A ratio-type hydrogen peroxide fluorescent probe based on aggregation-induced emission and a preparation method and application thereof

Abstract

The application discloses a ratio type hydrogen peroxide fluorescent probe based on an aggregation-induced emission and a preparation method and application thereof, wherein the ratio type hydrogen peroxide fluorescent probe is constructed by taking tetraphenyl ethylene as a fluorescent core, taking a pyridine quaternary ammonium salt as an electron acceptor and taking boric acid as an H2O2 recognition group, and further constructing a D-pi-A type intramolecular charge transfer system. The probe has a fast response to H2O2 and a strong anti-interference capability, can emit 584 nm fluorescence by itself, and after reacting with H2O2, the boric acid is oxidized and cleaved, the fluorescence is blue-shifted to 540 nm, and then based on the ratio value and the concentration, the high-selectivity quantitative detection of H2O2 is realized; in addition, the probe has good tissue penetration and biocompatibility, can reflect the plant oxidative stress level in real time, realizes the visualization of H2O2 in the plant body, and has important significance for the research of plant oxidative stress and new plant stress-resistant varieties.

Description

Technical Field

[0001] This invention relates to a detection technique for reactive oxygen species in plants, and in particular to a ratiometric hydrogen peroxide fluorescent probe based on aggregation-induced emission, its preparation method, and its application. Background Technology

[0002] Hydrogen peroxide (H2O2) is one of the most important reactive oxygen species (ROS) signaling molecules in plants. Under normal physiological conditions, it acts as a second messenger in the regulation of programmed cell death, stomatal movement, and growth and development. However, abiotic stresses such as high temperature, drought, salinity, and heavy metals can disrupt the dynamic balance of ROS in plants, leading to excessive accumulation of H2O2 and subsequent oxidative damage, which severely affects crop yield and quality. Therefore, real-time dynamic monitoring of H2O2 levels in plants is of great significance for the study of plant stress resistance mechanisms and the breeding of stress-resistant varieties.

[0003] Currently, the main methods for detecting H2O2 include histochemical staining (such as DAB staining), spectrophotometry, chromatography, and electrochemical analysis. However, these methods generally suffer from drawbacks such as cumbersome operation, high sample destructiveness, and low spatiotemporal resolution, making it difficult to meet the needs of in-situ monitoring of live organisms.

[0004] Fluorescent probe technology has been widely used for the detection of bioactive molecules in organisms due to its advantages such as ease of operation, high sensitivity, good selectivity, and the ability to achieve visualization imaging. However, traditional fluorescent probes (such as the commercial probe DCFH-DA) face two major bottlenecks in practical applications: First, most traditional fluorophores exhibit aggregation-induced quenching (ACQ) effects, which easily lead to fluorescence self-quenching in plant tissues with high water content or in high-concentration labeled environments, reducing the signal-to-noise ratio; second, single-wavelength fluorescent probes are easily affected by factors such as probe concentration, excitation light intensity, photobleaching, and sample autofluorescence, resulting in poor quantitative accuracy. Ratiometric fluorescent probes, by measuring the ratio of fluorescence intensities at two different wavelengths, can effectively eliminate the above interferences and improve the reliability of quantitative analysis.

[0005] In recent years, the emergence of aggregation-induced emission (AIE) materials has provided a novel approach to solving the ACQ problem. AIE molecules exhibit weak fluorescence in dilute solutions, but emit strong fluorescence in their aggregated state due to restricted intramolecular rotation, making them highly suitable for stable imaging in high-water-content plant environments. However, there are currently no reports of near-infrared fluorescent probes that combine the AIE mechanism with ratiometric detection modes for H2O2 imaging within plants. Summary of the Invention

[0006] In view of this, the present invention proposes a ratiometric hydrogen peroxide fluorescent probe based on aggregation-induced emission, a method for preparing the ratiometric hydrogen peroxide fluorescent probe based on aggregation-induced emission, and an application of the ratiometric hydrogen peroxide fluorescent probe based on aggregation-induced emission.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: The aggregation-induced emission-based ratiometric hydrogen peroxide fluorescent probe of this invention uses tetraphenylethylene as the fluorescent core, pyridine quaternary ammonium salt as the electron acceptor, and boric acid as the H2O2 recognition group, with the following structural formula: .

[0008] This invention also proposes a method for preparing a ratiometric hydrogen peroxide fluorescent probe based on aggregation-induced emission, the method comprising the following steps: In the first step, 4-(1,2,2-triphenylvinyl)benzaldehyde and 4-pyridineacetonitrile were dissolved in anhydrous ethanol, and Knoevenagel condensation reaction was carried out under reflux conditions with piperidine as a catalyst to obtain the intermediate. The structural formula of the intermediate is as follows: ; In the second step, the intermediate obtained in the first step and 4-(bromomethyl)phenylboronic acid are dissolved in acetonitrile and reacted under reflux conditions with stirring. After the reaction is completed, the ratiometric hydrogen peroxide fluorescent probe (TPE-H) is obtained.

[0009] The beneficial effects are as follows: The probe of this invention uses tetraphenylethylene as the fluorescent core, pyridine quaternary ammonium salt as the electron acceptor, and boric acid as the H2O2 recognition group, thereby constructing a D-π-A type intramolecular charge transfer system. It emits 584 nm fluorescence on its own. After reacting with H2O2, the boric acid is oxidized and cleaved, and the fluorescence shifts to 540 nm, realizing ratiometric detection. It can realize the quantitative detection and fluorescence imaging of H2O2, and thus realize the visualization of H2O2 in plants. It is of great significance for the study of plant oxidative stress and the research of new plant stress-resistant varieties.

[0010] Furthermore, the probe of this invention has a simple synthesis route, high yield, and is easy to separate and purify, allowing for scale-up synthesis and facilitating mass production and commercialization. Experiments show that the probe of this invention exhibits significantly enhanced fluorescence in water-soluble systems with increasing water volume fraction, overcoming the fluorescence quenching limitation of traditional probes in the high-water-content microenvironment of plants and ensuring a high signal-to-noise ratio in imaging.

[0011] The probe of this invention exhibits high selective recognition ability for hydrogen peroxide, and common interfering substances such as reactive oxygen species, reactive nitrogen species, amino acids, and anions do not cause significant changes in fluorescence ratio. Anti-interference experiments show that the probe's response to H2O2 is basically unaffected even in the presence of complex interfering substances, indicating that the probe has good anti-interference properties.

[0012] Preferably, in the first step, the molar ratio of 4-(1,2,2-triphenylvinyl)benzaldehyde and 4-pyridineacetonitrile is 1:(1.0-1.5); the heating reflux conditions are: temperature 80-90℃, reaction time 6-12h.

[0013] Preferably, in the first step, the molar amount of piperidine is 5% to 10% of the sum of the molar amounts of 4-(1,2,2-triphenylvinyl)benzaldehyde and 4-pyridineacetonitrile.

[0014] Preferably, in the first step, after the Knoevenagel condensation reaction is completed, column chromatography purification is performed, and the eluent is dichloromethane and methanol, with a volume ratio of dichloromethane to methanol of (20-50):1.

[0015] Preferably, in the second step, the molar ratio of the intermediate to 4-(bromomethyl)phenylboronic acid is 1:(1.0-1.5), and the heating and reflux stirring conditions are as follows: temperature 80-90℃, stirring for 10-15 h.

[0016] Preferably, in the first step, the molar ratio of 4-(1,2,2-triphenylvinyl)benzaldehyde to 4-pyridineacetonitrile is 1:1.2; in the second step, the molar ratio of the intermediate to 4-(bromomethyl)phenylboronic acid is 1:1.2.

[0017] Preferably, in the second step, after the reaction is completed under heating and reflux conditions with stirring, column chromatography purification is performed, with dichloromethane and methanol as the eluents, and the volume ratio of dichloromethane to methanol being (10-30):1.

[0018] The present invention also proposes the application of the ratiometric hydrogen peroxide fluorescent probe of the present invention in the detection of H2O2 fluorescence imaging in plants for non-disease diagnostic purposes, wherein the application quantitatively or semi-quantitatively determines the H2O2 concentration by detecting the ratio of the fluorescence intensity of the ratiometric hydrogen peroxide fluorescent probe at 540 nm and 584 nm.

[0019] In a preferred embodiment of the present invention, the application of a ratiometric hydrogen peroxide fluorescent probe in H2O2 fluorescence imaging within plants is also proposed. This application involves acquiring dual-channel fluorescence images using a laser confocal microscope at an excitation wavelength of 420 nm. In the probe of the present invention, the boric acid group is specifically oxidized and cleaved by H2O2 to generate a phenolic hydroxyl intermediate, which subsequently undergoes a 1,6-elimination reaction to remove the pyridine quaternary ammonium salt moiety. This results in a weakening of the intramolecular charge transfer (ICT) effect, causing a blue shift in fluorescence emission from 584 nm to 540 nm, thereby achieving a ratiometric response. This allows the probe of the present invention to assess the oxidative stress level of plants under abiotic stress conditions using dual-channel fluorescence images.

[0020] Compared with the prior art, the advantages of the present invention are as follows: 1. The probe of this invention uses tetraphenylethylene as the fluorescent core, pyridine quaternary ammonium salt as the electron acceptor, and boric acid as the H2O2 recognition group, thus constructing a D-π-A type intramolecular charge transfer system. It can emit fluorescence at 584 nm. After reacting with H2O2, the boric acid undergoes oxidative cleavage, shifting the fluorescence to 540 nm, enabling ratiometric detection and quantitative detection of H2O2. It also effectively eliminates interference from factors such as probe concentration, excitation light intensity, photobleaching, and sample autofluorescence, significantly improving quantitative accuracy. Furthermore, the probe of this invention has dual channels, enabling visualization of H2O2 within plants, which is of great significance for research on plant oxidative stress.

[0021] 2. The probe of this invention has a simple synthesis route, high yield, and is easy to separate and purify. It can be scaled up for synthesis, which is conducive to quantitative production and commercialization. Experiments show that the fluorescence of the probe of this invention in a water-soluble system increases significantly with the increase of water volume fraction, thus overcoming the shortcomings of traditional probes in fluorescence quenching in the high water content microenvironment of plants and ensuring the imaging signal-to-noise ratio.

[0022] 3. The probe of this invention exhibits high selective recognition ability for hydrogen peroxide, and common interfering substances such as reactive oxygen species, reactive nitrogen species, amino acids, and anions do not cause significant changes in fluorescence ratio. Anti-interference experiments show that the probe's response to H2O2 is basically unaffected under the coexistence of complex interfering substances, indicating that the probe has good anti-interference properties.

[0023] 4. The probe of this invention can achieve a rapid response to hydrogen peroxide (reaching reaction equilibrium within 15 min), exhibits strong fluorescence in the pH range of 6 to 9, and can adapt to a wide pH range; in addition, the probe of this application has a detection limit of 1.30 μM for hydrogen peroxide and exhibits good linearity in the range of 0-50 μM, thus meeting the quantitative detection needs of endogenous H2O2 concentration fluctuations under plant abiotic stress.

[0024] 5. Through practice, this invention has successfully applied the probe to the heavy metal chromium (Cr). 3+ Laser confocal imaging of endogenous H2O2 in the roots of tobacco seedlings under stress indicates that the probe has good tissue penetration and biocompatibility, and can reflect the level of plant oxidative stress in real time, providing a new detection approach for plant stress resistance physiology research. Attached Figure Description

[0025] Figure 1 This is the 1H NMR spectrum of the fluorescent probe TPE-H of this invention.

[0026] Figure 2 This is the mass spectrometry of the fluorescent probe TPE-H of this invention.

[0027] Figure 3 This is a schematic diagram of the response mechanism of the fluorescent probe TPE-H of the present invention.

[0028] Figure 4 The AIE performance test diagram of the fluorescent probe TPE-H of the present invention (fluorescence spectrum and fluorescence intensity change curve corresponding to different water volume fractions).

[0029] Figure 5 This is a graph showing the changes in the UV-Vis absorption spectra of the fluorescent probe TPE-H of this invention for interfering substances and H2O2.

[0030] Figure 6 The fluorescence spectra of the fluorescent probe TPE-H of this invention against interfering substances and H2O2 are shown.

[0031] Figure 7 This is a bar chart showing the fluorescence competition between the fluorescent probe TPE-H of this invention and H2O2 (the black bars represent the I-bars of the mixed system of interferon and probe). 540 / I 584 The ratio, with the red bars representing the I ratio of the mixture of interferon, probe, and H2O2. 540 / I 584 ratio).

[0032] Figure 8 This is a kinetic curve of the fluorescent probe TPE-H of the present invention in response to H2O2.

[0033] Figure 9 This is a pH range diagram for the fluorescent probe TPE-H of the present invention (probe solutions at different pH and probe solutions at different pH + H2O2). 540 / I 584 ratio).

[0034] Figure 10 The fluorescence titration spectrum of the fluorescent probe TPE-H of this invention on H2O2 (0-200 μM H2O2).

[0035] Figure 11 The standard curve of the fluorescent probe TPE-H against H2O2 in this invention (I) 540 / I 584 (Linear fitting of concentration).

[0036] Figure 12 This is a confocal fluorescence imaging image (bright field, 540 nm channel, 584 nm channel and overlay) of the fluorescent probe TPE-H of the present invention for endogenous H2O2 under chromium stress in the roots of tobacco seedlings. Detailed Implementation

[0037] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that the chemical reagents used in this application are all commercially available conventional reagents, and the experimental instruments used in this application are all standard laboratory instruments.

[0038] This invention proposes a ratiometric hydrogen peroxide fluorescent probe based on aggregation-induced emission. This ratiometric hydrogen peroxide fluorescent probe uses tetraphenylethylene as the fluorescent core, pyridine quaternary ammonium salt as the electron acceptor, and boric acid as the H₂O₂ recognition group. The specific synthesis steps include the following: The first step involves dissolving 4-(1,2,2-triphenylvinyl)benzaldehyde and 4-pyridineacetonitrile (molar ratio of 4-(1,2,2-triphenylvinyl)benzaldehyde to 4-pyridineacetonitrile 1:(1.0–1.5)) in anhydrous ethanol. Piperidine (molar amount of which is 5%–10% of the total molar amount of 4-(1,2,2-triphenylvinyl)benzaldehyde and 4-pyridineacetonitrile) is used as a catalyst. The mixture is heated to reflux and stirred at 80–90 °C for 6–12 h. After the reaction, excess ethanol is removed by rotary evaporation. The reaction product is then purified by column chromatography (using a mixture of dichloromethane and methanol with a volume ratio of (20–50):1) as the eluent). The purified product is an orange solid intermediate. The process flow is as follows: ; In the second step, the intermediate obtained in the first step and 4-(bromomethyl)phenylboronic acid were dissolved in acetonitrile at a molar ratio of 1:(1.0–1.5). The mixture was heated under reflux at 80–90 °C with stirring for 10–15 h. After the reaction, the acetonitrile was removed by rotary evaporation and purified by column chromatography. The eluent for column chromatography was dichloromethane and methanol at a volume ratio of (10–30):1. After column chromatography purification, a purple solid target product, a ratiometric hydrogen peroxide fluorescent probe based on aggregation-induced emission, was obtained, denoted as TPE-H. The process flow is as follows: .

[0039] Example 1: A ratiometric hydrogen peroxide fluorescent probe based on aggregation-induced emission The preparation process of the ratiometric hydrogen peroxide fluorescent probe in this embodiment is as follows: In the first step, 4-(1,2,2-triphenylvinyl)benzaldehyde (36.0 mg, 0.1 mmol) and 4-pyridineacetonitrile (14.2 mg, 0.12 mmol) were dissolved in 5.0 mL of anhydrous ethanol, and 0.1 mL of piperidine was added. The mixture was heated under reflux and stirred at 80–90 °C for 8 h. The reaction was monitored by TLC during the process. After the reaction was completed, excess ethanol was removed by rotary evaporation. The crude product was subjected to silica gel column chromatography (dichloromethane:methanol = 30:1, v / v) to obtain an orange solid intermediate (42.5 mg, yield 88.5%). In the second step, the intermediate (46.1 mg, 0.1 mmol) and 4-(bromomethyl)phenylboronic acid (25.8 mg, 0.12 mmol) were dissolved in 5.0 mL of acetonitrile and refluxed at 80 °C for 12 h. The reaction was monitored by TLC during the process. After the reaction was completed, rotary evaporation was performed, and the crude product was purified by silica gel column chromatography (dichloromethane:methanol = 15:1, v / v) to obtain the purple solid probe TPE-H (39.8 mg, yield 58.9%, based on the intermediate).

[0040] Structural validation of TPE-H (in conjunction with) Figure 1 and Figure 2 The specific confirmation is as follows: ¹H NMR (600 MHz, DMSO- d 6) δ 9.22 (d, J = 7.1 Hz, 2H), 8.59(s, 1H), 8.41(d J = 7.1 Hz 2H). 8.15 (s, 2H), 7.86 (m, 4H), 7.47 (d, J = 8.0 Hz 2H), 7.23 -7.14 (m, 11H), 7.05-6.99 (m, 6H), 5.85 (s, 2H). Mass spectrometry (MS) confirmed the molecular ion peak of the target product [M-Br]. + m / z calculated value C 41 H 32 BN2O2 + The measured value was 595.3, while the actual value was 595.2, indicating the successful synthesis of the target product of this invention.

[0041] Example 2: Scale-up Synthesis of TPE-H This embodiment performed a 10-fold scale-up experiment according to the molar ratio of Example 1, yielding 420 mg of the intermediate (yield 87.5%) and 385 mg of TPE-H (yield 58.0%). The target product after scale-up synthesis was confirmed by: ¹H NMR (600MHz, DMSO- d 6) δ 9.22 (d, J = 7.1 Hz, 2H), 8.59(s, 1H), 8.41 (d J = 7.1 Hz 2H). 8.15 (s, 2H), 7.86 (m, 4H), 7.47 (d, J = 8.0 Hz 2H), 7.23 -7.14 (m, 11H), 7.05-6.99 (m, 6H), 5.85 (s, 2H); Mass spectrometry (MS) confirmed the molecular ion peak of the target product [M-Br]. + m / z calculated value C 41 H 32 BN2O2⁺: 595.3, measured value 595.2, consistent with Example 1, the results prove the successful amplification of the probe.

[0042] The probe TPE-H synthesized in this invention uses tetraphenylethylene as the fluorescent core, pyridine quaternary ammonium salt as the electron acceptor, and boric acid as the H2O2 recognition group. In the actual reaction, the boric acid group of probe TPE-H is specifically oxidized and cleaved by H2O2 to generate a phenolic hydroxyl intermediate. Subsequently, a 1,6-elimination reaction occurs to remove the pyridine quaternary ammonium salt moiety, resulting in a weakening of the intramolecular charge transfer (ICT) effect. The fluorescence emission shifts from 584 nm (the fluorescence emitted by the probe itself) to 540 nm, thus achieving a ratiometric response. The specific reaction mechanism is described in [details omitted]. Figure 3 .

[0043] Example 3 Performance analysis of the probe TPE-H of the present invention It should be noted that the probe stock solutions used in this embodiment were all prepared in the following manner: the probe TPE-H synthesized in Example 1 was dissolved in DMSO to prepare a probe stock solution with a concentration of 1 mM.

[0044] 1. AIE performance of probe TPE-H PBS buffer (pH 7.4) and ethanol were mixed in different proportions to dilute the probe stock solution to a final concentration of 10 μM; the water volume fraction (f) of the probe solution was [not specified]. w The concentrations were 0%, 10%, 30%, 50%, 70%, 80%, 90%, 95%, and 100%, respectively. The fluorescence spectrum of each probe solution was measured (emission scan 430–800 nm, excitation wavelength 420 nm). The results are shown in [Figure number missing]. Figure 4 .

[0045] Depend on Figure 4 It can be seen that when the water volume fraction f of the probe solution... w When the water volume fraction is ≤60%, the fluorescence of the probe solution is very weak. When the water volume fraction of the probe solution increases to 70%, a significant fluorescence emission peak appears at 610 nm. Furthermore, the fluorescence intensity continues to increase with the increase of the water volume fraction, indicating that the probe has typical AIE characteristics.

[0046] 2. Selectivity and anti-interference properties of probe TPE-H for hydrogen peroxide Dilute the probe stock solution to 10 μM with PBS buffer (pH 7.4); take multiple equal volumes of probe solution, add 200 μM H2O2 to one of the probe solutions, and add an interfering substance at a concentration of 100 μM to each of the remaining probe solutions. The interfering substances are F. - Cl - ,Br - I - AcO - HCO3 - NO3 - NO2 - SO4 2- ,Cys,Hcy,GSH,GSSG,HS - S 2- HClO - ONOO - O2· - HNO3, NO2; after reacting for 15 min, the mixed solution was analyzed to obtain the UV-Vis absorption spectrum, fluorescence spectrum, and I2O spectrum of the mixed solution. 540 / I 584 The ratio, the results are shown in Figure 5 and Figure 6 .

[0047] Repeat the above steps, using a 10 μM probe solution as a blank, and add 100 μM of interfering substance to each probe solution. Then, add 200 μM H2O2 to the probe solution corresponding to each interfering substance. After reacting for 15 min, perform fluorescence detection and analysis, and calculate Ig. 540 / I 584 The ratio, the results are shown in Figure 7 ( Figure 7 The numbers 1-21 are blank, F respectively. - Cl - ,Br - I - AcO - HCO3 -NO3 - NO2 - SO4 2- ,Cys,Hcy,GSH,GSSG,HS - S 2- HClO, ONOO - O2· - HNO, NO).

[0048] The results showed that, according to the ultraviolet spectrum, TPE-H alone had a characteristic absorption peak at 350 nm, with only H2O2 significantly reducing its absorbance. Other interfering substances remained unchanged. Figure 5 In the fluorescence spectrum, TPE-H alone exhibits a distinct emission peak at 584 nm. This peak remains unchanged after the addition of various interfering substances (100 μM), but the addition of H2O2 (200 μM) quenches the 584 nm peak, while a new peak at 540 nm is enhanced, exhibiting a ratiometric response. This is attributed to the reduction in ICT caused by the specific cleavage of borate esters by H2O2. (See [link to relevant documentation]). Figure 6 In the competitive test, the ratio of probe solutions with added interfering substances was consistent with that without added interfering substances. After adding H₂O₂ to the blank group and each interfering substance, I... 540 / I 584 The ratio increased significantly (see Figure 7 This demonstrates that the probe of the present invention exhibits excellent selectivity and anti-interference ability for H2O2.

[0049] 3. The response capability of probe TPE-H to hydrogen peroxide and its applicable pH range. a. Dilute the 1 mM probe stock solution to 10 μM with PBS buffer (pH 7.4), add 200 μM H2O2 to the probe solution, and then perform fluorescence detection every 1 min, recording I. 540 / I 584 The ratios were as follows: The results showed that the ratio of the probe solution with added hydrogen peroxide increased rapidly from 0 to 5 min and stabilized at 15 min, while the ratio of the blank control (i.e., 10 μM probe solution) remained unchanged within 20 min (see [reference needed]). Figure 8 ).

[0050] b. Dilute the 1 mM probe stock solution to 10 μM using PBS buffers of different pH values, with the corresponding pH values ​​being 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13. Use the probe solutions diluted with different pH buffers as controls. Add 200 μM H2O2 to the probe solution corresponding to each pH value, react for 15 min, and then measure the I-value of the control and the solution after adding H2O2. 540 / I 584The ratio change. The results show that, for the probe solution, the pH of the probe solution has a significant impact on its I... 540 / I 584 The ratio had virtually no effect; however, after the addition of H2O2, the probe solution only showed a significant response in the pH range of 6–9, with the strongest response at pH 7.4 (see...). Figure 9 Therefore, when using the probe pair of the present invention for H2O2 fluorescence detection and analysis, the pH of the probe solution is preferably controlled in the range of 6-9, with pH = 7.4 being optimal.

[0051] 4. Fluorescent titration and detection limit of hydrogen peroxide by probe TPE-H Dilute the probe stock solution to 10 μM with PBS solution (pH 7.4). Take multiple equal volumes of probe solution and add 0 (blank sample), 2.5, 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, 150, and 200 μM H2O2 sequentially. After reacting for 15 min, measure the fluorescence spectrum (see [link to sample]). Figure 10 ), determine I 540 / I 584 The ratios were calculated and a standard curve was plotted. The results showed that the H2O2 concentration exhibited good linearity in the range of 0–50 μM (see...). Figure 11 The linear equation is Y = 0.01334X + 0.3381 (R²). 2 = 0.9982); Based on the standard deviation σ and slope k of multiple measurements of blank samples, the detection limit of the TPE-H probe for H2O2 was calculated to be 1.30 μM according to the formula LOD = 3σ / k.

[0052] Application Example 1: Application of the TPE-H probe of the present invention in H2O2 fluorescence imaging analysis in plants. This invention uses tobacco seedlings as the analytical object. After pre-culturing the tobacco seedlings in Hogrange nutrient solution for 3 days, the seedlings were randomly divided into three groups: a control group, a first treatment group, and a second treatment group. The control group and the experimental group were further cultured for 3 days, with the nutrient solution changed daily. The control group only had its Hogrange nutrient solution changed, while the first treatment group used a solution containing Cr. 3+ Hogland nutrient solution (Cr 3+ The concentration was 50 μM), and the second treatment group used Cr-containing... 3+ Hogland nutrient solution (Cr 3+ (The concentration is 100 μM). After cultivation, roots of tobacco seedlings from each group were collected and incubated with 10 μM probe solution in the dark for 60 min. After incubation, the roots were washed twice with PBS and imaged under a laser confocal microscope (excitation at 420 nm, collection at 540 nm (yellow) and 584 nm (red) channels). The results are shown in the figure. Figure 12 .Depend on Figure 12 It can be seen that the red fluorescence of the control group is strong and the yellow fluorescence is weak; with the increase of Cr 3+ As the concentration increases, the red fluorescence weakens while the yellow fluorescence strengthens, directly reflecting the accumulation of endogenous H2O2 induced by chromium stress. The results indicate that the probe of this invention possesses good tissue penetration and biocompatibility, and can reflect the level of oxidative stress in plants in real time, providing a new detection approach for plant stress resistance physiology research.

[0053] In summary, the probe of this invention uses tetraphenylethylene as the fluorescent core, pyridine quaternary ammonium salt as the electron acceptor, and boric acid as the H2O2 recognition group to construct a D-π-A type intramolecular charge transfer system. It emits fluorescence at 584 nm. After reacting with H2O2, the boric acid undergoes oxidative cleavage, shifting the fluorescence to 540 nm, thus achieving ratiometric detection of H2O2. This also effectively eliminates interference from factors such as probe concentration, excitation light intensity, photobleaching, and sample autofluorescence, significantly improving quantitative accuracy and meeting the quantitative detection needs for fluctuations in endogenous H2O2 concentration under plant stress. Furthermore, the probe of this invention has good tissue penetration and biocompatibility, enabling real-time reflection of plant oxidative stress levels and visualization of H2O2 within plants. This is of great significance for research on plant oxidative stress and the development of new stress-resistant plant varieties.

Claims

1. A ratiometric hydrogen peroxide fluorescent probe based on aggregation-induced emission, characterized in that: The ratiometric hydrogen peroxide fluorescent probe uses tetraphenylethylene as the fluorescent core, pyridine quaternary ammonium salt as the electron acceptor, and boric acid as the H2O2 recognition group. The specific structural formula is as follows: 。 2. A method for preparing a ratiometric hydrogen peroxide fluorescent probe based on aggregation-induced emission as described in claim 1, characterized in that: The preparation method Includes the following steps: In the first step, 4-(1,2,2-triphenylvinyl)benzaldehyde and 4-pyridineacetonitrile were dissolved in anhydrous ethanol, and Knoevenagel condensation reaction was carried out under reflux conditions with piperidine as a catalyst to obtain the intermediate. The structural formula of the intermediate is as follows: ； In the second step, the intermediate obtained in the first step and 4-(bromomethyl)phenylboronic acid are dissolved in acetonitrile and stirred under reflux conditions. After the reaction is completed, the ratiometric hydrogen peroxide fluorescent probe is obtained.

3. The method for preparing a ratiometric hydrogen peroxide fluorescent probe based on aggregation-induced emission according to claim 2, characterized in that: In the first step, the molar ratio of 4-(1,2,2-triphenylvinyl)benzaldehyde and 4-pyridineacetonitrile is 1:(1.0-1.5); the heating and reflux conditions are: temperature 80-90℃, reaction time 6-12h.

4. The method for preparing a ratiometric hydrogen peroxide fluorescent probe based on aggregation-induced emission according to claim 3, characterized in that: In the first step, the molar amount of piperidine is 5% to 10% of the sum of the molar amounts of 4-(1,2,2-triphenylvinyl)benzaldehyde and 4-pyridineacetonitrile.

5. The method for preparing a ratiometric hydrogen peroxide fluorescent probe based on aggregation-induced emission according to claim 2, characterized in that: In the first step, after the Knoevenagel condensation reaction is completed, column chromatography purification is performed, with dichloromethane and methanol as the eluents, and the volume ratio of dichloromethane to methanol is (20-50):

1.

6. The method for preparing a ratiometric hydrogen peroxide fluorescent probe based on aggregation-induced emission according to claim 3, characterized in that: In the second step, the molar ratio of the intermediate to 4-(bromomethyl)phenylboronic acid is 1:(1.0-1.5), and the heating and reflux stirring conditions are as follows: temperature 80-90℃, stirring for 10-15 h.

7. The method for preparing a ratiometric hydrogen peroxide fluorescent probe based on aggregation-induced emission according to claim 6, characterized in that: In the first step, the molar ratio of 4-(1,2,2-triphenylvinyl)benzaldehyde to 4-pyridineacetonitrile is 1:1.2; in the second step, the molar ratio of the intermediate to 4-(bromomethyl)phenylboronic acid is 1:1.

2.

8. The method for preparing a ratiometric hydrogen peroxide fluorescent probe based on aggregation-induced emission according to claim 2, characterized in that: In the second step, after the reaction is completed under heating and reflux conditions with stirring, column chromatography is performed for purification. The eluents are dichloromethane and methanol, with a volume ratio of dichloromethane to methanol of (10-30):

1.

9. The application of the ratiometric hydrogen peroxide fluorescent probe according to claim 1 or the ratiometric hydrogen peroxide fluorescent probe prepared according to any one of claims 2-8 in the detection of H2O2 fluorescence in plants for non-disease diagnostic purposes, wherein, The application quantitatively or semi-quantitatively determines the H2O2 concentration by detecting the ratio of fluorescence intensity at 540 nm and 584 nm of a ratiometric hydrogen peroxide fluorescent probe.

10. The application of the ratiometric hydrogen peroxide fluorescent probe according to claim 1 or the ratiometric hydrogen peroxide fluorescent probe prepared according to any one of claims 2-8 in H2O2 fluorescence imaging in plants, wherein, The application involves acquiring dual-channel fluorescence images using a laser confocal microscope at an excitation wavelength of 420 nm.

Images