Upconversion fluorescent nanoprobes with biodegradable hetero-nanostructure for super-resolution fluorescence microscopy imaging and preparation method thereof

Abstract

This invention discloses a biodegradable heterogeneous core-shell upconversion fluorescent nanoprobe for super-resolution fluorescence microscopy and its preparation method. The probe comprises a rare-earth-doped hexagonal metal fluoride core layer with an average particle size of less than 5.5 nm, surrounded by a rare-earth and group IVB transition metal-doped cubic alkaline earth metal fluoride shell, forming a heterogeneous core-shell structure. The rare-earth ion energy levels in the core layer are adapted to stimulated emission loss and photon avalanche mechanisms, enabling nanoscale bioimaging. The degradation products have a particle size of less than 5.5 nm and can be excreted via glomerular metabolism. The shell hydrolysis rate can be synergistically controlled by the shell thickness and the doping amount of group IVB elements. This probe combines controllable biodegradation with super-resolution imaging capabilities, achieving a loss efficiency of 92% under 820 nm loss light and an imaging spatial resolution of less than 100 nm.

Description

Technical Field

[0001] This invention relates to the fields of bionanomaterials and upconversion fluorescence imaging technology, specifically to a biodegradable heterogeneous core-shell upconversion fluorescent nanoprobe for super-resolution fluorescence microscopy and its preparation method. Background Technology

[0002] Fluorescence microscopy is a crucial technique for studying cell structure, molecular distribution, and biological processes. However, due to the optical diffraction limit, traditional fluorescence microscopy struggles to resolve small-scale biological structures at similar spatial distances. Stimulated emission loss (STED) microscopy utilizes the synergistic effect of excitation light and spatially modulated loss light to compress the effective fluorescence emission region, thereby improving spatial resolution. However, fluorescent probe materials that meet these performance requirements often exhibit some degree of biotoxicity, making them unsuitable for clinical medical applications and in vivo imaging. This contradiction is particularly pronounced in the field of using upconversion nanomaterials as fluorescent probes.

[0003] Upconversion fluorescent nanomaterials possess characteristics such as narrow emission bands, strong resistance to photobleaching, low background autofluorescence, and tunable rare-earth ion energy level structures. Among them, rare-earth-doped fluorides based on matrices such as NaGdF4 exhibit low phonon energies and excellent upconversion luminescence performance. By selecting the types and doping ratios of sensitizers and luminescent center ions, energy transfer and emission modulation channels suitable for stimulated emission loss processes can be constructed, thus showing potential applications in the field of super-resolution fluorescence microscopy.

[0004] However, rare-earth fluorides with excellent luminescence properties typically possess relatively stable crystal structures, making them difficult to decompose in aqueous phases and physiological environments. To reduce surface quenching and improve luminescence intensity, existing upconversion nanoparticles often employ methods such as increasing particle size or coating with an inert shell, which further increases the overall size and structural stability of the particles. Once these particles enter the body, they tend to accumulate in organs such as the liver and spleen, and are difficult to rapidly convert into removable ions, molecules, or smaller particles. Therefore, how to maintain the luminescence properties required for super-resolution imaging while enabling controllable degradation of the probe after imaging is a pressing problem that rare-earth upconversion fluorescent probes need to solve for bioimaging applications.

[0005] To improve the degradation performance of traditional inorganic fluorescent probes, current research has attempted to utilize fluorine halides and soft-lattice fluorides containing zirconium and hafnium. For example, materials such as K3ZrF7:Yb,Er and Na3ZrF7:Yb,Er,Ce can undergo hydrolysis through the dissolution of zirconium-containing fluorine coordination units. However, some systems degrade too rapidly under near-physiological conditions, making it difficult to maintain a long imaging window; simultaneously, their luminescence intensity is significantly lower than that of rigid-lattice rare-earth fluorides. Therefore, simply using easily hydrolyzed soft lattices as the luminescent matrix cannot simultaneously achieve a balance between luminescence efficiency, imaging stabilization time, and degradation behavior.

[0006] Since the luminescence properties of the biodegradable matrix itself are limited, it is advisable to allocate luminescence and degradation functions to different spatial regions. Existing research has achieved this by constructing a core-shell structure: coating the luminescent core with a biodegradable shell containing a Group IVB transition metal element, thus separating the luminescent and degradation regions. Specifically, coating the cubic NaYF4 luminescent core with a cubic CaF2 shell can suppress surface quenching and improve luminescence intensity; introducing Zr into the outer shell... 4+ This can alter the hydrolysis behavior of the shell. Although this homogeneous core-shell structure with the same crystal phase successfully combines optical performance and biodegradability, its luminescent core is also cubic with high crystal symmetry, resulting in insufficient upconversion efficiency and low loss efficiency during STED imaging, making it difficult to meet the performance requirements of super-resolution fluorescence microscopy for the luminescent core.

[0007] Therefore, the choice of crystal phase between the core and shell has a decisive influence on super-resolution performance. If hexagonal rare-earth fluorides, which exhibit superior upconversion luminescence performance and higher crystal asymmetry, are better suited to the stimulated emission loss mechanism, are retained as the luminescent core, while a cubic alkaline earth metal fluoride is used as the degradable shell, it is possible to achieve controlled in vivo degradation while maintaining the excellent upconversion luminescence and super-resolution imaging performance of the hexagonal luminescent core. Therefore, providing a heterogeneous core-shell structured upconversion fluorescent nanoprobe that retains the optical properties of a rare-earth-doped hexagonal luminescent core, possesses luminescence modulation capabilities suitable for super-resolution imaging, and allows for adjustment of hydrolysis behavior through shell composition and structural parameters, is of great significance for improving the problem of simultaneously achieving luminescence performance, super-resolution imaging stability, and biodegradability in existing technologies. Summary of the Invention

[0008] The technical problem to be solved by the invention is to overcome the defects of the prior art and provide a biodegradable heterostructured upconversion fluorescent nanoprobe for super-resolution fluorescence microscopy and its preparation method, so as to solve the problems that traditional upconversion fluorescent probes are non-degradable in biological environments and that it is difficult to balance luminescence performance and biodegradability.

[0009] To solve the above-mentioned technical problems, the technical solution provided by the present invention is: a biocontrollable degradable heterogeneous core-shell structure upconversion fluorescent nanoprobe for super-resolution fluorescence microscopy, comprising a nanoprobe designed with a heterogeneous core-shell structure composed of a hexagonal core layer and a cubic shell layer. The core layer is composed of hexagonal metal fluoride nanoparticles formed by rare earth element doping, and the average particle size of the core layer nanoparticles is less than 5.5 nm. The core metal fluoride has a hexagonal crystal phase, which is one of NaYF4, NaGdF4, NaYbF4, and NaLuF4. The core layer has an energy level structure formed by rare earth ion doping. The energy level structure is used to regulate the photoluminescence of the core layer through the stimulated emission depletion mechanism under the action of excitation light and depletion light, so as to realize fluorescence microscopy imaging with nanoscale spatial resolution. The core layer rare earth element doping also includes an activator and a sensitizer, with the activator being Tm. 3+ Er 3+ 、Nd 3+ Ho 3+ Pr 3 + One of them, with a doping concentration of 0.120 mol%; the sensitizer is Yb. 3+ or Nd 3+ Its doping concentration is 160 mol.

[0010] Furthermore, the shell is composed of cubic alkaline earth metal fluorides doped with rare earth elements and group IVB transition metal elements; the cubic alkaline earth metal fluorides of the shell are one or more combinations of CaF2, SrF2, and BaF2.

[0011] Furthermore, the shell coating thickness is 110 nm, and the overall particle size of the nanoprobe is 715 nm; the group IVB transition metal element is one or more of Ti, Zr, and Hf, wherein the molar ratio of the transition metal doping is 120 mol%; the rare earth element doped in the shell is Yb. 3+ Gd 3+ 、Tb 3+ 、Nd 3+ One or more of them.

[0012] Furthermore, the hydrolysis rate of the shell is jointly regulated by the shell thickness and the amount of group IVB transition metal doping; in an aqueous environment with pH 6.9, the degradation time of the core-shell structure is 4872 h.

[0013] A method for preparing the above-mentioned biodegradable heterostructured upconversion fluorescent nanoprobe for super-resolution fluorescence microscopy includes the following steps: (a): The rare earth compound is dispersed in a solvent: the rare earth compound is one or more of Ln(CF3COO)3, Ln(CH3COO)3, LnCl3, LnPO4, and Ln(NO3)3; the solvent is an anhydrous mixture of oleic acid, octadecene, and oleylamine; (b): Disperse the fluorine source and sodium source in the solvent: the fluorine source is one of NH4F, NH4HF2, NaF, KF, and the sodium source is one of Na2CO3, NaOH, NaHCO3, NaNO3, Na2SO4; the solvent is anhydrous methanol, anhydrous ethanol, or anhydrous cyclohexane. (c) The solution obtained in (a) is heated and kept at that temperature for a certain period of time to dissolve the rare earth elements, and then cooled to room temperature; (d) Then add the solution of (b) according to the stoichiometric ratio and keep it at a temperature above room temperature for a period of time to remove the low-boiling-point solvent, and then remove the residual low-boiling-point solvent by vacuuming. (e) The liquid obtained in (d) without low-boiling-point solvent is heated under nitrogen atmosphere and kept at high temperature for a period of time; finally, hexagonal rare earth doped metal fluoride core nanoparticles are obtained. (f) Preparation of shell precursor solution: Rare earth metal salts, alkaline earth metal salts, and Group IVB metal salts are dispersed in an anhydrous mixed solvent of oleic acid and octadecene; (g) The hexagonal rare earth-doped metal fluoride core nanoparticles obtained in (e) are dispersed in a solvent; the solvent is an anhydrous mixture of oleic acid and octadecene. (h) Coating with a biodegradable shell, the liquid in (e) is heated under a nitrogen atmosphere and injected in batches into it at high temperature. After each injection, the reaction is repeated for a period of time. After the reaction is completed, heterogeneous core-shell structured nanoparticles with shell thickness adjustable according to the number of injections are obtained.

[0014] Furthermore, Ln mentioned in (a) is one or more of Y, Yb, Gd, Tm, Er, Ho, Eu, Tb, and Nd; The rare earth metal salts mentioned in (f) are one or more of Yb(CF3COO)3, Ln(CF3COO)3, Ln(CH3COO)3, LnCl3, LnPO4, and Ln(NO3)3; The alkaline earth metal salts are M(CF3COO)2, M(CH3COO)2, MCl2, and M(NO3)2, wherein M is one or more of Ca, Sr, Ba, and Mg; The group IVB metal salt is IVBX4, wherein IVB is one or more of Zr, Hf, and Ti, and X is CH3COO, Cl, NO3, alkoxy, or an organic ligand.

[0015] Furthermore, it also includes the use of surfactants to synthesize nanoparticles, wherein the surfactants include one or more of oleic acid, oleylamine, octadecene, acetylacetonate, and lauric acid.

[0016] Furthermore, in step (c), the reaction temperature is 140-160°C, and the holding time is 30-60 minutes; In step (e), the reaction temperature is 240-260℃ and the holding time is 20-45 minutes; The reaction temperature in step (h) is 290-300℃.

[0017] Furthermore, in step (a), the volume ratio of oleic acid to octadecene is 2:5; In step (d), the volume ratio of oleic acid to octadecene is (13):(25). In step (e), the volume ratio of oleic acid to octadecene is (13):(25).

[0018] Furthermore, in (f), the total amount of rare earth metal salts, alkaline earth metal salts, and Group IVB metal salts is in the molar ratio of the corresponding anhydrous mixed solvent to 1:(520).

[0019] With the above structure, the present invention has the following advantages:

[0020] 1. This invention employs a hexagonal rare-earth-doped metal fluoride as the luminescent core layer, and primarily incorporates Group IVB transition metals for promoting hydrolysis within a cubic alkaline earth metal fluoride shell, thus spatially separating the luminescent region from the degradation control region. The hexagonal core layer provides a relatively stable lattice environment for both the sensitizer ions and the luminescent center ions, which is beneficial for maintaining the energy level transitions required for upconversion energy transfer. Simultaneously, it reduces the direct disturbance of the local lattice environment of the luminescent center by the Group IVB transition metals, thereby balancing upconversion luminescence intensity, photostability, and loss response performance.

[0021] 2. This invention, by doping a cubic alkaline earth metal fluoride shell with a Group IVB transition metal element, enables the shell to gradually hydrolyze in an aqueous phase or physiological environment. Simultaneously, by synergistically adjusting the shell thickness and the doping amount of the Group IVB transition metal element, the hydrolysis rate of the shell can be controlled, allowing the nanoprobe to maintain the necessary structural stability during the imaging stage and gradually deconstruct after imaging to form smaller core particles or degradation products. This improves the problems of traditional rare earth nanoprobes, such as structural instability, difficulty in degradation, and potential long-term retention.

[0022] 3. The heterogeneous core-shell structured upconversion fluorescent nanoprobe described in this invention has significant advantages in super-resolution microscopy. By designing a heterogeneous core-shell structured fluorescent probe, the loss efficiency of 820 nm laser in stimulated emission loss mode can reach over 90%, while the spatial resolution can be less than 100 nm. It can be used for super-resolution fluorescence microscopy imaging of cells, tissues, and living samples, and different imaging time windows can be adapted by adjusting the shell composition and thickness. Therefore, this nanoprobe has application potential in biomarker tracing, observation of cellular substructures, monitoring of dynamic life processes, and preparation of biomedical imaging reagents. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the preparation and degradation process of the degradable heterogeneous core-shell structured upconversion nanoparticles of the present invention and its STED imaging.

[0024] Figure 2 (a) is a transmission electron microscope image of the nanoparticles prepared in Example 1; Figure 2 (b) is a transmission electron microscope image of the heterogeneous core-shell nanoparticles prepared in Example 2.

[0025] Figure 3 The image shows the X-ray diffraction pattern of the heterogeneous core-shell nanoparticles prepared in Example 2.

[0026] Figure 4 The upconversion luminescence spectra of the heterogeneous core-shell structure nanoparticles with different shell thicknesses prepared in Example 3 are shown.

[0027] Figure 5 The upconversion spectrum of the heterogeneous core-shell nanoparticles in Example 3 as a function of degradation time in an aqueous environment is shown.

[0028] Figure 6 The emission spectrum of the degradable heterogeneous core-shell structured upconversion nanoparticles in Example 4 under 980nm laser excitation and their loss behavior under 820nm laser excitation are shown.

[0029] Figure 7 This is a super-resolution microscopic image of the degradable heterogeneous core-shell structured upconversion nanoparticles in Example 4. Detailed Implementation

[0030] The present invention will now be described in further detail with reference to the accompanying drawings.

[0031] This invention relates to a biodegradable heterostructured upconversion fluorescent nanoprobe for super-resolution fluorescence microscopy and its preparation method, comprising the following steps: (1) Preparation of core MLnF4 nanoparticles [(Ln=Y, Gd, Yb, Tm, Nd, Ho, Pr), (M=Li, Na, K)]: Small-sized MLnF4 nanoparticles were synthesized in a high-boiling-point solvent under high-temperature conditions. The core layer particles exhibit an irregular, approximately spherical morphology with an average particle size of less than 5.5 nm, enabling them to be filtered by living glomeruli and metabolized by the organism. The energy levels of the doped rare earth elements are used in conjunction with super-resolution microscopy imaging mechanisms.

[0032] (2) Preparation of heterogeneous biodegradable shell MF2:Ln,Ti / Zr / Hf (M=Ca,Sr,Ba): The generated MLnF4 nanoparticles were dispersed in an anhydrous mixed solution composed of various surfactants. A mixed solution of Ln(CF3COO)3, M(CF3COO)2 (M=Ca,Sr,Ba) and acetic acid solution of group IVB elements was mixed in the mixed solution composed of various surfactants and treated with water displacement and oxygen removal as a precursor. The nanoparticles obtained in step (1) were dispersed in an anhydrous mixed solution composed of various surfactants, and then injected into the precursor solution in batches at high temperature. The reaction time was several minutes after each injection, and a total of several injections were performed. The core nanoparticles began to gradually grow a heterogeneous biodegradable shell MF2:Ln,Ti / Zr / Hf, forming a controllable and biodegradable outer layer. The biodegradable shell can block the fluorescence quenching caused by surface defects of nanoparticles and significantly improve the upconversion fluorescence brightness.

[0033] Specifically, the probe employs a core-shell structure. Its core consists of hexagonal metal fluoride nanoparticles doped with rare earth elements, the combination of which is adapted to achieve super-resolution fluorescence microscopy. The shell is a cubic metal fluoride simultaneously doped with Group IVB transition metals (Ti, Zr, or Hf) and rare earth elements. The core nanoparticles have an average particle size of approximately 5.5 nm, ensuring that they can be filtered through the glomeruli after in vivo imaging and metabolism, achieving rapid renal clearance and in vivo metabolism.

[0034] The core is composed of a hexagonal metal fluoride, which is one of NaYF4, NaGdF4, NaYbF4, and NaLuF4, and has a stable hexagonal crystal structure. The optimal combination of rare earth elements is selected to form a core structure suitable for super-resolution microscopy, with the rare earth element combination being Yb. 3+ / Tm 3+ Yb 3+ / Er 3+ 、Nd 3+ / Yb 3+ / Er 3+ Yb 3+ / Tm 3+ / Er 3+ 、Nd 3+Pr 3+ Tm 3+ One type of rare earth energy level structure can form an efficient upconversion energy transfer path and regulate light emission behavior through stimulated emission loss mechanism to achieve nanoscale spatial resolution. The preferred sensitizer is ytterbium (Yb). 3+ ) or neodymium (Nd) 3+ The doping concentration is 1-60 mol%, and the activator is thulium (Tm). 3 + ), Erbium 3+ ), neodymium (Nd) 3+ ), holmium (Ho 3+ ) or praseodymium (Pr 3+ The preferred doping concentration is 0.1-20 mol%. Example 1 This embodiment prepares a hexagonal NaGdF4:Yb phase for super-resolution fluorescence microscopy. 3+ / Tm 3+ Core nanoparticles.

[0035] Weigh out 176.20 mg of gadolinium acetate, 168.89 mg of ytterbium acetate, and 36.41 mg of thulium acetate, wherein Yb 3+ The molar doping ratio relative to the total amount of rare earth elements is 40%, Tm 3+ The doping ratio relative to the total amount of rare earth elements is 10%. The above substances are added to an anhydrous mixed solvent composed of oleic acid and octadecene, wherein the volume ratio of oleic acid to octadecene is 2:3, and the total volume of the mixed solvent is 25 mL.

[0036] The solution was heated to 110°C until it became clear and transparent, indicating that excess water had been removed. The temperature was then raised to 150°C and held for 40 minutes to allow the rare earth precursors to fully dissolve and form a homogeneous rare earth precursor solution. Heating was then stopped, and the resulting solution was allowed to cool to room temperature.

[0037] Separately, 162.98 mg of fluorine source and 40.00 mg of sodium source were prepared, both of which had been pre-dissolved in methanol. The fluorine source was NH4F, and the sodium source was NaOH. The methanol solution containing the fluorine and sodium sources was added to the aforementioned rare earth precursor solution, and the mixture was stirred at 45°C for 60 minutes until the solution became clear and transparent, ensuring thorough mixing of the fluorine, sodium, and rare earth precursors.

[0038] After the solution became clear and transparent, it was heated to 90°C and evacuated at 0.01 mbar to remove methanol and other low-boiling-point components. Nitrogen gas was then introduced into the reaction system as a protective atmosphere, and the reaction temperature was raised to 240°C and maintained for 45 minutes to allow the formation of nanocrystal nuclei and complete the ripening reaction. After the reaction was complete, the reaction solution was allowed to cool naturally to room temperature.

[0039] Anhydrous ethanol was added to the resulting reaction solution to precipitate the product. The mixture was then centrifuged at 7500 rpm for 5 minutes, the supernatant was discarded, and the precipitate was collected. The precipitate was washed three times with anhydrous ethanol and cyclohexane to remove unreacted precursors and surfactants. Finally, the resulting product was dispersed in cyclohexane to obtain hexagonal NaGdF4:40%Yb. 3+ 10%Tm 3+ Core nanoparticle dispersion.

[0040] The obtained core nanoparticles were characterized by transmission electron microscopy. The results showed that the obtained nanoparticles were approximately spherical or irregularly spherical in shape, with an average particle size of less than 5.5 nm.

[0041] Further phase analysis of the obtained product was performed using powder X-ray diffraction. The obtained diffraction peaks matched the standard diffraction data of hexagonal NaGdF4, indicating that the obtained NaGdF4:Yb 3+ / Tm 3+ The core nanoparticles have a hexagonal phase crystal structure.

[0042] The hexagonal NaGdF4:Yb prepared in this embodiment 3+ / Tm 3+ The core nanoparticles can serve as the luminescent core for subsequent heterogeneous core-shell nanoprobes, and can be used to further coat a biodegradable shell containing alkaline earth metal fluorides and Group IVB transition metal elements.

[0043] Example 2 This embodiment uses the hexagonal NaGdF4:Yb prepared in Example 1. 3+ / Tm 3+ The core nanoparticles serve as seed crystals, with Yb doped onto their surface. 3+ and Zr 4+ To prepare NaGdF4:Yb with a cubic CaF2 shell. 3+ / Tm 3+ @CaF2:Yb 3+ ,Zr 4+ Heterogeneous core-shell structured upconversion fluorescent nanoprobe.

[0044] Preparation of shell precursor solution 168.3 mg of calcium trifluoroacetate, 177.6 mg of ytterbium trifluoroacetate, and 6.3 mg of zirconium acetate were weighed out respectively. In the shell precursor, Yb... 3+ The molar ratio of Zr relative to the total amount of shell metal ions is 40%. 4+ The molar ratio of the above substances to the total amount of shell metal ions is 5%. The above substances are added to an anhydrous mixed solvent composed of oleic acid and octadecene, wherein the volume ratio of oleic acid to octadecene is 1:1, and the total volume of the mixed solvent is 10 mL.

[0045] The solution was heated to 110°C and evacuated under nitrogen protection and stirring at 0.01 mbar for 15 minutes to remove moisture, oxygen, and other low-boiling-point components, yielding a clear shell precursor solution. The solution was then cooled and stored at 4°C for later use.

[0046] (2) Coating with a heterogeneous biodegradable shell Take 0.5 mmol of NaGdF4:Yb prepared in Example 1 3+ / Tm 3+ The core nanoparticles were dispersed in an anhydrous mixed solvent consisting of oleic acid and octadecene. The volume ratio of oleic acid to octadecene was 1:2, and the total volume of the mixed solvent was 15 ml. The solution was heated to 110 °C to remove moisture, oxygen, and low-boiling-point components. Under nitrogen protection and continuous stirring, the solution was then heated to 300 °C.

[0047] After the reaction temperature stabilizes, the shell precursor solution prepared in step (1) is injected into the core nanoparticle solution in batches.

[0048] Each batch was injected with 1 ml of the shell precursor solution. After each injection, the reaction was continued at 300°C for 15 minutes to allow the shell material to grow on the surface of the core nanoparticles. In this example, a total of 8 injections were performed.

[0049] After the final injection, continue the reaction at 300°C for 15 minutes. Once the reaction is complete, stop heating and allow the reaction solution to cool naturally to room temperature.

[0050] Anhydrous ethanol was added to the resulting reaction solution to precipitate the product. The mixture was then centrifuged at 7500 rpm for 5 minutes, the supernatant was discarded, and the precipitate was collected. The precipitate was washed three times with anhydrous ethanol and cyclohexane to remove unreacted precursors and surfactants. Finally, the obtained product was dispersed in cyclohexane to obtain a dispersion of heterogeneous core-shell upconversion fluorescent nanoprobes.

[0051] Transmission electron microscopy images show that the core layer of the hexagonal phase is covered with a biodegradable cubic shell layer. By adjusting the precursor concentration and the number of injections, the shell thickness can be precisely controlled, thereby adjusting the hydrolysis rate.

[0052] Example 3 Building upon Example 2, spatial decoupling of luminescence performance and degradation behavior was further achieved by constructing a core-shell structure and controlling the shell thickness and doping composition. Specifically, a cubic alkaline earth metal fluoride co-doped with rare earth elements and Group IVB transition metals was used as the degradable shell, which can shield core surface defects and surface quenching channels, thereby helping to maintain or enhance the upconversion luminescence intensity of the core. In an aqueous environment, the degradable shell will gradually hydrolyze, and the final degradation products and small core can be excreted through glomerular filtration. By adjusting the Zr content in the shell... 4+ The hydrolysis rate of the nanoprobe can be controlled by adjusting the doping amount and shell thickness. Through the synergistic adjustment of the above parameters, the hydrolysis time of the nanoprobe under preset test conditions can be controlled within the range of approximately 3-72 hours, thereby adapting to the super-resolution fluorescence microscopy imaging requirements of different time windows.

[0053] Different Zr 4+ The doped nanoprobes were placed in water and allowed to stand at 25°C. The upconversion emission spectra were measured at 0h, 4h, 8h, 12h, 24h, and 48h. As the degradation time increased, the emission brightness gradually decreased, and eventually, after 48h, the emission was almost invisible.

[0054] Example 4 To assess the super-resolution fluorescence microscopy performance of the nanoprobe, samples were tested using a super-resolution microscope. Under near-infrared light irradiation, the nanoprobe absorbs photons through the sensitizer, causing electrons to transition from the ground state to an excited state and transfer energy to activator ions, ultimately resulting in the particle emitting upconversion visible light.

[0055] The nanoparticles were diluted 1000 times and then spin-coated onto a glass slide, which was then placed on the microscope stage.

[0056] At high power density (700kW·cm) 2 Excitation by a 980 nm continuous wave (CW) laser, the fluorescent probe emits a dominant blue emission at 475 nm, while simultaneously generating several weaker emission bands at other Tm ion emission bands: 455 nm, 512 nm, and 650 nm. These emission bands belong to the Tm ion emission bands. 3+ of 1 D2→ 3 F4 (455nm) 1 G4→ 3 H6 (475nm) 1 D2→ 3 H5 (512nm) 1 G4→ 3 F4 (650nm) transition.

[0057] When irradiated by an 820nm continuous wave (CW) laser, the most intense blue emission light (475nm) mentioned earlier will be greatly quenched, with a depletion efficiency of up to 92%, thus enabling spatial resolution of less than 100 nanometers for super-resolution fluorescence microscopy.

[0058] The present invention and its embodiments have been described above. This description is not restrictive, and the actual structure is not limited thereto. In conclusion, if those skilled in the art, inspired by this description, design similar structures and embodiments without departing from the spirit of the invention, such designs should fall within the scope of protection of this invention.

Claims

1. A biocontrollably degradable upconversion fluorescent nanoprobe with a heterogeneous core-shell structure for super-resolution fluorescence microscopy, characterized in that: Including nanoprobes designed with a heterogeneous core-shell structure consisting of a hexagonal core layer and a cubic shell layer; The core layer is composed of hexagonal metal fluoride nanoparticles formed by rare earth element doping, and the average particle size of the core layer nanoparticles is less than 5.5 nm. The core metal fluoride has a hexagonal crystal phase, which is one of NaYF4, NaGdF4, NaYbF4, and NaLuF4. The core layer has an energy level structure formed by rare earth ion doping. The energy level structure is used to regulate the photoluminescence of the core layer through the stimulated emission depletion mechanism under the action of excitation light and depletion light, so as to realize fluorescence microscopy imaging with nanoscale spatial resolution. The core rare earth element doping also includes an activator and a sensitizer, with the activator being Tm. 3+ Er 3+ 、Nd 3+ Ho 3+ Pr 3+ One of them, with a doping concentration of 0.120 mol%; the sensitizer is Yb. 3+ or Nd 3+ Its doping concentration is 160 mol.

2. The upconversion fluorescent nanoprobe with a biocontrollable degradable heterogeneous core-shell structure for super-resolution fluorescence microscopy as described in claim 1, characterized in that: The shell is composed of cubic alkaline earth metal fluorides doped with rare earth elements and group IVB transition metal elements; the cubic alkaline earth metal fluorides of the shell are one or more combinations of CaF2, SrF2, and BaF2.

3. The upconversion fluorescent nanoprobe with a biocontrollable degradable heterogeneous core-shell structure for super-resolution fluorescence microscopy as described in claim 2, characterized in that: The shell coating thickness is 110 nm, and the overall particle size of the nanoprobe is 715 nm; the group IVB transition metal element is one or more of Ti, Zr, and Hf, wherein the molar ratio of the transition metal doping is 120 mol%; the rare earth element doped in the shell is Yb. 3+ Gd 3+ 、Tb 3+ 、Nd 3+ One or more of them.

4. The upconversion fluorescent nanoprobe for super-resolution fluorescence microscopy with a biocontrollable degradable heterogeneous core-shell structure according to claim 3, characterized in that: The hydrolysis rate of the shell is controlled by the shell thickness and the amount of group IVB transition metal doping; in an aqueous environment with pH 6.9, the degradation time of the core-shell structure is 4872 h.

5. A method for preparing the biodegradable heterostructured upconversion fluorescent nanoprobe for super-resolution fluorescence microscopy as described in any one of claims 14, characterized in that, Includes the following steps: (a): The rare earth compound is dispersed in a solvent: the rare earth compound is one or more of Ln(CF3COO)3, Ln(CH3COO)3, LnCl3, LnPO4, and Ln(NO3)3; the solvent is an anhydrous mixture of oleic acid, octadecene, and oleylamine; (b): Disperse the fluorine source and sodium source in the solvent: the fluorine source is one of NH4F, NH4HF2, NaF, KF, and the sodium source is one of Na2CO3, NaOH, NaHCO3, NaNO3, Na2SO4; the solvent is anhydrous methanol, anhydrous ethanol, or anhydrous cyclohexane. (c) The solution obtained in (a) is heated and kept at that temperature for a certain period of time to dissolve the rare earth elements, and then cooled to room temperature; (d) Then add the solution of (b) according to the stoichiometric ratio and keep it at a temperature above room temperature for a period of time to remove the low-boiling-point solvent, and then remove the residual low-boiling-point solvent by vacuuming. (e) The liquid obtained in (d) without low-boiling-point solvent is heated under nitrogen atmosphere and kept at high temperature for a period of time; finally, hexagonal rare earth doped metal fluoride core nanoparticles are obtained. (f) Preparation of shell precursor solution: Rare earth metal salts, alkaline earth metal salts, and Group IVB metal salts are dispersed in an anhydrous mixed solvent of oleic acid and octadecene; (g) The hexagonal rare earth-doped metal fluoride core nanoparticles obtained in (e) are dispersed in a solvent; the solvent is an anhydrous mixture of oleic acid and octadecene. (h) Coating with a biodegradable shell, the liquid in (e) is heated under a nitrogen atmosphere and injected in batches into it at high temperature. After each injection, the reaction is repeated for a period of time. After the reaction is completed, heterogeneous core-shell structured nanoparticles with shell thickness adjustable according to the number of injections are obtained.

6. The method for preparing the biodegradable heterogeneous core-shell structured upconversion fluorescent nanoprobe according to claim 5, characterized in that: (a) Ln is one or more of Y, Yb, Gd, Tm, Er, Ho, Eu, Tb, and Nd; The rare earth metal salts mentioned in (f) are one or more of Yb(CF3COO)3, Ln(CF3COO)3, Ln(CH3COO)3, LnCl3, LnPO4, and Ln(NO3)3; The alkaline earth metal salts are M(CF3COO)2, M(CH3COO)2, MCl2, and M(NO3)2, wherein M is one or more of Ca, Sr, Ba, and Mg; The group IVB metal salt is IVBX4, wherein IVB is one or more of Zr, Hf, and Ti, and X is CH3COO, Cl, NO3, alkoxy, or an organic ligand.

7. The method for preparing the biodegradable heterogeneous core-shell structured upconversion fluorescent nanoprobe according to claim 5, characterized in that: It also includes the use of surfactants to synthesize nanoparticles, wherein the surfactants include one or more of oleic acid, oleylamine, octadecene, acetylacetonate, and lauric acid.

8. The method for preparing the biodegradable heterogeneous core-shell structured upconversion fluorescent nanoprobe according to claim 5, characterized in that: In step (c), the reaction temperature is 140-160℃ and the holding time is 30-60 minutes; In step (e), the reaction temperature is 240-260℃ and the holding time is 20-45 minutes; The reaction temperature in step (h) is 290-300℃.

9. The method for preparing the biodegradable heterogeneous core-shell structured upconversion fluorescent nanoprobe according to claim 5, characterized in that: In step (a), the volume ratio of oleic acid to octadecene is 2:5; In step (d), the volume ratio of oleic acid to octadecene is (13):(25). In step (e), the volume ratio of oleic acid to octadecene is (13):(25).

10. The method for preparing the biodegradable heterogeneous core-shell structured upconversion fluorescent nanoprobe according to claim 5, characterized in that: The total amount of rare earth metal salts, alkaline earth metal salts, and Group IVB metal salts in (f) is in the ratio of the amount of the corresponding anhydrous mixed solvent to 1:(520).

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