Tyramine signal amplification system based on rare earth fluorescent material and application of tyramine signal amplification system in immunodetection

Abstract

The invention discloses a tyramine signal amplification system based on a rare earth fluorescent material and application of the tyramine signal amplification system in immunodetection. Firstly, the preparation method of the tyrosine functionalized rare earth fluorescent nanoparticles is provided, and the prepared tyrosine functionalized rare earth fluorescent nanoparticles can realize a tyramine signal amplification system without combination of a biotin-avidin system, so that the biotin-avidin combination step of traditional tyramine signal amplification can be simplified; and meanwhile, the detection cost is also reduced. The invention further provides a method for hypersensitive digital fluorescence immunodetection based on tyramine signal amplification, synchronous detection of multiple targets can be realized, fluorescence images of the multiple targets can be synchronously acquired under the same excitation light source, and tedious operation of frequent optical filter switching in multiple digital immunodetection is avoided. Furthermore, the rare earth fluorescent material doped based on different lanthanide ions can be subjected to digital detection through time resolution.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunoassay, and more specifically, relates to a tyramide signal amplification system based on rare earth fluorescent materials and an application thereof in immunoassay. Background Art

[0002] Digital immunoassay technology is an immunoassay technology that uses the number of immune complexes as a quantitative method. The growing demand for ultrasensitive and multi-target detection has promoted the rapid development of digital immunoassay technology, especially digital immunoassay based on microdroplets or encoded fluorescent magnetic beads (Magnetite Bead, MB). This method partitions the signals of single or multiple reporters and reads them independently, which not only improves the sensitivity but also achieves absolute quantification of the target. The limitations of fluorescence detection channels and the accurate decoding of encoded microspheres are bottlenecks that digital immunoassays still face in practical applications.

[0003] Digital immunoassays mainly rely on fluorescent markers to identify targets, but are limited by the number of fluorescent channels, which limits the ability of a single test to simultaneously identify multiple targets. In the multiple digital immunoassays that have been reported so far, multiple fluorescent pigments or quantum dots are often used to achieve multi-target detection. Due to the large differences in the fluorescence spectra of these materials, different filters need to be frequently switched during the detection process, and software processing is relied on to synthesize the final image. In addition, the formation of microdroplets often relies on fine physical separation or additional microfluidics and microarray chip technology, which not only increases the cost of detection, but also increases the complexity of operation. Encoded microspheres are used to load immune complexes, and it is a technical challenge to accurately decode a large number of encoded microspheres in the microscope field. The images generated at the digital immunoassay site will have blurred objects, irregular shapes, different sizes, and large intensity differences. These factors make it difficult to accurately identify and quantify the information in the image using conventional image processing software, and it is impossible to effectively perform multi-dimensional analysis; in addition, image processing based on conventional software needs to be performed one by one, and each processing takes several minutes, and the process is relatively complex and cumbersome. The above factors limit the efficiency of digital immunoassay technology in practical applications.

[0004] Tyramide signal amplification (TSA) technology is an enzymatic detection method that uses horseradish peroxidase (HRP) to perform high-density in situ labeling of target proteins or nucleic acids. TSA is based on HRP, which can convert the phenolic group of tyramine into a short-lived highly reactive free radical in the presence of H2O2, and then quickly covalently bind to HRP and the tyrosine residues of nearby proteins without increasing nonspecific signals. The fluorescently labeled tyramide molecules are activated by the HRP labeled with the secondary antibody in the H2O2 environment, producing a large number of enzymatic reactions, so that the fluorescein binds to the protein residues around the tissue at the antigen-antibody binding site, forming a large amount of fluorescein deposition, and realizing signal amplification. TSA can fluorescently label multiple protein antigens by using different coupling dyes for multiple cycles of experiments, and realize the co-staining of multiple proteins on a tissue section. At present, in the tyramide signal amplification system, the surface modification of fluorescein with tyramine is all through the biotin-avidin system, which is cumbersome and costly.

[0005] Rare earth fluorescent materials are fluorescent materials whose main components are rare earth elements (a general term for 17 elements such as the scandium series in the IIIB group of the periodic table). With the development of nanotechnology, rare earth materials have been successfully prepared into nano fluorescent probes and have been used in immunoassays. However, rare earth fluorescent materials have not yet been used in tyramide signal amplification systems. Since there are no convenient modification groups on the surface of rare earth materials, direct binding to tyramide is prone to self-polymerization by relying solely on the amino groups of tyramide, and tyramide needs to be dissolved in an organic solvent, DMSO, which is not conducive to the use of rare earth fluorescent materials. Therefore, it is necessary to improve the tyramide signal amplification technology, research and develop a new type of tyramide signal amplification system based on rare earth fluorescent materials and its multi-target digital immunoassay technology, which can avoid frequent filter switching and complex microdroplet formation process, and overcome the limitations of on-site image precision analysis. Summary of the invention

[0006] The purpose of the present invention is to overcome the above-mentioned defects and shortcomings in the prior art and to provide a method for preparing tyrosine-functionalized rare earth fluorescent nanoparticles.

[0007] The second object of the present invention is to provide tyrosine-functionalized rare earth fluorescent nanoparticles prepared by the preparation method.

[0008] The third object of the present invention is to provide a tyramide signal amplification system based on rare earth fluorescent materials.

[0009] The fourth object of the present invention is to provide the application of tyrosine-functionalized rare earth fluorescent nanoparticles or tyramide signal amplification system in immunoassay.

[0010] The fifth object of the present invention is to provide a method for ultra-sensitive multi-color or time-resolved digital fluorescence immunoassay based on tyramide signal amplification.

[0011] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0012] The invention provides a method for preparing tyrosine (Tyr) functionalized rare earth fluorescent nanoparticles. In the preparation of the rare earth fluorescent nanoparticles, a surfactant O-phosphoethanolamine is added to prepare amino-modified rare earth fluorescent nanoparticles, and then tyrosine is added to modify the surface of the rare earth fluorescent nanoparticles with tyrosine through an activated ester method to obtain tyrosine-functionalized rare earth fluorescent nanoparticles. The rare earth fluorescent nanoparticles are down-conversion rare earth fluorescent chelates.

[0013] The present invention introduces surfactant O-phosphoethanolamine in the preparation process of rare earth fluorescent nanoparticles, so that the surface of the rare earth fluorescent nanoparticles is modified with amino groups, and then uses tyrosine instead of tyramine, so that tyrosine (Tyr) can be directly modified on the surface of the rare earth fluorescent nanoparticles, and tyrosine-functionalized rare earth fluorescent nanoparticles can be prepared. The tyrosine-functionalized rare earth fluorescent nanoparticles can achieve tyramide signal amplification without being combined with a biotin-avidin system, and can be directly mixed with hydrogen peroxide under the catalysis of horseradish peroxidase to achieve tyramide signal amplification, thereby obtaining a novel tyramide signal amplification system based on rare earth fluorescent materials. Therefore, the tyrosine-functionalized rare earth fluorescent nanoparticles provided by the present invention are applied in immunoassays based on tyramide signal amplification strategies, which can simplify the steps of traditional tyramide signal amplification biotin-avidin combination and also reduce the detection cost. At the same time, the present invention uses rare earth fluorescent nanoparticles as down-conversion rare earth fluorescent chelates, which are down-conversion rare earth fluorescent materials. They can be excited to produce fluorescence at wavelengths in the near-ultraviolet region. Down-conversion rare earth fluorescent materials have the advantages of stable luminescence, low toxicity, resistance to photochemical degradation, and long fluorescence lifetime. Therefore, they have advantages in application in the field of immunofluorescence detection.

[0014] Furthermore, the ligand in the down-conversion rare earth fluorescent chelate can be selected from one of NaYF4 and Y2O3, and the doped rare earth metal element is cerium and another lanthanide rare earth element, namely NaYF4:Ce, Ln or Y2O3:Ce, Ln.

[0015] Preferably, the other lanthanide rare earth element may be selected from terbium, erbium, thulium, samarium, europium or dysprosium.

[0016] Furthermore, the rare earth fluorescent nanoparticles are NaYF4 fluorescent nanoparticles co-doped with cerium and another lanthanide rare earth element. 3+ ,Ln 3+ .

[0017] Preferably, the rare earth fluorescent nanoparticles are NaYF4:0.5%Ce 3+ ,(0.5~80%)Ln 3+ , of which 0.5% Ce 3+ ,(0.5~80%)Ln 3+ Ce 3+ ,Ln 3+ The amount ratio of the substance in rare earth fluorescent nanoparticles.

[0018] NaYF4:Ce 3+ ,Ln 3+ The excited state lifetime is long, which is 10 times that of general fluorescent materials. 4 times, which can eliminate the short-life fluorescence background interference and help improve the signal-to-noise ratio in detection. 3+ )-doped nanoparticles have a longer excited state lifetime (Eu 3+ 、Sm 3+ and Dy 3+ The fluorescence peaks are sharp, with little overlap with the excitation spectrum, and the fluorescence spectrum is rich.

[0019] Preferably, the other lanthanide rare earth element is samarium, europium or dysprosium; that is, the rare earth fluorescent nanoparticles are NaYF4:Ce 3+ ,Ln 3+ (Ln=Eu, Sm or Dy). 3+ ,Ln 3+ (Ln = Eu, Sm or Dy) can produce red, green and blue fluorescence respectively under the same excitation wavelength of 265nm. Therefore, images with up to three fluorescences coexisting at the same time can be collected under the same excitation light source, and up to three targets can be detected synchronously. Furthermore, multi-target detection can also be performed by time resolution.

[0020] Preferably, the amino-modified NaYF4:Ce 3+ ,Ln 3+ The preparation method of (Ln = Eu, Sm or Dy) is as follows: O-phosphoethanolamine, NaCl, NH4F, YCl3·6H2O, CeCl3·6H2O and LnCl3·6H2O are added to ethylene glycol, mixed evenly, and subjected to hydrothermal reaction to obtain NaYF4:Ce,Ln.

[0021] The invention adopts rare earth fluorescent nanoparticles NaYF4:Ce 3+ ,Ln 3+ The surfactant O-phosphoethanolamine was introduced into the preparation of NaYF4:Ce 3+ ,Ln3+ The surface has amino groups, so NaYF4:Ce 3+ ,Ln 3+ Capable of directly surface modifying tyrosine.

[0022] Preferably, the molar ratio of NH4F, YCl3·6H2O, CeCl3·6H2O and LnCl3·6H2O is 1-20:1:1:1-160, that is, amino-modified rare earth fluorescent nanoparticles NaYF4:0.5%Ce 3+ ,(0.5~80%)Ln 3+ .

[0023] Preferably, the molar ratio of NH4F, YCl3·6H2O, CeCl3·6H2O and LnCl3·6H2O is 18:1:1:1, that is, the amino-modified rare earth fluorescent nanoparticles NaYF4:0.5%Ce 3+ , 0.5% Ln 3+ .

[0024] Preferably, the hydrothermal reaction is carried out at 180° C. for 4 hours.

[0025] Furthermore, the activated ester method is as follows: dissolving the amino-modified rare earth fluorescent nanoparticles in water, adding tyrosine (Tyr), mixing, adding EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) and sulfo-NHS (N-hydroxysulfosuccinimide), mixing, and reacting to obtain tyrosine-functionalized rare earth fluorescent nanoparticles.

[0026] Preferably, the mass ratio of the amino-modified rare earth fluorescent nanoparticles to tyrosine is 1:1.

[0027] The present invention provides tyrosine-functionalized rare earth fluorescent nanoparticles prepared by any of the above-mentioned preparation methods.

[0028] Preferably, the tyrosine-functionalized rare earth fluorescent nanoparticles are Tyr-NaYF4:Ce 3+ ,Ln 3+ , more preferably Tyr-NaYF4:Ce 3+ ,Ln 3+ (Ln=Eu, Sm or Dy).

[0029] The present invention also provides a tyramide signal amplification system based on rare earth fluorescent materials, comprising any of the above-mentioned tyrosine-functionalized rare earth fluorescent nanoparticles, horseradish peroxidase and hydrogen peroxide.

[0030] The present invention also provides the use of any of the above-mentioned tyrosine-functionalized rare earth fluorescent nanoparticles or the tyramide signal amplification system in immunoassay.

[0031] Furthermore, the immunoassay is a multiplex fluorescent immunoassay.

[0032] The present invention provides a method for ultra-sensitive multicolor or time-resolved digital fluorescence immunoassay based on tyramide signal amplification, which is achieved by combining TSA strategy and ultra-sensitive digital fluorescence immunoassay technology, and specifically comprises the following steps:

[0033] S1. Modifying carboxyl magnetic beads with capture antibody cAb to obtain magnetic capture probe MB@cAb, incubating the target to be detected and MB@cAb, and magnetically separating unbound substances; the capture antibody cAb is an antibody that can specifically recognize the target to be detected;

[0034] S2. Adding a detection antibody dAb-HRP carrying a horseradish peroxidase label, the detection antibody specifically binds to the capture antibody cAb in S1 to form an immune complex MB@cAb-target-dAb-HRP, and magnetically separating unbound substances;

[0035] S3. adding any of the above tyrosine-functionalized rare earth fluorescent nanoparticles and H2O2, and causing a TSA reaction after HRP catalysis, wherein the tyrosine-functionalized rare earth fluorescent nanoparticles cover the surface of the immune complex described in step S2 to form magnetic rare earth fluorescent composite nanoparticles, and magnetically separating unbound substances;

[0036] S4. Under the excitation light source, the magnetic rare earth fluorescent composite nanoparticles obtained in step S3 generate fluorescence, and images are collected and analyzed.

[0037] Furthermore, the magnetic capture probe MB@cAb is synthesized by an activated ester method, and the specific steps are: taking carboxyl magnetic beads and washing them with MES (morpholineethanesulfonic acid buffer), adding EDC and sulfo-NHS to mix, adding cAb (mass ratio of carboxyl magnetic beads: cAb = 10:1), mixing for reaction, adding a blocking agent (glycine), mixing for reaction, washing and resuspending with MES to obtain MB@cAb.

[0038] Preferably, the particle size of the hydroxy magnetic beads MB is 2 μm to 4 μm.

[0039] Furthermore, the MB@cAb concentration in step S1 is 100-500 μg / mL.

[0040] Furthermore, the co-incubation time in step S1 is 30 to 60 minutes, and the temperature is 25 to 40°C.

[0041] Furthermore, the magnetic separation time in step S1 is 1 to 2 minutes.

[0042] Furthermore, the dAb-HRP in step S2 is used by diluting the stock solution (1 mg / mL) by 3000 to 6000 times.

[0043] Furthermore, the concentration of the tyrosine-functionalized rare earth fluorescent nanoparticles in step S3 is 500-5000 μg / mL.

[0044] Furthermore, the amount of H2O2 in step S3 is 0.01% to 0.03%.

[0045] Furthermore, the TSA reaction in step S3 is carried out at room temperature for 10 to 20 minutes.

[0046] Furthermore, the above reactions were all carried out in PBS (phosphate buffer, 0.01 M, pH 7.4).

[0047] Furthermore, steps S1 to S3 are repeated multiple times to obtain a plurality of magnetic rare earth fluorescent composite nanoparticles containing different targets to be detected, and the magnetic rare earth fluorescent composite nanoparticles containing different targets to be detected are made to generate fluorescence under an excitation light source, and images are collected and analyzed; that is, in step S3, tyrosine-functionalized rare earth fluorescent nanoparticles that generate different fluorescence are used respectively, so that a plurality of different targets to be detected can be detected simultaneously.

[0048] Preferably, in step S3, tyrosine-functionalized rare earth fluorescent nanoparticles that can produce different fluorescence at the same excitation wavelength are used respectively to realize the acquisition of images with multiple fluorescences existing simultaneously under the same excitation light source, thereby synchronously detecting multiple targets to be detected; further preferably, in step S3, tyrosine-functionalized rare earth fluorescent nanoparticles Tyr-NaYF4:Ce 3+ ,Ln 3+ (Ln=Eu, Sm or Dy), the Tyr-NaYF4:Ce 3+ ,Ln 3+ (Ln = Eu, Sm or Dy) can produce three colors of fluorescence at the same excitation wavelength, so it can detect three different targets to be tested, and collect images with three kinds of fluorescence at the same time under the same excitation light source, without frequently switching filters to collect images. Furthermore, multi-target detection can also be performed through time resolution.

[0049] Furthermore, the analysis in step S4 includes image processing, feature extraction, cluster analysis and regression fitting, and the analysis is all performed based on artificial intelligence algorithms.

[0050] Furthermore, the image processing is to first reduce the dimension of the color image and convert it into a grayscale image, then use a high-pass filtering algorithm to perform edge enhancement and convert it into a binary image, and finally perform semantic segmentation on the binary image to separate the magnetic rare earth fluorescent composite nanoparticles from the background.

[0051] Furthermore, the feature extraction is to extract the mean, standard deviation, skewness, 95th percentile, peak value and energy.

[0052] Furthermore, the cluster analysis is linear discriminant analysis, K-nearest neighbor or Gaussian mixture model analysis.

[0053] In the method of ultra-sensitive multicolor or time-resolved digital fluorescence immunoassay based on tyramide signal amplification of the present invention, the tyrosine-functionalized rare earth fluorescent nanoparticles realize the specific deposition of rare earth fluorescent nanoparticles on the surface of the immune complex carrying the horseradish peroxidase label based on the tyrosine signal amplification strategy, thereby realizing the amplification of the fluorescence signal. Further, in combination with carboxylated magnetic beads, a droplet-free digital fluorescence immunoassay method based on magnetic rare earth fluorescent composite nanoparticles is established; at the same time, the magnetic rare earth fluorescent composite nanoparticles formed by immune binding of tyrosine-functionalized rare earth fluorescent nanoparticles that can produce different fluorescence at the same excitation wavelength are used to realize the synchronous detection of multiple targets, and the fluorescent images of multiple targets are synchronously collected under the same excitation light source, avoiding the cumbersome operation of frequent filter switching in multiple digital immunoassays. Alternatively, the time-resolved image is obtained by controlling the fluorescence image acquisition time. Then, the advantage of the artificial intelligence algorithm that can quickly parse and batch process images is used to establish a portable detection platform based on artificial intelligence to directly perform image processing and feature extraction of the results on site, so as to realize accurate and efficient clustering analysis and concentration prediction of the target.

[0054] The present invention also provides an artificial intelligence-based portable detection platform for implementing the above method, specifically, placing the magnetic rare earth fluorescent composite nanoparticles obtained in step S3 of the above method on the portable detection platform, exciting the magnetic rare earth fluorescent composite nanoparticles to produce fluorescence, and collecting and analyzing images.

[0055] Specifically, the portable detection platform includes a dark box shooting platform equipped with a stage, an inverted fluorescence microscope, an excitation light source, a filter, an objective lens, etc.; the camera can be connected to a laptop computer and perform image processing, feature extraction, cluster analysis and regression fitting based on the artificial intelligence algorithm in the laptop computer.

[0056] Preferably, the dark box is made of readily available polystyrene material and is fully enclosed inside to provide a stable and excellent image acquisition environment.

[0057] Preferably, the height of the loading platform is 20 cm.

[0058] Preferably, the wavelength of the excitation light source is 250-280 nm.

[0059] Preferably, the camera model is MSHOT MS60.

[0060] Compared with the prior art, the present invention has the following beneficial effects:

[0061] (1) The present invention provides a tyrosine-functionalized rare earth fluorescent nanoparticle, which can realize a tyramide signal amplification system without binding through a biotin-avidin system. Therefore, the tyrosine-functionalized rare earth fluorescent nanoparticle provided by the present invention is applied in an immunoassay based on a tyramide signal amplification strategy, which can simplify the biotin-avidin binding step of the traditional tyramide signal amplification and also reduce the detection cost.

[0062] (2) The present invention also provides a method for ultra-sensitive multicolor or time-resolved digital fluorescence immunoassay based on tyramide signal amplification. The method is based on the magnetic rare earth fluorescent nanocomposite particles formed by the TSA strategy, which abandons the complex and cumbersome microdroplet generation steps in conventional digital immunoassay technology and eliminates the dependence on devices such as microfluidics and microarray chips. Further, by using tyrosine-functionalized rare earth fluorescent nanoparticles that can produce different fluorescence at the same excitation wavelength to form magnetic rare earth fluorescent nanocomposite particles through immune binding, it is possible to achieve synchronous detection of multiple targets, synchronously collect fluorescent images of multiple targets under the same excitation light source and can be resolved by time, avoiding the cumbersome operation of frequent filter switching in multiple digital immunoassays. In addition, the portable detection platform based on artificial intelligence performs image processing, cluster analysis and concentration analysis, overcomes the limitations of conventional image software on-site detection, and can achieve efficient, accurate and multi-dimensional result analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 This is the digital fluorescent immunoassay process based on magnetic rare earth fluorescent composite nanoparticles described in the present invention.

[0064] Figure 2 The portable detection platform (A) of the present invention and a schematic diagram of its internal structure (B).

[0065] Figure 3 (A) UV-visible full wavelength scanning spectrum, (B) magnetic adsorption and (C) Zeta potential diagram of MB@cAb prepared in Example 4 before and after modification.

[0066] Figure 4 Bright field (A) and dark field (B) observation results of MB@NaYF4:Ce,Sm prepared in Example 5 under an inverted fluorescence microscope (scale bar: 10 μm). DETAILED DESCRIPTION

[0067] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0068] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0069] Example 1 Tyrosine (Tyr) functionalized NaYF4:Ce, Ln (Tyr-NaYF4:Ce 3+ ,Ln 3+ )

[0070] 1. Amino-modified NaYF4:Ce 3+ ,Ln 3+ Preparation of (Ln=Eu, Sm or Dy):

[0071] In 30 mL of ethylene glycol (99.7 wt%), 1 mmol of O-phosphoethanolamine, 1 mmol of NaCl and a certain molar ratio (18:1:1:1) of NH4F, YCl3·6H2O, CeCl3·6H2O, and LnCl3·6H2O (Ln = Eu, Sm, and Dy) were added; the mixture was fully mixed for 5 min, and the mixture was transferred to a hydrothermal reactor and reacted at 180 °C for 4 h to obtain NaYF4:Ce 3+ ,Ln 3+ ; It can be dried at 60℃ for 8h to obtain fluorescent powder for long-term storage.

[0072] Specifically, the preparation method of amino-modified NaYF4:Ce, Sm is as follows: 0.14 g of O-phosphoethanolamine, 0.0586 g of NaCl, and 30 mL of ethylene glycol are added to a beaker in sequence, and magnetic stirring is performed to mix; 0.3477 g of YCl3·6H2O, 0.022 g of CeCl3·6H2O, and 0.023 g of SmCl3·6H2O are added to the above-mentioned mixed system in sequence, and magnetic stirring is performed to mix; 0.148 g of NH4F is weighed and dissolved in 10 mL of ethylene glycol, and added to the above-mentioned mixed system, and magnetic stirring is performed for 5 min, and then the entire mixed system is transferred to a hydrothermal reactor and reacted at 180°C for 4 h; after cooling to room temperature, the hydrothermal reactor is opened, and the product is transferred to a centrifuge tube, and washed twice with ethanol and pure water to obtain amino-modified rare earth fluorescent nanoparticles NaYF4:0.5%Ce 3+ ,0.5%Sm 3+ .

[0073] Adjust the amount of NH4F, YCl3·6H2O, CeCl3·6H2O and CeCl3·6H2O to prepare NaYF4:0.5%Ce 3+ ,(0.5~80%)Sm3+ .

[0074] Amino-modified NaYF4:Ce 3+ ,Eu 3+ 、NaYF4:Ce 3+ ,Dy 3+ The synthesis can refer to the preparation method of the above-mentioned amino-modified NaYF4:Ce,Sm.

[0075] 2. Tyr-functionalized NaYF4:Ce 3+ ,Ln 3+ (Tyr-NaYF4:Ce 3+ ,Ln 3+ ) is prepared by the activated ester method, which can be prepared according to the following steps:

[0076] 5 mg of amino-modified NaYF4:Ce 3+ ,Ln 3+ Dissolve in pure water, treat with ultrasound for 15 minutes, add 5 mg of Tyr while stirring magnetically, mix well, add 50 μg of cold EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) and 0.55 mg of sulfo-NHS (N-hydroxysulfosuccinimide), mix well for 15 minutes, react at low temperature for 3 hours, wash with ethanol and pure water in turn to obtain the product.

[0077] According to the above steps, Tyr-NaYF4:Ce 3+ ,Sm 3+ 、Tyr-NaYF4:Ce 3+ ,Eu 3+ 、Tyr-NaYF4:Ce 3+ ,Dy 3+ .

[0078] Example 2 Tyrosine (Tyr) functionalized NaYF4:Ce, Ln (Tyr-NaYF4:Ce 3+ ,Ln 3+ )

[0079] 1. Amino-modified NaYF4:Ce 3+ ,Ln 3+ Preparation of (Ln = Tb, Er or Tm):

[0080] In 30 mL of ethylene glycol (99.7 wt%), 1 mmol of O-phosphoethanolamine, 1 mmol of NaCl and a certain molar ratio (18:1:1:1) of NH4F, YCl3·6H2O, CeCl3·6H2O, and LnCl3·6H2O (Ln = Tb, Er or Tm) were added; the mixture was fully mixed for 5 min, and the mixture was transferred to a hydrothermal reactor and reacted at 180 °C for 4 h to obtain NaYF4:Ce 3+ ,Ln 3+ (Ln=Tb, Er or Tm); it can be dried at 60℃ for 8h to obtain phosphor for long-term storage.

[0081] 2. Tyr-functionalized NaYF4:Ce 3+ ,Ln 3+ (Tyr-NaYF4:Ce 3+ ,Ln 3+ ) is prepared by the activated ester method, which can be prepared according to the following steps:

[0082] 5 mg of the amino-modified NaYF4:Ce prepared above was added 3+ ,Ln 3+ (Ln = Tb, Er or Tm) was dissolved in pure water, and after ultrasonic treatment for 15 minutes, 5 mg of Tyr was added while magnetic stirring, and after mixing, 50 μg of cold EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) and 0.55 mg of sulfo-NHS (N-hydroxysulfosuccinimide) were added and mixed for 15 minutes. After reacting at low temperature for 3 hours, it was washed with ethanol and pure water in turn to obtain the product.

[0083] According to the above steps, Tyr-NaYF4:Ce 3+ ,Tb 3+ 、Tyr-NaYF4:Ce 3+ ,Er 3+ 、Tyr-NaYF4:Ce 3+ ,Tm 3+ .

[0084] Example 3 Tyrosine (Tyr) functionalized Y2O3:Ce, Ln (Tyr-Y2O3:Ce 3+ ,Ln 3+ )

[0085] 1. Amino-modified Y2O3:Ce 3+ ,Ln 3+ Preparation of (Ln = Eu, Tb or Tm):

[0086] In 30 mL of ethylene glycol (99.7 wt%), 1 mmol of O-phosphoethanolamine, 1 mmol of NaCl and a certain molar ratio (18:1:1) of YCl3·6H2O, CeCl3·6H2O, and LnCl3·6H2O (Ln = Eu, Tb or Tm) were added; the mixture was fully mixed for 5 min, and the mixture was transferred to a hydrothermal reactor and reacted at 180 °C for 4 h to obtain Y2O3:Ce 3+ ,Ln 3+ ; It can be dried at 60℃ for 8h to obtain fluorescent powder for long-term storage.

[0087] Specifically, the preparation method of amino-modified Y2O3:Ce,Eu is as follows: 0.14g of O-phosphoethanolamine, 0.0586g of NaCl, and 30mL of ethylene glycol are added to a beaker in sequence, and magnetic stirring is performed to mix; 0.3477g of YCl3·6H2O, 0.022g of CeCl3·6H2O, and 0.023g of EuCl3·6H2O are added to the above mixed system in sequence, and magnetic stirring is performed to mix for 5 minutes, and then the entire mixed system is transferred to a hydrothermal reactor and reacted at 180°C for 4 hours; after cooling to room temperature, the hydrothermal reactor is opened, the product is transferred to a centrifuge tube, and washed twice with ethanol and pure water to obtain amino-modified rare earth fluorescent nanoparticles Y2O3:0.5%Ce 3+ ,0.5%Eu 3+ .

[0088] Amino-modified Y2O3:0.5%Ce 3+ ,0.5%Tb 3+ 、Y2O3:0.5%Ce 3+ ,0.5%Tm 3+ The synthesis can refer to the preparation method of the above-mentioned amino-modified NaYF4:Ce,Sm.

[0089] 2. Tyr-functionalized Y2O3:Ce 3+ ,Ln 3+ (Tyr-Y2O3:Ce 3+ ,Ln 3+ ) is prepared by the activated ester method, which can be prepared according to the following steps:

[0090] 5 mg of the amino-modified Y2O3:Ce prepared above was added 3+ ,Ln 3+ (Ln=Eu, Tb or Tm) was dissolved in pure water, and after ultrasonic treatment for 15 minutes, 5 mg of Tyr was added while magnetic stirring, and after mixing, 50 μg of cold EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) and 0.55 mg of sulfo-NHS (N-hydroxysulfosuccinimide) were added and mixed for 15 minutes. After reacting at low temperature for 3 hours, it was washed with ethanol and pure water in turn to obtain the product.

[0091] According to the above steps, Tyr-Y2O3:Ce 3+ ,Eu 3+ 、Tyr-Y2O3:Ce 3+ ,Tb 3+ 、Tyr-Y2O3:Ce 3+ ,Tm 3+ .

[0092] Example 4 Ultrasensitive multicolor digital fluorescence immunoassay technology based on magnetic rare earth fluorescent composite nanoparticles and artificial intelligence

[0093] S1. Preparation of magnetic capture probe: Take 1 mg of carboxyl MB in a clean glass bottle and wash it twice with 500 μL MES (morpholineethanesulfonic acid buffer, 0.05 M, pH 6); add 50 μL cold EDC (4 mg / mL) and 0.55 mg sulfo-NHS and mix for 15 min; add 0.05 mg cAb (anti-African swine fever p30 / p54 / p72 primary antibody) and shake at RT for 4 h; add 50 μL blocking agent (glycine) and shake at RT for 0.5 h; after washing, resuspend it with 100 μL MES.

[0094] S2. Incubate MB@cAb and p30 / p54 / p72 in a 500 μL centrifuge tube with low adsorption capacity for 60 min, and then separate them on a magnetic stand for 2 min to remove unbound substances.

[0095] S3. Add 100 μL dAb-HRP (3000-fold dilution; anti-African swine fever p30 / p54 / p72 secondary antibody (1 mg / mL) to form an immune complex MB@cAb-p30 / p54 / p72-dAb-HRP, and separate the unbound substances by magnetic separation on a magnetic rack;

[0096] S4. Add 100 μL of NaYF4:0.5% Ce prepared in Example 1 3+ ,0.5%Sm 3+ and H2O2 mixture, TSA reaction occurs after HRP catalysis, a large amount of NaYF4:0.5%Ce 3+ ,0.5%Sm 3+ Covering the surface of immune complexes to form magnetic rare earth fluorescent composite nanoparticles, the unbound substances are magnetically separated by a magnetic frame;

[0097] S5. Transfer the above-mentioned magnetic rare earth fluorescent composite nanoparticles to a glass slide, place it on the stage of a portable detection platform to collect images, and process the images based on an artificial intelligence algorithm.

[0098] The above detection method process is as follows Figure 1The portable detection platform is as shown. Figure 2 As shown, it includes a dark box shooting platform equipped with a stage, an inverted fluorescence microscope, an excitation light source, filters, an objective lens, etc. The camera can be connected to a laptop computer and perform image processing, feature extraction, cluster analysis and regression fitting based on artificial intelligence algorithms.

[0099] Among them, the height of the stage is 20 cm, the wavelength of the excitation light source is 250-280 nm, and the camera model is MSHOT MS60; the dark box uses readily available polystyrene material and is fully enclosed inside to provide a stable and excellent image acquisition environment.

[0100] When performing image processing based on artificial intelligence algorithms, the color image is first reduced in dimension and converted into a grayscale image, then edge enhancement is performed using a high-pass filtering algorithm to convert it into a binary image, and finally the binary image is semantically segmented to separate the magnetic rare earth fluorescent composite nanoparticles from the background.

[0101] The image features extracted using AI are mean, standard deviation, skewness, 95th percentile, peak, and energy.

[0102] Use artificial intelligence to perform linear discriminant analysis, K-nearest neighbor and Gaussian mixture model clustering analysis and regression fitting.

[0103] Figure 3 Provides the carboxyl MB before and after binding to cAb ( Figure 3 A) UV-Visible full wavelength scanning spectrum, ( Figure 3 B)Magnetic adsorption and ( Figure 3 C) Zeta potential diagram. The changes in the UV absorption peak and Zeta potential indicate that the cAb was successfully modified onto the carboxyl MB and the magnetic capture probe was successfully synthesized; after adding the magnetic field, the dispersion state of the magnetic capture probe in the buffer solution changed, indicating that magnetic separation can be performed based on MB@cAb to shorten the detection time.

[0104] Further, in step S4, Tyr-NaYF4:Ce 3+ ,Ln 3+ (Ln = Sm) is replaced by Tyr-NaYF4:Ce prepared in Example 1, Example 2 or 3 3+ ,Ln 3+ (Ln=Eu or Dy), Tyr-NaYF4:Ce 3+ ,Ln 3+ (Ln=Tb, Er or Tm), Y2O3:Ce 3+ ,Ln 3+ Any one of (Ln=Eu, Tb or Tm) can also achieve tyramide signal amplification and detection of the target to be detected.

[0105] Example 5 Ultrasensitive time-resolved digital fluorescence immunoassay technology based on magnetic rare earth fluorescent composite nanoparticles and artificial intelligence

[0106] S1. Preparation of magnetic capture probe: Take 1 mg of carboxyl MB in a clean glass bottle and wash it twice with 500 μL MES (morpholineethanesulfonic acid buffer, 0.05 M, pH 6); add 50 μL cold EDC (4 mg / mL) and 0.55 mg sulfo-NHS and mix for 15 min; add 0.05 mg cAb (anti-African swine fever p30 / p54 / p72 primary antibody) and shake at RT for 4 h; add 50 μL blocking agent (glycine) and shake at RT for 0.5 h; after washing, resuspend it with 100 μL MES.

[0107] S2. Incubate MB@cAb and p30 / p54 / p72 in a 500 μL centrifuge tube with low adsorption capacity for 60 min, and then separate them on a magnetic stand for 2 min to remove unbound substances.

[0108] S3. Add 100 μL dAb-HRP (3000-fold dilution; anti-African swine fever p30 / p54 / p72 secondary antibody (1 mg / mL) to form an immune complex MB@cAb-p30 / p54 / p72-dAb-HRP, and separate the unbound substances by magnetic separation on a magnetic rack;

[0109] S4. Add 100 μL of NaYF4:0.5% Ce prepared in Example 1 3+ ,0.5%Ln 3+ and H2O2 mixture, TSA reaction occurs after HRP catalysis, a large amount of NaYF4:0.5%Ce 3+ ,0.5%Ln 3+ Covering the surface of immune complexes to form magnetic rare earth fluorescent composite nanoparticles, the unbound substances are magnetically separated by a magnetic frame;

[0110] S5. Transfer the above-mentioned magnetic rare earth fluorescent composite nanoparticles to a glass slide, place it on the stage of a portable detection platform, collect fluorescence images at 1ms, 3ms, and 10ms after excitation, and process the images based on an artificial intelligence algorithm.

[0111] Wherein, the portable detection platform described in step S5 is the portable detection platform described in Example 4.

[0112] When performing image processing based on artificial intelligence algorithms, the color image is first reduced in dimension and converted into a grayscale image, then edge enhancement is performed using a high-pass filtering algorithm to convert it into a binary image, and finally the binary image is semantically segmented to separate the magnetic rare earth fluorescent composite nanoparticles from the background.

[0113] The image features extracted using AI are mean, standard deviation, skewness, 95th percentile, peak, and energy.

[0114] Use artificial intelligence to perform linear discriminant analysis, K-nearest neighbor and Gaussian mixture model clustering analysis and regression fitting.

[0115] Further, in step S4, Tyr-NaYF4:Ce 3+ ,Ln 3+ (Ln = Sm) was replaced with Tyr-NaYF4:Ce prepared in Example 2 or 3 3+ ,Ln 3+ (Ln=Tb, Er or Tm), Y2O3:Ce 3+ ,Ln 3+ Any of the above (Ln=Eu, Tb or Tm) can also achieve tyramide signal amplification and detection of the target to be detected.

[0116] Example 6 Ultrasensitive digital fluorescence immunoassay technology based on magnetic rare earth fluorescent composite nanoparticles and artificial intelligence

[0117] S1. Preparation of magnetic capture probe: Take 1 mg of carboxyl MB in a clean glass bottle and wash twice with 500 μL MES; add 50 μL cold EDC (4 mg / mL) and 0.55 mg sulfo-NHS and mix for 15 min; add 0.05 mg cAb (anti-human IgM primary antibody) and shake at RT for 4 h; add 50 μL blocking agent (glycine) and shake at RT for 0.5 h; after washing, resuspend with 100 μL MES.

[0118] S2. MB@cAb and IgM were co-incubated in a 500 μL centrifuge tube with low adsorption capacity. After 60 min, the unbound substances were removed by magnetic separation on a magnetic stand for 2 min.

[0119] S3. Add 100 μL dAb-HRP (3000-fold dilution, anti-human IgM secondary antibody) to form the immune complex MB@cAb-IgM-dAb-HRP, and separate the unbound substances by magnetic separation on a magnetic rack;

[0120] S4. Add 100 μL Tyr-NaYF4:0.5% Ce 3+ ,0.5%Sm 3+ and H2O2 mixture, TSA reaction occurs after HRP catalysis, and a large amount of Tyr-NaYF4:Ce 3+ ,Sm 3+ Covering the surface of immune complexes to form magnetic rare earth fluorescent composite nanoparticles, the unbound substances are magnetically separated by a magnetic frame;

[0121] S5. Transfer the magnetic rare earth fluorescent composite nanoparticles to a glass slide and place it on the stage of a portable detection platform to collect images.

[0122] When performing image processing based on artificial intelligence algorithms, the color image is first reduced in dimension and converted into a grayscale image, then edge enhancement is performed using a high-pass filtering algorithm to convert it into a binary image, and finally the binary image is semantically segmented to separate the magnetic rare earth fluorescent composite nanoparticles from the background.

[0123] The image features extracted using AI are mean, standard deviation, skewness, 95th percentile, peak, and energy.

[0124] Use artificial intelligence to perform linear discriminant analysis, K-nearest neighbor and Gaussian mixture model clustering analysis and regression fitting.

[0125] MB@NaYF4:Ce,Sm was observed by fluorescence microscopy under bright field ( Figure 4 A) The dispersed magnetic rare earth fluorescent composite nanoparticles are visible, and the dispersion is good. Figure 4 B) The fluorescence of magnetic rare earth fluorescent composite nanoparticles can be observed and the quantity can be counted.

[0126] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing tyrosine-functionalized rare earth fluorescent nanoparticles, characterized in that: In the preparation of rare earth fluorescent nanoparticles, surfactant O-phosphoethanolamine is added to prepare amino-modified rare earth fluorescent nanoparticles, and then tyrosine is added to modify the surface of the rare earth fluorescent nanoparticles with tyrosine through an activated ester method to obtain tyrosine-functionalized rare earth fluorescent nanoparticles; the rare earth fluorescent nanoparticles are down-conversion rare earth fluorescent chelates.

2. The preparation method according to claim 1, characterized in that: The ligand in the down-conversion rare earth fluorescent chelate is one of NaYF4 and Y2O3, and the doped rare earth metal element is cerium and another lanthanide rare earth element.

3. The preparation method according to claim 2, characterized in that: The other lanthanide rare earth element is terbium, erbium, thulium, samarium, europium or dysprosium.

4. The preparation method according to claim 1, characterized in that: The rare earth fluorescent nanoparticles are NaYF4 fluorescent nanoparticles co-doped with cerium and another lanthanide rare earth element. 3+ ,(0.5~80%)Ln 3+ .

5. The preparation method according to claim 1, characterized in that: The activated ester method is to dissolve the amino-modified rare earth fluorescent nanoparticles in water, add tyrosine, mix well, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysulfosuccinimide, mix well, and obtain tyrosine-functionalized rare earth fluorescent nanoparticles.

6. Tyrosine-functionalized rare earth fluorescent nanoparticles prepared by the preparation method according to any one of claims 1 to 5.

7. A tyramide signal amplification system based on rare earth fluorescent materials, characterized in that: The method comprises the tyrosine-functionalized rare earth fluorescent nanoparticles according to claim 6, horseradish peroxidase and hydrogen peroxide.

8. Use of the tyrosine-functionalized rare earth fluorescent nanoparticles according to claim 6 or the tyramide signal amplification system according to claim 7 in immunoassay.

9. A method for ultrasensitive multicolor or time-resolved digital fluorescence immunoassay based on tyramide signal amplification, characterized in that: The following steps are involved: S1. Modifying carboxyl magnetic beads with capture antibody cAb to obtain magnetic capture probe MB@cAb, incubating the target to be detected with MB@cAb, and magnetically separating unbound substances; the cAb specifically recognizes the target to be detected; S2. Adding a detection antibody dAb-HRP carrying a horseradish peroxidase label, the detection antibody can specifically bind to the capture antibody cAb in S1 to form an immune complex MB@cAb-target-dAb-HRP, and magnetically separating unbound substances; S3. Add the tyrosine-functionalized rare earth fluorescent nanoparticles and H2O2 as claimed in claim 6, and a TSA reaction occurs after HRP catalysis, wherein the tyrosine-functionalized rare earth fluorescent nanoparticles are covered on the surface of the immune complex described in S2 to form magnetic rare earth fluorescent composite nanoparticles, and unbound substances are magnetically separated; S4. Under the excitation light source, the magnetic rare earth fluorescent composite nanoparticles obtained in step S3 generate fluorescence, and images are collected and analyzed.

10. The method according to claim 9, characterized in that: Repeat steps S1 to S3 to obtain a plurality of magnetic rare earth fluorescent composite nanoparticles containing different targets to be detected, make the magnetic rare earth fluorescent composite nanoparticles containing different targets to be detected generate fluorescence under an excitation light source, collect multicolor images or time-resolved images and analyze them.

Images