A photonic crystal coding microsphere with a multi-level dendritic structure and its preparation method and application

Abstract

The present invention discloses a photonic crystal microsphere with a multi-level dendritic structure, a preparation method thereof, and an application thereof. The preparation method comprises: (1) preparing SiO2 nanoparticles (D-mSiO2NPs) with a dendritic mesoporous structure; (2) preparing photonic crystal microspheres (DMSPCM) with a dendritic mesoporous structure using microfluidic technology; (3) cyclically modifying the surface of existing DMSPCM using vinyl-thiol click chemistry and esterification reactions to form a high-density carboxyl group with adjustable dendrite generation; and (4) coupling functional groups with specific capture effects. The multi-level DMSPCM prepared by the present invention has abundant functional molecular binding sites, structural colors discernible to the naked eye, and the characteristics of simple and rapid separation, which provides an effective platform for achieving rapid separation of primary cells in complex samples and lays the foundation for downstream proteomic analysis.

Description

Technical Field

[0001] The present invention relates to a photonic crystal coding microsphere and a preparation method and application thereof, and in particular to a photonic crystal coding microsphere with a multi-level dendritic structure and a preparation method and application thereof. Background Art

[0002] With the continuous deepening of research in proteomics, transcriptomics, genomics, and metabolomics, this will promote the progress of early precision medicine diagnosis research. Among them, cells are the foundation of life sciences, and especially the results of cell heterogeneity research are very important for discovering the mechanism of disease occurrence or finding new markers. Cell separation is a prerequisite for studying individual cell heterogeneity or population heterogeneity. The main methods for separating and extracting primary cells from tissues include density gradient centrifugation, flow cytometer sorting, magnetic bead sorting, adherent growth sorting and other technologies. However, these methods are difficult to simultaneously separate different types of cells in complex and trace biological samples. It is well known that exosomes are signal carriers for intercellular communication. The specific and efficient separation of exosomes is the prerequisite for early diagnosis and treatment of diseases. Finding an efficient exosome separation method is also a hot topic of current research.

[0003] Photonic crystal-encoded microspheres are materials with artificial periodic dielectric structures that exhibit photonic band gap properties. Due to their ability to generate distinct structural colors, they are often used as encoding carriers for high-throughput detection of biological samples. Currently, these microspheres have been successfully used in applications such as co-culture of multiple cells and capturing circulating tumor cells. However, the isolation of cells and exosomes still presents numerous challenges that need to be addressed. Summary of the Invention

[0004] Purpose of the invention: The present invention aims to provide a photonic crystal-encoded microsphere with a multi-level dendritic structure that helps improve the separation and capture efficiency of various primary cells or exosomes; another purpose of the present invention is to provide a method for preparing the photonic crystal-encoded microsphere to reduce experimental costs and increase experimental feasibility; another purpose of the present invention is to provide an application of the photonic crystal-encoded microsphere in the capture of primary cells in tissues and exosomes in body fluids.

[0005] Technical solution: The photonic crystal encoding microspheres with a multi-level dendritic structure described in the present invention are composed of D-mSiO2 MNPs (SiO2 nanoparticles with a dendritic mesoporous structure) or polymer-encapsulated D-mSiO2 MNPs.

[0006] Furthermore, the surface of the dendritic photonic crystal microspheres is easy to graft and modify. First, the surface of the microspheres can be modified with vinyl groups by means of a rapid hydrolysis reaction of trichlorovinylsilane. Second, the vinyl groups on the surface of the microspheres react with the thiol groups of thioglycerol in a light-induced click chemistry reaction, thereby grafting thioglycerol onto the surface of the microspheres. Subsequently, the alcoholic hydroxyl groups on the surface of the microspheres undergo an esterification reaction with 4-pentenoic acid. At this time, mercaptopropionic acid can be grafted onto the surface of the vinyl-modified microspheres by click chemistry. If you want to obtain more carboxyl groups with a higher density on the surface, you need to repeat the two steps of click chemistry and esterification to increase the effective reactive sites of the functional groups. The photonic crystal encoded microspheres have the characteristics of high specific surface area, controllable size, bright structural color, good stability, and easy separation. The modification of the surface functionalized dendritic molecules can be quickly completed by a cyclic method combining click chemistry and esterification or directly connected to the dendritic macromolecules, which will provide rich binding sites for antibodies, aptamers and other functional molecules that can be used for cell capture.

[0007] Furthermore, the constituent units of the photonic crystal are D-mSiO2 MNPs, and the pore size is controllable between 10 and 30 nm.

[0008] Furthermore, the constituent units of the photonic crystal are D-mSiO2 MNPs, and the porogen can be any combination of CTAB and sodium citrate or CTAC and sodium citrate.

[0009] Furthermore, the building blocks of the photonic crystal can be polymer-coated D-mSiO2 MNPs, which retain a mesoporous structure even after polymer coating. The polymer coating the mesoporous silica can be any of polydopamine, polymethacrylate, polylysine, or polyaniline. Polydopamine-coated SiO2 can enhance the structural stability and color saturation of the photonic crystal encoding microspheres.

[0010] Furthermore, the surface of the polymer-coated D-mSiO2 MNPs contains abundant reactive sites, is easy to be grafted and modified, and may contain abundant vinyl groups, amino groups, aldehyde groups, epoxy groups or phenolic hydroxyl groups.

[0011] Furthermore, the surface of the photonic crystal microspheres can be subjected to multiple grafting modifications, which can be accomplished through any one or more of click chemistry, esterification reaction, and hydrolysis polymerization reaction.

[0012] Furthermore, the surface of the photonic crystal microspheres can also be directly modified with dendrimers, and the dendrimers can be any one of PAMAM (G0, G1, G2, G3, G4, G5) modified with different functional groups.

[0013] Furthermore, the surface of the photonic crystal microsphere is coupled to any one of antibodies, polypeptides, aptamers or biotin.

[0014] Furthermore, the size of the photonic crystal microspheres can be adjusted between 20 μm and 500 μm, and the color can be edited in the visible light range.

[0015] The method for preparing the photonic crystal coding microspheres with a multi-level dendritic structure comprises the following steps:

[0016] (1) Preparation of D-mSiO2 MNPs: The surfactant and catalyst are mixed and dispersed in an alcohol / water solution, a silicon source is added, and after mixing, the mixture reacts to form D-mSiO2 MNPs of uniform size;

[0017] (2) Preparation of photonic crystal encoding microspheres: an aqueous solution containing mesoporous silica particles or polymer-coated silica nanoparticles is injected from the inlet A of a microfluidic generation device, and hexadecane containing 2296 as a shear phase is injected from the external phase inlet B, and microspheres of uniform size are generated at the outlet C of the microfluidic device; the microspheres are collected on a stainless steel plate treated with hydrophobicity, heated on a heating table, and naturally evaporated and assembled into microspheres with dense structure;

[0018] The preparation process of the polymer-coated D-mSiO2 MNPs is as follows: polymer monomers are dispersed in a suitable polymerization environment, an appropriate amount of dendritic mesoporous silica nanoparticles are added, and ultrasonic dispersion is performed uniformly, so that the polymer forms a polymerization layer on the surface of the dendritic mesoporous silica, and the thickness of the polymerization layer should not be too thick;

[0019] (3) Preparation of grafted modified photonic crystal coding microspheres: Silica microspheres with dendritic mesoporous structures need to be activated by surface groups after high-temperature curing, while polymer-coated mesoporous silica microspheres can omit this step. The grafting modification of the surface of photonic crystal microspheres can be achieved by connecting the grafted groups to the surface of the microspheres repeatedly through click chemistry, esterification reaction and / or hydrolysis polymerization, or by directly connecting the dendritic polymer containing functional groups to the surface of the microspheres.

[0020] (4) Preparation of functionalized multi-level dendritic structured photonic crystal encoding microspheres: The microspheres prepared in step (3) are dispersed in a buffer solution with a suitable pH, directly coupled with antibodies, peptides, aptamers or biotin in the presence of EDC and NHS, and washed to remove uncoupled functional molecules.

[0021] Furthermore, in step (2), the concentration of the aqueous solution containing D-mSiO2 MNPs or polymer-encapsulated D-mSiO2 MNPs is 2-13 wt%; the concentration of 2296 contained in the hexadecane is 1-5 wt%; and the heating temperature of the heating stage is 60-80°C.

[0022] Furthermore, the coupling reaction in step (4) is carried out at 2-8°C.

[0023] The photonic crystal microspheres with a multi-level dendritic structure can be used to capture primary tissue cells or exosomes in body fluids.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) It helps to improve the efficiency of specific separation and capture of primary cells or exosomes. The present invention proposes to use a photonic crystal encoding microsphere with a multi-level dendritic structure as a multifunctional platform for cell or exosome separation and detection. This platform will provide more possibilities for the efficient separation of specific targets in complex biological samples; (2) The photonic crystal encoding microsphere with a multi-level dendritic structure is composed of dendritic mesoporous SiO2 nanoparticles to form a photonic crystal structural unit. No further etching is required. The surface of the microsphere is rough and covered with gullies, which effectively increases the specific surface area for further modification; (3) The surface of the photonic crystal encoding microsphere with a multi-level dendritic structure is easy to graft and modify. After grafting and modification, the dendritic macromolecules formed can further expand the coupling sites of functional groups; (4) The photonic crystal encoding microsphere with a multi-level dendritic structure is controllable in size, bright in color, can be quickly separated by its own gravity, and is easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic structural diagram of the multi-level dendritic structure photonic crystal coding microspheres of the present invention;

[0026] Figure 2 Generate a plot for the microsphere device. DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0028] like Figure 1As shown, in an embodiment of the present invention, the multi-level dendritic structure photonic crystal coding microspheres are prepared by first synthesizing dendritic mesoporous silica nanoparticles or polymer-coated silica nanoparticles, and then these nanoparticles are self-assembled by evaporation to form brightly colored photonic crystal microspheres with a rough surface. Subsequently, the surface of the shell is further grafted and modified to increase the binding sites of the functional groups. Finally, microspheres of different colors are respectively coupled with characteristic functional molecules for identifying different cells, thereby realizing the coding preparation work for cell capture photonic crystal microspheres. Figure 1 In the figure, 1 represents dendritic mesoporous silica nanoparticles or polymer-encapsulated silica nanoparticles, 2 represents photonic crystal microspheres formed by evaporation self-assembly, 3 represents grafted dendritic molecules, and 4 represents functional molecules. Figure 2 The A end is the inner phase inlet end, the B end is the outer phase inlet end, and the C end is the microsphere outlet end.

[0029] Example 1

[0030] Prepare blue dendritic mesoporous photonic crystal coding microspheres as follows:

[0031] 1. Measure 25 mL of aqueous solution, add 0.068 g of triethanolamine, and heat with stirring at 80°C. Subsequently, add a mixture of 380 mg of hexadecyl ammonium bromide and 168 mg of sodium citrate and continue stirring for 1 hour. Directly add 4 mL of tetraethyl silicate and continue the reaction for 4 hours. Stop the reaction and repeat the ethanol wash. Transfer the washed particles to a mixture of ethanol and concentrated hydrochloric acid and perform Soxhlet extraction under reflux for 10 hours, three times.

[0032] 2. The nanoparticles were prepared into a 6.5 wt% aqueous dispersion and passed through a microfluidic device with a 100 μm outlet diameter for the internal phase and a 500 μm outlet diameter for the external phase at flow rates of 0.4 mL / h for the internal phase and 6 mL / h for the external phase, respectively. Hexadecane containing 2 wt% 2296 was used as the shearing solution for the external phase. The resulting droplets were collected on a hydrophobic-treated stainless steel plate, allowed to stand, and then heated at 70°C overnight to allow the water to evaporate, forming blue photonic crystal microspheres. The resulting droplets were then solidified by calcination at 700°C.

[0033] 3. The calcined particles were plasma treated, soaked in piranha solution overnight, thoroughly washed with water, and dried. The microspheres were then immersed in 40 mL of dichloromethane, and 50 mg of 4-dimethylaminopyridine, 200 μL of triethanolamine, and 100 μL of trichlorovinylsilane were added. The reaction was allowed to proceed at room temperature, and the particles were washed with ethanol and acetone and dried with nitrogen.

[0034] 4. The particles were immersed in 800 μL of DMF solution containing 50 μL of thioglycerol, 3 mg of DMPA photoinitiator was added, and irradiated in a 360 nm light box for 30 min, washed with ethanol, and dried.

[0035] 5. The microspheres treated in step 4 were immersed in 10 mL of acetone solution consisting of 11.2 mg of 4-dimethylaminopyridine, 25 μL of pentenoic acid, and 18 μL of DIC, reacted at room temperature for 4 h, washed with acetone, and dried.

[0036] 6. Repeat steps 4 and 5 twice, increasing the concentrations of monothioglycerol, DMPA, 4-dimethylaminopyridine, pentenoic acid, and DIC each time.

[0037] 7. The ends of the multi-level dendritic structure photonic crystal microspheres obtained in each cycle are connected to β-mercaptopropionic acid to form a carboxylated surface.

[0038] 8. The grafted carboxyl-modified photonic crystal encoding microspheres were immersed in a coupling reagent solution (10 mg / mL EDC, 10 mg / mL NHS, pH 6.2, MES buffer) for 0.5 h at 4°C. This was followed by immersion in 0.2 mg / mL antibody (F4 / 80) at 37°C. Unbound antibody was washed with PBS, and the microspheres were blocked overnight with 5% BSA. The microspheres were then washed thoroughly with PBS and set aside.

[0039] In this embodiment, the color of the photonic crystal coding microspheres is blue and the size is 150 μm.

[0040] In this embodiment, the structural units constituting the photonic crystal microspheres are silicon dioxide nanoparticles with a dendritic mesoporous structure, with a particle size of about 180 nm and a pore diameter of about 30 nm.

[0041] In this embodiment, two layers of dendritic molecules are grafted onto the surface of the photonic crystal encoding microspheres.

[0042] In this embodiment, the antibody linked to the photonic crystal encoded microspheres is the F4 / 80 antibody.

[0043] Example 2

[0044] Prepare green dendritic mesoporous photonic crystal encoding microspheres as follows:

[0045] 1. Measure 25 mL of aqueous solution, add 0.068 g of triethanolamine, and heat with stirring at 80°C. Subsequently, add a mixture of 380 mg of hexadecyl ammonium bromide and 168 mg of sodium citrate and continue stirring for 1 hour. Add 8 mL of tetraethyl silicate and continue the reaction for 4 hours. Stop the reaction and repeat the ethanol wash. Transfer the washed particles to a mixture of ethanol and concentrated hydrochloric acid and perform Soxhlet extraction under reflux for 10 hours, three times.

[0046] 2. The nanoparticles were prepared as a 6.5% aqueous dispersion and passed through a microfluidic device with a 100 μm outlet diameter for the internal phase and a 500 μm outlet diameter for the external phase at flow rates of 0.4 mL / h for the internal phase and 6 mL / h for the external phase, respectively. Hexadecane containing 2% 2296 was used as the shearing solution for the external phase. The resulting droplets were collected on a hydrophobic-treated stainless steel plate, allowed to stand, and then heated at 70°C overnight to allow the water to evaporate, forming blue photonic crystal microspheres. The resulting droplets were then solidified by calcination at 700°C.

[0047] 3. The calcined particles were plasma treated, soaked in piranha solution overnight, thoroughly washed with water, and dried. The microspheres were then immersed in 40 mL of dichloromethane, and 50 mg of 4-dimethylaminopyridine, 200 μL of triethanolamine, and 100 μL of trichlorovinylsilane were added. The reaction was allowed to proceed at room temperature, and the particles were washed with ethanol and acetone and dried with nitrogen.

[0048] 4. The particles were immersed in 800 μL of DMF solution containing 50 μL of thioglycerol, 3 mg of DMPA photoinitiator was added, and irradiated in a 360 nm light box for 30 min, washed with ethanol, and dried.

[0049] 5. The microspheres treated in step 4 were immersed in 10 mL of acetone solution consisting of 11.2 mg of 4-dimethylaminopyridine, 25 μL of pentenoic acid, and 18 μL of DIC, reacted at room temperature for 4 h, washed with acetone, and dried.

[0050] 6. Repeat steps 4 and 5 twice, increasing the concentrations of monothioglycerol, DMPA, 4-dimethylaminopyridine, pentenoic acid, and DIC each time.

[0051] 7. The ends of the multi-level dendritic structure photonic crystal microspheres obtained in each cycle are connected to β-mercaptopropionic acid to form a carboxylated surface.

[0052] 8. The grafted carboxyl-modified photonic crystal encoding microspheres were immersed in a coupling reagent solution (10 mg / mL EDC, 10 mg / mL NHS, pH 6.2, MES buffer) for 0.5 h at 4°C. This was followed by immersion in 0.2 mg / mL antibody (CD146) at 37°C. Unbound antibody was washed with PBS, and the microspheres were blocked overnight with 5% BSA. The microspheres were then washed thoroughly with PBS and set aside.

[0053] In this embodiment, the color of the photonic crystal coding microspheres is green and the size is 150 μm.

[0054] In this embodiment, the structural units constituting the photonic crystal microspheres are silicon dioxide nanoparticles with a dendritic mesoporous structure, with a particle size of about 260 nm and a pore diameter of about 30 nm.

[0055] In this embodiment, two layers of dendritic molecules are grafted onto the surface of the photonic crystal encoding microspheres.

[0056] In this embodiment, the antibody linked to the photonic crystal encoded microspheres is CD146 antibody.

[0057] Example 3

[0058] Preparation of green dendritic polydopamine-coated mesoporous photonic crystal encoding microspheres, the steps are as follows:

[0059] 1. Measure 25 mL of aqueous solution, add 0.068 g of triethanolamine, and heat with stirring at 80°C. Subsequently, add a mixture of 380 mg of hexadecyl ammonium bromide and 168 mg of sodium citrate and continue stirring for 1 hour. Directly add 4 mL of tetraethyl silicate and continue the reaction for 4 hours. Stop the reaction and repeat the ethanol wash. Transfer the washed particles to a mixture of ethanol and concentrated hydrochloric acid and perform Soxhlet extraction under reflux for 10 hours, three times.

[0060] 2. Take 1.5 mL of a 6.5% aqueous solution of dendritic mesoporous nanoparticles, add 8 mL of a 2 mM dopamine Tis solution, stir at room temperature overnight, and wash with water.

[0061] 3. The nanoparticles obtained above were prepared into a 6.5% aqueous dispersion and passed through a microfluidic device with a 100 μm outlet diameter for the internal phase and a 500 μm outlet diameter for the external phase at flow rates of 0.4 mL / h for the internal phase and 6 mL / h for the external phase, respectively. Hexadecane containing 2% 2296 was used as the shearing solution for the external phase. The resulting droplets were collected on a hydrophobic-treated stainless steel plate and heated at 70°C to allow the water to evaporate naturally, forming photonic crystal microspheres.

[0062] 4. The particles were soaked in 10 mL of water containing 50 μL of thioglycerol, aerated with nitrogen overnight, washed, washed with water, and dried.

[0063] 5. The microspheres treated in step 4 were immersed in 10 mL of acetone solution consisting of 11.2 mg of 4-dimethylaminopyridine, 25 μL of pentenoic acid, and 18 μL of DIC, reacted at room temperature for 4 h, washed with acetone, and dried.

[0064] 6. Repeat steps 4 and 5 twice.

[0065] 7. Microspheres grafted with carboxyl groups on their molecular surfaces were immersed in a coupling reagent solution (10 mg / mL EDC, 10 mg / mL NHS, pH 5.0, PBS buffer) for 3 hours at 37°C. This was followed by an incubation in 0.2 mg / mL antibody (F4 / 80) at 37°C. Unbound antibody was washed with PBS, and the microspheres were blocked overnight with 5% BSA. The microspheres were then washed thoroughly with PBS and set aside.

[0066] In this embodiment, the color of the photonic crystal coding microsphere is green and the size is 160 μm.

[0067] In this embodiment, the mesopore shape of the silicon dioxide constituting the photonic crystal microspheres is dendritic, the particle size is 260 nm, and the pore diameter is about 30 nm.

[0068] In this embodiment, two layers of dendritic molecules are grafted onto the surface of the photonic crystal encoding microspheres.

[0069] In this embodiment, the antibody linked to the photonic crystal encoded microspheres is the F4 / 80 antibody.

[0070] Application Examples

[0071] The blue F4 / 80 antibody-modified multi-level dendritic photonic crystal-encoded microspheres prepared in Example 1 and the green CD146 antibody-modified multi-level dendritic photonic crystal-encoded microspheres prepared in Example 2 were used to capture mouse liver macrophages and hepatic sinusoidal endothelial cells, respectively. The anti-mouse F4 / 80 antibody specifically recognizes Kupffer cells in mouse liver, while the anti-mouse CD146 antibody specifically recognizes hepatic sinusoidal endothelial cells in mouse liver. These two cell types have similar densities, making them difficult to separate using a single centrifugation.

[0072] Mouse nonparenchymal hepatocytes were obtained by in situ perfusion digestion of the liver and incubated with an FcR antibody blocker for 15 minutes. The blue and green functionalized photonic crystal encoded microspheres prepared in Examples 1 and 2 were co-incubated with the nonparenchymal cells for 2 hours. Nonspecifically bound cells were washed with PBS, and the blue photonic crystal microspheres that captured Kupffer cells and the green photonic crystal microspheres that captured liver sinusoidal endothelial cells were visually selected. The captured cells were lysed and digested, and analyzed using liquid chromatography-mass spectrometry to identify new specific markers for the two cell types. The entire capture process took only 3 hours, and the separation speed using the photonic crystals prepared in this invention was faster than magnetic separation.

Claims

1. A photonic crystal coding microsphere with a multi-level dendritic structure, characterized by: The constituent units of the photonic crystal encoding microspheres are D-mSiO2 MNPs or polymer-encapsulated D-mSiO2 MNPs, and the D-mSiO2 MNPs are SiO2 nanoparticles with a dendritic mesoporous structure. The method for preparing the photonic crystal microspheres with a multi-level dendritic structure comprises the following steps: (1) Preparation of D-mSiO2 MNPs: The surfactant and catalyst are mixed and dispersed in an alcohol / water solution, a silicon source is added, and after mixing evenly, the mixture reacts to form uniform D-mSiO2 MNPs; (2) Preparation of photonic crystal coding microspheres: an aqueous solution containing D-mSiO2 MNPs or polymer-coated D-mSiO2 MNPs is injected from the inlet A of a microfluidic generation device, and hexadecane containing 2296 is injected as a shear phase from the external phase inlet B, and microspheres of uniform size are generated at the outlet C of the microfluidic device; the microspheres are collected, heated, and naturally volatilized to form microspheres with dense structural arrangement; the preparation process of the polymer-coated D-mSiO2 MNPs is as follows: the polymer monomer is dispersed in a polymerization environment, the D-mSiO2 MNPs are added, and the dispersion is uniformly performed by ultrasonication, and the polymer forms a polymerization layer on the surface of the D-mSiO2 MNPs; (3) Preparation of grafted photonic crystal coding microspheres: D-mSiO2 MNPs need to be activated by surface groups after high-temperature curing, while polymer-coated D-mSiO2 MNPs can omit this step; the grafted modification adopts click chemistry, esterification reaction and / or hydrolysis polymerization to repeatedly connect the grafted groups to the surface of the microspheres or directly connect them to the dendrimers with functional groups to form grafted photonic crystal coding microspheres; (4) Preparation of photonic crystal encoding microspheres with functionalized multi-level dendritic structure: The microspheres prepared in step (3) are dispersed in a buffer solution, directly coupled with antibodies, peptides, aptamers or biotin, and washed to remove uncoupled functional molecules, finally obtaining photonic crystal microspheres with functionalized multi-level dendritic structure.

2. The photonic crystal encoding microsphere with a multi-level dendritic structure according to claim 1, characterized in that: The pore size of the D-mSiO2 MNPs is controllable between 10 and 30 nm.

3. The photonic crystal encoding microsphere with a multi-level dendritic structure according to claim 1, characterized in that: The porogen of the D-mSiO2 MNPs is any combination of CTAB and sodium citrate or CTAC and sodium citrate.

4. The photonic crystal encoding microsphere with a multi-level dendritic structure according to claim 1, characterized in that: The polymer in the polymer-encapsulated D-mSiO2 MNPs is polydopamine, polymethacrylate, polylysine or polyaniline.

5. The photonic crystal encoding microsphere with a multi-level dendritic structure according to claim 1, characterized in that: The surface of the polymer-wrapped D-mSiO2 MNPs contains abundant reactive sites, is easy to be grafted and modified, and contains vinyl, amino, aldehyde, epoxy or phenolic hydroxyl groups on the surface.

6. The photonic crystal encoding microsphere with a multi-level dendritic structure according to claim 1, characterized in that: The surface dendritic molecules of the dendritic structure photonic crystal microspheres are completed by multiple grafting modifications, the number of grafting modifications is flexibly adjusted according to demand, and the grafting method is completed through click chemistry, esterification reaction, and hydrolysis polymerization cycle reaction.

7. The photonic crystal encoding microsphere with a multi-level dendritic structure according to claim 1, characterized in that: The dendritic polymer on the surface of the dendritic structure photonic crystal microsphere is any one of PAMAMs modified with different functional groups and with different grafting degrees.

8. The photonic crystal encoding microsphere with a multi-level dendritic structure according to claim 1, characterized in that: Any one of antibodies, polypeptides, aptamers or biotin is coupled to the surface of the photonic crystal microspheres.

9. The photonic crystal encoding microsphere with a multi-level dendritic structure according to claim 1, characterized in that: The size of the photonic crystal microspheres is controllable between 20 μm and 500 μm, and the color is editable within the visible light range.

10. Use of the photonic crystal microspheres having a multi-level dendritic structure according to any one of claims 1 to 9 in capturing primary cells in tissues or exosomes in body fluids.

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