An integrated sensor for extracellular vesicle isolation and membrane protein detection thereof

Abstract

The application discloses an integrated sensor for extracellular vesicle separation and membrane protein detection, comprising a membrane probe and magnetic beads, one end of the membrane probe is used for inserting into the membrane of extracellular vesicles, the other end is combined with the magnetic beads, so that the extracellular vesicles are captured to the surface of the magnetic beads and separated out through magnetism, and the integrated sensor further comprises an antibody-nucleic acid probe, the antibody-nucleic acid probe can specifically recognize and combine the target protein on the separated extracellular vesicles through antigen-antibody interaction, the nucleic acid is amplified through a primer exchange reaction, and then is detected through a Cas12a system, and finally the quantitative detection of the target protein is realized through the fluorescence signal of a detection reaction solution. The sensor of the application can quickly and efficiently capture the extracellular vesicles on the surface of the magnetic beads by using the membrane probe and the magnetic beads, and realizes the detection of the surface protein of the extracellular vesicles by combining the antibody nucleic acid probe and the two-step nucleic acid amplification reaction.

Description

Technical Field

[0001] This invention relates to the field of protein detection, and more particularly to an integrated sensor for the separation of extracellular vesicles and the detection of their membrane proteins. Background Technology

[0002] Extracellular vesicles (EVs) are vesicle-like structures secreted by cells outside the cell. Enclosed by a phospholipid bilayer membrane, they range in size from 50 to 2000 nm, carry various biomolecules inherited from their parent cells (including nucleic acids, proteins, lipids, and metabolites), and play an important role in intercellular communication. [1] Studies have shown that extracellular vesicles are involved not only in stem cell maintenance but also in other aspects of life. [2] Tissue repair [3] Immune surveillance [4] Blood clotting [5] Extracellular vesicles are essential for maintaining normal physiological activities and are widely involved in the pathological processes of various diseases, including cancer, neurodegenerative diseases, cardiovascular diseases, and autoimmune diseases. Almost all mammalian cell types (including neurons, endothelial cells, mesenchymal stem cells, and epithelial cells) can secrete extracellular vesicles. Therefore, extracellular vesicles are widely present in bodily fluids such as blood, urine, bile, breast milk, synovial fluid, tears, semen, and saliva. [6] .

[0003] Due to the important role of extracellular vesicles in disease development, they are emerging as a new target in liquid biopsy. [7] Compared to other targets in liquid biopsy (circulating tumor cells, free tumor marker nucleic acids and proteins, etc.), the membrane-coated structure of extracellular vesicles is more stable than that of free nucleic acids and proteins, protecting their carried substances from degradation. Secondly, extracellular vesicles are more concentrated than circulating tumor cells, making them easier to detect, and their distribution in various bodily fluids makes them more suitable for minimally invasive or even non-invasive diagnostic needs. Currently, research on disease diagnosis based on extracellular vesicles is booming, with numerous studies using tumor-specific proteins on the surface of extracellular vesicles as biomarkers for early cancer diagnosis. However, the isolation of extracellular vesicles and the detection of proteins face significant challenges due to the small size and complex composition of clinical samples.

[0004] Among existing methods for separating extracellular vesicles, ultracentrifugation is considered the gold standard, but it is time-consuming, labor-intensive, and requires a large number of samples. [8] Other methods, such as ultrafiltration, polymer-based precipitation, and immunoaffinity capture, typically suffer from low extracellular vesicle yield and / or poor purity and integrity of extracellular vesicles. [9]For these reasons, they are not practical in clinical applications when only trace amounts of biological samples are available. Similarly, traditional methods for analyzing extracellular vesicle proteins, such as Western blotting and enzyme-linked immunosorbent assays (ELISA), are also impractical.

[10] Their low sensitivity and the need for prior separation of extracellular vesicles limit their application in clinical diagnosis.

[0005] To enable the analysis of extracellular vesicle proteins in trace samples, researchers have developed various platforms (such as fluorescence)

[11] Surface plasmon resonance

[12] Electrochemistry

[13] and colorimetric method

[14] A biosensor based on nucleic acid aptamers. Yu et al. reported a fluorescence competition method based on nucleic acid aptamers to detect extracellular vesicles in serum.

[15] They pre-hybridized magnetic beads modified with CD63 protein aptamers with Cy3-labeled nucleic acid probes. In the presence of extracellular vesicles, the CD63 protein on the surface of the extracellular vesicles specifically binds to the CD63 protein aptamers, causing the Cy3-labeled nucleic acid probes to be released into the supernatant. After magnetic separation, the extracellular vesicles were quantified by detecting the fluorescence intensity of the free Cy3 in the supernatant. This method can detect vesicles as low as 10⁻⁶. 5 Extracellular vesicles (EVs) can be detected at concentrations of 100 vesicles per μl. However, methods that simply use fluorescence to directly or indirectly reflect the concentration of EVs have limited sensitivity. To further improve the sensitivity of EV detection, researchers have combined fluorescence detection with signal amplification techniques, achieving higher sensitivity. For example, Wang et al. used a competitive nucleic acid aptamer to bind to the signal amplification effect of deoxyribonuclease I, achieving a detection limit as low as 1.9 × 10² EVs per μl.

[16] Huang and others

[17] After specifically capturing extracellular vesicles using antibody magnetic beads, a detection limit as low as 1.0 × 10⁻⁶ was achieved using rolling circle amplification and cyclic enzymatic digestion with nucleating endonucleases. 2 The sample size is 1 / μl, making it ideal for detecting trace clinical samples.

[0006] The detection approach based on electrochemistry is similar to that of fluorescence detection, except that the readout platform is changed from fluorescence to a sensitive electrochemical detection platform, aiming to achieve highly sensitive detection of extracellular vesicles. For example, Zhou et al. developed an electrochemical sensor using a nucleic acid aptamer of the CD63 protein on the surface of exosomes.

[18] The method involves immobilizing nucleic acid aptamers on the surface of a gold electrode and hybridizing a probe chain pre-labeled with redox molecules with the aptamer molecules, then immobilizing it on the electrode surface. When extracellular vesicles are present, the aptamer binds to proteins on the surface of the extracellular vesicles and releases the probe chain carrying redox molecules, leading to a decrease in the electrochemical signal. By observing the change in the electrochemical signal, highly sensitive quantitative analysis of extracellular vesicles can be achieved; this method can detect vesicles as low as 1.0 × 10⁻⁶. 3 Extracellular vesicles per μl. Similarly, leveraging the efficient replication and amplification capabilities of nucleic acid aptamers, electrochemical platforms can be combined with signal amplification strategies to achieve more sensitive detection. For example, An et al. established an ultra-sensitive detection platform by combining electrochemical analysis with hybridization chain reaction (HCR).

[19] The principle is as follows: an electrode modified with a CD63 protein aptamer specifically captures extracellular vesicles, which are then labeled with alkynyl-4-ONE. A copper (I)-catalyzed click chemistry reaction couples an azide-modified DNA probe to the surface of the extracellular vesicles. Upon addition of biotin-labeled single-stranded DNA (H1 and H2), the DNA anchored to the surface of the extracellular vesicles triggers an HCR reaction, forming a self-assembled DNA tandem that can be used for signal amplification. Subsequently, through the specific interaction between biotin and streptavidin, a large amount of streptavidin-labeled peroxidase is bound, catalyzing the oxidation of o-phenylenediamine. The extracellular vesicles are quantitatively detected by monitoring the subsequent reduction current signal, achieving detection of extracellular vesicles at a minimum concentration as low as 96 vesicles / μl.

[0007] Colorimetric detection of extracellular vesicles allows for direct visual observation without the need for specialized instruments, making it a simple, rapid, and highly portable method suitable for use outside of laboratories. For example, Xia et al.

[20] A method for visually detecting extracellular vesicles was developed using single-walled carbon nanotubes (s-SWCNTs) with good water solubility combined with nucleic acid aptamers. It is known that nucleic acid aptamers for CD63 protein are adsorbed onto the surface of s-SWCNTs (s-SWCNTs) via a non-covalent reaction, enhancing the peroxidase activity of s-SWCNTs and effectively catalyzing the H₂O₂-mediated oxidation of TMB, causing the solution to change from colorless to blue. Therefore, in the presence of extracellular vesicles, based on affinity, the nucleic acid aptamers detach from the s-SWCNT surface and bind to the CD63 protein on the surface of the extracellular vesicles, leading to a decrease in peroxidase activity and a change in solution color. Thus, the quantification of extracellular vesicles can be achieved by monitoring the color change of the solution with visual observation or UV-Vis spectroscopy, thereby constructing an intuitive and simple method for detecting extracellular vesicles with a detection limit of 5.2 × 10⁻⁶. 5 / μl. Subsequently, Wang et al.

[21] Studies have found that ssDNA adsorption onto the surface of graphitic carbon nitride nanosheets (g-C3N4NSs) can enhance the intrinsic peroxidase-like activity of g-C3N4NSs, catalyzing H2O2-mediated TMB oxidation, with a maximum reaction rate at least 4 times faster than g-C3N4NSs without ssDNA adsorption. Using a similar design, the nucleic acid aptamer of the CD63 protein was used to bind to g-C3N4NSs, achieving visualized detection of extracellular vesicles. While the sensitivity of visualized extracellular vesicle detection is not as high as other methods, it is more portable and suitable for off-laboratory detection scenarios, such as in areas with limited medical resources.

[0008] Surface-enhanced Raman scattering (SERS) possesses high specificity and sensitivity, and many researchers have already designed and synthesized SERS nanoprobes for highly sensitive analysis of extracellular vesicles. Kwizera et al.

[22] This technique, combining gold nanorod labeling with a microcapture platform, was applied to the detection and analysis of extracellular vesicles. An antibody array was prepared on a gold-coated standard glass microscope slide, and targeted specific antibodies were used to capture extracellular vesicles on the gold-coated slide. Subsequently, utilizing the electrostatic interaction between the extracellular vesicles and the gold nanorods (extracellular vesicles carrying a negative charge (zeta potential around -10 mV) and the gold nanorods carrying a positive charge (zeta potential around +35 mV), the gold nanorods were adsorbed onto the extracellular vesicles. Changes in the SERS signal were recorded using a portable Raman spectrometer, achieving a resolution of 2.0 × 10⁻⁶. 3The detection of extracellular vesicles per μl. Sensors based on surface plasmon resonance (SPR) operate on a similar principle; for example, Wang et al. constructed an SPR biosensor based on a two-dimensional metallic framework for the detection of extracellular vesicles.

[23] A two-dimensional metallic framework material (Cu-TCPP) was synthesized using a simple hydrothermal method. This material exhibits excellent conductivity, charge mobility, and efficient photogenerated carriers, which can enhance the excitation electric field and surface plasmon resonance interface of the sensor. Secondly, as an excellent optoelectronic material, Cu-TCPP can enhance the absorption of incident light, thereby enhancing signal output. Thirdly, Cu-TCPP has a layered structure with orderly arrangement, and its own SPW is coupled with the SPR of the gold film. Finally, as a two-dimensional nanosheet material, Cu-TCPP has a larger specific surface area, providing more active sites to bind analytes. The multi-element carbon ring structure of the porphyrin ligand can better bind to carbon-based biomolecules, thereby capturing more signal molecules. The researchers coupled a peptide segment on the material surface that can specifically interact with extracellular vesicles containing PD-L1, achieving highly sensitive detection with a detection limit of 16.7 vesicles / ml. This method has good scalability and broad prospects, providing possibilities for the clinical detection of trace target biomolecules.

[0009] While these sensors can achieve sensitive detection of extracellular vesicles to a certain extent, they all have certain limitations in use. For example, colorimetric methods, although simple to operate, have low sensitivity and are not suitable for sensitive detection of trace samples; while electrochemical and surface plasmon resonance methods have high sensitivity, they are complex to operate, have high instrument requirements, and do not meet the requirements of routine analytical tests. In addition, these sensors all use antigen-specific capture methods to separate extracellular vesicles, resulting in low separation efficiency and a tendency to miss information from extracellular vesicles that do not express the antigen, leading to incomplete analysis. Therefore, those skilled in the art are dedicated to developing a sensor that can efficiently separate extracellular vesicles and sensitively detect their proteins.

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[0033] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is how to quickly and efficiently separate extracellular vesicles and accurately detect their surface proteins.

[0034] To achieve the above objectives, the present invention provides an integrated sensor for the separation of extracellular vesicles and the detection of their membrane proteins, characterized in that it includes a membrane probe and a magnetic bead, wherein one end of the membrane probe is used to insert into the membrane of the extracellular vesicle, and the other end can bind to the magnetic bead, thereby capturing the extracellular vesicles to the surface of the magnetic bead and separating them by magnetism.

[0035] In a preferred embodiment of the present invention, the membrane probe is a molecule consisting of a hydrophobic lipid at one end and a biotin at the other end, connected by polyethylene glycol. The hydrophobic lipid end is used to insert into the membrane of extracellular vesicles, while the biotin at the other end is bound to magnetic beads.

[0036] In another preferred embodiment of the present invention, the magnetic beads are streptavidin magnetic beads.

[0037] In another preferred embodiment of the invention, the sensor further includes an antibody-nucleic acid probe, which can specifically recognize and bind to target proteins on isolated extracellular vesicles through antigen-antibody interaction.

[0038] In another preferred embodiment of the present invention, the nucleic acid in the antibody-nucleic acid probe is an oligonucleotide, which can be amplified into a long single strand with long repeating units through a primer exchange reaction, and the reaction system of the primer exchange reaction does not contain dGTP.

[0039] In another preferred embodiment of the present invention, the antibody of the antibody-nucleic acid probe is a CD81 antibody, and the nucleic acid is an oligonucleotide with 5'-SH modification, the sequence of which is as follows:

[0040] 5'-SH C6-TTTTTTTTTTACCCTCTCAA-3'.

[0041] In another preferred embodiment of the present invention, a dGTP scavenging sequence, namely the Clean.G sequence, and a hairpin template are added to the primer exchange reaction system, the sequences of which are as follows:

[0042] SED ID NO.1Clean.G sequence:

[0043] 5'-CCCCGAAAGTGGCCTCGGGCCTTTGGCCCGAGGCCACTTTCG-3';

[0044] SED ID NO.2 card template sequence:

[0045] 5'-ACATCATCATGGGCCTTTGGCCCATGATGATGTATGATGATG-Inverted dT-3'.

[0046] In another preferred embodiment of the present invention, the sensor further includes a Cas12a system for detecting amplified long single-stranded DNA with long repeat units. The Cas12a system includes a Cas12a enzyme, a Cas12a system guide RNA (crRNA), and a reporter molecule. One end of the crRNA binds to the Cas12a enzyme, and the other end is completely complementary to the repeat unit on the long single-stranded DNA, i.e., the target nucleic acid sequence. The reporter molecule is a fluorescent molecule and a quencher molecule with base linkages of short-chain deoxyribonucleic acid. The Cas12a enzyme can recognize the target nucleic acid sequence under the action of CrRNA, thereby activating its trans-cleavage property and non-specifically cleaving the surrounding single-stranded DNA, causing the short-chain nucleic acid of the reporter molecule to be cleaved and release fluorescence. The sensor ultimately achieves quantitative detection of the target protein by detecting the fluorescence signal of the reaction solution.

[0047] In another preferred embodiment of the present invention, the sequences of the crRNA and reporter molecule in the Cas12a system are as follows:

[0048] SED ID NO.3crRNA:

[0049] 5'-UAAUUUCUAACUAAGGUAGUAGAGGGUUUGAGAGGGUUUG-3';

[0050] SED ID NO.4 Reporting Molecule:

[0051] 5'-HEX-AAAAAAAAAAAA-BHQ1-3'.

[0052] In another preferred embodiment of the present invention, the target nucleic acid sequence is two repeating units of a long single strand with long repeating units after amplification.

[0053] Technical effect

[0054] The sensor of this invention uses membrane probes and magnetic beads to rapidly and efficiently capture extracellular vesicles on the surface of magnetic beads, and combines antibody nucleic acid probes and a two-step nucleic acid amplification reaction to achieve detection of extracellular vesicle surface proteins.

[0055] 1. One end of the membrane probe can be inserted into an extracellular vesicle, and the other end can be bound to a magnetic bead to fix the extracellular vesicle to the surface of the magnetic bead in a membrane-specific manner.

[0056] 2. Antibody nucleic acid probes recognize proteins on the surface of extracellular vesicles, converting protein detection into nucleic acid detection;

[0057] 3. A two-step amplification method (primer exchange reaction and Cas12a enzyme cleavage reaction) is used to amplify nucleic acids and quantify them using fluorescence.

[0058] The sensor of this invention can achieve a separation efficiency of 51.45% within 1 hour; taking CD81 protein detection as an example, it achieves a sensitivity as low as 10 extracellular vesicles per microliter.

[0059] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0060] Figure 1 This is a schematic diagram of the principle of an integrated sensor for extracellular vesicle separation and membrane protein detection according to a preferred embodiment of the present invention.

[0061] Figure 2 This is a schematic diagram illustrating the principle of primer exchange reaction (PER) according to a preferred embodiment of the present invention;

[0062] Figure 3 This is a scanning electron microscope image of a preferred embodiment of the present invention before and after streptomycin affinity magnetic beads capture extracellular vesicles;

[0063] Figure 4 This is a graph showing the results of Western blotting evaluation of the separation efficiency of extracellular vesicles according to a preferred embodiment of the present invention.

[0064] Figure 5 This is a verification diagram of the antibody oligonucleotide probe coupling results of a preferred embodiment of the present invention;

[0065] Figure 6 This is a diagram showing the detection limit of an integrated sensor for extracellular vesicle separation and membrane protein detection according to a preferred embodiment of the present invention.

[0066] Figure 7This is a schematic diagram illustrating the principle and results of the application of an integrated sensor for extracellular vesicle separation and membrane protein detection in cell lines according to a preferred embodiment of the present invention. Detailed Implementation

[0067] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0068] In the accompanying drawings, components with the same structure are indicated by the same numerical designation, and components with similar structures or functions are indicated by similar numerical designations. The dimensions and thicknesses of each component shown in the drawings are arbitrary, and the present invention does not limit the dimensions and thicknesses of each component. To make the illustrations clearer, the thickness of some components has been appropriately exaggerated in the drawings.

[0069] like Figure 1 As shown, this invention provides an integrated sensor for the separation and detection of extracellular vesicles and their membrane proteins, comprising a membrane probe and magnetic beads. One end of the membrane probe is inserted into the membrane of the extracellular vesicle, and the other end can bind to the magnetic beads, thereby capturing the extracellular vesicles onto the surface of the magnetic beads and separating them magnetically. Preferably, the sensor first uses a membrane probe (DSPE-PEG2K-biotin) and streptavidin-coated magnetic beads (SA-MBs) to separate and capture extracellular vesicles (EVs). The membrane probe is a molecule composed of polyethylene glycol (PEG2K) linked to a hydrophobic lipid (DSPE) at one end and biotin at the other end. Its lipid end can insert into the membrane of the extracellular vesicle, and its biotin end can bind to the bead, thereby capturing the extracellular vesicles onto the surface of the magnetic beads, and finally separating them magnetically.

[0070] The sensor also includes an antibody-nucleic acid probe, which specifically recognizes and binds to target proteins on isolated extracellular vesicles through antigen-antibody interaction. The nucleic acid in the antibody-nucleic acid probe is preferably an oligonucleotide, which can be amplified into a long single strand with long repeating units through a primer exchange reaction. Specifically, the antibody-nucleic acid probe is incubated with extracellular vesicles (EVs) captured by magnetic beads. Through antigen-antibody interaction, the probe can specifically recognize and bind to the target protein, thereby converting protein detection into nucleic acid detection. The antibody-oligonucleotide probe is amplified using a primer exchange reaction (PER), such as... Figure 2As shown, the principle of this primer exchange reaction is as follows: a primer with the sequence domain α (in this invention, the oligonucleotide on the antibody-oligonucleotide) binds to the α* on its hairpin (step 1) and begins to extend with the help of chain displacement polymerase (step 2). After extending another α domain, the polymerase stops at the GC pair because there is no dGTP in the reaction. Then, the α domain on the hairpin competes with the synthesized α domain on the primer strand through branch migration, restoring the original hairpin structure (step 3). Once the synthesized α domain is replaced, the extended primer can spontaneously dissociate from the hairpin (step 4) and start the next cycle. By repeating this cycle multiple times, under the action of the enzyme and the hairpin template, the oligonucleotide can be amplified into a long single strand with long repeating units.

[0071] The sensor also includes a Cas12a system for detecting amplified long single-stranded DNA with long repeating units. The detection principle is as follows: the Cas12a system comprises three parts: the Cas12a enzyme, the Cas12a guide RNA (crRNA), and a reporter molecule. One end of the guide RNA can bind to the Cas12a enzyme, and the other end is completely complementary to the target nucleic acid sequence, thus guiding the Cas12a enzyme to specifically bind to the target sequence. The reporter molecule is a fluorescent molecule and a quencher molecule linked together by 10-12 deoxyribonucleic acid bases. Normally, when the fluorescent molecule and the quencher molecule are linked by short-stranded nucleic acids, the fluorescence is quenched; however, when the Cas12a enzyme recognizes the target nucleic acid under the action of the guide RNA, Cas12a is activated by its trans-cleavage property and begins non-specific cleavage of the surrounding single-stranded DNA, thus cleaving the short-stranded nucleic acid of the reporter molecule and releasing fluorescence. Preferably, the two repeating units of the long-stranded nucleic acid amplified in the previous step are used as the target sequence, and the Cas12a system can initiate the enzymatic cleavage reaction to perform the second step of signal amplification and quantification. The fluorescence intensity of the final solution is positively correlated with the concentration of the target sequence, so the target protein can be quantitatively detected by detecting the fluorescence of the solution.

[0072] Example 1: Separation of extracellular vesicles using membrane probes and streptavidin magnetic beads, and evaluation of the process and separation efficiency:

[0073] The membrane probe was dissolved in anhydrous ethanol to a concentration of 5 mM as a storage solution and stored at -20°C.

[0074] The membrane probe was diluted to 2 μM using dilution buffer C and incubated with a sample containing extracellular vesicles at 4 °C for 30 min. Then, 20 μl of 50 mg / ml streptavidin magnetic beads was added and incubated for another 30 min at room temperature.

[0075] After incubation, the magnetic beads were washed three times with phosphate-buffered saline (PBS) and finally resuspended in PBS.

[0076] The streptavidin beads before the reaction and after capturing extracellular vesicles were characterized using scanning electron microscopy (SEM). Figure 3 As shown, the results indicate that extracellular vesicles (white dashed box) with a size of 30-200 nm can be completely captured on the surface of magnetic beads.

[0077] 6×10 10 Each extracellular vesicle was divided into three portions. One portion was used directly as a standard, and the other two portions were mixed with PBS buffer containing 10% fetal bovine serum from which extracellular vesicles had been removed to serve as simulants. Extracellular vesicles in these simulants were separated using the above method and ultracentrifugation, respectively. The three portions of extracellular vesicles were then lysed and concentrated to 40 μl. 10 μl of the concentrate was used for Western blotting experiments to detect the intensity of CD63 and CD81 protein bands in each sample.

[0078] The separation efficiency of extracellular vesicles was quantified by the intensity ratio of histone bands in the experimental group to those in the standard group. Results are as follows: Figure 4 As shown, using CD63 and CD81 as target proteins, the EV separation efficiencies of experimental group 1 (this method) were 50.0±4.8% and 52.9±2.8%, respectively, while the separation efficiencies of experimental group 2 (ultracentrifugation) were 43.0±4.7% and 42.5±2.4%. Taking the average, the efficiency of this method in separating extracellular vesicles was 51.45%, which is 8.7% higher than that of ultracentrifugation (42.75%).

[0079] Example 2: Antibody-oligonucleotide probe conjugation (taking CD81 antibody as an example) and verification.

[0080] 1) Resuspend the antibody in PBS at a concentration of 1 mg / ml;

[0081] 2) Take 20 μL of antibody (1 mg / ml) and add 2 μL of 4-(N-maleimide methyl)cyclohexane-1-carboxylic acid sulfonyl succinimide sodium salt (Sulfo-SMCC) (4 mM) for activation. After mixing, incubate at 4℃ for 2 h with intermittent shaking 3 times.

[0082] 3) Dissolve the 5'-SH modified oligonucleotide (5'-SHC6-TTTTTTTTTTACCCTCTCAA-3') to 100 μM using coupling buffer; C6 is a type of modified molecule composed of a straight carbon chain or ethylene glycol, usually used to establish a distance between oligonucleotides or between oligonucleotides and other functional groups to avoid steric hindrance, reduce unfavorable interactions between groups, and increase flexibility; here, a 6-carbon chain is incorporated during synthesis to reduce steric hindrance and facilitate subsequent reactions;

[0083] 4) Add 200 μL of 0.015 M TCEP to 20 μL of 100 μM oligonucleotide, and incubate with shaking at room temperature for 1 h to reduce its 5'-SH.

[0084] 5) Take 50kDa and 3kDa ultrafiltration tubes, add 400μL of coupling buffer to rinse them once, and then centrifuge at 12000g for 5 minutes;

[0085] 6) The incubated antibody and nucleic acid were purified using 50kDa and 3kDa ultrafiltration tubes, respectively. They were washed three times with coupling buffer, 100μL each time, and centrifuged at 15000g for 5 minutes.

[0086] 7) Mix the activated antibody and the reduced nucleic acid in a 1:3 molar ratio, mix thoroughly, and incubate overnight at 4°C in the dark.

[0087] 8) After incubation, the probe is purified using a 50k centrifuge column. After centrifugation at 15000g for 5 min, the probe is washed 5 times with 100μl of coupling buffer to obtain the purified antibody-nucleic acid probe.

[0088] 9) Characterize the coupled probes using SDS-PAGE, such as... Figure 5 As shown, the results demonstrate that the antibody and oligonucleotide were successfully conjugated. Figure 5 In the diagram, lane 1 is the protein marker, lane 2 is the CD81 antibody (within the box), and lane 3 is the conjugation result of the CD81 antibody and nucleic acid (within the box).

[0089] Example 3: Antibody-oligonucleotide probes recognize and label extracellular vesicle proteins and perform two-step amplification and quantification to explore the detection limit of this method.

[0090] Take extracellular vesicles at different concentrations (10-10) 7 CD81 protein was detected after being separated by the method described in Example 1 (samples / μL).

[0091] The synthesized antibody nucleic acid probe was diluted 1:200 with PBS buffer containing 0.1% bovine serum albumin and 0.1 mg / ml salmon sperm DNA. 100 μL of the diluted solution was incubated with extracellular vesicles captured on the surface of magnetic beads at room temperature for 1 hour.

[0092] After incubation, wash three times with PBS to remove unbound antibody-nucleic acid probes. Incubate 50 μl of the primer amplification reaction mixture at 37°C for 20 minutes to remove any dGTP that may be present in the mixture. The final concentrations of the components in this mixture are as follows: 0.8 U / μl Bst large fragment, 10 mM MgSO4, 1 mM dATP / dCTP / dTTP, 0.1 μM Clean.G sequence, and 1 μM hairpin sequence. Add the pre-incubated primer amplification reaction mixture to the washed magnetic bead mixture and incubate at 37°C for 2 hours. The Clean.G sequence is a dGTP removal sequence. To prevent interference from dGTP in the added dATP / dCTP / dTTP mixture, this sequence is added beforehand to react with the dATP / dCTP / dTTP mixture and remove dGTP from the solution.

[0093] Clean.G:

[0094] 5'-CCCCGAAAGTGGCCTCGGGCCTTTGGCCCGAGGCCACTTTCG-3';

[0095] Card issuance sequence:

[0096] 5'-ACATCATCATGGGCCTTTTGGCCCATGATGATGTATGATGATG-InverteddT-3'.

[0097] Inverted dT is a nucleic acid modification method that typically modifies the 3' end of oligonucleotides by inverting the last T base, creating a 3'-3' cross-linked end that inhibits 3' exonuclease degradation and DNA polymerase elongation. After the reaction, the beads were washed three times with PBS and resuspended in 50 μl of PBS. 5 μl of the magnetic bead mixture was mixed with 45 μl of Cas12a enzyme detection reaction solution (50 nM LbaCas12a, 50 nM crRNA, 100 nM reporter molecule, 1×NEBuffer 2.1). This mixture was placed in a qPCR tube and reacted. The reaction process was monitored using a qPCR instrument, and the real-time fluorescence signal was read. The qPCR instrument was set to a constant temperature of 37°C, a reaction time of 2 hours, and the fluorescence measurement channel was set to HEX, with a fluorescence signal readout per minute.

[0098] crRNA:

[0099] 5'-UAAUUUCUAACUAAGGUAGUAGAGGGUUUGAGAGGGUUUG-3';

[0100] Reporting molecule: 5'-HEX-AAAAAAAAAAAA-BHQ1-3'.

[0101] The results are attached. Figure 6 As shown, the results indicate that this method can detect CD81 on the surface of extracellular vesicles as low as 10 cells / μL, and exhibits a good linear relationship.

[0102] Example 4: This integrated sensor for extracellular vesicle isolation and membrane protein detection was used to detect proteins in different cell lines, and the results were compared with fluorescence imaging methods. The principle is as follows: Figure 7 As shown in Figure A.

[0103] 1) Take 10 10 Extracellular vesicles secreted by breast cancer cell lines SK-BR-3, MCF-7, MDA-MB-231 and normal cell line MCF-10A were used as samples to detect the target proteins HER2, EpCAM, and EGFR.

[0104] 2) The fluorescence imaging method was as follows: First, the primary antibody and the corresponding fluorescent secondary antibody were diluted 1:200 in PBS buffer containing 2% BSA. 100 μl of the primary antibody solution was reacted with extracellular vesicles and incubated overnight at 4°C. The cells were then washed three times using a 300 kDa ultrafiltration tube and PBS, centrifuged at 6000 g for 5 min. Next, 100 μl of the corresponding fluorescent secondary antibody solution was added and incubated for another 2 hours at room temperature. The cells were then washed three times using a 300 kDa ultrafiltration tube and PBS, centrifuged at 6000 g for 5 min. The solution was then resuspended in PBS and stored in the dark. Confocal fluorescence microscopy was then used to observe and statistically analyze the fluorescence intensity of these three proteins in extracellular vesicles derived from different cell lines. Figure 7 As shown in B.

[0105] 3) The method for detecting extracellular vesicle proteins using this integrated sensor for extracellular vesicle isolation and membrane protein detection is the same as in the above embodiments, and will not be repeated here. The detection results are as follows: Figure 7 As shown in C, a correlation analysis was performed with the results of the fluorescence imaging method, as follows: Figure 7 As shown in D, the results indicate that the detection effect of this method is consistent with that of fluorescence imaging, and it can be used for the detection of membrane proteins related to extracellular vesicles.

[0106] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. An integrated sensor for the separation of extracellular vesicles and the detection of their membrane proteins, characterized in that, Includes membrane probes and streptavidin magnetic beads, CD81 antibody-thiol modified nucleic acid probes, dGTP-free PER amplification components, and a Cas12a detection system containing specific crRNA and fluorescent reporter molecules; The membrane probe is made of polyethylene glycol with a hydrophobic lipid end at one end and a biotin molecule at the other end. The hydrophobic lipid end is used to insert into the membrane of extracellular vesicles, and the biotin at the other end binds to magnetic beads, thereby capturing extracellular vesicles to the surface of magnetic beads and separating them by magnetism. The CD81 antibody-thiol-modified nucleic acid probe contains a 5'-SH modified oligonucleotide, and the oligonucleotide sequence is SED ID NO.1; The PER amplification reaction kit includes a Clean.G clearance sequence and a hairpin template. The PER primer exchange reaction system does not contain dGTP raw materials. The primer exchange reaction can amplify oligonucleotides into long single strands containing multiple repeating units. The Clean.G sequence is shown in SED ID NO.2, and the hairpin template sequence is shown in SED ID NO.

3. The Cas12a system includes the Cas12a enzyme, the Cas12a system guide RNA (crRNA), and a reporter molecule. One end of the crRNA binds to the Cas12a enzyme, and the other end is completely complementary to the repeating unit on the long single strand, i.e., the target nucleic acid sequence. The reporter molecule is a fluorescent molecule and a quencher molecule linked by the bases of a short-chain deoxyribonucleic acid. Under the action of the crRNA, the Cas12a enzyme recognizes the target nucleic acid sequence, thereby activating its trans-cleavage property and non-specifically cleaving the surrounding single-stranded DNA. This causes the short-chain nucleic acid of the reporter molecule to be cleaved and release fluorescence. The sensor ultimately achieves quantitative detection of the target protein by detecting the fluorescence signal of the reaction solution. The sequences of the crRNA and the reporter molecule are shown in SED ID NO.4 and SED ID NO.5, respectively. The target nucleic acid sequence is two repeating units of a long single strand with a long repeating unit after amplification. The integrated sensor relies on PER to generate a long single-chain multi-repetitive unit to activate the output of Cas12a fluorescence signal, and directly performs quantitative detection of membrane proteins in unpurified body fluid extracellular vesicle lysate.

2. The sensor as described in claim 1, characterized in that, The sensor also includes an antibody-nucleic acid probe, which can specifically recognize and bind to target proteins on isolated extracellular vesicles through antigen-antibody interactions.

3. The sensor as described in claim 2, characterized in that, The nucleic acid in the antibody-nucleic acid probe is an oligonucleotide. The oligonucleotide can be amplified into a long single strand with long repeating units through a primer exchange reaction. The primer exchange reaction system does not contain dGTP.

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