50 mL two-necked flask. Add 9 mL of N,N-dimethylformamide to dissolve it. Subsequently, add 276.95 mg (0.85 mmol) of cesium carbonate, and introduce nitrogen to make the reaction system under nitrogen protection. After stirring the reaction system in an ice-water bath for 25 minutes, add 143.08 mg (0.65 mmol) of 4-hydroxyphenylboronic acid pinacol ester, and then restore to room temperature and react for 10 h. After the reaction is completed, add 25 mL of water to the reaction system, extract the reaction system with 25 mL of DCM, wash the organic phase 2-3 times with water, collect the organic phase, dry it with anhydrous magnesium sulfate, rotary evaporate to remove DCM, and purify the obtained oily liquid by silica gel column chromatography (the eluent used is dichloromethane / methanol, V / V = 20:1) to obtain 339.67 mg of the fluorescent probe (yield: 49.78%).

[0039] The fluorescent probe obtained in this example has the same characterization results as those in Example 1.

[0040] Example 3 Take 1180.55 mg (1 mmol) of compound Cy-Cl p-toluenesulfonate and place it in a 100 mL two-necked flask. Add 20 mL of N,N-dimethylformamide to dissolve it. Subsequently, add 651.64 mg (2 mmol) of cesium carbonate, and introduce nitrogen to make the reaction system under nitrogen protection. After stirring the reaction system in an ice-water bath for 30 minutes, add 330.19 mg (1.5 mmol) of 4-hydroxyphenylboronic acid pinacol ester, and then restore to room temperature and react for 12 h. After the reaction is completed, add 50 mL of water to the reaction system, extract the reaction system with 50 mL of DCM, wash the organic phase 2-3 times with water, collect the organic phase, dry it with anhydrous magnesium sulfate, rotary evaporate to remove DCM, and purify the obtained oily liquid by silica gel column chromatography (the eluent used is dichloromethane / methanol, V / V = 20:1) to obtain 688.35 mg of the fluorescent probe (yield: 50.44%).

[0041] The fluorescent probe obtained in this example has the same characterization results as those in Example 1.

[0042] Application Example 1 Add hydrogen peroxide with different concentrations to the fluorescent probe to test the fluorescence emission spectrum: The fluorescent probe prepared in Example 1 was dissolved in ethanol to prepare a test mother liquor with a probe concentration of 1 mM. 30 μL was taken from the probe mother liquor, and then added to PBS buffer (pH = 7.4), and different amounts of hydrogen peroxide solution were added respectively (the final concentrations of hydrogen peroxide were 0 μM, 10 μM, 20 μM, 40 μM, 60 μM, 80 μM, 100 μM), keeping the total test volume at 3.0 mL. The total volume of the test system was 3 mL, the probe concentration was 10 μM, and the volume percentage of ethanol was 1%. Using 808 nm as the excitation wavelength, the test results are as Figure 4 shown. As can be seen from Figure 4 , when no hydrogen peroxide was added, the fluorescence of the probe was relatively weak. As the concentration of hydrogen peroxide increased, the fluorescence intensity of the test system in the range of 900 - 1200 nm (peak at 932 nm) gradually increased. The fluorescence intensity at 932 nm was linearly fitted with the hydrogen peroxide concentration to obtain the fitting curve Y = 14.00441*X + 133.56223, R 2 = 0.995, as shown Figure 5 below. According to the formula for the lowest detection limit LOD = 3σ / K (LOD is the lowest detection limit, σ is the standard deviation of multiple measurements (≥20 times) of the blank sample (i.e., the probe solution with a hydrogen peroxide concentration of 0 μM), and K is the slope of the fitting curve), the lowest detection limit of this probe was calculated to be 0.84 μM. The above test results show that the fluorescent probe of the present invention has a good response effect on hydrogen peroxide, and can realize the characterization of the change in hydrogen peroxide concentration through the change in fluorescence intensity, and is suitable for the detection of hydrogen peroxide in plants.

[0043] Application Example 2 Test on the response time of the fluorescent probe to hydrogen peroxide: 30 μL of the probe mother liquor in Application Example 1 was added to a centrifuge tube, and then PBS buffer (pH = 7.4) was added, and hydrogen peroxide solution (the final concentration of the hydrogen peroxide aqueous solution was 100 μM) was added. The total volume of the test system was 3 mL. After incubation at room temperature for 5 min, 10 min, 15 min, 20 min, 30 min, 40 min, 50 min and 60 min respectively, the test was carried out. The test results are as Figure 6 、 Figure 7 shown. As can be seen from Figure 6 , the fluorescence intensity of the test system in the range of 900 - 1200 nm (peak at 932 nm) gradually increased with the increase of time and tended to be stable after 40 min. As can be seen from Figure 7 , the fluorescence intensity at 932 nm gradually increased with the increase of time and basically no longer changed after 40 min, indicating that the response time of the probe to hydrogen peroxide was 40 min.

[0044] Application Example 3 Selective test of the fluorescent probe for hydrogen peroxide: Add 30 μL of the probe mother liquor in Application Example 1 to a centrifuge tube, then add PBS buffer (pH = 7.4), and add the following analytes respectively: a. blank (i.e., the probe solution without adding any analyte), b. H2O2 (100 μM), c. glutathione (1 mM), d. cysteine (1 mM), e. leucine (1 mM), f. glutamic acid (1 mM), g. tryptophan (1 mM), h. alanine (1 mM), i. Fe 3+ (1 mM), j. Hg 2+ (1 mM), k. K + (1 mM), l. Al 3+ (1 mM), m. Zn 2+ (1 mM), n. NaNO2 (1 mM), o. ONOO - (1 mM). The total volume of each system is 3 mL, and the concentration of the fluorescent probe in each test sample is 10 μM. The final concentration of each analyte in the test system is the concentration marked in parentheses. During the selective test, incubate for 40 min at room temperature and then conduct the test. The test results are as Figure 8 (the fluorescence intensity is the fluorescence intensity at the peak of 932 nm) shown. It can be seen from Figure 8 that when other analytes are added, the fluorescence intensity of the test group does not change significantly; only when hydrogen peroxide is added, the fluorescence intensity of the test group increases significantly. The experimental results show that the probe has good selectivity for hydrogen peroxide, which will be beneficial to its application in the detection of hydrogen peroxide in plants.

[0045] Application Example 4 Application of the fluorescent probe in detecting hydrogen peroxide in plants: Disinfect rice seeds and germinate them in an environment of 25 °C for three days. Then place the seeds on a seedling tray filled with clear water and change the clear water every day. After four days, select seedlings with similar growth conditions for subsequent treatment. To induce plant oxidative stress, incubate rice seedlings with HgCl2 solutions at different concentrations (50 μM, 100 μM, 150 μM) for 12 h respectively. Additionally, set up a control group without HgCl2 solution, and a treatment group containing 150 μM HgCl2 and 150 mg / L of the antioxidant N-acetyl-L-cysteine (NAC) solution. Before imaging, incubate the rice seedlings with the fluorescent probe for 2 h, and then place the rice seedlings into the imaging chamber of the NIR-II imaging system for imaging. In the imaging experiment, the selected laser excitation wavelength is 808 nm, and the power is 60 mW / cm 2, The wavelength range of the emission filter is 900 - 1700 nm. The test results are as Figure 9 shown. It can be seen from Figure 9 that in the fluorescence imaging of the control group, almost no fluorescence can be observed; while in the experimental group, obvious fluorescence can be observed, and the fluorescence intensity increases with the increase of the HgCl2 concentration. After adding NAC for treatment, the fluorescence decreases significantly. The experimental results show that this fluorescence probe can effectively detect hydrogen peroxide in plants and reflect the degree of oxidative stress induced by metal ions in plants through the change of fluorescence intensity. This probe provides an effective solution for imaging and tracking the oxidative stress of plants exposed to metal pollutants, demonstrating its potential in promoting plant health assessment and environmental monitoring.

[0046] The above examples are the preferred embodiments of the present invention, but the embodiments of the invention are not limited by the above examples. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A near-infrared second near-infrared fluorescence probe for detecting hydrogen peroxide, characterized in that, The molecular formula of the probe is C 71 H 94 BN2O 13 + , and it has the following structural formula: 。 2. The preparation method of the near-infrared second-region fluorescent probe for detecting hydrogen peroxide according to claim 1, characterized in that, Comprising the following steps: Dissolve the compound Cy-Cl p-toluenesulfonate shown below in N,N-dimethylformamide, add cesium carbonate, introduce an inert gas to make the reaction system in an inert atmosphere, stir under an ice-water bath, and then add 4-hydroxyphenylboronic acid pinacol ester for reaction; after the reaction is completed, separate and purify to obtain the near-infrared second-region fluorescent probe. 。 3. The preparation method of the near-infrared second-region fluorescence probe for detecting hydrogen peroxide according to claim 2, characterized in that, The molar ratio of the compound Cy-Cl to cesium carbonate is 1:(1.5 - 2).

4. The preparation method of the near-infrared second near-infrared fluorescence probe for detecting hydrogen peroxide according to claim 2, wherein, The molar ratio of the compound Cy-Cl to 4-hydroxyphenylboronic acid pinacol ester is 1:(1 - 1.5).

5. The preparation method of the near-infrared second-region fluorescence probe for detecting hydrogen peroxide according to claim 2, wherein, The amount of N,N-dimethylformamide added per mmol of the compound Cy-Cl is 15 - 20 mL.

6. The preparation method of the near-infrared second-region fluorescence probe for detecting hydrogen peroxide according to claim 2, characterized in that, The inert gas is nitrogen.

7. The preparation method of the near-infrared second-region fluorescent probe for detecting hydrogen peroxide according to claim 2, characterized in that, The stirring reaction time under the ice-water bath is 20 - 30 minutes; the reaction time is 8 - 12 h.

8. The preparation method of the near-infrared second-region fluorescence probe for detecting hydrogen peroxide according to claim 2, wherein, The purification method is silica gel column chromatography.

9. The preparation method of the near-infrared second-region fluorescence probe for detecting hydrogen peroxide according to claim 8, characterized in that, The eluent for the silica gel chromatography is dichloromethane / methanol.

10. Use of the near-infrared second-region fluorescence probe for detecting hydrogen peroxide according to claim 1, characterized in that, Detect hydrogen peroxide generated in plants during the metal-induced oxidative stress process.

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