A choline phosphorylation-functionalized nanohydrogel structure, an efficient method for extracting exosomes, and its applications.

Abstract

This invention discloses a choline phosphate-functionalized nanohydrogel structure, prepared as follows: choline phosphate monomers are modified onto the surface of nanomaterials via free radical polymerization, resulting in a choline phosphate-functionalized nanohydrogel structure. The CP on the surface of this hydrogel magnetic sphere interacts with phosphatidylcholine on the exosome membrane surface through intermolecular multivalent coordination, efficiently and with high purity extracting exosomes from various body fluids. Combined with clinically commonly used methods such as SDS-PAGE, Western blotting, NTA, and cryo-electron microscopy, the enrichment purity and efficiency of exosomes are identified, thus establishing a novel method for exosome extraction. The implementation of this invention will provide a completely new approach and tool for liquid biopsy screening of malignant tumors, helping to improve the early diagnosis rate of cancer patients in my country.

Description

Technical Field

[0001] This invention belongs to the field of compound and material technology, and in particular to a choline phosphorylation-functionalized nanohydrogel structure, an efficient extraction method for exosomes, and their applications. Background Technology

[0002] Malignant tumors are one of the leading causes of death among Chinese residents. Early screening can effectively prevent and detect malignant tumors. Surgical biopsy is the gold standard for tumor screening, but it is inefficient and invasive, resulting in low participation rates among the population.

[0003] Early-stage cancer patients often experience no symptoms, and routine checkups typically don't include high-resolution CT scans. Therefore, by the time most patients are diagnosed, the cancer has already progressed to the middle or late stages. The five-year survival rate for many advanced-stage cancers drops dramatically to around 10%. This demonstrates that early-stage cancer detection and surgical removal significantly improve survival rates.

[0004] In recent years, the rapid advancements in in vitro diagnostic (IVD) technology have brought new opportunities for the widespread adoption of malignant tumor screening. IVD refers to the technology of obtaining clinical diagnostic information by performing in vitro testing on patient samples (such as blood, urine, and stool). Its advantages include minimal invasiveness, avoiding the risks of organ biopsy; it also features high sensitivity, rapid detection, and repeatability, and has been widely used in clinical diagnosis. Detection of circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and protein biomarkers in serum can be used for the auxiliary diagnosis and prognostic assessment of cancer. However, serological tests lack specificity and sensitivity, and their widespread clinical application depends on more high-level evidence-based medicine and the discovery of more biomarkers.

[0005] Due to the unique correlation between various body fluids and tumor lesions, abnormal indicators detected through in vitro biopsies are of great value for the early diagnosis of tumors. During the occurrence and development of malignant tumors, the lesion site continuously releases biomarkers into body fluids. Detecting specific biomarkers in body fluids makes early tumor detection possible.

[0006] Exosomes are phospholipid bilayer vesicles secreted by cells, with a diameter of 30–150 nm. All mammalian cells secrete exosomes, which carry important molecules such as specific membrane proteins, nucleic acids, and lipids of the mother cell, and are widely involved in processes such as cell communication, cell migration, angiogenesis, and tumor cell growth. Because normal epithelial cells and tumor cells have different molecular biological behaviors, the exosomes secreted by them have different molecular "fingerprints." Large-scale protein, nucleic acid, and metabolite analyses of exosomes in body fluid samples from cancer patients are performed using molecular diagnostic techniques such as immunoassay, nucleic acid detection, and mass spectrometry to screen and identify exosome-specific "fingerprints" for colorectal cancer. Based on these fingerprints, early lesions can be detected and surgically removed, effectively preventing and treating colorectal cancer.

[0007] However, the composition of body fluids is extremely complex, making it difficult to efficiently and effectively separate, enrich, and identify exosomes. Currently, commonly used methods for exosome separation and enrichment include ultracentrifugation, density gradient centrifugation, immunomagnetic beading, and polymer precipitation. Ultracentrifugation is the most commonly used method for exosome separation and purification. Its separation principle is based on the size, density, and sedimentation rate of the target analyte. This method is simple but very time-consuming, has low separation efficiency, requires large sample volumes, and is difficult to conduct large-scale clinical sample studies. Density gradient centrifugation is also a common exosome separation technique. This method typically combines ultracentrifugation with a sucrose density gradient to separate exosomes from particles of different densities. This method offers high purity but is complex to operate and relies on large equipment. Immunomagnetic beading involves antibodies or aptamers modified on magnetic particles binding to specific proteins on the surface of exosomes, achieving rapid separation of exosomes under the influence of an external magnetic field. Immunomagnetic beading is simple to operate and highly specific; however, due to the heterogeneity of exosome protein expression, this method has a low capture rate and high cost. Polymer precipitation utilizes the differences in solubility and dispersibility between polymers (usually polyethylene glycol) and various substances in the sample to precipitate exosomes. This method is simple to operate and has a high yield, but it suffers from low purity and is prone to contamination with other proteins. In summary, to accurately obtain molecular information about exosomes in various body fluids, there is an urgent need to develop a reliable, efficient, and large-scale clinical exosome extraction and identification technology.

[0008] A search revealed no patent publications related to this invention's patent application. Summary of the Invention

[0009] The purpose of this invention is to overcome the shortcomings of the prior art and provide a choline phosphorylation-functionalized nanohydrogel structure, an efficient extraction method for exosomes, and their applications.

[0010] The technical solution adopted by this invention to solve its technical problem is:

[0011] A choline phosphorylation-functionalized nanohydrogel structure is prepared by the following steps:

[0012] Choline phosphate monomers are modified onto the surface of nanomaterials using free radical polymerization, resulting in choline phosphate-functionalized nanohydrogel structures.

[0013] Furthermore, the nanomaterial is a magnetic sphere, a silicon sphere, a protein, colloidal gold, cerium dioxide, platinum nanoparticles, or quantum dots;

[0014] Furthermore, when the nanomaterials are magnetic spheres or silicon spheres, the preparation steps of the nanohydrogel structure are as follows:

[0015] (1) Preparation of choline phosphate derivative C=C-CP

[0016] Choline phosphate (CP) derivatives were prepared using 2-chloro-2-oxo-1,3,2-dioxophosphazene as a raw material.

[0017] First, 2-chloro-2-oxo-1,3,2-dioxophosphanecyclopentane undergoes a substitution reaction with an organic reagent under the catalysis of dimethylaminoethyl methacrylate to generate 2-methoxy-2-oxo-1,3-dioxophosphanecyclopentane. Subsequently, it undergoes a ring-opening reaction with dimethylaminoethyl methacrylate in a solution at 70°C to synthesize a CP derivative containing a double bond.

[0018] (2) Preparation of nanohydrogel structures

[0019] Uniform magnetic nanoparticles (MB) or silicon spheres were prepared by hydrothermal method. C=C bonds were then introduced on the surface of the magnetic nanoparticles by silanizing agent to obtain MB-C=C. Finally, using azobisisobutyronitrile as an initiator, the synthesized CP derivative monomer and crosslinking agent were polymerized onto the surface of MB by free radical polymerization to obtain MB@CPs, which are choline phosphorylation functionalized hydrogel magnetic spheres.

[0020] When the nanomaterial is a protein, the preparation steps of the nanohydrogel structure are as follows:

[0021] (1) Choline phosphate derivatives

[0022] Choline phosphate (CP) derivatives were prepared using 2-chloro-2-oxo-1,3,2-dioxophosphazene as a raw material.

[0023] First, 2-chloro-2-oxo-1,3,2-dioxophosphanecyclopentane undergoes a substitution reaction with an organic reagent under the catalysis of dimethylaminoethyl methacrylate to generate 2-methoxy-2-oxo-1,3-dioxophosphanecyclopentane. Subsequently, it undergoes a ring-opening reaction with dimethylaminoethyl methacrylate in a solution at 70°C to synthesize a CP derivative containing a double bond.

[0024] (2) Preparation of choline phosphorylation functionalized nanohydrogel structures

[0025] Horseradish peroxidase (HRP) is a commercially available material;

[0026] HRP-C=C was obtained by introducing C=C bonds onto the surface of horseradish peroxidase using N-hydroxysuccinimide acrylate. Finally, using ammonium persulfate and TEMED as initiators, the synthesized CP derivative monomers and cross-linking agents were polymerized onto the protein surface via free radical polymerization, resulting in a choline phosphorylation-functionalized nanohydrogel structure.

[0027] When the nanomaterials are colloidal gold, cerium dioxide, nano-platinum, or quantum dots, the preparation steps of the nano-hydrogel structure are as follows:

[0028] (1) Choline phosphate derivatives

[0029] Choline phosphate (CP) derivatives were prepared using 2-chloro-2-oxo-1,3,2-dioxophosphazene as a raw material.

[0030] First, 2-chloro-2-oxo-1,3,2-dioxophosphanecyclopentane undergoes a substitution reaction with an organic reagent under the catalysis of dimethylaminoethyl methacrylate to generate 2-methoxy-2-oxo-1,3-dioxophosphanecyclopentane. Subsequently, it undergoes a ring-opening reaction with dimethylaminoethyl methacrylate in a solution at 70°C to synthesize a CP derivative containing a double bond.

[0031] (2) Preparation of choline phosphorylation functionalized nanohydrogel structures

[0032] Prepare 5 nanomolar colloidal gold solution, cerium dioxide solution, nano-platinum solution, and quantum dot solution;

[0033] First, the pH of the above nanomaterial solution was adjusted to 9 using a sodium hydroxide solution at 1 mol / L. Then, N-(3-aminopropyl)methacrylamide hydrochloride with a final concentration of 10 μmol was added to the nanomaterial solution to introduce carbon-carbon double bonds on the surface of the nanomaterial. Finally, using ammonium persulfate and TEMED as initiators, the synthesized CP derivative monomer and crosslinking agent were polymerized onto the surface of the nanomaterial via free radical polymerization, resulting in a choline phosphorylation-functionalized nanohydrogel structure.

[0034] Furthermore, in step (2) of the preparation of hydrogel nanomaterials, the particle size of the magnetic spheres is 50-500 nm, the particle size of the silicon spheres is 100-1000 nm, and the particle size of the colloidal gold, cerium dioxide, nano-platinum, and quantum dots is 1-200 nm.

[0035] Furthermore, when the nanomaterials are magnetic spheres or silicon spheres, the specific preparation steps for the nanohydrogel structure are as follows:

[0036] (1) Synthesis steps of a series of choline phosphate groups

[0037] Organic reagents, dimethylaminoethyl methacrylate, hydroquinone monomethyl ether, and acetonitrile were added to a single-necked flask under argon protection, and then the temperature was lowered to -78°C. Subsequently, 2-chloro-2-oxo-1,3,2-dioxophosphazenecyclopentane was added dropwise over 2 hours, and the reaction was continued for 8 hours. After the reaction system was transferred to room temperature overnight, it was incubated at -20°C for 30 minutes. After filtering the precipitate, the reaction temperature was raised to 70-75°C and the reaction was carried out for 96 hours. After the reaction was completed, the mixture was cooled to room temperature, and a white solid was precipitated with excess diethyl ether. The solid was then washed 3-5 times with tetrahydrofuran. After the supernatant was clear, it was dried under vacuum to obtain choline phosphate monomer.

[0038] The ratio of the organic reagents: dimethylaminoethyl methacrylate: hydroquinone monomethyl ether: acetonitrile: 2-chloro-2-oxo-1,3,2-dioxophosphazenecyclopentane, in moles: moles: milligrams: milliliters: moles was 0.08:0.12:200:50:0.05.

[0039] (2) Surface modification of double bonds in nanomaterials

[0040] Nanomaterials were added to a mixed solution of ethanol and isopropanol, followed by the addition of ammonia and 3-(trimethoxysilyl)propyl methacrylate, and reacted at 60 degrees Celsius for 2 hours. Unreacted raw materials were then removed by washing with ethanol multiple times to obtain magnetic spheres with modified double bonds.

[0041] The ratio of nanomaterials: ethanol: isopropanol: ammonia: 3-(trimethoxysilyl)propyl methacrylate (mg: ml: ml: ml: ml) is 50:45:15:3:0.45.

[0042] (3) Synthesizing choline phosphate-modified nanohydrogel structures using precipitation polymerization.

[0043] Nanomaterials with modified double bonds were added to an acetonitrile solution, followed by the addition of choline phosphate monomer, methylenebisacrylamide, and azobisisobutyronitrile. The reaction was stopped after heating at 100 degrees Celsius for 1 hour. The product was then washed several times with ethanol and water to obtain a choline phosphate-functionalized nanohydrogel structure.

[0044] The ratio of acetonitrile: modified double-bonded nanomaterial: choline phosphate monomer: methylenebisacrylamide: azobisisobutyronitrile (mL): mg: mg: mg: mg: mg is 40:50:200:20:4.5.

[0045] When the nanomaterial is a protein, the specific preparation steps for the nanohydrogel structure are as follows:

[0046] (1) Synthesis steps of a series of choline phosphate groups

[0047] Organic reagents, dimethylaminoethyl methacrylate, hydroquinone monomethyl ether, and acetonitrile were added to a single-necked flask under argon protection, and then the temperature was lowered to -78°C. Subsequently, 2-chloro-2-oxo-1,3,2-dioxophosphazenecyclopentane was added dropwise over 2 hours, and the reaction was continued for 8 hours. After the reaction system was transferred to room temperature overnight, it was incubated at -20°C for 30 minutes. After filtering the precipitate, the reaction temperature was raised to 70-75°C and the reaction was carried out for 96 hours. After the reaction was completed, the mixture was cooled to room temperature, and a white solid was precipitated with excess diethyl ether. The solid was then washed 3-5 times with tetrahydrofuran. After the supernatant was clear, it was dried under vacuum to obtain choline phosphate monomer.

[0048] The ratio of the organic reagents: dimethylaminoethyl methacrylate: hydroquinone monomethyl ether: acetonitrile: 2-chloro-2-oxo-1,3,2-dioxophosphazenecyclopentane, in moles: moles: milligrams: milliliters: moles was 0.08:0.12:200:50:0.05.

[0049] (2) Surface modification of double bonds in nanomaterials

[0050] Horseradish peroxidase was added to a 50 mmol sodium bicarbonate solution at pH 8.5, followed by the addition of N-hydroxysuccinimide acrylate. The mixture was reacted at room temperature for 2 hours and then dialyzed for 24 hours to obtain HRP with modified double bonds.

[0051] The ratio of sodium bicarbonate solution: horseradish peroxidase: N-hydroxysuccinimide acrylate (mL: mg: mg) was 4:10:3.4.

[0052] (3) Synthesis of choline phosphate-modified HRP by free radical polymerization

[0053] Choline phosphate monomer, methylene bisacrylamide, ammonium persulfate and TEMED were added to the HRP solution with modified double bonds. The reaction was stopped after stirring at room temperature for one hour. Then, the reaction was dialyzed for 24 hours to remove excess monomer and initiator, and the product choline phosphate functionalized nanohydrogel structure was obtained.

[0054] The ratio of modified double-bond nanomaterials: choline phosphate monomer: methylenebisacrylamide: ammonium persulfate: TEMED (mg: mg: mg: mg: ml) was 5:50:2:2:0.005.

[0055] When the nanomaterials are colloidal gold, cerium dioxide, nano-platinum, or quantum dots, the specific preparation steps for the nano-hydrogel structure are as follows:

[0056] (1) Synthesis steps of a series of choline phosphate groups

[0057] Organic reagents, dimethylaminoethyl methacrylate, hydroquinone monomethyl ether, and acetonitrile were added to a single-necked flask under argon protection, and then the temperature was lowered to -78°C. Subsequently, 2-chloro-2-oxo-1,3,2-dioxophosphazenecyclopentane was added dropwise over 2 hours, and the reaction was continued for 8 hours. After the reaction system was transferred to room temperature overnight, it was incubated at -20°C for 30 minutes. After filtering the precipitate, the reaction temperature was raised to 70-75°C and the reaction was carried out for 96 hours. After the reaction was completed, the mixture was cooled to room temperature, and a white solid was precipitated with excess diethyl ether. The solid was then washed 3-5 times with tetrahydrofuran. After the supernatant was clear, it was dried under vacuum to obtain choline phosphate monomer.

[0058] The ratio of the organic reagents: dimethylaminoethyl methacrylate: hydroquinone monomethyl ether: acetonitrile: 2-chloro-2-oxo-1,3,2-dioxophosphazenecyclopentane, in moles: moles: milligrams: milliliters: moles was 0.08:0.12:200:50:0.05.

[0059] (2) Surface modification of double bonds in nanomaterials

[0060] N-(3-aminopropyl)methacrylate amino hydrochloride was added to colloidal gold solution, cerium dioxide solution, nanoplatinum solution and quantum dot solution at pH 9, and the reaction was carried out at room temperature for 1 hour to obtain nanomaterials with modified double bonds.

[0061] The ratio of nanomaterial to N-(3-aminopropyl)methacrylate amino hydrochloride was 5:0.01 (mL to molar concentration).

[0062] (3) Synthesizing choline phosphate-modified nanohydrogel structures using free radical polymerization.

[0063] Choline phosphate monomer, methylenebisacrylamide, ammonium persulfate and TEMED were added to colloidal gold solution, cerium dioxide solution, nanoplatinum solution and quantum dot solution with modified double bonds. The reaction was stopped after stirring at room temperature for one hour. Then, the reaction was stopped by dialyzing for 24 hours to remove excess monomer and initiator, and the product choline phosphate functionalized nanohydrogel structure was obtained.

[0064] The ratio of modified double-bond nanomaterials: choline phosphate monomer: methylenebisacrylamide: ammonium persulfate: TEMED in milliliters: milligrams: milligrams: milligrams: milliliters was 5:50:2:2:0.005.

[0065] Furthermore, the organic reagent in step (1) during the preparation of the nano-hydrogel structure is methanol, ethanol, isopropanol, methoxyethanol or n-butanol;

[0066] Alternatively, in step (2) of the preparation of the nano-hydrogel structure, the particle size of the magnetic spheres is 50-500 nm, the particle size of the silicon spheres is 100-1000 nm, and the particle size of the colloidal gold, cerium dioxide, nano-platinum, and quantum dots is 1-200 nm.

[0067] In step (2) of the preparation of the nanohydrogel structure, the protein includes all commercially available proteins.

[0068] The application of choline phosphorylation-functionalized nanohydrogel structures as described above in the recognition or extraction of exosomes in body fluids.

[0069] The efficient method for identifying or extracting exosomes from body fluids using choline phosphorylation-functionalized nanohydrogel structures, as described above, comprises the following steps:

[0070] The identification method is as follows:

[0071] After capturing exosomes expressing disease-specific proteins in cell culture supernatant or body fluid by microspheres, magnetic beads, ELISA plates or microarrays modified with disease-specific protein antibodies, nanohydrogel structures are added and incubated at room temperature for two hours. After incubation, excess choline phosphorylation-functionalized nanohydrogel structures are removed by washing, and finally, color development is performed using a colorimetric solution.

[0072] The extraction method is as follows:

[0073] Collect body fluid, centrifuge, add hydrogel magnetic beads, and shake at room temperature for two hours. After the process, remove excess impurities by magnetic washing. Finally, place the magnetic beads in PBS and shake at 42 degrees Celsius for 30 minutes to release exosomes.

[0074] Furthermore, the specific steps for extraction are as follows:

[0075] 1) The extraction steps for exosomes from serum are as follows:

[0076] Serum was centrifuged at 3000g for 10 minutes at 4 degrees Celsius, and then 2 mg / mL of hydrogel magnetic beads MB@CPs were added and shaken at room temperature for 2 hours. After the operation, excess impurities were removed by magnetic washing. Finally, the magnetic beads were placed in PBS and shaken at 42 degrees Celsius for 30 minutes to release exosomes.

[0077] The volume ratio of serum: hydrogel magnetic beads: PBS was 300:0.5:300 (µL:mL:µL).

[0078] 2) The extraction steps for exosomes from urine are as follows:

[0079] Urine was centrifuged at 5000g for 30 minutes at 4 degrees Celsius, then 2 mg / mL of hydrogel magnetic beads MB@CPs were added and shaken at room temperature for two hours. After the centrifugation, excess impurities were removed by magnetic washing. Finally, the magnetic beads were placed in PBS and shaken at 42 degrees Celsius for 30 minutes to release exosomes.

[0080] The volume ratio of urine: hydrogel magnetic beads: PBS (mL:mL:µL) was 10:1:300.

[0081] 3) The extraction steps for exosomes from saliva are as follows:

[0082] Saliva was centrifuged at 3000g for 10 minutes at 4 degrees Celsius, then 2 mg / mL of hydrogel magnetic beads MB@CPs were added and shaken at room temperature for 2 hours. After the process, excess impurities were removed by magnetic washing. Finally, the magnetic beads were placed in PBS and shaken at 42 degrees Celsius for 30 minutes to release exosomes.

[0083] The volume ratio of saliva: hydrogel magnetic beads: PBS was 1:1:300 (mL:mL:µL).

[0084] The advantages and positive effects of this invention are as follows:

[0085] 1. This invention innovatively designs a choline phosphate (CP)-functionalized nanohydrogel structure. The CP on its surface interacts with phosphatidylcholine (PC) on the exosome membrane surface through intermolecular multivalent coordination, enabling the efficient and high-purity extraction of exosomes from various body fluids. Combined with clinically commonly used methods such as SDS-PAGE, Western blotting, NTA, and cryo-electron microscopy, the enrichment purity and efficiency of exosomes are identified, thereby establishing a novel method for exosome extraction. Figure 10 The implementation of this invention will provide a novel approach and tool for liquid biopsy screening of malignant tumors, helping to improve the early diagnosis rate of cancer patients in my country.

[0086] 2. This invention addresses the significant national need for early cancer screening by innovatively proposing the use of exosomes for this purpose, overcoming the technical bottleneck of non-invasive cancer screening. Exosomes are vesicles secreted by almost all mammalian cells, carrying important molecular information such as cell-specific membrane proteins, nucleic acids, small molecule metabolites, and lipids, participating in various physiological and pathological processes including cell communication, cell migration, angiogenesis, and tumor growth and metastasis. Malignant tumor cells are heterogeneous cells, and the exosomes they secrete have distinctly different molecular information from those secreted by normal cells. By identifying these different molecular information, it is hoped that tumor exosome markers can be discovered from body fluids. However, the composition of body fluids is extremely complex, and currently, there is a lack of exosome extraction and detection methods suitable for large-scale clinical research. This invention proposes a novel exosome extraction strategy, extracting exosomes from the body fluids of cancer patients, combining bioinformatics with molecular diagnostic technology to screen and identify tumor-specific exosome markers, and establishing a new cancer screening method, contributing to the popularization of early cancer screening in my country.

[0087] 3. Phosphatidylcholine (PC) is an important component of the cell membrane of all eukaryotic cells and is also distributed on the surface of exosomes released from eukaryotic cells. Numerous studies have confirmed that PC on the cell membrane can form a strong coordination structure with choline phosphate (CP), which has the opposite charge direction, through electrostatic interactions. This invention provides a series of CP-modified nanohydrogel structures in which the CP group is firmly bound to PC on the exosome membrane surface through intermolecular electrostatic interactions, thereby enabling the efficient extraction of exosomes from clinical samples under an applied magnetic field, achieving highly efficient exosome capture.

[0088] Meanwhile, CP can bind water molecules through ionic solvation, forming a robust hydration layer on the surface, thereby endowing the nano-hydrogel structure with anti-protein adsorption properties, which helps to improve the purity of exosome capture. Testing showed that the nano-hydrogel structure of this invention can effectively extract exosomes from serum, with extraction efficiency and purity reaching 96.4% and 94.2%, respectively, far exceeding conventional methods in the prior art.

[0089] 4. The nano-hydrogel structure of this invention can be used to establish a new method for early tumor screening by detecting exosome molecular information carried in the patient's body fluids. Attached Figure Description

[0090] Figure 1 This is a preparation route diagram of choline phosphorylation-functionalized hydrogel magnetic beads (MB@CPs) in this invention; wherein, A is a two-step method for preparing CP derivatives containing double bonds, B is for preparing hydrogel magnetic beads containing double bonds, and C is for preparing hydrogel magnetic beads using free radical polymerization.

[0091] Figure 2 This is a stability diagram of MB@CPs modified with different substituents observed by a nanoparticle size analyzer in a complex environment in this invention; wherein, the compounds from top to bottom are n-butyl, methoxyethanol, isopropyl, ethyl, methyl, carbon-carbon double bond and bare magnetic sphere;

[0092] Figure 3 This is a graph showing the anti-protein adsorption capacity of MB@CPs modified with different substituents observed by protein adsorption experiments in this invention; where M is the protein molecular weight standard, C=C is a magnetic sphere modified with carbon-carbon double bond, Me is methyl, Et is ethyl, iPr is isopropyl, and MOE is methoxyethanol.

[0093] Figure 4 The image shows the capture efficiency of exosomes by MB@CPs modified with different substituents in this invention; where Et is ethyl, iPr is isopropyl, and MOE is methoxyethanol.

[0094] Figure 5 The image shows the morphology of MB@CPs in this invention, proving that MB@CPs were successfully synthesized.

[0095] Figure 6 This invention demonstrates, through energy dispersive spectroscopy analysis of the elemental composition of MB@CPs, that the CP derivative was successfully modified onto the surface of the magnetic spheres.

[0096] Figure 7 The graphs show the hysteresis curves of MB@CPs before and after synthesis in this invention, proving that the MB@CPs process does not affect the magnetism of the magnetic spheres; where MB is a pure magnetic sphere, MB-C=C is a magnetic sphere modified with carbon-carbon double bonds, and MB@CPs is a magnetic sphere modified with CP hydrogel structure.

[0097] Figure 8 This is a graph showing the adsorption efficiency of magnetic beads on vesicles of different concentrations, as observed by exosome adsorption experiments in this invention.

[0098] Figure 9 This is a diagram showing how MB@CPs can release exosomes adsorbed on the surface of magnetic spheres at around 42°C, as observed by laser confocal microscopy in this invention.

[0099] Figure 10 This invention demonstrates how MB@CPs can efficiently and with high purity extract exosomes from various bodily fluids; the left image is a Western blotting result, and the right image is an SDS-PAGE image, with serum, urine, and saliva appearing from top to bottom.

[0100] Figure 11This diagram demonstrates the high recognition efficiency of the HRP-based nanohydrogel structure for exosomes in this invention; it can achieve highly efficient recognition of disease-related exosomes in 20 μL of serum.

[0101] Figure 12 This diagram demonstrates the high recognition efficiency of the nano-hydrogel structure with platinum nanoparticles as its core in this invention for exosomes; it can simultaneously recognize different types of exosomes in cell culture supernatant.

[0102] Figure 13 This diagram demonstrates the high recognition efficiency of the quantum dot-based nanohydrogel structure for exosomes in this invention; it can achieve single-particle recognition of tumor-derived exosomes in urine. Detailed Implementation

[0103] The embodiments of the present invention are described in detail below. It should be noted that these embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0104] Unless otherwise specified, the raw materials used in this invention are all conventional commercially available products; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0105] A choline phosphorylation-functionalized nanohydrogel structure is prepared by the following steps:

[0106] Choline phosphate monomers are modified onto the surface of nanomaterials using free radical polymerization, resulting in choline phosphate-functionalized nanohydrogel structures.

[0107] Preferably, the nanomaterial is a magnetic sphere, a silicon sphere, a protein, colloidal gold, cerium dioxide, platinum nanoparticles, or quantum dots;

[0108] Preferably, when the nanomaterial is a magnetic sphere or a silicon sphere, the preparation steps of the nanohydrogel structure are as follows:

[0109] (1) Preparation of choline phosphate derivative C=C-CP

[0110] Choline phosphate (CP) derivatives were prepared using 2-chloro-2-oxo-1,3,2-dioxophosphazene as a raw material.

[0111] First, 2-chloro-2-oxo-1,3,2-dioxophosphanecyclopentane undergoes a substitution reaction with an organic reagent under the catalysis of dimethylaminoethyl methacrylate to generate 2-methoxy-2-oxo-1,3-dioxophosphanecyclopentane. Subsequently, it undergoes a ring-opening reaction with dimethylaminoethyl methacrylate in a solution at 70°C to synthesize a CP derivative containing a double bond.

[0112] (2) Preparation of MB nanohydrogel structure

[0113] Uniform magnetic nanoparticles (MB) or silicon spheres were prepared by hydrothermal method. C=C bonds were then introduced on the surface of the magnetic nanoparticles using a silanizing agent to obtain MB-C=C. Finally, using azobisisobutyronitrile as an initiator, the synthesized CP derivative monomers and crosslinking agents were polymerized onto the surface of MB by free radical polymerization to obtain MB@CPs, which are choline phosphorylation functionalized hydrogel magnetic spheres.

[0114] When the nanomaterial is a protein, the preparation steps of the nanohydrogel structure are as follows:

[0115] (1) Choline phosphate derivatives

[0116] Choline phosphate (CP) derivatives were prepared using 2-chloro-2-oxo-1,3,2-dioxophosphazene as a raw material.

[0117] First, 2-chloro-2-oxo-1,3,2-dioxophosphanecyclopentane undergoes a substitution reaction with an organic reagent under the catalysis of dimethylaminoethyl methacrylate to generate 2-methoxy-2-oxo-1,3-dioxophosphanecyclopentane. Subsequently, it undergoes a ring-opening reaction with dimethylaminoethyl methacrylate in a solution at 70°C to synthesize a CP derivative containing a double bond.

[0118] (2) Preparation of choline phosphorylation functionalized nanohydrogel structures

[0119] Horseradish peroxidase (HRP) is a commercially available material;

[0120] HRP-C=C was obtained by introducing C=C bonds onto the surface of horseradish peroxidase using N-hydroxysuccinimide acrylate. Finally, using ammonium persulfate and TEMED as initiators, the synthesized CP derivative monomers and cross-linking agents were polymerized onto the protein surface via free radical polymerization, resulting in a choline phosphorylation-functionalized nanohydrogel structure.

[0121] When the nanomaterials are colloidal gold, cerium dioxide, nano-platinum, or quantum dots, the preparation steps of the nano-hydrogel structure are as follows:

[0122] (1) Choline phosphate derivatives

[0123] Choline phosphate (CP) derivatives were prepared using 2-chloro-2-oxo-1,3,2-dioxophosphazene as a raw material.

[0124] First, 2-chloro-2-oxo-1,3,2-dioxophosphanecyclopentane undergoes a substitution reaction with an organic reagent under the catalysis of dimethylaminoethyl methacrylate to generate 2-methoxy-2-oxo-1,3-dioxophosphanecyclopentane. Subsequently, it undergoes a ring-opening reaction with dimethylaminoethyl methacrylate in a solution at 70°C to synthesize a CP derivative containing a double bond.

[0125] (2) Preparation of choline phosphorylation functionalized nanohydrogel structures

[0126] Prepare 5 nanomolar colloidal gold solution, cerium dioxide solution, nano-platinum solution, and quantum dot solution;

[0127] First, the pH of the above nanomaterial solution was adjusted to 9 using a sodium hydroxide solution at 1 mol / L. Then, N-(3-aminopropyl)methacrylamide hydrochloride with a final concentration of 10 μmol was added to the nanomaterial solution to introduce carbon-carbon double bonds on the surface of the nanomaterial. Finally, using ammonium persulfate and TEMED as initiators, the synthesized CP derivative monomer and crosslinking agent were polymerized onto the surface of the nanomaterial via free radical polymerization, resulting in choline phosphorylation-functionalized hydrogel nanomaterials.

[0128] Preferably, in step (2) of preparing hydrogel nanomaterials, the particle size of the magnetic spheres is 50-500 nm, the particle size of the silicon spheres is 100-1000 nm, and the particle size of the colloidal gold, cerium dioxide, nano-platinum, and quantum dots is 1-200 nm.

[0129] Preferably, when the nanomaterial is a magnetic sphere or a silicon sphere, the specific preparation steps of the nanohydrogel structure are as follows:

[0130] (1) Synthesis steps of a series of choline phosphate groups

[0131] Organic reagents, dimethylaminoethyl methacrylate, hydroquinone monomethyl ether, and acetonitrile were added to a single-necked flask under argon protection, and then the temperature was lowered to -78°C. Subsequently, 2-chloro-2-oxo-1,3,2-dioxophosphazenecyclopentane was added dropwise over 2 hours, and the reaction was continued for 8 hours. After the reaction system was transferred to room temperature overnight, it was incubated at -20°C for 30 minutes. After filtering the precipitate, the reaction temperature was raised to 70-75°C and the reaction was carried out for 96 hours. After the reaction was completed, the mixture was cooled to room temperature, and a white solid was precipitated with excess diethyl ether. The solid was then washed 3-5 times with tetrahydrofuran. After the supernatant was clear, it was dried under vacuum to obtain choline phosphate monomer.

[0132] The ratio of the organic reagents: dimethylaminoethyl methacrylate: hydroquinone monomethyl ether: acetonitrile: 2-chloro-2-oxo-1,3,2-dioxophosphazenecyclopentane, in moles: moles: milligrams: milliliters: moles was 0.08:0.12:200:50:0.05.

[0133] (2) Surface modification of double bonds in nanomaterials

[0134] Nanomaterials were added to a mixed solution of ethanol and isopropanol, followed by the addition of ammonia and 3-(trimethoxysilyl)propyl methacrylate, and reacted at 60 degrees Celsius for 2 hours. Unreacted raw materials were then removed by washing with ethanol multiple times to obtain magnetic spheres with modified double bonds.

[0135] The ratio of nanomaterials: ethanol: isopropanol: ammonia: 3-(trimethoxysilyl)propyl methacrylate (mg: ml: ml: ml: ml) is 50:45:15:3:0.45.

[0136] (3) Synthesizing choline phosphate-modified nanohydrogel structures using precipitation polymerization.

[0137] Nanomaterials with modified double bonds were added to an acetonitrile solution, followed by the addition of choline phosphate monomer, methylenebisacrylamide, and azobisisobutyronitrile. The reaction was stopped after heating at 100 degrees Celsius for 1 hour. The product was then washed several times with ethanol and water to obtain a choline phosphate-functionalized nanohydrogel structure.

[0138] The ratio of acetonitrile: modified double-bonded nanomaterial: choline phosphate monomer: methylenebisacrylamide: azobisisobutyronitrile (mL): mg: mg: mg: mg: mg is 40:50:200:20:4.5.

[0139] When the nanomaterial is a protein, the specific preparation steps for the nanohydrogel structure are as follows:

[0140] (1) Synthesis steps of a series of choline phosphate groups

[0141] Organic reagents, dimethylaminoethyl methacrylate, hydroquinone monomethyl ether, and acetonitrile were added to a single-necked flask under argon protection, and then the temperature was lowered to -78°C. Subsequently, 2-chloro-2-oxo-1,3,2-dioxophosphazenecyclopentane was added dropwise over 2 hours, and the reaction was continued for 8 hours. After the reaction system was transferred to room temperature overnight, it was incubated at -20°C for 30 minutes. After filtering the precipitate, the reaction temperature was raised to 70-75°C and the reaction was carried out for 96 hours. After the reaction was completed, the mixture was cooled to room temperature, and a white solid was precipitated with excess diethyl ether. The solid was then washed 3-5 times with tetrahydrofuran. After the supernatant was clear, it was dried under vacuum to obtain choline phosphate monomer.

[0142] The ratio of the organic reagents: dimethylaminoethyl methacrylate: hydroquinone monomethyl ether: acetonitrile: 2-chloro-2-oxo-1,3,2-dioxophosphazenecyclopentane, in moles: moles: milligrams: milliliters: moles was 0.08:0.12:200:50:0.05.

[0143] (2) Surface modification of double bonds in nanomaterials

[0144] Horseradish peroxidase was added to a 50 mmol sodium bicarbonate solution at pH 8.5, followed by the addition of N-hydroxysuccinimide acrylate. The mixture was reacted at room temperature for 2 hours and then dialyzed for 24 hours to obtain HRP with modified double bonds.

[0145] The ratio of sodium bicarbonate solution: horseradish peroxidase: N-hydroxysuccinimide acrylate (mL: mg: mg) was 4:10:3.4.

[0146] (3) Synthesis of choline phosphate-modified HRP by free radical polymerization

[0147] Choline phosphate monomer, methylene bisacrylamide, ammonium persulfate and TEMED were added to the HRP solution with modified double bonds. The reaction was stopped after stirring at room temperature for one hour. Then, the reaction was dialyzed for 24 hours to remove excess monomer and initiator, and the product choline phosphate functionalized nanohydrogel structure was obtained.

[0148] The ratio of modified double-bond nanomaterials: choline phosphate monomer: methylenebisacrylamide: ammonium persulfate: TEMED (mg: mg: mg: mg: ml) was 5:50:2:2:0.005.

[0149] When the nanomaterials are colloidal gold, cerium dioxide, nano-platinum, or quantum dots, the specific preparation steps for the nano-hydrogel structure are as follows:

[0150] (1) Synthesis steps of a series of choline phosphate groups

[0151] Organic reagents, dimethylaminoethyl methacrylate, hydroquinone monomethyl ether, and acetonitrile were added to a single-necked flask under argon protection, and then the temperature was lowered to -78°C. Subsequently, 2-chloro-2-oxo-1,3,2-dioxophosphazenecyclopentane was added dropwise over 2 hours, and the reaction was continued for 8 hours. After the reaction system was transferred to room temperature overnight, it was incubated at -20°C for 30 minutes. After filtering the precipitate, the reaction temperature was raised to 70-75°C and the reaction was carried out for 96 hours. After the reaction was completed, the mixture was cooled to room temperature, and a white solid was precipitated with excess diethyl ether. The solid was then washed 3-5 times with tetrahydrofuran. After the supernatant was clear, it was dried under vacuum to obtain choline phosphate monomer.

[0152] The ratio of the organic reagents: dimethylaminoethyl methacrylate: hydroquinone monomethyl ether: acetonitrile: 2-chloro-2-oxo-1,3,2-dioxophosphazenecyclopentane, in moles: moles: milligrams: milliliters: moles was 0.08:0.12:200:50:0.05.

[0153] (2) Surface modification of double bonds in nanomaterials

[0154] N-(3-aminopropyl)methacrylate amino hydrochloride was added to colloidal gold solution, cerium dioxide solution, nanoplatinum solution and quantum dot solution at pH 9, and the reaction was carried out at room temperature for 1 hour to obtain nanomaterials with modified double bonds.

[0155] The ratio of nanomaterial to N-(3-aminopropyl)methacrylate amino hydrochloride was 5:0.01 (mL to molar concentration).

[0156] (3) Synthesizing choline phosphate-modified nanohydrogel structures using free radical polymerization.

[0157] Choline phosphate monomer, methylenebisacrylamide, ammonium persulfate and TEMED were added to colloidal gold solution, cerium dioxide solution, nanoplatinum solution and quantum dot solution with modified double bonds. The reaction was stopped after stirring at room temperature for one hour. Then, the reaction was stopped by dialyzing for 24 hours to remove excess monomer and initiator, and the product choline phosphate functionalized nanohydrogel structure was obtained.

[0158] The ratio of modified double-bond nanomaterials: choline phosphate monomer: methylenebisacrylamide: ammonium persulfate: TEMED in milliliters: milligrams: milligrams: milligrams: milliliters was 5:50:2:2:0.005.

[0159] Preferably, the organic reagent in step (1) of the preparation of the nano-hydrogel structure is methanol, ethanol, isopropanol, methoxyethanol or n-butanol;

[0160] Alternatively, in step (2) of the preparation of the nano-hydrogel structure, the particle size of the magnetic spheres is 50-500 nm, the particle size of the silicon spheres is 100-1000 nm, and the particle size of the colloidal gold, cerium dioxide, nano-platinum, and quantum dots is 1-200 nm.

[0161] In step (2) of the preparation of the nanohydrogel structure, the protein includes all commercially available proteins.

[0162] The application of choline phosphorylation-functionalized nanohydrogel structures as described above in the recognition or extraction of exosomes in body fluids.

[0163] The efficient method for identifying or extracting exosomes from body fluids using choline phosphorylation-functionalized nanohydrogel structures, as described above, comprises the following steps:

[0164] The identification method is as follows:

[0165] After capturing exosomes expressing disease-specific proteins in cell culture supernatant or body fluid by microspheres, magnetic beads, ELISA plates or microarrays modified with disease-specific protein antibodies, nanohydrogel structures are added and incubated at room temperature for two hours. After incubation, excess choline phosphorylation-functionalized nanohydrogel structures are removed by washing, and finally, color development is performed using a colorimetric solution.

[0166] The extraction method is as follows:

[0167] Collect body fluid, centrifuge, add hydrogel magnetic beads, and shake at room temperature for two hours. After the process, remove excess impurities by magnetic washing. Finally, place the magnetic beads in PBS and shake at 42 degrees Celsius for 30 minutes to release exosomes.

[0168] Preferably, the specific steps for extraction are as follows:

[0169] 1) The extraction steps for exosomes from serum are as follows:

[0170] Serum was centrifuged at 3000g for 10 minutes at 4 degrees Celsius, and then 2 mg / mL of hydrogel magnetic beads MB@CPs were added and shaken at room temperature for 2 hours. After the operation, excess impurities were removed by magnetic washing. Finally, the magnetic beads were placed in PBS and shaken at 42 degrees Celsius for 30 minutes to release exosomes.

[0171] The volume ratio of serum: hydrogel magnetic beads: PBS was 300:0.5:300 (µL:mL:µL).

[0172] 2) The extraction steps for exosomes from urine are as follows:

[0173] Urine was centrifuged at 5000g for 30 minutes at 4 degrees Celsius, then 2 mg / mL of hydrogel magnetic beads MB@CPs were added and shaken at room temperature for two hours. After the centrifugation, excess impurities were removed by magnetic washing. Finally, the magnetic beads were placed in PBS and shaken at 42 degrees Celsius for 30 minutes to release exosomes.

[0174] The volume ratio of urine: hydrogel magnetic beads: PBS (mL:mL:µL) was 10:1:300.

[0175] 3) The extraction steps for exosomes from saliva are as follows:

[0176] Saliva was centrifuged at 3000g for 10 minutes at 4 degrees Celsius, then 2 mg / mL of hydrogel magnetic beads MB@CPs were added and shaken at room temperature for 2 hours. After the process, excess impurities were removed by magnetic washing. Finally, the magnetic beads were placed in PBS and shaken at 42 degrees Celsius for 30 minutes to release exosomes.

[0177] The volume ratio of saliva: hydrogel magnetic beads: PBS was 1:1:300 (mL:mL:µL).

[0178] Specifically, the relevant preparation and detection examples are as follows:

[0179] Example 1

[0180] The preparation steps of a choline phosphorylation-functionalized nanohydrogel magnetic sphere are as follows:

[0181] 1.1 Synthetic steps of a series of choline phosphate groups

[0182] 0.08 mol methanol (or equimolar amounts of ethanol, isopropanol, methoxyethanol, and n-butanol), 0.12 mol dimethylaminoethyl methacrylate (CAS No.: 2867-47-2), 200 mg hydroquinone methyl ether, and 50 mL acetonitrile were added to a single-necked flask under argon protection, and then cooled to -78°C. Subsequently, 0.05 mol 2-chloro-2-oxo-1,3,2-dioxophosphazenecyclopentane was added dropwise over 2 hours, and the reaction was continued for 8 hours. After the reaction system was transferred to room temperature overnight, it was incubated at -20°C for 30 minutes. After filtering the precipitate, the reaction temperature was increased to 70–75°C and the reaction was carried out for 96 hours. After the reaction was completed, the mixture was cooled to room temperature, and a white solid was precipitated with excess diethyl ether, followed by washing with tetrahydrofuran 3–5 times. After the washing supernatant became clear, the product was dried under vacuum to obtain the product, with a yield of 40%–60%.

[0183] 1.2 Surface modification of double bonds on 400 nm magnetic spheres

[0184] 50 mg of magnetic beads were added to a mixture of 45 mL of ethanol and 15 mL of isopropanol, followed by the addition of 3 mL of ammonia and 0.45 mL of 3-(trimethoxysilyl)propyl methacrylate. The mixture was reacted at 60°C for 2 hours. Unreacted reactants were then removed by repeated washing with ethanol to obtain magnetic beads with modified double bonds.

[0185] 1.3 Synthesis of choline phosphate-modified nanogel magnetic spheres by precipitation polymerization

[0186] 50 mg of modified double-bonded magnetic spheres were added to 40 mL of acetonitrile solution, followed by 200 mg of choline phosphate monomer, 20 mg of methylenebisacrylamide, and 4.5 mg of initiator. The reaction was stopped after heating at 100°C for 1 hour. The product was then obtained by washing several times with ethanol and water.

[0187] Example 2

[0188] A choline phosphorylation-functionalized nanohydrogel structure is prepared by the following steps:

[0189] (1) Synthesis steps of a series of choline phosphate groups

[0190] Organic reagents, dimethylaminoethyl methacrylate, hydroquinone monomethyl ether, and acetonitrile were added to a single-necked flask under argon protection, and then the temperature was lowered to -78°C. Subsequently, 2-chloro-2-oxo-1,3,2-dioxophosphazenecyclopentane was added dropwise over 2 hours, and the reaction was continued for 8 hours. After the reaction system was transferred to room temperature overnight, it was incubated at -20°C for 30 minutes. After filtering the precipitate, the reaction temperature was raised to 70-75°C and the reaction was carried out for 96 hours. After the reaction was completed, the mixture was cooled to room temperature, and a white solid was precipitated with excess diethyl ether. The solid was then washed 3-5 times with tetrahydrofuran. After the supernatant was clear, it was dried under vacuum to obtain choline phosphate monomer.

[0191] The ratio of the organic reagents: dimethylaminoethyl methacrylate: hydroquinone monomethyl ether: acetonitrile: 2-chloro-2-oxo-1,3,2-dioxophosphazenecyclopentane, in moles: moles: milligrams: milliliters: moles was 0.08:0.12:200:50:0.05.

[0192] (2) Surface modification of double bonds in nanomaterials

[0193] Horseradish peroxidase was added to a 50 mmol sodium bicarbonate solution at pH 8.5, followed by the addition of N-hydroxysuccinimide acrylate. The mixture was reacted at room temperature for 2 hours and then dialyzed for 24 hours to obtain HRP with modified double bonds.

[0194] The ratio of sodium bicarbonate solution: horseradish peroxidase: N-hydroxysuccinimide acrylate (mL: mg: mg) was 4:10:3.4.

[0195] (3) Synthesis of choline phosphate-modified HRP by free radical polymerization

[0196] Choline phosphate monomer, methylene bisacrylamide, ammonium persulfate and TEMED were added to the HRP solution with modified double bonds. The reaction was stopped after stirring at room temperature for one hour. Then, the reaction was dialyzed for 24 hours to remove excess monomer and initiator, and the product choline phosphate functionalized nanohydrogel structure was obtained.

[0197] The ratio of modified double-bond nanomaterials: choline phosphate monomer: methylenebisacrylamide: ammonium persulfate: TEMED (mg: mg: mg: mg: ml) is 5:50:2:2:0.005.

[0198] Example 3

[0199] The specific preparation steps of a choline phosphorylation-functionalized hydrogel nanomaterial are as follows:

[0200] (1) Synthesis steps of a series of choline phosphate groups

[0201] Organic reagents, dimethylaminoethyl methacrylate, hydroquinone monomethyl ether, and acetonitrile were added to a single-necked flask under argon protection, and then the temperature was lowered to -78°C. Subsequently, 2-chloro-2-oxo-1,3,2-dioxophosphazenecyclopentane was added dropwise over 2 hours, and the reaction was continued for 8 hours. After the reaction system was transferred to room temperature overnight, it was incubated at -20°C for 30 minutes. After filtering the precipitate, the reaction temperature was raised to 70-75°C and the reaction was carried out for 96 hours. After the reaction was completed, the mixture was cooled to room temperature, and a white solid was precipitated with excess diethyl ether. The solid was then washed 3-5 times with tetrahydrofuran. After the supernatant was clear, it was dried under vacuum to obtain choline phosphate monomer.

[0202] The ratio of the organic reagents: dimethylaminoethyl methacrylate: hydroquinone monomethyl ether: acetonitrile: 2-chloro-2-oxo-1,3,2-dioxophosphazenecyclopentane, in moles: moles: milligrams: milliliters: moles was 0.08:0.12:200:50:0.05.

[0203] (2) Surface modification of double bonds in nanomaterials

[0204] N-(3-aminopropyl)methacrylate amino hydrochloride was added to colloidal gold solution, cerium dioxide solution, nanoplatinum solution and quantum dot solution at pH 9, and the reaction was carried out at room temperature for 1 hour to obtain nanomaterials with modified double bonds.

[0205] The ratio of nanomaterial to N-(3-aminopropyl)methacrylate amino hydrochloride was 5:0.01 (mL to molar concentration).

[0206] (3) Synthesis of choline phosphate-modified nanomaterials by free radical polymerization

[0207] Choline phosphate monomer, methylenebisacrylamide, ammonium persulfate and TEMED were added to colloidal gold solution, cerium dioxide solution, nanoplatinum solution and quantum dot solution with modified double bonds. The reaction was stopped after stirring at room temperature for one hour. Then, the reaction was stopped by dialyzing for 24 hours to remove excess monomer and initiator, and the product was choline phosphate functionalized hydrogel nanomaterial.

[0208] The ratio of modified double-bond nanomaterials: choline phosphate monomer: methylenebisacrylamide: ammonium persulfate: TEMED in milliliters: milligrams: milligrams: milligrams: milliliters was 5:50:2:2:0.005.

[0209] Detection Example:

[0210] I. Effects of different substituents:

[0211] Considering the high-salt, high-protein environments in which exosomes exist, such as blood and urine, this invention examines the effects of different substituents by observing their stability, anti-protein adsorption capacity, and adsorption efficiency on exosomes in complex environments.

[0212] 1) The stability of magnetic spheres modified with different substituents in complex environments was observed using a nanoparticle size analyzer.

[0213] like Figure 2 As shown, magnetic spheres modified with different monomers were incubated in PBS, and their particle size distribution was observed using a nanoparticle size analyzer. Magnetic spheres modified with methyl, ethyl, isopropyl, and methoxyethanol monomers showed good dispersibility in PBS, with a particle size of approximately 500 nm, consistent with experimental expectations. Magnetic spheres modified with n-butyl monomers exhibited some degree of aggregation but still retained some adsorption capacity for exosomes.

[0214] 2) Observe the anti-protein adsorption ability of magnetic beads modified with different substituents through protein adsorption experiments.

[0215] Choline phosphate monomers possess excellent anti-adsorption capabilities due to their amphiphilic ionic properties. For example... Figure 3 As shown, magnetic beads modified with different monomers were incubated in PBS containing 10% fetal bovine serum, and their anti-protein adsorption capacity was observed by SDS-PAGE. Magnetic beads modified with ethyl, isopropyl, and methoxyethanol monomers showed good anti-protein adsorption capacity. Although the methyl monomer-modified magnetic beads had slightly weaker anti-protein adsorption capacity, they still had some adsorption capacity for exosomes.

[0216] 3) Capture efficiency of exosomes by magnetic beads modified with different substituents

[0217] Exosomes were stained with DiO2 for fluorescence and then co-incubated with magnetic beads modified with different substituents. Changes in fluorescence intensity before and after incubation were observed to calculate the enrichment efficiency of the magnetic beads for exosomes. Figure 4 As shown, magnetic spheres modified with different substituents exhibit high enrichment efficiency for exosomes. Among them, isopropyl-modified magnetic spheres show the highest enrichment efficiency for exosomes; therefore, isopropyl-modified monomers are preferred for subsequent verification.

[0218] Example 2: Relevant Characterization Methods and Results

[0219] 1) such as Figure 5 As shown, TEM results indicate the presence of an organic shell on the surface of the magnetic sphere.

[0220] 2) such as Figure 6 As shown, the energy spectrum reveals iron and oxygen elements originating from the magnetic sphere, silicon elements originating from silicon dioxide, and phosphorus elements originating from phosphocholine.

[0221] 3) such as Figure 7 As shown, the hysteresis loop data indicates that the magnetism of the modified magnetic sphere still exists.

[0222] 4) such as Figure 8 As shown, 3×10 9 Exosomes were stained with DiO and resuspended in 100 μL of PBS, then 100 μL of 2 mg / mL MB@CPs were added and the solution was shaken at room temperature. Magnetic enrichment was performed after stopping shaking at different time points. The enrichment time of the magnetic beads on exosomes was tested by comparing the changes in DiO fluorescence intensity before and after magnetic enrichment. The experimental results showed that the magnetic beads could achieve an enrichment efficiency of over 95% for exosomes within 5 minutes.

[0223] 5) 3×10 9 Exosomes were stained with DiO and resuspended in 100 μL PBS. Then, 100 μL of 2 mg / mL MB@CPs was added, and the mixture was shaken at room temperature for 30 minutes before magnetic enrichment to remove excess fluorescent dye. The magnetic beads containing the exosomes were resuspended in 1 mL PBS and placed in a confocal culture dish. The confocal culture dish was then placed in a temperature-adjustable live-cell workstation, and the fluorescence intensity of the vesicles adsorbed on the surface of the magnetic beads was observed under a fluorescence microscope at different temperatures. The experimental results are as follows: Figure 9 As shown, the results indicate that incubation at 42 degrees Celsius for 20 minutes can release more than 95% of the exosomes on the surface of the magnetic spheres.

[0224] II. Methods for extracting exosomes from different body fluids:

[0225] 1) Exosomes in serum

[0226] Centrifuge 300 μL of serum at 3000g for 10 minutes at 4°C, then add 0.5 mL of 2 mg / mL MB@CPs and shake at room temperature for 2 hours. After the experiment, remove excess impurities by magnetic washing. Finally, place the magnetic beads in 300 μL of PBS and shake at 42°C for 30 minutes to release exosomes.

[0227] 2) Exosomes in urine

[0228] 10 mL of urine was centrifuged at 5000g for 30 minutes at 4°C, then 1 mL of 2 mg / mL MB@CPs was added and shaken at room temperature for 2 hours. After the experiment, excess impurities were removed by magnetic washing. Finally, the magnetic beads were placed in 300 μL of PBS and shaken at 42°C for 30 minutes to release exosomes.

[0229] 3) Exosomes in saliva

[0230] Centrifuge 1 mL of saliva at 3000g for 10 minutes at 4°C, then add 1 mL of 2 mg / mL MB@CPs and shake at room temperature for 2 hours. After the experiment, remove excess impurities by magnetic washing. Finally, place the magnetic beads in 300 μL of PBS and shake at 42°C for 30 minutes to release exosomes.

[0231] 4) Comparison of extraction efficiency and purity of different extraction methods

[0232] This invention compared three different exosome enrichment methods: ultracentrifugation (UC), polymer precipitation (PEG), and hydrogel nanosphere enrichment. CD9, CD63, and CD81 are marker proteins on exosomes, while the other proteins are the most common impurity proteins in different body fluids. The results are as follows: Figure 10 As shown, the Western blot results in the left figure demonstrate that exosomes enriched by the magnetic bead method have the highest efficiency (the bands of the three marker proteins are the darkest) and the highest purity (no impurity proteins are present in the enriched exosome solution). The SDS-PAGE results in the right figure also show that the enriched exosome solution contains the fewest impurity proteins.

[0233] 5) The performance comparison of various exosome separation and enrichment techniques is shown in the table. As can be seen from Table 1, the nano-hydrogel structure of the present invention can effectively extract exosomes from serum, with extraction efficiency and purity reaching 96.4% and 94.2% respectively, which is much higher than the extraction efficiency of conventional methods in the prior art, which is 30% to 60%, and the extraction purity is 5% to 70%.

[0234] Table 1. Performance Comparison of Various Exosome Isolation and Enrichment Technologies

[0235] Separation and enrichment techniques High efficiency High purity Short processing time Easy to operate Good integrity Ultracentrifugation × √ × √ × Density gradient centrifugation × √ √ × × Immunomagnetic bead method × × √ √ √ Polymer precipitation method √ × √ √ √ Method of the present invention √ √ √ √ √

[0236] III. Identification methods for exosomes associated with different diseases

[0237] 1) Microsphere enrichment

[0238] Serum was centrifuged at 3000g for 10 minutes at 4°C, then 0.1 mg of microspheres was added and shaken at room temperature for 2 hours. After the experiment, excess impurities were removed by repeated centrifugation and washing. Next, a nano-hydrogel structure was added and incubated at room temperature for 2 hours. After incubation, excess choline phosphate-functionalized nano-hydrogel structure was removed by washing, and finally, color development was performed using a colorimetric reagent. Figure 11 As shown, only 20 microliters of serum are needed to achieve specific recognition of exosomes.

[0239] 2) ELISA plate enrichment

[0240] Exosomes were obtained from the supernatant of HeLa cell culture medium by ultracentrifugation, and their concentration was quantitatively calculated using BCA. Different concentrations of exosomes were added to ELISA plates modified with CD63, MUC-1, CD81, and CD9 antibodies, respectively, and incubated at 37°C for 2 hours. Excess impurities were then removed by repeated washing. Next, a nanogel structure was added and incubated at room temperature for 2 hours. After incubation, excess choline phosphate-functionalized nanogel structure was removed by washing, and finally, color development was performed using a chromogenic buffer. Figure 12 As shown, the nano-hydrogel structure of this invention can serve as a universal exosome dye, enabling the relative quantification of the concentrations of different proteins on the surface of exosomes.

[0241] The above 1) and 2) apply to colloidal gold, colloidal platinum, cerium dioxide and protein-modified nanohydrogel structures.

[0242] 3) Microarray enrichment

[0243] Ten microliters of urine were dropped onto the surface of a microarray modified with CD63 antibody and agitated at 37°C for two hours. Excess impurities were then removed by repeated washing. Next, a nanohydrogel structure (only applicable to quantum dot-modified nanohydrogels) was added and incubated at room temperature for two hours. After incubation, excess choline phosphate-functionalized nanohydrogel structure was removed by washing. Finally, imaging was performed using fluorescence microscopy. Results are as follows: Figure 13 As shown, this method enables the identification of single-particle exosomes.

[0244] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A choline phosphate functionalized nanohydrogel, characterized in that: The nanomaterials are magnetic balls, silicon balls, and the preparation steps of the nanohydrogel are as follows: (1) Preparation of choline phosphate derivative C=C-CP 2-Chloro-2-oxo-1,3,2-dioxaphospholane is selected as raw material to prepare choline phosphate CP derivative: Firstly, 2-chloro-2-oxo-1,3,2-dioxaphospholane is subjected to substitution reaction with organic reagent under catalysis of dimethylaminoethyl methacrylate to generate 2-methoxy-2-oxo-1,3-dioxaphospholane, and then ring-opening reaction with dimethylaminoethyl methacrylate in solution at 70°C to synthesize CP derivative containing double bond; (2) Preparation of nanohydrogel Uniform-sized magnetic nanoparticles or silicon balls are prepared by hydrothermal method: then C=C bond is introduced on the surface of the magnetic nanoparticles or silicon balls by silane reagent, to obtain MB-C=C or silicon ball-C=C, finally, CP derivative monomer and crosslinking agent synthesized above are polymerized on the surface of MB or silicon ball by free radical polymerization method with azobisisobutyronitrile as initiator, to obtain MB@CPs or silicon ball@CPs, i.e. choline phosphate functionalized hydrogel magnetic balls and silicon balls.

2. The nanohydrogel of claim 1, wherein: The preparation steps are as follows: (1) Choline phosphate monomer synthesis step The organic reagent, dimethylaminoethyl methacrylate, hydroquinone monomethyl ether and acetonitrile are added into a single-neck flask under argon protection, and then cooled to-78°C; Then, 2-chloro-2-oxo-1,3,2-dioxaphospholane is added dropwise in 2 hours, and the reaction is continued for 8 hours; after the reaction system is transferred to room temperature overnight, the reaction system is incubated at-20°C for 30 minutes, the precipitate is filtered, and then the reaction temperature is increased to 70-75°C for 96 hours; after the reaction is completed, the reaction temperature is cooled to room temperature, and the reaction system is precipitated with excess ethyl ether to obtain white solid, which is then washed with tetrahydrofuran for 3-5 times; after the supernatant is clear, the product is vacuum dried to obtain choline phosphate monomer; The ratio of the organic reagent, dimethylaminoethyl methacrylate, hydroquinone monomethyl ether, acetonitrile and 2-chloro-2-oxo-1,3,2-dioxaphospholane is 0.08:0.12:200:50:0.05 (mole:mole:milligram:milliliter:mole); (2) Double bond modification on the surface of nanomaterials The nanomaterials are added into a mixed solution of ethanol and isopropyl alcohol, and then ammonia water and 3(trimethoxysilyl)propyl methacrylate are added, and the reaction is carried out at 60°C for 2 hours; then the unreacted raw materials are removed by washing with ethanol for several times to obtain the magnetic balls and silicon balls modified with double bond; The ratio of the nanomaterials, ethanol, isopropyl alcohol, ammonia water and 3(trimethoxysilyl)propyl methacrylate is 50:45:15:3:0.45 (milligram:milliliter:milliliter:milliliter:milliliter); (3) Synthesis of choline phosphate modified nanohydrogel by precipitation polymerization The nanomaterials modified with double bond are added into acetonitrile solution, and then choline phosphate monomer, methylenebisacrylamide and azobisisobutyronitrile are added, and the reaction is stopped after heating at 100°C for 1 hour; then the product, choline phosphate functionalized nanohydrogel, is obtained by washing with ethanol and water for several times. The ratio of acetonitrile: modified double bond nanomaterial: choline phosphate monomer: methylene bisacrylamide: azobisisobutyronitrile is 40:50:200:20:4.

5.

3. The nanohydrogel according to claim 1 or 2, characterized in that: The particle size of the magnetic ball in step (2) is 50-500 nm, and the particle size of the silicon ball is 100-1000 nm. Alternatively, the organic reagent in step (1) is methanol, ethanol, isopropanol, methoxy ethanol or n-butanol.

4. A choline phosphate functionalized nanohydrogel, characterized in that: The nanomaterial is a protein, and the preparation steps of the nanohydrogel are as follows: (1) Choline phosphate derivative 2-Chloro-2-oxo-1,3,2-dioxaphospholane is used as raw material to prepare choline phosphate CP derivative: First, 2-chloro-2-oxo-1,3,2-dioxaphospholane is substituted with organic reagent under the catalysis of dimethylaminoethyl methacrylate to generate 2-methoxy-2-oxo-1,3-dioxaphospholane, and then ring-opening reaction with dimethylaminoethyl methacrylate in solution at 70°C to synthesize CP derivative containing double bond; (2) Preparation of choline phosphate functionalized nanohydrogel C=C bond is introduced on the surface of horseradish peroxidase by N-hydroxysuccinimide acrylate, and HRP-C=C is obtained; finally, the CP derivative monomer and crosslinking agent synthesized above are polymerized on the surface of the protein by free radical polymerization method using ammonium persulfate and TEMED as initiator, and choline phosphate functionalized nanohydrogel is obtained.

5. The nanohydrogel of claim 4, wherein: The specific preparation steps are as follows: (1) Choline phosphate monomer synthesis step The organic reagent, dimethylaminoethyl methacrylate, hydroquinone monomethyl ether and acetonitrile are added to a single-neck flask under argon protection, and then cooled to-78°C; Then, 2-chloro-2-oxo-1,3,2-dioxaphospholane is added dropwise in 2 hours, and the reaction is continued for 8 hours; after the reaction system is transferred to room temperature overnight, the reaction system is incubated at-20°C for 30 minutes, the precipitate is filtered, and the reaction temperature is increased to 70-75°C for 96 hours; after the reaction is completed, it is cooled to room temperature, and an excess of diethyl ether is used to precipitate white solid, which is then washed with tetrahydrofuran for 3-5 times; after the supernatant is clear, it is vacuum dried to obtain choline phosphate monomer; The ratio of organic reagent: dimethylaminoethyl methacrylate: hydroquinone monomethyl ether: acetonitrile: 2-chloro-2-oxo-1,3,2-dioxaphospholane: mole: mole: milligram: milliliter: mole is 0.08:0.12:200:50:0.05; (2) Double bond modification on the surface of nanomaterial Horseradish peroxidase is added to 50 mmol sodium bicarbonate solution with pH of 8.5, and then N-hydroxysuccinimide acrylate is added, and the reaction is carried out at room temperature for 2 hours, and then dialyzed for 24 hours to obtain modified double bond HRP; The ratio of sodium bicarbonate solution: horseradish peroxidase: N-hydroxysuccinimide acrylate is 4:10:3.4; (3) Synthesis of choline phosphate modified HRP by free radical polymerization method The HRP solution with modified double bond is added with choline phosphate monomer, methylene bisacrylamide, ammonium persulfate and TEMED, the reaction is stopped after stirring at room temperature for one hour; then, the excess monomer and initiator are removed by dialysis for 24 hours to obtain the product choline phosphate functionalized nanohydrogel; The ratio of the nanomaterial with modified double bond, choline phosphate monomer, methylene bisacrylamide, ammonium persulfate and TEMED is 5:50:2:2:0.005 mg:mg:mg:mg:ml.

6. The nanohydrogel according to claim 4 or 5, wherein: The organic reagent in step (1) is methanol, ethanol, isopropyl alcohol, methoxy ethanol or n-butanol.

7. A choline phosphate functionalized nanohydrogel, characterized in that: The nanomaterial is colloidal gold, cerium dioxide, nano platinum, quantum dots or nanohydrogel, and the preparation steps of the nanohydrogel are as follows: (1) Choline phosphate derivative 2-chloro-2-oxo-1,3,2-dioxaphospholane is selected as raw material to prepare choline phosphate CP derivative: Firstly, 2-chloro-2-oxo-1,3,2-dioxaphospholane is subjected to substitution reaction with organic reagent under catalysis of dimethylaminoethyl methacrylate to generate 2-methoxy-2-oxo-1,3-dioxaphospholane, and then subjected to ring-opening reaction with dimethylaminoethyl methacrylate in solution at 70°C to synthesize CP derivative containing double bond; (2) Preparation of choline phosphate functionalized nanohydrogel Prepare 5 nanomolar colloidal gold solution, cerium dioxide solution, nano platinum solution and quantum dot solution; Firstly, the pH of the above nanomaterial solution is adjusted to 9 by using 1 mole per liter sodium hydroxide solution; then, N-(3-aminopropyl) methacrylamide hydrochloride with a final concentration of 10 micromole is added to the nanomaterial solution to introduce carbon-carbon double bond on the surface of the nanomaterial; finally, the CP derivative monomer and crosslinking agent synthesized above are polymerized on the surface of the nanomaterial by free radical polymerization method with ammonium persulfate and TEMED as initiators, thereby obtaining choline phosphate functionalized nanohydrogel.

8. The nanohydrogel of claim 7, wherein: The specific preparation steps are as follows: (1) Choline phosphate monomer synthesis step The organic reagent, dimethylaminoethyl methacrylate, hydroquinone monomethyl ether and acetonitrile are added to a single-neck flask under argon protection, and then cooled to-78°C; Then, 2-chloro-2-oxo-1,3,2-dioxaphospholane is added dropwise within 2 hours, and the reaction is continued for 8 hours; after the reaction system is transferred to room temperature overnight, the reaction system is incubated at-20°C for 30 minutes, the precipitate is filtered, and then the reaction temperature is increased to 70-75°C for 96 hours; after the reaction is completed, the reaction system is cooled to room temperature, and the white solid is precipitated by using excess ether, and then washed with tetrahydrofuran for 3-5 times; after the supernatant is clear, the choline phosphate monomer is obtained by vacuum drying; The ratio of the organic reagent, dimethylaminoethyl methacrylate, hydroquinone monomethyl ether, acetonitrile and 2-chloro-2-oxo-1,3,2-dioxaphospholane is 0.08:0.12:200:50:0.05 mole:mole:mg:ml:mole; (2) Double bond modification on the surface of nanomaterial In the colloidal gold solution, cerium dioxide solution, nano platinum solution, quantum dot solution, N- (3- aminopropyl) methacrylic acid amine hydrochloride is added, and the reaction is carried out at room temperature for 1 hour to obtain a nano material with modified double bonds; The ratio of the nano material: N- (3- aminopropyl) methacrylic acid amine hydrochloride is 5:0.01; (3) Synthesis of choline phosphate modified nanohydrogel by using free radical polymerization method In the colloidal gold solution with modified double bonds, cerium dioxide solution, nano platinum solution, quantum dot solution, choline phosphate monomer, methylene bisacrylamide, ammonium persulfate and TEMED are added, and the reaction is stopped after stirring at room temperature for one hour; then, the excess monomer and initiator are removed by dialysis for 24 hours to obtain the product choline phosphate functionalized nanohydrogel; The ratio of the nano material with modified double bonds: choline phosphate monomer: methylene bisacrylamide: ammonium persulfate: TEMED is 5:50:2:2:0.

005.

9. The nanohydrogel according to claim 7 or 8, wherein: The particle size of the colloidal gold, cerium dioxide, nano platinum and quantum dots is 1-200 nm; Alternatively, the organic reagent in step (1) is methanol, ethanol, isopropanol, methoxyethanol or n-butanol.

10. A highly efficient method for recognizing or extracting exosomes in body fluids using the choline phosphate functionalized nanohydrogel according to claim 1, characterized in that: The steps are as follows: The method of identification is: After capturing the exosomes expressing disease-specific proteins in the cell culture supernatant or body fluid by modifying the microspheres, magnetic spheres, ELISA well plates or microarrays with disease-specific protein antibodies, the nanohydrogel is added, incubated at room temperature for two hours, and then the excess choline phosphate functionalized nanohydrogel is removed by washing. Finally, color development is performed using color developing solution; The extraction method is: Take the body fluid, centrifuge, add nanohydrogel magnetic spheres, shake at room temperature for two hours, and then remove the excess impurities by magnetic washing. Finally, the magnetic spheres are placed in PBS at 42 degrees Celsius for 30 minutes to release the exosomes.

11. The method of claim 10, wherein: The specific steps of extraction are as follows: 1) The extraction steps for exosomes in serum are as follows: The serum is centrifuged at 3000g at 4 degrees Celsius for 10 minutes, then 2mg / mL nanohydrogel magnetic spheres MB@CPs are added, shaken at room temperature for two hours, and then the excess impurities are removed by magnetic washing. Finally, the magnetic spheres are placed in PBS at 42 degrees Celsius for 30 minutes to release the exosomes; The volume ratio of serum: nanohydrogel magnetic spheres: PBS is 300:0.5:300; 2) The extraction steps for exosomes in urine are as follows: The urine is centrifuged at 5000g at 4 degrees Celsius for 30 minutes, then 2mg / mL nanohydrogel magnetic spheres MB@CPs are added, shaken at room temperature for two hours, and then the excess impurities are removed by magnetic washing. Finally, the magnetic spheres are placed in PBS at 42 degrees Celsius for 30 minutes to release the exosomes; The volume ratio of urine: nanohydrogel magnetic spheres: PBS is 10:1:300; 3) The extraction steps for exosomes in saliva are as follows: Saliva was centrifuged at 3000g for 10 minutes at 4 degrees Celsius, then 2 mg / mL hydrogel magnetic beads MB@CPs were added and shaken at room temperature for two hours. After the end, the excess impurities were removed by magnetic washing, and finally the magnetic beads were placed in PBS and shaken at 42 degrees Celsius for 30 minutes to release the exosomes. Among them, the volume ratio of saliva: hydrogel magnetic beads: PBS is 1:1:300.

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