A probe set and a method for analyzing sialic acid on the surface of exosomes by combining the probe set with a 3D printed microfluidic chip

Abstract

The application discloses a kind of probe set and its combination 3D printing microfluidic chip to the method for analyzing specific glycoprotein sialic acid on exosome surface, belong to biochemical analysis technical field.The present application is based on 3D printing microfluidic chip combined with three-probe co-localization signal amplification strategy, nucleic acid signal is converted into fluorescent signal, by directly reading fluorescent signal intensity, realize the analysis of sialic acid signal on the glycoprotein on the surface of exosome, which provides a new method for overcoming the defects and limitations of exosome clinical application in the prior art, reduces the loss of sample, improves the sensitivity of reaction, and provides a feasible method for exploring the possible relationship between different exosomes and their parent cell specific glycoprotein sialic acid and disease development, and using sialic acid for tumor screening and disease course determination.

Description

Technical Field

[0001] This invention relates to the field of biochemical analysis technology, and in particular to a probe set and a method for analyzing sialic acid, a specific glycoprotein on the surface of exosomes, by combining it with a 3D-printed microfluidic chip. Background Technology

[0002] Exosomes are lipid bilayer nanovesicles with a particle size of 30-150 nm, secreted by different types of cells and widely distributed in various body fluids. To date, exosomes have been shown 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. Secreted by the parent cell itself, exosomes have low immunogenicity. Their stable lipid bilayer structure protects the activity of their contents from degradation, and they also hold great potential in areas such as liquid biopsy and intracellular chemical drug or gene delivery. Exosomes carry various biomolecules derived from the parent cell, such as proteins, nucleic acids, and lipids. The structure, biological characteristics, and biological functions of exosomes depend to a great extent on these biomolecules. Effective bioanalysis of exosomes can enable tumor screening and predict cancer progression and prognosis.

[0003] Besides proteins and nucleic acids, the glycosides contained in exosomes also play an important role in biogeneration, cell recognition, and disease progression. Sialic acid, attached to the distal end of the exosome membrane surface glycan, has recently been shown to be highly correlated with malignant behaviors such as tumorigenesis and invasion. Cellular heterogeneity is often accompanied by abnormal sialic acid modification of proteins; therefore, sialic acid on the exosome surface can serve as a highly promising new disease biomarker with wide applications in disease diagnosis and prognosis.

[0004] Existing methods for analyzing sialic acid glycans on the surface of exosomes mainly focus on lectin arrays, liquid chromatography, and mass spectrometry. While these methods can effectively analyze sialic acid on the exosome surface, they often damage the physiological structure of the exosomes, which is detrimental to biological research. Therefore, converting glycan information into nucleic acid information will help maintain the integrity of exosomes during the detection process.

[0005] Furthermore, due to the unique bioformation process of exosomes, biological samples obtained from laboratories and clinics are complex in composition and have low exosome content. Therefore, the isolation of exosomes is the first step in effective exosome analysis. Traditional exosome isolation methods usually rely on the physicochemical properties of exosomes, such as buoyancy density, particle size, and hydrophilicity / hydrophobicity differences. In addition, in recent years, researchers have developed a series of strategies and new technical platforms for enrichment targeting specific exosome surface sites. However, due to the low concentration of exosomes in untreated biological samples, the detection of exosome biomarkers currently used in clinical practice is usually not very specific or sensitive, which greatly limits the clinical application of exosomes. Summary of the Invention

[0006] The purpose of this invention is to provide a probe set and a method for analyzing sialic acid of specific glycoproteins on the surface of exosomes using a 3D-printed microfluidic chip, in order to solve the problems existing in the prior art. By constructing a universal three-probe co-localization signal amplification strategy, the invention accurately identifies and amplifies the differences in glycosylation and sialylation among multiple targets, and establishes a method for analyzing the degree of sialylation of glycoproteins on the surface of exosomes. The invention detects the content of sialic acid of specific glycoproteins in different exosomes and their parent cells, analyzes the possible relationship between sialic acid and disease development, and provides a reference detection method for using sialic acid for tumor screening and disease progression assessment.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] The present invention provides a probe set for the analysis of sialic acid of specific glycoproteins on the surface of exosomes, the probe set comprising a first probe combination participating in a signal amplification strategy and a second probe combination capable of binding to the same specific glycoprotein on the surface of exosomes;

[0009] The first probe assembly includes a backbone probe with a nucleotide sequence as shown in SEQ ID NO. 9, a first linker probe as shown in SEQ ID NO. 10, a second linker probe as shown in SEQ ID NO. 11, and a fluorescent reporter probe as shown in SEQ ID NO. 12; the second probe assembly includes an aptamer probe that specifically recognizes a specific glycoprotein on the surface of an exosome, a sialic acid conversion probe that specifically recognizes sialic acid on the specific glycoprotein, and a glycan conversion probe.

[0010] Optionally, 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, with nucleotide sequences as shown in SEQ ID NO.1-5, respectively.

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

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

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

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

[0015] A probe system was prepared by pretreating the framework probe, the first connection probe, and the second connection probe.

[0016] In a microfluidic chip containing four cylindrical reaction chambers, magnetic beads and exosomes are added to chamber I, magnetic bead washing solution is added to chamber II, aptamer probes, sialic acid conversion probes, glycan conversion probes, PBA-PEG-N3 and probe system are added to chamber III, and magnetic bead elution solution is added to chamber IV. The four chambers are connected by channels for the flow of magnetic beads and reaction system.

[0017] The microfluidic chip is oscillated to obtain magnetic beads that adsorb exosomes. Then, a magnet is placed below chamber I to move the magnetic beads from the aqueous phase to the oil phase. The magnet is then moved to move the magnetic beads from chamber I to chamber II and placed below chamber II to move the magnetic beads from the oil phase to the aqueous phase. The same method is used to move the magnetic beads to chamber III for amplification reaction, and then to chamber IV for elution to obtain the eluent.

[0018] The fluorescence intensity of the eluent was measured, and the sialic acid content was analyzed based on the fluorescence intensity.

[0019] Optionally, the pretreatment is as follows: mixing 3 μL of a 20 μM framework probe, 3 μL of a 20 μM first ligation probe, and 3 μL of a 20 μM second ligation probe with a solution containing 10 mM Mg. 2+ The probe system was obtained by mixing the phosphate buffer solution, annealing at 95°C for 10 min, and cooling to room temperature.

[0020] Optionally, 20 μL Tim4 magnetic beads and 10 μg exosomes are added to 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 probe system obtained by annealing and cooling are added to chamber III, and then the volume is supplemented to 80 μL with phosphate buffer solution.

[0021] Optionally, the oscillation conditions are: 25℃, 500-1000rpm oscillation for 1-3 hours.

[0022] Optionally, in chamber III, the amplification reaction comprises the following steps: adding 1 μL T4 DNA ligase, 3 μL T4 DNA ligase buffer, and 2 μL 0.5% BSA to chamber III and reacting at room temperature for 1-2 hours. Then, adding 2 μL Phi29 DNA polymerase, 4 μL dNTPs, 5 μL DNA polymerase buffer, and 2 μL 0.5% BSA to chamber III and incubating at room temperature for 1-3 hours. Finally, adding 2 μL of a fluorescent reporter probe to chamber III and incubating for another 1-2 hours.

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

[0024] This invention develops a highly specific in situ amplification strategy based on three-probe adjacency linkage. By using a sensing element to identify three adjacent target sites, it induces rapid assembly and signal output between probes, thereby highly sensitively converting the sensed target information into amplified fluorescence signals. Ultimately, it obtains information related to the sialylation of glycoproteins on the surface of different cells and exosomes. This invention overcomes the deficiencies and limitations of existing technologies in the clinical application of exosomes, providing a feasible method for exploring the possible relationship between sialic acid, a specific glycoprotein from different exosomes and their parent cells, and disease development, as well as for using sialic acid for tumor screening and disease progression assessment.

[0025] This invention utilizes 3D printing technology to fabricate a microfluidic chip for this analytical measurement, integrating the entire process into the chip. The chip contains four compartments for exosome separation, washing, amplification, and elution. Magnetic beads control the movement of the sample between the four compartments, thereby reducing sample loss and improving reaction sensitivity. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is a schematic diagram of the microchip structure and the Tri-PLA-RCA in the microchip used for the analysis of sialic acid of specific glycoproteins on the surface of exosomes;

[0028] Figure 2 The results show the fluorescence intensity detection after the Tri-PLA-RCA reaction and after incubation with the Reporter probe; 1-6 in the figure and 1-6 in Table 1 are group numbers with the same meaning.

[0029] Figure 3 The results of sialic acid signal analysis for different exosomal proteins: (A) HepG2 exosome, (B) MCF-7 exosome, (C) MDA-MB-231 exosome, (D) HEK293T exosome;

[0030] Figure 4 The results of sialic acid analysis on exosomal glycoproteins using Tri-PLA-RCA are as follows: (A) Sialic acid analysis on MUC1 protein of MCF-7 exosomal protein with different particle concentrations; (B) Standard curve of Tri-PLA-RCA fluorescence intensity versus MCF-7 exosomal particle concentration; (C) Sialic acid analysis on MUC1 protein of HEK293T exosomal protein with different particle concentrations; (D) Standard curve of Tri-PLA-RCA fluorescence intensity versus HEK293T exosomal particle concentration. Detailed Implementation

[0031] Various exemplary embodiments of the present invention will now 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, features, and embodiments of the present invention.

[0032] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0034] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0035] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0036] Based on the problems and limitations of exosomes in clinical applications, and to ensure the integrity of exosome structure and achieve non-destructive detection of sialic acid, a glycoprotein on the exosome surface, for use in clinical sample analysis based on exosome sialic acid detection, this invention develops a three-probe colocalization signal amplification strategy (Tri-PLA-RCA) to analyze sialic acid signals on exosome surface glycoproteins. Glycoproteins contain abundant cis-diol sites, which can act as acceptors and undergo affinity reactions with phenylboronic acid (PBA) groups, forming cyclic phenylborates in a slightly alkaline aqueous medium. This invention utilizes a PBA-PEG-N3 compound as an intermediate. The PBA group in the compound covalently binds to the cis-diol sites on the glycoprotein, and the N3 in the compound undergoes a click reaction with a DNA probe modified with DBCO at its 5' end, linking the DNA probe to the cis-diol sites on the oligosaccharide chain, constructing a glycan conversion probe to convert glycan information into nucleic acid information. The co-localization mechanism of specific borate affinity oligosaccharide conversion probes, sialic acid conversion probes, and protein aptamer probes improves the accuracy and specificity of the reaction. The fact that the three probes act on the same target protein leads to their DNA probes being spatially close, inducing an adjacent linkage (RCA) reaction. The amplified long DNA then binds complementary to the Reporter fluorescent signal, realizing the conversion of nucleic acid information into a fluorescent signal, which can be directly read by a microplate reader.

[0037] Simultaneously, this invention utilizes 3D printing technology to fabricate a microfluidic chip for this analytical measurement, integrating the entire process into the microfluidic chip. This chip comprises four chambers (named sequentially from one end as Chamber I, Chamber II, Chamber III, and Chamber IV, with identical liquid channels on the walls of adjacent chambers for liquid flow; these channels can be located in the middle or near the lower end of the wall, and the channel aperture size is not strictly limited, as long as it allows for normal and smooth liquid flow, and can be configured according to requirements), used for exosome separation, washing, amplification reaction, and exosome elution, respectively. Magnetic beads are used to control the movement of the sample between the four chambers, thereby reducing sample loss and reaction sensitivity.

[0038] The technical solution of the above-mentioned mechanism of action of the present invention will be described in detail below with specific embodiments.

[0039] Example 1

[0040] First, magnetic bead enrichment was used to separate and purify exosomes and elute excess probes. Then, backbone probes, connector probes (Connector 1 & Connector 2), and a reporter probe modified with a fluorescent group were designed for the Tri-PLA-RCA amplification reaction (Table 2). Exosomes are rich in glycoproteins, which contain not only sialylation sites but also oligosaccharide chains with numerous exposed cis-diol sites. Cis-diols can specifically bind to phenylboronic acid (PBA). Therefore, aptamer probes that specifically recognize exosome surface proteins, sialic acid conversion probes (Siaprobes) that specifically recognize sialic acid, and glycan conversion probes (PBAprobes) were designed (Table 2). The three functional probes specifically bind to the same exosome glycoprotein, shortening the spatial distance between probes. Complementary to the backbone and connector probes, T4 DNA ligase was introduced, and adjacent ligation and hybridization formed a DNA quaternary structure that can serve as a template for RCA amplification. Following this, phi29 DNA polymerase is added to catalyze the RCA amplification reaction, amplifying the original template into long single-stranded DNA with hundreds or thousands of repeating sequences. After terminating the reaction, a Reporter signal probe is added to hybridize with the long single-stranded DNA, converting the nucleic acid amplification signal. The fluorescence intensity is measured at 520 nm using a microplate reader. The specific experimental procedures are as follows:

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

[0042] The feasibility of the Tri-PLA-RCA reaction was determined by mixing the sample with the conditions shown in Table 1.

[0043] Table 1

[0044]

[0045]

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

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

[0048] Meanwhile, the same tests were also performed 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 this invention is feasible.

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

[0050] (1) Using 3D printing technology, the required microfluidic chip is drawn and then 3D printed. The microfluidic chip contains four cylindrical reaction chambers arranged side-by-side, each with a radius of 3 mm and a height of 5 mm, capable of holding 140 μL of liquid. The four chambers are connected by a 3 mm high channel, allowing for the flow of magnetic beads (see...). Figure 1 ).

[0051] (2) Before loading the system into the chip, the probes are pretreated by taking 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 containing 10 mg of Mg. 2+ After mixing with phosphate buffer, the mixture was annealed at 95°C for 10 min and then gradually cooled to room temperature to obtain the probe system for later use.

[0052] Before running the chip, a portion of the liquid from the magnetic bead kit (purchased from Beijing TransGen Biotech Co., Ltd.) 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 buffer 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 are added to chamber III, and the volume is supplemented to 80 μL with PBS buffer containing 1% FBS; 100 μL of magnetic bead elution buffer is added to chamber 4 to recover the exosomes. First, the chip is placed on a heated shaker and shaken at 25°C and 750 rpm for 1.5 h to obtain magnetic beads that adsorb 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. A magnet was then placed below chamber II, causing the magnetic beads to move from the oil phase to the aqueous phase. The beads were cleaned in chamber II and transferred to chamber III using the same procedure. The reaction process involved adding 1 μL L4 DNA ligase, 3 μL L4 DNA ligase buffer, and 2 μL 0.5% BSA to chamber III and incubating at room temperature for 1.5 h. Then, 2 μL phi29 DNA polymerase, 4 μL dNTP (10 mM), 5 μL DNA polymerase buffer, and 2 μL 0.5% BSA were added to chamber III and incubated at room temperature for 3 h. Finally, 2 μL Reporter fluorescent probe (50 mM) was added to chamber III and incubated for 1 h, resulting in fluorescently labeled magnetic beads. The beads were then transferred to chamber IV using the same procedure, incubated with shaking for 1 min, and 100 μL of eluent was added to a 384-well plate. The fluorescence intensity at 520 nm was measured using 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 other colored sequences are probe pairing regions; all aptamers are protein-specific aptamers that have been partially modified to adapt to the amplification system.

[0057] The sialic acid content of proteins from different exosomes (HepG2 exosomes, MCF-7 exosomes, MDA-MB-231 exosomes, and HEK293T exosomes) was analyzed using the above method, and the results are as follows: Figure 3As shown, the Tri-PLA-RCA strategy can distinguish differences in sialic acid abundance on different exosomal glycoproteins. Exosomal surface proteins secreted by HEK293T (normal cells) also exhibit sialylation, but have lower sialic acid abundance compared to tumor cell-derived exosomals. The sialic acid abundance of EpCAM is generally higher on tumor-derived exosomals than on healthy-derived exosomals, suggesting that increased sialic acid abundance on EpCAM may be associated with epithelial tumorigenesis.

[0058] Using the same method, the sensitivity of Tri-PLA-RCA for sialic acid analysis on exosomal proteins was determined. MCF-7 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. The relationship between Tri-PLA-RCA fluorescence intensity and exosome particle concentration was also analyzed, and a standard curve was constructed. Results are as follows: Figure 4 As shown, the quantitative detection results of sialic acid on the surface of MCF-7 exosome MUC1 protein showed a LOD value of 2.81 × 10⁻⁶. 6 Particles / mL. Quantitative detection of sialic acid on the surface of HEK293T exosome MUC1 protein showed a LOD value of 1.26 × 10⁻⁶. 6 Particles / mL.

[0059] Differences in glycosylation and sialylation of exosome surface proteins are often used as indicators for judging the occurrence and progression of cancer. The universal Tri-PLA-RCA provided by this invention can accurately identify and amplify the differences in glycosylation and sialylation among multiple targets, and establish a method for analyzing the degree of sialylation of exosome surface glycoproteins. It can detect the sialic acid capping rate of specific glycoproteins in different exosomes and their parent cells, explore the possible relationship between them and disease development, and provide a reference detection method for using sialic acid for tumor screening and disease progression assessment.

[0060] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A non-diagnostic method for analyzing sialic acid, a specific glycoprotein on the surface of exosomes, based on a 3D-printed microfluidic chip combined with a probe array, characterized in that... The probe set includes a first probe combination that participates in the signal amplification strategy and a second probe combination that can bind to a specific glycoprotein on the surface of the same exosome. The first probe assembly includes a backbone probe with a nucleotide sequence as shown in SEQ ID NO. 9, a first linker probe as shown in SEQ ID NO. 10, a second linker probe as shown in SEQ ID NO. 11, and a fluorescent reporter probe as shown in SEQ ID NO. 12; the second probe assembly includes an aptamer probe that specifically recognizes a specific glycoprotein on the surface of an exosome, a sialic acid conversion probe that specifically recognizes sialic acid on the specific glycoprotein, and a glycan conversion probe. 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, with nucleotide sequences as shown in SEQ ID NO.1-5, respectively. The nucleotide sequence of the sialic acid conversion probe is shown in SEQ ID NO.6, and the nucleotide sequence of the glycan conversion probe is shown in SEQ ID NO.7; The 5' end of the glycan inversion probe is modified with DBCO; The method includes the following steps: A probe system was prepared by pretreating the framework probe, the first connection probe, and the second connection probe. Magnetic beads and exosomes are added to chamber I of a microfluidic chip containing four cylindrical reaction chambers; magnetic bead washing solution is added to chamber II; aptamer probes, sialic acid conversion probes, glycan conversion probes, PBA-PEG-N3, and the probe system are added to chamber III; and magnetic bead elution solution is added to chamber IV. The four chambers are connected by channels for the flow of magnetic beads and the reaction system. The microfluidic chip is oscillated to obtain magnetic beads that adsorb exosomes. Then, a magnet is placed below chamber I to move the magnetic beads from the aqueous phase to the oil phase. The magnet is then moved to move the magnetic beads from chamber I to chamber II and placed below chamber II to move the magnetic beads from the oil phase to the aqueous phase. The same method is used to move the magnetic beads to chamber III for amplification reaction, and then to chamber IV for elution to obtain the eluent. The fluorescence intensity of the eluent was measured, and the sialic acid content was analyzed based on the fluorescence intensity.

2. The method as described in claim 1, characterized in that, The pretreatment consisted of mixing 3 μL of a 20 μM framework probe, 3 μL of a 20 μM first ligation probe, and 3 μL of a 20 μM second ligation probe with a solution containing 10 mM Mg. 2＋ The probe system was obtained by mixing the phosphate buffer solution, annealing at 95 °C for 10 min, and cooling to room temperature.

3. The method as described in claim 2, characterized in that, 20 μL of Tim4 magnetic beads and 10 μg of exosomes are added to 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 probe system are added to chamber III, and then the volume is increased to 80 μL with phosphate buffer solution.

4. The method as described in claim 1, characterized in that, The oscillation conditions are: 25℃, 500-1000 rpm for 1-3 hours.

5. The method as described in claim 1, characterized in that, In chamber III, the amplification reaction includes the following steps: 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 1-2 h. Then, 2 μL of phi29 DNA polymerase, 4 μL of dNTP, 5 μL of DNA polymerase buffer, and 2 μL of 0.5% BSA are added to chamber III and incubated at room temperature for 1-3 h. Finally, 2 μL of fluorescent reporter probe is added to chamber III and incubated for 1-2 h.

6. A probe set for the analysis of sialic acid of specific glycoproteins on the surface of exosomes, characterized in that, The probe set includes a first probe combination that participates in the signal amplification strategy and a second probe combination that can bind to a specific glycoprotein on the surface of the same exosome. The first probe assembly includes a backbone probe with a nucleotide sequence as shown in SEQ ID NO. 9, a first linker probe as shown in SEQ ID NO. 10, a second linker probe as shown in SEQ ID NO. 11, and a fluorescent reporter probe as shown in SEQ ID NO. 12; the second probe assembly includes an aptamer probe that specifically recognizes a specific glycoprotein on the surface of an exosome, a sialic acid conversion probe that specifically recognizes sialic acid on the specific glycoprotein, and a glycan conversion probe. 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, with nucleotide sequences as shown in SEQ ID NO.1-5, respectively. The nucleotide sequence of the sialic acid conversion probe is shown in SEQ ID NO.6, and the nucleotide sequence of the glycan conversion probe is shown in SEQ ID NO.7; The 5' end of the glycan inversion probe is modified with DBCO.

7. The use of the probe set as described in claim 6 in the preparation of products for the analysis of sialic acid of specific glycoproteins on the surface of exosomes.

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