Preparation method and application of water-soluble near-infrared two-region fluorescent nano sensor
By preparing water-soluble fluorescent nanosensors assembled by near-infrared second-zone fluorescent nanomicrospheres and polyoxylates, the problem of long and weak signal detection of plant stress signal in the prior art is solved, real-time monitoring of plant stress information is realized, and it is suitable for lossless real-time detection of plant health status.
Patent Information
- Application Number
- CN202510633364.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-16
- Publication Date
- 2025-07-01
AI Technical Summary
The existing plant stress signal detection methods take a long time, have a high background and cannot be monitored in real time. The fluorescent nanosensor signal is weak and easy to photobleach, making it impossible to realize real-time in-situ monitoring of plant stress information.
Water-soluble fluorescent nanosensors were prepared by assembly of near-infrared two-zone fluorescent nanomicrospheres and polymetallic acid salts. The self-assembly of the nanosensors was realized through electrostatic adsorption, and the fluorescent signal was enhanced by combining aggregation-induced luminescence (AIE) molecules to avoid photobleaching, and real-time monitoring of plant stress signals was used to utilize the advantages of near-infrared two-zone.
Real-time in-situ monitoring of plant stress information is realized, and nanosensors with uniform particle size, good light stability and strong fluorescence signal can non-destructively monitor the health of living plants and are suitable for precision agriculture.
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Figure CN120232864A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of fluorescence imaging, and in particular to a preparation method and application of a water-soluble near-infrared second-zone fluorescence nanosensor. Background Art
[0002] Detecting stress-induced signals in living plants is challenging because these signals are present at low concentrations and often coexist with various other active substances. Current methods for sensing stress-induced signals mainly rely on histochemical reagents after separation and purification of plant extracts, which are usually destructive and cannot track endogenous dynamic signals in real time. Genetically encoded stress signal molecular sensors, as a non-destructive method, mainly focus on stress-specific signal transduction pathways triggered in model plants, and phenotypic expression and screening are time-consuming.
[0003] Given that most crops are non-model plants, studying the stress signal patterns of these crops places higher demands on existing detection technologies. Sensors based on nanotechnology are species-independent and can monitor plant health in real time through electronic devices, thereby detecting stress responses of various wild-type plants without the need for genetic engineering. However, existing fluorescent nanosensor technologies for stress-specific signal detection have defects and shortcomings such as weak fluorescence signals, easy photobleaching of probes, and strong plant autofluorescence background. Summary of the invention
[0004] In view of the above-mentioned deficiencies and defects in the prior art, the present invention proposes a preparation method and application of a water-soluble near-infrared second-zone fluorescence nanosensor, which overcomes the shortcomings of existing plant stress perception methods such as long time consumption, high background, and inability to perform real-time monitoring, and realizes real-time in-situ monitoring of plant stress information.
[0005] The present invention provides the following technical solutions:
[0006] The method of the invention uses near-infrared second-zone fluorescent nano-microspheres and polyoxometalates as main components, and realizes self-assembly of near-infrared second-zone fluorescent nano-sensors through electrostatic adsorption. The prepared nano-sensor has the advantages of uniform particle size, good photostability, strong fluorescent signal, etc.
[0007] The present invention aims to prepare a near-infrared second-zone fluorescent nanosensor with uniform particle size, which mainly includes the preparation of nano-microspheres with near-infrared second-zone fluorescent signals, the preparation of polyoxometalates with fluorescence quenching performance, and the assembly of fluorescent nano-microspheres and polyoxometalates.
[0008] The method comprises the following steps:
[0009] (1) Synthesize positively charged near-infrared second-zone fluorescent nanospheres by organic solvent swelling;
[0010] (2) Synthesize polyoxometalates using ammonium molybdate;
[0011] (3) Combine positively charged near-infrared second-region fluorescent nanospheres and polyoxometalates to assemble and prepare a nanosensor.
[0012] The specific steps of step (1) are as follows:
[0013] (11) Dissolve polystyrene microspheres in an aqueous solution containing a surfactant. After ultrasonic dispersion, add AIE dyes dissolved in an organic solvent, and perform swelling by magnetic stirring at room temperature to form a reaction mixture solution. Then, centrifuge and wash the reaction mixture solution with ultrapure water to obtain preliminary near-infrared fluorescent polystyrene nanospheres;
[0014] The molecular structural formula of the AIE dye is as follows:
[0015]
[0016] (12) Disperse the preliminary near-infrared fluorescent polystyrene nanospheres in an aqueous solution of polyethyleneimine and perform ultrasonic dispersion again. Then, wash with ultrapure water to remove the uncoated polyethyleneimine to obtain positively charged near-infrared fluorescent polystyrene nanospheres.
[0017] Preferably, the surfactant is polyoxyethylene octadecanol, polyoxyethylene cetyl alcohol, sodium dodecyl sulfate, sodium dodecyl sulfonate, sodium dodecylbenzenesulfonate, N-hexadecyl-N-ethyl sodium sulfate, etc. The volume ratio between the surfactant and the polystyrene microspheres is (1 - 10):1000;
[0018] Preferably, the organic solvent used to dissolve the AIE dye is chloroform, dichloromethane, acetone, methanol, toluene, n-hexane, 1-methoxy-2-propanol, etc.
[0019] Preferably, in step (11), the volume ratio between the organic solution containing the AIE dye and the aqueous solution containing the polystyrene microspheres and the surfactant is (2 - 10):(1 - 2), the magnetic stirring swelling time is 30 - 120 min, the ultrasonic power is 50 - 200 w, and the ultrasonic time is 3 - 10 min;
[0020] Preferably, in step (12), the molecular weight of polyethyleneimine is 800 - 25000, the concentration of polyethyleneimine in the aqueous solution of polyethyleneimine is 1 - 10 mg / mL, the ultrasonic power is 50 - 200 w, and the ultrasonic time is 30 - 60 min.
[0021] The specific steps of step (2) are as follows:
[0022] (21) Dissolve ammonium molybdate in ultrapure water, add other metal aqueous solutions respectively under continuous stirring, and then add ascorbic acid solution to the above solution, and continue stirring and reacting;
[0023] Here are the synthetic products of ammonium molybdate and other metals respectively. For example, ammonium molybdate and FeCl3 synthesize polyoxometalates, and ammonium molybdate and CuCl2 synthesize polyoxometalates.
[0024] (22) Then add ethanol to precipitate polyoxometalates, then centrifuge to collect the precipitate, wash the precipitate with ultrapure water and ethanol, and dry it in a freeze dryer to obtain polyoxometalates.
[0025] Preferably, the other metal aqueous solution is an aqueous solution such as FeCl3 or CuCl2, the molar ratio of ammonium molybdate to the metal in the other metal aqueous solution is (1 - 3):(1 - 3), and the concentration of ascorbic acid in the ascorbic acid solution is 50 - 200 mg / mL.
[0026] The specific step (3) is: dissolve the prepared near-infrared second-region fluorescent nanospheres in ultrapure water, and simultaneously add the prepared polyoxometalates under magnetic stirring. Through electrostatic adsorption, the polyoxometalates are adsorbed on the surface of the nanospheres to assemble into a nanosensor; preferably, the mass ratio of the near-infrared second-region fluorescent nanospheres to the polyoxometalates is (1 - 2):(10 - 200), and the electrostatic adsorption time is 6 - 24 h.
[0027] The near-infrared second region of the present invention refers to 1000 - 1700 nm.
[0028] The water-soluble near-infrared second-region fluorescent nanosensor is used to detect hydrogen peroxide in in vivo fluorescence imaging of plants.
[0029] The water-soluble near-infrared second-region fluorescent nanosensor prepared by the method of the present invention can monitor the hydrogen peroxide signal generated by stress in real time in in vivo fluorescence imaging of plants, and can provide a new optical tool for precision agriculture.
[0030] The present invention mainly includes polystyrene aggregation-induced fluorescence microspheres and polyoxometalates. The preparation method includes the following steps: encapsulate the aggregation-induced emission dye with near-infrared second-region fluorescence emission inside the polystyrene microspheres by the swelling method to prepare nanospheres with near-infrared second-region fluorescence signals; mix the fluorescent nanospheres with polyoxometalates, fully stir, then centrifuge to take the precipitate, wash and dry to obtain the water-soluble near-infrared second-region fluorescent nanosensor.
[0031] The present invention provides near-infrared II region (1000 - 1700 nm) fluorescence imaging. Since the autofluorescence of pigments in plants in the near-infrared II region is weak, it can effectively avoid signal confusion during monitoring, enabling sensitive sensing of the stress response of plants. Compared with traditional visible light (400 - 700 nm) and near-infrared I region (700 - 900 nm) fluorescence, photons can penetrate plant tissues more deeply, significantly improving the spatial resolution and signal-to-noise ratio of imaging.
[0032] This method utilizes the significant enhancement of fluorescence of aggregation-induced emission (AIE) molecules in the aggregated state, which not only solves the problem of signal attenuation caused by the aggregation quenching effect of fluorescence probes but also exhibits stronger anti-photobleaching and photostability, making it suitable for long-term dynamic monitoring in plants. Thus, co-assembling near-infrared II region AIE nanospheres with a fluorescence quencher responsive to stress signal molecules to develop an activatable fluorescence sensor is of great significance for intuitively displaying the stress information of plants.
[0033] Compared with the prior art, the present invention has the following advantages and beneficial effects:
[0034] (1) The present invention uses polystyrene microspheres as templates and prepares near-infrared II region fluorescent nanospheres by the swelling method. The synthesized microspheres have uniform particle sizes, strong fluorescence signals, good stability, and the potential for large-scale synthesis, meeting the requirements of batch production.
[0035] (2) The present invention uses AIE molecules as the fluorescence signal source, avoiding the problem of fluorescence quenching that occurs when ordinary fluorescent dyes reach a certain concentration, and having stronger fluorescence signals and stronger photostability.
[0036] (3) The nanosensor synthesized by the present invention has near-infrared II region fluorescence activatable by hydrogen peroxide. Compared with sensors in the visible light and near-infrared I regions, it has better tissue penetration effects and higher spatial resolution.
[0037] (4) The present invention assembles near-infrared II region fluorescent nanospheres and polyoxometalates into a nanosensor, which can non-destructively monitor the hydrogen peroxide signal generated by living plants, and can continuously monitor the health status of plants through an electronic device, thereby detecting the stress responses of various plants without the need for complex genetic engineering. BRIEF DESCRIPTION OF THE DRAWINGS
[0038] Figure 1 It is a schematic diagram of the synthesis of the near-infrared II region fluorescent nanosensor according to the present invention;
[0039] Figure 2 It is a transmission electron microscope image of the near-infrared II region fluorescent nanospheres prepared in Example 1;
[0040] Figure 3Absorption spectrum and fluorescence emission spectrum of the near-infrared second-region fluorescent nanospheres prepared in Example 1;
[0041] Figure 4 Photostability diagrams of the near-infrared second-region fluorescent nanospheres and ICG prepared in Example 1 in an aqueous solution environment;
[0042] Figure 5 Transmission electron microscopy image of the polyoxometalate prepared in Example 2;
[0043] Figure 6 Absorption spectrum of the polyoxometalate prepared in Example 2;
[0044] Figure 7 Transmission electron microscopy image of the near-infrared second-region fluorescent nanosensor prepared in Example 3;
[0045] Figure 8 Schematic diagram of the principle for the nanosensor of the present invention to detect hydrogen peroxide;
[0046] Figure 9 Imaging diagram of the near-infrared second-region fluorescent nanosensor prepared in Example 3 for real-time monitoring of hydrogen peroxide generated under stress in lettuce leaves;
[0047] Figure 10 Comparison diagram of the imaging effects of the fluorescent nanospheres in plant leaves. Detailed implementation mode
[0048] The present invention will be further described below with reference to the accompanying drawings. It should be noted that the following detailed description is illustrative and is 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 those of ordinary skill in the technical field to which the present application belongs.
[0049] Embodiments of the present invention are as follows:
[0050] Example 1:
[0051] (1) Preparation of near-infrared second-region fluorescent nanospheres
[0052] The synthesis route of the near-infrared second-region fluorescent nanospheres described in the present invention is as Figure 1 shown. Dissolve 50 mg of 200-nm polystyrene microspheres in 5 mL of ultrapure aqueous solution containing 1 mg / mL of surfactant, and ultrasonically disperse to obtain solution A; dissolve 2 mg of AIE dye in 15 mL of 1-methoxy-2-propanol, and ultrasonically disperse to obtain solution B; rapidly inject solution B into solution A under stirring at room temperature, and continuously stir and react for 30 min to obtain a polystyrene nanosphere solution containing AIE dye.
[0053] The above reaction solution was centrifuged and washed three times with ultrapure water to obtain near-infrared second-region fluorescent nanospheres.
[0054] 50 mg of the fluorescent nanospheres were dispersed in 10 mL of an aqueous solution of polyethyleneimine with a molecular weight of 25,000 at a concentration of 2 mg / mL, and ultrasonicated for 30 min at an ultrasonic power of 120 w. The obtained product was washed three times with ultrapure water to remove the uncoated polyethyleneimine, and positively charged near-infrared fluorescent polystyrene nanospheres were obtained.
[0055] The morphology of the fluorescent nanospheres prepared in Example 1 was observed by transmission electron microscopy, as Figure 2 shown. The fluorescent nanospheres were homogeneous spherical structures with a particle size of 208 ± 2 nm. The prepared fluorescent nanospheres were characterized by absorption spectra and emission spectra using a UV-Vis-NIR spectrophotometer and a fluorescence spectrometer, as Figure 3 shown. The characteristic absorption peak of the fluorescent nanospheres was at 722 nm, and the maximum fluorescence emission wavelength was at 1035 nm. Fluorescent nanospheres and ICG solution with the same concentration were taken, continuously irradiated with an 808 nm laser, and the near-infrared second-region fluorescence signal intensity was continuously measured. As Figure 4 shown, the fluorescence signal of the fluorescent nanospheres remained almost stable after continuous irradiation for 60 min, while the fluorescence signal of ICG decreased significantly. It shows that the near-infrared second-region fluorescent nanospheres of the present invention have better photostability compared with commercial ICG.
[0056] (2) Preparation of polyoxometalates
[0057] 1.2 g of ammonium molybdate tetrahydrate and 0.05 g of copper(II) chloride dihydrate were dissolved in 20 mL of ultrapure water. Under continuous stirring, 2 mL of a 100 mg / mL L-ascorbic acid solution was added, and the mixture was stirred at room temperature for 2 h.
[0058] 20 mL of ethanol was added to precipitate the polyoxometalates, which were collected by centrifugation and washed three times with ultrapure water and ethanol. Finally, the sample was dried in a freeze dryer.
[0059] The polyoxometalates prepared in Example 2 were observed by transmission electron microscopy, as Figure 5 shown. The polyoxometalates were homogeneous spherical structures with a particle size of 5 ± 1 nm. The prepared polyoxometalates were characterized by absorption spectra using a UV-Vis-NIR spectrophotometer, as Figure 6 shown. The characteristic absorption peaks of the polyoxometalates were at 748 nm and 1096 nm.
[0060] (3) Preparation of near-infrared second-region fluorescent nanosensors
[0061] Dissolve 1 mg of the prepared near-infrared second-region fluorescent nanospheres in 10 mL of ultrapure water. Under magnetic stirring, add 100 mg of the prepared polyoxometalate and continuously stir and react for 12 h. Wash the above solution with ultrapure water to obtain a near-infrared second-region fluorescent nanosensor.
[0062] Observe the near-infrared second-region fluorescent nanosensor prepared in Example 3 through a transmission electron microscope, as Figure 7 shown. The nanosensor has fluorescent nanospheres as the center, and polyoxometalates are uniformly distributed on the outside. The particle size is 228 ± 6 nm.
[0063] Application:
[0064] Use the near-infrared second-region fluorescent nanosensor prepared in Example 1 to conduct in vivo optical monitoring of the hydrogen peroxide signal in lettuce leaves.
[0065] Take 1 mg of the prepared nanosensor and dissolve it in 10 mL of MES buffer. Then, use a 1 mL needleless syringe to infiltrate 10 μL of fluorescent microspheres and 10 μL of nanosensor into two regions of the lettuce leaf separated by the leaf vein respectively. Apply slight pressure to the leaf during the infiltration process to prevent mechanical damage. Subsequently, wash away the residual nanoparticles on the leaf surface with water and place the infiltrated leaf for 1 h.
[0066] Subsequently, scratch the leaf at a position 1 cm away from the infiltration point. Immediately use a near-infrared imaging system to observe the generation of endogenous hydrogen peroxide in the leaf. Conduct real-time fluorescence imaging of the lettuce leaf under 808 nm laser excitation. The near-infrared second-region fluorescent signal of the nanosensor in the leaf is detected by an InGaAs imaging system equipped with a 900 nm long-pass optical filter.
[0067] As Figure 8 shown, under stress conditions, hydrogen peroxide oxidizes the polyoxometalate, thereby weakening the quenching effect of the polyoxometalate on the fluorescent microspheres and activating the near-infrared second-region fluorescent signal of the fluorescent microspheres.
[0068] The real-time monitoring results of the fluorescence imaging of lettuce are as Figure 9 shown. Neither the control group nor the touched leaf triggered a signal response. Within a few minutes after injury, the fluorescence intensity of the nanosensor in the region on the right side of the leaf vein increased rapidly, and the fluorescent microspheres in the region on the left side of the leaf vein served as a reference sensor, with the fluorescence intensity remaining unchanged. This proves the feasibility of the near-infrared second-region fluorescent nanosensor in real-time monitoring of plant endogenous stress signal molecules.
[0069] Comparison situation:
[0070] Conduct optical imaging of the fluorescent nanospheres prepared in Example 1 in plant leaves.
[0071] The preparation methods of Example 1 were used to prepare visible light fluorescent nanospheres, near-infrared region I fluorescent nanospheres, and near-infrared region II fluorescent nanospheres respectively. 1 mg of the prepared fluorescent nanospheres were separately dissolved in 10 mL of MES buffer, and then 10 μL of the fluorescent nanospheres were infiltrated into pepper leaves using a 1 mL needleless syringe. Slight pressure was applied to the leaves during the infiltration process to prevent mechanical damage. Subsequently, the residual nanospheres on the leaf surface were rinsed off with water, and the infiltrated leaves were left for 1 h.
[0072] A small animal in vivo imager was used to observe the imaging effect of the visible light fluorescent nanospheres in the leaves. Real-time fluorescence imaging of the leaves was performed under 430 nm laser excitation, and the fluorescence signals of the leaves and the nanospheres were detected by a CCD camera. A small animal in vivo imager was used to observe the imaging effect of the near-infrared region I fluorescent nanospheres in the leaves. Fluorescence imaging of the leaves was performed under 640 nm laser excitation, and the fluorescence signals of the leaves and the nanospheres were detected by a CCD camera. A near-infrared imaging system was used to observe the imaging effect of the near-infrared region II fluorescent nanospheres in the leaves. Fluorescence imaging of lettuce leaves was performed under 808 nm laser excitation, and the fluorescence signals of the leaves and the nanospheres were detected by an InGaAs imaging system equipped with a 900 nm long-pass optical filter.
[0073] As Figure 10 shown, the plant leaves injected with visible light and near-infrared region I fluorescent nanospheres had a relatively high autofluorescence background of the plants and a relatively low fluorescence signal of the nanospheres, indicating that the nanomaterials in these two fluorescence bands were not suitable for optical imaging of plant leaves; while the plant leaves injected with near-infrared region II fluorescent nanospheres had almost no interference from the plant autofluorescence background, and the fluorescence signal of the nanospheres was relatively strong, indicating that the use of fluorescent nanomaterials in the near-infrared region II band was helpful for reducing background interference and improving imaging quality.
[0074] It can be seen from this implementation that the water-soluble near-infrared region II nanosensor prepared by the present invention has the advantages of uniform particle size, good optical stability, strong fluorescence signal, etc. When applied to the real-time monitoring of stress signal molecules in plants, it overcomes the disadvantages of the existing plant stress perception methods, such as long time consumption, high background, and inability to perform real-time monitoring, and realizes the real-time in-situ monitoring of plant stress information.
[0075] The above-described embodiments have detailed the technical solutions and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the present invention. Any modifications, supplements, equivalent replacements, etc. made within the principle scope of the present invention should be included within the protection scope of the present invention.
Claims
1. A method for preparing a water-soluble near-infrared second-zone fluorescent nanosensor, characterized in that: The method is to use near-infrared second-region fluorescent nano-microspheres and polyoxometalates as main components and realize self-assembly of near-infrared second-region fluorescent nano-sensors through electrostatic adsorption.
2. The method for preparing a water-soluble near-infrared second-region fluorescent nanosensor according to claim 1, characterized in that: The method comprises the following steps: (1) Synthesize positively charged near-infrared second-zone fluorescent nanospheres by organic solvent swelling; (2) Synthesis of polyoxometalates using ammonium molybdate; (3) Nanosensors are prepared by combining positively charged near-infrared second-zone fluorescent nanospheres and polyoxometalates.
3. The method for preparing a water-soluble near-infrared second-region fluorescent nanosensor according to claim 2, characterized in that: The step (1) is specifically: (11) dissolving polystyrene microspheres in an aqueous solution containing a surfactant, and then adding an AIE dye dissolved in an organic solvent after ultrasonic dispersion, and swelling the mixture under magnetic stirring at room temperature to form a reaction mixture solution, and then washing the reaction mixture solution with ultrapure water by centrifugation to obtain near-infrared fluorescent polystyrene nano-microspheres; The molecular structure of the AIE dye is as follows: (12) Dispersing the near-infrared fluorescent polystyrene nanoparticles in a polyethyleneimine aqueous solution and ultrasonically dispersing them again, the nanoparticles are then washed with ultrapure water to remove uncoated polyethyleneimine, thereby obtaining positively charged near-infrared fluorescent polystyrene nanoparticles.
4. The method for preparing a water-soluble near-infrared second-region fluorescent nanosensor according to claim 3, characterized in that: The surfactant is polyoxyethylene octadecyl alcohol, polyoxyethylene hexadecyl alcohol, sodium dodecyl sulfate, sodium dodecyl sulfonate, sodium dodecylbenzene sulfonate, N-hexadecyl-N-ethyl sodium sulfate, etc., and the volume ratio between the surfactant and the polystyrene microspheres is (1-10):1000; The organic solvent used to dissolve the AIE dye is chloroform, dichloromethane, acetone, methanol, toluene, n-hexane, 1-methoxy-2-propanol, etc.
5. The method for preparing a water-soluble near-infrared second-region fluorescent nanosensor according to claim 3, characterized in that: In the step (11), the volume ratio of the organic solution containing the AIE dye to the aqueous solution containing polystyrene microspheres and a surfactant is (2-10):(1-2), the magnetic stirring swelling time is 30-120 min, the ultrasonic power is 50-200 w, and the ultrasonic time is 3-10 min; In the step (12), the molecular weight of polyethyleneimine is 800-25000, the concentration of polyethyleneimine in the polyethyleneimine aqueous solution is 1-10 mg / mL, the ultrasonic power is 50-200w, and the ultrasonic time is 30-60min.
6. The method for preparing a water-soluble near-infrared second-region fluorescent nanosensor according to claim 2, characterized in that: The step (2) is specifically: (21) dissolving ammonium molybdate in ultrapure water, adding other metal aqueous solutions respectively under continuous stirring, and then adding ascorbic acid solution, and continuing to stir the reaction; (22) Then, ethanol was added to precipitate the polyoxometalate, and the precipitate was collected by centrifugation. The precipitate was washed with ultrapure water and ethanol, and then dried in a freeze dryer to obtain the polyoxometalate.
7. The method for preparing a water-soluble near-infrared second-region fluorescent nanosensor according to claim 6, characterized in that: The molar ratio of ammonium molybdate to the metal in the other metal aqueous solution is (1-3):(1-3), and the ascorbic acid concentration in the ascorbic acid solution is 50-200 mg / mL.
8. The method for preparing a water-soluble near-infrared second-region fluorescent nanosensor according to claim 1, characterized in that: The step (3) is specifically as follows: dissolving the prepared near-infrared zone II fluorescent nano-microspheres in ultrapure water, adding the prepared polyoxometalate at the same time under magnetic stirring, and adsorbing the polyoxometalate on the surface of the nano-microspheres through electrostatic adsorption to assemble into a nanosensor; the mass ratio of the near-infrared zone II fluorescent nano-microspheres to the polyoxometalate is (1-2):(10-200), and the electrostatic adsorption time is 6-24h.
9. A water-soluble near-infrared second-zone fluorescent nanosensor, characterized in that: The invention is prepared by the method according to any one of claims 1 to 8.
10. An application of the water-soluble near-infrared second-region fluorescence nanosensor according to claim 9, characterized in that: Used for detecting hydrogen peroxide in in vivo fluorescence imaging of plants.