Preparation method and application of hydrogen sulfide-responsive NIRIIb ratiometric fluorescent nanoprobe

Abstract

The invention discloses a preparation method and application of a hydrogen sulfide responsive NIR IIb ratiometric fluorescent nanoprobe. The dye is mainly composed of a specific hydrogen sulfide response dye IR-820 dihexanoic acid and lanthanide doped down-conversion nanoparticles with NIR IIb emission characteristics, and the preparation method comprises the following steps: synthesizing a nanocrystal nucleus by pyrolyzing lanthanide acetate, and preparing core-shell structure nanoparticles with multiband absorption characteristics; and modifying the surfaces of the PEI functionalized lanthanide nanoparticles with IR-820 dihexanoic acid in a covalent coupling manner, and centrifuging and washing to obtain a probe product. The nanoprobe disclosed by the invention combines a ratio-type fluorescence response mechanism and a deep tissue penetration characteristic of an NIR IIb window, effectively overcomes background interference and an autofluorescence effect of complex biomolecules in a plant body, remarkably improves sensitivity and spatial resolution of hydrogen sulfide detection, provides a novel nano optical tool, and has a very good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of near-infrared fluorescence imaging, and in particular to a preparation method and application of a hydrogen sulfide-responsive NIR IIb ratiometric fluorescence nanoprobe. Background Art

[0002] Real-time monitoring of endogenous signaling molecules in living plants faces numerous challenges, primarily due to their low concentrations, dynamic nature, and interference from complex biological environments. Current traditional detection methods typically require tissue destruction for sampling, making in situ monitoring of targets difficult. While conventional fluorescent probe imaging techniques can achieve non-invasive detection of targets, they still face challenges with strong autofluorescence interference from plant tissues and insufficient tissue penetration when imaging in the visible light window, resulting in limited detection sensitivity and accuracy.

[0003] Near-infrared fluorescence imaging technology, with its low tissue scattering and background interference, provides a new solution for the real-time monitoring of endogenous signaling molecules in living plants. Since most current crops are non-model plants, their diverse physiological and metabolic characteristics make traditional detection methods lack sufficient universality in monitoring various crop stress signals. At the same time, the complexity of plant physiological environments and composition increases the difficulty of using fluorescent probes to accurately identify specific stress signaling molecules in complex plant systems. These bottlenecks significantly affect the accuracy of crop stress signal acquisition. 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 of a hydrogen sulfide-responsive near-infrared II b-window (NIR IIb) ratiometric fluorescent nanoprobe and its application in plant stress monitoring.

[0005] Compared with existing plant stress detection technologies that have problems such as low signal-to-noise ratio, poor timeliness, and difficulty in dynamic monitoring, the probe of the present invention uses near-infrared two-zone fluorescence imaging technology to achieve real-time, in situ, and highly specific detection of endogenous hydrogen sulfide signals in plants, providing a new visualization tool for real-time tracking of plant stress response signals and a new nano-optical tool for in situ dynamic monitoring of endogenous hydrogen sulfide signal molecules in plants. It has important application prospects in the field of real-time perception of plant stress information.

[0006] The present invention provides the following technical solutions:

[0007] The present invention mainly comprises two parts: hydrogen sulfide-responsive dye IR-820 dihexanoic acid and down-conversion lanthanide nanoparticles with NIR IIb emission characteristics. The preparation mainly includes the following three steps: synthesizing nanocrystal cores by high-temperature thermal decomposition of rare earth element precursor salts, and preparing core-shell structured lanthanide nanoparticles with multi-band absorption by using a layer-by-layer growth strategy; surface modification of the lanthanide nanoparticles by using polyethyleneimine; using NHS / EDC to activate the carboxyl end of IR-820 dihexanoic acid, and relying on the modified polyethyleneimine to covalently connect to the surface of the lanthanide nanoparticles, and obtaining a hydrogen sulfide-responsive NIR IIb ratio fluorescent nanoprobe after sufficient centrifugation and washing.

[0008] 1. Preparation method of a hydrogen sulfide-responsive NIR IIb ratiometric fluorescent nanoprobe:

[0009] More specifically, the method of the present invention uses NIR IIb-emitting lanthanide nanoparticles and IR-820 dihexanoic acid as the main components, and assembles hydrogen sulfide-responsive NIR IIb ratiometric fluorescent nanoprobes through covalent linkage. The prepared nanofluorescent probe has the advantages of small particle size, strong anti-interference ability, and is not affected by tissue autofluorescence.

[0010] The present invention aims to prepare a hydrogen sulfide-responsive NIR IIb ratiometric fluorescent nanoprobe, which mainly includes the preparation of lanthanide nanoparticles with NIR IIb emission characteristics, polyethyleneimine modification of the surface of the lanthanide nanoparticles, activation of IR-820 dihexanoic acid with hydrogen sulfide responsive performance, and assembly of the polyethyleneimine-modified lanthanide nanoparticles and the activated IR-820 dihexanoic acid.

[0011] 2. A method for preparing a hydrogen sulfide-responsive NIR IIb ratiometric fluorescent nanoprobe, the method comprising the following steps:

[0012] (1) Preparation of core-shell lanthanide nanoparticles by thermal decomposition of rare earth element precursor salts;

[0013] (2) using polyethyleneimine (PEI) to modify the surface of lanthanide nanoparticles to obtain PEI-modified lanthanide nanoparticles;

[0014] (3) IR-820 dihexanoic acid was activated by EDC / NHS and assembled with PEI-modified lanthanide nanoparticles to prepare a nanofluorescent probe.

[0015] NHS is N-hydroxysuccinimide, and EDC is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

[0016] The step (1) is specifically as follows:

[0017] (11) mixing the first rare earth metal precursor salt solution with octadecene and oleic acid, stirring and fully mixing at room temperature, then heating the system to above 130° C. for a period of time to fully remove water, and cooling to room temperature after the heating is completed;

[0018] Then, methanol solution of ammonium fluoride and sodium hydroxide was added to the mixed solution and stirred continuously at 45°C to form nuclei;

[0019] The nucleated solution was then heated to boiling to remove methanol, and nitrogen was introduced into the solution system under vacuum to ensure that no alcohol remained.

[0020] Finally, the solution system was heated from room temperature to 300°C for continuous reaction. After the reaction, it was cooled to room temperature, the precipitate was collected by centrifugation and washed with ethanol several times to obtain the preliminary NaGdF4:Yb,Er,Ce nanocrystal core.

[0021] (12) mixing the second rare earth metal precursor salt solution with octadecene and oleic acid, stirring and fully mixing at room temperature, then heating the system to above 130° C. for a period of time to fully remove water, and cooling to room temperature after the heating is completed;

[0022] Then, NaGdF4:Yb, Er, Ce nanocrystal cores and a methanol solution of ammonium fluoride and sodium hydroxide were added to the mixed solution. The molar ratio of the crystal cores to the second rare earth metal precursor salt in the second rare earth metal precursor salt aqueous solution was 1:1, and the mixture was stirred continuously at 50°C for nucleation.

[0023] The nucleated solution was then heated to boiling to remove methanol, and nitrogen was introduced into the solution system under vacuum to ensure that no alcohol remained.

[0024] Finally, the solution system was heated from room temperature to 300°C for continuous reaction. After the reaction, it was cooled to room temperature. The precipitate was collected by centrifugation and washed with ethanol several times to obtain NaGdF4:Yb,Er,Ce@NaGdF4:Yb,Nd core-shell structured nanocrystals.

[0025] Preferably, in step (11), the rare earth metal precursor salts contained in the first rare earth metal precursor salt aqueous solution are gadolinium (III) acetate, ytterbium (III) acetate, erbium (III) acetate and cerium (III) acetate, wherein the molar ratio of the lanthanide metals Gd:Yb:Er:Ce is 0.73:0.2:0.02:0.05, and the molar concentrations of all rare earth metal precursor salts of gadolinium (III) acetate, ytterbium (III) acetate, erbium (III) acetate and cerium (III) acetate in the aqueous solution are 29.2 mmol / L, 8.0 mmol / L, 0.8 mmol / L and 2.0 mmol / L respectively;

[0026] In step (12), the rare earth metal precursor salts contained in the second rare earth metal precursor salt aqueous solution are gadolinium (III) acetate, ytterbium (III) acetate and neodymium (III) acetate, wherein the molar ratio of the lanthanide metals Gd:Yb:Nd is 0.7:0.1:0.2, and the molar concentrations of all rare earth metal precursor salts of gadolinium (III) acetate, ytterbium (III) acetate and neodymium (III) acetate in the aqueous solution are 28 mmol / L, 4.0 mmol / L and 8.0 mmol / L respectively;

[0027] Preferably, in steps (11) and (12), the volume ratio of octadecene to oleic acid is 3:2, the concentrations of ammonium fluoride and sodium hydroxide in the methanol solution of ammonium fluoride and sodium hydroxide are 0.4 mol / L and 0.5 mol / L, respectively, and the volume ratio added to the reaction system is 1.65:1.

[0028] Preferably, in steps (11) and (12), the heating temperature range for heating to remove water is 130-150°C, which refers to the temperature range of the heating source. The heating temperature for heating to remove alcohol is 100°C, which refers to the temperature range of the heating source. The heating rate during the heating process is lower than 5°C / min and the temperature is raised slowly to prevent bumping.

[0029] The heating nucleation time is 1 h, and the nucleation temperature does not exceed 50°C;

[0030] Preferably, the nitrogen gas is introduced under vacuum for more than three times to ensure that other gases in the system are evacuated;

[0031] The conditions for collecting the precipitated product by centrifugation were 6000 rpm for 10 min;

[0032] After collecting the precipitate by centrifugation and before washing with ethanol, the precipitate was fully resuspended in cyclohexane as a solvent under 250W ultrasound.

[0033] The step (2) is specifically as follows:

[0034] (1) The surface ligands of NaGdF4:Yb,Er,Ce@NaGdF4:Yb,Nd were removed by acetone precipitation and acid washing and dissolved in ethanol. Polyethylenimine solution was continuously added dropwise to the system under ultrasound. The reaction solution was stirred at room temperature for 12 hours. After the reaction, it was centrifuged at high speed and washed with water to remove excess polyethyleneimine to obtain polyethyleneimine-modified lanthanide nanoparticles.

[0035] Preferably, after adding acetone to precipitate the lanthanide nanoparticles, the conditions for centrifuging the product are 12000 rpm for 4 min;

[0036] For pickling, add concentrated hydrochloric acid with a mass fraction of 36%-38% to the separated product, ultrasonicate at 100-250W for 30 minutes, and then let it stand for more than 2 hours.

[0037] Preferably, the molecular weight of polyethyleneimine is 800-10000 Da, the ultrasonic power is 100-250 W, the ultrasonic time is 20-40 min, and the high-speed centrifugation condition is 15000-20000 rpm, 30 min.

[0038] The step (3) is specifically as follows: fully dissolving NHS / EDC in ethanol, adding IR-820 dihexanoic acid to the mixed solution and stirring at room temperature for 2 hours, adding PEI-modified lanthanide nanoparticles to the system, stirring at room temperature for 12 hours, and then centrifuging, washing with water and washing with alcohol in sequence to obtain a hydrogen sulfide-responsive NIR IIb ratiometric fluorescent nanoprobe;

[0039] Preferably, the molar ratio of PEI-modified lanthanide nanoparticles to IR-820 dihexanoic acid is (360-2650):1; the concentrations of NHS and EDC solutions are 0.05 mM and 0.05 mM, respectively; and the centrifugal separation parameters are 15000-20000 rpm, 30 min.

[0040] NIR IIb in the present invention refers to 1500-1700 nm.

[0041] The hydrogen sulfide-responsive NIR IIb ratiometric fluorescent nanoprobe is used in plant stress monitoring, specifically for in situ fluorescence imaging of hydrogen sulfide signaling molecules in living plants.

[0042] The hydrogen sulfide-responsive NIR IIb ratiometric fluorescent nanoprobe prepared by the method of the present invention can track the dynamic changes of hydrogen sulfide signals generated by stress in real time during in vivo fluorescence imaging of plants, and can provide a new monitoring tool for the development of intelligent agriculture.

[0043] The probe is composed of the specific hydrogen sulfide-responsive dye IR-820 dihexanoic acid and lanthanide-doped down-conversion nanoparticles with NIR IIb emission characteristics. IR-820 dihexanoic acid is covalently modified onto the surface of polyethyleneimine (PEI)-functionalized lanthanide nanoparticles. The probe product is obtained after centrifugation and washing. This nanoprobe combines a ratiometric fluorescence response mechanism with the deep tissue penetration properties of the NIR IIb window, effectively overcoming background interference and autofluorescence from complex biomolecules in plants, significantly improving the sensitivity and spatial resolution of hydrogen sulfide detection.

[0044] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0045] (1) The present invention adopts the strategy of rare earth metal precursor decomposition and layer-by-layer growth to synthesize core-shell structured lanthanide nanocrystals, which have unique multi-band narrow absorption, excellent optical stability and tunability, and have broad application prospects in the field of bio-optical imaging.

[0046] (2) The present invention uses lanthanide nanoparticles with NIR IIb emission characteristics as the fluorescence signal source, avoiding the problem that the absorption and emission windows of traditional visible light probes are greatly affected by the autofluorescence of plant tissues, and has an ultra-high imaging signal-to-noise ratio.

[0047] (3) The nanofluorescent probe designed in the present invention has NIR IIb fluorescence that can be activated by hydrogen sulfide. Compared with the visible light and near-infrared I window, it has higher tissue penetration and spatial resolution.

[0048] (4) The present invention assembles lanthanide nanoparticles with NIR IIb emission characteristics and IR-820 dihexanoic acid into a nanofluorescent probe, which can realize non-destructive detection of endogenous hydrogen sulfide signals in living plants and monitor the physiological conditions of plants in real time through optical equipment, thereby realizing the early detection of plant stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Schematic diagram of the synthesis of the hydrogen sulfide-responsive NIR IIb ratiometric fluorescent nanoprobe of the present invention;

[0050] Figure 2 is a transmission electron microscopy image of the lanthanide nanoparticles prepared in Example 1;

[0051] Figure 3 is a fluorescence emission spectrum of the lanthanide nanoparticles prepared in Example 1;

[0052] Figure 4 TEM image of the PEI-modified lanthanide nanoparticles prepared in Example 1;

[0053] Figure 5 TEM image of the hydrogen sulfide-responsive NIR IIb ratiometric fluorescent nanoprobe prepared in Example 1;

[0054] Figure 6 is the absorption spectrum of the lanthanide nanoparticles prepared in Example 1 and IR-820 dihexanoic acid;

[0055] Figure 7 This is a schematic diagram of the principle of the nano fluorescent probe for detecting hydrogen sulfide according to the present invention;

[0056] Figure 8 This is a graph showing the selectivity of the nano fluorescent probe of the present invention to hydrogen sulfide and common substances in plants;

[0057] Figure 9 This is an imaging image of the NIR IIb ratiometric fluorescent nanoprobe prepared in Example 3 monitoring hydrogen sulfide produced by stress in lettuce leaves in real time;

[0058] Figure 10 These are images of commonly used cyanine dyes and lanthanide nanoparticles in lettuce leaves under laser excitation of different wavelengths. DETAILED DESCRIPTION

[0059] 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 art to which this application belongs.

[0060] The embodiments of the present invention are as follows:

[0061] Example 1:

[0062] (1) Preparation of NIR IIb window emitting lanthanide nanoparticles

[0063] The synthesis route of the hydrogen sulfide responsive NIR IIb ratiometric fluorescent nanoprobe of the present invention is as follows: Figure 1 As shown, an aqueous solution of gadolinium (III) acetate, ytterbium (III) acetate, erbium (III) acetate, and cerium (III) acetate in a molar ratio of 0.73:0.2:0.02:0.05 is fully dispersed in a mixture of 4 mL of oleic acid and 6 mL of octadecene, and then heated at 130-150 ° C to fully remove water and cool to room temperature. A methanol solution of ammonium fluoride and sodium hydroxide is added to the mixed solution, and the nucleation is continuously stirred at 45-50 ° C for 1 hour. After the end, it is heated to 100 ° C to remove alcohol, and nitrogen is passed into the system under vacuum. The mixed solution is quickly heated to 300 ° C and reacted for 1.5 hours. After the end, it is cooled to room temperature, the product is collected by centrifugation and washed with ethanol multiple times to obtain NaGdF4:Yb,Er,Ce nanocrystal cores.

[0064] An aqueous solution of gadolinium (III) acetate, ytterbium (III) acetate, and neodymium (III) acetate in a molar ratio of 0.7:0.1:0.2 was mixed with octadecene and oleic acid, stirred thoroughly at room temperature, mixed thoroughly, and heated to remove water. After cooling to room temperature, the mixture was quickly added with NaGdF4:Yb, Er, Ce lanthanide nanocrystal cores and a methanol solution of ammonium fluoride and sodium hydroxide. The mixture was stirred continuously at 50°C for nucleation. After the reaction, the alcohol was fully heated to remove alcohol. Nitrogen was introduced under vacuum to ensure that no alcohol remained. The system was quickly heated to 300°C and the reaction was continued. After the reaction was completed, it was cooled to room temperature, the precipitate was collected by centrifugation, and washed with ethanol several times to obtain NaGdF4:Yb, Er, Ce@NaGdF4:Yb, Nd core-shell structure nanocrystals.

[0065] The morphology of the lanthanide nanocrystals prepared in Example 1 was observed by transmission electron microscopy. Figure 2 As shown, the lanthanide nanocrystals have a uniform particle structure, the particle size of the crystal core is 10.98±0.16nm, and the particle size of the core-shell structure crystal after layer-by-layer growth is 13.09±0.08nm. The emission spectrum of the prepared core-shell structure nanocrystals was characterized by fluorescence spectrometer, as shown in FIG. Figure 3 As shown, the core-shell structured lanthanide nanocrystals have obvious emission peaks at 986nm, 1060nm, 1350nm and 1550nm.

[0066] (2) Preparation of polyethyleneimine surface-modified lanthanide nanoparticles

[0067] The surface ligands of NaGdF4:Yb,Er,Ce@NaGdF4:Yb,Nd were removed by acetone sedimentation and acid washing and dissolved in ethanol. Polyethyleneimine solution was continuously added dropwise to the system under ultrasound. The reaction solution was stirred at room temperature for 12 hours. After completion, it was centrifuged at high speed and washed with water to remove excess polyethyleneimine to obtain polyethyleneimine-modified lanthanide nanoparticles.

[0068] The PEI-modified lanthanide nanocrystals prepared in Example 2 were observed by transmission electron microscopy. Figure 4 As shown, the composite material has a granular structure with a particle size of 16.91±0.29 nm, and polyethyleneimine wrapping increases the particle size of lanthanide nanocrystals.

[0069] (3) Preparation of hydrogen sulfide-responsive NIR IIb ratiometric fluorescent nanoprobes

[0070] NHS / EDC was fully dissolved in ethanol, IR-820 dihexanoic acid was added to the mixed solution and stirred at room temperature for 2 hours, PEI-modified lanthanide nanoparticles were added to the system, stirred at room temperature for 12 hours, and then centrifuged, washed with water and alcohol to obtain a hydrogen sulfide-responsive NIR IIb ratiometric fluorescent nanoprobe.

[0071] The hydrogen sulfide-responsive NIR IIb ratiometric fluorescent nanoprobe prepared in Example 2 was observed by transmission electron microscopy. Figure 5 As shown, the composite material has a granular structure with a particle size of 16.94±0.26nm. The particle size of the composite material does not change much after IR-820 dihexanoic acid is connected to the surface polyethyleneimine.

[0072] The affinity of lanthanide nanoparticles with IR-820 dihexanoic acid was determined by UV-visible-infrared absorption spectroscopy. Figure 6As shown, both lanthanide nanoparticles and IR-820 dihexanoic acid have absorption peaks at 808 nm, while lanthanide nanoparticles have an obvious absorption peak at 980 nm.

[0073] like Figure 7 As shown in the figure, when the IR-820 dihexanoic acid on the surface of the composite nano fluorescent probe reacts with hydrogen sulfide with nucleophilic reaction, its absorption at 808nm will decrease significantly. At this time, the fluorescence emission of the lanthanide nanoparticles in the probe that was shielded at 1550nm will be restored, while the emission of the lanthanide nanoparticles at 980nm remains unchanged. By measuring the emission ratio of 1550nm under 808nm and 980nm laser excitation, the fluorescence emission of the lanthanide nanoparticles at 1550nm is significantly reduced. 808 / I 980 The level of hydrogen sulfide can be monitored.

[0074] application:

[0075] like Figure 8 As shown, the prepared hydrogen sulfide-responsive NIR IIb ratiometric fluorescent nanoprobe has good selectivity for hydrogen sulfide and good anti-interference ability for common components in plants and biological thiols.

[0076] The hydrogen sulfide-responsive NIR IIb ratiometric fluorescent nanoprobe prepared in Example 1 was used to perform in vivo optical monitoring of hydrogen sulfide signals in lettuce leaves.

[0077] The prepared nanofluorescent probes were dispersed in MES buffer. Then, 10 μL of the nanofluorescent probes were infiltrated into lettuce leaves using a 1 mL needleless syringe. Gentle pressure was applied to the leaves during infiltration to prevent mechanical damage. Residual nanoparticles on the leaf surface were thoroughly rinsed with water, and the injected leaves were left to rest for 1 hour.

[0078] Subsequently, mechanical injury or heat shock stress was applied 1-2 cm from the probe infiltration point, and Cd 2+ Before stress treatment, plants were watered with a cadmium chloride solution. After stress treatment, endogenous hydrogen sulfide production in the leaves was recorded using a NIR II imaging system. Real-time fluorescence imaging was performed on lettuce leaves under 808nm laser excitation. The NIR IIb fluorescence signal of the nanoprobes within the lettuce leaves was monitored using an InGaAs imaging system equipped with a 1400nm longpass optical filter.

[0079] The results of real-time fluorescence imaging of lettuce leaves are as follows: Figure 9 As shown, the control group did not show obvious signal response, but heat shock, mechanical damage and Cd 2+ The fluorescence ratio signal of the nanofluorescent probe in the stress group increased significantly with the imaging time, which demonstrated the feasibility of the prepared hydrogen sulfide-responsive NIR IIb ratiometric fluorescent nanoprobe for in situ monitoring of endogenous hydrogen sulfide signaling molecules in plants.

[0080] Comparative Example 1:

[0081] The traditional cyanine fluorescent probe and the NIR IIb fluorescent probe prepared in Example 1 were used for in vivo imaging in plant leaves.

[0082] NIR IIb ratiometric fluorescent nanoprobes were prepared using the method described in Example 1. 0.1 mM concentrations of commercial Cy-5, Cy-7.5, IR-808, IR-820, IR-1048, and IR-1060 probes, along with the prepared NIR IIb ratiometric fluorescent nanoprobes, were dispersed in 10 mL of MES buffer. A needleless syringe was used to infiltrate 10 μL of each probe into lettuce leaves. Finally, the leaves were rinsed with deionized water to remove any residual probes, and the leaves were left to rest for 1 hour.

[0083] The imaging effects of different probes in lettuce leaves were observed using a near-infrared imaging system. Real-time fluorescence imaging of the leaves was performed under 635nm, 808nm, and 980nm laser excitation. The fluorescence signals of the probes in the leaves were captured using an InGaAs imaging system equipped with a 600nm or 1400nm longpass filter.

[0084] like Figure 10 As shown, under 635nm visible light excitation, the InGaAs imaging system captured obvious background autofluorescence of lettuce leaves, which had a significant impact on the imaging of dyes that absorb in the visible light band, such as Cy-5, Cy-7.5, and IR-1048, confirming that fluorescent probes in this absorption band are not suitable for in vivo imaging of plant leaves. However, under 808nm and 980nm laser excitation, lettuce leaves injected with NIR IIb ratiometric fluorescent nanoprobes did not show obvious plant autofluorescence interference, and the fluorescence signal of the nanoprobe was strong. This confirms that 808nm and 980nm laser excitation does not bring tissue autofluorescence interference to leaf imaging, and that nanofluorescent probes with emission bands in the NIR IIb window have higher in vivo imaging resolution.

[0085] From this implementation, it can be seen that the hydrogen sulfide-responsive NIR IIb ratiometric fluorescent nanoprobe prepared by the present invention has the advantages of strong anti-interference ability and high imaging resolution. It is used for real-time monitoring of stress signal molecules in plants, overcoming the shortcomings of existing plant stress perception methods such as long time consumption, high background, and inability to perform real-time monitoring, and realizing real-time in situ monitoring of plant stress information.

[0086] The embodiments described above provide a detailed description of 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 intended to limit the present invention. Any modifications, supplements and equivalent substitutions made within the scope of the principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a hydrogen sulfide-responsive NIR IIb ratiometric fluorescent nanoprobe, characterized by: The method is to assemble a hydrogen sulfide-responsive NIR IIb ratiometric fluorescent nanoprobe using NIR IIb-emitting lanthanide nanoparticles and IR-820 dihexanoic acid as the main components through covalent linkage.

2. The method for preparing a hydrogen sulfide-responsive NIR IIb ratiometric fluorescent nanoprobe according to claim 1, wherein: The method comprises the following steps: (1) Preparation of core-shell lanthanide nanoparticles; (2) using polyethyleneimine (PEI) to modify the surface of lanthanide nanoparticles to obtain PEI-modified lanthanide nanoparticles; (3) IR-820 dihexanoic acid was activated by EDC / NHS and assembled with PEI-modified lanthanide nanoparticles to prepare a nanofluorescent probe.

3. The method for preparing a hydrogen sulfide-responsive NIR IIb ratiometric fluorescent nanoprobe according to claim 2, characterized in that: The step (1) is specifically as follows: (11) mixing the first rare earth metal precursor salt solution with octadecene and oleic acid, stirring and fully mixing at room temperature, then heating to above 130° C., and cooling to room temperature after the heating is completed; Then, methanol solution of ammonium fluoride and sodium hydroxide was added to the mixed solution and stirred continuously at 45°C to form nuclei; The nucleated solution is then heated to boiling, and nitrogen is introduced into the solution under vacuum; Finally, the solution was heated from room temperature to 300°C for continuous reaction. After the reaction, it was cooled to room temperature, the precipitate was collected by centrifugation and washed with ethanol several times to obtain the NaGdF4:Yb,Er,Ce nanocrystal core. (12) mixing the second rare earth metal precursor salt solution with octadecene and oleic acid, stirring and fully mixing at room temperature, then heating to above 130° C., and cooling to room temperature after the heating is completed; Then, NaGdF4:Yb, Er, Ce nanocrystal cores and a methanol solution of ammonium fluoride and sodium hydroxide were added to the mixed solution. The molar ratio of the crystal cores to the second rare earth metal precursor salt was 1:1, and the mixture was stirred continuously at 50°C for nucleation. The nucleated solution is then heated to boiling, and nitrogen is introduced into the solution under vacuum; Finally, the solution was heated from room temperature to 300°C for continuous reaction. After the reaction, it was cooled to room temperature. The precipitate was collected by centrifugation and washed with ethanol several times to obtain NaGdF4:Yb,Er,Ce@NaGdF4:Yb,Nd core-shell structured nanocrystals.

4. The method for preparing a hydrogen sulfide-responsive NIR IIb ratiometric fluorescent nanoprobe according to claim 3, characterized in that: In step (11), the rare earth metal precursor salts contained in the first rare earth metal precursor salt aqueous solution are gadolinium (III) acetate, ytterbium (III) acetate, erbium (III) acetate and cerium (III) acetate, wherein the molar ratio of the lanthanide metals Gd:Yb:Er:Ce is 0.73:0.2:0.02:0.05, and the molar concentrations of all the rare earth metal precursor salts in the aqueous solution are 29.2 mmol / L, 8.0 mmol / L, 0.8 mmol / L and 2.0 mmol / L; In step (12), the rare earth metal precursor salts contained in the second rare earth metal precursor salt aqueous solution are gadolinium (III) acetate, ytterbium (III) acetate and neodymium (III) acetate, wherein the molar ratio of the lanthanide metals Gd:Yb:Nd is 0.7:0.1:0.2, and the molar concentrations of all rare earth metal precursor salts in the aqueous solution are 28 mmol / L, 4.0 mmol / L and 8.0 mmol / L; In steps (11) and (12), the volume ratio of octadecene to oleic acid is 3:2, the concentrations of ammonium fluoride and sodium hydroxide in the methanol solution of ammonium fluoride and sodium hydroxide are 0.4 mol / L and 0.5 mol / L, respectively, and the volume ratio added to the reaction system is 1.65:

1.

5. The method for preparing a hydrogen sulfide-responsive NIR IIb ratiometric fluorescent nanoprobe according to claim 3, characterized in that: In the steps (11) and (12), the heating temperature for heating to remove water is in the range of 130-150°C, the heating temperature for heating to remove alcohol is 100°C, and the heating rate during the heating process is less than 5°C / min; The heating nucleation time is 1 h, and the nucleation temperature does not exceed 50°C; Repeat the operation of introducing nitrogen under vacuum for more than three times; The conditions for collecting the precipitated product by centrifugation were 6000 rpm for 10 min; After collecting the precipitate by centrifugation and before washing with ethanol, the precipitate was fully resuspended in cyclohexane as a solvent under 250W ultrasound.

6. The method for preparing a hydrogen sulfide-responsive NIR IIb ratiometric fluorescent nanoprobe according to claim 2, characterized in that: The step (2) is specifically as follows: (1) The surface ligands of NaGdF4:Yb,Er,Ce@NaGdF4:Yb,Nd were removed by acetone precipitation and acid washing and dissolved in ethanol. Polyethyleneimine solution was continuously added dropwise to the system under ultrasound. The reaction solution was stirred at room temperature for 12 hours. After the reaction, it was centrifuged and washed with water to obtain polyethyleneimine-modified lanthanide nanoparticles.

7. The method for preparing a hydrogen sulfide-responsive NIR IIb ratiometric fluorescent nanoprobe according to claim 6, characterized in that: After adding acetone to precipitate the lanthanide nanoparticles, the products were separated by centrifugation at 12000 rpm for 4 min; For pickling, add concentrated hydrochloric acid with a mass fraction of 36%-38% to the separated product, ultrasonicate at 100-250W for 30 minutes, and then let it stand for more than 2 hours. The molecular weight of polyethyleneimine is 800-10000 Da, the ultrasonic power is 100-250 W, the ultrasonic time is 20-40 min, and the high-speed centrifugation condition is 15000-20000 rpm, 30 min.

8. The method for preparing a hydrogen sulfide-responsive NIR IIb ratiometric fluorescent nanoprobe according to claim 1, characterized in that: The step (3) is specifically as follows: NHS / EDC is fully dissolved in ethanol, IR-820 dihexanoic acid is added to the mixed solution and stirred at room temperature for 2 hours, PEI-modified lanthanide nanoparticles are added to the system, stirred at room temperature for 12 hours, and then centrifuged, washed with water, and washed with alcohol to obtain a hydrogen sulfide-responsive NIR IIb ratiometric fluorescent nanoprobe; the molar ratio of the PEI-modified lanthanide nanoparticles to the IR-820 dihexanoic acid is (360-2650):1; the concentrations of the NHS and EDC solutions are 0.05 mM and 0.05 mM, respectively; and the centrifugal separation parameters are 15000-20000 rpm and 30 min.

9. A hydrogen sulfide-responsive NIR IIb ratiometric fluorescent nanoprobe, characterized by: The invention is prepared by the method according to any one of claims 1 to 8.

10. Use of the hydrogen sulfide-responsive NIR IIb ratiometric fluorescent nanoprobe according to claim 9, characterized in that: Applications in plant stress monitoring.

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