Rare earth composite nano material and preparation method and application thereof

Abstract

The invention belongs to the technical field of preparation of organic-inorganic hybrid nano materials, and discloses a rare earth composite nano material, a preparation method of the rare earth composite nano material and application of the rare earth composite nano material in preparation of peroxynitrite fluorescent nano probes. The surface of the fluorescent probe is modified with a cyanine dye containing sulfonate for sensitizing rare earth nano-particles, and the rare earth nano-particles are wrapped with an amphiphilic surfactant for surface modification, so that the water-soluble excitation ratio type fluorescent nano-probe is obtained. The fluorescent nanoprobe can be applied to high-resolution imaging of in-vivo blood vessels; meanwhile, the dye in the nanoprobe can generate specific response with OONO <-> ions, the fluorescence emission intensity under the excitation of 808nm laser is reduced, but the fluorescence emission intensity under the excitation of 980nm laser is almost unchanged, and the ratio-type nano fluorescent probe can be constructed by using the two luminous ratio-type changes F1550 and 980Ex / F1550 and 808Ex, and is applied to the detection of OONO <-> ions in basic models such as inflammation and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of preparation of organic-inorganic hybrid nanomaterials, and specifically relates to a rare earth composite nanomaterial and a preparation method thereof, and application of the composite nanomaterial in preparing peroxynitrite fluorescent nanoprobes. Background Art

[0002] Peroxynitrite (ONOO – ) is the product of the rapid combination of nitric oxide and superoxide anion. It is a strong oxidant produced in organisms, which can mediate the modification and damage of biological macromolecules including DNA, proteins and lipids. – The concentration in the body is closely related to the pathogenic process of many diseases in the human body, such as autoimmune diseases and inflammatory diseases. – It is of great significance in pathological and physiological research and can be used to diagnose ONOO – related diseases.

[0003] Compared with visible region fluorescent probes, near-infrared optical probes have higher spatial resolution and penetration depth due to less influence of light absorption and scattering, and are more suitable for deep in vivo ONOO – Detection and analysis, and in order to eliminate the interference of background fluorescence, etc., the introduction of a self-calibrated reference signal, such as the construction of a ratio fluorescence imaging detection method, can provide more accurate and reliable quantitative information. See the public 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. A rare earth composite probe Cy7-PEI-UCNPs is disclosed for detecting ONOO, a marker of liver poisoning in mice. – The probe is composed of rare earth core-shell nanocrystals NaYF4:Yb, Tm@NaYF4, cyanine dye Cy7 and polyethyleneimine PEI. As an energy donor, Tm-UCNPs can emit 800nm ​​near-infrared fluorescence under the action of 980nm excitation light. At the same time, since the absorption of Cy7 (energy donor) is also near 800nm, the LRET effect between the two causes the fluorescence quenching of the probe. In the mouse liver poisoning model, due to the overexpression of ONOO in the liver, – It can degrade with Cy7, and the quenched fluorescence can gradually recover. The intensity of fluorescence in vivo is similar to that of ONOO– The concentration of Tm ions is positively correlated, so the near-infrared fluorescence intensity of Tm ions at 800 nm can effectively and qualitatively determine the liver poisoning of mice. – However, the probe can only achieve fluorescence emission in the near-infrared region.

[0004] Imaging technology in the near-infrared region II (NIR-II, 1000-1700nm) provides a new research direction for biological imaging due to its high penetration and low autofluorescence. In the NIR-II region, NIR-IIb (1500-1700nm) is an ideal imaging region, where light scattering is further reduced and the influence of the tissue's own background fluorescence can be ignored, thereby obtaining imaging with higher resolution and signal-to-noise ratio. Therefore, the development of NIR-IIb fluorescent probes is of great significance.

[0005] The above-mentioned public scheme uses the same excitation light source and different emission light strategy for ratiometric detection. This ratiometric strategy makes it difficult to achieve emission light bands in the near-infrared region II. At the same time, the penetration ability of light of different wavelengths in the infrared region II in biological tissues varies greatly. Therefore, ratiometric probes based on different emission wavelengths will be affected by external factors such as optical path and probe position, and the detection accuracy may deteriorate. In order to further improve the accuracy of in vivo optical sensing, it is necessary to develop a new excitation ratio near-infrared region II fluorescent probe for ONOO – detection and analysis.

[0006] Rare earth nanomaterials are one of the few fluorescent probes that can reach the NIR-IIb region in terms of emission wavelength. They are also a hot topic in the current research of fluorescent nanoprobes because of their excellent properties such as being difficult to decompose, convenient preparation methods, high luminescence efficiency, and low biological toxicity. However, although rare earth nanomaterials have extremely high quantum yields, their luminescence intensity is not outstanding when used in the same dosage. This is because the ff transition of rare earth ions is a forbidden transition, and its absorption coefficient is low. This also makes the luminescence brightness of rare earth particles low when used as fluorescent nanoprobes for biological imaging.

[0007] Dye sensitization is an effective strategy to improve the luminescence intensity of rare earth fluorescent probes. It can be used to enhance the absorption of photons by materials, giving them better optical properties. At the same time, dyes with specific response characteristics can specifically identify relevant ROS in the body after combining with rare earth particles, and specifically detect specific ROS. However, existing commercial dyes have low specificity, can respond to a variety of substances, and have low recognition reliability for different models. Therefore, how to improve the specific recognition of rare earth nanoparticles after dye sensitization is also a technical problem that needs to be solved.

[0008] There are limited methods for nanoprobes combined with dyes to achieve high sensitization efficiency in aqueous solution. The distance between the dye and the rare earth ion has a great influence on the sensitization effect, and the dye molecule should be directly modified on the surface of the rare earth particles. In the past, most methods were to remove the ligand first and then combine with the dye, select cyanine dye molecules containing sulfonic acid groups, and use the strong interaction between sulfonic acid groups and rare earths to modify the dye molecules on the surface of rare earth nanoparticles. However, this method cannot control the modification amount of the dye. Excessive dye modification cannot achieve limited sensitization of rare earths due to the self-quenching of the dye, and it will also affect the dispersibility of the nanoparticles. Partial ligand exchange on the basis of retaining the original ligand is an effective means to solve this problem. However, due to the polarity of sulfonates, these commercial cyanine dyes are usually insoluble in solvents such as dichloromethane and tetrahydrofuran used to disperse rare earth nanoparticles, which is not conducive to ligand exchange. There are also studies that propose the use of amphiphilic surfactants to simultaneously encapsulate nanomaterials and dyes, but this will make the sensitization effect of the dye worse, and may also cause problems with low encapsulation efficiency. Summary of the invention

[0009] In order to overcome the deficiencies of the above-mentioned prior art, the present invention provides a rare earth composite nanomaterial, which is a water-soluble composite nanomaterial based on rare earth nanoparticles obtained by modifying the surface of rare earth nanoparticles with a double-layer core-shell structure of a specific component and containing cyanine dye molecules with sulfonate, and then using an amphiphilic surfactant to modify the surface of the rare earth nanoparticles; the dye is bound to the surface of the rare earth nanoparticles by partial ligand exchange, and the colloidal stability of the rare earth material in an organic solvent is maintained under the premise of ensuring that the dye is fully modified. The dye-modified rare earth nanoparticles are then coated with an amphiphilic surfactant, which can not only maintain the dispersibility of the composite rare earth nanomaterial in an aqueous solution, but also improve its sensitization efficiency in an aqueous solution; the material can produce stronger fluorescence emission at 1550nm than the nanoprobe without modified dye under 808nm laser excitation, and is used for high-resolution imaging of blood vessels in the body; at the same time, the material can specifically respond to peroxynitrite ions (ONOO - ), through the changes in the fluorescence emission at 1550nm produced by the probe under the excitation of 808nm laser and 980nm laser, it is used for high-resolution imaging detection of diseases such as inflammation.

[0010] In order to achieve the above object, 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 amphiphilic surfactants, wherein 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;

[0012] The rare earth nanoparticles are of a double-layer core-shell structure, including a core and a shell, wherein the shell is grown on the surface of the core by an epitaxial growth method; the core is composed of rare earth nanoparticles with Yb, Ce, and Er doped with NaYF4 as a matrix, and the diameter of the core is in the range of 15-20nm; the single shell is composed of NaYF4 as a matrix doped with Nd or doped with two rare earth elements, Yb and Nd, and the thickness of the single shell is 2-4nm.

[0013] In the core of the rare earth composite nanomaterial, NaYF4 is the matrix, the molar percentage of doped Ce in the total amount of all rare earth elements in the core is not higher than 25%, the molar percentage of doped Er in the total amount of all rare earth elements in the core is not higher than 5%, and the molar percentage of doped Yb in the total amount of all 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 all rare earth elements in the shell is not higher than 40%, and the molar percentage of doped Yb in the total amount of all rare earth elements in the shell is 0% to 20%.

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

[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 purchased from Merck Life Sciences; the tetraphenylethylene-modified cyanine dye IR783-TPE is prepared by modifying tetraphenylethylene with IR783 as a raw material, with reference to the synthesis steps disclosed in the 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 by 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 Life Sciences; PMA refers to the synthesis steps of the amphiphilic polymer disclosed in the "Amphiphilic Polymer Synthesis" section of the "Supporting information" file in the open document [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.], and its structural formula is 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 as follows

[0020]

[0021] PMA-PEG structure

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

[0023] In order to improve the luminescence intensity of rare earth nanoparticles in the NIR-IIb region, and to be able to react with specific ONOO in vivo - For specific detection, the rare earth composite material provided by the present invention is based on the rare earth nanoparticles NaYF4:Yb,Ce,Er@NaYF4:Yb,Nd with a core-shell structure, and the rare earth nanoparticles are surface-modified with cyanine dye molecules containing sulfonates, and then modified with amphiphilic surfactants to obtain a fluorescent nanoprobe in the NIR-IIb region. The dye on the surface of the rare earth nanoparticles can absorb excitation light of about 800nm ​​and transfer it to the Nd shell through a non-radiative energy transfer process. 3+ (If the shell contains Yb 3+ The energy of the ions is also transferred to Yb 3+ ions), and the energy is ultimately transferred to the Er in the core 3+ ions, enhancing its luminescence around 1550nm. 3+The absorption of ions is at 980nm, so the prepared nanoprobe can be directly excited by the 980nm excitation light to produce emission light around 1550nm. Under 808nm laser excitation, the luminescence of the rare earth composite nanomaterial at 1550nm is much higher than that of rare earth nanoparticles that have not been sensitized by dyes.

[0024] Therefore, in the second aspect, the present invention also provides the use of the rare earth composite nanomaterial described in the first aspect in the preparation of peroxynitrite fluorescent nanoprobes. The rare earth composite nanomaterial is used as a peroxynitrite fluorescent nanoprobe, and the sulfonate-containing cyanine dye molecules used can be oxidized by specific reactive oxygen species, changing the original structure and properties, so that the prepared nano fluorescent probe can produce a specific response to reactive oxygen species. The probe has a long blood circulation time in the body and can be used for high-resolution imaging of blood vessels. After the nano fluorescent probe is injected into the body of a mouse with abdominal inflammation through the abdomen, the mouse abdominal injection site is irradiated with 808nm and 980nm lasers respectively. Due to the surface modification of the nano fluorescent probe and the ONOO produced in the inflammatory site of the mouse, the nano fluorescent probe is irradiated with the ONOO produced in the inflammatory site of the mouse. - The ions produced a specific response, and the sensitization of the dye to the nanofluorescent probe decreased, resulting in a continuous decrease in the luminescence intensity under 808nm excitation, while the emission light intensity under 980nm excitation remained basically unchanged. 1550,980Ex / F 1550,808Ex Ratio-type changes can be used to specifically identify inflammatory models and perform high-resolution imaging.

[0025] In summary, the rare earth composite nanomaterial provided by the present invention can be used as a nano fluorescent probe to generate 1550nm near-infrared IIb region luminescence under 808nm laser irradiation for high-resolution biological imaging, and can also be irradiated by different lasers of 808 and 980nm to specifically identify the inflammation model by utilizing the ratio change of fluorescence intensity.

[0026] Dye sensitization is often used to enhance the optical strength and optical properties of inorganic materials, but the surface bond energy between the dye and the rare earth element is weak, and it is very unstable. It also shows a certain degree of self-quenching effect at high concentrations. In order to reduce the influence of the nanomaterial water-modified dye on the dispersibility of the nanomaterial and improve the specificity of peroxynitrite detection, preferably, the sulfonate-containing cyanine dye molecule is IR783-TPE.

[0027] The rare earth nanoparticles of core-shell structure are hydrophobic materials, and the rare earth nanoparticles need to be surface modified, and the outer layer is coated with an amphiphilic surfactant. In order to reduce the influence of the water solubility modification of the nanomaterials of the present invention on the dispersibility of the nanomaterials, preferably, the amphiphilic surfactant is PMA. PMA has good dispersibility in aqueous solution, and can make the nanomaterials have good water solubility while maintaining the dye sensitization effect in aqueous solution.

[0028] As a preferred preparation method of the rare earth composite nanomaterial of the present invention, the method comprises the following steps:

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

[0030] Prepare a 1 mg / mL IR783-TPE tetrahydrofuran solution, mix the 20 mg / mL rare earth nanoparticle solution with the IR783-TPE tetrahydrofuran solution, stir for 2 hours at 50°C under a nitrogen atmosphere, add ethanol for precipitation, centrifuge and disperse in tetrahydrofuran to obtain a dye-modified rare earth nanoparticle solution of about 4 mg / mL; wherein the raw material configuration volume ratio of the rare earth nanoparticle solution and the IR783-TPE tetrahydrofuran solution is 1:(0.04-0.34);

[0031] A 6 mg / mL PMA aqueous solution is prepared, wherein a NaOH solution is added to adjust the pH value to about 12 so that the PMA is dissolved in water; under ultrasonic conditions, the 4 mg / mL dye-sensitized rare earth nanoparticle solution is quickly injected into the PMA aqueous solution, the volume ratio of the rare earth nanoparticle solution to the PMA aqueous solution is (0.6-1.8):9, and the ultrasonic treatment is maintained for 3 minutes; tetrahydrofuran in the aqueous solution is blown away with nitrogen, and PMA-modified dye-sensitized rare earth nanoparticles are obtained after purification by ultrafiltration.

[0032] The beneficial effects of the present invention are:

[0033] The rare earth composite nanomaterial provided by the present invention is a rare earth nanoparticle with a core-shell structure constructed by rationally designing rare earth element doping, and is combined with a cyanine dye containing a sulfonate to achieve high-intensity NIR-IIb luminescence through dye sensitization. At the same time, it can be used as a peroxynitrite fluorescent nanoprobe to detect OONO in vivo. - ions for specific detection, obtaining clear in vivo vascular imaging images, and using fluorescent probes in F 1550,980Ex / F 1550,808ExRatio-type changes are used to image and detect diseases such as inflammation.

[0034] The peroxynitrite fluorescent nanoprobe constructed by the present invention is based on the unique optical properties of rare earth materials and can produce NIR-IIb luminescence of the same wavelength under different wavelengths of excitation light. This method of changing the excitation light does not require switching detectors during detection, making detection more convenient. In addition, it can also overcome the problem of detection reliability caused by the difference in penetration ability of light of different wavelengths in traditional emission ratio detection. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0036] Figure 2 The fluorescent nanoprobe in Example 1 at different concentrations of OONO - The fitting of the luminescence intensity ratio of 808nm and 980nm excitation;

[0037] Figure 3 This is a fluorescence imaging picture 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 ratio of different experimental groups after the fluorescent nanoprobes were injected in Example 1;

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

[0040] Figure 6 The fluorescence intensity change at 1550 nm after the rare earth nanoparticles in Example 1 were modified with dyes of different concentrations. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is described in detail below with reference to the accompanying drawings and specific implementation methods.

[0042] The raw materials used in the following examples or comparative examples are as follows:

[0043] DSPE-PEG was purchased from Merck; PMA was obtained from the “Amphiphilic Polymer” in the “Supporting information” file of the open 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.]. Synthesis"; PMA-PEG is synthesized according to the preparation process of paragraph

[0037] in the public 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 is prepared by modifying tetraphenylethylene with IR783 as a raw material, referring to the synthesis steps disclosed in the public 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 refers to the public document [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 double-layer core-shell rare earth nanoparticles used in the following examples is: NaYF4: 20% Yb, 10% Ce, 2% Er@NaYF 4: 30%Nd10%Yb:

[0046] The preparation method specifically includes the following steps: 0.68mmol Y(CH3COO)3, 0.2mmol Yb(CH3COO)3, 0.1mmol Ce(CH3COO)3 and 0.02mmol Er(CH3COO)3 are heated to 150°C under nitrogen protection and dissolved in 7mL oleic acid and 15mL octadecene, and cooled to room temperature after being completely dissolved. 10mL methanol solution containing 4.0mmol NH4F and 2.5mmol sodium oleate is added thereto, and stirred at 50 degrees for 30min. After heating and evaporating the methanol, the temperature is raised to 290°C under nitrogen protection and maintained for 1.5h, cooled and ethanol is added for precipitation, centrifuged and washed twice with ethanol, and then dispersed in 7mL chloroform to obtain NaYF4:20%Yb10%Ce2%Er rare earth nanoparticles. As above, 0.3mmol Y(CH3COO)3, 0.05mmol Yb(CH3COO)3 and 0.15mmol Nd(CH3COO)3 were dissolved in 7mL oleic acid and 15mL octadecene, 3.5mL of the nanocore prepared above and 10mL methanol solution containing 2.0mmol NH4F and 1.25mmol sodium oleate were added, and the low boiling point solvent was evaporated after stirring at 50 degrees for 30min. The temperature was raised to 280℃ under nitrogen protection and maintained for 1.5h. After ethanol sedimentation and centrifugal washing, it was dispersed in 7mL chloroform to obtain core-shell structured rare earth nanoparticles NaYF4: 20% Yb, 10% Ce, 2% Er@NaYF4: 30% Nd10% Yb; the concentration was about 20mg / mL. During the synthesis process, rare earth acetates were purchased from Alfa Aesar, and other reagents were purchased from Sigma-Aldrich.

[0047] Embodiment 1

[0048] A 1 mg / mL IR783-TPE tetrahydrofuran solution was prepared, and the 20 mg / mL rare earth nanoparticle solution and the 30 μg / mL IR783-TPE tetrahydrofuran solution were mixed and stirred at 50° C. for 2 hours under a nitrogen atmosphere, and then ethanol was added for precipitation. After centrifugation, the mixture was dispersed in tetrahydrofuran to obtain a dye-modified rare earth nanoparticle solution of about 4 mg / mL; wherein the raw material configuration volume ratio of the rare earth nanoparticle solution and the IR783-TPE tetrahydrofuran solution was 1:0.168;

[0049] A 6 mg / mL PMA aqueous solution is prepared, wherein a NaOH solution is added to adjust the pH value to about 12 so that the PMA is dissolved in water; under ultrasonic conditions, the 4 mg / mL dye-sensitized rare earth nanoparticle solution is quickly injected into the PMA aqueous solution, the volume ratio of the rare earth nanoparticle solution to the PMA aqueous solution is (0.6-1.8):9, and the ultrasonic treatment is maintained for 3 minutes; tetrahydrofuran in the aqueous solution is blown away with nitrogen, and PMA-modified dye-sensitized rare earth nanoparticles are obtained after purification by ultrafiltration.

[0050] The experimental results show that: Figure 1 As shown, the particle size of the obtained rare earth composite nanomaterial is about 20nm; the rare earth composite nanomaterial is in OONO - Different ion concentrations have different absorption spectra, making the fluorescent probe sensitive to OONO - Ions have specific responses; e.g. Figure 2 As shown, with OONO - As the concentration increases, the luminescence intensity under 808nm excitation decreases continuously, while the luminescence intensity under 980nm excitation remains basically unchanged. It can also be seen from the ratio image that F 808 nm / F 980nm The luminous intensity of F 980nm / F 808nm The intensity values ​​of the nanoprobe were fitted, and the fitting coefficient R2 = 0.996, which proves that the ratio of the down-conversion luminescence intensity of the nanoprobe under 808nm and 980nm excitation is similar to that of OONO - There is a linear relationship between the concentrations of ions; e.g. Figure 3 As shown, in the NIR-IIb region after 1500nm, the imaging signal-to-noise ratio and imaging quality of the rare earth nanocomposite material are relatively high; Figure 4 As shown, the abdomen of mice injected with lipopolysaccharide (LPS) developed inflammation. 980nm / F 808 nm The fluorescence intensity ratio increased approximately linearly, while the mice injected with phosphate buffer solution (PBS) and the mice injected with LPS and glutathione (GSH) at the same time, GSH consumed the peroxynitrite produced by inflammation in the mice, and F 980nm / F 808 nm The fluorescence intensity ratio did not change, indicating that the nano-fluorescent probe can respond to peroxynitrite in a ratiometric manner and can achieve high-resolution imaging. Different substances were added to the nano-fluorescent probe to detect its response to different ROS including ClO - 、H2O2、·OH、 1 O2 and other interferents such as glutathione, dithiothreitol, camptothecin, vitamin C, Fe3+ 、Ag + responsiveness, such as Figure 5 As shown, the nanofluorescent probe can detect OONO - Produce a specific response and have little response to other ROS or interferents. Figure 6 As shown, the concentration of the modified dye was changed, and the change in fluorescence intensity at 1550 nm was measured after rare earth nanoparticles were modified with dyes of different concentrations. The optimal dye modification concentration was 30 μg / mL.

[0051] Embodiment 2

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

[0053] The experimental results show that after dye sensitization, the outer layer of the nanoparticles does not contain Yb 3+ After the structure is excited by 808nm laser, the fluorescence intensity value and the ratio of improvement after sensitization are both lower than those in Example 1, but can meet the basic requirements of in vivo optical imaging.

[0054] Embodiment 3

[0055] The only difference between Example 3 and Example 1 is that PMA-PEG of the same concentration is used to replace PMA in Example 1, and 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 as PMA and slightly increased fluorescence intensity, but the cost is higher.

[0057] Comparative Example 1

[0058] The difference between this 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 other conditions remain unchanged, to prepare rare earth composite nanomaterials as nanoprobes.

[0059] The experimental results show that the undoped Nd 3+ Although the structure of the ions can also increase the luminescence intensity several times after dye sensitization, due to the lack of Nd 3+The doping of ions makes its own luminescence intensity at 1530nm lower under 808nm excitation, resulting in 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 is used to replace the IR783-TPE tetrahydrofuran solution in Example 1, and other conditions remain unchanged to prepare rare earth composite nanomaterials as nanoprobes.

[0062] The experimental results show that the dye has poor solubility in the ligand exchange reaction system and has low sensitization effect in aqueous solution after modification. However, IR783-TPE, because of the modification of tetraphenylethylene groups, can show better dispersibility in non-polar solutions such as dichloromethane and tetrahydrofuran, and can also better achieve ligand exchange, which commercial dyes cannot achieve. At the same time, IR808 has poor sensitization to ClO - It also has a certain responsiveness, making it easy to - The test lacks specificity.

[0063] Comparative Example 3

[0064] The only difference between Comparative Example 3 and Example 1 is that DSPE-PEG of equal concentration is used to replace PMA in Example 1, and other conditions remain unchanged to prepare rare earth composite nanomaterials as nanoprobes.

[0065] The experimental results show that after using DSPE-PEG instead of PMA to coat rare earth nanoparticles, the fluorescence intensity of the fluorescent probe decreases significantly.

[0066] In summary, the present invention designs a composite rare earth nanoparticle for preparing a water-soluble fluorescent nanoprobe for ONOO - It has specific response capability and can perform excitation ratio imaging detection of inflammatory sites.

Claims

1. A rare earth composite nanomaterial, characterized in that: The invention comprises rare earth nanoparticles, cyanine dye molecules containing sulfonate and amphiphilic surfactants, wherein the cyanine dye molecules containing sulfonate 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 are double-layer core-shell structures, including a core and a shell, wherein 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 a matrix, and the diameter of the core is in the range of 15-20 nm; the single shell is composed of NaYF4 as a matrix doped with Nd or doped with two rare earth elements, Yb and Nd, and the thickness of the single shell is 2-4 nm; In the core of the rare earth composite nanomaterial, NaYF4 is the matrix, the molar percentage of doped Ce in the total amount of all rare earth elements in the core is not higher than 25%, the molar percentage of doped Er in the total amount of all rare earth elements in the core is not higher than 5%, and the molar percentage of doped Yb in the total amount of all 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 all rare earth elements in the shell is not higher than 40%, and the molar percentage of doped Yb in the total amount of all rare earth elements in the shell is 0% to 20%.

2. A rare earth composite nanomaterial according to claim 1, characterized in that: The molar percentage of Ce doped in the core is 10% of the total amount of all rare earth elements in the core, the molar percentage of Er doped in the core is 2%, and the molar percentage of Yb doped in the core is 20%; the molar percentage of Nd doped in the shell is 30% of the total amount of all rare earth elements in the shell, and the molar percentage of Yb doped in the shell is 10%.

3. A rare earth composite nanomaterial according to claim 1, characterized in that: The sulfonate-containing cyanine dye molecule is selected from any one of IR783, IR783-TPE, IR806 or IR806-TPE.

4. The rare earth composite nanomaterial according to claim 1, characterized in that: The amphiphilic surfactant is selected from any one of F127, PMA or PMA-PEG.

5. The method for preparing the rare earth composite nanomaterial according to claim 1, characterized in that: The preparation method comprises 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, and then preparing a single shell NaYF4:Yb, Nd on the surface of the core by epitaxial growth to obtain the rare earth nanoparticles, and dispersing the rare earth nanoparticles in chloroform to prepare a rare earth nanoparticle solution with a concentration of 20 mg / mL; A 1 mg / mL IR783-TPE tetrahydrofuran solution is prepared, and the 20 mg / mL rare earth nanoparticle solution is mixed with the IR783-TPE tetrahydrofuran solution, and then stirred at 50° C. for 2 hours under a nitrogen atmosphere, and then ethanol is added for precipitation. After centrifugation, the mixture is dispersed in tetrahydrofuran to obtain a dye-modified rare earth nanoparticle solution of about 4 mg / mL; wherein the raw material configuration volume ratio of the rare earth nanoparticle solution and the IR783-TPE tetrahydrofuran solution is 1:(0.04-0.34); A 6 mg / mL PMA aqueous solution is prepared, wherein a NaOH solution is added to adjust the pH value to about 12 so that the PMA is dissolved in water; under ultrasonic conditions, the 4 mg / mL dye-sensitized rare earth nanoparticle solution is quickly injected into the PMA aqueous solution, the volume ratio of the rare earth nanoparticle solution to the PMA aqueous solution is (0.6-1.8):9, and the ultrasonic treatment is maintained for 3 minutes; tetrahydrofuran in the aqueous solution is blown away with nitrogen, and PMA-modified dye-sensitized rare earth nanoparticles are obtained after purification by ultrafiltration.

6. Use of the rare earth composite nanomaterial according to any one of claims 1 to 4 in the preparation of peroxynitrite fluorescent nanoprobes.

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