Hydrogen peroxide and salicylic acid double-response fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a hydrogen peroxide and salicylic acid double-response fluorescent probe as well as a preparation method and application thereof, and belongs to the technical field of fluorescent probes. The preparation method comprises the following steps: dissolving (E)-2-amino-6 '-(diethylamino)-4'-[2-((E)-1, 3, 3-trimethylindoline-2-subunit) ethylidene]-1 ', 2', 3 ', 4'-tetrahydrospiro [isoindoline-1, 9 '-xanthene]-3-ketone in anhydrous acetonitrile, adding NaH and DMAP, uniformly stirring, then adding 3-[6-(4, 4, 5, 5-tetramethyl-1, 3, 2-boron dioxolane-2-yl)-1, 2, 4-triazole-2-yl)-1, 3, 4-triazole-2-yl]-1, 3, 4-triazole-2-yl]-1, 3, 4-triazole-2-yl]-1, 3, 4-triazole-2- adding 2, 3-dioxo-1H-benzo [de] isoquinoline-2 (3H)-yl] propionyl chloride, and reacting to obtain the target fluorescent probe. According to the probe, H2O2 and SA recognition sites are orthogonally coupled to the same parent nucleus, so that it is ensured that all recognition units synchronously reach a target microenvironment at a constant stoichiometric ratio, high sensitivity and near-infrared emission characteristics are achieved, autofluorescence interference of plant tissues is effectively avoided, and the detection sensitivity is high. Accurate in-situ imaging and dynamic analysis of the space-time evolution process of H2O2 and SA in plant cells are achieved, and the method has wide application prospects in the fields of plant immunology, analytical chemistry, life science and the like.

Description

A hydrogen peroxide and salicylic acid dual-responsive fluorescent probe, its preparation method and application Technical Field

[0001] This invention relates to the field of fluorescent probe technology, and more specifically, to a hydrogen peroxide and salicylic acid dual-response fluorescent probe, its preparation method, and its application. Background Technology

[0002] In the plant immune system, crosstalk between reactive oxygen species (ROS) and plant hormones constitutes a core signaling axis in plant responses to environmental stress. The transient burst of hydrogen peroxide (H2O2) is one of the earliest universal signaling characteristics of plant responses to environmental stress, while salicylic acid (SA) is crucial for coordinating local defenses and establishing systemically acquired resistance (SAR). Studies have shown that these two substances synergistically regulate the plant's defense network through mutually reinforcing feedback loops. Therefore, real-time, dynamic monitoring of the spatiotemporal distribution of H2O2 and SA in vivo has significant scientific and applied value for in-depth analysis of plant immune signal transduction mechanisms.

[0003] However, existing plant signal sensing technologies still face significant limitations in resolving such complex interactions. First, most traditional fluorescent probes are designed for single analytes, making simultaneous monitoring of multiple signals difficult. Although research on fluorescent probes for H2O2 is relatively mature (covering various types from visible to near-infrared), attempts to achieve multi-target imaging by physically mixing them with SA probes often fail because the inherent differences in the uptake efficiency, diffusion rate, and subcellular localization of different probe molecules within plant tissues prevent ensuring that each probe is simultaneously enriched in the same cellular microenvironment in a constant proportion, leading to mismatches and distortions in spatiotemporal correlation signals. Second, existing SA probes generally suffer from low sensitivity (detection limits typically above 1 μM) and short emission wavelengths (λ). em The problem of <600 nm makes it difficult to accurately capture the dynamic changes of trace level signals in the early immune response in plant tissues with strong autofluorescence background.

[0004] Therefore, developing a multifunctional probe that integrates multiple recognition sites into a single molecular scaffold is a pressing problem that needs to be solved. Summary of the Invention

[0005] The technical problem to be solved by the invention is to address the shortcomings of the existing technology. The invention aims to provide a hydrogen peroxide and salicylic acid dual-response fluorescent probe, its preparation method and application. This probe orthogonally couples H2O2 and SA (salicylic acid) recognition sites to the same core, which not only ensures that each recognition unit arrives at the target microenvironment synchronously with a constant stoichiometric ratio, but also has high sensitivity and near-infrared emission characteristics, effectively avoiding the interference of autofluorescence in plant tissues.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a dual-response fluorescent probe for hydrogen peroxide and salicylic acid, named RhCy-NI, which has the following general structural formula: This probe orthogonally couples H2O2 and SA recognition sites to the same nucleus, ensuring that each recognition unit arrives at the target microenvironment synchronously with a constant stoichiometric ratio. It also features high sensitivity and near-infrared emission characteristics, effectively avoiding autofluorescence interference in plant tissues. This successfully enables precise in-situ imaging and dynamic analysis of the spatiotemporal evolution of H2O2 and SA in plant cells.

[0007] Further fluorescent probes, their reaction formulas are as follows: .

[0008] The above-mentioned method for preparing the fluorescent probe involves dissolving (E)-2-amino-6'-(diethylamino)-4'-[2-((E)-1,3,3-trimethylindoline-2-ylidene)ethylylidene]-1',2',3',4'-tetrahydrospiro[isoindoline-1,9'-xanthones]-3-one in anhydrous acetonitrile, adding NaH and DMAP, stirring until homogeneous, and then adding 3-[6-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl]propionyl chloride. After the reaction, the target fluorescent probe RhCy-NI is obtained.

[0009] A further preparation method is used, wherein the molar ratio of (E)-2-amino-6'-(diethylamino)-4'-[2-((E)-1,3,3-trimethylindoline-2-ylidene)ethylylidene]-1',2',3',4'-tetrahydrospiro[isoindoline-1,9'-xanthon]-3-one: NaH: DMAP: 3-[6-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl]propionyl chloride is 1:5:1:3.

[0010] A further preparation method involves stirring at room temperature for 2 hours.

[0011] Further preparation method: During the reaction, the color of the reaction solution gradually changed from brown to blue; after the reaction was completed, the solvent was removed by vacuum concentration, and the crude product was purified by silica gel column chromatography to obtain the green solid target probe RhCy-NI.

[0012] In a further preparation method, the volume ratio of dichloromethane to methanol used as the eluent for separation and purification is 30:1.

[0013] The aforementioned fluorescent probes are specifically used in in-situ dynamic imaging of hydrogen peroxide and salicylic acid in plant roots under abiotic stress conditions. The detection mechanism is based on a two-site orthogonal reaction: hydrogen peroxide (H₂O₂) specifically oxidizes and breaks the borate ester bonds in the probe, triggering yellow fluorescence emission; while salicylic acid (SA) induces specific ring-opening of the rhodamine-spironolactam structure, generating near-infrared fluorescence, thus enabling independent and simultaneous detection of the two target analytes.

[0014] Further applications of the fluorescent probe revealed that, in the H2O2 concentration range of 0–25 μM, the fluorescence intensity exhibited a good linear relationship with the concentration.

[0015] Further applications show that the optical response performance of salicylic acid exhibits a good linear relationship between fluorescence intensity and concentration within the salicylic acid concentration range of 0–90 μM.

[0016] The beneficial effects of the technical solution provided by this invention are as follows compared with the prior art: (1) The hydrogen peroxide and salicylic acid dual-response fluorescent probe of this invention can achieve the detection limit of H2O2 and SA at the nanomolar level, and has excellent sensitivity; it has dual fluorescence emission channels that do not interfere with each other, and can realize the independent detection of two target analytes; (2) The hydrogen peroxide and salicylic acid dual-response fluorescent probe of this invention can accurately identify H2O2 and SA, and is not affected by other common reactive oxygen species (ROS), reactive nitrogen species (RNS), reactive sulfur species (RSS) and SA structural analogs, and has excellent selectivity; (3) The hydrogen peroxide and salicylic acid dual-response fluorescent probe of this invention can be used in living cells and living organisms. Imaging, thereby achieving simultaneous in-situ detection of H2O2 and SA; (4) The hydrogen peroxide and salicylic acid dual-response fluorescent probe of the present invention can be used to monitor the dynamic changes of H2O2 and SA in the roots of rice and other plants under abiotic stress conditions such as salt stress and cadmium stress in real time; (5) The hydrogen peroxide and salicylic acid dual-response fluorescent probe and its preparation method of the present invention, by orthogonally coupling the H2O2 and SA recognition sites to the same parent nucleus, not only ensures that each recognition unit arrives at the target microenvironment synchronously with a constant stoichiometric ratio, but also has high sensitivity and near-infrared emission characteristics, effectively avoiding the interference of autofluorescence in plant tissues, and successfully realizing accurate in-situ imaging and dynamic analysis of the spatiotemporal evolution of H2O2 and SA in plant cells. In addition, the synthesis route of the present invention is simple, the raw materials are cheap and readily available, and the reaction yield is high. It is particularly suitable for real-time monitoring of the spatiotemporal dynamic evolution of H2O2 and SA in cells and plants (such as rice) under abiotic stress conditions such as salt stress and heavy metal stress, and has broad application prospects in the fields of plant immunology, analytical chemistry and life sciences. Attached Figure Description

[0017] Figure 1 shows the optical response of the fluorescent probe RhCy-NI to H2O2 in a specific embodiment; Figure 2 shows the optical response of the fluorescent probe RhCy-NI to SA in a specific embodiment; Figure 3 shows the intracellular imaging performance analysis of the fluorescent probe RhCy-NI in a specific embodiment; Figure 4 shows the fluorescence imaging of rice roots when different concentrations of exogenous H2O2 and SA are added to the fluorescent probe RhCy-NI in a specific embodiment; Figure 5 shows the fluorescence imaging of H2O2 and SA in rice roots under abiotic stress by the fluorescent probe RhCy-NI in a specific embodiment. Detailed Implementation

[0018] To further understand the content of this invention, a detailed description of the invention is provided in conjunction with the accompanying drawings.

[0019] Example: The synthesis and preparation method of the hydrogen peroxide and salicylic acid dual-response fluorescent probe in this example is as follows: S1, (E)-2-amino-6'-(diethylamino)-4'-[2-((E)-1,3,3-trimethylindoline-2-yl)ethylyl]-1',2',3',4'-tetrahydrospiro[isoindoline-1,9'-xanthones]-3-one (57.2 mg, 0.1 mmol), sodium hydride (NaH, 12.0 mg, 0.5 mmol), and 4-dimethylaminopyridine (DMAP, 12.2 mg, 0.1 mmol) are dissolved in anhydrous acetonitrile (10... S2, then add freshly prepared compound 3-[6-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl]propionyl chloride (123.9 mg, 0.3 mmol), and continue stirring the mixture at room temperature for 2 h.

[0020] S3. During the reaction, the color of the reaction solution gradually changed from brown to blue. The specific reaction formula is as follows: .

[0021] S4. After the reaction, the solvent was removed by concentration under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: dichloromethane: methanol = 30:1, v / v) to obtain the green solid target fluorescent probe RhCy-NI, which was detected to be 24.8 mg, with a yield of 26%; melting point (mp): 166.2–166.7°C; S5. Its nuclear magnetic resonance hydrogen spectrum (… 1 H NMR) and carbon nuclear magnetic resonance (NMR) 13 The specific data for C NMR are as follows: 1H NMR (400 MHz, Methanol-d4) δ 9.08 (dd,J= 8.4, 1.2 Hz, 1H), 8.46(dd,J= 7.3, 1.1 Hz, 2H), 8.42 (d,J= 7.3 Hz, 1H), 8.21 (d,J= 7.3 Hz, 1H),8.10 – 8.05 (m, 1H), 7.96 – 7.82 (m, 1H), 7.76 (dd,J= 8.5, 7.3 Hz, 2H), 7.70– 7.63 (m, 1H), 7.52 (d,J= 7.4 Hz, 1H), 7.46 – 7.36 (m, 1H), 7.28 (dd,J=17.6, 8.2 Hz, 1H), 7.11 (s, 1H), 6.97 (d,J= 7.7 Hz, 2H), 6.84 – 6.64 (m,1H), 6.33 (d,J= 24.0 Hz, 1H), 5.32 (d,J= 4.9 Hz, 1H), 4.38 (t,J= 7.5 Hz,2H), 3.30 (s, 4H), 3.24 (s, 3H), 3.14 (td,J= 6.6, 3.7 Hz, 2H), 2.69 (t,J=8.0 Hz, 2H), 1.84 (t,J= 3.1 Hz, 2H), 1.77 (d,J= 11.3 Hz, 2H), 1.45 (d,J=7.6 Hz, 18H), 1.27 (s, 6H); 13 C NMR (101 MHz, CDCl3) δ 179.6, 171.7, 170.2, 169.8, 166.6, 165.1,164.0, 159.1, 157.3, 153.3, 149.5, 148.8, 145.3, 143.9, 138.5, 135.7, 134.8,132.7, 130.7, 129.2, 128.1, 127.7, 127.0, 124.4, 123.9, 122.2, 121.6, 120.8,119.3, 113.5, 108.2, 105.6, 104.0, 103.2, 97.9, 97.8, 92.4, 84.6, 83.6, 80.7,75.0, 68.3, 45.2, 43.9, 37.0, 33.2, 29.7, 28.3, 25.0, 14.1, 12.6。

[0022] HRMS (High Resolution Mass Spectrometry): m / z calculated for RhCy-NI, molecular formula: C 58 H 60 BN5O7, ionic mode: [M + H] + (Protonated molecular ion); Theoretical value: 950.4659; Measured value: 950.4659; Error: 0.0 ppm; Perfect match, confirming the correct molecular structure.

[0023] The application of the hydrogen peroxide and salicylic acid dual-response fluorescent probe (RhCy-NI) in this embodiment is based on a two-site orthogonal reaction: hydrogen peroxide (H2O2) can specifically oxidize and break the borate ester bond in the probe, triggering yellow fluorescence emission; while salicylic acid (SA) induces a specific ring-opening of the rhodamine-hydrazide spironolactam structure, generating near-infrared fluorescence, thereby achieving independent and simultaneous detection of the two target analytes.

[0024] Application Example 1: Evaluation of the Optical Response Performance of H2O2. The detection performance of the probe RhCy-NI was evaluated in a DMF / H2O (1:1, v / v) system. As shown in Figure 1: (A) Changes in the absorption spectrum of the probe to different concentrations of H2O2; (B) Changes in the fluorescence emission spectrum; (C) Linear relationship between fluorescence intensity and H2O2 concentration; (D) Response time of the probe to H2O2; (E) Stability of the probe's response to H2O2 under different pH conditions; (F) Selectivity of the probe to H2O2. The probe concentration was 20 μM, the H2O2 concentration was 100 μM, the detection system was a mixture of DMF and water (volume ratio 1:1), and the excitation wavelength was 466 nm.

[0025] As shown in Figures 1A and 1B, the background fluorescence signal of the probe itself is weak and negligible. Upon addition of H₂O₂, a new and distinct absorption band appears at 466 nm, and the fluorescence intensity at 562 nm increases significantly by approximately 25-fold. Within the H₂O₂ concentration range of 0–25 μM, the fluorescence intensity exhibits a good linear relationship with the H₂O₂ concentration, and the calculated detection limit is 96 nM (Figure 1C), indicating that the probe RhCy-NI has the ability to monitor the dynamic changes in trace H₂O₂ levels during early immune signaling events. Kinetic analysis results show that the fluorescence signal of the reaction system reaches a plateau within approximately 40 min (Figure 1D). Furthermore, the probe exhibits good stability over a wide pH range (5.0–9.0, Figure 1E) and can specifically recognize H₂O₂, while showing no significant response to other common reactive oxygen species, reactive nitrogen species, biothiols, and metal ions, confirming its excellent selectivity (Figure 1F).

[0026] Application Example 2: Evaluation of the Optical Response Performance of SA. The RhCy-NI probe was used to evaluate the SA detection performance in a DMF / H2O (1:1, v / v) system. As shown in Figure 2: (A) Changes in the absorption spectrum of the probe for different concentrations of SA; (B) Changes in the fluorescence emission spectrum; (C) Linear relationship between fluorescence intensity and SA concentration; (D) Response time of the probe to SA; (E) Stability of the probe response to SA under different pH conditions; (F) Selectivity of the probe to SA. The probe concentration was 20 μM, the SA concentration was 160 μM, the detection system was a mixture of DMF and water (volume ratio 1:1), and the excitation wavelength was 720 nm.

[0027] The probe RhCy-NI achieves SA detection based on a specific spirocyclic ring-opening mechanism of the rhodamine-hydrazide spironolactam structure. As shown in Figures 2A and 2B, SA induces a structural transition in RhCy-NI in the near-infrared channel, resulting in strong near-infrared fluorescence emission at 751 nm. Notably, the induced fluorophore exhibits a high quantum yield, with a significant 250-fold increase in fluorescence intensity. A good linear relationship is observed between fluorescence intensity and SA concentration within the range of 0–90 μM (Figure 2C), with a calculated limit of detection (LOD) of 24 nM and an extremely short response time (<1 min) (Figure 2D), indicating that RhCy-NI possesses the sensitivity to monitor dynamic changes in trace SA levels in vivo. For biological applications, this probe maintains a stable fluorescence signal within the physiological pH range (6.0–8.0, Figure 2E), indicating that it is less affected by pH fluctuations in the physiological environment. Furthermore, selective experiments confirmed that the probe can specifically recognize SA without being affected by other structural analogs and potential interfering substances (Figure 2F), thus ensuring its high reliability in the detection of complex biological samples.

[0028] Application Example 3: Evaluation of Intracellular Imaging Performance of Probe RhCy-NI As shown in Figure 3, this example utilizes confocal laser scanning microscopy and flow cytometry to analyze 293T cells, aiming to verify the imaging capability of the probe RhCy-NI in a cellular environment. Experimental conditions were set as follows: probe concentration was 20 μM, and the concentration gradients of H2O2 and SA were set to 0, 100, and 200 μM respectively. Fluorescence channel settings were as follows: H2O2 response was set to the yellow channel (λ). ex = 488 nm, λ em = 575–590 nm); SA response is in the red channel (λ ex = 638 nm, λ em = 700–725 nm); the nuclear-directed dye DAPI is in the blue channel (λ). ex = 405 nm, λ em= 427–472 nm); The experiment divided 293T cells into six groups for imaging analysis: Blank control group: After adding different concentrations of H2O2 and SA exogenously, no probe was added, and only DAPI was used for nuclear staining to locate the cell nucleus. Experimental group: After adding the same concentration of H2O2 and SA, the cells were co-incubated with 20 μM probe RhCy-NI for 0.5 h before imaging. The results showed (Figures 3A, 3B, 3E, 3F): No obvious probe channel fluorescence signal was observed in the blank control group under any concentration conditions; while the experimental group showed a significant concentration dependence. With the increase of H2O2 and SA concentration, the fluorescence intensity of the corresponding yellow channel (responding to H2O2) and red channel (responding to SA) were significantly enhanced. In addition, flow cytometry analysis further confirmed (Figures 3C, 3D, 3G, 3H) that the average fluorescence intensity of cells and the cell positivity rate increased synchronously with the increase of the analyte concentration, proving that the probe RhCy-NI has excellent sensitivity and detection ability at the cellular level.

[0029] Application Example 4: Detection of Exogenous H2O2 and SA in Rice Roots. Figure 4 shows the fluorescence imaging results of rice roots when different concentrations of exogenous H2O2 and SA were added using the fluorescent probe RhCy-NI. Experimental conditions were set as follows: probe concentration was 100 μM, and the concentration gradients of H2O2 and SA were set to 0, 100, and 200 μM. Fluorescence channel settings were as follows: yellow channel excitation wavelength was 488 nm, and the emission wavelength collection range was 500–580 nm; red channel excitation wavelength was 640 nm, and the emission wavelength collection range was 650–700 nm. Data significance markers: *** indicates P < 0.001, **** indicates P < 0.0001; scale bar: 100 μm.

[0030] A key criterion for multiplex imaging is the ability to maintain spectral orthogonality in heterogeneous plant tissues. To verify this, in situ fluorescence imaging experiments were conducted on live rice roots (Figure 4A). Roots pretreated with different concentrations of H2O2 or SA showed significant dose-dependent fluorescence enhancement in their respective fluorescence channels (yellow channel for H2O2, red channel for SA), with negligible inter-channel interference (Figures 4B–E). More importantly, under mixed treatment conditions with both analytes present, the probe RhCy-NI successfully achieved independent signal separation, confirming the ability to simultaneously detect two analytes while maintaining strict spectral orthogonality (Figures 4F–G). The good linearity observed in the in situ experiments validated the in vitro experimental results, indicating that the probe's response was not significantly affected by the complex intracellular environment. These in situ experimental results confirm the effectiveness of the probe RhCy-NI as a reliable, high-fidelity detection tool, enabling it to resolve the precise spatiotemporal dynamics of exogenous H2O2 and SA.

[0031] Application Example 5: Imaging Analysis of Rice Roots under Abiotic Stress. Figure 5 shows the imaging results of H2O2 and SA in rice roots under abiotic stress using the fluorescent probe RhCy-NI. The probe concentration was 100 μM. Single stress conditions: salt stress concentration of 200 mM and cadmium stress concentration of 200 μM; mixed stress conditions: salt stress concentration of 100 mM + cadmium stress concentration of 100 μM. The fluorescence channels were set as follows: yellow channel excitation wavelength of 488 nm, emission wavelength collection range of 500–580 nm; red channel excitation wavelength of 640 nm, emission wavelength collection range of 650–700 nm. Scale bar: 100 μm.

[0032] The RhCy-NI probe was used to reveal the endogenous dynamic interaction between H2O2 and SA in rice roots under salt stress (NaCl) and heavy metal stress (CdCl2). Under salt stress (200 mM NaCl), the RhCy-NI probe exhibited a significantly asynchronous signal spectrum, characterized by a clear temporal hierarchy (Figs. 5A and 5B). Specifically, the H2O2 signal (yellow channel) showed a rapid "burst," reaching its peak within 1 h. Similarly, a similar signal trend was observed under heavy metal stress (200 μM CdCl2) (Figs. 5C and 5D). Under both conditions, the oxidative burst was consistent with the rapid activation of membrane-bound NADPH oxidase or Fenton-like reactions. In stark contrast, the SA signal (red channel) showed a significant lag phase, only beginning to significantly enhance after 3 h. The probe’s dual-channel output clearly revealed a time difference of about 2 hours, which confirms the existing biological consensus that reactive oxygen species act as an immediate biochemical signal, while the accumulation of SA depends on the de novo synthesis of biosynthetic enzymes, a process involving a time delay caused by transcriptional regulation.

[0033] Application Example 6: Imaging Analysis of Rice Roots under Combined Abiotic Stress In natural and agricultural environments, soil salinization often occurs simultaneously with heavy metal pollution, especially in wastewater-irrigated areas or near industrial mining zones. This combined pollution poses a severe agronomic challenge, typically triggering unique signal responses different from those triggered by single stresses. As shown in Figure 5, under combined stress (100 mM NaCl and 100 μM CdCl2), traditional single-analyte probes only reflect an increase in signal intensity, while the dual-channel readings of the RhCy-NI probe successfully revealed a nonlinear amplification of the stress signal (Figures 5E and 5F). Specifically, the co-treatment group exhibited a unique H2O2 "secondary rebound peak" at 6 h, a feature not observed in single stress treatments. It is speculated that this secondary peak marks a critical "physiological tipping point": a shift from controlled signal transduction to systemic oxidative exhaustion and structural damage, driven by a synergistic effect of ion homeostasis imbalance and antioxidant system collapse. Meanwhile, the SA signal intensity in the combined treatment group significantly exceeded the arithmetic sum of the individual treatments (an increase of >2-fold), indicating that the defense pathway was synergistically activated. This synergistic effect was clearly visible in the integrated time-series analysis (Figure 5G), which compared signal evolution at different time points. The data confirmed that the signal intensity "state transition" induced by combined stress significantly exceeded the simple superposition prediction of single stress effects, demonstrating that the probe RhCy-NI is a sophisticated tool capable of resolving the complex nonlinear dynamics of plant adaptation.

[0034] The hydrogen peroxide and salicylic acid dual-response fluorescent probe provided by this invention can be used for the detection of H2O2 and SA. The probe has independent dual-channel fluorescence emission characteristics, excellent selectivity and good biocompatibility (low cytotoxicity), and can be widely used for the simultaneous detection of H2O2 and SA in cells and plants, and has high commercial application value.

[0035] The foregoing description is merely illustrative of the invention and its embodiments and should be understood as non-limiting. The accompanying drawings show only one embodiment of the invention, and the actual structure and manufacturing steps are not limited thereto. Therefore, for those skilled in the art, any structural methods and embodiments similar to this technical solution that can be derived without departing from the concept of the invention should fall within the protection scope of this invention.

Claims

1. A dual-responsive fluorescent probe for hydrogen peroxide and salicylic acid, characterized in that, The fluorescent probe has the following general structural formula: 。 2. The hydrogen peroxide and salicylic acid dual-response fluorescent probe according to claim 1, characterized in that, Its reaction formula is: 。 3. A method for preparing the hydrogen peroxide and salicylic acid dual-responsive fluorescent probe according to claim 1, characterized in that, (E)-2-amino-6'-(diethylamino)-4'-[2-((E)-1,3,3-trimethylindoline-2-ylidene)ethylidene]-1',2',3',4'-tetrahydrospiro[isoindoline-1,9'-xanthones]-3-one was dissolved in anhydrous acetonitrile. NaH and DMAP were added and stirred until homogeneous. Then, 3-[6-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl]propionyl chloride was added. After the reaction, the target fluorescent probe RhCy-NI was obtained.

4. The preparation method according to claim 3, characterized in that, The molar ratio of (E)-2-amino-6'-(diethylamino)-4'-[2-((E)-1,3,3-trimethylindoline-2-yl)ethyl]-1',2',3',4'-tetrahydrospiro[isoindoline-1,9'-xanthon]-3-one: NaH: DMAP: 3-[6-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl]propionyl chloride is 1:5:1:

3.

5. The preparation method according to claim 4, characterized in that, The reaction was carried out under the condition of stirring at room temperature for 2 hours.

6. The preparation method according to claim 5, characterized in that, During the reaction, the color of the reaction solution gradually changed from brown to blue. After the reaction was completed, the solvent was removed by vacuum concentration. The crude product was purified by silica gel column chromatography to obtain the green solid target probe RhCy-NI.

7. The preparation method according to claim 6, characterized in that, The volume ratio of dichloromethane to methanol used as the eluent for separation and purification is 30:

1.

8. The application of the hydrogen peroxide and salicylic acid dual-response fluorescent probe according to claim 1, characterized in that, The application is in the in-situ dynamic imaging of hydrogen peroxide and salicylic acid in plant roots under abiotic stress conditions.

9. The application of the hydrogen peroxide and salicylic acid dual-response fluorescent probe according to claim 8, characterized in that, The probe has a linear detection range of 0–25 μM for hydrogen peroxide.

10. The application of the hydrogen peroxide and salicylic acid dual-response fluorescent probe according to claim 8, characterized in that, The probe has a linear detection range of 0–90 μM for salicylic acid.

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