A rare earth down-conversion nanometer fluorescent probe, a preparation method and application thereof

Abstract

The application relates to the technical field of nanobiomaterials, and specifically discloses a rare earth down-conversion nanofluorescent probe and a preparation method and application thereof. The nanofluorescent probe has a core-shell structure, and comprises a core structure taking NaYF4 as a main body, a shell structure taking NaNdF4 as a main body which is coated outside the core structure, and a group structure or layer structure used for quantitative detection of a to-be-detected substance. The rare earth down-conversion nanofluorescent probe provided by the application has higher sensitivity, and is favorable for improving the accuracy of detection results.

Description

Technical Field

[0001] This application relates to the technical field of nanobiomaterials, and more specifically, to a rare-earth downconversion nanofluorescent probe, its preparation method, and its application. Background Technology

[0002] Fluorescence detection technology is a visualization technique that uses changes in the fluorescence properties of fluorescent probes as detection signals. It offers advantages such as simple operation and high resolution. By using luminescent probe labeling, dynamic detection of signals from analyte targets within cells and biological tissues can be achieved, enabling qualitative, quantitative, and localized analysis. Time-resolved fluorescence (TRF), as an emerging fluorescence detection method, offers advantages over steady-state fluorescence techniques, including higher resolution and lower background. TRF can control the excitation light to excite the fluorescent probe in pulses and selectively receive only the time region of delayed fluorescence, filtering out excitation light signals and solving the problem of excitation light interference.

[0003] Currently, various long-lived fluorescent probes have been studied in the field of time-resolved fluorescence bioimaging and detection. However, most fluorescent probes suffer from problems such as low energy conversion efficiency, weak fluorescence intensity, and poor biocompatibility. Therefore, there is an urgent need to develop novel rare-earth downconversion nanofluorescent probes suitable for early quantitative detection. Summary of the Invention

[0004] To improve the sensitivity of nanofluorescent probes and thus enhance the accuracy of detection results, this application provides a rare-earth downconversion nanofluorescent probe, its preparation method, and its application.

[0005] This application designs and synthesizes novel rare-earth-doped downconversion fluorescent nanoprobes by fine-tuning the matrix structure and co-doping with other ions, and studies and improves their emission and downconversion luminescence properties. The novel rare-earth-doped downconversion luminescent materials of this application are coated with different shells, and their morphology and luminescence properties are improved. They can be used for reagent kit development.

[0006] Firstly, this application provides a rare-earth downconversion nanofluorescent probe, employing the following technical solution:

[0007] A rare-earth downconversion nanofluorescent probe, characterized in that the nanofluorescence has a core-shell structure, comprising a core structure mainly composed of NaYF4; a shell structure mainly composed of NaNdF4 covering the core structure; and a group structure or layer structure for quantitative detection of the analyte.

[0008] In one specific embodiment, the method for preparing the functional group structure for quantitative detection of the analyte is as follows: removing the oleic acid ligand from the surface of the core-shell structure nanoparticles, modifying the shell structure surface with carboxyl groups, activating and coupling the antibody.

[0009] Optionally, a silicon coating process is included after removing the oleic acid ligand and before modifying the carboxyl group.

[0010] Alternatively, hydrochloric acid can be used to remove oleic acid ligands from the surface of core-shell structured nanoparticles.

[0011] Optionally, carboxyl groups can be modified on the surface of the shell structure using APTES and succinic anhydride.

[0012] Optionally, TEOS can be used for silicon encapsulation.

[0013] In one specific embodiment, the nanofluorescent probe has a core-shell structure: from the inside out, it consists of: a core structure mainly composed of NaYF4; a first shell structure mainly composed of NaNdF4 covering the core structure; a second shell structure mainly composed of C covering the first shell structure; and a third shell structure mainly composed of PEI and FA covering the second shell structure.

[0014] This application utilizes matrix modulation and elemental doping to prepare downconversion fluorescent nanoprobes with excellent downconversion luminescence properties. By coating the fluorescent nanoprobes with different shells, a high-performance downconversion fluorescent nanoprobe is ultimately developed.

[0015] The rare earth downconversion nanofluorescent probe provided in this application has a core structure based on NaYF4, which has better sensitivity and can more accurately measure the content of SAA in the sample than the detection result based on NaGdF4, thus improving the accuracy of the detection result.

[0016] In addition, the first shell structure of the rare earth downconversion nanofluorescent probe provided in this application is based on NaNdF4, which has better sensitivity and can more accurately measure the content of SAA in the sample than the detection results based on NaYF4, NaYbF4, NaErF4, and NaGdF4, thus improving the accuracy of the detection results.

[0017] The rare-earth downconversion nanofluorescent probes of this application have a second-shell structure dominated by C and a third-shell structure dominated by PEI and FA, respectively. This makes the rare-earth downconversion nanofluorescent probes more sensitive and can more accurately measure the content of SAA in the sample than the detection results of the second-shell structure dominated by NaYF4, NaYbF4, NaErF4, or NaNdF4, thus improving the accuracy of the detection results.

[0018] In one specific embodiment, the core structure is NaYF4:Er,Yb,Nd nanoparticles.

[0019] In one specific embodiment, the nanofluorescent probe has a particle size of less than 100 nm.

[0020] In one specific embodiment, the nanofluorescent probe has a particle size in the range of 65-95 nm.

[0021] In one specific embodiment, the nanofluorescent probe has a particle size in the range of 70-90 nm.

[0022] In one specific embodiment, the nanofluorescent probe has a particle size in the range of 75-85 nm.

[0023] The fluorescent nanoprobes of this application have small and uniform particle sizes, below 100 nm, and emit light within the second near-infrared window. When the particle size of the fluorescent nanoprobes is within the above range, they exhibit better sensitivity and higher accuracy in detection results.

[0024] In one specific implementation, the particle size of the core structure is 10-50 nm.

[0025] In one specific embodiment, the thickness of the first shell structure is 20-30 nm.

[0026] In one specific embodiment, the thickness of the second shell structure is 10-20 nm.

[0027] In one specific embodiment, the thickness of the third shell structure is 10-30 nm.

[0028] The presence of a second or third shell structure in the rare-earth downconversion nanofluorescent probe provided in this application can effectively improve the sensitivity and accuracy of the detection results. When the nanofluorescent probe lacks a second or third shell structure, the obtained detection results have low sensitivity and are inaccurate. Furthermore, when the thickness of the second shell structure is controlled within the range of 10-20 nm, it exhibits better sensitivity and higher accuracy compared to second shell thicknesses less than 10 nm and greater than 20 nm. Similarly, when the thickness of the third shell structure is controlled within the range of 10-30 nm, it also exhibits better sensitivity and higher accuracy compared to second shell thicknesses less than 10 nm and greater than 30 nm.

[0029] Secondly, this application provides a method for preparing rare-earth downconversion nanofluorescent probes, employing the following technical solution:

[0030] A method for preparing rare-earth downconversion fluorescent nanoprobes includes the following steps: synthesis of NaYF4:Er,Yb,Nd nanoparticles, synthesis of NaYF4:Er,Yb,Nd@NaNdF4 nanoparticles, synthesis of carbon-shell-coated NaYF4:Er,Yb,Nd@NaNdF4@C nanoparticles, and synthesis of PEI and FA-coated DCNPs@C nanoparticles.

[0031] Among them, the NaYF4:Er,Yb,Nd nanoparticles and the NaYF4:Er,Yb,Nd@NaNdF4 nanoparticles were prepared by high-temperature thermal decomposition method, respectively.

[0032] The synthesis of the carbon-shell-coated NaYF4:Er,Yb,Nd@NaNdF4@C nanoparticles is specifically as follows: the NaYF4:Er,Yb,Nd@NaNdF4 nanoparticles obtained in step (2) of the oil phase are converted into NaYF4:Er,Yb,Nd@NaNdF4 nanoparticles in the aqueous phase using a ligand exchange method; then uniformly dispersed in a glucose aqueous solution and hydrothermally treated at 170-190℃ for 2.5-3.5h; the system is cooled to 30-35℃, and the product is collected by centrifugation to obtain the carbon-shell-coated NaYF4:Er,Yb,Nd@NaNdF4@C nanoparticles.

[0033] The synthesis of the PEI and FA-coated DCNPs@C nanoparticles is specifically as follows: PEI aqueous solution is mixed with the carbon-shell-coated NaYF4:Er,Yb,Nd@NaNdF4@C nanoparticles, and the mixture is magnetically stirred at room temperature for 24 hours. The precipitate is collected by centrifugation, washed, and dispersed in deionized water to obtain DCNPs@C@PEI aqueous solution. The carboxyl groups of FA are activated in an acidic environment and added to the DCNPs@C@PEI aqueous solution. After magnetic stirring at room temperature for 24 hours, the precipitate is collected by centrifugation, washed, and dispersed in deionized water to obtain PEI and FA-coated DCNPs@C nanoparticles, i.e., rare earth downconversion fluorescent nanoprobes.

[0034] In one specific embodiment, in the synthesis of the NaYF4:Er,Yb,Nd nanoparticles, the molar ratio of Y, Er, Yb, and Nd is (0.75-0.80):(0.01-0.04):(0.15-0.20):(0.01-0.03).

[0035] In one specific embodiment, in the synthesis of the NaYF4:Er,Yb,Nd nanoparticles, the molar amount of Nd is 8-12 times the molar amount of Nd in the NaYF4:Er,Yb,Nd nanoparticles.

[0036] Thirdly, this application provides an application of a rare earth downconversion nanofluorescent probe, which can be used as a fluorescent marker in the field of biolabeling or in the process of biofluorescence imaging, and can also be used in the preparation of kits and reagent cards for quantitative detection of analytes.

[0037] In one specific implementation, the organism includes any one or more of protein molecules and / or nucleic acid molecules.

[0038] In summary, this application has the following beneficial effects:

[0039] This application utilizes matrix modulation and elemental doping to prepare novel downconversion fluorescent nanoprobes with excellent downconversion luminescence properties. By coating the fluorescent nanoprobes with different shells, a novel downconversion fluorescent nanoprobe with superior performance was ultimately developed. The prepared probes possess the following characteristics: 1. Particle size below 100 nm; 2. Emission located in the second near-infrared window (1000-1700 nm); 3. Downconversion luminescence intensity reaching 10... 4 above.

[0040] By combining novel rare-earth-doped downconversion fluorescent nanoprobes with immunochromatography, highly sensitive detection of analytes can be achieved, with the aim of enabling early screening for diseases.

[0041] The nano-fluorescent probe designed and synthesized in this application has a small and uniform particle size and emits light in the second near-infrared window. Compared with other fluorescent probes, it has higher sensitivity, which is beneficial to improving the accuracy of detection results and is suitable for high-sensitivity biological detection applications. Attached Figure Description

[0042] Figure 1 This is the standard curve for human serum amyloid A.

[0043] Figure 2 The zeta potential detection results of the rare earth downconversion nanofluorescent probe provided in this application.

[0044] Figure 3 The FTIR detection results of the rare earth downconversion nanofluorescent probe provided in this application.

[0045] Figure 4 Stability test results of the rare earth downconversion nanofluorescent probe provided in this application in different solutions (black and white).

[0046] Figure 5 Stability test results of the rare earth downconversion nanofluorescent probe provided in this application in different solutions (color).

[0047] Figure 6 Stability test results of the rare earth downconversion nanofluorescent probe provided in this application in different solutions (black and white).

[0048] Figure 7 Stability test results of the rare earth downconversion nanofluorescent probe provided in this application in different solutions (color).

[0049] Figure 8 The fluorescence spectral detection results of the rare earth downconversion nanofluorescent probe provided in this application. Detailed Implementation

[0050] This application provides a rare-earth downconversion nanofluorescent probe, its preparation method, and its application.

[0051] I. Preparation methods of rare earth downconversion nanofluorescent probes and their application in the preparation of in vitro diagnostic reagent strips.

[0052] Regarding rare earth downconversion nanofluorescent probes

[0053] This rare-earth downconversion fluorescent nanoprobe has a core-shell structure, consisting of a core structure primarily composed of NaYF4 and a shell structure primarily composed of NaNdF4 surrounding the core structure. Carboxyl groups are modified on the surface of the shell structure to activate and conjugate an antibody.

[0054] Preparation methods of rare earth downconversion nanofluorescent probes

[0055] The preparation method of this rare-earth downconversion nanofluorescent probe specifically includes the following steps:

[0056] (1) Synthesis of core NaYF4:Er,Yb,Nd nanoparticles

[0057] Spherical NaYF4:Er,Yb,Nd nanoparticles were prepared by high-temperature thermal decomposition and used as the core structure of rare-earth downconversion fluorescent nanoprobes. The molar ratio of Y, Er, Yb, and Nd was (0.75-0.80):(0.01-0.04):(0.15-0.20):(0.01-0.03).

[0058] (2) Synthesis of NaYF4:Er,Yb,Nd@NaNdF4 (DCNPs) nanoparticles

[0059] NaYF4:Er,Yb,Nd@NaNdF4 (DCNPs) nanoparticles were prepared by high-temperature thermal decomposition. The nanoparticles have a core-shell structure, consisting of, from the inside out: the NaYF4:Er,Yb,Nd nanoparticle core prepared in step (1) and a NaNdF4 shell structure. The molar amount of Nd is 8-12 times that in the NaYF4:Er,Yb,Nd nanoparticles.

[0060] (3) Deoleic acid

[0061] The oleic acid ligands on the surface of NaYF4:Er,Yb,Nd@NaNdF4 (DCNPs) nanoparticles were removed by hydrochloric acid, and then the nanoparticles were dispersed in ethanol for later use.

[0062] (4) Modify carboxyl groups on the surface of the shell structure

[0063] APTES and succinic anhydride were used to modify the carboxyl groups of nanoparticles. Specifically, APTES (aminopropyltriethoxysilane) was added dropwise to an ethanol solution containing nanoparticles under oil bath conditions and ultrasonically stirred. Then, a succinic anhydride acetone solution was added and ultrasonically stirred to obtain carboxyl-modified fluorescent nanoprobes.

[0064] In one specific embodiment, the modification process is as follows: the above-treated nanoparticles are dispersed in ethanol, and 20-40 μL of APTES (aminopropyltriethoxysilane, 5 times the volume of TEOS) is slowly added dropwise to the ethanol solution containing nanoparticles under oil bath conditions at 50-70℃, followed by ultrasonic stirring for 4-6 hours.

[0065] The ultrasonically treated reaction solution was centrifuged three times (first time with ethanol, second and third times with acetone), tilting the tube as much as possible to sonicate off the nanoparticles from the tube wall. The nanoparticles were then re-dissolved in acetone and sonicated again, followed by rinsing with acetone. 0.5-1.5 mL of a 50 mg / mL succinic anhydride-acetone solution was added, and the mixture was sonicated and stirred overnight. After the reaction was complete, the nanoparticles were centrifuged, washed, and the precipitate was dispersed in a small amount of water to obtain the carboxyl-modified fluorescent nanoprobe.

[0066] (5) Activation and conjugation of antibodies

[0067] The carboxyl-modified fluorescent nanoprobe was sonicated for 1-2 min, centrifuged at high speed (12000-14000 rpm) for 5-15 min, and the precipitate was washed with 10-100 mM MES solution with a pH of 5.0-7.0. The nanoprobe was then sonicated for 2-3 min. 20-100 mg / mL carbodiimide was added and mixed for 5-10 min. Then 20-100 mg / mL NHS was added and mixed for 10-20 min. The nanoprobe was then centrifuged at 12000-14000 rpm for 5-15 min. The precipitate was then washed with 10-100 mM MES solution with a pH of 5.0-7.0 to obtain the activated fluorescent nanoprobe.

[0068] After sonicating the activated fluorescent nanoprobe for 1-2 min, add SAA monoclonal antibody 1 at a ratio of 50-200 μg / 200 μl, mix well for 1-3 h, block with 10-50 mM Tris-HCl blocking buffer containing 0.5% BSA at pH 7.5-8.5 for 0.5-1 h, centrifuge at high speed (12000-14000 rpm) for 5-15 min, wash and resuspend with 10-50 mM Tris-HCl storage buffer containing 0.5% NaCl, 0.5% BSA, and 0.2% Tween-20 at pH 7.5-8.5, and the nanoprobe is obtained. Store at 4 °C in the dark.

[0069] Furthermore, in the above preparation method, after the deoleic acid step in step (3) and before the carboxyl group modification step on the shell structure surface in step (4), a silicon coating step may also be included, specifically as follows: The deoleic acid-treated nanoparticles are placed in the following solution and stirred for 8 hours, and centrifuged to obtain the silicon-coated nanoparticles. The solution is a mixture of ethanol, water, ammonia, and 5% TEOS (ethyl silicate) ethanol solution in a volume ratio of 10:1:0.2:0.05.

[0070] II. Preparation methods of rare earth downconversion nanofluorescent probes and their application in the preparation of products for in vivo detection.

[0071] Regarding rare earth downconversion nanofluorescent probes

[0072] This rare-earth downconversion fluorescent nanoprobe has a core-shell structure, consisting of, from the inside out: a core structure dominated by NaYF4; a first shell structure dominated by NaNdF4 covering the core structure; a second shell structure dominated by C covering the first shell structure; and a third shell structure dominated by PEI and FA covering the second shell structure.

[0073] Preparation methods of rare earth downconversion nanofluorescent probes

[0074] The preparation method of this rare-earth downconversion nanofluorescent probe specifically includes the following steps:

[0075] (1) Synthesis of core NaYF4:Er,Yb,Nd nanoparticles

[0076] Spherical NaYF4:Er,Yb,Nd nanoparticles were prepared by high-temperature thermal decomposition and used as the core structure of rare-earth downconversion fluorescent nanoprobes. The molar ratio of Y, Er, Yb, and Nd was (0.75-0.80):(0.01-0.04):(0.15-0.20):(0.01-0.03).

[0077] (2) Synthesis of NaYF4:Er,Yb,Nd@NaNdF4 (DCNPs) nanoparticles

[0078] NaYF4:Er,Yb,Nd@NaNdF4 (DCNPs) nanoparticles were prepared by high-temperature thermal decomposition. The nanoparticles have a core-shell structure, consisting of, from the inside out: the NaYF4:Er,Yb,Nd nanoparticle core prepared in step (1) and a NaNdF4 shell structure. The molar amount of Nd is 8-12 times that in the NaYF4:Er,Yb,Nd nanoparticles.

[0079] (3) Synthesis of carbon-shell-coated NaYF4:Er,Yb,Nd@NaNdF4@C (DCNPs@C) nanoparticles The above NaYF4:Er,Yb,Nd@NaNdF4@C nanoparticles have a core-shell structure, which consists of the following from the inside out: the NaYF4:Er,Yb,Nd nanoparticle core prepared in step (1), the NaNdF4 shell structure, and the carbon shell structure.

[0080] The core-shell nanoparticles (oil phase) obtained in step (2) were converted into core-shell nanoparticles (aqueous phase) using the ligand exchange method; the core-shell nanoparticles (aqueous phase) were then uniformly dispersed in a glucose aqueous solution and hydrothermally treated at 170-190℃ for 2.5-3.5h; the system was cooled to 30-35℃, and the product was collected by centrifugation to obtain carbon-shell coated DCNPs (DCNPs@C) nanoparticles for later use.

[0081] (4) Synthesis of DCNPs@C (DCPF) nanoparticles coated with polyethyleneimine (PEI) and folic acid (FA)

[0082] The DCNPs@C nanoparticles coated with polyethyleneimine (PEI) and folic acid (FA) have a core-shell structure, which consists of the following from the inside out: the NaYF4:Er,Yb,Nd nanoparticle core prepared in step (1), the NaNdF4 shell structure, the carbon shell structure, and the shell structure with PEI and FA as the main components.

[0083] Mix the PEI aqueous solution with the DCNPs@C nanoparticles obtained in step (3), stir magnetically at room temperature for 24 h, collect the precipitate by centrifugation, wash and disperse in deionized water to obtain DCNPs@C@PEI aqueous solution for later use.

[0084] The carboxyl groups of FA were activated in an acidic environment and added to an aqueous solution of DCNPs@C@PEI. After stirring magnetically for 24 hours at room temperature, the precipitate was collected by centrifugation, washed, and dispersed in deionized water to obtain PEI and FA-coated DCNPs@C(DCPF) nanoparticles, i.e. rare earth downconversion fluorescent nanoprobes.

[0085] Applications of rare earth downconversion nanofluorescent probes

[0086] The rare-earth downconversion nanofluorescent probes provided in this application can be used as fluorescent markers in the field of biolabeling or in biofluorescence imaging. Herein, "biolabeling" includes any one or more of protein molecules and / or nucleic acid molecules. This application also provides the application of the above-mentioned rare-earth downconversion nanofluorescent probes in the preparation of kits and reagent cards for the quantitative detection of analytes.

[0087] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0088] The present application will be further described in detail below with reference to embodiments, comparative examples and performance test results.

[0089] Example

[0090] Example 1

[0091] This embodiment provides a rare-earth downconversion fluorescent nanoprobe. This rare-earth downconversion fluorescent nanoprobe can be used for in vitro detection of reagent strips.

[0092] The preparation method of the above-mentioned rare-earth downconversion nanofluorescent probes specifically includes the following steps:

[0093] (1) Synthesis of core NaYF4:Er,Yb,Nd nanoparticles

[0094] Spherical NaYF4:Er,Yb,Nd nanoparticles (specifically NaYF4:2%Er,18%Yb,1%Nd nanoparticles) were prepared using a high-temperature thermal decomposition method and used as the core structure for a rare-earth downconversion fluorescent nanoprobe. The particle size of the core structure was 20 nm.

[0095] The specific steps are as follows: First, add 0.79 mmol Y(CH3COO)3·xH2O, 0.02 mmol Er(CH3COO)3·xH2O, 0.18 mmol LYb(CH3COO)3·xH2O, and 0.01 mmol Nd(CH3COO)3·xH2O to a three-necked flask containing 6 mL OA (oleic acid) and 15 mL ODE (octadecene) to obtain a reaction mixture. Under a nitrogen atmosphere, heat the reaction mixture to 120°C and stir for 20 min until completely dissolved. Then cool the solution to 50-55°C to obtain a cooled solution.

[0096] Dissolve 2.5 mmol NaOH and 4 mmol NH4F in 5 mL methanol (MeOH), and add the solution to the cooled solution. Stir the mixture at 50 °C for 30 min. Raise the temperature to 100 °C to remove excess MeOH.

[0097] The mixture was then heated to 300℃ under nitrogen protection and reacted for 90 min. After cooling to room temperature, the product was collected by centrifugation at 6000 rpm for 10 min, yielding core NaYF4:Er,Yb,Nd nanoparticles. The product was washed three times with an ethanol-cyclohexane mixture and dispersed in 4 mL of cyclohexane for later use.

[0098] (2) Synthesis of NaYF4:Er,Yb,Nd@NaNdF4 (DCNPs) nanoparticles

[0099] NaYF4:Er,Yb,Nd@NaNdF4 (DCNPs) nanoparticles were prepared by high-temperature thermal decomposition. The nanoparticles have a core-shell structure, consisting of, from the inside out: the NaYF4:Er,Yb,Nd nanoparticle core prepared in step (1) and the NaNdF4 shell structure. The thickness of the NaNdF4 shell structure is 25 nm.

[0100] The specific steps are as follows: First, add 0.5 mmol Nd(CH3COO)3·xH2O to a three-necked flask containing 6 mL OA and 15 mL ODE to obtain a reaction mixture. Under a nitrogen atmosphere, heat the reaction mixture to 120°C and stir for 20 min until completely dissolved. Then cool the solution to 50-55°C to obtain a cooled solution.

[0101] Add 2 mL of cyclohexane solution containing NaYF4:Er,Yb,Nd nanoparticles to the cooled solution above; then dissolve 1.25 mmol NaOH and 2 mmol NH4F in 5 mL MeOH and quickly add it to the cooled solution above, keep at 50°C and stir again for 30 min; raise the temperature to 100°C to remove excess MeOH.

[0102] The mixture was then heated to 300℃ under nitrogen protection and reacted for 90 min. After cooling to room temperature, the product was collected by centrifugation at 6000 rpm for 10 min, yielding core-shell structured NaYF4:Er,Yb,Nd@NaNdF4 nanoparticles. The nanoparticles were washed three times with an ethanol-cyclohexane mixture, and then dispersed in 4 mL of cyclohexane for later use.

[0103] (3) Deoleic acid

[0104] The prepared NaYF4:Er,Yb,Nd@NaNdF4 (DCNPs) nanoparticles were dispersed in 0.1M HCl solution and allowed to stand at room temperature for 1 day to remove oleic acid ligands on the surface of the nanoparticles. The nanoparticles were then extracted by centrifugation at 14000 rpm, allowed to stand for 30 min, and then dispersed in ethanol for later use.

[0105] (4) Modify carboxyl groups on the surface of the shell structure

[0106] The treated nanoparticles were dispersed in ethanol and centrifuged at 7300 rpm for 10 min, repeated three times. Finally, the nanoparticles were dispersed in 5 mL of ethanol. The ethanol solution containing the nanoparticles was added to a round-bottom flask under oil bath conditions at 60 °C and stirred. 30 μL of LAPTES (aminopropyltriethoxysilane, 5 times the volume of TEOS) was slowly added dropwise, and the flask was covered with plastic wrap and ultrasonically stirred for 5 h.

[0107] The ultrasonically treated reaction solution was centrifuged three times (first time with ethanol, second and third times with acetone), tilting the tube as much as possible to sonicate the nanoparticles off the tube wall. 3 ml of the solution was reconstituted and sonicated again, followed by rinsing with 3 ml of acetone. 6 ml of the solution was transferred to a flat-bottomed vial, and 1 ml of a 50 mg / mL succinic anhydride-acetone solution was added. The mixture was sonicated and stirred overnight. After the reaction was complete, the solution was centrifuged at 7000 rpm for 10 min, three times (first time with acetone, second and third times with water). The precipitate was dispersed in a small amount of water to obtain the carboxyl-modified fluorescent nanoprobe.

[0108] (5) Activation and conjugation of antibodies

[0109] The carboxyl-modified fluorescent nanoprobe was sonicated for 2 min, centrifuged at 14000 rpm for 10 min, and the precipitate was washed with 50 mM MES solution at pH 7.0. The nanoprobe was then sonicated for 2 min. 50 mg / mL carbodiimide was added and mixed for 10 min. Then 50 mg / mL NHS was added and mixed for 10 min. The nanoprobe was then centrifuged at 14000 rpm for 10 min. The precipitate was washed with 50 mM MES solution at pH 7.0 to obtain the activated fluorescent nanoprobe.

[0110] After sonicating the activated fluorescent nanoprobe for 2 min, add SAA monoclonal antibody 1 at a ratio of 100 μg / 200 μl, mix for 2 h, block with 50 mM Tris-HCl blocking buffer containing 0.5% BSA at pH 7.5 for 1 h, centrifuge at high speed (14000 rpm) for 10 min, wash and resuspend with 50 mM Tris-HCl storage buffer containing 0.5% NaCl, 0.5% BSA, and 0.2% Tween-20 at pH 7.5 to obtain the antibody-conjugated fluorescent nanoprobe, and store at 4 °C in the dark.

[0111] Example 2

[0112] This embodiment provides a rare-earth downconversion fluorescent nanoprobe. The difference between this embodiment and Embodiment 1 is that, after the oleic acid removal step in step (3) and before the carboxyl group modification step on the shell structure surface in step (4), a silicon coating step may be included, specifically as follows: The oleic acid-treated nanoparticles are placed in the following solution and stirred for 8 hours, then centrifuged to obtain the silicon-coated nanoparticles. This solution is obtained by mixing ethanol, water, ammonia, and 5% TEOS (ethyl silicate) ethanol solution in a volume ratio of 10:1:0.2:0.05.

[0113] Example 3

[0114] This embodiment provides a rare-earth downconversion fluorescent nanoprobe. This rare-earth downconversion fluorescent nanoprobe can be used to prepare products for in vivo detection.

[0115] The preparation method of the above-mentioned rare-earth downconversion nanofluorescent probes specifically includes the following steps:

[0116] (1) Synthesis of core NaYF4:Er,Yb,Nd nanoparticles

[0117] Spherical NaYF4:Er,Yb,Nd nanoparticles (specifically NaYF4:2%Er,18%Yb,1%Nd nanoparticles) were prepared using a high-temperature thermal decomposition method and used as the core structure for a rare-earth downconversion fluorescent nanoprobe. The particle size of the core structure was 20 nm.

[0118] The specific steps are as follows: First, add 0.79 mmol Y(CH3COO)3·xH2O, 0.02 mmol Er(CH3COO)3·xH2O, 0.18 mmol LYb(CH3COO)3·xH2O, and 0.01 mmol Nd(CH3COO)3·xH2O to a three-necked flask containing 6 mL OA (oleic acid) and 15 mL ODE (octadecene) to obtain a reaction mixture. Under a nitrogen atmosphere, heat the reaction mixture to 120°C and stir for 20 min until completely dissolved. Then cool the solution to 50-55°C to obtain a cooled solution.

[0119] Dissolve 2.5 mmol NaOH and 4 mmol NH4F in 5 mL methanol (MeOH), and add the solution to the cooled solution. Stir the mixture at 50 °C for 30 min. Raise the temperature to 100 °C to remove excess MeOH.

[0120] Then, under nitrogen protection, the mixture was heated to 300℃ and reacted for 90 min. After cooling to room temperature, the product was collected by centrifugation at 6000 rpm for 10 min to obtain the core NaYF4:Er,Yb,Nd nanoparticles. The nanoparticles were washed three times with an ethanol-cyclohexane mixture, and the resulting product was dispersed in 4 mL of cyclohexane for later use.

[0121] (2) Synthesis of NaYF4:Er,Yb,Nd@NaNdF4 (DCNPs) nanoparticles

[0122] NaYF4:Er,Yb,Nd@NaNdF4 (DCNPs) nanoparticles were prepared by high-temperature thermal decomposition. The nanoparticles have a core-shell structure, consisting of, from the inside out: the NaYF4:Er,Yb,Nd nanoparticle core prepared in step (1) and the NaNdF4 shell structure. The thickness of the NaNdF4 shell structure is 25 nm.

[0123] The specific steps are as follows: First, add 0.5 mmol Nd(CH3COO)3·xH2O to a three-necked flask containing 6 mL OA and 15 mL ODE to obtain a reaction mixture. Under a nitrogen atmosphere, heat the reaction mixture to 120°C and stir for 20 min until completely dissolved. Then cool the solution to 50-55°C to obtain a cooled solution.

[0124] Add 2 mL of cyclohexane solution containing NaYF4:Er,Yb,Nd nanoparticles to the cooled solution above; then dissolve 1.25 mmol NaOH and 2 mmol NH4F in 5 mL MeOH and quickly add it to the cooled solution above, keep at 50°C and stir again for 30 min; raise the temperature to 100°C to remove excess MeOH.

[0125] The mixture was then heated to 300℃ under nitrogen protection and reacted for 90 min. After cooling to room temperature, the product was collected by centrifugation at 6000 rpm for 10 min, yielding core-shell structured NaYF4:Er,Yb,Nd@NaNdF4 nanoparticles. The nanoparticles were washed three times with an ethanol-cyclohexane mixture, and then dispersed in 4 mL of cyclohexane for later use.

[0126] (3) Synthesis of carbon-shell-coated NaYF4:Er,Yb,Nd@NaNdF4@C (DCNPs@C) nanoparticles

[0127] The NaYF4:Er,Yb,Nd@NaNdF4@C nanoparticles described above have a core-shell structure, consisting of, from the inside out: the NaYF4:Er,Yb,Nd nanoparticle core prepared in step (1), the NaNdF4 shell structure, and the carbon shell structure. The thickness of the carbon shell structure is 10 nm.

[0128] The specific steps are as follows: The core-shell structured nanoparticles (oil phase) obtained in step (2) are converted into core-shell structured nanoparticles (aqueous phase) by ligand exchange method: The core-shell structured nanoparticles (oil phase) obtained in step (2) are dispersed in 6 mL HCl (0.1 M) and stirred for 1.5 h; 5 mL anhydrous diethyl ether is added for extraction for 10 min, repeated three times, the upper organic phase is discarded, the lower aqueous phase product is collected by centrifugation, and washed 3 times with deionized water to obtain core-shell structured nanoparticles (aqueous phase).

[0129] The core-shell structured nanoparticles (aqueous phase) were then uniformly dispersed in a 0.2M glucose aqueous solution, maintaining a concentration of 2 mg / mL, to obtain a mixed solution. The mixed solution was then transferred to a 25 mL autoclave and hydrothermally treated at 180 °C for 3 h. Finally, the system was cooled to 30-35 °C, and the product was collected by centrifugation. After washing three times with deionized water by centrifugation, the product was dispersed in 4 mL of deionized water to obtain an aqueous solution of carbon-shell-coated DCNPs (DCNPs@C) nanoparticles for later use.

[0130] (4) Synthesis of DCNPs@C (DCPF) nanoparticles coated with polyethyleneimine (PEI) and folic acid (FA)

[0131] The aforementioned polyethyleneimine (PEI) and folic acid (FA) coated DCNPs@C nanoparticles have a core-shell structure, consisting of, from the inside out: the NaYF4:Er,Yb,Nd nanoparticle core prepared in step (1), a NaNdF4 shell structure, a carbon shell structure, and a shell structure dominated by PEI and FA. The thickness of the shell structure dominated by PEI and FA is 20 nm.

[0132] Add the PEI aqueous solution (concentration of 500 mg / L) to the aqueous solution of the carbon shell-coated DCNPs (DCNPs@C) nanoparticles obtained in step (3), stir magnetically for 24 h at room temperature, and collect the precipitate by centrifugation at 10000 rpm for 10 min; wash three times with deionized water by centrifugation, and disperse in 4 mL of deionized water to obtain the DCNPs@C@PEI aqueous solution for later use.

[0133] FA, EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide), and NHS (N-hydroxythiosuccinimide) were then mixed in a molar ratio of 1:1:3. The carboxyl groups of folic acid were activated in an acidic environment for 1.5 h. The mixture was then added to an aqueous solution of DCNPs@C@PEI and magnetically stirred at room temperature for 24 h. The precipitate was collected by centrifugation at 10,000 rpm for 10 min. After washing three times with deionized water, the precipitate was dispersed in 4 mL of deionized water to obtain PEI and FA-coated DCNPs@C (DCPF) nanoparticles, which are rare earth downconversion fluorescent nanoprobes.

[0134] Example 4

[0135] Example 4 provides a rare-earth downconversion fluorescent nanoprobe. Its difference from Example 1 lies in that the core structure is predominantly NaYF4:Er,Yb. Details are shown in Table 1.

[0136] The preparation method of the above rare earth downconversion nanofluorescent probe differs from that of Example 1 in that Nd(CH3COO)3·xH2O is not added in step (1).

[0137] Example 5

[0138] Example 5 provides a rare-earth downconversion fluorescent nanoprobe. Its difference from Example 1 lies in that the core structure is predominantly NaYF4:Er. Details are shown in Table 1.

[0139] The preparation method of the above rare earth downconversion nanofluorescent probe differs from that of Example 1 in that Nd(CH3COO)3·xH2O and Yb(CH3COO)3·xH2O are not added in step (1).

[0140] Example 6

[0141] Example 6 provides a rare-earth downconversion fluorescent nanoprobe. Its difference from Example 1 lies in that the core structure is predominantly NaYF4. Details are shown in Table 1.

[0142] The preparation method of the above rare earth downconversion nanofluorescent probe differs from that of Example 1 in that Nd(CH3COO)3·xH2O, Yb(CH3COO)3·xH2O and Er(CH3COO)3·xH2O are not added in step (1).

[0143] Comparative Example

[0144] Comparative Example 1

[0145] Comparative Example 1 provides a rare-earth downconversion fluorescent nanoprobe. Its difference from Example 6 lies in that the first shell structure is primarily composed of NaErF4. Details are shown in Table 1.

[0146] The preparation method of the above rare earth downconversion nanofluorescent probe differs from that of Example 6 in that Er(CH3COO)3·xH2O is used instead of Nd(CH3COO)3·xH2O in step (2).

[0147] Comparative Example 2

[0148] Comparative Example 2 provides a rare-earth downconversion fluorescent nanoprobe. Its difference from Example 6 lies in that the first shell structure is primarily composed of NaYbF4. Details are shown in Table 1.

[0149] The preparation method of the above rare earth downconversion nanofluorescent probe differs from that of Example 6 in that Yb(CH3COO)3·xH2O is used instead of Nd(CH3COO)3·xH2O in step (2).

[0150] Comparative Example 3

[0151] Comparative Example 3 provides a rare-earth downconversion fluorescent nanoprobe. Its difference from Example 6 lies in that the first shell structure is primarily composed of NaYF4. Details are shown in Table 1.

[0152] The preparation method of the above rare earth downconversion nanofluorescent probe differs from that of Example 6 in that Y(CH3COO)3·xH2O is used instead of Nd(CH3COO)3·xH2O in step (2).

[0153] Comparative Example 4

[0154] Comparative Example 4 provides a rare-earth downconversion fluorescent nanoprobe. Its difference from Example 6 lies in that the first shell structure is primarily composed of NaGdF4. Details are shown in Table 1.

[0155] The preparation method of the above rare earth downconversion nanofluorescent probe differs from that of Example 6 in that Gd(CH3COO)3·xH2O is used instead of Nd(CH3COO)3·xH2O in step (2).

[0156] Comparative Example 5

[0157] Comparative Example 5 provides a rare-earth downconversion fluorescent nanoprobe. Its difference from Example 1 lies in that its core structure is predominantly NaGdF4. Details are shown in Table 1.

[0158] The preparation method of the above rare earth downconversion nanofluorescent probe differs from that of Example 1 in that only Gd(CH3COO)3·xH2O is added in step (1).

[0159] Table 1. Differences in the rare-earth downconversion nanofluorescent probe structures of Examples 1, 4-6 and Comparative Examples 1-5

[0160]

[0161] Performance Test Experiment 1

[0162] The rare-earth downconversion nanofluorescent probes provided in Examples 1-2, 4-6 and Comparative Examples 1-5 were used in the human serum amyloid A (SAA) assay kit (time-resolved fluorescence immunochromatography) to detect SAA (a sensitive indicator of early inflammation in infectious diseases) in isolated fresh serum samples.

[0163] 1. Components of the kit

[0164] The kit includes a test card, sample diluent, and ID card (calibration curve). The assays were performed using the AFS1000 dry fluorescence immunoassay analyzer from Guangzhou Lanbo Biotechnology Co., Ltd.

[0165] The test card is sequentially coated with a nitrocellulose membrane containing mouse anti-SAA monoclonal antibody (Hangzhou Huakui Jinpei Biotechnology Co., Ltd.) and goat anti-mouse IgG antibody (Jiangsu Bio-Tech Medical Instruments Technology Co., Ltd.), a glass fiber conjugate pad containing fluorescent microspheres of mouse anti-SAA monoclonal antibody (Hangzhou Huakui Jinpei Biotechnology Co., Ltd.), and absorbent paper. The fluorescent microspheres are the rare-earth downconversion fluorescent nanoprobes provided in the above examples and comparative examples.

[0166] The sample diluent was pH 8.0 Tris-HCl.

[0167] The calibration curve shown on the ID card is as follows: Figure 1 As shown. The specific method for preparing the calibration curve is as follows:

[0168] Human serum amyloid A antigen was measured and quantitatively diluted to a concentration of 150 mg / L with 20 mmol / L PBS containing 1% BSA. The SAA reference stock solution was removed and allowed to return to room temperature. The SAA reference stock solution was diluted with freshly prepared BSA-PBS buffer to obtain linear references of different concentrations. The linear references of different concentrations were tested, and a standard curve was plotted.

[0169] Lowest detection limit comparison: Perform 20 repeated measurements using a zero-value sample, calculate the mean M and standard deviation SD of the 20 results, and report the method's detection limit (M+2SD) as the blank mean plus twice the standard deviation.

[0170] 2. The detection method is as follows:

[0171] Preheat the fluorescence immunoassay analyzer for 5 minutes and place the test kit and fresh serum sample at room temperature until they reach room temperature before use. Insert the ID card into the analyzer. Use a pipette to add 5 μL of fresh serum sample to 300 μL of sample diluent and mix well. Add 80 μL of the mixed sample to the sample well of the test card, taking care to avoid generating significant air bubbles during aspiration and addition. Insert the test card into the test slot and start the detection program. The analyzer will automatically scan the test card (please strictly control the time from sample addition to detection to 15 minutes) and read the test results.

[0172] 3. Test Results

[0173] The test results are shown in Tables 2 and 3.

[0174] Table 2 SAA Standard Curve

[0175]

[0176] Table 3. Detection results of the examples and comparative examples.

[0177]

[0178] As shown in Table 4, by comparing the results of Examples 1, 4-6, and Comparative Example 5, it can be seen that the rare earth downconversion nanofluorescent probe provided in this application, with NaYF4 as the main core structure, has better sensitivity and can more accurately measure the SAA content in the sample than the one with NaGdF4 as the main core structure, thus improving the accuracy of the detection results. Furthermore, comparing the detection results of Examples 1 and 4-6, it can be seen that when the nanofluorescent probe has NaYF4:Er,Yb,Nd as the main core structure, it has even better sensitivity and obtains more accurate detection results.

[0179] By comparing the results of Example 1 and Comparative Examples 1-4, it can be seen that the shell structure of the rare earth downconversion nanofluorescent probe provided in this application is mainly composed of NaNdF4, which has better sensitivity. Compared with the detection results mainly composed of NaYF4, NaYbF4, NaErF4, and NaGdF4, it can more accurately measure the content of SAA in the sample to be tested, thus improving the accuracy of the detection results.

[0180] Performance Test 2

[0181] The following detections were performed using the rare-earth downconversion nanofluorescent probes provided in Example 3 above.

[0182] (1) Zeta potential and FTIR detection were performed on the following nanofluorescent probes respectively. The detection results are shown in Table 2 and... Figure 3 As shown.

[0183] The detected nanofluorescent probes are as follows:

[0184] NPs@C@PEI@FA — the nanofluorescent probe prepared in Example 3.

[0185] NPs@C@PEI – This differs from the nanofluorescent probe prepared in Example 3 in that its outermost layer is only coated with PEI.

[0186] NPs@C—that is, the difference from the nanofluorescent probe prepared in Example 3 is that the carbon shell is not coated with PEI and FA.

[0187] FEI can improve the bioidentity of fluorescent nanoprobes, while FA can enhance their targeting of tumors. Figure 2 and Figure 3It is evident that further coating with PEI and FA can result in a lower negative potential for the nanofluorescent probe while simultaneously increasing the intensity of the vibrational peak. From Figure 3 Furthermore, it can be seen that the fluorescent nanoparticles exhibit strong NH bending vibration peaks at 1650 nm, 1770-1750 nm, and 3000 nm. Specifically, the peak at 1650 nm represents a primary amine group, the peak at 1770-1750 nm represents a carboxyl group, and the peak at 3000 nm represents an amide group. The vibrational peaks of NPs@C@PEI and NPs@C@PEI@FA at 1770-1750 nm indicate that coating with PEI and FA can further enhance the carboxyl groups on the fluorescent nanoprobes.

[0188] (2) The nanofluorescent probes prepared in Example 3 were dispersed in different solutions (H2O, PBS, DMEM, FBS) and placed for 24 h. The detection results are as follows: Figure 4-7 As shown.

[0189] Depend on Figure 4-7 It can be seen that the fluorescent nanoprobes prepared in this application exhibit good stability within the experimental range. This further demonstrates that the fluorescent nanoprobes prepared in this application possess excellent stability.

[0190] (3) The nanofluorescent probes prepared in Example 3 were characterized and analyzed, such as... Figure 8 As shown.

[0191] Figure 8 The spectrum of the nanofluorescent probe is shown by... Figure 8 It is known that the nanofluorescent probe provided in this application has a relatively broad emission peak in the near-infrared II region, indicating that it has the potential for imaging in the NIRII region, and therefore has good potential for tumor imaging.

[0192] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A method for preparing a rare-earth downconversion nanofluorescent probe, characterized in that, The preparation method specifically includes the following steps: (1) Synthesis of core NaYF4:2%Er,18%Yb,1%Nd nanoparticles Spherical NaYF4:2%Er,18%Yb,1%Nd nanoparticles were prepared by high-temperature thermal decomposition and used as the core structure of rare earth downconversion fluorescent nanoprobes. (2) Synthesis of NaYF4:2%Er,18%Yb,1%Nd@NaNdF4 nanoparticles Core-shell nanoparticles of NaYF4:2%Er,18%Yb,1%Nd@NaNdF4 were prepared by high-temperature thermal decomposition. The molar amount of Nd in the shell was 8-12 times that in the NaYF4:2%Er,18%Yb,1%Nd nanoparticles. (3) Deoleic acid The oleic acid ligands on the surface of NaYF4:2%Er,18%Yb,1%Nd@NaNdF4 nanoparticles were removed by hydrochloric acid, and then the nanoparticles were dispersed in ethanol for later use. (4) Modify carboxyl groups on the surface of the shell structure Carboxyl groups of core-shell nanoparticles were modified using APTES and succinic anhydride. Specifically, APTES was added dropwise to an ethanol solution containing nanoparticles under oil bath conditions and ultrasonically stirred. Then, succinic anhydride acetone solution was added and ultrasonically stirred to obtain carboxyl-modified fluorescent nanoprobes. (5) Activation and conjugation of antibodies The carboxyl-modified fluorescent nanoprobe was sonicated for 1-2 min, centrifuged at 12000-14000 rpm for 5-15 min, and the precipitate was washed with 10-100 mM MES solution at pH 5.0-7.

0. The nanoprobe was then sonicated for 2-3 min. 20-100 mg / mL carbodiimide was added and mixed for 5-10 min, followed by 20-100 mg / mL NHS. The mixture was then centrifuged at 12000-14000 rpm for 5-15 min, and the precipitate was washed with 10-100 mM MES solution at pH 5.0-7.0 to obtain the activated fluorescent nanoprobe. The activated fluorescent nanoprobe was sonicated for 1-2 min, and SAA monoclonal antibody 1 was added at a ratio of 50-200 μg / 200 μl. The mixture was mixed for 1-3 h, and the nanoprobe was washed with 10-50 mM MES solution containing 0.5% BSA at pH 5.0-7.

0. After blocking with 7.5–8.5% Tris-HCl blocking buffer for 0.5–1 h, centrifuge at 12,000–14,000 rpm for 5–15 min. Wash and resuspend with 10–50 mM Tris-HCl preservation buffer containing 0.5% NaCl, 0.5% BSA, and 0.2% Tween-20 at pH 7.5–8.5 to obtain rare earth downconversion fluorescent nanoprobes, which should be stored at 4 °C in the dark.

2. The preparation method according to claim 1, characterized in that, After removing the oleic acid ligand and before modifying the carboxyl group, a silicon coating process is included, which is performed using TEOS.

3. The preparation method according to claim 1, characterized in that, The nanofluorescent probe has a particle size of less than 100 nm.

4. The rare earth downconversion fluorescent nanoprobe prepared by the preparation method according to any one of claims 1-3.

5. The application of the rare-earth downconversion nanofluorescent probe according to claim 4 in the preparation of fluorescent markers or in the process of biofluorescence imaging.

6. The application of the rare earth downconversion nanofluorescent probe according to claim 4 in the preparation of reagent kits and reagent cards for quantitative detection of analytes.

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