Probe group and method for analyzing specific glycoprotein sialic acid on exosome surface by combining probe group with 3D printing micro-fluidic chip

Abstract

The invention discloses a probe group and a method for analyzing specific glycoprotein sialic acid on the surface of an exosome by combining with a 3D printing micro-fluidic chip, and belongs to the technical field of biochemical analysis. On the basis of a 3D printing micro-fluidic chip combined with a three-probe co-localization signal amplification strategy, a nucleic acid signal is converted into a fluorescence signal, and the sialic acid signal on glycoprotein on the surface of the exosome is analyzed by directly reading the intensity of the fluorescence signal; the invention provides a novel method for overcoming the defects and limitations of the clinical application of the exosome in the prior art, reduces the loss of a sample, improves the sensitivity of reaction, and provides a foundation for researching the possible relationship between different exosomes and specific glycoprotein sialic acid of parent cells thereof and disease development. And a feasible method is provided for tumor screening and disease course judgment by using sialic acid.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical analysis, and particularly to a probe set and a method for analyzing specific glycoprotein sialic acid on the surface of exosomes by combining with a 3D printed microfluidic chip. Background Art

[0002] Exosomes are nanovesicles with a lipid bilayer, with a particle size ranging from 30 to 150 nm. They can be secreted by different types of cells and are widely present in various body fluids. To date, exosomes have been proven to be involved in the transport of various bioactive substances such as proteins, nucleic acids, and lipids, and can mediate intercellular communication, metabolic regulation, or reprogramming. Exosomes are autosecreted by parental cells and have low immunogenicity. Their stable lipid bilayer structure can protect the activity of their contents from degradation, and they also have great potential in the fields of liquid biopsy and intracellular chemical drug or gene delivery. Exosomes carry various biomolecules derived from parental cells, such as proteins, nucleic acids, lipids, etc. The structure, biological characteristics, and biological functions of exosomes largely depend on these biomolecules. Effective biological analysis of exosomes can achieve tumor screening and predict cancer progression and prognosis.

[0003] In addition to proteins and nucleic acids, the carbohydrate compounds contained in exosomes are also of great significance in biogenesis, cell recognition, and disease progression. Sialic acid is attached to the distal end of the glycan on the exosome membrane surface and has been proven to be highly correlated with malignant behaviors such as tumorigenesis and invasion in recent years. Heterogeneous transformation of cells is often accompanied by abnormal sialic acid modification of proteins. Therefore, sialic acid on the surface of exosomes can be used as a highly potential new type of disease biomarker and is widely used in disease diagnosis and prognosis judgment.

[0004] Existing methods for analyzing sialic acid glycans on the surface of exosomes mainly focus on lectin arrays, liquid chromatography, and mass spectrometry. Although they can effectively analyze sialic acid on the surface of exosomes, they often destroy the physiological structure of exosomes themselves, which is not conducive to biological research. Therefore, converting glycan information into nucleic acid information will help maintain the integrity of exosomes during the detection process.

[0005] In addition, affected by the unique biogenesis process of exosomes, the components of biological samples obtained in the laboratory and clinic are complex, and the exosome content is low. Therefore, the isolation of exosomes is the first step for effective exosome analysis. Traditional exosome isolation methods usually rely on the physical and chemical properties of exosomes, such as separation based on buoyant density, particle size, hydrophilic-hydrophobic differences, etc. In addition, in recent years, researchers have developed a series of strategies for enriching exosomes targeting specific surface targets and some new technical platforms. However, due to the low concentration of exosomes in untreated biological samples, the detection of exosome markers currently applied in clinics usually has low specificity and poor sensitivity, which greatly limits the clinical application of exosomes. Summary of the Invention

[0006] The object of the present invention is to provide a probe set and a method for analyzing specific glycoprotein sialic acid on the surface of exosomes by combining with a 3D printed microfluidic chip, so as to solve the problems existing in the above-mentioned prior art. By constructing a general three-probe co-localization signal amplification strategy, the differences between glycosylation and sialylation among multiple targets are accurately identified and amplified, and a method for analyzing the sialylation degree of glycoproteins on the surface of exosomes is established. The sialic acid content of specific glycoproteins in different exosomes and their parental cells is detected, and the possible relationship between them and disease development is analyzed, providing a detectable method for tumor screening and disease course judgment using sialic acid.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a probe set for analyzing specific glycoprotein sialic acid on the surface of exosomes, which includes a first probe combination participating in a signal amplification strategy and a second probe combination that can bind to specific glycoproteins on the surface of the same exosome;

[0009] Among them, the first probe combination includes a backbone probe with a nucleotide sequence as shown in SEQ ID NO.9, a first ligation probe as shown in SEQ ID NO.10, a second ligation probe as shown in SEQ ID NO.11, and a fluorescent reporter probe as shown in SEQ ID NO.12; the second probe combination includes an aptamer probe that specifically recognizes specific glycoproteins on the surface of exosomes, a sialic acid conversion probe that specifically recognizes sialic acid on the specific glycoprotein, and a sugar chain conversion probe.

[0010] Optionally, the aptamer probe that specifically recognizes specific glycoproteins on the surface of exosomes includes aptamer probes that specifically recognize PTK7, MUC1, EpCAM, HER2, and PD-L1, and the nucleotide sequences are as shown in SEQ ID NOs.1-5 respectively.

[0011] Optionally, the nucleotide sequence of the sialic acid conversion probe is as shown in SEQ ID NO.6, and the nucleotide sequence of the glycan conversion probe is as shown in SEQ ID NO.7.

[0012] Optionally, the 5'-end of the glycan conversion probe is modified with DBCO.

[0013] The present invention also provides the application of the probe set in the preparation of products for analyzing specific glycoprotein sialic acid on the surface of exosomes.

[0014] The present invention also provides a method for analyzing specific glycoprotein sialic acid on the surface of exosomes based on a 3D printed microfluidic chip combined with the probe set, comprising the following steps:

[0015] Pre-treat the backbone probe, the first ligation probe, and the second ligation probe to prepare a probe system;

[0016] Add magnetic beads and exosomes to chamber I of the microfluidic chip containing 4 cylindrical reaction chambers, add magnetic bead washing solution to chamber II, add the aptamer probe, sialic acid conversion probe, glycan conversion probe, PBA-PEG-N3, and the probe system to chamber III, and add magnetic bead eluent to chamber IV; wherein, channels are provided between the four chambers for the circulation of magnetic beads and the reaction system;

[0017] Oscillate the microfluidic chip to obtain magnetic beads adsorbed with exosomes, then place a magnet under chamber I, move the magnetic beads from the aqueous phase to the oil phase, and then move the magnet to move the magnetic beads from chamber I to chamber II, and place the magnet under chamber II to move the magnetic beads from the oil phase to the aqueous phase. In the same way, move the magnetic beads to chamber III in turn for amplification reaction, and then move to chamber IV for elution to obtain an eluate;

[0018] Measure the fluorescence intensity of the eluate, and analyze the content of sialic acid according to the fluorescence intensity.

[0019] Optionally, the pre-treatment is: mix 3 μL of 20 μM backbone probe, 3 μL of 20 μM first ligation probe, and 3 μL of 20 μM second ligation probe with a phosphate buffer containing 10 mM Mg 2+ anneal at 95 °C for 10 min, and cool to room temperature to obtain the probe system.

[0020] Optionally, 20 μL of Tim4 magnetic beads and 10 μg of exosomes are added to the chamber I; and / or 2 μL of 20 μM aptamer probe, 2 μL of 20 μM sialic acid conversion probe, 2 μL of 20 μM glycan conversion probe, 2 μL of 2 μM PBA-PEG-N3 and the obtained probe system after annealing and cooling are added to the chamber III, and then supplemented to 80 μL with phosphate buffer solution.

[0021] Optionally, the conditions for shaking are: shaking at 25 °C and 500 - 1000 rpm for 1 - 3 h.

[0022] Optionally, in the chamber III, the amplification reaction includes the following steps: Add 1 μL of T4 DNA ligase, 3 μL of T4 DNA ligase buffer, and 2 μL of 0.5% BSA to the chamber III, and react at room temperature for 1 - 2 h. Then add 2 μL of phi29 DNA polymerase, 4 μL of dNTP, 5 μL of DNA polymerase buffer, and 2 μL of 0.5% BSA to the chamber III, incubate at room temperature for 1 - 3 h, and finally add 2 μL of fluorescent reporter probe to the chamber III, and the incubation continues for 1 - 2 h.

[0023] The present invention discloses the following technical effects:

[0024] The present invention has developed a highly specific in-situ amplification strategy based on triple-probe proximity ligation, which induces rapid assembly and signal output between probes through the recognition of three adjacent targets by the sensing element, thereby highly sensitively converting the sensed target information into amplified fluorescence signals, and finally obtaining information related to the sialylation of glycoproteins on the surfaces of different cells and exosomes. The present invention overcomes the defects and limitations in the clinical application of exosomes in the prior art, and provides a feasible method for exploring the possible relationship between the specific glycoprotein sialic acid of different exosomes and their parental cells and the development of diseases, as well as using sialic acid for tumor screening and disease course judgment.

[0025] The present invention uses 3D printing technology to fabricate a microfluidic chip for this analysis and measurement. The entire process can be integrated in the chip, and the chip contains 4 chambers, which are respectively used for the separation, washing, amplification reaction, and elution of exosomes. Magnetic beads are used to control the movement of the sample between the 4 chambers, thereby reducing sample loss and the sensitivity of the reaction. Description of the Drawings

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0027] Figure 1 Schematic diagram of the microchip structure and Tri-PLA-RCA in the microchip for the analysis of specific glycoprotein sialic acid on the surface of exosomes;

[0028] Figure 2 Fluorescence intensity detection results after incubation with the Reporter probe at the end of the Tri-PLA-RCA reaction; 1-6 in the figure and 1-6 in Table 1 are both grouping numbers with the same meaning;

[0029] Figure 3 Analysis results of sialic acid signals of different exosome proteins; (A) HepG2 exosomes, (B) MCF-7 exosomes, (C) MDA-MB-231 exosomes, (D) HEK293T exosomes;

[0030] Figure 4 Analysis results of Tri-PLA-RCA for sialic acid on exosome glycoproteins; (A) Analysis of sialic acid on the MUC1 protein of MCF-7 exosomes with different particle concentrations, (B) Standard curve of Tri-PLA-RCA fluorescence intensity and MCF-7 exosome particle concentration, (C) Analysis of sialic acid on the MUC1 protein of HEK293T exosomes with different particle concentrations, (D) Standard curve of Tri-PLA-RCA fluorescence intensity and HEK293T exosome particle concentration. Detailed implementation manners

[0031] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0034] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0035] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0036] Based on the problems and limitations existing in the clinical application of exosomes, in order to ensure the integrity of the exosome structure and achieve non-destructive detection of the glycoprotein sialic acid on the exosome surface, and thus for the analysis of clinical samples based on exosome sialic acid detection, the present invention has developed a triple-probe co-localization signal amplification strategy (Tri-PLA-RCA) to achieve the analysis of sialic acid signals on glycoproteins on the exosome surface. There are a high abundance of cis-diol sites on glycoproteins, and these exposed diol sites can act as receptors to undergo an affinity reaction with phenylboronic acid (PBA) groups to form cyclic phenyl borates in a slightly alkaline aqueous medium. The present invention uses a PBA-PEG-N3 compound as an intermediate. The PBA group in the compound covalently binds to the cis-diol site on the glycoprotein, and the N3 in the compound undergoes a click reaction with a DNA probe modified at the 5'-end with DBCO, connecting the DNA probe to the cis-diol site on the oligosaccharide chain to construct a sugar chain conversion probe to convert glycan information into nucleic acid information. The co-localization mechanism of the specific borate affinity oligosaccharide conversion probe, sialic acid conversion probe, and protein aptamer probe improves the accuracy and specificity of the reaction. The action sites of the three probes on the same target protein will cause the DNA probes to be close in spatial distance, inducing a neighboring ligation RCA reaction. The amplified long-chain DNA is complementary to the Reporter fluorescence reporting signal, realizing the conversion of nucleic acid information into fluorescence signals, and the fluorescence intensity value can be directly read out by an enzyme-linked immunosorbent assay (ELISA) reader.

[0037] Meanwhile, the present invention uses 3D printing technology to fabricate a microfluidic chip for this analysis and measurement. All processes can be integrated in the microfluidic chip, which includes 4 chambers (named chamber I, chamber II, chamber III, and chamber IV in sequence from one end. Liquid channels with the same liquid are provided on the walls of adjacent chambers for liquid flow. The channels can be set in the middle of the wall or near the lower part. There is no clear limit on the aperture size of the channels, as long as they can supply the normal and smooth flow of liquid, and can be set according to requirements), which are respectively used for the separation, washing, amplification reaction, and elution of exosomes. Magnetic beads are used to control the movement of the sample between the 4 chambers, thereby reducing sample loss and the sensitivity of the reaction.

[0038] The following uses specific examples to elaborate in detail on the technical solutions of the above action mechanism of the present invention.

[0039] Example 1

[0040] First, the method of magnetic bead enrichment separation is adopted to achieve the separation and purification of exosomes in the system and the elution of excess probes. Then, the backbone probe, connector probes (Connector’1, Connector’2), and Reporter probe modified with a fluorescent group participating in the Tri-PLA-RCA amplification reaction are designed, as shown in Table 2. The surface of exosomes is rich in glycoproteins, which not only have sialylation sites but also oligosaccharide chains with a large number of exposed cis-diol sites. Cis-diols can specifically bind to phenylboronic acid (PBA). Therefore, aptamer probes that can specifically recognize exosome surface proteins, sialic acid conversion probes (Siaprobe) that specifically recognize sialic acid, and sugar chain conversion probes (PBAprobe) are designed, as shown in Table 2. The three functional probes specifically bind to the same exosome glycoprotein, shortening the spatial distance between the probes, being complementary to the backbone and connector probes, introducing T4 DNA ligase, and undergoing adjacent ligation and hybridization to form a DNA quaternary structure that can serve as an RCA amplification template. After that, phi29 DNA polymerase is added to catalyze the occurrence of the RCA amplification reaction, amplifying the original template into a long single-stranded DNA with hundreds or thousands of repeat sequences. After terminating the reaction, the Reporter signal probe is added to hybridize with the long single-stranded DNA to achieve the conversion of nucleic acid amplification signals, and the fluorescence intensity value is measured at 520 nm with an enzyme-labeled instrument. The specific experimental operations are as follows:

[0041] 1. Verify the feasibility of the Tri-PLA-RCA reaction occurring on the surface of exosomes

[0042] Mix with the sample to be tested according to the conditions shown in Table 1, and measure the feasibility of the Tri-PLA-RCA reaction.

[0043] Table 1

[0044]

[0045]

[0046] Note: All of the above systems contain T4 ligase and Phi29 Polymerase.

[0047] The results are as Figure 2 shown. Taking HepG2 exosomes as an example for analysis, the results show that compared with the use of various probes alone, the specific Tri-PLA-RCA reaction system constructed in the present invention (i.e., Group 6) has strong fluorescence and is feasible, and can be used for exosome detection.

[0048] Meanwhile, the same detections were also carried out on MCF-7 exosomes, MDA-MB-231 exosomes, and HEK293T exosomes, and the results were consistent, all indicating that the specific Tri-PLA-RCA reaction system constructed in the present invention is feasible.

[0049] 2. Tri-PLA-RCA is used for the analysis of sialic acid on exosomal glycoproteins

[0050] (1) Using 3D printing technology, draw the diagram of the required microfluidic chip, and then obtain it by 3D printing. Among them, the microfluidic chip contains 4 cylindrical reaction chambers arranged side by side, with a radius of 3 mm and a height of 5 mm, which can accommodate 140 μL of liquid. The 4 chambers are connected by channels with a height of 3 mm, allowing magnetic beads to flow through (see Figure 1 ).

[0051] (2) Before loading the system into the chip, pre-treat the probes first. Take 3 μL of 20 μM backbone probe, 3 μL of 20 μM first connector probe (Connector’1), and 3 μL of 20 μM second connector probe (Connector’2), and mix them with phosphate buffer containing 10 mM Mg 2+ . After annealing at 95 °C for 10 min and gradually cooling to room temperature, a probe system is obtained for standby.

[0052] Before running the chip, a part of the liquid in the magnetic bead kit (purchased from TransGen Biotech Co., Ltd., Beijing) needs to be added to the chip. Specifically, 20 μL of Tim4 magnetic beads and 10 μg of exosomes are added to Chamber I, 100 μL of magnetic bead washing solution is added to Chamber II, 2 μL of 20 μM protein aptamer probe, 2 μL of 20 μM Sia probe, 2 μL of 20 μM PBA probe, 2 μL of 2 μM PBA-PEG-N3 and the annealed and cooled probe system mentioned above are added to Chamber III, and it is supplemented to 80 μL with PBS buffer containing 1% FBS. 100 μL of magnetic bead eluent is added to Chamber 4 to recover exosomes. First, the chip is placed on a thermal shaker and shaken at 25 °C and 750 rpm for 1.5 h to obtain magnetic beads adsorbed with exosomes. A magnet is placed under Chamber I to move the magnetic beads from the aqueous phase to the oil phase. Subsequently, the magnet is moved to move the magnetic beads from Chamber I to Chamber II. Then the magnet is placed under Chamber II, and the magnetic beads move from the oil phase to the aqueous phase. The magnetic beads are cleaned in Chamber II and moved to Chamber III for reaction through the same steps. The reaction process is as follows: 1 μL of T4 DNA ligase, 3 μL of T4 DNA ligase buffer and 2 μL of 0.5% BSA are added to Chamber III and reacted at room temperature for 1.5 h. Then 2 μL of phi29 DNA polymerase, 4 μL of dNTP (10 mM), 5 μL of DNA polymerase buffer and 2 μL of 0.5% BSA are added to Chamber 3 and incubated at room temperature for 3 h. Finally, 2 μL of Reporter fluorescent reporter probe (50 mM) is added to Chamber III and the incubation continues for 1 h. At this time, the obtained magnetic beads are fluorescently labeled. The magnetic beads are moved to Chamber IV through the same operation, and 100 μL of eluent is obtained after shaking and incubating for 1 min and added to a 384-well plate, and the fluorescence intensity at 520 nm is detected with a microplate reader.

[0053] Table 2 Sequence information of nucleic acid probes required for the experiment

[0054]

[0055]

[0056] Note: The blue underlined sequences in the table are aptamers, and the sequences of other colors are probe pairing regions; all aptamers are sequences of protein-specific aptamers that have been partially modified to adapt to the amplification system.

[0057] The sialic acid of proteins in different exosomes (HepG2 exosomes, MCF-7 exosomes, MDA-MB-231 exosomes, HEK293T exosomes) was analyzed by the above method, and the results are as Figure 3As shown in the figure. The Tri-PLA-RCA strategy can distinguish the differences in sialic acid abundance on different exosomal glycoproteins. The surface proteins of exosomes secreted by HEK293T (normal cells) are also sialylated, with a lower sialic acid abundance compared to exosomes derived from tumor cells. The sialic acid abundance of EpCAM is generally higher on exosomes derived from tumors than on those from healthy sources, suggesting that the increase in sialic acid abundance on EpCAM may be related to the occurrence of epithelial tumors.

[0058] Using the same method, the sensitivity of Tri-PLA-RCA for sialic acid analysis on exosomal proteins was determined. MCF-7 exosomes and HEK293T exosomes with different particle concentrations were prepared, and the sialic acid content on the MUC1 protein of exosomes with different particle concentrations was analyzed using the above method, as well as the relationship between the fluorescence intensity of Tri-PLA-RCA and the exosomal particle concentration, to construct a standard curve. The results are as Figure 4 shown. The quantitative detection results of sialic acid on the surface of the MUC1 protein of MCF-7 exosomes showed that the LOD value was 2.81×10 6 Particles / mL. The quantitative detection results of sialic acid on the surface of the MUC1 protein of HEK293T exosomes showed that the LOD value was 1.26×10 6 Particles / mL.

[0059] The different glycosylation and sialylation differences of exosomal surface proteins are often used as indicators for judging the occurrence and progression of cancer. The universal Tri-PLA-RCA provided by the present invention can accurately identify and amplify the differences between glycosylation and sialylation among multiple targets, and establish a method for analyzing the degree of sialylation of exosomal surface glycoproteins, detect the sialic acid capping rate of specific glycoproteins of different exosomes and their parental cells, and explore the possible relationship between them and disease development, providing a detectable method for tumor screening and disease course judgment using sialic acid.

[0060] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A probe set for analyzing sialic acid of specific glycoprotein on the surface of exosomes, characterized in that: The probe set includes a first probe combination involved in a signal amplification strategy and a second probe combination that can bind to a specific glycoprotein on the surface of the same exosome; Among them, the first probe combination includes a backbone probe with a nucleotide sequence as shown in SEQ ID NO.9, a first ligation probe as shown in SEQ ID NO.10, a second ligation probe as shown in SEQ ID NO.11 and a fluorescent reporter probe as shown in SEQ ID NO.12; the second probe combination includes an aptamer probe that specifically recognizes a specific glycoprotein on the surface of exosomes, a sialic acid conversion probe and a sugar chain conversion probe that specifically recognize sialic acid on the specific glycoprotein.

2. The probe set according to claim 1, characterized in that: The aptamer probes that specifically recognize specific glycoproteins on the surface of exosomes include aptamer probes that specifically recognize PTK7, MUC1, EpCAM, HER2 and PD-L1, and the nucleotide sequences are shown in SEQ ID NO.1-5 respectively.

3. The probe set according to claim 1, characterized in that The nucleotide sequence of the sialic acid conversion probe is shown in SEQ ID NO.6, and the nucleotide sequence of the sugar chain conversion probe is shown in SEQ ID NO.

7.

4. The probe set according to claim 1, characterized in that: The 5' end of the sugar chain conversion probe was modified with DBCO.

5. Use of the probe group according to any one of claims 1 to 4 in preparing a product for analyzing sialic acid of specific glycoproteins on the surface of exosomes.

6. A method for analyzing sialic acid of specific glycoprotein on the surface of exosomes based on a 3D printed microfluidic chip combined with the probe set according to any one of claims 1 to 4, characterized in that: The following steps are involved: Pre-treating the backbone probe, the first ligation probe, and the second ligation probe to prepare a probe system; Magnetic beads and exosomes are added to chamber I of a microfluidic chip comprising four cylindrical reaction chambers, a magnetic bead washing solution is added to chamber II, an aptamer probe, a sialic acid conversion probe, a sugar chain conversion probe, PBA-PEG-N3 and a probe system are added to chamber III, and a magnetic bead elution solution is added to chamber IV; wherein, channels are provided between the four chambers for the magnetic beads and the reaction system to flow; The microfluidic chip is shaken to obtain magnetic beads adsorbing exosomes, and then a magnet is placed under chamber I to move the magnetic beads from the water phase to the oil phase, and then the magnet is moved to move the magnetic beads from chamber I to chamber II, and the magnet is placed under chamber II to move the magnetic beads from the oil phase to the water phase. In the same way, the magnetic beads are moved to chamber III for amplification reaction, and then moved to chamber IV for elution to obtain an eluate; The fluorescence intensity of the eluate is measured, and the content of sialic acid is analyzed according to the fluorescence intensity.

7. The method according to claim 6, characterized in that The pretreatment is as follows: 3 μL of 20 μM backbone probe, 3 μL of 20 μM first ligation probe and 3 μL of 20 μM second ligation probe are mixed with 10 mM Mg 2+ The mixture was mixed with phosphate buffer, annealed at 95° C. for 10 min, and cooled to room temperature to obtain the probe system.

8. The method according to claim 7, characterized in that 20 μL of Tim4 magnetic beads and 10 μg of exosomes were added to the chamber I; and / or 2 μL of 20 μM aptamer probe, 2 μL of 20 μM sialic acid conversion probe, 2 μL of 20 μM sugar chain conversion probe, 2 μL of 2 μM PBA-PEG-N3 and the probe system obtained by annealing and cooling were added to the chamber III, and then supplemented to 80 μL with phosphate buffer solution.

9. The method according to claim 6, characterized in that The shaking conditions are: 25° C., 500-1000 rpm, shaking for 1-3 h.

10. The method according to claim 6, characterized in that In the chamber III, the amplification reaction includes the following steps: 1 μL T4 DNA ligase, 3 μL T4 DNA ligase buffer and 2 μL 0.5% BSA are added to the chamber III and reacted at room temperature for 1-2 hours. Then 2 μL phi29 DNA polymerase, 4 μL dNTP, 5 μL DNA polymerase buffer and 2 μL 0.5% BSA are added to the chamber III and incubated at room temperature for 1-3 hours, and finally 2 μL fluorescent reporter probe is added to the chamber III and incubated for 1-2 hours.

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