Hydrophilic modified upconversion luminescent nanoparticle and its preparation method and applications

Abstract

The present invention belongs to bio-detection technology and specifically relates to a hydrophilic modified upconversion luminescent nanoparticle and its preparation method and applications. It is fabricated through the following steps: a lanthanide metal salt is mixed with oleic acid and l—octadecene to create an oleic acid precursor; this precursor is then combined with ammonium fluoride and sodium hydroxide in methanol to form a nanocore, subsequently yielding core upconversion nanoparticles via thermal decomposition. These core particles are mixed with additional oleic acid precursor, ammonium fluoride, and sodium hydroxide in methanol, leading to the formation of upconversion luminescent nanoparticles through thermal decomposition. Finally, hydrophilic modification is achieved by substituting the oleic acid on the upconversion luminescent nanoparticles with a carboxylic acid containing a sulfi1ydryl group. These nanoparticles replace HRP-labelled detection antibodies in a doubleantibody sandwich method for target analyte detection, offering enhanced stability, specificity, sensitivity, and re-testability.

Description

Technical Field The present invention belongs to the eld of bio-detection technology, and specically relates to a hydrophilic modied upconversion luminescent nanoparticle and its preparation method and applications. Background Technology Conventional antigen / antibody detection methods mainly include enzyme-linked immunosorbent assay (ELISA), chemiluminescence, and immunouorescence. ELISA is a routine clinical and laboratory diagnostic tool for detecting a wide range of diseases from infectious diseases to cancer biomarkers, and it is a precise, sensitive, versatile, and quantiable diagnostic method. However, ELISA has the disadvantages of time- consuming and high cost due to its complexity, long experimental operation time, and lack of multiplicity and reproducible detection capability in practical high-throughput assays. ln addition, ELISA has relatively low detection sensitivity, which is insufcient for early diagnosis and screening of specic diseases (such as Alzheimer's disease and acute myocardial infarction). Although chemiluminescence and immunouorescence are more sensitive, the decay of the luminescent substrate of chemiluminescence is faster, and the inconsistency of the luminescent substrate spiking time in the case of a large number of samples will lead to more serious systematic bias, which will affect the accuracy of the results. ln addition, chemiluminescence and immunouorescence both have the problem of photobleaching and instability, which is the biggest defect restricting their development. Immunouorescence usually uses organic uorescent dyes as markers, but the poor photostability of traditional organic uorescent dyes, serious photobleaching and photodegradation, and short uorescence lifetime limit the clinical application of immunouorescence. ln recent years, lanthanide-doped upconversion luminescent nanoparticles (UCNPs) have become a new generation of uorescent markers due to their excellent photostability, long uorescence lifetime, narrow emission bandwidth and high quantum yield, absence of autouorescence in near-infrared radiation, and low optical noise background. In addition, the uorescence intensity of UCNPs can be improved by changing the type and content of doping elements and the surface coating strategy to enhance the detection signal and signicantly improve the sensitivity, which has a broad application prospect in the eld of antigen / antibody detection. Based on this, the present invention intends to provide a new upconversion nanomaterial as an indication system for a highly sensitive and stable immunouorescence detection method, providing a new idea and a new technology for clinical detection. Content ofthe Invention ln response to the deciencies of the above prior art, the present invention provides a hydrophilic modied upconversion luminescent nanoparticle, with which the upconversion luminescent nanoparticle is used instead of horseradish peroxidase (HRP)-labelled detection antibody, to establish an analytical method for detecting a target analyte with high sensitivity. The specic technical solutions provided by the present invention are as follows: In a rst aspect of the present invention, there is provided a hydrophilic modied upconversion luminescent nanoparticle which is prepared according to the following steps: Lanthanide metal salts were mixed with oleic acid (OA) and 1-octadecene (OED) in a coordination reaction to obtain lanthanide-doped OA precursors; The lanthanide dopedOA precursorswere mixed withammonium uoride (NH4F) methanol solution and sodium hydroxide (NaOH) methanol solution for complex decomposition reaction and chemical adsorption to generate nanocores, followed by thermal decomposition to obtain on-core conversion nanoparticles; The lanthanide-doped OA precursor was mixed with the core upconversion nanoparticles, NH4F methanol solution and NaOH methanol solution to carry out a complexdecomposition reaction and chemical adsorption reaction, and then prepared by thermal decomposition to obtain upconversion luminescent nanoparticles with core-shell structure; Displacing oleic acid on the surface of the core-shell structured upconversion luminescent nanoparticles using a sul1ydryl-containing carboxylic acid to obtain the hydrophilic modied upconversion luminescent nanoparticles. Preferably, the lanthanide metal salt is one or a combination of gadolinium, ytterbium, thulium, erbium or lutetium salts. Preferably, the sul1ydryl-containing carboxylic acid is selected from any of mercapto polyethylene glycol carboxylate (HS-PEG1000COOH), mercaptoacetic acid or mercaptoundecanoic acid. The sul1ydryl group can react with the lanthanide metal ions on the nanoparticles in a coordination reaction, so that the sul1ydryl-containing carboxylic acid and the nanoparticles are more tightly bound by coordination, and the carboxylic acid group can be further activated to be coupled with the biomolecule, such as a detection antibody, to obtain hydrophilic-modied upconversion luminescent nanoparticles labelled with the detection antibody, which is stable, and can be used for the high sensitivity and specicity detection of the biomolecule. The mercapto polyethylene glycol carboxylate may be further preferred as a substance for displacing oleic acid due to the optimal water solubility of the mercapto polyethylene glycol carboxylate. Preferably, the coordination reaction is 20-60 min in an inert environment, at 100- 200°C; The complex decomposition reaction and chemisorption reaction are reacted in an inert environment at 30~75°C for 20~45 min; All of the thermal decomposition methods described are reacted in an inert environment, at ZOO-500°C for 1.02.0h. Preferably, the replacement is carried out according to the following steps: After adding the sul1ydryl-containing carboxylic acid to the dispersion of the core-shell structured upconversion luminescent nanoparticles, stirring at room temperature for 30-72h, removing the solvent and the excess of the sul1ydryl- containing carboxylic acid, the hydrophilic modied upconversion luminescent nanoparticles are obtained. In a second aspect of the present invention, there is provided an upconversion luminescence detection reagent obtained by crosslinking the hydrophilically modied upconversion luminescent nanoparticles with a detection antibody. Preferably, the detection antibody is a C-reactive protein (CRP) detection antibody or a Staphylococcus aureus enterotoxin B (SEB) detection antibody. Preferably, the C-reactive protein detection antibody is FIVlU-CRP-l and the Staphylococcus aureus enterotoxin B detection antibody is FIVlU-SEB-l. Preferably, the crosslinking is performed by maintaining the pH of the mixed system at 5.05.5 using a pH stabiliser, and forming a covalent bond with the free amino group on the detection antibody after activating the carboxyl group on the surface of the hydrophilic modied upconversion luminescent nanoparticles using an amide bond coupling agent. The amide bond coupling agent can be selected from 1- (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and succinimide (NHS, with the formula C4H5N03). When EDC and NHS are used as the amide bond coupling agent, the molar ratio of both of them is preferably 1:1, but this is not a specic limitation on the amount of both of them and the person skilled in the eld can make specic choices according to the actual situation in use. However, this is not a specic limitation on the dosage of the two, and the person skilled in the art may choose according to the actual situation. In a fourth aspect of the present invention, there is provided an immunouorescence detection method comprising the steps of: The capture antibodywas added into the opaque ELISA plate and left for l6~24 h to immobilise the capture antibody on the ELISA plate, after which different concentrations of antigen standards were added, incubated, and upconversion luminescence detection reagent was added with the upconversion luminescence detection reagent, incubated so that the upconversion luminescence detection reagent, the capture antibody, and the antigen standards formed a sandwich complex immobilised on the ELISA plate, and each experimental step was separated from each other with Phosphate buffer solution (PBST) containing Tween-20 was washed 3 times between each experimental step; The sandwich complexes immobilised on an ELISA plate are subjected to uorescence analysis, the uorescence intensity of the upconversion luminescent nanoparticles is detected, and a standard curve is plotted in terms ofthe uorescence intensity and the concentration of the antibody standard; Calculatingthe concentration ofthe substance to be tested based on the standard curve and the uorescence intensity of the substance to be tested. Wherein the detection of the uorescence intensity of the substance to be measured is the same as the detection of the uorescence intensity of the antigen standard, as follows: Capture antibody was added to an opaque ELISA plate and left for 16~24 h to immobilise the capture antibody on the ELISA plate, followed by the addition of the substance to be tested, incubation, the addition of the upconversion luminescence detection reagent, and the incubation to immobilise the upconversion luminescence detection reagent, the capture antibody, and the antigens in the substance to be tested in a sandwich complex on the ELISA plate, and washed 3 times with phosphate buffer containing Tween-20 between each experimental step. -20 in phosphate buffer for 3 times between each experimental step; the sandwich complex immobilised on the ELISA plate was subjected to uorescence analysis to detect the uorescence intensity of the substance to be measured. Compared with the prior art, the present invention has the following benecial technical effects: 1. The present invention firstly prepares an upconversion luminescent nanoparticle NaGdF4:Yb / Tm / Er@NaLuF4 (NGF@NLF), which has a uniform particle size, good dispersion, and a core-shell structure, and is able to significantly increase the fluorescence intensity to amplify the detection signals, thereby increasing the lower limit of detection ofthe target analytes, and effectively improving the sensitivity and stability of detection. The hydrophilicity of the upconversion luminescent nanoparticles was further modified by the use of sulfhydryl carboxylic acid to obtain carboxylated NGF@NLF, and the sulfhydryl carboxylic acid and the upconversion luminescent nanoparticles were more tightly bonded through the coordination between sulfhydryl groups and metal ions; the carboxylic group in the sulfhydryl carboxylic acid could be further activated to couple with biomolecules (such as detection antibodies) to obtain the NGF@NLF-labelled detection antibodies, which are stable in nature and have the ability to detect the target analytes. NGF@NLF labelled antibody can be prepared, which is stable and can be stored for a long time. 2. The present invention successfully establishes a stable immunouorescence method for CRP detection antibody using hydrophilic modied upconversion luminescent nanoparticles, which is a method based on CRP-specic monoclonal antibody, which is an antibody that recognises a single epitope, and so the detection method of the present invention is highly specic. By using hydrophilic modied NGF@NLF-labelled detection antibody instead of HRP-labelled detection antibody, the sensitivity can be increased to 0.015 ng / mL, which is a 66-fold increase in sensitivity as compared to the lower limit of detection of commercially available ELISA kits of 1 ng / mL. 3. The stable hydrophilic modied NGF@NLF coupled detection antibody established by the present invention can also be used for SEB detection by immunouorescence, with a lower limit of detection of up to 0.006 ng / mL, which is a 120fold increase in sensitivity as compared to the commercially available ELISA kits, which have a lower limit of detection of 0.75 ng / mL. 4. Although the existing chemiluminescence method and immunofluorescence method have high sensitivity, the luminescent substrate of chemiluminescence method decays faster, and the inconsistency of the luminescent substrate spiking time will lead to serious systematic biaswhen the amount of samples is more, which affects the accuracy of the results. Immunofluorescence method usually uses organic fluorescent dyes as markers, however, traditional organic fluorescent dyes have poor photostability, serious photobleaching and photodegradation phenomena, and short fluorescence lifetimes, which limit the clinical application of immunofluorescence method. The hydrophilic modified upconversion luminescent nanoparticles provided by the present invention can solve the problems of low detection sensitivity of ELISA and photobleaching, fluorescence quenching and photobleaching of chemiluminescence and immunofluorescence due to their excellent photostability, long fluorescence lifetime, narrow emission bandwidth and high quantum yield . 5. After the detection is completed using the assay constructed by the present invention, it is xed by adding glutaraldehyde to the opaque ELISA plate, and the preservation time is up to 1 year or more, and it can be applied to re-testing, which is completely impossible to achieve with the existing HRP-labelled or uorescence- labelled immunoassay methods. 6. The detection method constructed by the present invention is characterised by good stability, high specicity, high sensitivity, and can be retested at any time. 7. The hydrophilic modied upconversion luminescent nanoparticles provided by the present invention have a simpler structure and are easy to prepare. Description ofthe Figures FIG.1: aow chart of the immunouorescence method using UCNPs as indicators; FIG.2: the transmission electron microscopy (TEM) analysis (A) of the core structure NGF and the particle size distribution (B) of the core structure NGF; FIG.3: the energy spectrum of the core structure NGF; FIG.4: the measured elemental distribution ofthe core structure NGF; FIG.5: the TEM image (A) and the particle size distribution (B) of the core-shell structure NGF@NLF; FIG.6: the energy spectrum of the core-shell structure NGF@NLF; FIG.7: the measured elemental distribution of the core-shell structure NGF@NLF; FIG.8: the IR spectra of NGF@NLF before and after modication and HS'PEGmOO' COOH; FIG.9: the photostability energy proles of NGF@NLF (A) and NGF@NLF / HS- PEGloooCOOH (B); FIG.10: the uorescence spectra of NGF, NGF@NLF before and after modication; FIG.11: the standard curves of uorescence intensity versus concentration ofCRP standards by ELISA (a) and with UCNPs (b) as the indicator, (b) The small plot in the upper left corner of the graph shows the standard curve at low concentrations; FIG.12: the standard curve of uorescence intensity versus concentration of SEB standards using UCNPs as indicators; the small graph in the upper left corner of the gure shows the standard curve at low concentrations. Specic Embodiments The technical programme of the present invention is described in further detail below in connection with the accompanying drawings and embodiments. The present invention provides a hydrophilic modied upconversion luminescent nanoparticle, which is prepared according to the following steps: Lanthanide metal salts were mixed with oleic acid and 1-octadecene in a coordination reaction to obtain lanthanide-doped oleic acid precursors; The lanthanide doped oleic acid precursor was mixed with NH4F methanol solution and NaOH methanol solution for complex decomposition reaction and chemisorption to generate nanocores, followed by thermal decomposition to obtain core upconversion nanoparticles; The lanthanide-doped oleic acid precursor was mixed with the core upconversion nanoparticles, NH4F methanol solution and NaOH methanol solution to carry out a complex decomposition reaction and a chemical adsorption reaction, and then the upconversion luminescent nanoparticles with core-shell structure were prepared by thermal decomposition method; Displacement of oleic acid on the surface of the core-shell structured upconversion luminescent nanoparticles using mercapto-containing carboxylic acid to obtain the hydrophilic modied upconversion luminescent nanoparticles. The present invention further employs a mercapto-containing carboxylic acid such as HS'PEGmOO' COOH, mercaptoacetic acid or mercaptoundecanoic acid to modify the hydrophilicity of the upconversion luminescent nanoparticles to obtain carboxylated NGF@NLF. As a further preferred embodiment, HS-PEGlooo-COOH is selected as the hydrophilicity modifying substance, and compared to other carboxylic acid-containing substances such as mercaptoacetic acid, mercaptoundecanoic acid, etc., HS-PEG -COOH is selected to be the hydrophilic modifying substance. alkanoic acid, etc., HSPEGmOO'COOH has better hydrophilicity, which enables better dispersion of the modied upconversion luminescent nanoparticles, and facilitates the subsequent activation of the carboxyl groups and coupling ofthe UCNPs with biomolecules (e.g., detection antibodies). Example 1 Preparation of hydrophilic modied upconversion luminescent nanoparticles (UCNPQ 1. Preparation of homogeneous and well dispersed B-NaGdF4:Yb / Tm / Er (NGF) nanoparticles (NPs) with core structure. The specific steps are as follows: A 100 mL three-necked round-bottomed flask was filled with 2 mL of 0.2 M Gd(CH3COz)3 solution, 1.915 mL of 0.2 M Yb(CH3COz)3 solution, 5 uL of 0.2 M Tm(CH3COZ)3 solution and 80 uL of 0.2M Er(CH3C02)3 solution, and then 8 mL ofOA and 12 mLofOEDwere added. 150 °C and reacted for40 min to obtain the lanthanide- doped OA precursor. After reduced to room temperature, a mixture of 5.6 mL, 0.4 M ofNH4F methanol solution and 2 mL, 1M ofNaOH methanol solutionwas slowlyadded to the vial and reacted at 50 °C for 30 min to generate nanocores. Subsequently, the temperature was raised to 100 °C for evacuation operation to remove methanol and water from the solution. When bubbles no longer appeared in the solution, the temperature was set to 300 °C, the argon rate was adjusted to the minimum, and the reaction was carried out at this temperature for 1.5 h to finally generate B- NaGdF4:Yb / Tm / Er nanoparticles. After centrifugation to remove excess OA and ODE, the precipitate was dispersed with 8 mL of cyclohexane and set aside. 2. Preparation of homogeneous and well dispersed B-NaGdF4:Yb / Tm / Er@NaLuF4 luminescent nanoparticles (NGF@NLF) with core-shell structure.The specific steps are as follows: A 100 mL three-necked round-bottomed flask was taken, to which 4 mL, 0.2M of Lu(CH3COz)3 solution and 8 mL of OA and 12 mL of OED were added, and the temperature was raised to 150 °C and reacted for 40 min under argon protection to synthesize theOA precursor. Subsequently, a mixture of 5.6 mL, 0.4M NH4F methanol solution and 2 mL, 1 M NaOH methanol solution was added, and 7 mL of B- NaGdF4:Yb / Tm / Er (NGF) solution prepared above was continued to be added, and the reaction was carried out at 50 °C for 30 min in order to generate the nanocores. Subsequently, the temperature was raised to 100 °C for evacuation operation to remove methanol and water from the solution. When bubbles no longer appeared in the solution, the temperature was set to 300 °C, the argon rate was adjusted to the minimum, and the reaction was carried out at this temperature for 1.5 h to finally obtain B-NaGdF4:Yb / Tm / Er@NaLuF4 (NGF@NLF). After centrifugation to remove excess OA and ODE, the precipitate was dispersed with 8 mL of cyclohexane to obtain B-NaGdF4:Yb / Tm / Er@NaLuF4 luminescent nanoparticles with core-shell structure (NGF@NLF). 3. Hydrophilic modified B-NaGdF4:Yb / Tm / Er@NaLuF4 luminescent nanoparticles. The specific preparation process is as follows: The cyclohexane solution containing 20 mg ofNGF@NLF prepared in step 2 was taken, and after the cyclohexane was evaporated to dryness, 3 mL ofDMSO was added to it to make it completely dispersed in DMSO, and 24 mg of HS-PEGlOOO-COOH was added, and stirring was carried out at room temperature for 48 h. The solution after the end of the reaction was transferred to a dialysis membrane with a molecular weight cut-off of 7000, and dialyzed for 24 h to remove the DMSO and excess HS- PEGlOOO-COOH, and finally the HS-PEGlOOO-COOH modified NGF@NLFwas obtained, which was placed in 4 °C cold storage and set aside. Example 2 Preparation of hydrophilic modied upconversion luminescent nanoparticles (UCNPQ The difference from Example 1 is that Step 1 and Step 2 are different, specically: 1. Preparation of homogeneous and well dispersed B-NaGdF4:Yb / Tm / Er (NGF) nanoparticles (NPs) with core structure. The specific steps are as follows: A 100 mL three-necked round-bottomed flask was filled with 2 mL of 0.2 M Gd(CH3COz)3 solution, 1.915 mL of 0.2 M Yb(CH3COz)3 solution, 5 uL of 0.2 M Tm(CH3C02)3 solution and 80 uL of 0.2 M Er(CH3C02)3 solution, and then 8 mL of OA and 12 mLofOEDwere added. 100 °C and reacted for60 min to obtain the lanthanide- doped OA precursor. After reduced to room temperature, a mixture of 5.6 mL, 0.4 M ofNH4F methanol solution and 2 mL, 1M ofNaOH methanol solutionwas slowlyadded to the vial and reacted at 50 °C for 30 min to generate nanocores. Subsequently, the temperature was raised to 100 °C for evacuation operation to remove methanol and water from the solution. When bubbles no longer appeared in the solution, the temperature was set to 300 °C, the argon rate was adjusted to the minimum, and the reaction was carried out at this temperature for 1.5 h to finally generate ß- NaGdF4:Yb / Tm / Er nanoparticles. After centrifugation to remove excess OA and ODE, the precipitate was dispersed with 8 mL of cyclohexane and set aside. 2. Preparation of homogeneous and well dispersed B-NaGdF4:Yb / Tm / Er@NaLuF4 luminescent nanoparticles (NGF@NLF) with core-shell structure.The specific steps are as follows: A 100 mL three-necked round-bottomed flask was taken, to which 4 mL, 0.2M of Lu(CH3COz)3 solution and 8 mL of OA and 12 mL of OED were added, and the temperature was raised to 150 °C and reacted for 20 min under the protection of argon to synthesize the OA precursor. Subsequently, a mixture of 5.6 mL, 0.4 M NH4F methanol solution and 2 mL, 1M NaOH methanol solution was added, and 7 mL of ß- NaGdF4:Yb / Tm / Er (NGF) solution prepared above was continued to be added, and the reaction was carried out at 50 °C for 30 min in order to generate the nanocores. Subsequently, the temperature was raised to 100 °C for evacuation operation to remove methanol and water from the solution. When bubbles no longer appeared in the solution, the temperature was set to 300 °C, the argon rate was adjusted to the minimum, and the reaction was carried out at this temperature for 1.5 h to finally obtain B-NaGdF4:Yb / Tm / Er@NaLuF4 (NGF@NLF). After centrifugation to remove excess OA and ODE, the precipitate was dispersed with 8 mL of cyclohexane to obtain B-NaGdF4:Yb / Tm / Er@NaLuF4 luminescent nanoparticles with core-shell structure (NGF@NLF). Example 3 Preparation of hydrophilic modied upconversion luminescent nanoparticles (UCNPQ The difference from Example 1 is that step 1 is different, specically: 1. Preparation of homogeneous and well dispersed B-NaGdF4:Yb / Tm / Er (NGF) nanoparticles (NPs) with core structure. The specific steps are as follows: A 100 mL three-necked round-bottomed flask was filled with 2 mL of 0.2 M Gd(CH3COz)3 solution, 1.915 mL of 0.2 M Yb(CH3COz)3 solution, 5 uL of 0.2 M Tm(CH3C02)3 solution and 80 uL of 0.2 M Er(CH3C02)3 solution, and then 8 mL of OA and 12 mLofOEDwere added. 200 °C and reacted for 20 min to obtain the lanthanide- doped OA precursor. After reduced to room temperature, a mixture of 5.6 mL, 0.4 M ofNH4F methanol solution and 2 mL, 1M ofNaOH methanol solutionwas slowlyadded to the vial and reacted at 50 °C for 30 min to generate nanocores. Subsequently, the temperature was raised to 100 °C for evacuation operation to remove methanol and water from the solution. When bubbles no longer appeared in the solution, the temperature was set to 300 °C, the argon rate was adjusted to the minimum, and the reaction was carried out at this temperature for 1.5 h to finally generate ß- NaGdF4:Yb / Tm / Er nanoparticles. After centrifugation to remove excess OA and ODE, the precipitate was dispersed with 8 mL of cyclohexane and set aside. Example 4 Preparation of hydrophilic modied upconversion luminescent nanoparticles (UCNPQ The difference from Example 1 is that Step 1 and Step 2 are different, specically: 1. Preparation of homogeneous and well dispersed B-NaGdF4:Yb / Tm / Er (NGF) nanoparticles (NPs) with core structure. The specific steps are as follows: A 100 mL three-necked round-bottomed flask was filled with 2 mL of 0.2 M Gd(CH3COz)3 solution, 1.915 mL of 0.2 M Yb(CH3COz)3 solution, 5 uL of 0.2 M Tm(CH3C02)3 solution and 80 uL of 0.2 M Er(CH3C02)3 solution, and then 8 mL of OA and 12 mLofOEDwere added. 150 °C and reacted for40 min to obtain the lanthanide- doped OA precursor. After reduced to room temperature, a mixture of 5.6 mL, 0.4 M ofNH4F methanol solution and 2 mL, 1M ofNaOH methanol solutionwas slowlyadded to the vial and reacted at 30 °C for 45 min to generate nanocores. Subsequently, the temperature was raised to 100 °C for evacuation operation to remove methanol and water from the solution. When bubbles no longer appeared in the solution, the temperature was set to 300 °C, the argon rate was adjusted to the minimum, and the reaction was carried out at this temperature for 1.5 h to finally generate ß- NaGdF4:Yb / Tm / Er nanoparticles. After centrifugation to remove excess OA and ODE, the precipitate was dispersed with 8 mL of cyclohexane and set aside. 2. Preparation of homogeneous and well dispersed B-NaGdF4:Yb / Tm / Er@NaLuF4 luminescent nanoparticles (NGF@NLF) with core-shell structure.The specific steps are as follows: A 100 mL three-necked round-bottomed flask was taken, to which 4 mL, 0.2M of Lu(CH3COz)3 solution and 8 mL of OA and 12 mL of OED were added, and the OA precursor was synthesized by raising the temperature to 150 °C and reacting for 40 min under argon protection. Subsequently, a mixture of 5.6 mL, 0.4M NH4F methanol solution and 2 mL, 1 M NaOH methanol solution was added, and 7 mL of ß- NaGdF4:Yb / Tm / Er (NGF) solution prepared above was continued to be added, and the reaction was carried out at 50 °C for 20 min in order to generate the nanocores. Subsequently, the temperature was raised to 100 °C for evacuation operation to remove methanol and water from the solution. When bubbles no longer appeared in the solution, the temperature was set to 300 °C, the argon rate was adjusted to the minimum, and the reaction was carried out at this temperature for 1.5 h to finally obtain B-NaGdF4:Yb / Tm / Er@NaLuF4 (NGF@NLF). After centrifugation to remove excess OA and ODE, the precipitate was dispersed with 8 mL of cyclohexane to obtain B-NaGdF4:Yb / Tm / Er@NaLuF4 luminescent nanoparticles with core-shell structure (NGF@NLF). Example 5 Preparation of hydrophilic modied upconversion luminescent nanoparticles (UCNPs) The difference from Example 1 is that Step 1 and Step 2 are different, specically: 1. Preparation of homogeneous and well dispersed B-NaGdF4:Yb / Tm / Er (NGF) nanoparticles (NPs) with core structure. The specific steps are as follows: A 100 mL three-necked round-bottomed flask was filled with 2 mL of 0.2 M Gd(CH3COz)3 solution, 1.915 mL of 0.2 M Yb(CH3COz)3 solution, 5 uL of 0.2 M Tm(CH3C02)3 solution and 80 uL of 0.2 M Er(CH3C02)3 solution, and then 8 mL ofOA and 12 mLofOEDwere added. 150 °C and reacted for40 min to obtain the lanthanide- doped OA precursor. After reduced to room temperature, a mixture of 5.6 mL, 0.4M ofNH4F methanol solution and 2 mL, 1M ofNaOH methanol solutionwas slowlyadded to the vial and reacted at 75 °C for 30 min to generate nanocores. Subsequently, the temperature was raised to 100 °C for evacuation operation to remove methanol and water from the solution. When bubbles no longer appeared in the solution, the temperature was set to 300 °C, the argon rate was adjusted to the minimum, and the reaction was carried out at this temperature for 1.5 h to finally generate B- NaGdF4:Yb / Tm / Er nanoparticles. After centrifugation to remove excess OA and ODE, the precipitate was dispersed with 8 mL of cyclohexane and set aside. 2. Preparation of homogeneous and well dispersed B-NaGdF4:Yb / Tm / Er@NaLuF4 luminescent nanoparticles (NGF@NLF) with core-shell structure.The specific steps are as follows: A 100 mL three-necked round-bottomed flask was taken, to which 4 mL, 0.2M of Lu(CH3COz)3 solution and 8 mL of OA and 12 mL of OED were added, and the OA precursor was synthesized by raising the temperature to 150 °C and reacting for 40 min under argon protection. Subsequently, a mixture of 5.6 mL, 0.4M NH4F methanol solution and 2 mL, 1 M NaOH methanol solution was added, and 7 mL of B- NaGdF4:Yb / Tm / Er (NGF) solution prepared above was continued to be added, and the reaction was carried out at 30 °C for 45 min in order to generate the nanocores. Subsequently, the temperature was raised to 100 °C for evacuation operation to remove methanol and water from the solution. When bubbles no longer appeared in the solution, the temperature was set to 300 °C, the argon rate was adjusted to the minimum, and the reaction was carried out at this temperature for 1.5 h to finally obtain B-NaGdF4:Yb / Tm / Er@NaLuF4 (NGF@NLF). After centrifugation to remove excess OA and ODE, the precipitate was dispersed with 8 mL of cyclohexane to obtain B-NaGdF4:Yb / Tm / Er@NaLuF4 nanoparticles with core-shell structure (NGF@NLF). Example 6 Preparation of hydrophilic modied upconversion luminescent nanoparticles (UCNPQ The difference from Example 1 is that Step 1 and Step 2 are different, specically: 1. Preparation of homogeneous and well dispersed B-NaGdF4:Yb / Tm / Er (NGF) nanoparticles (NPs) with core structure. The specific steps are as follows: A 100 mL three-necked round-bottomed flask was filled with 2 mL of 0.2 M Gd(CH3COz)3 solution, 1.915 mL of 0.2 M Yb(CH3COz)3 solution, 5 uL of 0.2 M Tm(CH3C02)3 solution and 80 uL of 0.2 M Er(CH3C02)3 solution, and then 8 mL of OA and 12 mLofOEDwere added. 150 °C and reacted for40 min to obtain the lanthanide- doped OA precursor. After reduced to room temperature, a mixture of 5.6 mL, 0.4 M ofNH4F methanol solution and 2 mL, 1M ofNaOH methanol solutionwas slowlyadded to the vial and reacted at 50 °C for 30 min to generate nanocores. Subsequently, the temperature was raised to 100 °C for evacuation operation to remove methanol and water from the solution. When bubbles no longer appeared in the solution, the temperature was set to 200 °C, the argon rate was adjusted to the minimum, and the reaction was carried out at this temperature for 1.5 h to finally generate ß- NaGdF4:Yb / Tm / Er nanoparticles. After centrifugation to remove excess OA and ODE, the precipitate was dispersed with 8 mL of cyclohexane and set aside. 2. Preparation of homogeneous and well dispersed ß-NaGdF4:Yb / Tm / Er@NaLuF4 luminescent nanoparticles (NGF@NLF) with core-shell structure.The specific steps are as follows: A 100 mL three-necked round-bottomed flask was taken, to which 4 mL, 0.2M of Lu(CH3COz)3 solution and 8 mL of OA and 12 mL of OED were added, and the OA precursor was synthesized by raising the temperature to 150 °C and reacting for 40 min under argon protection. Subsequently, a mixture of 5.6 mL, 0.4M NH4F methanol solution and 2 mL, 1 M NaOH methanol solution was added, and 7 mL of ß- NaGdF4:Yb / Tm / Er (NGF) solution prepared above was continued to be added, and the reaction was carried out at 50 °C for 30 min in order to generate the nanocores. Subsequently, the temperature was raised to 100 °C for evacuation operation to remove methanol and water from the solution. When bubbles no longer appeared in the solution, the temperaturewas set to 450 °C and the argon rate was adjusted to the minimum, and the reaction was carried out at this temperature for 1.0 h to finally obtain ß-NaGdF4:Yb / Tm / Er@NaLuF4 (NGF@NLF). After centrifugation to remove excess OA and ODE, the precipitate was dispersed with 8 mL of cyclohexane to obtain ß-NaGdF4:Yb / Tm / Er@NaLuF4 luminescent nanoparticles with core-shell structure (NGF@NLF). Example 7 Preparation of hydrophilic modied upconversion luminescent nanoparticles (UCNPQ The difference from Example 1 is that Step 1 and Step 2 are different, specically: 1. Preparation of homogeneous and well dispersed B-NaGdF4:Yb / Tm / Er (NGF) nanoparticles (NPs) with core structure. The specific steps are as follows: A 100 mL three-necked round-bottomed flask was filled with 2 mL of 0.2 M Gd(CH3COz)3 solution, 1.915 mL of 0.2 M Yb(CH3COz)3 solution, 5 uL of 0.2 M Tm(CH3C02)3 solution and 80 uL of 0.2 M Er(CH3C02)3 solution, and then 8 mL of OA and 12 mLofOEDwere added. 150 °C and reacted for40 min to obtain the lanthanide- doped OA precursor. After reduced to room temperature, a mixture of 5.6 mL, 0.4 M ofNH4F methanol solution and 2 mL, 1M ofNaOH methanol solutionwas slowlyadded to the vial and reacted at 50 °C for 30 min to generate nanocores. Subsequently, the temperature was raised to 100 °C for evacuation operation to remove methanol and water from the solution. When bubbles no longer appeared in the solution, the temperature was set to 300 °C, the argon rate was adjusted to minimum, and the reaction was carried out at this temperature for 2.0 h to finally generate ß- NaGdF4:Yb / Tm / Er nanoparticles. After centrifugation to remove excess OA and ODE, the precipitate was dispersed with 8 mL of cyclohexane and set aside. 2. Preparation of homogeneous and well dispersed ß-NaGdF4:Yb / Tm / Er@NaLuF4 luminescent nanoparticles (NGF@NLF) with core-shell structure.The specific steps are as follows: A 100 mL three-necked round-bottomed flask was taken, to which 4 mL, 0.2M of Lu(CH3COz)3 solution and 8 mL of OA and 12 mL of OED were added, and the OA precursor was synthesized by raising the temperature to 150 °C and reacting for 40 min under argon protection. Subsequently, a mixture of 5.6 mL, 0.4M NH4F methanol solution and 2 mL, 1 M NaOH methanol solution was added, and 7 mL of ß- NaGdF4:Yb / Tm / Er (NGF) solution prepared above was continued to be added, and the reaction was carried out at 50 °C for 30 min in order to generate the nanocores. Subsequently, the temperature was raised to 100 °C for evacuation operation to remove methanol and water from the solution. When bubbles no longer appeared in the solution, the temperature was set to 500 °C, the argon rate was adjusted to the minimum, and the reaction was carried out at this temperature for 1.5 h to finally obtain ß-NaGdF4:Yb / Tm / Er@NaLuF4 (NGF@NLF). After centrifugation to remove excess OA and ODE, the precipitate was dispersed with 8 mL of cyclohexane to obtain ß-NaGdF4:Yb / Tm / Er@NaLuF4 luminescent nanoparticles with core-shell structure (NGF@NLF). Example 8 Preparation of hydrophilic modied upconversion luminescent nanoparticles (UCNPs) The difference from Example 1 is that Step 1 and Step 2 are different, specically: 1. Preparation of homogeneous and well dispersed B-NaGdF4:Yb / Tm / Er (NGF) nanoparticles (NPs) with core structure. The specific steps are as follows: A 100 mL three-necked round-bottomed flask was filled with 5 mL of 0.2 M Gd(CH3COz)3 solution, 1.915 mL of 0.2 M Yb(CH3COz)3 solution, 5 uL of 0.2 M Tm(CH3C02)3 solution and 100 uL of 0.2M Er(CH3C02)3 solution, and then 10 mL ofOA and 12 mL of OED were added. The temperature was raised to 150 °C under argon protection and reacted for 40 min to obtain the lanthanide-doped OA precursor. After the temperaturewas lowered to room temperature, 5.6 mL of a mixture of 0.4M NH4F methanol solution and 2 mL of 1M NaOH methanol solution were slowly added to the vial, and the reaction was carried out at 50 °C for 30 min to generate nanocores. Subsequently, the temperature was raised to 100 °C for evacuation operation to remove methanol and water from the solution. When bubbles no longer appeared in the solution, the temperature was set to 300 °C, the argon rate was adjusted to the minimum, and the reaction was carried out at this temperature for 1.5 h to finally generate B-NaGdF4:Yb / Tm / Er nanoparticles. After centrifugation to remove excessOA and ODE, the precipitate was dispersed with 8 mL of cyclohexane and set aside. 2. Preparation of homogeneous and well dispersed ß-NaGdF4:Yb / Tm / Er@NaLuF4 luminescent nanoparticles (NGF@NLF) with core-shell structure.The specific steps are as follows: A 100 mL three-necked round-bottomed flask was taken, to which 3 mL, 0.2M of Lu(CH3COz)3 solution and 10 mL of OA and 12 mL of OED were added, and the OA precursor was synthesized by raising the temperature to 150 °C and reacting for 40 min under argon protection. Subsequently, a mixture of 5.6 mL, 0.4M NH4F methanol solution and 2 mL, 1 M NaOH methanol solution was added, and 7 mL of ß- NaGdF4:Yb / Tm / Er (NGF) solution prepared above was continued to be added, and the reaction was carried out at 50 °C for 30 min in order to generate the nanocores. Subsequently, the temperature was raised to 100 °C for evacuation operation to remove methanol and water from the solution. When bubbles no longer appeared in the solution, the temperature was set to 300 °C, the argon rate was adjusted to the minimum, and the reaction was carried out at this temperature for 1.5 h to finally obtain ß-NaGdF4:Yb / Tm / Er@NaLuF4 (NGF@NLF). After centrifugation to remove excess OA and ODE, the precipitate was dispersed with 8 mL of cyclohexane to obtain ß-NaGdF4:Yb / Tm / Er@NaLuF4 luminescent nanoparticles with core-shell structure (NGF@NLF). Example 9 Preparation of hydrophilic modied upconversion luminescent nanoparticles (UCNPQ The difference from Example 1 is that Step 1 and Step 2 are different, specically: 1. Preparation of homogeneous and well dispersed B-NaGdF4:Yb / Tm / Er (NGF) nanoparticles (NPs) with core structure. The specific steps are as follows: A 100 mL three-necked round-bottomed flask was filled with 2 mL of 0.2 M Gd(CH3COz)3 solution, 1.915 mL of 0.2 M Yb(CH3COz)3 solution, 20 uL of 0.2 M Tm(CH3C02)3 solution and 80 uL of 0.2 M Er(CH3C02)3 solution, and then 8 mL of OA and 10mLofOEDwere added. 150 °C and reacted for40 min to obtain the lanthanide- doped OA precursor. After reduced to room temperature, a mixture of 5.6 mL, 0.4 M ofNH4F methanol solution and 2 mL, 1M ofNaOH methanol solutionwas slowlyadded to the vial and reacted at 50 °C for 30 min to generate nanocores. Subsequently, the temperature was raised to 100 °C for evacuation operation to remove methanol and water from the solution. When bubbles no longer appeared in the solution, the temperature was set to 300 °C, the argon rate was adjusted to the minimum, and the reaction was carried out at this temperature for 1.5 h to finally generate ß- NaGdF4:Yb / Tm / Er nanoparticles. After centrifugation to remove excess OA and ODE, the precipitate was dispersed with 8 mL of cyclohexane and set aside. 2. Preparation of homogeneous and well dispersed ß-NaGdF4:Yb / Tm / Er@NaLuF4 luminescent nanoparticles (NGF@NLF) with core-shell structure.The specific steps are as follows: A 100 mL three-necked round-bottomed flask was taken, to which 5 mL, 0.2M of Lu(CH3COz)3 solution and 8 mL of OA and 8 mL of OED were added, and the OA precursor was synthesized by raising the temperature to 150 °C and reacting for 40 min under argon protection. Subsequently, a mixture of 5.6 mL, 0.4M NH4F methanol solution and 2 mL, 1 M NaOH methanol solution was added, and 7 mL of ß- NaGdF4:Yb / Tm / Er (NGF) solution prepared above was continued to be added, and the reaction was carried out at 50 °C for 30 min in order to generate the nanocores. Subsequently, the temperature was raised to 100 °C for evacuation operation to remove methanol and water from the solution. When bubbles no longer appeared in the solution, the temperature was set to 300 °C, the argon rate was adjusted to the minimum, and the reaction was carried out at this temperature for 1.5 h to finally obtain ß-NaGdF4:Yb / Tm / Er@NaLuF4 (NGF@NLF). After centrifugation to remove excess OA and ODE, the precipitate was dispersed with 8 mL of cyclohexane to obtain ß-NaGdF4:Yb / Tm / Er@NaLuF4 luminescent nanoparticles with core-shell structure (NGF@NLF). Example 10 Preparation of hydrophilic modied upconversion luminescent nanoparticles (UCNPs) The difference from Example 1 is that Step 1 and Step 2 are different, specically: 1. Preparation of homogeneous and well dispersed B-NaGdF4:Yb / Tm / Er (NGF) nanoparticles (NPs) with core structure. The specific steps are as follows: A 100 mL three-necked round-bottomed flask was filled with 2 mL of 0.2 M Gd(CH3COz)3 solution, 2.25 mL of 0.2 M Yb(CH3COz)3 solution, 5 uL of 0.2 M Tm(CH3C02)3 solution, and 80 uL of 0.2 M Er(CH3C02)3 solution, and then 12 mL ofOA and 12 mLofOEDwere added. 150 °C and reacted for40 min to obtain the lanthanide- doped OA precursor. After reduced to room temperature, a mixture of 5.6 mL, 0.4 M ofNH4F methanol solution and 4 mL, 1M ofNaOH methanol solutionwas slowlyadded to the vial and reacted at 50 °C for 30 min to generate nanocores. Subsequently, the temperature was raised to 100 °C for evacuation operation to remove methanol and water from the solution. When bubbles no longer appeared in the solution, the temperature was set to 300 °C, the argon rate was adjusted to the minimum, and the reaction was carried out at this temperature for 1.5 h to finally generate B- NaGdF4:Yb / Tm / Er nanoparticles. After centrifugation to remove excess OA and ODE, the precipitate was dispersed with 8 mL of cyclohexane and set aside. 2. Preparation of homogeneous and well dispersed ß-NaGdF4:Yb / Tm / Er@NaLuF4 luminescent nanoparticles (NGF@NLF) with core-shell structure.The specific steps are as follows: A 100 mL three-necked round-bottomed flask was taken, to which 5 mL, 0.2M of Lu(CH3COz)3 solution and 10 mL of OA and 12 mL of OED were added, and the OA precursor was synthesized by raising the temperature to 150 °C and reacting for 40 min under argon protection. Subsequently, a mixture of 7.8 mL, 0.4M NH4F methanol solution and 2 mL, 1 M NaOH methanol solution was added, and 7 mL of B- NaGdF4:Yb / Tm / Er (NGF) solution prepared above was continued to be added, and the reaction was carried out at 50 °C for 30 min in order to generate the nanocores. Subsequently, the temperature was raised to 100 °C for evacuation operation to remove methanol and water from the solution. When bubbles no longer appeared in the solution, the temperature was set to 300 °C, the argon rate was adjusted to the minimum, and the reaction was carried out at this temperature for 1.5 h to finally obtain ß-NaGdF4:Yb / Tm / Er@NaLuF4 (NGF@NLF). After centrifugation to remove excess OA and ODE, the precipitate was dispersed with 8 mL of cyclohexane to obtain ß-NaGdF4:Yb / Tm / Er@NaLuF4 luminescent nanoparticles with core-shell structure (NGF@NLF). Example 11 Preparation of hydrophilic modied upconversion luminescent nanoparticles (UCNPq 1. Preparation of homogeneous and well dispersed ß-NaGdF4:Yb / Tm / Er (NGF) nanoparticles (NPs) with core structure. The specific steps are as follows: A 100 mL three-necked round-bottomed flask was filled with 1.5 mL of 0.2 M Gd(CH3COz)3 solution, 1.5 mL of0.2M Yb(CH3COz)3 solution, 5 uL of0.2M Tm(CH3C02)3 solution and 80 uL of 0.2 M Er(CH3C02)3 solution, and then 8 mL ofOA and 15 mL of OED were added. 150 °C and reacted for 40 min to obtain the lanthanide-doped OA precursor. After reduced to room temperature, a mixture of 5.6 mL, 0.4 M of NH4F methanol solution and 3 mL, 1M of NaOH methanol solution was slowly added to the vial and reacted at 50 °C for 30 min to generate nanocores. Subsequently, the temperature was raised to 100 °C for evacuation operation to remove methanol and water from the solution. When bubbles no longer appeared in the solution, the temperature was set to 300 °C, the argon rate was adjusted to the minimum, and the reaction was carried out at this temperature for 1.5 h to finally generate ß- NaGdF4:Yb / Tm / Er nanoparticles. After centrifugation to remove excess OA and ODE, the precipitate was dispersed with 8 mL of cyclohexane and set aside. 2. Preparation of homogeneous and well dispersed ß-NaGdF4:Yb / Tm / Er@NaLuF4 luminescent nanoparticles (NGF@NLF) with core-shell structure.The specific steps are as follows: A 100 mL three-necked round-bottomed flask was taken, to which 4 mL, 0.2M of Lu(CH3COz)3 solution and 8 mL of OA and 15 mL of OED were added, and the OA precursor was synthesized by raising the temperature to 150 °C and reacting for 40 min under argon protection. Subsequently, a mixture of 5.6 mL, 0.4M NH4F methanol solution and 3 mL, 1 M NaOH methanol solution was added, and 7 mL of ß- NaGdF4:Yb / Tm / Er (NGF) solution prepared above was continued to be added, and the reaction was carried out at 50 °C for 30 min in order to generate the nanocores. Subsequently, the temperature was raised to 100 °C for evacuation operation to remove methanol and water from the solution. When bubbles no longer appeared in the solution, the temperature was set to 300 °C, the argon rate was adjusted to the minimum, and the reaction was carried out at this temperature for 1.5 h to finally obtain ß-NaGdF4:Yb / Tm / Er@NaLuF4 (NGF@NLF). After centrifugation to remove excess OA and ODE, the precipitate was dispersed with 8 mL of cyclohexane to obtain ß-NaGdF4:Yb / Tm / Er@NaLuF4 luminescent nanoparticles with core-shell structure (NGF@NLF). 3. Hydrophilic modified ß-NaGdF4:Yb / Tm / Er@NaLuF4 luminescent nanoparticles. The specific preparation process is as follows: The cyclohexane solution containing 20 mg ofNGF@NLF prepared in step 2 was taken, and the cyclohexane was evaporated to dryness, to which 3 mL of DMSO was added to make it completely dispersed in DMSO, and 15 mg of mercaptoacetic acid was added, and it was stirred at room temperature for 30 h. The solution at the end of the reaction was transferred to a dialysis membrane with a molecular weight cut-off of 7000, and dialysed for 24 h to remove the DMSO and the excess mercaptoacetic acid, and finally obtain the NGF@NLF modified by mercaptoacetic acid, which was refrigerated at 4 °C and set aside. Acetic acid, the final NGF@NLF modified with mercaptoacetic acid was obtained, which was refrigerated at 4 °C and set aside. Example 12 Preparation of hydrophilic modied upconversion luminescent nanoparticles (UCNPQ 1. Preparation of homogeneous and well dispersed ß-NaGdF4:Yb / Tm / Er (NGF) nanoparticles (NPs) with core structure. The specific steps are as follows: A 100 mL three-necked round-bottomed flask was filled with 5 mL of 0.2 M Gd(CH3COz)3 solution, 1.5 mL of0.2M Yb(CH3COz)3 solution, 5 uL of0.2M Tm(CH3C02)3 solution and 50 uL of 0.2M Er(CH3C02)3 solution, and then added with 8 mL ofOA and 12 mL of OED. The reaction was carried out by increasing the temperature to 150 °C for 40 min under argon protection to obtain the OA precursor. °C and reacted for 40 min to obtain lanthanide-doped OA precursors. After reduced to room temperature, a mixture of 5.6 mL, 0.4M of NH4F methanol solution and 2 mL, 1 M of NaOH methanol solution was slowly added to the vial and reacted at 50 °C for 30 min to generate nanocores. Subsequently, the temperature was raised to 100 °C for evacuation operation to remove methanol and water from the solution. When bubbles no longer appeared in the solution, the temperature was set to 300 °C, the argon rate was adjusted to the minimum, and the reaction was carried out at this temperature for 1.5 h to finally generate ß-NaGdF4:Yb / Tm / Er nanoparticles. After centrifugation to remove excess OA and ODE, the precipitate was dispersed with 8 mL of cyclohexane and set aside. 2. Preparation of homogeneous and well dispersed ß-NaGdF4:Yb / Tm / Er@NaLuF4 luminescent nanoparticles (NGF@NLF) with core-shell structure.The specific steps are as follows: A 100 mL three-necked round-bottomed flask was taken, to which 5 mL, 0.2M of Lu(CH3COz)3 solution and 8 mL of OA and 12 mL of OED were added, and the OA precursor was synthesized by raising the temperature to 150 °C and reacting for 40 min under argon protection. Subsequently, a mixture of 5.6 mL, 0.4M NH4F methanol solution and 3 mL, 1 M NaOH methanol solution was added, and 7 mL of B- NaGdF4:Yb / Tm / Er (NGF) solution prepared above was continued to be added, and the reaction was carried out at 50 °C for 30 min in order to generate the nanocores. Subsequently, the temperature was raised to 100 °C for evacuation operation to remove methanol and water from the solution. When bubbles no longer appeared in the solution, the temperature was set to 300 °C, the argon rate was adjusted to the minimum, and the reaction was carried out at this temperature for 1.5 h to finally obtain ß-NaGdF4:Yb / Tm / Er@NaLuF4 (NGF@NLF). After centrifugation to remove excess OA and ODE, the precipitate was dispersed with 8 mL of cyclohexane to obtain ß-NaGdF4:Yb / Tm / Er@NaLuF4 luminescent nanoparticles with core-shell structure (NGF@NLF). 3. Hydrophilic modified ß-NaGdF4:Yb / Tm / Er@NaLuF4 luminescent nanoparticles. The specific preparation process is as follows: The cyclohexane solution containing 20 mg ofNGF@NLF prepared in step 2 was taken, and after the cyclohexane was evaporated dry, 3 mL of DMSO was added to it to make it completely dispersed in DMSO, and 36 mg of mercaptoundecanoic acid was added, and it was stirred at room temperature for 72 h. After the reaction was completed, the solution was transferred to a dialysis membrane with a molecular weight cut-off of 7000, and dialysed for 24 h to remove the DMSO and the excess of mercaptoundecanoic acid, and finally obtained mercaptoundecanoic acid-modified NGF@NLF, which was refrigerated at 4 °C for spare parts. Since the structures and properties of the NPs, NGF@NLF and hydrophilic modified NGF@NLF obtained by the preparation of Examples 1 to 12 are basically the same, the following is only an example of the effects of the NPs, NGF@NLF and hydrophilic modified NGF@NLF prepared in Example 1. TEM and energy analyser (EDS) were used to characterize the morphology and elemental composition. Figures 24 show, in turn, the TEM images, particle size distributions, energy spectra and elemental distribution measurements of the NPs prepared in Example 1.The TEM analysis of the NPs showed that the NPs were uniformly spherical and possessed good dispersion. The average particle size of the NPswas 11.74nm 1 0.43 nmwhen 100 NPswere calculated bythe particle size analysis software (FIG.2), and the particle size showed a concentrated distribution trend. In addition, the energy spectrum analysis and elemental energy spectrum distribution diagrams obtained from the EDS test showed that the NPs contained only six chemical elements, Na, Gd, F, Yb, Tm and Er (FIGs. 3 and 4). TEM and EDS were used to characterise the morphology and elemental composition. Figures 5~7 show, in turn, the TEM images, particle size distribution, energy spectra and elemental distribution measurements of NGF@NLF prepared in Example 1.TheTEM analysis ofNGF@NLF showed that the NGF@NLF was circular, with a regular morphology and good dispersion (FIG.5A). The average particle size of the NGF@NLF was 22.30 nm 1 1.03 nm when 100 NGF@NLF were calculated by the particle size analysis software (FIG.SB). From the EDS results, it was learnt that the constituent elements of this NGF@NLF added a new element, i.e., Lu, in addition to Na, Gd, F, Yb, Tm and Er as mentioned above (FIGs. 6 and 7). FIG.8 shows the IR spectra of NGF@NLF before and after modification and HS- PEGlOOO'COOH. Before modification of the detection antibody, the surface group of NGF@NLF is a carboxyl group (-COO' ), and after modification of the antibody this group is changed to an amide bond, i.e. -CO-NH- bond. Therefore, based on the absorption peak positions in FIG.8, it can be concluded that the VC=O and ôsN-H in the IR absorption peaks of NGF@NLF after antibody conjugation replaced the -COOH absorption peaks (1460 cm'1 and 1557 cm'1 ) on HS-PEGlOOO'COOH, which proved the generation ofamide bond, indicating the successful modification of the antibody. A fluorescence spectrometer was used to detect the relative fluorescence intensity of NGF and NGF@NLF before and after modification, and the photostability ofNGF@NLF beforeand after modificationwas verified. FIG.9showsthe photostability performance curves of NGF@NLF and NGF@NLF / HS-PEGlOOO'COOH. After 1 h of laser irradiation, the luminescence intensity ofNGF@NLF and NGF@NLF / HS-PEGlOOO'COOH decreased slightly (<1%) due to the influence ofsolvothermal warming caused by laser irradiation. Therefore, NGF@NLF and NGF@NLF / HS-PEGlOOO'COOH have excellent photostability and are ideal fluorescent markers. FIG.10 shows the uorescence spectra of NGF and NGF@NLF before and after modication, and no signicant changes were observed in their uorescence spectra before and after modication of NGF and NGF@NLF. Example of experiment 1 NGF@NLF (labelled as UCNPs) based immunouorescence assay for the determination of CRP content (shown in FIG.1). CRP-detecting antibody (FMU-CRP1) was used to label modified NGF@NLF. A mixture of 40 uL EDC (1 mM) and 40 uL NHS (1 mM) was added to 1 mL of HS'PEGlooo' COOH-modified NGF@NLF solution and stirred for 2 h at room temperature, and then the mixture was centrifuged to remove unreacted NHS, EDC, and by-products. . The solution was redispersed in 1 mL of 5 mM phosphate buffer (PB 7.4). Then, 50 ug of FMUCRP 1 (a kind gift from Prof. Boquan JIN of the People's Liberation Army Air Force Medical University(AFMU), China) was added, and the reaction was maintained overnight at 4 °C to obtain UCNP / HSPEGloooCOOH / FMUCRP1. The UCNP / HS PEG1000COOH / FMU-CRP1 was washed 3 times with deionised water to remove unbound antibody and resuspended with phosphate buffer (PBS, 8 mM phosphate buffer plus 145mM NaCl, pH 7.4) containing 0.02% NaN3 (w / v) to obtain upconversion luminescent reagents and stored at 4 °C for further use. The modification of the detected antibodies was characterised by Fourier transform infrared spectrophotometry (FTIR). 2. Establish the process of double antibody sandwich method. The specic steps are as follows: The CRP concentration was adjusted to 15.1625, 31.25, 62.5, 125, 250, 500, and 1000 pg / mL with 0.5% sodium caseinate solution, and then the above upconversion luminescence reagents were diluted in a 1:320 ratio. Next, the FMU-CRP-7 ((a kind gift from Prof. Boquan JIN of the People's Liberation Army Air Force Medical University(AFMU), China) capture antibody was diluted to 5 ug / mL with 0.05 M carbonate buffer (pH 9.6), and 100 uL of 5 ug / mL of the FMU-CRP7 capture antibody was added to an opaque ELISA well plate, placed at 4 °C and incubated overnight. The plate was washed 3 times with PBST, and then 100 uL of CRP standard and 10 human plasma samples (1:50,000 dilution) at concentrations of 15.1625, 31.25, 62.5, 125, 250, 500, and 1,000 pg / mL were added sequentially, and then incubated at 37 °C for 1 h. The plate was washed 3 times with PBST. Then, 100 uL of upconversion luminescent reagent UCNP / HSPEGlooo-COOH / FMU-CRP1 diluted with 0.5% sodium caseinate was added into the wells and incubated at 37 °C for 1 h. The wells were washed 3 times with PBST. Subsequently, a uorescence analyserwas used to collect 10 s uorescence signals at 650 nm in order to obtain the standard curve ofuorescence intensity versus CRP concentration. FIG.11 shows the standard curve of uorescence intensity versus concentration of CRP standard by ELISA method and using UCNPs as indicator. In the ELISA method, CRP showed good linearity in the range of 1~32 ng / mL with a linear equation of Y=0.0667X+0.269, a correlation coefcient of R2 =0.9935, and a lower limit ofdetection of 1 ng / mL. The linearity ofCRP was good in the range of 0.015~1 ng / mL, with a linear equation of Y=430.61X+8812.9. The correlation coefcient R2 =0.9944, and the lower limit of detection was 0.015 ng / mL. The results of human plasma samples are shown in Table 1. Glutaraldehyde was added to the opaque ELISA plates and xed at room temperature for 4 h. The plates were washed 3 times with PBST, air-dried at room temperature for 12 h, and then sealed and stored at room temperature and protected from light. 105 uorescence signals were collected at 650 nm by a uorescence analyser after 1 year, and the average attenuation rate of uorescence signals was 2.14%. Table 1 Results of Human Plasma Samples Sample fluorescence CRP concentration CRP concentration number intensity (pg / ml) in plasma samples (mg / L) 1 73986 151.33 7.57 2 42491 78.19 3.91 3 109589 234.01 11.70 4 57300 112.58 5.63 5 93381 196.37 9.82 6 224842 501.66 25.08 7 168789 371.49 18.57 8 84842 176.54 8.83 9 106278 226.32 11.32 10 81238 168.17 8.41 Example 2 Determination of SEB content by immunouorescence based on UCNPs SEB detection antibody (FMU-SEB -1) was used to label modified NGF@NLF. A mixture of40 uL EDC (1 mM) and 40 uL NHS (1 mM) was added to 1 mL of HS-PEGlOOO' COOH-modified NGF@NLF solution and stirred at room temperature for 2 h. The mixture was then centrifuged to remove unreacted NHS, EDC, and by-products . The solution was redispersed in 1 mL of 5 mM phosphate buffer (PB 7.4). Then, 50 ug of FMU-SEB -1 was added and the reaction was maintained overnight at 4 °C to obtain UCNP / HS-PEGlOOO'COOH / FMUSEBl. UCNP / HS-PEGlOOO'COOH / FMUSEBl was washed 3 times with deionised water to remove unconjugated antibodies and washed with phosphate buffer containing 0.02% NaN3 (w / v) ( PBS, 8mM phosphate buffer plus 145 mM NaCl, pH 7.4) to remove unconjugated antibodies and resuspended and stored at 4 °C for further use. 2. Establish the process of double-antibody sandwich method (as shown in FIG.1). The specific steps are as follows: The SEB concentration was adjusted to 6.25, 12.5, 25, 50, 100, 200, and 400 pg / mL with 0.5% sodium caseinate solution, and then the upconversion luminescent reagent was diluted in a 1:320 ratio. Next, the FMU-SEB-2 capture antibody (a kind gift from Prof. Boquan JIN of the People's Liberation Army Air Force Medical University(AFMU), China) was diluted to 5 ug / mL with 0.05 M carbonate buffer (pH 9.6), and 100 uL of 5 ug / mL of the FMU-SEB-2 capture antibody was added to a 96- well plate, which was placed at 4 °C and incubated overnight. The plate was washed 3 times with PBST, and then 100 uL of SEB standard and 10 SEB-contaminated milk samples (1:100 dilution) at concentrations of 6.25, 12.5, 25, 50, 100, 200, and 400 pg / mLwere added sequentially, and the platewas incubated at 37 °C for 1 h. The plate was washed 3 times with PBST. Then, 100 uL of upconversion luminescence detection reagent dilution was added into the wells, incubated at 37 °C for 1 h, and washed 3 times with PBST. Subsequently, a uorescence analyser was used to collect the uorescence signal at 650 nm for 10 s in order to obtain the standard curve of uorescence intensity versus SEB concentration. The results of SEB detection in milk samples are shown in Table 2. FIG.12 shows the standard curve of uorescence intensity versus the concentration of SEB standard with UCNPs as the indicator. SEB showed good linearity in the range of 6.25~400 pg / mL with the linear equation of Y=387.16X3786.7, correlation coefcient R2 =0.9956, and the lower limit of detection was 6.25 pg / mL. Glutaraldehyde was added to the opaque ELISA plates and xed at room temperature for 4 h. The plates were washed 3 times with PBST, air-dried at room temperature for 12 h, and then sealed and stored at room temperature and protected from light. 105 uorescence signals were collected at 650 nm by a uorescence analyser after 1 year, and the average attenuation rate of the uorescence signals was 1.98%. Table ZSEB Results of Milk Samples Sample Fluorescence SEB concentration Milk sample SEB number intensity (pg / ml) concentration (ug / L) 1 8916 32.81 3.28 2 7205 28.39 2.84 3 639 11.43 1.14 4 2745 16.87 1.69 5 17786 55.72 5.57 6 29745 86.61 8.66 7 34662 99.31 9.93 8 65379 178.65 17.87 9 12950 43.23 4.32 10 26071 77.12 7.71 Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention, and are not intended to be a limitation of the embodiments of the present invention. For those of ordinary skill in the art, other variations or changes in different forms may be made on the basis of the above description. It is neither necessary nor possible to exhaust all the embodiments herein. Any modications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall be included in the scope of protection of the claims of the present invention.

Claims

1. Hydrolyzed up-conversion luminescent nanoparticles, characterized by being prepared according to the following steps: Mixing lanthanide metal salts with oleic acid and 1-octadecene in a coordination reaction to produce an oleic acid precursor doped with lanthanide elements to acquire; Mixing the oleic acid precursor doped with lanthanide elements with an ammonium fluoride methanol solution and a sodium hydroxide methanol solution for performing a double decomposition reaction and a chemical adsorption reaction to form nanonuclei, followed by thermolysis to obtain core-upconversion nanoparticles; Mixing the oleic acid precursor doped with lanthanide elements with the core upconversion nanoparticles, ammonium fluoride methanol solution and sodium hydroxide methanol solution for performing a double decomposition reaction and a chemical adsorption reaction, followed by thermolysis to to obtain a core-shell structure up-conversion luminescent nanoparticles; Replacing the oleic acid on the surface of the core-shell structure up- conversion of luminescent nanoparticles by a mercapto-containing carboxylic acid to the To obtain hydrolyzed up-conversion luminescent nanoparticles.

2. Up-conversion luminescent nanoparticles according to claim 1, characterized because the lanthanide metal salt is a combination of one or more of gadolinium salt, ytterbium salt, thulium salt, erbium salt or lutetium salt.

3. Up-conversion luminescent nanoparticles according to claim 1 or 2, characterized because the mercapto-containing carboxylic acid is selected from mercaptopolyethylene glycol carboxylic acid, mercaptoacetic acid or mercapto- undecanoic acid.

4. Up-conversion luminescent nanoparticles according to claim 1, 2 or 3, characterized by: The coordination reaction takes place in an inert environment, at 100200°C for 20- 60 minutes; The double decomposition reaction and chemical adsorption reaction take place in a inert environment, at 30-75°C for 2045 minutes; The thermolysis takes place in an inert environment, at 200500°C for 1.0-2.0 o'clock.

5. Up-conversion luminescent nanoparticles according to claim 1, 2, 3 or 4, characterized in that the replacement is carried out as follows: Adding the mercapto-containing carboxylic acid to the dispersion of the core-shell structure up-conversion luminescent nanoparticles, stirring at room temperature for 30-72 hours, removing the solvent and the excess mercapto-containing carboxylic acid to the hydrolyzed modified up- conversion to obtain luminescent nanoparticles.

6. Method for obtaining hydroelectric modified upconversion luminescent nanoparticles, characterized by the following steps: Mixing lanthanide metal salts with oleic acid and 1-octadecene in a coordination reaction to produce an oleic acid precursor doped with lanthanide elements to acquire; Mixing the oleic acid precursor doped with lanthanide elements with an ammonium fluoride methanol solution and a sodium hydroxide methanol solution for performing a double decomposition reaction and a chemical adsorption reaction to form nanonuclei, followed by thermolysis to obtain core-upconversion nanoparticles; Mixing the oleic acid precursor doped with lanthanide elements with the core upconversion nanoparticles, ammonium fluoride methanol solution and sodium hydroxide methanol solution for performing a double decomposition reaction and a chemical adsorption reaction, followed by thermolysis to to obtain a core-shell structure up-conversion luminescent nanoparticles; Replacing the oleic acid on the surface of the core-shell structure up- conversion of luminescent nanoparticles by a mercapto-containing carboxylic acid to the To obtain hydrolyzed up-conversion luminescent nanoparticles.

7. A method according to claim 6, characterized in that the lanthanide metal salt is a combination of one or more of gadolinium salt, ytterbium salt, thulium salt, erbium salt or lutetium salt.

8. Method according to claim 6 or 7, characterised in that the mercapto- containing carboxylic acid is selected from mercaptopolyethylene glycol carboxylic acid, mercaptoacetic acid or mercapto-undecanoic acid.

9. Method according to claim 6, 7 or 8, characterised in that: The coordination reaction takes place in an inert environment, at 100200°C for 20- 60 minutes; The double decomposition reaction and chemical adsorption reaction take place in a inert environment, at 30-75°C for 2045 minutes; The thermolysis takes place in an inert environment, at 200500°C for 1.0-2.0 o'clock.

10. Method according to claim 6, 7, 8 or 9, characterised in that the replacement is performed as follows: Adding the mercapto-containing carboxylic acid to the dispersion of the core-shell structure up-conversion luminescent nanoparticles, stirring at room temperature for 30-72 hours, removing the solvent and the excess mercapto-containing carboxylic acid to the hydrolyzed modified up- conversion to obtain luminescent nanoparticles.

11. An up-conversion luminescent detection reagent characterized by being obtained by hydrolyzed up-conversion luminescent nanoparticles according to any one of claims 1 to 5 or obtained according to the method to be coupled to a detection antibody according to any one of claims 6 to 10.

12. Up-conversion luminescent detection reagent according to claim 11, characterized in that the detection antibody is a C-reactive protein detection- antibody or a Staphylococcus aureus enterotoxin B detection antibody. Up-conversion luminescent detection reagent according to claim 12, characterized by the C-reactive protein detection antibody being FIVlU-CRP-l and the Staphylococcus aureus enterotoxin B detection antibody FlVlU-SEB-1 is. Up-conversion luminescent detection reagent according to claim 11, 12 or 13 characterized by the fact that the coupling is performed by activating the carboxyl groups on the surface of the hydrolytically modified up-conversion luminescent nanoparticles with an amid bond coupling reagent and then a to form a covalent bond with the amino groups on the detection antibody.

15. An immunofluorescence detection method characterized by the following steps: Adding a capture antibody to an opaque polystyrene plate and let it stand so that the capture antibody attaches to the polystyrene plate, then adding the sample to be tested or antigen standards of different concentrations and incubate, adding of the up-conversion luminescent detection reagent according to any one of claims 11 to and with 14 and incubate to form a sandwich complex of the capture antibody, the up-conversion luminescent detection reagent and the antigen of the sample to be tested or to form antigen standards that adhere to the polystyrene plate; Performing a fluorescent analysis of the polystyrene plate fixed sandwich complex, measuring the fluorescent intensity of the up- conversion luminescent detection reagent and preparation of a standard curve with the fluorescence intensity and the concentration of the antigen standards; 5 Calculating the concentration of the sample to be tested based on the standard curve and the fluorescence intensity of the sample to be tested.