Rare earth composite nanomaterial, preparation method and application thereof

Abstract

The application belongs to the technical field of organic-inorganic hybrid nanomaterials, and discloses a rare earth composite nanomaterial, a preparation method thereof and application of the nanomaterial in preparation of a peroxynitrite fluorescent nanoprober. The rare earth composite nanomaterial contains core-shell structure rare earth nanoparticles with a specific doping ratio, the surface of the nanoparticles is modified with sulfonate-containing cyanine dye for sensitizing the rare earth nanoparticles, and the nanoparticles are surface-modified by using a surfactant with amphiphilic properties to obtain a water-soluble excitation ratio type fluorescent nanoprober. The fluorescent nanoprober can be used for high-resolution imaging of blood vessels in vivo. The dye in the nanoprober produces a specific response with OONO ‑ ions, and the fluorescence emission intensity under 808nm laser excitation decreases, while the fluorescence emission intensity under 980nm laser excitation almost does not change. The ratio type change F 1550,980Ex / F 1550,808Ex of the two kinds of luminescence can be used to construct a ratio type nanofluorescent prober for detecting OONO ‑ ions in basic models such as inflammation.

Description

Technical Field

[0001] This invention belongs to the field of organic-inorganic hybrid nanomaterial preparation technology, specifically relating to a rare earth composite nanomaterial, its preparation method, and its application in the preparation of peroxynitrite fluorescent nanoprobes. Background Technology

[0002] peroxynitrite (ONOO) – nitric oxide (NO) is a product of the rapid combination of nitric oxide and superoxide anions. It is a strong oxidant produced in living organisms and can mediate the modification and damage of biological macromolecules, including DNA, proteins, and lipids. – Its concentration in the body is closely related to the pathogenesis of many diseases, such as autoimmune diseases and inflammatory diseases. Therefore, ONOO – Research in pathology and physiology is of great significance and can be used to diagnose ONOO. – Related diseases.

[0003] Compared to visible-area fluorescent probes, near-infrared optical probes have higher spatial resolution and penetration depth due to less light absorption and scattering, making them more suitable for deep intracellular imaging. – In order to eliminate interference from background fluorescence and other factors, a self-calibrated reference signal is introduced. For example, a ratiometric fluorescence imaging detection method can provide more accurate and reliable quantitative information. See the published literature Peng J, Samanta A, Zeng X et al. Real-Time In Vivo Hepatotoxicity Monitoring through Chromophore-Conjugated Photon-Upconverting Nanoprobes[J]. Angewandte Chemie International Edition, 2017, 56(15):4165-4169. It discloses a rare earth composite probe Cy7-PEI-UCNPs that can be used to detect the marker ONOO for liver toxicity in mice. – The probe is composed of rare-earth core-shell nanocrystals NaYF4:Yb,Tm@NaYF4, cyanine dye Cy7, and polyethyleneimine (PEI). Tm-UCNPs act as energy donors, emitting near-infrared fluorescence (NIIR) at 800 nm under 980 nm excitation. Simultaneously, due to the absorption of Cy7 (energy donor) also near 800 nm, the two undergo LRET interaction, leading to fluorescence quenching of the probe. In a mouse liver toxicity model, due to the overexpression of ONOO in the liver... – It can undergo a degradation reaction with Cy7, and the quenched fluorescence can gradually recover. The intensity of the fluorescence in vivo is similar to that of ONOO.– The concentration showed a positive correlation, therefore, the near-infrared fluorescence intensity of Tm ions at 800 nm can effectively qualitatively determine the liver toxicity in mice. Cy7 and Tm-doped nanocrystals were used to construct an LRET probe for ONOO. – It can sense fluorescence, but the probe can only achieve fluorescence emission in the near-infrared region.

[0004] Near-infrared II (NIR-II, 1000-1700 nm) imaging technology offers a new research direction for bioimaging due to its high penetration and low autofluorescence. Within the NIR-II region, NIR-IIb (1500-1700 nm) is a more ideal imaging area, with further reduced light scattering and negligible influence from tissue background fluorescence, resulting in higher resolution and signal-to-noise ratio imaging. Therefore, the development of NIR-IIb fluorescent probes is of great significance.

[0005] The aforementioned disclosed scheme employs a strategy of using the same excitation source but different emitted light for ratiometric detection. This ratiometric strategy struggles to ensure that all emitted light wavelengths fall within the near-infrared II region. Furthermore, the penetrating power of light of different wavelengths in biological tissue varies significantly within the infrared II region. Therefore, ratiometric probes based on different emission wavelengths will be affected by external factors such as optical path length and probe position, potentially leading to decreased detection accuracy. To further improve the accuracy of in vivo optical sensing, it is necessary to develop novel excitation ratiometric near-infrared II fluorescent probes for ONOO. – Detection and analysis.

[0006] Rare earth nanomaterials are among the few fluorescent probes capable of emitting wavelengths in the NIR-IIb region. Their excellent properties, such as low decomposition, convenient preparation methods, high luminescence efficiency, and low biotoxicity, have made them a current research hotspot for fluorescent nanoprobes. However, although rare earth nanomaterials possess extremely high quantum yields, their luminescence intensity is not outstanding at the same dosage. This is due to the fact that the ff transition of rare earth ions is a forbidden transition, resulting in a low absorption coefficient. This also leads to lower luminescence brightness of rare earth particles when used as fluorescent nanoprobes for bioimaging.

[0007] Dye sensitization is an effective strategy for improving the luminescence intensity of rare-earth fluorescent probes, as it can enhance the absorption of photons by materials, thereby improving their optical properties. Simultaneously, dyes with specific responsive properties, after binding with rare-earth particles, can specifically recognize relevant reactive oxygen species (ROS) in biological systems, enabling specific detection of specific ROS. However, existing commercially available dyes have low specificity, responding to a wide range of substances and exhibiting low reliability in recognizing different models. Therefore, improving the specific recognition of dye-sensitized rare-earth nanoparticles is a technical problem that needs to be solved.

[0008] There are limited methods for achieving high sensitization efficiency in aqueous solutions using dye-bound nanoprobes. The distance between the dye and rare earth ions significantly affects the sensitization effect; the dye molecules should be directly modified onto the surface of rare earth particles. Past methods often involved removing ligands before binding with the dye, selecting cyanine dye molecules containing sulfonic acid groups, and utilizing the strong interaction between sulfonate groups and rare earth elements to modify the surface of rare earth nanoparticles. However, this method cannot control the amount of dye modification; excessive dye modification, due to dye self-quenching, fails to achieve limited sensitization of rare earth elements and also affects the dispersibility of nanoparticles. Partial ligand exchange while retaining the original ligands is an effective way to solve this problem. However, due to the polarity of sulfonates, these commercially available cyanine dyes are usually insoluble in solvents such as dichloromethane and tetrahydrofuran used to disperse rare earth nanoparticles, which is detrimental to ligand exchange. Some studies have proposed using amphiphilic surfactants to simultaneously encapsulate nanomaterials and dyes; however, this can worsen the dye's sensitization effect and may also result in low encapsulation efficiency. Summary of the Invention

[0009] To overcome the shortcomings of the prior art, this invention provides a rare-earth composite nanomaterial. This rare-earth composite nanomaterial is a water-soluble composite nanomaterial based on rare-earth nanoparticles, prepared by modifying the surface of sulfonate-containing cyanine dye molecules on rare-earth nanoparticles with a specific double-layer core-shell structure, followed by modification with an amphiphilic surfactant. The dye is bound to the surface of the rare-earth nanoparticles through partial ligand exchange, maintaining the colloidal stability of the rare-earth material in organic solvents while ensuring sufficient dye modification. The dye-modified rare-earth nanoparticles are then coated with an amphiphilic surfactant, which maintains the dispersibility of the composite rare-earth nanomaterial in aqueous solution and improves its sensitization efficiency in aqueous solution. This material exhibits stronger fluorescence emission at 1550 nm under 808 nm laser excitation than unmodified dye nanoprobes, making it suitable for high-resolution imaging of blood vessels in vivo. Simultaneously, this material specifically responds to peroxynitrite ions (ONOO). - The changes in fluorescence emission at 1550nm generated by the probe under 808nm and 980nm laser excitation can be used for high-resolution imaging detection of diseases such as inflammation.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] In a first aspect, the present invention provides a rare earth composite nanomaterial comprising rare earth nanoparticles, cyanine dye molecules containing sulfonates and an amphiphilic surfactant, wherein the cyanine dye molecules containing sulfonates are modified on the surface of the rare earth nanoparticles and the amphiphilic surfactant is coated on the surface of the dye-modified rare earth nanoparticles.

[0012] The rare earth nanoparticles have a double core-shell structure, including a core and a shell. The shell is grown on the surface of the core by epitaxial growth. The core is composed of rare earth nanoparticles doped with Yb, Ce, and Er using NaYF4 as the matrix, and the diameter of the core ranges from 15 to 20 nm. The single shell is composed of Nd doped with NaYF4 as the matrix or doped with both Yb and Nd rare earth elements, and the thickness of the single shell is 2 to 4 nm.

[0013] In the core of the rare earth composite nanomaterial, NaYF4 is the matrix, the molar percentage of Ce doping in the total amount of rare earth elements in the core is no more than 25%, the molar percentage of Er doping in the total amount of rare earth elements in the core is no more than 5%, and the molar percentage of Yb doping in the total amount of rare earth elements in the core is 10% to 40%.

[0014] In the shell of the rare earth composite nanomaterial, NaYF4 is the matrix, the molar percentage of doped Nd in the total amount of rare earth elements in the shell is no more than 40%, and the molar percentage of doped Yb in the total amount of rare earth elements in the shell is 0% to 20%.

[0015] Preferably, as a preferred embodiment of this application, the molar percentage of Ce doped in the core is 10% of the total molar percentage of all rare earth elements in the core, the molar percentage of Er doped in the core is 2% of the total molar percentage of all rare earth elements in the core, and the molar percentage of Yb doped in the core is 20% of the total molar percentage of all rare earth elements in the core; the molar percentage of Nd doped in the shell is 30% of the total molar percentage of all rare earth elements in the shell, and the molar percentage of Yb doped in the shell is 10% of the total molar percentage of all rare earth elements in the shell.

[0016] Preferably, the sulfonate-containing cyanine dye molecule is selected from any one of IR783, IR783-TPE, IR806, or IR806-TPE; wherein IR783 and IR806 are both purchased from Merck Life Sciences; the tetraphenylethylene-modified cyanine dye IR783-TPE is prepared by modifying tetraphenylethylene with IR783 as a raw material, referring to the synthesis steps disclosed in the published patent [Fan Quli, Wang Xu, Wang Zhen, et al. A rare earth composite nanomaterial and its preparation method and application [P]. Jiangsu Province: CN202311098376.6, 2024-05-24.]. IR806-TPE is synthesized by replacing the raw material IR783 with IR806 using the same method.

[0017] The amphiphilic polymer is selected from any one of F127, PMA, or PMA-PEG; wherein, the amphiphilic polymer F127 is purchased from Merck Biotechnology; the synthesis steps of the amphiphilic polymer disclosed in the "Amphiphilic Polymer Synthesis" section of the "Supporting information" file of the published literature [Hessel CM, P. Pattani V, Rasch M, et al. Copper Selenide Nanocrystals for Photothermal Therapy[J]. Nano Letters, 2011, 11(6):2560-2566. DOI:10.1021 / nl201400z.] are as follows:

[0018]

[0019] Among them, PMA-PEG was synthesized with reference to the published patent [Fan Quli, Wang Xu, Wang Zhen, et al. A rare earth composite nanomaterial and its preparation method and application [P]. Jiangsu Province: CN202311098376.6, 2024-05-24.], and its structural formula is shown below.

[0020]

[0021] PMA-PEG structure

[0022] It should be noted that the value of m in the above PMA and PMA-PEG structural formulas ranges from 6 to 7.

[0023] To enhance the luminescence intensity of rare earth nanoparticles in the NIR-IIb region, and simultaneously enable specific ONOO in vivo... - For specific detection, the rare earth composite material provided by this invention, based on core-shell structured rare earth nanoparticles NaYF4:Yb,Ce,Er@NaYF4:Yb,Nd, is obtained by surface modification of the rare earth nanoparticles with sulfonate-containing cyanine dye molecules, followed by modification with amphiphilic surfactants. It can serve as a fluorescent nanoprobe in the NIR-IIb region. The dye on the surface of the rare earth nanoparticles can absorb excitation light at approximately 800 nm and transfer it to the Nd in the shell through a non-radiative energy transfer process. 3+ (If the shell contains Yb) 3+ The ions also transfer energy to Yb. 3+ (ions), and finally the energy is transferred to the Er in the core. 3+ Ions enhance its luminescence near 1550 nm. This is due to the Yb in rare earth nanoparticles. 3+Ion absorption occurs at 980 nm, therefore the prepared nanoprobe can be directly excited by 980 nm excitation light, producing emission light at around 1550 nm. Under 808 nm laser excitation, the luminescence of this rare earth composite nanomaterial at 1550 nm is much higher than that of rare earth nanoparticles that have not undergone dye sensitization.

[0024] Therefore, in a second aspect, the present invention also provides the application of the rare earth composite nanomaterials described in the first aspect in the preparation of peroxynitrite fluorescent nanoprobes. The rare earth composite nanomaterials, as peroxynitrite fluorescent nanoprobes, utilize sulfonate-containing cyanine dye molecules that can be oxidized by specific reactive oxygen species, altering their original structure and properties, thereby enabling the prepared fluorescent nanoprobes to exhibit a specific response to reactive oxygen species. This probe exhibits a long blood circulation time in vivo, allowing for high-resolution imaging of blood vessels. After injecting the fluorescent nanoprobes into mice with peritoneal inflammation via abdominal injection, the injection sites were irradiated with 808nm and 980nm lasers, respectively. Due to the interaction between the dyes modified on the surface of the fluorescent nanoprobes and the ONOO generated at the inflamed sites in the mice... - The ions produced a specific response, reducing the sensitization of the dye to the nano-fluorescent probe, leading to a continuous decrease in its luminescence intensity under 808 nm excitation, while the emission intensity under 980 nm excitation remained essentially unchanged. Therefore, the nano-fluorescent probe can be used to detect the emission intensity at F... 1550,980Ex / F 1550,808Ex The ratio-dependent changes are used to specifically identify inflammatory models and perform high-resolution imaging.

[0025] In summary, the rare earth composite nanomaterials provided by this invention, as nanofluorescent probes, can generate near-infrared IIb region emission of 1550nm under 808nm laser irradiation for high-resolution bioimaging. They can also be used to specifically identify inflammatory models by irradiation with different lasers of 808nm and 980nm and utilizing the ratio change of fluorescence intensity.

[0026] Dye sensitization is commonly used to enhance the optical intensity and properties of inorganic materials, but the surface bond energy between dyes and rare earth elements is weak, making them highly unstable. At high concentrations, they also exhibit a certain degree of self-quenching effect. To reduce the impact of water-modified dyes on the dispersibility of the nanomaterials described in this invention, and to improve the specificity of peroxynitrite detection, preferably, the sulfonate-containing cyanine dye molecule is IR783-TPE.

[0027] Core-shell structured rare earth nanoparticles are hydrophobic materials, requiring further surface modification by coating them with an amphiphilic surfactant. To minimize the impact of water-soluble modification on the dispersibility of the nanomaterials described in this invention, the amphiphilic surfactant is preferably PMA. PMA exhibits good dispersibility in aqueous solutions and enables the nanomaterials to maintain dye sensitization while also possessing good water solubility.

[0028] A preferred preparation method for the rare earth composite nanomaterials of the present invention includes the following steps:

[0029] The rare earth nanoparticles were prepared by doping Yb, Ce, and Er with NaYF4 as a matrix. The core of the rare earth nanoparticles was NaYF4:Yb,Ce,Er. Then, a single shell NaYF4:Yb,Nd was prepared on the surface of the core by epitaxial growth. The rare earth nanoparticles were then dispersed in chloroform to prepare a rare earth nanoparticle solution with a concentration of 20 mg / mL.

[0030] To prepare a 1 mg / mL IR783-TPE tetrahydrofuran solution, the 20 mg / mL rare earth nanoparticle solution was mixed with the IR783-TPE tetrahydrofuran solution and stirred at 50°C for 2 hours under a nitrogen atmosphere. Ethanol was then added for precipitation, and the mixture was centrifuged and dispersed in tetrahydrofuran to obtain a dye-modified rare earth nanoparticle solution of approximately 4 mg / mL. The volume ratio of the rare earth nanoparticle solution to the IR783-TPE tetrahydrofuran solution was 1:(0.04–0.34).

[0031] A 6 mg / mL PMA aqueous solution was prepared by adding NaOH solution to adjust the pH to approximately 12 to dissolve the PMA in the water. Under ultrasonic conditions, a 4 mg / mL dye-sensitized rare earth nanoparticle solution was rapidly injected into the PMA aqueous solution at a volume ratio of (0.6-1.8):9, and ultrasonication was maintained for 3 min. Tetrahydrofuran was removed from the aqueous solution with nitrogen gas, and the PMA-modified dye-sensitized rare earth nanoparticles were obtained after ultrafiltration purification.

[0032] The beneficial effects of this invention are:

[0033] The rare-earth composite nanomaterials provided by this invention utilize rationally designed rare-earth element doping to construct core-shell structured rare-earth nanoparticles, which are then combined with sulfonate-containing anthocyanin dyes to achieve high-intensity NIR-IIb luminescence through dye sensitization. Simultaneously, as a peroxynitrite fluorescent nanoprobe, it can detect OONO in vivo. - Ions are specifically detected to obtain clear in vivo vascular imaging images, and fluorescent probes are used to detect them at F... 1550,980Ex / F 1550,808ExThe ratio-type changes are used to perform imaging detection of diseases such as inflammation.

[0034] Furthermore, the peroxynitrite fluorescent nanoprobe constructed in this invention, based on the unique optical properties of rare-earth materials, can generate NIR-IIb luminescence of the same wavelength under different excitation wavelengths. This method of changing the excitation light eliminates the need to switch detectors during detection, making the detection more convenient. In addition, it can overcome the reliability problem caused by the difference in light penetration ability at different wavelengths in traditional emission ratio detection. Attached Figure Description

[0035] Figure 1 TEM image of the rare earth nanoparticles obtained in Example 1;

[0036] Figure 2 The fluorescent nanoprobes in Example 1 were tested at different concentrations of OONO. - Fitting of the ratio of luminescence intensity excited at 808nm and 980nm;

[0037] Figure 3 This is a fluorescence imaging image of the fluorescent nanoprobe in Example 1 after tail vein injection under a 1500nm long-pass filter;

[0038] Figure 4 This is a graph showing the fluorescence intensity ratios of different experimental groups after the fluorescent nanoprobes were injected in Example 1.

[0039] Figure 5 The ratio of fluorescence intensity of the fluorescent nanoprobe under excitation at 808 nm and 980 nm in different substances in Example 1;

[0040] Figure 6 The fluorescence intensity at 1550 nm is shown in Example 1 after the rare earth nanoparticles were modified with different concentrations of dye. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0042] The raw materials used in the following examples or comparative examples are shown below:

[0043] DSPE-PEG was purchased from Merck Life Sciences; PMA was obtained from the "Supporting information" file of the publicly available literature [Hessel CM, P. Pattani V, Rasch M, et al. Copper Selenide Nanocrystals for Photothermal Therapy[J]. NanoLetters, 2011, 11(6):2560-2566. DOI:10.1021 / nl201400z.] "Amphiphilic Polymer The amphiphilic polymer was synthesized according to the steps disclosed in the "Synthesis" section; PMA-PEG was synthesized according to the preparation process in the

[0037] section of the published patent [Fan Quli, Wang Xu, Wang Zhen, et al. A rare earth composite nanomaterial and its preparation method and application [P]. Jiangsu Province: CN202311098376.6, 2024-05-24.]; the tetraphenylethylene-modified cyanine dye IR783-TPE was prepared by modifying tetraphenylethylene with IR783 as raw material, referring to the synthesis steps disclosed in the

[0035] section of the published patent [Fan Quli, Wang Xu, Wang Zhen, et al. A rare earth composite nanomaterial and its preparation method and application [P]. Jiangsu Province: CN202311098376.6, 2024-05-24.]; the sulfonate-containing cyanine dye molecule IR808 is described in the published literature [Wu X, Lee H, Bilsel O, et al. Tailoring Dye-sensitized Upconversion Nanoparticles Excitation]. Bands towards Excitation WavelengthSelective Imaging[J].Nanoscale,2015,7(44).DOI:10.1039 / c5nr05437k.】Synthesis.

[0044] Preparation of rare earth nanoparticles

[0045] The chemical composition of the rare earth nanoparticles with a double core-shell structure used in the following examples is: NaYF4: 20% Yb, 10% Ce, 2% Er@NaYF 4: 30% Nd 10% Yb:

[0046] The specific preparation steps include: 0.68 mmol Y(CH3COO)3, 0.2 mmol Yb(CH3COO)3, 0.1 mmol Ce(CH3COO)3, and 0.02 mmol Er(CH3COO)3 were heated to 150 °C under nitrogen protection and dissolved in 7 mL oleic acid and 15 mL octadecene. After complete dissolution, the solution was cooled to room temperature. A 10 mL methanol solution containing 4.0 mmol NH4F and 2.5 mmol sodium oleate was added, and the mixture was stirred at 50 °C for 30 min. After evaporating the methanol by heating, the temperature was raised to 290 °C under nitrogen protection and maintained for 1.5 h. After cooling, ethanol was added to precipitate the nanoparticles. After centrifugation and washing twice with ethanol, the precipitate was dispersed in 7 mL chloroform to obtain NaYF4:20%Yb10%Ce2%Er rare earth nanoparticles. As above, 0.3 mmol Y(CH3COO)3, 0.05 mmol Yb(CH3COO)3, and 0.15 mmol Nd(CH3COO)3 were dissolved in 7 mL of oleic acid and 15 mL of octadecene. 3.5 mL of the nanocore prepared above and 10 mL of methanol solution containing 2.0 mmol NH4F and 1.25 mmol sodium oleate were added. The mixture was stirred at 50°C for 30 min, then heated to evaporate the low-boiling-point solvent. Under nitrogen protection, the temperature was raised to 280°C and maintained for 1.5 h. After ethanol precipitation and centrifugation washing, the nanoparticles were dispersed in 7 mL of chloroform to obtain core-shell structured rare earth nanoparticles NaYF4:20%Yb,10%Ce,2%Er@NaYF4:30%Nd10%Yb; the concentration was approximately 20 mg / mL. All rare earth acetates used in the synthesis were purchased from Alfa Aesar, and other reagents were purchased from Sigma-Aldrich.

[0047] Example 1

[0048] A 1 mg / mL IR783-TPE tetrahydrofuran solution was prepared. The 20 mg / mL rare earth nanoparticle solution was mixed with the 30 μg / mL IR783-TPE tetrahydrofuran solution, and stirred at 50°C for 2 hours under a nitrogen atmosphere. Ethanol was added for precipitation, and after centrifugation, the mixture was dispersed in tetrahydrofuran to obtain a dye-modified rare earth nanoparticle solution of approximately 4 mg / mL. The volume ratio of the rare earth nanoparticle solution to the IR783-TPE tetrahydrofuran solution was 1:0.168.

[0049] A 6 mg / mL PMA aqueous solution was prepared by adding NaOH solution to adjust the pH to approximately 12 to dissolve the PMA in the water. Under ultrasonic conditions, a 4 mg / mL dye-sensitized rare earth nanoparticle solution was rapidly injected into the PMA aqueous solution at a volume ratio of (0.6-1.8):9, and ultrasonication was maintained for 3 min. Tetrahydrofuran was removed from the aqueous solution with nitrogen gas, and the PMA-modified dye-sensitized rare earth nanoparticles were obtained after ultrafiltration purification.

[0050] Experimental results show that: Figure 1 As shown, the particle size of the obtained rare earth composite nanomaterial is approximately 20 nm; the rare earth composite nanomaterial is used in OONO - Different ion concentrations result in different absorption spectra, thus allowing the micron fluorescent probe to detect OONO. - Ions have specific responses; such as Figure 2 As shown, with OONO - With increasing concentration, the luminescence intensity under 808nm excitation continuously decreases, while the luminescence intensity under 980nm excitation remains essentially unchanged. This can also be seen from the ratio graph. 808 nm / F 980nm The luminescence intensity is continuously decreasing. Meanwhile, for F... 980nm / F 808nm The intensity values ​​were fitted, and the fitting coefficient R² = 0.996, proving that the ratio of the downconversion luminescence intensity of the nanoprobe under excitation at 808 nm and 980 nm is related to the OONO intensity. - There is a linear relationship between the concentrations of ions; for example... Figure 3 As shown, the rare-earth nanocomposite material exhibits high imaging signal-to-noise ratio and imaging quality in the NIR-IIb region beyond 1500 nm; for example... Figure 4 As shown, mice injected with lipopolysaccharide (LPS) developed abdominal inflammation, F 980nm / F 808 nm The fluorescence intensity ratio increased approximately linearly. In mice injected with phosphate-buffered saline (PBS), and in mice simultaneously injected with LPS and glutathione (GSH), GSH depleted peroxynitrite produced in the mice due to inflammation. 980nm / F 808 nm The fluorescence intensity ratio remained essentially unchanged, indicating that the nanofluorescent probe can respond ratiometrically to peroxynitrite and achieve high-resolution imaging; the nanoprobe was further tested for its response to different ROS, including ClO₂, by adding different substances. - H2O2, ·OH 1 O2 and other interfering substances such as glutathione, dithiothreitol, camptothecin, vitamin C, and Fe3+ Ag + Response capability, such as Figure 5 As shown, the nano-fluorescent probe can detect OONO - It produces a specific response and is almost unresponsive to other ROS or interfering substances. For example... Figure 6 As shown, the concentration of the modifying dye was changed, and the fluorescence intensity at 1550 nm was measured after the rare earth nanoparticles were modified with different concentrations of dye. The optimal dye modification concentration was 30 μg / mL.

[0051] Example 2

[0052] The only difference between Example 2 and Example 1 is that NaYF4:Yb,Er,Ce@NaYF4:Nd of equal concentration is used instead of NaYF4:Yb,Er,Ce@NaYF4:Nd,Yb in Example 1. The Nd doping ratio is 30%, and the other conditions remain unchanged to prepare rare earth composite nanomaterials as nanoprobes.

[0053] Experimental results show that, after dye sensitization, the outer layer of the nanoparticles does not contain Yb. 3+ After the structure was excited by an 808nm laser, the fluorescence intensity and the rate of increase after sensitization were both lower than those in Example 1, but it was still able to meet the basic requirements of in vivo optical imaging.

[0054] Example 3

[0055] The only difference between Example 3 and Example 1 is that PMA-PEG of the same concentration is used instead of PMA in Example 1, while the other conditions remain unchanged, to prepare rare earth composite nanomaterials as nanoprobes.

[0056] Experimental results show that the material has similar dispersibility in aqueous solution to PMA, with a slight increase in fluorescence intensity, but at a higher cost.

[0057] Comparative Example 1

[0058] The only difference between Comparative Example 1 and Example 1 is that NaYF4:Yb,Er,Ce@NaYF4:Yb of equal concentration is used instead of NaYF4:Yb,Er,Ce@NaYF4:Nd,Yb in Example 1, wherein the doping ratio of Yb is 10%, and the other conditions remain unchanged, to prepare rare earth composite nanomaterials as nanoprobes.

[0059] Experimental results show that undoped Nd 3+ Although the structure of the ion can increase the luminescence intensity several times after dye sensitization, the lack of Nd... 3+Ion doping results in a lower luminescence intensity at 1530 nm under 808 nm excitation, leading to a lower luminescence intensity after dye sensitization, which is insufficient for in vivo optical imaging.

[0060] Comparative Example 2

[0061] The only difference between Comparative Example 2 and Example 1 is that an equal amount of IR808 tetrahydrofuran solution was used instead of the IR783-TPE tetrahydrofuran solution in Example 1, while the other conditions remained unchanged, to prepare rare earth composite nanomaterials as nanoprobes.

[0062] Experimental results show that this dye has poor solubility in the ligand exchange reaction system, and its sensitization effect in aqueous solution is low after modification. In contrast, IR783-TPE, modified with tetraphenylethylene groups, exhibits better dispersibility in nonpolar solutions such as dichloromethane and tetrahydrofuran, and achieves better ligand exchange, which commercially available dyes cannot. Meanwhile, IR808 also shows improved sensitization of ClO₂. - It also has a certain degree of responsiveness, making it suitable for OONO - The detection lacks specificity.

[0063] Comparative Example 3

[0064] The only difference between Comparative Example 3 and Example 1 is that an equal concentration of DSPE-PEG was used instead of PMA in Example 1, while all other conditions remained the same, to prepare rare earth composite nanomaterials as nanoprobes.

[0065] Experimental results show that the fluorescence intensity of the fluorescent probe decreased significantly after replacing PMA with DSPE-PEG to coat rare earth nanoparticles.

[0066] In summary, this invention designs a composite rare-earth nanoparticle for preparing water-soluble fluorescent nanoprobes for ONOO. - It has specific response capabilities and can perform excitation ratio imaging detection on inflamed sites.

Claims

1. The application of rare earth composite nanomaterials in the preparation of peroxynitrite fluorescent nanoprobes, characterized in that, The rare earth composite nanomaterial includes rare earth nanoparticles, cyanine dye molecules containing sulfonates, and amphiphilic surfactants. The cyanine dye molecules containing sulfonates are modified on the surface of the rare earth nanoparticles, and the amphiphilic surfactants are coated on the surface of the dye-modified rare earth nanoparticles. The rare earth nanoparticles have a double core-shell structure, including a core and a shell. The shell is grown on the surface of the core by epitaxial growth. The core is composed of rare earth nanoparticles doped with Yb, Ce and Er using NaYF4 as the matrix, and the diameter of the core ranges from 15 to 20 nm. The shell is composed of rare earth elements doped with Yb and Nd using NaYF4 as the matrix, and the thickness of the shell is 2 to 4 nm. In the core of the rare earth composite nanomaterial, NaYF4 is the matrix, the molar percentage of Ce doping in the total amount of rare earth elements in the core is no more than 25%, the molar percentage of Er doping in the total amount of rare earth elements in the core is no more than 5%, and the molar percentage of Yb doping in the total amount of rare earth elements in the core is 10% to 40%. In the shell of the rare earth composite nanomaterial, NaYF4 is the matrix, the molar percentage of doped Nd in the total amount of rare earth elements in the shell is no more than 40%, and the molar percentage of doped Yb in the total amount of rare earth elements in the shell is 0%~20%. The sulfonate-containing cyanine dye molecule is IR783-TPE; The amphiphilic surfactant is PMA or PMA-PEG.

2. The application according to claim 1, characterized in that, The core contains 10% Ce, 2% Er, and 20% Yb. The shell contains 30% Nd and 10% Yb.

3. The application according to claim 1, characterized in that, The preparation method of the rare earth composite nanomaterial includes the following steps: using NaYF4 as a matrix to dope Yb, Ce, and Er to prepare the core NaYF4:Yb, Ce, Er of the rare earth nanoparticles; then preparing the shell NaYF4:Yb, Nd on the surface of the core by epitaxial growth to obtain the rare earth nanoparticles; and dispersing them in chloroform to prepare a rare earth nanoparticle solution with a concentration of 20 mg / mL. A 1 mg / mL IR783-TPE tetrahydrofuran solution was prepared. The 20 mg / mL rare earth nanoparticle solution was mixed with the IR783-TPE tetrahydrofuran solution and stirred at 50°C for 2 hours under a nitrogen atmosphere. Ethanol was added for precipitation, and after centrifugation, the mixture was dispersed in tetrahydrofuran to obtain a 4 mg / mL dye-modified rare earth nanoparticle solution. The volume ratio of the rare earth nanoparticle solution to the IR783-TPE tetrahydrofuran solution was 1:(0.04~0.34). A 6 mg / mL PMA aqueous solution was prepared by adding NaOH solution to adjust the pH to 12 to dissolve the PMA in the water. Under ultrasonic conditions, a 4 mg / mL solution of dye-modified rare earth nanoparticles was rapidly injected into the PMA aqueous solution at a volume ratio of (0.6-1.8):

9. The mixture was sonicated for 3 min. Tetrahydrofuran was removed from the aqueous solution by nitrogen purging, and the rare earth composite nanomaterial was obtained after ultrafiltration purification.

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