A method for isolation and detection of extracellular biological macromolecules

Abstract

The application provides a method for separating and detecting extracellular biological macromolecules, and belongs to the technical field of molecular biology. The method comprises the following steps: labeling target extracellular biological macromolecules by using a fluorescent dye; separating the extracellular biological macromolecules by using a magnetic capture agent and an external magnetic field; and performing single-nanoparticle analysis by using a particle analysis detection device, wherein the capture agent is a capture agent capable of binding to the target extracellular biological macromolecules. The method has the advantages of fewer operation steps, high accuracy, less damage to the biological characteristics of the extracellular biological macromolecules, and the like, and has great potential for clinical application.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a method for the isolation and detection of extracellular biological macromolecules. Background Technology

[0002] Extracellular biomolecules are extracellular nucleic acids, proteins, carbohydrates, lipids, or combinations thereof. Examples include extracellular proteins that can serve as tumor markers, such as serum tumor marker proteins like CEA, AFP, CA19-9, SCC, NSE, CK19, CA125, CA 15-3, and CA 19-9; extracellular nucleic acids with clinical diagnostic value, such as extracellular deoxyribonucleic acid (DNA); free DNA released from circulating tumor cells in plasma; extracellular biomolecules with plasma membrane structures, such as extracellular vesicles; extracellular nucleoprotein particles with vital characteristics; pathogenic or infectious agents in body fluids, such as blood-borne infectious viruses, such as HIV, hepatitis B virus, hepatitis C virus, and cytomegalovirus in the blood.

[0003] In recent years, with a deeper understanding of extracellular macromolecules, their detection has become a novel and promising liquid biopsy technology widely used in clinical research institutions for non-invasive diagnosis, treatment, and monitoring of tumors and diseases. Efficient extraction of extracellular macromolecules is key to the routine clinical application of this emerging liquid biopsy technology. As one of the three major areas of liquid biopsy, extracellular macromolecules possess numerous potential biological targets, but the ability to simultaneously extract trace amounts of nucleic acids and proteins typically requires the application of exocrine separation techniques to obtain high-purity extracellular macromolecules.

[0004] Traditional methods for enriching and separating extracellular macromolecules include ultracentrifugation and polymer precipitation. These methods generally suffer from poor purity, low yield, and large batch-to-batch variability. Immunomagnetic bead adsorption can overcome these drawbacks and has potential for clinical application. However, the aforementioned techniques, including traditional immunomagnetic bead adsorption, can only separate one component of extracellular macromolecules (e.g., total extracellular macromolecules or total extracellular vesicles), and cannot separate specific subgroups of extracellular macromolecules (e.g., extracellular biological macromolecules). Therefore, it cannot achieve the goal of using a specific subgroup of extracellular macromolecules to reflect the severity of disease. Other limitations of traditional immunomagnetic bead adsorption include:

[0005] 1. It cannot be washed away, and even if it can be washed away, it requires extreme physical conditions (high temperature, strong alkali and strong acid), which undoubtedly causes the loss or destruction of extracellular biological macromolecules;

[0006] 2. Currently popular methods for separating extracellular biomolecules using immunomagnetic beads can only harvest total extracellular biomolecules. This means that specific extracellular biomolecules carrying disease biomarkers are interfered with by the total extracellular biomolecules, limiting their detection sensitivity. In today's era of precision medicine advancements, accurate typing of extracellular macromolecules based on different cell origins has become an urgent problem to solve. This requirement represents the first step in the development and clinical application of liquid biopsy.

[0007] 3. Current non-destructive elution methods for magnetic beads all require elution reagents, which are often in excess. High concentrations of these excess reagents lead to high background, affecting detection. Furthermore, excessively high concentrations of some elution reagents may elute and destroy meaningful components on extracellular biomolecules. Therefore, a highly efficient elution method with controllable reagent concentrations is needed to be compatible with downstream single-particle analysis.

[0008] Therefore, there is still an urgent need for a method that is highly accurate, easy to operate, and capable of separating and detecting single particles of extracellular biomolecules. Summary of the Invention

[0009] To address the above problems, this invention provides a method for the isolation and detection of extracellular biomacromolecules.

[0010] A method for separating and detecting extracellular biomolecules in a biological fluid includes: labeling target extracellular biomolecules with a fluorescent dye; separating extracellular biomolecules using a magnetic trap and an external magnetic field; and performing single nanoparticle analysis using a particle analysis detection device. The trap is a trap capable of binding to the target extracellular biomolecule. The magnetic trap comprises a solid support, a connecting medium, and a trap, or the magnetic trap comprises a solid support and a trap.

[0011] In some embodiments, the fluorescent dye contains an adaptor structure capable of binding to target extracellular biomolecules.

[0012] In some embodiments, the trapping agent in the magnetic trapping device is a trapping agent capable of binding to the target extracellular biomolecule. In some embodiments, the trapping agent in the magnetic trapping device is a trapping agent capable of specifically binding to the target extracellular biomolecule.

[0013] In some embodiments of the present invention, a method for separating and detecting extracellular biomacromolecules in a biological fluid includes:

[0014] Step S1: The fluorescent dye contains an adaptor structure that can bind to the target extracellular biomolecule, enabling the fluorescent dye to bind to the target extracellular biomolecule through the adaptor structure.

[0015] Step S2: The trapping agent in the magnetic trapping molecule is a trapping agent that can bind to the target extracellular biomolecule, so that the magnetic trapping molecule binds to the target extracellular biomolecule through the trapping agent;

[0016] Step S3: Separate the target extracellular biomolecules from the biological fluid using an external magnetic field;

[0017] Step S4: Use elution buffer to release the magnetic trapping molecule from the target extracellular biomolecule and remove the solid-phase carrier;

[0018] Step S5: After steps S1, S2, S3 and S4, the target extracellular biomolecule bound to the fluorescent dye is obtained. Single nanoparticle analysis is performed on the target extracellular biomolecule bound to the fluorescent dye using a particle analysis detection device.

[0019] The order of steps S1, S2, S3, S4 and S5 is as follows: (1) Step S1 → Step S2 → Step S3 → Step S4 → Step S5; or (2) Step S2 → Step S1 → Step S3 → Step S4 → Step S5; or (3) Step S1 and Step S2 are performed simultaneously → Step S3 → Step S4 → Step S5; or (4) Step S2 → Step S3 → Step S1 → Step S4 → Step S5.

[0020] In some preferred embodiments of the present invention, the order of steps S1, S2, S3, S4, and S5 is: step S2 → step S3 → step S1 → step S4 → step S5. This embodiment eliminates the need for ultracentrifugation to remove dyes, avoids damage to the target extracellular biomolecules caused by ultracentrifugation, and has advantages such as fewer operational steps, higher accuracy, and less damage to the biological characteristics of extracellular biomolecules.

[0021] In some preferred embodiments of the present invention, the order of steps S1, S2, S3, S4, and S5 is: step S1 → step S2 → step S3 → step S4 → step S5. This embodiment eliminates the need for ultracentrifugation to remove dyes, avoids damage to the target extracellular biomolecules caused by ultracentrifugation, and has advantages such as fewer operational steps, higher accuracy, and less damage to the biological characteristics of extracellular biomolecules.

[0022] In some preferred embodiments of the present invention, the order of steps S1, S2, S3, S4, and S5 is as follows: steps S1 and S2 are performed simultaneously → step S3 → step S4 → step S5. This embodiment eliminates the need for ultracentrifugation to remove dyes, avoids damage to the target extracellular biomolecules caused by ultracentrifugation, and has advantages such as fewer operational steps, higher accuracy, and less damage to the biological characteristics of extracellular biomolecules.

[0023] In some preferred embodiments of the present invention, the order of steps S1, S2, S3, S4, and S5 is: step S2 → step S1 → step S3 → step S4 → step S5. This embodiment eliminates the need for ultracentrifugation to remove dyes, avoids damage to the target extracellular biomolecules caused by ultracentrifugation, and has advantages such as fewer operational steps, higher accuracy, and less damage to the biological characteristics of extracellular biomolecules.

[0024] The solid support may include nanomagnetic beads or solid-phase chips.

[0025] The solid support can be superparamagnetic.

[0026] The solid support may be loaded with functional groups.

[0027] The nanomagnetic beads can be nanomagnetic beads with superparamagnetism.

[0028] The particle size of the solid support can be 10 nm to 2000 nm. In some embodiments, the particle size of the solid support is 100 nm to 1200 nm. In some embodiments, the particle size of the solid support is 30 nm to 200 nm.

[0029] In some preferred embodiments of the present invention, a method for separating and detecting extracellular biomolecules in a biological liquid includes: mixing a fluorescent dye with a biological liquid, wherein the fluorescent dye contains an adaptor structure capable of binding to a target extracellular biomolecule; incubating the mixture to allow the fluorescent dye to bind to the target extracellular biomolecule through the adaptor structure; adding a magnetic trapping molecule, wherein the trapping agent in the magnetic trapping molecule is a trapping agent capable of binding to the target extracellular biomolecule; incubating the mixture to allow the magnetic trapping molecule to bind to the target extracellular biomolecule through the trapping agent; applying an external magnetic field for separation and enrichment; removing the supernatant; washing the mixture to separate the target extracellular biomolecule from the biological liquid, obtaining the target extracellular biomolecule bound to the fluorescent dye and the magnetic trapping molecule; eluting the magnetic trapping molecule from the target extracellular biomolecule with an eluent and removing the solid-phase support, obtaining the target extracellular biomolecule bound to the fluorescent dye; and performing single-nanoparticle analysis on the target extracellular biomolecule containing the fluorescent dye using a particle analysis detection device.

[0030] In some preferred embodiments of the present invention, a method for separating and detecting extracellular biomolecules in a biological fluid includes: mixing a magnetic trapping molecule with the biological fluid, wherein the trapping agent in the magnetic trapping molecule is a trapping agent capable of binding to the target extracellular biomolecule; incubating to allow the magnetic trapping molecule to bind to the target extracellular biomolecule through the trapping agent; adding a fluorescent dye, wherein the fluorescent dye contains an adaptor structure capable of binding to the target extracellular biomolecule; incubating to allow the fluorescent dye to bind to the target extracellular biomolecule through the adaptor structure; applying an external magnetic field for separation and enrichment; removing the supernatant; washing to separate the target extracellular biomolecule from the biological fluid, obtaining the target extracellular biomolecule bound to the fluorescent dye and the magnetic trapping molecule; eluting with an elution buffer to release the binding of the magnetic trapping molecule to the target extracellular biomolecule; removing the solid-phase support to obtain the target extracellular biomolecule bound to the fluorescent dye; and performing single-nanoparticle analysis on the target extracellular biomolecule containing the fluorescent dye using a particle analysis detection device.

[0031] In some preferred embodiments of the present invention, a method for separating and detecting extracellular biomolecules in a biological liquid includes: mixing a magnetic trapping molecule and a fluorescent dye with the biological liquid, wherein the trapping agent in the magnetic trapping molecule is a trapping agent capable of binding to the target extracellular biomolecule, and the fluorescent dye contains an adaptor structure capable of binding to the target extracellular biomolecule; incubating to allow the magnetic trapping molecule and the fluorescent dye to bind to the target extracellular biomolecule through the trapping agent; applying an external magnetic field for separation and enrichment; removing the supernatant; washing to separate the target extracellular biomolecule from the biological liquid, obtaining the target extracellular biomolecule bound to the fluorescent dye and the magnetic trapping molecule; eluting with an elution buffer to release the binding of the magnetic trapping molecule to the target extracellular biomolecule, and removing the solid-phase support, obtaining the target extracellular biomolecule bound to the fluorescent dye; and performing single-nanoparticle analysis on the target extracellular biomolecule containing the fluorescent dye using a particle analysis detection device.

[0032] In some embodiments of the present invention, a method for separating and detecting extracellular biomolecules in a biological fluid includes: mixing a magnetic trapping molecule with the biological fluid, wherein the trapping agent in the magnetic trapping molecule is a trapping agent capable of binding to the target extracellular biomolecule; incubating to allow the magnetic trapping molecule to bind to the target extracellular biomolecule through the trapping agent; applying an external magnetic field for separation and enrichment; removing the supernatant; washing to separate the target extracellular biomolecule from the biological fluid; mixing a fluorescent dye with the target extracellular biomolecule, wherein the fluorescent dye contains an adaptor structure capable of binding to the target extracellular biomolecule; incubating to allow the fluorescent dye to bind to the target extracellular biomolecule through the adaptor structure; removing the free fluorescent dye by centrifugation, ultrafiltration, or dialysis to obtain the target extracellular biomolecule bound to the fluorescent dye and the magnetic trapping molecule; using an eluent to release the binding between the magnetic trapping molecule and the target extracellular biomolecule, and removing the solid-phase support to obtain the target extracellular biomolecule bound to the fluorescent dye; and performing single-nanoparticle analysis on the target extracellular biomolecule bound to the fluorescent dye using a particle analysis detection device.

[0033] In some embodiments, the capture agent may include at least one or a combination thereof selected from peptides, proteins, antibodies, ligands and / or receptors, enzymes, growth factors, glycolipids, polysaccharides, and nucleic acids.

[0034] In some embodiments, the trapping agent is at least one of antibodies, lipids, polysaccharides, or a combination thereof.

[0035] In some embodiments, the antibody includes at least one or a combination of CD9 antibody, CD63 antibody, CD81 antibody, TIM4 protein, Annexin V protein, extracellular vesicle protein-specific antibody, disease marker protein-specific antibody, or virus-specific antibody.

[0036] In some embodiments, the disease includes tumors, inflammation, chronic diseases, or neurodegenerative diseases.

[0037] In some embodiments, the lipids include at least one or a combination thereof selected from distearylphosphatidylethanolamine, 1,2-di-O-octadecenyl-3-trimethylpropane, 1,2-dioleoyl-3-trimethyl-propane, 3β-[N-(N', dimethylaminoethane hydrochloride)-carbamoyl]cholesterol, 1,2-di-O-octadecenyl-3-dimethylpropane, 1,2-dioleoyl-3-dimethyl-propane, methionine, L-α-phosphatidylcholine, cholesterol, 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine, saturated fatty acids, 1,1,2-dioleoyl-sn-glycerol-3-phosphocholine, and 1,2-distearyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)].

[0038] In some embodiments, the polysaccharide includes at least one or a combination thereof selected from heparin, polylactosamine, α2,6-conjugated sialic acid, high-mannopolysaccharide, complex N-glycans.

[0039] In some embodiments, the nucleic acid includes at least one selected from nucleic acid aptamers, nucleic acid probes, or combinations thereof.

[0040] In some embodiments, the antibody may include an antibody selected from CD63.

[0041] In some embodiments, the surface of the solid support may be loaded with functional groups, including at least one or a combination of hydroxyl, carboxyl, amino, aldehyde, epoxy, maleimide, p-toluenesulfonyl, N-hydroxysuccinimide, or thiol groups.

[0042] In some embodiments, the trapping agent can be directly connected to the solid support. In some embodiments, the trapping agent can be connected to the solid support via a connecting medium.

[0043] In some embodiments, the capture agent can be directly connected to a solid support, and the capture agent is selected from TIM4, nucleic acid aptamers, and heparin.

[0044] In some embodiments, the trapping agent can be connected to a solid support via a linking medium, wherein the trapping agent is selected from TIM4, nucleic acid aptamers, and heparin. The linking medium comprises one or a combination of compounds, groups, and molecular materials that link the solid support and the trapping agent.

[0045] In a preferred embodiment, the trapping agent can be connected to a solid support via a linking medium, and the loaded functional groups on the surface of the solid support can be used to bind the trapping agent to the solid support. This implementation allows for the separation of the solid support from extracellular biomolecules by using a specific eluent to break down and / or depolymerize the linking medium under mild reaction conditions. If the linking medium cannot be broken down and / or depolymerized under mild conditions in either the direct or indirect connection method, a specific eluent can be used to competitively elute the solid support from the extracellular biomolecules under the mild reaction conditions.

[0046] In some embodiments, the linking medium may include a linker selected from oligonucleotide single strands, oligonucleotide double strands, oligonucleotide double strands containing specific restriction enzyme sites, or linkers containing disulfide bond bridging molecules.

[0047] In some embodiments, the linker molecule comprising the disulfide bond bridging molecule may include, selected from, distearate-phosphatidylethanolamine-polyethylene glycol-disulfide bond (DSPE-PEG-SS), biotin disulfide bond (NHS-SS-biotin), dithiobis(succinimide propionate) (DSP), dithiobis(sulfosuccinimide propionate) (DTSSP), disuccinimide succinate (DSS), sodium bis(thiosuccinimide) succinate (BS3), disuccinimide tartrate (DST), disulfosuccinimide tartrate (sulfo-DST), bis(2-(succinimideoxycarbonyloxy)ethyl) sulfone (BSOCOES), bis(2-(sulfosuccinimideoxycarbonyloxy)ethyl) sulfone (sulfo-BSOCOES), and ethyl... Diol-bis(succinimide succinimide) succinimide (EGS), ethylene glycol-bis(sulfosuccinimide succinimide) succinimide (sulfo-EGS), bis(succinimide glutarate) (DSG), N,N'-disuccinimide carbonate (DSC), dimethyl hexamethylenediimide (DMA), dimethyl heptamethimide (DMP), dimethyl octyl dinitrate (DMS), dimethyl 3,3'-dithiodipropionimine (DTBP), 1,4-di(3'-(2'-dithiopyridine)propionic acid amino)butane (DPDPB), dimaleimide hexane (BMH), difluorodinitrophenyl (DFDNB), difluorodinitrophenyl sulfone (DFDNPS), di(β-(4-azidosalicylic acid amino)ethyl)disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-Butanediol glycidyl ether, adipic acid dihydrazide (ADH), carbazide, diaminodimethylbiphenyl, p-diaminobiphenyl, nitrogen-succinate argonamine-3-(2-pyridinedithio)-ester (SPDP), long-chain nitrogen-succinate argonamine-3-(2-pyridinedithio)-ester (LC-SPDP), sulfonyl long-chain nitrogen-succinate argonamine-3-(2-pyridinedithio)-ester (sulfo-LC-SPDP), succinimide oxocarbonyl-methyl-(2-pyridylthio)benzene (SMPT), sulfonyl long-chain succinimide oxocarbonyl-methyl-(2-pyridylthio)benzene (sulfo-LC-SMPT), 4-(N-maleimide methyl 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid N-hydroxysuccinimide ester (SMCC), 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid 3-thio-N-succinimide sodium salt (sulfo-SMCC), maleimide benzoate succinimide ester (MBS), M-maleimide benzoate succinimide ester (sulfo-MBS), N-succinimide (4-iodoacetyl)aminobenzoic acid (SIAB), sulfonyl-N-succinimide (4-iodoacetyl)aminobenzoic acid (sulfo-SIAB), 4-(4-maleimidephenyl)butyrate succinimide ester (SMPB), sulfosuccinimide-4-(P-maleimide) Succinimide-6-((iodoacetyl)amino)hexanoate (SIAX), succinimide-6-(6-(((4-iodoacetyl)amino)hexanoate)amino)hexanoate (SIAXX), succinimide-4-((((iodoacetyl)amino)methyl)cyclohexane-1-carboxylic acid (SIAC), succinimide-6-((((4(iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (SIAXX) SIACX), 4-nitrobenzene iodoacetic acid (NPIA), 4-(4-N-maleimide benzyl ester) butyric acid hydrazide (MPBH), 4-N-maleimide methylcyclohexane-1-carboxylic acid hydrazide (M2C2H), 3-(2-pyridyl dithio)propionyl hydrazide (PDPH), N-hydroxysuccinimide-4-azidosalicylic acid (NHS-ASA), N-hydroxysulfosuccinimide-4-azidosalicylic acid (sulfo-NHS-ASA), sulfosuccinimide-4-azidosalicylic acid (sulfo-NHS-LC-ASA), sulfosuccinimide-2-(P-azido-salicylic acid)ethyl-1-3'-Dithiopropionate (SASD), succinimide-4-azidobenzoate (HSAB), sulfosuccinimide-4-azidobenzoate (sulfo-HSAB), N-succinimide-6-(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH), sulfosuccinimide-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-SANPAH), 5-azido-2-nitrobenzoic acid-N-succinimide (ANB-NOS), sulfosuccinimide-2-(M-nitroazido-benzamide)- Ethyl-1,3'-dithiopropionate (SAND), N-succinimide-(4-azidophenyl)-1,3'-dithiopropionate (SADP), N-sulfosuccinimide-(4-azidophenyl)-1,3'-dithiopropionate (sulfo-SADP), sulfosuccinimide-4-(P-azidophenyl)butyric acid (Sulfo-SAPB), sulfosuccinimide-2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3'-dithiopropionate (SAED), sulfosuccinimide-7-azido-4-methylcoumarin-3-acetamide Sulfo-SAMCA, p-nitrophenyl diazopyruvate (pNPDP), p-nitrophenyl-2-diazo-3,3,3-trifluoropropionic acid (PNP-DTP), 1-(p-azidosalicylamido)-4-(iodoacetamido)butane (ASIB), N-(4-(p-azidosalicylamido)butyl)-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimide, p-azidobenzoylhydrazine, 4-(p-azidosalicylamido)-butylamine (ASBA), p-azidophenylglyoxal (APG), 4-azido- At least one or a combination of the following: 2-nitrophenyl biotin-4-nitrophenyl ester (ABNP), sulfosuccinimide-2-(6-(biotinamido)-2-(p-azidobenzoylamino)hexanoylamino)ethyl-1,3-dithiopropionate (sulfo-SBED), methanethiosulfonate azidotetrafluoro-long-chain biotin (MTS-ATF-biotin), methanethiosulfonate azidotetrafluorobiotin (MTS-ATF-LC-biotin), tris(hydroxymethyl)phosphine (THP), tris(hydroxymethyl)phosphopropionic acid (THPP), and tris(2-carboxyethyl)phosphine hydrochloride.

[0048] In some embodiments, the functional group may include at least one or a combination thereof selected from hydroxyl (-OH), carboxyl (-COOH), amino (-NH2), aldehyde (-CHO), epoxy, maleimide, N-hydroxysuccinimide, tosyl, N-hydroxysuccinimide (-NHS), or thiol (-SH).

[0049] In some embodiments, the linking medium may undergo cleavage and / or depolymerization reactions under conditions using a specific eluent, or the linking medium may separate the trapping agent from the target extracellular biomolecule through competitive elution under conditions using a specific eluent; this is beneficial for improving the retention of the biological characteristics of the extracellular biomolecule. The linking medium is selected from ion exchange complexes, enzymes and their substrate complexes, antigen-antibody complexes, ligands and their receptor complexes, photocatalytically mediated chemicals capable of chemical bond cleavage, and reducing or oxidizing agents mediated by chemical bond cleavage.

[0050] In some preferred embodiments, the linker can undergo cleavage and / or depolymerization under conditions using a specific eluent, or the linker can separate the capture agent from the target extracellular biomolecule through competitive elution under conditions using a specific eluent; this is beneficial for improving the retention of the biological characteristics of the extracellular biomolecule. The linker is selected from nickel chloride or nickel sulfate with histone affinity structures, or glutathione with glutathione thiotransferase affinity structures. In some embodiments, the linker is selected from nickel chloride or nickel sulfate with histone affinity structures, and the specific eluent is selected from imidazole. In some embodiments, the linker is selected from glutathione with glutathione thiotransferase affinity structures, and the specific eluent is reduced glutathione. In some embodiments, the competitive elution may include elution using one or more of complementary sequences, structural analogs, chelating agents, and solvent displacement. In some embodiments, the linker is an oligonucleotide single strand, and the competitive elution involves eluting with a sequence complementary to the oligonucleotide single strand, thereby altering one or more of the affinities between linkers, between linkers and traps, and between traps and targets, thus achieving the elution effect. In some embodiments, the competitive elution involves applying an ion chelating agent to chelate specific ions in the solvent, thereby altering one or more of the affinities between linkers, between linkers and traps, and between traps and targets, thus achieving the elution effect. In some embodiments, the linker is dethiobiotin or its analogues and avidin protein, and the competitive elution involves applying one or more of biotin or its analogues to competitively elute the linker and / or the trap containing one or more of dethiobiotin or its analogues, thereby altering one or more of the affinities between linkers, between linkers and traps, and between traps and targets, thus achieving the elution effect. In some embodiments, the competitive elution is achieved by changing the solvent ionic strength through altering the competitive elution method, thereby changing one or more of the affinities between the linking media, between the linking media and the trapping agent, and between the trapping agent and the target, thus achieving the elution effect.

[0051] In some embodiments, the reaction temperature of the fracture and / or depolymerization reaction can be 0°C-55°C. In some embodiments, the reaction temperature of the fracture and / or depolymerization reaction is 4°C-42°C. In some embodiments, the reaction temperature of the fracture and / or depolymerization reaction is 20°C-37°C. In some embodiments, the reaction temperature of the fracture and / or depolymerization reaction is 0°C, 4°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 42°C, 45°C, 50°C, or 55°C.

[0052] In some embodiments, the reaction pH of the fracture and / or depolymerization reaction can be 5-8. In some embodiments, the reaction pH of the fracture and / or depolymerization reaction is 6.5-7.5. In some embodiments, the reaction pH of the fracture and / or depolymerization reaction is 5.0, 5.5, 6.0, 6.5, 7.0, or 7.5.

[0053] In some embodiments, the breaking and / or depolymerization reaction may include a selection of breaking and / or depolymerizing the connecting medium mediated by biological enzymes and / or chemical reagents.

[0054] In some embodiments, the bioenzyme may include a selection of nucleases or restriction endonucleases.

[0055] In some embodiments, the chemical reagent may include at least one or a combination thereof selected from trichloroethyl phosphate (TCEP), trichloropropyl phosphate, ethyl phosphate, 2-mercaptoethylamine (2-MEA), reduced glutamate, oxidized glutamate, 2-mercaptoethanol, dithiothreitol (DTT), cysteine, cystine, dithiobutylamine, or sodium sulfite.

[0056] In some embodiments, the breaking and / or depolymerization reaction may include using a nuclease to break and / or depolymerize the phosphodiester bonds on the linking medium having oligonucleotide single strands or oligonucleotide double strands.

[0057] In some embodiments, the breaking and / or depolymerization reaction may include breaking and / or depolymerization reactions using specific restriction enzyme sites contained on the linker having an oligonucleotide double strand with restriction endonuclease pairs.

[0058] In some embodiments, the breaking and / or depolymerization reaction may include using the eluent, comprising at least one or a combination of the following chemical reagents: trichloroethyl phosphate (TCEP), trichloropropyl phosphate, ethyl phosphate, 2-mercaptoethylamine (2-MEA), reduced glutamate, oxidized glutamate, 2-mercaptoethanol, dithiothreitol (DTT), cysteine, cystine, dithiobutylamine, or sodium sulfite, to break and / or depolymerize the disulfide bonds of the linking medium containing disulfide bridging molecules.

[0059] In some embodiments, the surface of the target extracellular biomacromolecule may contain substances that can specifically bind to the trapping agent.

[0060] In some embodiments, the substance that can specifically bind to the trapping agent may include at least one or a combination of antigens, nucleic acids, fats, carbohydrates, or other similar substances.

[0061] In some embodiments, the surface of the target extracellular biomacromolecule may contain CD63 antigen, and the capture agent may be a CD63 antibody.

[0062] In some embodiments, the surface of the target extracellular biomacromolecule may contain phosphatidylserine, and the trapping agent may be a member of the T-cell immunoglobulin domain mucin domain protein family, including TIM1, TM3, and TIM4 proteins.

[0063] In some embodiments, the surface of the target extracellular biomacromolecule may contain CD63 antigen and phosphatidylserine, and the trapping agent may be CD63 antibody and TIM4 protein.

[0064] In some embodiments, the target extracellular biomolecule is an extracellular vesicle, the surface of which contains a lipid membrane, and the scavenging agent is distearate phosphatidylethanolamine.

[0065] In some embodiments, the target extracellular biomolecule is an extracellular vesicle, the surface of which contains a lipid membrane and phosphatidylserine, and the scavenging agent is distearate phosphatidylethanolamine and TIM4 protein.

[0066] In some embodiments, the trapping agent and the solid support can be linked via polyethylene glycol disulfide bonds. In some embodiments, the trapping agent and the solid support can be linked via biotin and streptavidin protein.

[0067] In some embodiments, the CD63 antibody and the solid-phase carrier can be linked via polyethylene glycol-disulfide bonds, and the TIM4 protein and the solid-phase carrier can be linked via biotin and streptavidin.

[0068] In some embodiments, the solid support and the trapping agent are connected using avidin-biotin-disulfide-polyethylene glycol as a linking medium to obtain a magnetic trap with the structure of solid support-avidinin-biotin-disulfide-polyethylene glycol-trapping agent. In some embodiments, the solid support and the trapping agent are connected using streptavidin-biotin-disulfide-polyethylene glycol as a linking medium to obtain a magnetic trap with the structure of solid support-streptavidin-biotin-disulfide-polyethylene glycol-trapping agent.

[0069] In some embodiments, the solid support and the trapping agent are connected using streptavidin-biotin-disulfide-polyethylene glycol as a linking medium, and the trapping agent is distearylphosphatidylethanolamine, resulting in a magnetic trap with the structure solid support-streptavidin-biotin-disulfide-polyethylene glycol-distearylphosphatidylethanolamine. In some embodiments, the solid support and the trapping agent are connected using streptavidin-biotin-disulfide-polyethylene glycol as a linking medium, and the trapping agent is distearylphosphatidylethanolamine, resulting in a magnetic trap with the structure solid support-streptavidin-biotin-disulfide-polyethylene glycol-distearylphosphatidylethanolamine.

[0070] In some embodiments, the TIM4 protein is linked to a solid-phase support using biotin and streptavidin to obtain a magnetic trap with the structure solid-phase support-streptavidin-biotin-TIM4 protein.

[0071] In some embodiments, the capture agent and the solid support are connected via DTSSP-streptavidin protein-biotin as a linking medium.

[0072] In some embodiments, the eluent may be an aqueous solution containing an eluent.

[0073] In some embodiments, the capture agent and the solid support are connected via DTSSP-streptavidin-biotin as a linking medium, and the eluent is an aqueous solution containing an eluent. The eluent may include at least one or a combination thereof selected from trichloroethyl phosphate (TCEP), trichloropropyl phosphate, ethyl phosphate, 2-mercaptoethylamine (2-MEA), reduced glutamate, oxidized glutamate, 2-mercaptoethanol, dithiothreitol (DTT), cysteine, cystine, dithiobutylamine, or sodium sulfite.

[0074] In some embodiments, the capture agent and the solid support can be linked by streptavidin-biotin-disulfide bond-polyethylene glycol as a linking medium, and the eluent may include at least one or a combination thereof selected from trichloroethyl phosphate (TCEP), trichloropropyl phosphate, ethyl phosphate, 2-mercaptoethylamine (2-MEA), reduced glutamate, oxidized glutamate, 2-mercaptoethanol, dithiothreitol (DTT), cysteine, cystine, dithiobutylamine, or sodium sulfite.

[0075] In some embodiments, the capture agent may be a TIM4 protein, and the eluent may be an aqueous solution containing an eluent, which may include metal chelators selected from those capable of chelating calcium ions.

[0076] In some embodiments, the connecting medium is desulfurized biotin or its analogues and avidin protein, and the eluent is an aqueous solution containing an eluent, the eluent including biotin, biotin analogues and combinations thereof.

[0077] In some embodiments, the eluent is an aqueous solution containing an eluent, which may include at least one or a combination thereof selected from the following: trichloroethyl phosphate (TCEP), trichloropropyl phosphate, ethyl phosphate, 2-mercaptoethylamine (2-MEA), reduced glutamate, oxidized glutamate, 2-mercaptoethanol, dithiothreitol (DTT), cysteine, cystine, dithiobutylamine, or sodium sulfite. The content of the eluent, calculated by the total mass of the eluent, is 10 mM-500 mM, or 10 mM, 20 mM, 30 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, or 500 mM.

[0078] In some embodiments, the capturing agent may be TIM4 protein, the eluent is an aqueous solution containing an eluent, and the eluent may be a metal chelating agent capable of chelating calcium ions. Calculated by the total mass of the eluent, the content of the metal chelating agent capable of chelating calcium ions is 1mM-200mM or is 1mM, 5mM, 10mM, 15mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 150mM, or 200mM.

[0079] In some embodiments, the metal chelating agent capable of chelating calcium ions includes calcium edetate.

[0080] In some embodiments, the connecting medium is desulfurized biotin or its analogues and avidin protein, the eluent is an aqueous solution containing an eluent, the eluent including biotin, biotin analogues and combinations thereof, and the content of the eluent, calculated based on the total mass of the eluent, is 2mM-100mM or 2 mM, 5mM, 10mM, 15mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, or 100mM.

[0081] In some embodiments, the adapter structure may include at least one or a combination of CD9 antibody and PSA antibody.

[0082] In some embodiments, the fluorescent dye is a fluorescent molecule, a fluorescent material, or a combination thereof.

[0083] In some embodiments, the fluorescent dye includes at least one or a combination thereof selected from organic fluorescent molecules, fluorescent proteins, nucleic acid dyes, lipid membrane dyes, quantum dots, and polymer dots.

[0084] In some embodiments, the fluorescent group may include one selected from Alexa Fluor 488.

[0085] In some embodiments, the biological fluid may include at least one or a combination of blood, plasma, serum, urine, sputum, cerebrospinal fluid, cerebrospinal fluid, pleural effusion, nipple aspiration fluid, lymph, fluids from the respiratory tract, intestine and genitourinary tract, tears, saliva, breast milk, fluids from the lymphatic system, semen, cerebrospinal fluid, intra-organ system fluids, ascites, tumor cyst fluid, amniotic fluid, bacterial cell culture, mammalian cell culture, cell culture supernatant, cell-free protein transcription-translation reactants.

[0086] In some preferred embodiments, the magnetic trapping unit includes a magnetic trapping unit with a structure of solid-phase support-streptavidin-biotin-disulfide bond-polyethylene glycol-trapping agent and a magnetic trapping unit with a structure of solid-phase support-streptavidin-biotin-TIM4 protein. Using two types of magnetic trapping units is more conducive to the separation of extracellular biomolecules and to obtaining more accurate single-particle detection data of extracellular biomolecules.

[0087] In some preferred embodiments, the magnetic trapping molecule includes a magnetic trapping molecule with a structure of solid-phase support-streptavidin-biotin-disulfide bond-polyethylene glycol-distearate phosphatidylethanolamine and a magnetic trapping molecule with a structure of solid-phase support-streptavidin-biotin-TIM4 protein. Using two types of magnetic trapping molecule is more conducive to the separation of extracellular biomolecules and to obtaining more accurate single-particle detection data of extracellular biomolecules.

[0088] In some embodiments, the particle analysis and detection equipment may include the flow cytometry particle detection equipment or the nanoflow cytometry particle detection equipment.

[0089] The flow cytometer is a particle analysis and detection device that enables directional flow of sample streams.

[0090] In some embodiments, the particle analysis and detection device includes a directional fluid system, an optical system, and a particle detector.

[0091] In some embodiments, the directional fluid system may consist of a sample loading unit and a flow unit.

[0092] In some embodiments, the particle detector consists of a photoelectric sensor and a signal conditioning circuit with band-limited filtering function for high-frequency noise.

[0093] In some embodiments, the method for preparing the magnetic trap includes: mixing a solid carrier (e.g., streptavidin-magnetic nanobeads) connected to streptavidin protein with distearylphosphatidylethanolamine-polyethylene glycol-disulfide bond-biotin, incubating, adding biotin-labeled bovine serum albumin in Tris buffer for reaction, washing with PBS buffer, adding streptavidin-containing Tris buffer for reaction, washing with PBS buffer, and blocking with bovine serum albumin to obtain a magnetic trap with the structure of solid-phase carrier-streptavidin-biotin-disulfide bond-polyethylene glycol-distearylphosphatidylethanolamine.

[0094] In some embodiments, the method for preparing the magnetic trapping molecule includes: reacting carboxylated PEG, N-hydroxysuccinimide (NHS), 1,3-dicyclohexylcarbodiimide, dopamine hydrochloride, and anhydrous sodium carbonate in an organic solvent; then adding an oleic acid-coated solid support (e.g., magnetic nanobeads) to obtain a carboxylated solid support; removing the organic solvent; reacting the carboxylated solid support with streptavidin protein in 2-(N-morpholino)ethanesulfonic acid (MES) buffer; adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; ultrafiltration to remove excess reagents; resuspending in BB buffer containing streptavidin protein and reacting; separating with an external magnetic field; and precipitating with 100... Filtration with a pore size of nm yielded streptavidin protein-solid-phase carrier. The streptavidin protein-solid-phase carrier was then reacted with biotinylated TIM4 protein, separated by an external magnetic field, washed, and separated by an external magnetic field to obtain a magnetic trap with the structure of solid-phase carrier-streptavidin protein-biotin-TIM4 protein.

[0095] In some embodiments, the organic solvent may include at least one or a combination of chloroform, N,N-dimethylformamide (DMF).

[0096] In some embodiments, the fluorescent dye may include a dye selected from any one of: PE, PE-Cy5 fluorophore, Alexa Fluor488 fluorophore, or FITC (fluorescein isothiocyanate), PerCP-Cy5.5, PerCP-eFluor 710, PE-Cy7, APC, eFluor 660, Alexa Fluor647, ALexa Fluor700, and ALexa Fluor780.

[0097] On the other hand, the present invention provides another method for the separation and detection of extracellular biomolecules in biological fluids, comprising: mixing magnetic trapping molecules with biological fluids and incubating them to allow the magnetic trapping molecules to bind to the target extracellular biomolecules through a trapping agent; applying an external magnetic field for separation and enrichment, removing the supernatant, washing, and separating the target extracellular biomolecules from the biological fluids; using an eluent to release the binding of the magnetic trapping molecules to the target extracellular biomolecules and removing the solid support; mixing a fluorescent dye with the target extracellular biomolecules to allow the fluorescent dye to bind to the target extracellular biomolecules through an adaptor structure, and then removing the free fluorescent dye by centrifugation, ultrafiltration, or dialysis to obtain the target extracellular biomolecules bound to the fluorescent dye; and using a particle analysis detection device to perform single nanoparticle analysis on the target extracellular biomolecules bound to the fluorescent dye.

[0098] Beneficial effects

[0099] Compared with the prior art, one embodiment of the present invention has at least one or a combination of the following beneficial effects:

[0100] (1) Compared with the fluorescent dye staining after eluting the magnetic traps and the target extracellular biomolecules with the elution buffer, the present invention adopts the operation of completing the fluorescent dye labeling before elution, which is beneficial to reduce the interference of free fluorescent dye on detection and eliminates the operation step of removing free dye by ultracentrifugation, thus reducing the operation steps.

[0101] (2) The present invention uses an indirect connection to connect the capture agent and the solid support, which is beneficial to use a specific eluent to break and / or depolymerize the connection medium under mild reaction conditions before detection, thereby separating the solid support from the extracellular biomacromolecules, which is beneficial to the accuracy of single particle detection of extracellular biomacromolecules.

[0102] (3) Compared with other biological enzymes, using nucleases or restriction endonucleases is beneficial to avoid damaging the biological characteristics of extracellular biomolecules (such as extracellular vesicles, proteins, carbohydrates or lipids).

[0103] (4) The present invention uses two capture agents, which is more conducive to the separation of extracellular biomacromolecules and to obtaining more accurate single-particle detection data of extracellular biomacromolecules. For example, compared with using TIM4 protein alone or distearylphosphatidylethanolamine alone as a capture agent, using both TIM4 protein and distearylphosphatidylethanolamine as capture agents is more conducive to accurately detecting the number of PSA+ extracellular biomacromolecules in prostate cancer patient samples or healthy human samples.

[0104] (5) Compared with other capture agents, using TIM4 protein and distearate phosphatidylethanolamine as capture agents is more conducive to accurately detecting the number of PSA+ extracellular biomacromolecules in samples from prostate cancer patients or healthy individuals.

[0105] (6) Compared with the use of antibodies corresponding to other antigens on the surface of other prostate cancer cells for detection, the present invention uses distearate phosphatidylethanolamine and TIM4 protein as capture agents, which results in more accurate detection than other types of capture agents. Attached Figure Description

[0106] Figure 1 This is a schematic diagram illustrating the principle of the separation and detection of extracellular biomacromolecules in Example 4 (using AF488-PSA antibody for optical labeling).

[0107] Figure 2 A statistical chart showing the proportion of PSA+ exosomes obtained from the separation and detection method described in Example 4 from the serum pool of prostate cancer patients.

[0108] Figure 3 A statistical chart showing the average particle size of total exosomes obtained from the serum pool of prostate cancer patients by separation and detection using the method described in Example 4.

[0109] Figure 4 A statistical graph showing the concentration results of total exosomes obtained from the serum pool of prostate cancer patients by the separation and detection method described in Example 4.

[0110] Figure 5 ROC curves for the separation and detection methods of Examples 4, 5, 6 and 7.

[0111] Figure 6 The graphs show the inhibition effect curves of the three separation and detection methods in Example 4, Comparative Example 5, and Comparative Example 6.

[0112] Figure 7 The PSA levels obtained by detecting 30 biofluid samples from healthy individuals and 25 biofluid samples from prostate cancer patients using the separation and detection method described in Example 4. + Statistical analysis of extracellular biomolecule assay results.

[0113] Figure 8 PSMA was obtained by detecting 30 biofluid samples from prostate cancer patients at different Gleason stages using the separation and detection method described in Example 4. + Statistical analysis of extracellular biomolecule assay results.

[0114] Figure 9The image shown in Example 6 is an image of HIV captured and eluted by a nanomagnetic bead-streptavidin protein-biotin-desulfobiotin-VSV-G antibody under a transmission electron microscope.

[0115] Terminology Explanation

[0116] In this invention, "room temperature" refers to the ambient temperature, which can be 20℃-30℃; in some embodiments, it is 22℃-28℃; in some embodiments, it is 24℃-26℃; and in some embodiments, it is 25℃.

[0117] In the foregoing description of this invention, all figures disclosed herein, whether or not the words “approximately” or “about” are used, are approximate values. Based on the disclosed figures, the value of each figure may vary by less than ±10% or by a difference that is considered reasonable by those skilled in the art, such as ±1%, ±2%, ±3%, ±4%, or ±5%.

[0118] The term "extracellular biomacromolecules" refers to extracellular nucleic acids, proteins, carbohydrates, or lipids, or combinations thereof. Examples include extracellular proteins that can serve as tumor markers, such as serum tumor marker proteins like CEA, AFP, CA19-9, SCC, NSE, CK19, CA125, CA 15-3, and CA 19-9; extracellular nucleic acids with clinical diagnostic value, such as extracellular deoxyribonucleic acid (DNA); free DNA released from circulating tumor cells in plasma; extracellular biomacromolecules with plasma membrane structures, such as extracellular vesicles; extracellular nucleoprotein particles with vital characteristics; pathogenic or infectious agents in body fluids, such as blood-borne infectious viruses, such as HIV, hepatitis B virus, hepatitis C virus, and cytomegalovirus in the blood.

[0119] The term "atm" represents standard atmospheric pressure. For example, 1 atm represents the value of 1 standard atmosphere, which is 101325 Pa.

[0120] The term "aptamer structure" refers to antibodies, antibody fragments, antibody analogs, hemagglutinins, ligands and / or receptors, aptamers, nucleic acid aptamers, peptides, or combinations thereof.

[0121] The term "extracellular vesicle" refers to a small, membrane-bound vesicle, including exosomes, released from a cell into the extracellular matrix. The terms "optional," "discretionary," or "optionally" mean that the event or situation described subsequently may, but is not necessarily, occur. For example, "optional surfactant" means that the surfactant may or may not be present.

[0122] The term “weight percentage” or “percentage by weight” or “wt%” is defined as the weight of a single component in a composition divided by the total weight of all components in the composition and then multiplied by 100.

[0123] The term “and / or” should be understood to mean any one of the options or any combination of two or more of the options.

[0124] The term "SA" stands for streptavidin protein.

[0125] The term "Biotin" refers to biotin.

[0126] In this article, the term "treatment" refers to a clinical intervention intended to alter the natural course of a disease in an individual receiving treatment. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving prognosis.

[0127] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0128] In this application, the "composition" can be conveniently presented in unit dose form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with a carrier constituting one or more adjunct components. Typically, the composition is prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a finely pulverized solid carrier, or both. Detailed Implementation

[0129] To enable those skilled in the art to better understand the technical solutions of the present invention, some non-limiting embodiments are further disclosed below to provide a more detailed description of the present invention.

[0130] All reagents used in this invention can be purchased commercially or prepared by the methods described in this invention.

[0131] 1) Specimen source

[0132] 1.1) Biological liquid samples from prostate cancer patients

[0133] A serum pool (2.7 ml) was obtained from 9 ml of peripheral blood collected on the same day from a prostate cancer patient admitted to the Affiliated Hospital of Xiamen University. The peripheral blood was collected into a 13 * 75 mm BD vacuum blood collection tube containing 50 u / ml heparin sodium.

[0134] 1.2) Bio-liquid samples from healthy individuals

[0135] A serum pool (2.5 ml) was obtained from 8 ml of peripheral blood. The serum pool was provided by the sample library of the Department of Laboratory Medicine of Xiamen University Affiliated Hospital. The peripheral blood was collected into 13 * 75 mm BD vacuum blood collection tubes containing 50 u / ml heparin sodium.

[0136] 2) Main reagents and instruments

[0137] AF488-CD9 (AF488 mouse anti-human CD9), AF488-PSA (AF488 mouse anti-human PSA9) were from NanoFCM Inc.;

[0138] TIM4 (Human TIM-4 protein, human IgG1 Fc tag (active)) was purchased from Genetex;

[0139] Anti-VSV-G [8G5F11], Mouse IgG2a, and Kappa were purchased from absoluteantibody.

[0140] SA (Streptavidin Protein) and Biotin were purchased from MCE.

[0141] Distearate phosphatidylethanolamine was purchased from Aladdin Company;

[0142] Biotin-labeled bovine serum albumin was purchased from Sigma-Aldrich.

[0143] Washing solution: 10 mM TBS solution (10 mM triethanolamine buffer solution);

[0144] Elution buffer 1: 10 mM TBS solution containing 25 mM TCEP, purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0145] Elution buffer 2: 10 mM TBS solution containing 2 mM EDTA, purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0146] Elution buffer 3: 10 mM TBS solution containing 20 mM biotin, purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0147] EZ-Link® NHS-Biotin Reagents were purchased from Thermo Fisher.

[0148] Desulfurized biotin was purchased from Ruixi Biotechnology;

[0149] The magnetic nanobeads-streptavidin protein were purchased from Suzhou Beaver Biomedical Engineering Co., Ltd.

[0150] Flow NanoAnalyzer is from Xiamen Fuliu Biotechnology Co., Ltd.

[0151] The Centrifuge 5810R refrigerated centrifuge was purchased from Eppendorf.

[0152] The Optima™ Max-XP Tabletop Ultracentrifuge was purchased from Beckman Coulter.

[0153] 2) Flow NanoAnalyzer Detection Method

[0154] Detection was performed under the following conditions: laser detector 488nm+638nm; dual-channel detection SSC (488 / 10nm), FL2 (710 / 40nm); attenuation rate: 10%; detection pressure: 1kPa; signal type: small siginal. CD9+ extracellular macromolecules or PSA in the serum pool samples were recorded using two-dimensional scatter plots and histograms. + Detection data of extracellular biomolecules.

[0155] Example 1: Preparation of magnetic nanobeads-streptavidin protein-biotin-disulfide bond-polyethylene glycol-distearate phosphatidylethanolamine

[0156] Collect 150 µl of magnetic nanobeads-streptavidin protein (i.e., 1.3 × 10⁻⁶). 8Magnetic beads were washed with 99% ethanol to remove contaminants and increase the humidity of the nanobeads. Biotin-disulfide-polyethylene glycol-distearate phosphatidylethanolamine was added to the washed nanobeads-streptavidin protein, and incubated at room temperature for 12 h. Excess distearate phosphatidylethanolamine-polyethylene glycol-disulfide-biotin was eluted with deionized water. Then, 10 mM Tris buffer containing 1 mg / ml biotin-labeled bovine serum albumin was added to the nanobeads-streptavidin protein, and the reaction was carried out at 4°C for 1 h. Excess residue was washed with PBS buffer. The reaction was then carried out with 10 mM Tris buffer containing 200 µg / ml SA, and incubated at 4°C for 30 min. Excess residue was washed with PBS buffer. Finally, the reaction was carried out at 4°C for 1 h, and then blocked with 1% bovine serum albumin aqueous solution to prepare nano-magnetic beads-streptavidin protein-biotin-disulfide bond-polyethylene glycol-distearate phosphatidylethanolamine, which was stored at 4°C.

[0157] Example 2: Preparation of magnetic nanobeads-streptavidin-biotin-TIM4 protein

[0158] 1. Preparation of Biotinylated TIM4 Protein

[0159] The TIM4 protein was biotinylated according to the EZ-Link® NHS-Biotin Reagents instructions.

[0160] 2. Preparation of magnetic bead-streptavidin protein nanoparticles by magnetic bead labeling and modification

[0161] First, 60 mg of carboxylated PEG, 9 mg of NHS, 11 mg of DCC, 3.9 mg of dopamine hydrochloride, and 30 mg of anhydrous Na2CO3 were added to 6 ml of chloroform and 3 ml of DMF, and the mixture was stirred at 35 °C for 4 h. Then, 1.5 ml of oleic acid-coated Fe3O4 was added dropwise to the reaction product, and the mixture was stirred at 35 °C for 10 h. The carboxylated PEG-modified Fe3O4 nanoparticles were separated by an external magnetic field and dried under nitrogen. The carboxylated PEG-modified Fe3O4 nanoparticles were dispersed in distilled water and dialyzed using a 30 kDa dialysis bag to remove other residual reagents. The final Fe concentration in the carboxylated PEG-modified Fe3O4 nanoparticles was quantified to 1 mg / ml. 15 ml of the carboxylated PEG-modified Fe3O4 nanoparticles and 11.25 mg of SA were dissolved in 0.01 M MES buffer (pH=5.5) and reacted with shaking for 30 min. Then, 7.5 mg of EDC was added, and the reaction was continued at 4 °C with shaking for 4 h. Excess reagents in the above reactants were removed by ultrafiltration using a 30 kDa ultrafiltration tube, and the mixture was resuspended in BB buffer (pH=8.0) containing 11.25 mg SA and reacted with shaking at 4 °C for 12 h. The precipitate was separated by an external magnetic field and filtered through a syringe with a 100 nm pore size to obtain nanomagnetic beads-streptavidin protein.

[0162] 3. Preparation of magnetic nanobeads-streptavidin-biotin-TIM4 protein:

[0163] Transfer 60 μL of magnetic nanobeads-streptavidin protein to a 1.5 mL Eppendorf tube and add 500 µL of detergent, then vortex to mix. Centrifuge rapidly for 10 s, then place the Eppendorf tube on a magnetic rack and let it stand for 1 min to separate the magnetic beads from the solution; discard the supernatant. Add 500 µL of detergent and 10 µL of biotinylated TIM4 protein, and react at 2–10 °C for 10 min. Centrifuge rapidly for 10 s, then place the Eppendorf tube on a magnetic rack and let it stand for 1 min to separate the magnetic beads from the solution; discard the supernatant. Add 500 µL of detergent and vortex to mix. Centrifuge rapidly for 10 s, then place the Eppendorf tube on a magnetic rack and let it stand for 1 min to separate the magnetic beads from the solution; discard the supernatant. Add 500 µL of detergent and vortex to mix. Centrifuge quickly for 10 seconds, then place the Ep tube on a magnetic rack and let it stand for 1 minute to separate the magnetic beads from the solution. Discard the supernatant and store the obtained nano-magnetic beads-streptavidin protein-biotin-TIM4 protein at 4°C.

[0164] Example 3: Isolation and detection of extracellular biomacromolecules (continuous magnetic separation method with dual magnetic traps, labeling followed by elution, optical labeling with AF488-CD9 antibody)

[0165] 1. Sample pretreatment

[0166] The serum pool samples were preferentially centrifuged, a process consisting of two steps of differential centrifugation and filtration. The serum pool sample (2.5–2.7 ml volume) was centrifuged at 500 g for 10 min at 4°C to remove blood cells and cell debris, followed by centrifugation at 16500 g for 20 min at 4°C to remove microparticles and apoptotic bodies from the biological fluid. Filtration was then performed on a 0.45 µm filter membrane to remove protein aggregates larger than 450 nm, yielding the pretreated sample.

[0167] 2. Isolation and enrichment of extracellular biomacromolecules

[0168] 2.5 ml of the pretreated sample was mixed with 100 µl of a mixture of magnetic nanobeads-streptavidin-biotin-disulfide-polyethylene glycol-distearate phosphatidylethanolamine and magnetic nanobeads-streptavidin-biotin-TIM4 protein (volume ratio of magnetic nanobeads-streptavidin-biotin-disulfide-polyethylene glycol-distearate phosphatidylethanolamine and magnetic nanobeads-streptavidin-biotin-TIM4 protein 1:2.3) and incubated at room temperature with a rotating stirrer for 3 h to obtain a complex formed by the magnetic nanobeads-streptavidin-biotin-disulfide-polyethylene glycol-distearate phosphatidylethanolamine and magnetic nanobeads-streptavidin-biotin-TIM4 protein with extracellular biomacromolecules. The complex was separated and enriched using a magnetic rack, the supernatant was removed, and the precipitate was collected with 500 µl of... The above precipitate was washed five times with a buffer containing 10 mM PBS and 0.5% Tween for five minutes each time to obtain isolated and enriched extracellular biomolecules.

[0169] 3. Optical labeling of target extracellular biomolecules

[0170] The extracellular biomolecules isolated and enriched in step 2 were resuspended in a buffer containing 10 mM PBS and 0.5% Tween. After diluting 50-fold with 10 mM PBS buffer, 50 μL of the diluted and enriched extracellular biomolecules were incubated with 20 μL of AF488-CD9 antibody at 37°C for 30 min. The supernatant was then removed by magnetic adsorption. The complex was washed 5 times with 500 µL of washing buffer for 5 minutes each time, and then resuspended in 150 µL of 10 mM PBS buffer to obtain the optically labeled complex solution.

[0171] 4. Eluting and removing nano-magnetic beads

[0172] (1) Take the complex solution obtained in step 3 of Example 3 and mix it with 100 μl of elution solution 1. React at 37°C for 30 min. Then use a magnetic rack to separate the magnetic beads from the solution to obtain supernatant i and precipitated magnetic beads ii. Remove supernatant i.

[0173] (2) Take the precipitated magnetic beads ii and mix them with 50 μl of elution buffer 2, centrifuge quickly for 10 s, and then use a magnetic rack to separate the magnetic beads from the solution to obtain supernatant iii and precipitated magnetic beads iv. Add 50 μl of elution buffer 2 to precipitated magnetic beads iv, centrifuge quickly for 10 s, and then use a magnetic rack to separate the magnetic beads from the solution to obtain supernatant v. Combine supernatant iii and supernatant v to obtain a solution containing extracellular biomacromolecules labeled AF488-CD9 obtained by continuous magnetic separation using dual magnetic traps.

[0174] 5. Sample testing

[0175] The solution containing extracellular biomolecules labeled AF488-CD9 obtained by the continuous magnetic separation method using dual magnetic traps (i.e., the solution containing extracellular biomolecules labeled AF488-CD9 obtained in the above steps) was detected using a FlowNanoAnalyzer. The diagnostic analysis method for the results is described in Example 5.

[0176] Comparative Example 1: Isolation and detection of extracellular biomacromolecules (a continuous magnetic separation method using dual magnetic traps, followed by elution and labeling)

[0177] 1. Sample pretreatment

[0178] The serum pool samples were preferentially centrifuged, a process consisting of two steps of differential centrifugation and filtration. The serum pool sample (2.5–2.7 ml volume) was centrifuged at 500 g for 10 min at 4°C to remove blood cells and cell debris, followed by centrifugation at 16500 g for 20 min at 4°C to remove microparticles and apoptotic bodies from the biological fluid. Filtration was then performed on a 0.45 µm filter membrane to remove protein aggregates larger than 450 nm, yielding the pretreated sample.

[0179] 2. Isolation and enrichment of extracellular biomacromolecules

[0180] 2.5-2.7 ml of the pretreated sample was mixed with 100 µl of a mixture of magnetic nanobeads-streptavidin-biotin-disulfide-polyethylene glycol-distearate phosphatidylethanolamine and magnetic nanobeads-streptavidin-biotin-TIM4 protein (volume ratio of magnetic nanobeads-streptavidin-biotin-disulfide-polyethylene glycol-distearate phosphatidylethanolamine and magnetic nanobeads-streptavidin-biotin-TIM4 protein 1:2.3) and incubated at room temperature with stirring for 3 h to obtain a complex formed by magnetic nanobeads-streptavidin-biotin-disulfide-polyethylene glycol-distearate phosphatidylethanolamine and magnetic nanobeads-streptavidin-biotin-TIM4 protein with extracellular biomacromolecules. The complex was separated and enriched using a magnetic rack, the supernatant was removed, and the precipitate was obtained. The precipitate was then treated with 500 µl of a mixture containing 10 mM... The precipitate was washed five times with PBS and 0.5% Tween buffer for five minutes each time to obtain the isolated and enriched extracellular biomolecules.

[0181] 3. Elution to remove nano-magnetic beads

[0182] (1) Take the extracellular biomacromolecules separated and enriched in step 2 of Comparative Example 1 and mix them with 100 μl of elution buffer 1. React at 37°C for 30 min. Then use a magnetic rack to separate the magnetic beads from the solution to obtain supernatant a and precipitated magnetic beads b. Remove supernatant a.

[0183] (2) Mix precipitated magnetic beads b with 50 μl of elution buffer 2, centrifuge quickly for 10 s, and then use a magnetic rack to separate the magnetic beads from the solution to obtain supernatant c and precipitated magnetic beads d. Add 50 μl of elution buffer 2 to precipitated magnetic beads d, centrifuge quickly for 10 s, and then use a magnetic rack to separate the magnetic beads from the solution to obtain supernatant e. Combine supernatant c and supernatant e to obtain a solution containing extracellular biomacromolecules.

[0184] 4. Optical labeling of target extracellular biomolecules

[0185] The extracellular biomolecules isolated and enriched in step 3 above were resuspended in a buffer containing 10 mM PBS and 0.5% Tween. After diluting 50 times with 10 mM PBS buffer, 50 μL of the diluted extracellular biomolecules were incubated with 20 μL of AF488-CD9 antibody at 37°C for 30 min. The free AF488-CD9 antibody was removed by ultracentrifugation. The precipitate after ultracentrifugation was then resuspended in 100 μL of 10 mM PBS buffer to obtain the optically labeled complex solution.

[0186] 5. Sample testing

[0187] The optically labeled complex solution obtained in step 4 of Comparative Example 1 was taken and detected using a Flow NanoAnalyzer. The diagnostic analysis method for the results is described in Example 6.

[0188] Comparative Example 2: Magnetic separation method using a single magnetic trap (using magnetic nanobeads-streptavidin protein-biotin-TIM4 protein as magnetic traps)

[0189] 1. Sample pretreatment

[0190] The serum pool samples were preferentially centrifuged, a process consisting of two steps of differential centrifugation and filtration. The serum pool sample (2.5–2.7 ml volume) was centrifuged at 500 g for 10 min at 4°C to remove blood cells and cell debris, followed by centrifugation at 16500 g for 20 min at 4°C to remove microparticles and apoptotic bodies from the biological fluid. Filtration was then performed on a 0.45 µm filter membrane to remove protein aggregates larger than 450 nm, yielding the pretreated sample.

[0191] 2. Isolation and enrichment of extracellular biomacromolecules

[0192] 2.5-2.7 ml of the pretreated sample was incubated with 100 µl of magnetic nanobeads-streptavidin-biotin-TIM4 protein at room temperature for 3 h by rotary stirring to obtain a complex formed by magnetic nanobeads-streptavidin-biotin-TIM4 protein and extracellular biomolecules. The complex was separated and enriched using a magnetic rack, and the supernatant was removed to obtain the precipitate. The precipitate was washed 5 times with 500 µl of buffer containing 10 mM PBS and 0.5% Tween for 5 minutes each time to obtain the separated and enriched extracellular biomolecules.

[0193] 3. Optical labeling of target extracellular biomolecules

[0194] The extracellular biomolecules isolated and enriched in step 2 of Comparative Example 2 were resuspended in a buffer containing 10 mM PBS and 0.5% Tween. After diluting 50-fold with 10 mM PBS buffer, 50 μL of the diluted and enriched extracellular biomolecules were incubated with 20 μL of AF488-CD9 antibody at 37°C for 30 min. Then, the supernatant was removed by magnetic adsorption. The complex was washed 5 times with 500 µL of washing buffer for 5 minutes each time, and then resuspended in 150 µL of 10 mM PBS buffer to obtain the optically labeled complex solution.

[0195] 4. Detection after elution to remove nano-magnetic beads

[0196] Take the optically labeled complex solution obtained in the previous step and mix it with 50 μl of elution buffer 2. Centrifuge rapidly for 10 s, and then use a magnetic rack to separate the magnetic beads from the solution to obtain supernatant vi and precipitated magnetic beads vii. Add 50 μl of elution buffer 2 to the precipitated magnetic beads, centrifuge rapidly for 10 s, and then use a magnetic rack to separate the magnetic beads from the solution to obtain supernatant viii. Combine supernatant vi and supernatant viii to obtain a solution containing extracellular biomacromolecules labeled AF488-CD9. Detect the solution using a Flow NanoAnalyzer. The diagnostic analysis method for the results is described in Example 5.

[0197] Comparative Example 3: Magnetic separation method using a single magnetic trap (using magnetic nanobeads-streptavidin protein-biotin-disulfide bonds-polyethylene glycol-distearate phosphatidylethanolamine as magnetic traps)

[0198] 1. Sample pretreatment

[0199] The serum pool samples were preferentially centrifuged, a process consisting of two steps of differential centrifugation and filtration. The serum pool sample (2.5–2.7 ml volume) was centrifuged at 500 g for 10 min at 4°C to remove blood cells and cell debris, followed by centrifugation at 16500 g for 20 min at 4°C to remove microparticles and apoptotic bodies from the biological fluid. Filtration was then performed on a 0.45 µm filter membrane to remove protein aggregates larger than 450 nm, yielding the pretreated sample.

[0200] 2. Isolation and enrichment of extracellular biomacromolecules

[0201] 2.5-2.7 ml of the pretreated sample was incubated with 100 µl of a mixture of magnetic nanobeads-streptavidin-biotin-disulfide-polyethylene glycol-distearate phosphatidylethanolamine at room temperature for 3 h with rotary stirring to obtain a complex of magnetic nanobeads-streptavidin-biotin-disulfide-polyethylene glycol-distearate phosphatidylethanolamine and extracellular biomolecules. The complex was separated and enriched using a magnetic rack, and the supernatant was removed to obtain the precipitate. The precipitate was washed 5 times with 500 µl of buffer containing 10 mM PBS and 0.5% Tween for 5 minutes each time to obtain the separated and enriched extracellular biomolecules.

[0202] 3. Optical labeling of target extracellular biomolecules

[0203] The extracellular biomolecules isolated and enriched in step 2 of Comparative Example 3 were resuspended in a buffer containing 10 mM PBS and 0.5% Tween. After diluting 50-fold with 10 mM PBS buffer, 50 μL of the diluted and enriched extracellular biomolecules were incubated with 20 μL of AF488-CD9 antibody at 37°C for 30 min. Then, the supernatant was removed by magnetic adsorption. The complex was washed 5 times with 500 µL of washing buffer for 5 minutes each time, and then resuspended in 150 µL of 10 mM PBS buffer to obtain the optically labeled complex solution.

[0204] 4. Detection after elution to remove nano-magnetic beads

[0205] Take the optically labeled complex solution obtained in step 3 of Comparative Example 3 and mix it with 100 μl of elution buffer 1. React at 37°C for 30 min. Then use a magnetic rack to separate the magnetic beads from the solution to obtain the supernatant. Use a Flow NanoAnalyzer to detect the supernatant. The diagnostic analysis method of the results is shown in Example 5.

[0206] Comparative Example 4: Ultracentrifugation

[0207] 1) Take a plasma sample, centrifuge at 300 g for 5 min at 4℃ to remove large particles such as cells;

[0208] 2) Take the supernatant and centrifuge at 1200 g for 20 min at 4℃ to remove any potentially apoptotic cells and cell debris;

[0209] 3) Take the supernatant, centrifuge at 100,000 g for 80 min at 4℃, and discard the supernatant;

[0210] 4) Resuspend the precipitate in PBS, centrifuge at 100,000 g for 20 min at 4°C, and discard the supernatant;

[0211] 5) Resuspend the precipitate in 100 μL of 10 mM PBS buffer to obtain the extracellular biomacromolecules after ultracentrifugation;

[0212] 6) Take 100 μL of the extracellular biomacromolecules obtained from step 5) after ultracentrifugation, add 10 μL of 0.1 mg / ml AF488-CD9 antibody, and incubate at 37°C for 30 min. Centrifuge at 100,000 g for 80 min at 4°C, discard the supernatant, resuspend the precipitate with PBS, centrifuge at 100,000 g for 20 min at 4°C, discard the supernatant, add 100 μL of 10 mM PBS buffer to resuspend the precipitate, and obtain the solution of extracellular biomacromolecules containing AF488-CD9 after ultracentrifugation. Detect the solution using a Flow NanoAnalyzer. The diagnostic analysis method for the results is described in Example 5.

[0213] Example 4: Isolation and detection of extracellular biomacromolecules (optical labeling using AF488-PSA antibody)

[0214] Replace AF488-CD9 with AF488-PSA, and follow the same procedures as in Example 3. The schematic diagram for separation and detection in Example 4 is shown below. Figure 1 Figure a shows that, using the separation and detection method described in Example 4, the proportion of PSA+ exosomes in the serum pool of prostate cancer patients was 18.27% of the total extracellular biomolecules (see Figure a for details). Figure 1 (Figure b). The total exosome concentration in the serum pool of prostate cancer patients was determined to be 6.5 × 10⁻⁶ using the separation and detection method described in Example 4. 9 Particles / ml, average particle size 67.2±12nm (see results) Figure 1 (See Figure c in the original text). The diagnostic analysis method for the results is described in Example 5.

[0215] Comparative Example 5: Direct Chemiluminescence Method

[0216] 1) Reagent kits and instruments used

[0217] Prostate-Specific Antigen Assay Kit (Direct Chemiluminescence Method) [Registration Certificate No.] 20133402084 (CFDA Import Registration Certificate No.), ADVIA Centaur XP Fully Automated Chemiluminescence Immunoassay Analyzer purchased from Siemens Medical Diagnostics Products (Shanghai) Co., Ltd.

[0218] 2) Following the operating instructions, load the serum pool sample and prostate-specific antigen assay kit into the designated location on the ADVIA Centaur XP fully automated chemiluminescence immunoassay analyzer and run the system. The system will automatically execute the following steps:

[0219] a. Add 35 µL of serum sample to the cuvette.

[0220] b. Add 250 µL of solid-phase reagent and 100 µL of labeling reagent, and incubate at 37°C. 0 Incubate at C for 7.5 min.

[0221] c. Separate, aspirate, and wash the cuvette with washing solution.

[0222] d. Add 300 µL each of acidic and basic reagents to stimulate the chemiluminescence reaction.

[0223] e. Record the number of light quanta (RLUs) detected by the system and calculate the PSA content (ng / ml) of the patient's serum pool sample based on the PSA standard curve.

[0224] Comparative Example 6: Magnetic separation method using a single magnetic trap (using magnetic nanobeads-streptavidin-biotin-TIM4 protein as magnetic traps, with AF488-PSA optical labeling)

[0225] Replace AF488-CD9 with AF488-PSA, and perform the remaining operations as in Comparative Example 2. The diagnostic analysis method for the results is described in Example 5.

[0226] Comparative Example 7: Magnetic separation method using a single magnetic trap (magnetic nanobeads-streptavidin protein-biotin-disulfide bond-polyethylene glycol-distearate phosphatidylethanolamine, AF488-PSA optical labeling)

[0227] Replace AF488-CD9 with AF488-PSA, and perform the remaining operations as in Comparative Example 3. The diagnostic analysis method for the results is described in Example 5.

[0228] Example 5: Diagnostic Analysis Method for Results

[0229] 1) Using Origin 9.5 software, the concentration (particles / ml) of PSA+ extracellular macromolecules in biological fluid samples from prostate cancer patients or healthy individuals, measured using the methods in Examples 4, 5, 6, and 7, was statistically analyzed. ROC curves were then used to characterize the diagnostic efficacy of each method. Figure 5 .

[0230] 2) Using SPSS 11.0 software and in accordance with the Clinical and Laboratory Standards Institute (CLSI) EP12-A2 and CLSI EP15-A2 documents, a series of repeated tests were performed on samples diluted to a certain ratio so that the concentration was exactly at the critical value. The results were 50% negative and 50% positive. The sample with this analyte concentration was called C50. C5 and C95 were prepared by analogy. The above three methods were repeated 40 times (within the batch). The data were recorded and statistically analyzed. The intra-batch imprecision and total imprecision of the above three methods were compared. The results are shown in Table 2.

[0231] 3) SPSS 11.0 software and the dose-response test method according to the Clinical and Laboratory Standards Institute (CLSI) EP-7 document were used to evaluate the interference effect. C95 specimens were selected. The interfering agent and blank control were the Sysmex kit containing conjugated bilirubin, free bilirubin, chyle, and hemolysis. Mix the interfering agent with the specimen at a volume ratio of 1:9 to prepare sample A; mix the blank control with the specimen at a volume ratio of 1:9 to prepare sample B. Mix sample A and sample B at different dilution ratios: 10B (10 volumes of sample B), 9B+1A (9 volumes of sample B and 1 volume of sample A), 8B+2A (8 volumes of sample B and 2 volumes of sample A), 7B+3A (7 volumes of sample B and 3 volumes of sample A), 6B+4A (6 volumes of sample B and 4 volumes of sample A), 5B+5A (5 volumes of sample B and 5 volumes of sample A), 4B+6A (4 volumes of sample B and 6 volumes of sample A), 3B+7A (3 volumes of sample B and 7 volumes of sample A), 2B+8A (2 volumes of sample B and 8 volumes of sample A), 1B+9A (1 volume of sample B and 9 volumes of sample A), and 10A (10 volumes of sample A). Each sample was measured three times. A scatter plot of interfering substance concentration and interfering effect value was plotted. The concentrations of each interfering substance were calculated by fitting the dose-effect equation, and the relative bias was equal to the maximum bias of 10%. The results are shown in […]. Figure 3 .

[0232] 4) Biological fluid samples were collected from prostate cancer patients at different Gleason stages and from healthy individuals. These samples were separated and detected according to the method described in Example 4. The results were processed using Graphpad Prism 8.0 software, and a two-tailed t-test was applied for variance analysis to determine whether there were significant differences in the detection data of PSA+ extracellular macromolecules and whether they were associated with the progression of prostate cancer. The results are shown in […]. Figure 4 .

[0233] Results: The results are shown in Tables 1 and 2.

[0234]

[0235]

[0236] As shown in Table 1, the exosome extraction concentration of Example 3 is comparable to that of Comparative Example 4 and the other methods described in Table 1. Furthermore, the exosome extraction concentration of Example 3 is superior to the other methods described in Table 1 (e.g., Comparative Example 1, Comparative Example 2, Comparative Example 3 and Comparative Example 4) in terms of Cell mask dye positivity rate and AF488-CD9 positivity rate.

[0237] As shown in Table 2, the total CV values ​​for all concentration levels in Example 4 were significantly lower than the total CV value (15%) recommended by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). The separation and detection method provided in Example 4 was considered good and had a low level of imprecision (i.e., high precision), meeting the methodological standards for IVD kits. In contrast, the total CV values ​​for all concentration levels in Comparative Examples 6 and 7 were greater than the total CV value (15%) recommended by the IFCC, indicating poorer methodological performance.

[0238] Depend on Figure 5 As can be seen, using the method of Example 4 for separation and detection, and statistically analyzing the detection results of 52 serum pool samples, the AUC was 0.959, and the cutoff value for serum PSA+ exosome concentration was set to 5.4 × 10⁻⁶. 7 The particle count was [value missing], with a sensitivity of 93.3% and a specificity of 88%. The method described in Comparative Example 6 was used for separation and detection. Statistical analysis of 37 serum samples showed an AUC of 0.807, and the cutoff value for serum PSA+ exosome concentration was set at 7.5 × 10⁻⁶. 7 The particle count was 70% (particles / ml), with a sensitivity of 70% and a specificity of 72%. The method described in Comparative Example 7 was used for separation and detection. The results of 37 peripheral blood samples were analyzed, and the AUC was 0.527. The cutoff value for serum PSA+ exosome concentration was set at 4.8 × 10⁻⁶. 7 The particle / ml concentration showed a sensitivity of 53.3% and a specificity of 56%. Using the method described in Comparative Example 5 for separation and detection, and statistical analysis of 46 serum samples, the AUC was 0.645. The serum PSA concentration cutoff was set at 2.35 ng / mL, with a sensitivity of 67.3% and a specificity of 52%. Among the four methods mentioned above, the separation and detection based on extracellular biomacromolecules (using AF488-PSA antibody for optical labeling) (Example 4) showed the best diagnostic efficacy for prostate cancer.

[0239] Depend on Figure 6It can be seen that, using the dose-effect equation and the method described in Example 4 for separation and detection, when the relative bias of detection equals the maximum bias of 10%, the mass concentration of hemoglobin is 4263.2 mg / L, the concentration of chyle is 769.2 FTU, the mass concentration of free bilirubin is 246.3 mg / L, and the mass concentration of conjugated bilirubin is 279.9 mg / L; using the dose-effect equation and the method described in Comparative Example 5 for separation and detection, when the relative bias of detection equals the maximum bias of 10%, the mass concentration of hemoglobin is 4946.3 mg / L, the concentration of chyle is 823.2 FTU, the mass concentration of free bilirubin is 266.3 mg / L, and the mass concentration of conjugated bilirubin is 309.1 mg / L; using the dose-effect equation and the method described in Comparative Example 6 for separation and detection, when the relative bias of detection equals the maximum bias of 10%, the mass concentration of hemoglobin is 6824.6 mg / L. The concentrations of bilirubin, chyle, free bilirubin, and conjugated bilirubin were 1569.2 FTU, 642.8 mg / L, and 362.5 mg / L, respectively. Therefore, compared to other methods, the separation and detection method described in Example 4 of this invention exhibits the best anti-interference effect against common serum / peripheral blood interfering substances.

[0240] Depend on Figure 7 It can be seen that, by using the separation and detection method described in Example 4 to detect 30 biological fluid samples from healthy individuals and 25 biological fluid samples from prostate cancer patients, the obtained PSA... + The results of the extracellular biomolecule assay were analyzed using a two-tailed t-test ANOVA (t=12.61, df=24), and the results showed that P<0.0001, indicating a highly significant difference. This demonstrates that the separation and detection method described in Example 4 can significantly distinguish between healthy individuals and prostate cancer patients.

[0241] Depend on Figure 8 It can be seen that, by using the separation and detection method described in Example 4 to detect 30 biological fluid samples from prostate cancer patients at different Gleason stages, the obtained PSMA +The results of extracellular macromolecule assays were analyzed using multiple comparison ANOVA. Using 6 patients with Gleason stage I as a baseline, statistical analysis showed: no statistically significant difference between Gleason stage I and 5 patients with Gleason stage II (P=0.8761); statistically significant difference between Gleason stage I and 5 patients with Gleason stage III (P=0.0495); and highly statistically significant difference between Gleason stage IV and 5 patients with Gleason stage V (P<0.0001). This demonstrates the ability of the isolation and detection method provided in this invention to assist in disease subtyping of prostate cancer patients at different Gleason stages.

[0242] Example 6: Magnetic separation method using a single magnetic trap (using magnetic nanobeads-streptavidin protein-dethiobiotin-VSV-G antibody as magnetic traps)

[0243] 1. Sample pretreatment

[0244] 293T cells were transfected with HIV packaging plasmid and backbone plasmid for 48 h, and then cultured in 100 mm diameter culture dishes. 10 ml of cell culture supernatant was collected, and 10 ml of fresh cell culture medium was added. The cells were cultured for another 24-48 h. All cell culture supernatants containing HIV were collected and stored in a 4°C freezer.

[0245] 2. Desulfobiotin-labeled VSV-G antibody

[0246] 2.1 Calculation of dethiobiotin labeling amount: 10mM dethiobiotin was used to label 1mL of 1mg / ml VSV-G antibody (IgG, 58KD).

[0247] Based on a desulfurized biotin to antibody ratio of 20:1, the volume of desulfurized biotin added... =34.5µL.

[0248] 2.2 Preparation of VSV-G antibody solution: Take 1 mg of the VSV-G antibody to be labeled into an ultrafiltration tube and add labeling buffer not exceeding the maximum volume of the ultrafiltration tube. Centrifuge at 12,000 g for 10 min. Repeat this step 3 times. After the last ultrafiltration is completed, add an appropriate amount of labeling buffer to adjust the antibody concentration to 2 mg / mL.

[0249] 2.3 Dethiobiotin labeling: Add 34.5 µL of 10 mM dethiobiotin DMSO solution to the ultrafiltration tube and gently mix by pipetting. Incubate at 37°C in the dark for 30 min. Centrifuge at 12,000 x g for 10 min. Add an appropriate amount of labeling buffer to the ultrafiltration tube and gently mix by pipetting. Centrifuge at 12,000 g for 10 min. Repeat this step twice. Collect the solution in the ultrafiltration tube (i.e., the dethiobiotin-labeled VSV-G antibody), add an equal volume of storage solution, and store at -20°C.

[0250] 3. Preparation of magnetic nanobeads-streptavidin protein-biotin-desulfobiotin-VSV-G antibody

[0251] Collect 150 µl of magnetic nanobeads-streptavidin protein, wash the nanobeads with 99% ethanol to remove contaminants and increase the humidity of the nanobeads. Add 10 µl of dethiobiotin-labeled VSV-G antibody to the washed magnetic nanobeads-streptavidin protein and incubate at room temperature for 12 h. Then add 10 mM Tris buffer containing 1 mg / ml biotin-labeled bovine serum albumin to the magnetic nanobeads-streptavidin protein and react at 4°C for 1 h to prepare the magnetic nanobeads-streptavidin protein-biotin-dethiobiotin-VSV-G antibody, and store at 4°C.

[0252] 4. Isolation and enrichment of HIV-containing cell culture supernatants

[0253] 3.5 ml of the pretreated sample was incubated with 100 µl of a mixture of magnetic nanobeads-streptavidin-biotin-desulfobiotin-VSV-G antibody at room temperature for 3 h with rotary stirring to obtain a complex of the magnetic nanobeads-streptavidin-biotin-desulfobiotin-VSV-G antibody and HIV. The complex was then separated and enriched using a magnetic rack. The supernatant was removed, and the precipitate was washed five times with 500 µl of buffer containing 10 mM PBS and 0.5% Tween for 5 minutes each time to obtain the separated and enriched HIV. The separated and enriched HIV was mixed with 100 μl of elution buffer 3 and reacted at 37 °C for 30 min. The magnetic beads were then separated from the solution using a magnetic rack to obtain the supernatant.

[0254] 5. Images of HIV captured and eluted using nanomagnetic beads-streptavidin protein-biotin-dethiobiotin-VSV-G antibody were observed under a transmission electron microscope. The results are shown in [Figure number missing]. Figure 9 .

[0255] Conclusion: After incubation of HIV cell culture supernatant with magnetic nanobeads-streptavidin-dethiobiotin-VSV-G antibody, followed by elution with 20 mM biotin in 10 mM TBS solution, transmission electron microscopy revealed that the magnetic nanobeads-streptavidin-dethiobiotin-VSV-G antibody possesses the function of rapidly capturing and non-destructively releasing HIV. This method does not affect the structure and morphology of HIV, has high separation specificity and good reproducibility, and non-destructively releases captured HIV under mild conditions.

[0256] The method of this invention has been described through preferred embodiments. Those skilled in the art will readily be able to modify or appropriately alter and combine the methods and applications described herein within the scope, spirit, and context of this invention to implement and apply the technology of this invention. Those skilled in the art can refer to the content herein to appropriately improve process parameters. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of this invention.

Claims

1. A method for separating and detecting extracellular biomacromolecules in a biological fluid, comprising: steps S1: The target extracellular biomolecule is labeled with a fluorescent dye. The fluorescent dye contains an adaptor structure that can bind to the target extracellular biomolecule, so that the fluorescent dye binds to the target extracellular biomolecule through the adaptor structure. Step S2: The magnetic trapping molecule binds to the target extracellular biomolecule. The trapping agent in the magnetic trapping molecule is a trapping agent that can bind to the target extracellular biomolecule, so that the magnetic trapping molecule binds to the target extracellular biomolecule through the trapping agent. Step S3: Separate the target extracellular biomolecules using an external magnetic field. The target extracellular biomolecules are separated from the biological fluid by using an external magnetic field. Step S4: Use an elution buffer to break the binding of the target extracellular biomolecules to the magnetic trapping molecule and remove the solid-phase support; Step S5: Single nanoparticle analysis is performed using a particle analysis and detection device. After steps S1, S2, S3 and S4, the target extracellular biomolecule bound to the fluorescent dye is obtained. Single nanoparticle analysis is then performed on the target extracellular biomolecule bound to the fluorescent dye using a particle analysis and detection device. The order of steps S1, S2, S3, S4 and S5 is as follows: (1) Step S1 → Step S2 → Step S3 → Step S4 → Step S5; or (2) Step S2 → Step S1 → Step S3 → Step S4 → Step S5; or (3) Step S1 and Step S2 are performed simultaneously → Step S3 → Step S4 → Step S5; or (4) Step S2 → Step S3 → Step S1 → Step S4 → Step S5. The magnetic trapping element includes a solid support, a connecting medium, and a trapping agent; The eluent is an aqueous solution containing an eluent; The magnetic trap and eluent comprise any one of the following: (1) The solid support and the trapping agent are connected by avidin-biotin-disulfide bond-polyethylene glycol as the linking medium, and the trapping agent is distearylphosphatidylethanolamine, to obtain a magnetic trapping molecule with the structure of solid support-avidinin-biotin-disulfide bond-polyethylene glycol-distearylphosphatidylethanolamine; the eluent is trichloroethyl phosphate; (2) The TIM4 protein is linked to a solid-phase support using biotin and avidin to obtain a magnetic trap with the structure of solid-phase support-avidin-biotin-TIM4 protein; the eluent is a metal chelating agent capable of chelating calcium ions; (3) The magnetic trap of solid-phase carrier-avidin protein-biotin-disulfide bond-polyethylene glycol-distearate phosphatidylethanolamine and the magnetic trap of solid-phase carrier-avidin protein-biotin-TIM4 protein, wherein the eluent is trichloroethyl phosphate and a metal chelating agent capable of chelating calcium ions; (4) The structure of the magnetic trap is magnetic nanobeads-streptavidin protein-desulfobiotin-VSV-G antibody, and the eluent is biotin.

2. The method according to claim 1, comprising: Choose from any of the following methods: A fluorescent dye containing an adaptor structure capable of binding to a target extracellular biomolecule is mixed with a biological liquid. The mixture is then incubated to allow the fluorescent dye to bind to the target extracellular biomolecule through the adaptor structure. A magnetic trapping agent is then added, which is a trapping agent capable of binding to the target extracellular biomolecule. The mixture is incubated again to allow the magnetic trapping agent to bind to the target extracellular biomolecule through the trapping agent. An external magnetic field is applied for separation and enrichment. The supernatant is removed, and the mixture is washed to separate the target extracellular biomolecule from the biological liquid, yielding the target extracellular biomolecule bound to the fluorescent dye and the magnetic trapping agent. The binding of the magnetic trapping agent to the target extracellular biomolecule is then eluted with an elution buffer, and the solid-phase support is removed to obtain the target extracellular biomolecule bound to the fluorescent dye. Single nanoparticle analysis of target extracellular biomacromolecules containing fluorescent dyes is performed using particle analysis and detection equipment; or The process includes: mixing a magnetic trap with a biological liquid, wherein the trapping agent in the magnetic trap is a trapping agent capable of binding to the target extracellular biomolecule; incubating the mixture to allow the magnetic trap to bind to the target extracellular biomolecule through the trapping agent; adding a fluorescent dye containing an adaptor structure capable of binding to the target extracellular biomolecule; incubating the mixture to allow the fluorescent dye to bind to the target extracellular biomolecule through the adaptor structure; applying an external magnetic field for separation and enrichment; removing the supernatant; washing to separate the target extracellular biomolecule from the biological liquid, obtaining the target extracellular biomolecule bound to the fluorescent dye and the magnetic trap; eluting the magnetic trap to the target extracellular biomolecule with an elution buffer and removing the solid-phase support, obtaining the target extracellular biomolecule bound to the fluorescent dye; and performing single-nanoparticle analysis on the target extracellular biomolecule containing the fluorescent dye using a particle analysis detection device. or It includes: mixing magnetic trapping molecule and fluorescent dye with a biological liquid, wherein the trapping agent in the magnetic trapping molecule is a trapping agent capable of binding to the target extracellular biomolecule, and the fluorescent dye contains an adaptor structure capable of binding to the target extracellular biomolecule; incubating to allow the magnetic trapping molecule and fluorescent dye to bind to the target extracellular biomolecule through the trapping agent; applying an external magnetic field for separation and enrichment; removing the supernatant; washing to separate the target extracellular biomolecule from the biological liquid, obtaining the target extracellular biomolecule bound to the fluorescent dye and magnetic trapping molecule; eluting with an elution buffer to release the binding of the magnetic trapping molecule to the target extracellular biomolecule, and removing the solid-phase support, obtaining the target extracellular biomolecule bound to the fluorescent dye; and performing single-nanoparticle analysis on the target extracellular biomolecule containing the fluorescent dye using a particle analysis and detection device; or The process includes: mixing magnetic traps with a biological liquid, wherein the trapping agent in the magnetic traps is a trapping agent capable of binding to the target extracellular biomolecules; incubating to allow the magnetic traps to bind to the target extracellular biomolecules through the trapping agent; applying an external magnetic field for separation and enrichment; removing the supernatant; washing to separate the target extracellular biomolecules from the biological liquid; mixing a fluorescent dye with the target extracellular biomolecules, wherein the fluorescent dye contains an adaptor structure capable of binding to the target extracellular biomolecules; incubating to allow the fluorescent dye to bind to the target extracellular biomolecules through the adaptor structure, obtaining target extracellular biomolecules bound to both fluorescent dye and magnetic traps; removing free fluorescent dye by centrifugation, ultrafiltration, or dialysis; using an eluent to release the binding between the magnetic traps and the target extracellular biomolecules; and removing the solid-phase support to obtain target extracellular biomolecules bound to fluorescent dye; and performing single-nanoparticle analysis on the target extracellular biomolecules bound to fluorescent dye using a particle analysis detection device.

3. The method according to claim 1, wherein the surface of the solid support is loaded with functional groups, the functional groups including at least one or a combination of hydroxyl, carboxyl, amino, aldehyde, epoxy, maleimide, p-toluenesulfonyl, N-hydroxysuccinimide or thiol.

4. The method according to claim 1, wherein the target extracellular biomacromolecule contains a substance on its surface or inside that can specifically bind to the trapping agent, and the substance that can specifically bind to the trapping agent includes at least one or a combination of antigens, nucleic acids, fats, and carbohydrates.

5. The method according to claim 4, wherein the target extracellular biomolecule is an extracellular vesicle, the surface of the extracellular vesicle contains phosphatidylserine, and the scavenging agent is TIM4 protein.

6. The method according to claim 4, wherein the target extracellular biomolecule is an extracellular vesicle, the surface of the extracellular vesicle contains a lipid membrane, and the scavenging agent is distearate phosphatidylethanolamine.

7. The method according to claim 4, wherein the target extracellular biomolecule is an extracellular vesicle, the surface of the extracellular vesicle contains a lipid membrane and phosphatidylserine, and the scavenging agent is distearate phosphatidylethanolamine and TIM4 protein.

8. According to the method of claim 1, in group (1) or (3), the content of trichloroethyl phosphate is 10 mM-500 mM, calculated based on the total mass of the eluent.

9. According to the method of claim 1, in group (2) or (3), the content of the metal chelating agent capable of chelating calcium ions is 1 mM-200 mM, calculated based on the total mass of the eluent.

10. The method according to claim 1, wherein in group (4), the content of the eluent is 2 mM-100 mM, calculated based on the total mass of the eluent.

11. The method according to claim 1 or 2, wherein the fluorescent dye comprises a fluorescent molecule, a fluorescent material, or a combination thereof.

12. The method according to claim 1 or 2, wherein the fluorescent dye comprises at least one or a combination thereof of organic fluorescent molecules, fluorescent proteins, nucleic acid dyes, lipid membrane dyes, quantum dots, and polymer dots.

13. The method of claim 12, wherein the organic fluorescent molecule comprises one selected from Alexa Fluor 488.

14. The method according to claim 1, wherein the biological fluid comprises at least one or a combination thereof selected from blood, plasma, serum, urine, sputum, cerebrospinal fluid, cerebrospinal fluid, pleural effusion, nipple aspiration fluid, lymph, fluids from the respiratory tract, intestine and genitourinary tract, tears, saliva, breast milk, fluids from the lymphatic system, semen, intra-organ system fluids, ascites, tumor cyst fluid, amniotic fluid, bacterial cell culture, mammalian cell culture, cell culture supernatant, cell-free protein transcription-translation reactants.

15. The method according to claim 1, wherein the particle analysis and detection equipment includes a flow cytometer for particle detection.

16. The method according to claim 15, wherein the flow cytometer is a particle analysis and detection device capable of realizing directional flow of sample stream.

17. The method according to claim 1 or 15, wherein the particle analysis and detection device comprises a directional fluid system, an optical system, and a particle detector.

18. The method according to claim 17, wherein the directional fluid system comprises a sample loading unit and a flow unit.

19. The method according to claim 17, wherein the particle detector comprises a photoelectric sensor and a signal conditioning circuit with band-limited filtering function for high-frequency noise.

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