Exosome chip liquid biopsy method

Abstract

The present invention discloses an exosome chip liquid biopsy method, which utilizes an exosome enrichment and purification chip made by activation of special glass, can efficiently capture, purify and anchor exosomes in biological samples, efficiently promote target-specific binding, and utilize enzymatic color development reaction to ultra-sensitively detect targets on exosome membranes. The glass substrate is a special glass with ultra-thinness <0.19 mm, geometric mean roughness <100 nm, and excellent light transmittance. It is activated and functionalized through multiple steps to achieve efficient and high-density streptavidin immobilization. After surface polarization connection to streptavidin, it is linked to a biotinylated capture antibody, presenting a multi-stage amplification effect, and the enrichment and capture antibody coating efficiency is extremely high, so that more exosomes can be captured from a unit volume of biological samples, and specific target-bound exosomes are firmly anchored on the chip, and the enriched exosomes have a very high purity.

Description

Technical Field

[0001] The present invention belongs to the technical field of liquid biopsy, and specifically relates to an exosome chip liquid biopsy method (ExosomalChip Liquid Biopsy, abbreviated as ExoCLiB). Background Art

[0002] In 2015, liquid biopsy was selected as one of the top ten breakthrough technologies of the year by the MIT Technology Review. The three major markers of liquid biopsy are: circulating free DNA (cfDNA), circulating tumor cells (CTC) and extracellular vesicles (EVs). EVs refer to the collective name of membrane vesicles with a phospholipid bilayer structure and nanometer to micrometer size that are actively secreted by cells. They belong to the world's top scientific research field. The 2013 Nobel Prize in Physiology or Medicine was awarded to American scientists James Rothman and Randy Schekman, and German scientist Thomas Südhof in recognition of their discovery of the Regulation Mechanism of Cellular Vesicle Transport; EVs are mainly divided into three categories: exosomes (EXO), microvesicles (MVs) and apoptotic bodies. Exosomes are EVs with a diameter of 40-160nm formed by the fusion of multivesicular bodies and cell membranes; MVs are EVs with a diameter of 200-1000nm formed by budding from the cell membrane; apoptotic bodies are membrane-wrapped vesicular bodies with a diameter of 500-2000nm containing cytoplasm and organelles formed by cell shrinkage and fragmentation during apoptosis. Liquid biopsy is a non-invasive test of biological target fluids. It is easy, safe, convenient, and efficient to sample. It can monitor circulating tumor cells (CTCs), circulating free DNA (CfDNA) fragments or EVs and exosomes released into the blood by diseases or tumor metastases. It is a breakthrough technology for early disease screening, dynamic prognosis monitoring, and adjuvant therapy.

[0003] Compared with circulating free DNA (cfDNA) and circulating tumor cells (CTC), exosomes (EXO) have the advantages of higher concentration, better stability and presence in various types of liquid specimens in body fluid specimens. Exosomes can carry a variety of important information substances such as nucleic acids, proteins, lipids and small molecules to form intercellular communication, realize information interaction with the external microenvironment, and directly affect processes such as cell migration, cell growth and differentiation, angiogenesis and immune response. Exosomes can be secreted by almost all cells and are widely present in cell supernatants and various body fluids, such as peripheral blood, urine, saliva, peritoneal effusion, amniotic fluid, milk, cerebrospinal fluid, joint fluid, etc. It is different from other markers such as proteins and nucleic acids that are free and unprotected in the blood. Exosomes are special organelles with biological functions, which can timely and accurately reflect the cell origin and pathological information. They are circulating markers with considerable clinical diagnostic value. Exosomes, known as "biological express delivery", are wrapped in an outer membrane, so the substances they carry are not easily degraded by proteins like cytokines, thus completing the "mission" of long-distance cell communication and information transmission. Compared with cfDNA and CTC, exosomes have many unique advantages.

[0004] 1. Comprehensiveness of information. cfDNA only reflects DNA information and is a DNA fragment with little information and low quality. CTC is a complete cell and contains the most comprehensive cell information, but it is limited to tumor cells. Exosomes are secreted by all cells, including tumor cells and related immune cells, so they can reflect a variety of information such as the disease microenvironment;

[0005] 2. The timeline density of cfDNA and CTC is not enough, while exosomes flow in the body, and cells continuously send and receive exosomes, so they are active, forming the "Internet of Things and Internet" in the human body; there are only 1-10 CTCs per milliliter of blood, while exosomes contain 10 10-12The content is extremely high, and it can provide information on various levels such as proteins, nucleic acids, and metabolites. With so many "highlights", exosomes have become the most ideal analytical material for liquid biopsy. They are the medium for close-range communication between cells in health and disease, and are involved in all aspects of cell differentiation, immune response, infection metabolism, tumor metastasis, etc. They are expected to become markers for early diagnosis, treatment monitoring, and prognosis of various diseases. In the disease-related exosome research, tumor-related research accounts for the majority. More and more evidence shows that exosomes secreted by tumor cells induce normal cells to become cancerous, promote the formation of new blood vessels, improve the migration and invasion ability of tumor cells, and enhance drug resistance. These biological functions are closely related to the biological macromolecules carried by exosomes, such as proteins, abbreviated miRNA (miRNA), messenger RNA (mRNA), methylated DNA, and long non-coding RNA (lncRNA). By analyzing the expression differences of these biological macromolecules in exosomes, we can distinguish the different stages of the development of different diseases. They are a new type of target with great potential for liquid biopsy of diseases, especially tumors, and their own characteristics and detection advantages are very promising.

[0006] At present, there are few exosome target detection and biomarker development and verification. The separation and purification of exosomes is a bottleneck restricting the development of technology. Exosomes are easily contaminated by factors such as free proteins, free nucleic acids, lipoproteins, and intracellular vesicles. The separation and purification methods are complex and it is difficult to obtain a large number of high-quality exosome specimens. The existing methods for separation and purification of exosomes mainly include: ultracentrifugation (abbreviated as: ultracentrifugation) and PEG precipitation (abbreviated as: PEG). Ultracentrifugation: The purity of isolated exosomes is high, but there is still a certain degree of contamination by non-exosome-related proteins, and the throughput is small, time-consuming (6-12 hours), the ultracentrifuge is expensive to purchase, the consumables of the centrifuge tubes are expensive, the recovery rate is unstable, the loss is high, and it is easy to damage the exosome structure; PEG precipitation: low purity, more impurities (false positives), low recovery rate, uneven particle size, difficult-to-remove polymers, and the chemical components in the reagents may damage the exosome structure; after transmission electron microscopy verification, the exosomes enriched and purified by the ExoCLiB chip better retain the original structure, higher purity, uniform particle size, regular morphology, and intact (see for details). Figure 3Ultracentrifugation, PEG, ExoCLiB separation, and purification of exosomes. In terms of downstream analysis, the separated and purified exosomes are often screened by second-generation sequencing and gene chips in biomarker screening methods. Protein marker high-throughput screening technologies include labeling, non-labeling protein mass spectrometry, protein chips, etc. Researchers also use immunoblotting (Western Blot, WB or flow cytometry) to screen the biomarkers. cytometry (FCM) is used to identify marker proteins, but the RNA extraction process is complicated and lengthy, requires amplification, samples are easily contaminated, and the results are unstable; WB is time-consuming and labor-intensive, with complex processes and low throughput, requiring 1-3 days, and cannot strictly distinguish whether the protein is in the exosome or on the membrane; FCM requires the use of exosome capture magnetic beads, which are not stable, and the consumables and instruments are very expensive. The ExoCLiB technology uses special biochips to immobilize exosome capture antibodies to the greatest extent. First, the exosomes in the cell culture supernatant, blood or body fluid specimens are captured and enriched by monoclonal or polyclonal antibodies, and then enzyme-labeled monoclonal antibodies are used to detect other targets on the exosome membrane, realizing the simultaneous detection of multiple antigen targets of exosomes, providing a new platform for convenient and efficient detection of liquid biopsies. Summary of the invention

[0007] In view of the problems existing in the prior art, the purpose of the present invention is to design and provide an exosome chip liquid biopsy method, which is specifically implemented by the following technical solutions:

[0008] The exosome chip liquid biopsy method uses an exosome enrichment and purification chip for liquid biopsy. The chip uses ultra-thin, high-transmittance, low-fluorescence glass as a substrate, and is activated and functionalized to achieve high-efficiency and high-density streptavidin immobilization. The specific preparation method includes the following:

[0009] 1) Amination treatment of glass surface: After the glass substrate is thoroughly cleaned with piranha solution, it is treated with aminopropyl triethoxysilane with a concentration of 1 to 100 mM, and aminopropyl triethoxysilane is grafted onto the surface of the glass substrate through the reaction of aminopropyl triethoxysilane to achieve amination of the surface of the glass substrate; the preparation process of piranha solution is: slowly add 30% hydrogen peroxide to 98% or more concentrated sulfuric acid, mix well, and wait for at least 20 minutes until the solution cools to room temperature; thorough cleaning means gently placing the glass substrate in the piranha solution, standing for 30 minutes, taking out the glass substrate, and repeatedly cleaning it with ultrapure deionized water with a resistance greater than 18 megohms for at least 5 times, blowing it dry with nitrogen and then putting it aside for use, and the volume ratio of the concentrated sulfuric acid to the hydrogen peroxide is 3:1 to 7:1;

[0010] 2) Formation of electrolyte molecular membrane: A polymer electrolyte molecular membrane is formed by a layer-by-layer deposition method, wherein the electrolyte of the polymer electrolyte molecular membrane is polyallylamine hydrochloride with a concentration of 1 to 100 mM, and the following steps are specifically included: Step 1: At 24°C to 28°C, a sodium salt form of anionic polymer polymethylene succinic acid homopolymer PIA or polymethylene succinic acid sodium salt acrylic acid copolymer PIA-co-AA is selected as an electrolyte, wherein each structural unit of the polymethylene succinic acid homopolymer contains two carboxylic acid groups, and each structural unit of acrylic acid contains one carboxylic acid group. The polymethylene succinic acid homopolymer can form an electrolyte layer with ultra-high charge and functional group density; different The polymethylene succinic acid sodium salt acrylic acid copolymer can achieve further regulation of charge and functional group density; Step 2: Use EDC / NHS coupling reaction to covalently bond the polymer to the siloxane amino group, so that the negatively charged PIA or PIA-co-AA electrolyte molecular layer is fixed on the glass; Step 3: Use LBL to alternately deposit the positively charged polyallylamine hydrochloride PAH, PIA or PIA-co-AA on the chip according to the charge demand of the deposition layer to form a dense electrolyte molecular film; By controlling the number of depositions, polymer electrolyte concentration and solution ionic strength, polymer composition, the density, smoothness, and surface charge density of the molecular film can be controlled. The last deposited polymer electrolyte is PAH;

[0011] 3) Immobilization: Add glutaraldehyde GA and streptavidin to the chip after treatment in step 2) in sequence. The concentration of glutaraldehyde is between 2% and 25%, and the volume ratio of glutaraldehyde to streptavidin is 1:5 to 1:9. The amine group on the lysine in streptavidin reacts with glutaraldehyde to form a Schiff base, thereby achieving the immobilization of streptavidin on the chip. GA contains two aldehyde groups, which can quickly react with the amine group (NH3 + ) forms a Schiff base. In this way, one end of GA is connected to PAH, and the aldehyde group at the other end can continue to react; streptavidin contains lysine, and the amine group on lysine reacts with GA to form a Schiff base, so that streptavidin is immobilized on the chip, and the number of layers of the chip polymer dot layer can be adjusted according to the thickness requirements. Whether PIA or PIA-co-AA is used in each anion layer and what composition of PIA-co-AA can be determined according to the properties of the required molecular membrane. The biotinylated capture antibody can be coated on it at a ratio of 1 molecule of avidin to 4 molecules of biotin. According to the test requirements, it is equipped with a polystyrene skeleton and made into various specifications such as 8, 24, 48, and 96 reaction wells.

[0012] Furthermore, the thickness of the ultra-thin high light transmittance low fluorescence glass is less than 0.19 mm, and the roughness is less than 100 mm.

[0013] Furthermore, the antibodies captured by the biopsy chip are biotinylated antibodies of characteristic markers of exosomes or other targets expressed on the membrane of exosomes, such as biotinylated CD9, CD63, CD81, etc.

[0014] Furthermore, the detection target of the biopsy chip can be any antigen substance expressed on the exosome membrane, and the target detection antibody is labeled with HRP or alkaline phosphatase AP.

[0015] Furthermore, the capture antibody is a specific monoclonal or polyclonal antibody labeled with biotin and purified.

[0016] Furthermore, the detection antibody is a specific monoclonal or polyclonal antibody labeled with HRP and purified.

[0017] The exosome chip of the present invention uses ultra-thin, highly transparent, low-fluorescence glass as a substrate and undergoes multi-step activation and functionalization to achieve high-efficiency and high-density streptavidin immobilization, which is crucial for detection sensitivity.

[0018] Currently, the commonly used immobilization technology mostly uses the hydroxyl groups on the glass surface to connect functional groups, and then connects biotin or streptavidin. The immobilization density is limited by the low number of hydroxyl groups on the glass surface, so the immobilization density is very low. This greatly limits the detection sensitivity. The present application uses a polymer electrolyte with an extremely high functional group density and adopts a layer-by-layer self-assembly technology to form a multilayer molecular film. By controlling the number of depositions, the polymer electrolyte concentration and the solution ionic strength, and the polymer composition, the density, smoothness, surface charge density, and functional group density of the molecular film can be controlled. The chip has multiple functional molecular layers (organic silicon, polymer cation electrolyte, homopolymer and copolymer anion electrolyte independently developed by our company, etc.), and is made using a variety of processes, including chemical covalent bonding, layer-by-layer self-assembly (LBL), biocoupling and activation, etc. The super strong non-covalent binding ability of the biotin-avidin system is used to efficiently coat the capture antibody, and the specific target-bound exosomes are firmly anchored on the chip. The enzymatic color reaction is used to detect trace targets on the exosome membrane enriched and purified from various biological samples. The enzymatic color reaction uses substrates such as TMB, heterocyclic azine (ABST), o-phenylenediamine (OPD), p-nitrophenyl phosphate (p-nitrophenyphosate, pNPP), etc., and the reaction is terminated with solutions such as 2M sulfuric acid, 10M sodium azide, and 1.25% NaF. The results are read by a microplate reader with filters of wavelengths such as 450nm and 405nm, so as to qualitatively or quantitatively identify the target.

[0019] The present invention discloses an exosome enrichment and purification chip that can be made by using special glass activation, which can efficiently capture, purify and anchor exosomes in biological samples, efficiently promote target-specific binding, and use enzymatic color development reaction to ultrasensitively detect targets on trace exosome membranes in biological samples. LiquidBiopsy (ExoCLiB for short) uses complex and innovative physical, biological and chemical technologies to polarize glass carriers and immobilize specific exosome capture antibodies on them, maximizing the coating rate of capture antibodies and greatly improving the capture efficiency of exosomes in biological samples per unit volume. Combined with modern immunological technology, it realizes ultra-sensitive detection of targets on trace exosome membranes. It is a new ultra-sensitive liquid biopsy technology. ExoCLiB technology polarizes the surface of an ultra-thin (<0.19mm) special glass substrate with excellent light transmittance to connect streptavidin, and then link it with biotinylated capture antibodies, greatly improving the capture antibody coating efficiency, thereby capturing more exosomes from biological samples per unit volume, enriching and purifying exosomes. 20 samples of cell supernatant, 1 ml each, and 30 samples of plasma, 90 μl each, were used to enrich and purify exosomes using ExoCLiB, and all were detected with CD81 antibody and verified using total internal reflection fluorescence microscopy (TIRFM). 100 images were randomly taken for each sample to calculate the unit exosome density, signal-to-noise ratio, sensitivity and specificity. The density reached 1.78×10 per milliliter of cell supernatant. 6 mm 2 and 2.53 × 10 per 90 μl of plasma 6 mm 2 , the signal-to-noise ratio was 72, 61, the sensitivity was 47, 42, and the specificity was 8, 7, 9.5.

[0020] For details, see: Table 1: Density calculation table of exosomes enriched and purified from cell supernatant by ExoCLiB, Table 2: Density calculation table of exosomes enriched and purified from plasma by ExoCLiB, Table 3: Signal-to-noise ratio, sensitivity and specificity calculation table of exosomes enriched and purified by ExoCLiB chip; showing high density, high signal-to-noise ratio, sensitivity and specificity.

[0021] Table 1

[0022] SampleName <![CDATA[Density( / mm 2 )]]> <![CDATA[Density( / mm 2 )]]> 1 1601388.44 1.60E+06 2 1576398.075 1.58E+06 3 1682271.344 1.68E+06 4 1665240.524 1.67E+06 5 1736924.452 1.74E+06 6 1877875.491 1.88E+06 7 1760240.579 1.76E+06 8 1854156.368 1.85E+06 9 1963272.984 1.96E+06 10 1736059.375 1.74E+06 11 1663290.801 1.66E+06 12 1681374.069 1.68E+06 13 1827072.508 1.83E+06 14 1844160.609 1.84E+06 15 1602129.388 1.60E+06 16 1855252.546 1.86E+06 17 1789671.24 1.79E+06 18 1903408.309 1.90E+06 19 2092596.251 2.09E+06 20 1864341.939 1.86E+06 average 1778856.265 1.78E+08

[0023] Table 2

[0024]

[0025]

[0026] Table 3

[0027] Cell culture supernatant

[0028]

[0029] plasma

[0030]

[0031]

[0032] After ExoCLiB enriches and purifies exosomes, it uses horseradish peroxidase (HRP)-labeled detection antibodies to bind to targets on the exosome membrane, and then undergoes an enzymatic colorimetric reaction with substrates such as tetramethylbenzidine (TMB), heterocyclic azine (ABST), o-phenylenediamine (OPD), and p-nitrophenyl phosphate (pNPP). The optical density (OD) value of the reaction solution is read by a microplate reader to qualitatively or quantitatively identify the target. This technology utilizes the light transmittance of the glass substrate to minimize the loss of light signals. Since more exosomes are captured, the signal of the target on the membrane is also stronger, so the signal-to-noise ratio is higher, which further improves the sensitivity and specificity of target detection on the exosome membrane, making liquid biopsy of trace biomarkers on the nanoscale exosome membrane a reality.

[0033] The chip prepared by the present invention can efficiently capture the enriched exosomes and detect disease markers other than the characteristic exosome markers CD9 / CD63 / CD81 with HRP enzyme-labeled antibodies, which can amplify the originally weak signal. Since the chip can well distinguish the exosome target signals from blank, negative and positive control sources, it has great application value for early diagnosis and prognosis monitoring of diseases, especially cancer. Compared with traditional tissue biopsy, liquid biopsy has natural technical advantages. It is minimally invasive or even non-invasive, making it safer and more convenient. In addition, the protective effect of exosomes on pathological targets, the functions of these targets themselves and their dynamic changes at different stages, all make exosome target detection more reflective of the patient's disease status, and only liquid biopsy can safely, conveniently and efficiently monitor the entire course of the patient's disease.

[0034] Through the same capture antibody, the chip can efficiently capture exosomes from various disease sources. By simply replacing the detection antibodies of different targets, multiple pathological targets can be detected at the lowest cost. The RNA and membrane proteins in a single exosome can be observed using a high-definition total internal reflection fluorescence microscope (TIFRM), and the optical density OD value of the exosome membrane protein in a single sample well on the chip can be detected using an enzyme-labeled instrument. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the exosomal chip structure (Exosomal Chip);

[0036] Figure 2 Schematic diagram of Exosomal Chip Liquid Biopsy (ExoCLiB);

[0037] Figure 3 Transmission electron microscopy images of exosomes separated and purified by ultracentrifugation, PEG, and ExoCLiB;

[0038] Figure 4 TIRFM images of exosomes separated and purified by ultracentrifugation, PEG, and ExoCLiB;

[0039] Figure 5 Schematic diagram of the detection principle of ExoCLiB;

[0040] Figure 6 Schematic diagram of ExoCLiB HRP direct-labeled antibody detection;

[0041] Figure 7 Schematic diagram of ExoCLiB HRP non-directly labeled antibody detection;

[0042] Figure 8 ExoCLiB CD81HRP direct-labeled antibody was used to detect exosomes in the supernatant of A549 cells;

[0043] Fig. 9 ExoCLiB CD81HRP direct-labeled antibody is used to detect plasma exosomes;

[0044] Fig.10 ExoCLiB PDL1HRP non-directly labeled antibody was used to detect exosomes in the supernatant of A549 cells;

[0045] Fig.11 ExoCLiB PDL1HRP non-directly labeled antibody is used to detect plasma exosomes. DETAILED DESCRIPTION

[0046] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments to facilitate a better understanding of the present technical solution.

[0047] Preparation of exosome enrichment and purification chip

[0048] The present invention uses a special glass with a thickness of less than 0.19 mm and a roughness (RMS roughness) of less than 100 nm, which is ultra-thin, ultra-smooth, ultra-low fluorescence background, and ultra-high light transmittance. First, the glass substrate is thoroughly cleaned with piranha solution piranha treatment and then treated with aminopropyltriethoxysilane (APTES). Aminopropyltriethoxysilane (APTES) is grafted onto the glass surface through the reaction of hydroxyl and ethoxy groups to achieve surface amination. Next, a polymer electrolyte molecular membrane is formed by layer-by-layer deposition. The anionic polymer electrolyte uses a homopolymer of poly(itaconic acid), PIA, or a sodium salt of poly(itaconic acid-co-acrylic acid), PIA-co-AA electrolyte (both in the form of sodium salt). Each structural unit of PIA contains two carboxylic acid groups, and each structural unit of acrylic acid contains one carboxylic acid group. Therefore, PIA can form an electrolyte layer with ultra-high charge and functional group density. The use of PIA-co-AA with different compositions can further control the charge and functional group density (the higher the AA ratio, the lower the charge and carboxylic acid group density). The polymer is prepared into an aqueous solution. Next, the EDC / NHS coupling reaction is used to covalently bond the polymer to the siloxane amino group. In this way, the negatively charged PIA or PIA-co-AA electrolyte molecular layer is immobilized on the glass. Next, LBL is used to alternately deposit positively charged polyallylamine hydrochloride (PAH) and PIA (or PIA-co-AA, depending on the charge requirement of the deposition layer) onto the chip to form a dense electrolyte molecular film. By controlling the number of depositions, the concentration of the polymer electrolyte, the ionic strength of the solution, and the composition of the polymer, the density, smoothness, and surface charge density of the molecular film can be controlled. The last deposited polymer electrolyte is PAH. Next, glutaraldehyde (GA) is added. GA contains two aldehyde groups, which can quickly react with amine groups (NH3 + ) to form a Schiff base. In this way, one end of GA is connected to PAH, and the aldehyde group at the other end can still continue to react. Next, add streptavidin. Streptavidin contains lysine, and the amine group on lysine reacts with GA to form a Schiff base. In this way, streptavidin is immobilized on the chip. See the chip structure diagram for details. Figure 1 Please note that the four polymer electrolyte layers in the figure are only for illustration. In actual applications, the number of layers can be adjusted according to the thickness requirements. Whether PIA or PIA-co-AA is used in each anion layer and what composition of PIA-co-AA is used can be determined according to the properties of the desired molecular membrane.

[0049] like Figure 2As shown, the exosome chip liquid biopsy technology (Exosomal Chip Liquid Biopsy, referred to as ExoCLiB) of the present invention uses ultra-thin high-transmittance low-fluorescence glass as the substrate, which is activated and functionalized through multiple steps to achieve high-efficiency and high-density streptavidin immobilization. The present invention uses polymer electrolytes with extremely high functional group density and adopts layer-by-layer self-assembly technology to form a multilayer molecular film. By controlling the number of depositions, polymer electrolyte concentration, solution ionic strength, and polymer composition, the density, smoothness, surface charge density, and functional group density of the molecular film can be controlled. The chip has multiple functional molecular layers (organic silicon, polymer cationic electrolytes, homopolymer and copolymer anionic electrolytes independently developed by our company, etc.), and is made using a variety of processes, including chemical covalent bonding, layer-by-layer self-assembly (LBL), biocoupling and activation, etc. The super strong non-covalent binding ability of the biotin-avidin system is used to efficiently coat the capture antibodies, and the specific target-bound exosomes are firmly anchored on the chip. The enzymatic colorimetric reaction is used to detect trace targets on the membranes of exosomes enriched and purified from various biological samples.

[0050] The ExoCLiB chip of the present invention not only has the characteristics of high sensitivity, high specificity, and high signal-to-noise ratio, but also performs a softening treatment similar to a high-density molecular brush on the surface of the chip. While efficiently capturing exosomes, it can also completely retain the physiological structure of the exosomes, will not destroy the exosomes, and its target structure on the membrane will not be affected. It is very suitable for the detection of trace exosome targets. The existing exosome separation methods mainly include ultracentrifugation and PEG precipitation. Ultracentrifugation: low throughput, time-consuming (6-12 hours), high purchase cost of ultracentrifuge, expensive consumables for centrifuge-specific tubes, unstable recovery rate, high losses, and may cause damage to the exosome structure; PEG precipitation: low purity, more impurities (false positives), low recovery rate, uneven particle size, and the production of polymers that are difficult to remove. The chemical components in the reagents may cause damage to the exosome structure; after verification by transmission electron microscopy, the exosomes enriched and purified by the ExoCLiB chip better retain the original structure, higher purity, uniform particle size, regular and intact morphology, and clearer imaging pictures. See for details. Figure 3 :Transmission electron microscopy images of exosomes separated and purified by ultracentrifugation, PEG, and ExoCLiB. Figure 4 : TIRFM images of exosomes separated and purified by ultracentrifugation, PEG, and ExoCLiB.

[0051] The detection principle of ExoCLiB of the present invention is as follows Figure 5As shown: there are no exosomes in the blank control, so neither the capture antibody nor the detection antibody can bind to the target, so TMB does not show color; the negative control contains exosomes, but they are mainly derived from healthy cells and can be captured by the capture antibody, but do not contain diseased cell sources or contain a small amount of normally expressed characteristic markers. The detection antibody does not bind or binds to a small amount of targets, so TMB does not show color or shows color weakly; while the positive control sample contains a large number of exosomes derived from diseased cells, whose surface contains not only the characteristic markers CD9 / CD63 / CD81 of exosomes, but also characteristic markers of diseased cells, such as PD-L1, EGFR, GPC3, etc. The capture antibody binds to the characteristic CD9 / CD63 / CD81 molecules on the surface of exosomes, and the HRP enzyme-labeled detection antibody binds to pathological markers such as PD-L1, EGFR, GPC3, etc., to make the substrate TMB show color.

[0052] Example 1: Taking CD9 capture and CD81-HRP direct labeled antibody detection as an example, Figure 6 As shown, the specific steps are as follows:

[0053] 1) Take out the ExoCLiB chip and add 100 μL of Avidin diluted in PBS;

[0054] 2) Seal the plate, incubate at 400 rpm for 30 min at room temperature (cover the plate or apply sealing film);

[0055] 3) Remove the solution, wash with PBS for 3 times, and shake or tap the plate;

[0056] 4) Take 4 μL of CD9-Biotin capture antibody, add it to 396 μL 1X PBS, and vortex to mix;

[0057] 5) Add 100 μL of capture antibody dilution to each well, seal the plate with film, and incubate at 4°C overnight;

[0058] 6) Remove the solution, wash with PBS for 3 times, and shake or tap the plate;

[0059] 7) Add 300 μL 5% BSA and block at room temperature for 2 hours (cover the plate or apply sealing film);

[0060] 8) Remove the solution and wash with PBS for 3 times;

[0061] 9) Add 100 μL of sample, negative and positive controls to each well, seal the plate with film, and incubate at 37°C for 90 min;

[0062] 10) Remove the solution and wash with PBS for 3 times;

[0063] 11) Take 4 μL of CD81-HRP detection antibody, add it to 396 μL of 5% BSA, and mix well;

[0064] 12) Add 100 μL of detection antibody dilution to each well, seal the plate with film, and incubate at room temperature for 2 hours;

[0065] 13) Wash the plate three times with 300 μL 1X PBST (soak for 30 seconds each time and discard), and shake or tap the plate;

[0066] 14) Add 50 μL TMB colorimetric solution to each well, incubate at room temperature, away from light, at 400 rpm, and incubate for 5-30 min;

[0067] 15) Add 50 μL of stop solution to each well at an appropriate time, shake the plate for 10 seconds, and read the plate at 450 nm using an ELISA reader.

[0068] Example 2: Taking CD9 capture, PDL1 non-directly labeled primary antibody, and HRP-IgG enzyme-labeled secondary antibody detection as an example, Figure 7 As shown, the specific steps are as follows:

[0069] 1) Take out the ExoCLiB chip and add 100 μL of Avidin diluted in PBS;

[0070] 2) Seal the plate, incubate at 400 rpm for 30 min at room temperature (cover the plate or apply sealing film);

[0071] 3) Remove the solution, wash with PBS for 3 times, and shake or tap the plate;

[0072] 4) Take 4 μL of CD9-Biotin capture antibody, add it to 396 μL 1X PBS, and vortex to mix;

[0073] 5) Add 100 μL of capture antibody dilution to each well, seal the plate with film, and incubate at 4°C overnight;

[0074] 6) Remove the solution, wash with PBS for 3 times, and shake or tap the plate;

[0075] 7) Add 300 μL 5% BSA and block at room temperature for 2 hours (cover the plate or apply sealing film);

[0076] 8) Remove the solution and wash with PBS for 3 times;

[0077] 9) Add 100 μL of sample, negative and positive controls to each well, seal the plate with film, and incubate at 37°C for 90 min;

[0078] 10) Remove the solution and wash with PBS for 3 times;

[0079] 11) Take 4 μL of CD81 detection antibody, add it to 396 μL of 5% BSA, and mix well;

[0080] 12) Add 100 μL of detection antibody dilution to each well, seal the plate with film, and incubate at room temperature for 2 hours;

[0081] 13) Wash the plate three times with 300 μL 1X PBST (soak for 30 seconds each time and discard), and shake or tap the plate;

[0082] 14) Dilute HRP-IgG enzyme-labeled secondary antibody into 5% BSA at 1:5000 and mix well;

[0083] 15) Add 100 μL of enzyme-labeled secondary antibody dilution to each well, seal the plate with film, and incubate at room temperature for 2 hours;

[0084] 16) Wash the plate three times with 300 μL 1X PBST (soak for 30 seconds each time and discard), and shake or tap the plate for the last wash;

[0085] 17) Add 50 μL TMB colorimetric solution to each well, incubate at room temperature, away from light, at 400 rpm, and incubate for 5-30 min;

[0086] 18) Add 50 μL of stop solution to each well at an appropriate time, shake the plate for 10 seconds, and read the plate at 450 nm using an ELISA reader.

[0087] Test results

[0088] Ten negative controls (NC) and 10 positive controls (PC) were collected from the culture supernatant of A549 cells, a common lung cancer cell line, and 30 plasma samples from healthy subjects and patients with lung malignancies. ExoCLiB was used to detect the CD81-HRP labeled antibody and PDL1 non-labeled primary antibody, and HRP-IgG enzyme-labeled secondary antibody. The plate was read at 450nm using an enzyme reader. The results are as follows:

[0089] Figure 8 ExoCLiB CD81HRP direct-labeled antibody detected exosomes in the supernatant of A549 cells, P = 0.003;

[0090] Fig. 9 ExoCLiB CD81HRP direct labeled antibody detected plasma exosomes, P = 0.002;

[0091] Fig.10 ExoCLiB PDL1HRP non-directly labeled antibody detected exosomes in the supernatant of A549 cells, P = 0.001;

[0092] Fig.11 ExoCLiB PDL1HRP non-directly labeled antibody detected plasma exosomes, P = 0.003;

[0093] The test results showed that the OD values ​​of CD81 and PDL1 negative controls in the culture supernatant of lung cancer cell line A549 were significantly lower than those of positive controls, with P values ​​less than 0.05, showing significant differences. The OD values ​​of CD81 and PDL1 in plasma samples of healthy people were significantly lower than those in patients with lung malignant tumors, with P values ​​less than 0.05, showing significant differences. This verified that the exosome chip liquid biopsy technology (Exosomal Chip Liquid Biopsy, ExoCLiB) can efficiently, sensitively and specifically detect targets on the exosome membrane.

Claims

1. A method for preparing an exosome chip, characterized in that: For exosome enrichment and purification, the chip uses ultra-thin, highly transparent, low-fluorescence glass as the substrate, and is activated and functionalized to achieve high-efficiency and high-density streptavidin immobilization. The specific preparation method includes the following: 1) Amination treatment of glass surface: the glass substrate is thoroughly cleaned with piranha solution, and then treated with aminopropyl triethoxysilane with a concentration of 1 to 100 mM, and aminopropyl triethoxysilane is grafted onto the surface of the glass substrate through the reaction of aminopropyl triethoxysilane to achieve amination of the surface of the glass substrate; the preparation process of piranha solution is: slowly add 30% hydrogen peroxide to 98% or more concentrated sulfuric acid, mix well, and wait for at least 20 minutes until the solution cools to room temperature; thorough cleaning means gently placing the glass substrate in the piranha solution, leaving it for 30 minutes, taking out the glass substrate, and repeatedly cleaning it with ultrapure deionized water with a resistance greater than 18 megohms for at least 5 times, blowing it dry with nitrogen and then setting it aside, and the volume ratio of the concentrated sulfuric acid to the hydrogen peroxide is 3:1-7:1; 2) Formation of electrolyte molecular membrane: A polymer electrolyte molecular membrane is formed by a layer-by-layer deposition method, wherein the electrolyte of the polymer electrolyte molecular membrane is polyallylamine hydrochloride with a concentration of 1 to 100 mM; the formation of the electrolyte molecular membrane includes the following steps: Step 1: At 24°C to 28°C, a sodium salt form of anionic polymer polymethylene succinic acid homopolymer PIA or polymethylene succinic acid sodium salt acrylic acid copolymer PIA-co-AA is selected as an electrolyte. Each structural unit of the polymethylene succinic acid homopolymer contains two carboxylic acid groups, and each structural unit of acrylic acid contains one carboxylic acid group. The polymethylene succinic acid homopolymer can form an electrolyte layer with ultra-high charge and functional group density; the use of polymethylene succinic acid sodium salt acrylic acid copolymers with different compositions can achieve further regulation of charge and functional group density; Step 2: Use EDC / NHS coupling reaction to covalently bond the polymer to the siloxane amino group, so that the negatively charged PIA or PIA-co-AA electrolyte molecular layer is immobilized on the glass; Step 3: Using LBL, positively charged polyallylamine hydrochloride PAH, PIA or PIA-co-AA is alternately deposited on the chip according to the power requirement of the deposition layer to form a dense electrolyte molecular film; 3) Immobilization: Glutaraldehyde and streptavidin are added to the chip treated in step 2) in sequence, so that the amine group on the lysine in the streptavidin reacts with glutaraldehyde to form a Schiff base, thereby achieving the immobilization of streptavidin on the chip. The concentration of glutaraldehyde is between 2% and 25%, and the volume ratio of glutaraldehyde to streptavidin is 1:5 to 1:

9.

2. The method for preparing the exosome chip according to claim 1, characterized in that The ultra-thin high light transmittance low fluorescence glass has a thickness of less than 0.19 mm and a roughness of less than 100 mm.

3. The method for preparing the exosome chip according to any one of claims 1 to 2, characterized in that: The antibodies captured by the exosome enrichment and purification chip are biotinylated antibodies of characteristic markers of exosomes or other targets expressed on the exosome membrane.

4. The method for preparing the exosome chip according to any one of claims 1 to 2, characterized in that: The target of biopsy chip detection is any antigen expressed on the exosome membrane, and the target detection antibody is labeled with HRP or alkaline phosphatase AP.

5. The method for preparing the exosome chip according to claim 3, characterized in that: The capture antibody is a specific monoclonal or polyclonal antibody labeled with biotin and purified.

6. The method for preparing the exosome chip according to claim 3, characterized in that: The detection antibody is a specific monoclonal or polyclonal antibody labeled with HRP and purified.

Images