Near-infrared two-region fluorescent microneedle patch for detecting hydrogen peroxide signal induced by stress of plant in real time, method and application of near-infrared two-region fluorescent microneedle patch

Abstract

The invention discloses a near-infrared two-region fluorescent microneedle patch for detecting hydrogen peroxide signals induced by stress of plants in real time, a method and application. Comprising the following steps: casting a mixed solution of lanthanide nanoparticles doped with ytterbium, erbium, cerium and neodymium and emitting fluorescence in a near-infrared second region, polyoxomolybdate and polycaprolactone on a microneedle mold of polydimethylsiloxane, performing high-speed centrifugation to fill a needle tip with the solution, and casting a pure polyvinyl alcohol solution on the mold to form a stable substrate; detecting the mechanical property of the microneedle patch by using a texture analyzer, and evaluating the feasibility of the microneedle patch for detecting hydrogen peroxide; the microneedle patch is pressed into a petiole of a leaf with a thumb, and after plant stress is given, the intensity change of fluorescence emission signals before and after the microneedle patch is detected. The microneedle patch prepared by the invention has the advantages of uniform morphology, good light stability, strong fluorescence signal, high biocompatibility and the like, and is applied to real-time detection of stress signal molecules in plants.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of fluorescence imaging, and particularly relates to a near-infrared two-region fluorescence microneedle patch for real-time detection of hydrogen peroxide signals induced by plant stress, and a method and application thereof. BACKGROUND

[0002] Hydrogen peroxide in plants is a key signal molecule of plant stress response, and its increase is an important indicator of plant response to environmental stress. Therefore, timely sensing the hydrogen peroxide content in plants is conducive to sensing the stress state of plants and timely adjusting the environmental parameters of plants to promote the normal growth and development of plants. Due to the low content of hydrogen peroxide in plants, its quantitative detection often relies on large instruments. In addition, these techniques all need to destroy plant tissues to extract juice in the pretreatment process, and have the limitations of many steps, long time consumption and low efficiency. The micro-needle in-situ detection technology has broad application prospects in the detection of hydrogen peroxide content in plants due to its advantages of convenience and minimally invasive. Micro-needle refers to a needle with a length of 100-1000 μm, which originated from the medical field, such as blood collection, signal detection, human drug delivery, gene delivery, local anesthesia, etc. Compared with the animal field, the research of micro-needle in the plant field still needs to be developed. SUMMARY

[0003] In view of the above deficiencies of the prior art, the present application proposes a preparation method of a near-infrared two-region fluorescence microneedle patch and applies it to the early sensing of plant stress. The customized size micro-needle is used to minimally invasively pierce the plant petiole for accurate real-time detection, which has good in-vivo biocompatibility and does not affect the normal physiological activities of plants and does not leave fluorescent substances.

[0004] The technical scheme adopted by the present application is One, a near-infrared two-region fluorescence microneedle patch for real-time detection of hydrogen peroxide signals induced by plant stress The near-infrared two-region fluorescence microneedle patch is mainly composed of a needle tip and a microneedle body, the needle tip is arranged at the tip of the microneedle body and is integrally formed with the microneedle body, the needle tip is mainly composed of lanthanide nanoparticles doped with metal atoms, multi-metal molybdate and polycaprolactone material mixed preparation, and the microneedle body is mainly composed of polyvinyl alcohol material; the metal atoms doped in the lanthanide nanoparticles mainly consist of ytterbium, erbium, cerium and neodymium.

[0005] Two, a preparation method of a near-infrared two-region fluorescence microneedle patch for real-time detection of hydrogen peroxide signals induced by plant stress The preparation method comprises preparation of lanthanide doped nanoparticles emitting fluorescence signals in the near-infrared two-region, preparation of multi-metal molybdate with fluorescence quenching performance, and preparation of a microneedle patch.

[0006] The preparation method is as follows: 1) Synthesis of lanthanide nanoparticles emitting fluorescence signals in the near-infrared II region: NaGdF4:Yb,Er,Ce@NaGdF4:Nd Lanthanide nanoparticles were grown by co-precipitation in a binary solvent mixture of oleic acid and 1-octadecene.

[0007] 2) Synthesis of polyoxomolybdates: POM POM is obtained directly from molybdenum carbide ( β -Mo2C) powder is synthesized by a one-pot oxidation reaction.

[0008] 3) Synthetic microneedle patches: Lanthanide nanoparticles, polyoxomolybdate, and polycaprolactone (PCL) were mixed and cast, then centrifuged to form needle tips. The mixture was then cast and centrifuged again to form a substrate, and finally dried and demolded to obtain microneedle patches.

[0009] In step 1), the main process of synthesizing lanthanide nanoparticles is as follows: first, core nanoparticles NaGdF4:Yb,Er,Ce are synthesized; then, the doped NaGdF4:Yb,Er,Ce nanoparticles are used as seed crystals to epitaxially grow shells of Gd and Nd.

[0010] In step 1), preferably, the molar ratio of Gd, Yb, Er, and Ce in the core nanoparticles is 58:20:2:20; the molar ratio of Gd and Nd in the shell nanoparticles is 70:30; and the volume ratio of oleic acid to 1-octadecene in the binary solvent mixture is 2:3.

[0011] In step 2), the main process for synthesizing lanthanide nanoparticles is as follows: molybdenum carbide (… β Molybdenum carbide (Mo2C) was dissolved in ultrapure water, and hydrogen peroxide (H2O2) was added dropwise while the reaction was allowed to proceed overnight. When the reaction was complete, the molybdenum carbide was removed by centrifugation. β The residue (-Mo2C) was then freeze-dried to obtain a dark blue powder, which was named POM.

[0012] In step 2), 2 g of molybdenum carbide ( β -Mo2C) was dissolved in 15 mL of ultrapure water, and then 2 mL of 30% hydrogen peroxide (H2O2) was added dropwise; the volume ratio of the lanthanide nanoparticles, polyoxomolybdate and polycaprolactone (PCL) was 1:3:16; the centrifugation treatment was performed at 2000 rpm for 10 min.

[0013] In step 3), lanthanide nanoparticles, polyoxomolybdate, and polycaprolactone (PCL) are mixed in dichloromethane and cast onto a polydimethylsiloxane (PDMS) mold. After high-speed centrifugation, needle tips are formed. Pure polyvinyl alcohol (PVA) solution is cast onto the PDMS mold and centrifuged to form a stable substrate. After drying, the mold is demolded to obtain a complete microneedle patch. Preferably, the volume ratio of lanthanide nanoparticles to polyoxomolybdate is 1:3.

[0014] III. Application of near-infrared II fluorescent microneedle patches for real-time detection of stress-induced hydrogen peroxide signals in plants The near-infrared II fluorescent microneedle patch prepared by the method of this invention is used for real-time detection of hydrogen peroxide signals in plants during in vivo fluorescence imaging.

[0015] IV. A method for real-time detection of hydrogen peroxide signal in plants using a near-infrared II fluorescent microneedle patch for real-time detection of stress-induced hydrogen peroxide signal, characterized in that: The method is specifically as follows: The mechanical properties of the microneedle patch were tested using a texture analyzer to evaluate its feasibility for detecting hydrogen peroxide. The microneedle patch was immersed in hydrogen peroxide solutions of varying concentrations, ranging from 0 μM to 500 μM. The fluorescence emission intensity of the microneedle patch showed a linear relationship with the hydrogen peroxide concentration; that is, the stronger the fluorescence emission signal, the higher the hydrogen peroxide concentration. The microneedle patch was then pressed into the petiole of a leaf to induce plant stress. Changes in fluorescence emission intensity before and after the application of the microneedle patch were measured. These changes reflected changes in hydrogen peroxide concentration, thus enabling hydrogen peroxide concentration detection. The near-infrared II emission microneedle patch prepared in this invention possesses advantages such as uniform morphology, good photostability, strong fluorescence signal, and high biocompatibility. Its application in the real-time detection of stress signal molecules in plants provides a new optical tool for precision agriculture.

[0016] The beneficial effects of this invention are: (1) The present invention uses lanthanide nanoparticles doped with ytterbium, erbium, cerium and neodymium as a probe fluorescence signal source. Near-infrared II fluorescent lanthanide nanoparticles are prepared by co-precipitation method. The synthesized particles have uniform particle size, strong fluorescence signal and good stability, and have the potential for large-scale synthesis to meet the needs of mass production.

[0017] (2) The present invention uses polyoxomolybdate nanoclusters as sensing units, which have good selectivity and high chemical stability. They can specifically react with the hydrogen peroxide signal produced by living plants and modulate the fluorescence emission signal of lanthanide nanoparticles.

[0018] (3) The present invention combines lanthanide nanoparticles and polyoxomolybdate nanoclusters as microneedle tips, so that the microneedles have the advantages of both materials being able to react with the target molecule hydrogen peroxide, having good photostability and strong fluorescence signal, and being more convenient to act on plants as microneedles.

[0019] (4) The near-infrared II fluorescent microneedle patch prepared by the present invention has near-infrared II fluorescence that can be activated by hydrogen peroxide. Compared with the visible light and near-infrared I sensors, it can overcome the interference of fluorescence signals from the plant's own self-fluorescent substances and obtain a higher signal-to-noise ratio.

[0020] (5) The size design of the near-infrared II fluorescent microneedle patch prepared by the present invention is based on the actual plant tissue structure. The microneedle patch can be accurately positioned in the vascular bundles in the petiole of the plant, and quickly and accurately sense the changes in hydrogen peroxide signal generated by the plant after being stressed.

[0021] (6) The near-infrared II fluorescent microneedle patch prepared by the present invention is inserted into the petiole of the plant for in situ and real-time detection. It has good biocompatibility in vivo, does not affect the normal physiological activities of the plant, and does not leave fluorescent substances. Attached Figure Description

[0022] Figure 1 This is a transmission electron microscope image of the near-infrared II fluorescent lanthanide nanoparticles prepared in Example 1; Figure 2 The absorption and fluorescence emission spectra of the near-infrared II fluorescent lanthanide nanoparticles prepared in Example 1 are shown. Figure 3 This is a transmission electron microscope image of the polyoxomolybdate prepared in Example 1; Figure 4 The absorption spectrum of the polyoxomolybdate prepared in Example 1 is shown below. Figure 5 This is a photograph of the near-infrared II fluorescent microneedle patch prepared in Example 1; Figure 6 Optical microscope images of three different lengths of near-infrared II fluorescent microneedle patches prepared in Example 1 being inserted into plant petioles; Figure 7 This is a scanning electron microscope image of the near-infrared II fluorescent microneedle patch with a length of 950 μm prepared in Example 1. Figure 8 The images show fluorescence images of the near-infrared II fluorescent microneedle patch prepared in Example 1 after reacting with hydrogen peroxide of different concentrations, and a linear relationship between fluorescence intensity and hydrogen peroxide concentration. Figure 9The image shows the real-time fluorescence imaging of hydrogen peroxide produced in tomato leaves after stress using three different lengths of near-infrared II fluorescent microneedle patches prepared in Example 1. Figure 10 Fluorescence imaging of tomato leaves after removing the near-infrared II fluorescent microneedle patch prepared in Example 1; Figure 11 The image shows the detection of stem diameter, leaf nitrogen content, and chlorophyll content within seven days after the near-infrared II fluorescent microneedle patch prepared in Example 1 was inserted into tomato leaves. Figure 12 Fluorescence images of pure polycaprolactone microneedles dipped in plant juice, captured using a small animal in vivo imaging system and a near-infrared fluorescence imaging system. Detailed Implementation

[0023] The invention will be further described below with reference to the accompanying drawings. It should be noted that the following detailed description is illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0024] The embodiments of the present invention are as follows: Example 1: Synthesis of near-infrared II fluorescent microneedle patches 1) Synthesis of lanthanide nanoparticles emitting fluorescence signals in the near-infrared II region 1.1) The synthetic route for the near-infrared II fluorescent lanthanide nanoparticles NaGdF4:Yb,Er,Ce@NaGdF4:Nd of this invention is as follows: 2.32 mL of 0.2 M gadolinium acetate aqueous solution, 0.8 mL of ytterbium acetate aqueous solution, 0.08 mL of erbium acetate aqueous solution, 0.8 mL of cerium acetate aqueous solution, 8 mL of oleic acid, and 12 mL of 1-octadecene are added to a 100 mL flask. A thermocouple temperature sensor is installed on the flask, and the solution is heated to 150°C and maintained at this temperature for 40 minutes while stirring. The heating mantle is removed, and the reaction mixture is slowly cooled to room temperature while stirring.

[0025] 1.2) At room temperature, transfer 4 mL of 0.5 M sodium hydroxide in methanol and 6.6 mL of 0.4 M ammonium fluoride in methanol into a 15 mL centrifuge tube. Vortex for 10 seconds, then quickly transfer the mixture into a reaction flask. Raise the solution temperature to 50 °C and maintain this temperature for 30 minutes. Heat the solution to 100 °C. Next, connect the flask to a Srank pipe with a double vacuum / gas manifold, and maintain the solution under vacuum for 10 minutes.

[0026] 1.3) Adjust the stopcock position to fill the flask with argon gas, then raise the solution temperature to 300°C at a heating rate of 10°C / min and maintain it for 1.5 h. Remove the heating mantle and allow the solution to cool slowly to room temperature while stirring.

[0027] 1.4) Transfer the solution from the flask to a 15 mL centrifuge tube, add ethanol, and rotate the product at 6,000 rpm for 3 minutes at room temperature, then discard the supernatant. Add ethanol again, and rotate the product at 6,000 rpm for 3 minutes at room temperature, then discard the supernatant. Disperse the NaGdF4:Yb,Er,Ce core lanthanide nanoparticles stored in the centrifuge tube in 8 mL of cyclohexane.

[0028] 1.5) Add 2.8 mL of 0.2 M gadolinium acetate aqueous solution, 1.2 mL of neodymium acetate aqueous solution, 8 mL of oleic acid, and 12 mL of 1-octadecene to a 100 mL flask. Install a thermocouple temperature sensor on the flask, heat the solution to 150°C, and maintain this temperature for 40 minutes while stirring. Remove the heating mantle and allow the reaction mixture to cool slowly to room temperature while stirring.

[0029] 1.6) The previously synthesized NaGdF4:Yb,Er,Ce core lanthanide nanocrystals were injected into a flask. At room temperature, 4 mL of 0.5 M sodium hydroxide in methanol and 6.6 mL of 0.4 M ammonium fluoride in methanol were transferred into a 15 mL centrifuge tube. After vortexing for 10 seconds, the mixture was quickly injected into the reaction flask. The solution temperature was raised to 50 °C and maintained at this temperature for 30 minutes. The solution was then heated to 100 °C. Next, the flask was connected to a Srankk line with a double vacuum / gas manifold, and the solution was maintained under vacuum for 10 minutes.

[0030] 1.7) Adjust the stopcock position to fill the flask with argon gas, then raise the solution temperature to 300°C at a heating rate of 10°C / min and maintain it for 1.5 h. Remove the heating mantle and allow the solution to cool slowly to room temperature while stirring.

[0031] 1.8) Transfer the solution from the flask to a 15 mL centrifuge tube, add ethanol, and rotate the product at 6,000 rpm for 3 minutes at room temperature, then discard the supernatant. Add ethanol again, and rotate the product at 6,000 rpm for 3 minutes at room temperature, then discard the supernatant. Disperse the NaGdF4:Yb,Er,Ce@NaGdF4:Nd core-shell lanthanide nanoparticles stored in the centrifuge tube in 8 mL of cyclohexane.

[0032] The morphology of the NaGdF4:Yb,Er,Ce@NaGdF4:Nd core-shell lanthanide nanoparticles prepared in this embodiment was observed by transmission electron microscopy, such as...Figure 1 As shown, the lanthanide nanoparticles have a uniform spherical structure with a particle size of approximately 33.47 nm. The absorption and emission spectra of the prepared lanthanide nanoparticles were characterized using a UV-Vis-NIR spectrophotometer and a fluorescence spectrometer, as shown below. Figure 2 As shown, the lanthanide nanoparticles have a characteristic absorption peak at 808 nm and a fluorescence emission peak at 1550 nm.

[0033] 2) Synthesis of polyoxomolybdate nanoclusters (POM) The synthetic route of the polyoxomolybdate nanoclusters (POM) of this invention is as follows: 2 g of β-Mo2C powder is dispersed in 15 mL of ultrapure water. Under vigorous stirring, 2 mL of a 30% aqueous solution of hydrogen peroxide is added dropwise. The reaction is allowed to proceed overnight, and the product is removed by centrifugation at 3000 rpm. β -Mo2C residue, freeze-dried, yields a dark blue powder, which is POM.

[0034] The polyoxomolybdate prepared in this embodiment was observed using transmission electron microscopy, such as... Figure 3 As shown, the polyoxomolybdate exhibits a uniform spherical structure with a particle size of approximately 3.59 nm. The prepared polyoxomate was characterized by absorption spectroscopy using a UV-Vis-NIR spectrophotometer, as shown below. Figure 4 As shown, this polyoxomolybdate exhibits a strong absorption coefficient at 808 nm.

[0035] 3) Preparation of near-infrared II fluorescent microneedle patches 1 g of polycaprolactone was dissolved in 7 mL of dichloromethane. 1.6 mL of the mother liquor, 100 μL of core-shell lanthanide nanoparticles in the dichloromethane phase, and 300 μL of polyoxomolybdate in the dichloromethane phase were mixed thoroughly. The mixture was then cast into a pre-designed PDMS microneedle mold and centrifuged at 2000 rpm for 10 min. Residual solution above the needle tip was removed. Pure polyvinyl alcohol solution was cast into the mold and stored at 37 °C for 6 h to cure. The mold was then removed to obtain a near-infrared II fluorescent microneedle patch.

[0036] The near-infrared II fluorescent microneedle patch prepared in this embodiment was observed using an optical microscope. The tip lengths of the three microneedles were 950 μm, 550 μm, and 300 μm, respectively. An image of the 950 μm microneedle is shown below. Figure 5 As shown in the image. Three types of microneedle patches are inserted into the petioles of plants. Figure 6 As shown. Microneedle patches with lengths of 950 μm, 550 μm, and 300 μm were prepared and observed using a scanning electron microscope, as shown. Figure 7As shown. Near-infrared II fluorescent microneedle patches were incubated in 0 μM, 10 μM, 50 μM, 100 μM, 200 μM, and 500 μM hydrogen peroxide aqueous solutions, respectively. Figure 8 As shown, the fluorescence intensity of the microneedle patch is linearly related to the hydrogen peroxide concentration, with a detection limit of 2.454 μM, demonstrating its excellent detection capability.

[0037] Three different lengths of near-infrared II fluorescent microneedle patches prepared in this embodiment were used for in vivo optical monitoring of hydrogen peroxide signals in tomato leaves. The prepared microneedle patches were inserted into the petiole of tomato leaves and left to stand for 1.5 h. Subsequently, the leaves were scratched 1 cm from the infiltration point. The fluorescence response of the near-infrared II fluorescent microneedle patches was immediately observed using a near-infrared imaging system. Fluorescence imaging of the near-infrared II fluorescent microneedle patches was performed under 808 nm laser excitation, and the near-infrared II fluorescence signal was detected using an InGaAs imaging system equipped with a 1400 nm long-pass optical filter.

[0038] Real-time fluorescence imaging detection results of tomatoes are as follows Figure 9 As shown, the control group did not trigger a signal response. Within minutes of injury, the fluorescence intensity of the 950 μm long near-infrared II fluorescent microneedle patch increased rapidly, while the 550 μm and 300 μm long microneedle patches barely triggered a signal response. This demonstrates the feasibility of using a 950 μm long near-infrared II fluorescent microneedle patch inserted into the plant petiole for in vivo real-time detection of endogenous stress signaling molecules in plants.

[0039] The near-infrared II fluorescent microneedle patch prepared in this embodiment was inserted into tomato leaves and left for 10 min, 30 min, 1 h, 6 h, 12 h, 24 h, 48 h, and 72 h, respectively. After removal, fluorescence imaging was performed on the punctured areas of the tomato leaves under 808 nm laser excitation. The images were then detected using an InGaAs imaging system equipped with a 1400 nm long-pass optical filter. Figure 10 As shown, no fluorescent residue was observed on the leaves. The near-infrared II fluorescent microneedle patch prepared in Example 1 was inserted into the petiole of a tomato plant, and the stem diameter, nitrogen content, and chlorophyll content of the inserted leaves were continuously measured for seven days. Figure 11 As shown, no drastic fluctuations were observed. This demonstrates that the near-infrared II fluorescent microneedle patch prepared in Example 1 has good biocompatibility in vivo, does not affect the normal physiological activities of plants, and does not leave behind fluorescent substances.

[0040] Comparative Example 1: 1 g of polycaprolactone was dissolved in 7 mL of dichloromethane. 2 mL of the mother liquor was poured into a pre-designed PDMS microneedle mold and centrifuged at 2000 rpm for 10 min. The mother liquor was then poured into the mold and stored at 37 ℃ for 6 h to solidify. The mold was then removed to obtain a microneedle patch synthesized from pure polycaprolactone material. The imaging effect of the pure polycaprolactone microneedle patch after being dipped in plant juice was observed using a small animal in vivo imaging system. Real-time fluorescence imaging of the leaves was performed under 465 nm laser excitation, and the fluorescence signal of the microneedle patch was detected by a CCD camera. The imaging effect of the pure polycaprolactone microneedle patch after being dipped in plant juice was observed using a near-infrared imaging system, and real-time fluorescence imaging was performed under 808 nm laser excitation. Figure 12 As shown, microneedle patches excited by a 465 nm laser exhibit a significant plant autofluorescence background at 630-770 nm, indicating that the visible light band and the near-infrared I band are not suitable for optical imaging of microneedle patches used in plants. However, in the near-infrared II band, there is no plant autofluorescence background interference, indicating that optical imaging in the near-infrared II band helps to reduce background interference and improve imaging resolution.

[0041] As can be seen from the present invention, the near-infrared II fluorescent microneedle patch prepared by the present invention has the advantages of uniform morphology, good photostability, and strong fluorescence signal. When applied to the real-time detection of stress signal molecules in plants, it overcomes the shortcomings of existing plant stress sensing methods, such as long time consumption, large background interference, and inability to perform real-time detection, and realizes the real-time detection of plant stress information.

[0042] The embodiments described above provide a detailed explanation of the technical solutions and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, and equivalent substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A near-infrared II fluorescent microneedle patch for real-time detection of stress-induced hydrogen peroxide signals in plants, characterized in that: The near-infrared II fluorescent microneedle patch mainly consists of a needle tip and a microneedle body. The needle tip is located at the tip of the microneedle body and is integrally formed with the microneedle body. The needle tip is mainly prepared by mixing lanthanide nanoparticles doped with metal atoms, polyoxomolybdate, and polycaprolactone. The microneedle body is mainly composed of polyvinyl alcohol. The metal atoms doped by the lanthanide nanoparticles are mainly composed of ytterbium, erbium, cerium, and neodymium.

2. The method for preparing a near-infrared II fluorescent microneedle patch for real-time detection of hydrogen peroxide signals induced by stress in plants, as described in claim 1, is characterized in that: The preparation method includes the preparation of lanthanide nanoparticles, the preparation of polyoxomolybdates, and the preparation of microneedle patches.

3. The method for preparing a near-infrared II fluorescent microneedle patch for real-time detection of hydrogen peroxide signals induced by stress in plants, as described in claim 2, is characterized in that... The preparation method is as follows: 1) Synthesis of lanthanide nanoparticles: NaGdF4:Yb,Er,Ce@NaGdF4:Nd Lanthanide nanoparticles were grown by co-precipitation in a binary solvent mixture of oleic acid and 1-octadecene. 2) Synthesis of polyoxomolybdates: POM POM is synthesized through the oxidation reaction of molybdenum carbide powder; 3) Synthetic microneedle patches: Lanthanide nanoparticles, polyoxomolybdate, and polycaprolactone were mixed and cast, then centrifuged to form needle tips. The mixture was then cast and centrifuged again to form a substrate, and finally dried and demolded to obtain microneedle patches.

4. The method for preparing a near-infrared II fluorescent microneedle patch for real-time detection of hydrogen peroxide signals induced by stress in plants, as described in claim 3, is characterized in that... In step 1), the main process of synthesizing lanthanide nanoparticles is as follows: first, core nanoparticles NaGdF4:Yb,Er,Ce are synthesized; then, the doped NaGdF4:Yb,Er,Ce nanoparticles are used as seed crystals to epitaxially grow shells of Gd and Nd.

5. A method for preparing a near-infrared II fluorescent microneedle patch for real-time detection of hydrogen peroxide signals induced by stress in plants, as described in claim 4, characterized in that... In step 1), the molar ratio of Gd, Yb, Er, and Ce in the core nanoparticles is 58:20:2:20; the molar ratio of Gd and Nd in the shell nanoparticles is 70:30; and the volume ratio of oleic acid to 1-octadecene in the binary solvent mixture is 2:

3.

6. A method for preparing a near-infrared II fluorescent microneedle patch for real-time detection of hydrogen peroxide signals induced by stress in plants, as described in claim 3, characterized in that... In step 2), the main process for synthesizing lanthanide nanoparticles is as follows: molybdenum carbide is dissolved in ultrapure water, hydrogen peroxide is added dropwise, and the reaction is maintained overnight; when the reaction is complete, the molybdenum carbide residue is removed by centrifugation and then freeze-dried to obtain a dark blue powder, which is named POM.

7. A method for preparing a near-infrared II fluorescent microneedle patch for real-time detection of hydrogen peroxide signals induced by stress in plants, as described in claim 6, characterized in that... In step 2), 2 g of molybdenum carbide is dissolved in 15 mL of ultrapure water, and then 2 mL of hydrogen peroxide with a mass concentration of 30% is added dropwise; the volume ratio of the lanthanide nanoparticles, polyoxomolybdate, and polycaprolactone is 1:3:16; the centrifugation treatment is performed at 2000 rpm for 10 min.

8. A method for preparing a near-infrared II fluorescent microneedle patch for real-time detection of hydrogen peroxide signals induced by stress in plants, as described in claim 3, characterized in that... In step 3), lanthanide nanoparticles, polyoxomolybdate, and polycaprolactone are mixed in dichloromethane and cast onto a polydimethylsiloxane mold. After centrifugation, needle tips are formed. Pure polyvinyl alcohol solution is cast onto the polydimethylsiloxane mold and centrifuged to form a substrate. After drying, the microneedle patch is demolded. The volume ratio of lanthanide nanoparticles to polyoxomolybdate is 1:

3.

9. The application of a near-infrared II fluorescent microneedle patch for real-time detection of hydrogen peroxide signals induced by stress in plants according to claim 1, or a near-infrared II fluorescent microneedle patch for real-time detection of hydrogen peroxide signals induced by stress in plants prepared by any of claims 2-6, characterized in that: Application of real-time detection of hydrogen peroxide signals in plants in live-cell fluorescence imaging.

10. A real-time detection method for hydrogen peroxide signals in plants using a near-infrared II fluorescent microneedle patch for real-time detection of stress-induced hydrogen peroxide signals in plants, as described in claim 1, or a near-infrared II fluorescent microneedle patch prepared by the preparation method described in claims 2-8, characterized in that: The method specifically involves: using a texture analyzer to detect the mechanical properties of the microneedle patch and assessing its feasibility for detecting hydrogen peroxide; immersing the microneedle patch in hydrogen peroxide solutions of different concentrations; pressing the microneedle patch into the petiole of the leaf; applying stress to the plant; detecting the change in fluorescence emission signal intensity before and after the microneedle patch is applied; and using the change in fluorescence emission signal intensity to reflect the change in hydrogen peroxide concentration, thereby achieving hydrogen peroxide concentration detection.

Images