A method for simultaneously detecting multiple membrane proteins on the surface of extracellular vesicles

Abstract

The present disclosure relates to the field of biological detection technology, and in particular to a method for simultaneously detecting multiple membrane proteins on the surface of extracellular vesicles. The present disclosure provides an "electrical sandwich" assay method based on a biosensor, and increases the negative charge of the antibody by independently coupling negatively charged single-stranded DNA to antibodies to the second to nth membrane proteins to be detected, thereby enhancing its electrical response when binding to extracellular vesicle surface membrane proteins. The method can realize the detection of sEVs subtypes containing multiple extracellular vesicle surface membrane proteins at the same time, which is of great significance in the field of biological detection.

Description

Technical Field

[0001] The present disclosure relates to the field of biological detection technology, and in particular to a method for simultaneously detecting multiple membrane proteins on the surface of extracellular vesicles. Background Art

[0002] Extracellular vesicles (EVs) are heterogeneous, tiny biological vesicles with a phospholipid bilayer membrane that are released by cells into the extracellular fluid environment. Based on their size, EVs can be classified as small EVs (sEVs, <200 nm) and large EVs (lEVs, >200 nm). sEVs are widely present in cell culture supernatants and various body fluids (such as sweat, blood, urine, and cerebrospinal fluid). The sEV membrane contains numerous membrane proteins, the types and relative abundance of which vary depending on the parental cell type and microenvironment. This heterogeneity enables different sEV subpopulations to provide important biological information about disease states, holding the potential for application in disease diagnosis and detection.

[0003] However, the heterogeneity of sEVs and the complexity of body fluids present significant challenges for in-depth study of sEVs. Classifying and analyzing specific protein-positive sEV subpopulations is crucial for a more accurate understanding of the biological significance of sEVs. Detection methods for sEV subpopulations can be broadly divided into two categories. Label-free methods, such as cryo-electron microscopy (cryo-EM), atomic force microscopy (AFM), nanoparticle tracking analysis (NTA), Raman tweezers microscopy (RTM), and single-particle interferometer reflectance imaging sensors (SP-IRIS), primarily characterize sEV morphology, concentration, size distribution, and surface proteins. However, these methods suffer from low throughput, limited information, and low sensitivity. Fluorescent tag-based methods, such as high-resolution flow cytometry (hrFC), digital droplet PCR (ddPCR), super-resolution microscopy (SRM), and fluorescence microscopy, offer higher sensitivity and specificity than these label-free methods, but they still have limitations. For example, fluorescence observation is susceptible to background signal interference, and optical methods rely on complex optical systems. Furthermore, nanoflow cytometry often relies on complex signal amplification strategies such as hybridization chain reaction (HCR) and rolling circle amplification (RCA) to improve sensitivity. Therefore, the development of simpler and more direct detection methods is needed to achieve more convenient and sensitive detection of sEV subpopulations. Summary of the Invention

[0004] In order to solve the above technical problems, the present disclosure provides a method for simultaneously detecting multiple membrane proteins on the surface of extracellular vesicles. The method is based on the biosensor described in the present disclosure, and an "electrical sandwich" determination method is developed to achieve rapid label-free electrical detection of multiple extracellular vesicle membrane proteins at the same time, which has good application prospects.

[0005] The present disclosure provides a method for simultaneously detecting multiple membrane proteins on the surface of extracellular vesicles, wherein the method is performed using a biosensor, wherein the biosensor is formed by bonding a silicon nanowire device to a polydimethylsiloxane microfluidic channel, wherein silicon nanowires are provided on the surface of the silicon nanowire device, and the silicon nanowires are located in the polydimethylsiloxane microfluidic channel;

[0006] The method comprises the following steps:

[0007] (1) The silicon nanowire device in the biosensor is surface modified, and then the antibody of the first membrane protein to be detected is modified onto the silicon nanowire of the silicon nanowire device, and then the control solution is passed into the polydimethylsiloxane microfluidic channel of the biosensor for electrical detection to obtain the basic threshold voltage. ;

[0008] (2) A solution containing extracellular vesicles is passed into the polydimethylsiloxane microfluidic channel of the biosensor, so that the extracellular vesicles expressing the first membrane protein to be detected react with the antibody of the first membrane protein to be detected, and then electrical detection is performed to obtain a first threshold voltage. ;

[0009] , It reflects the expression level of the first membrane protein to be detected in extracellular vesicles;

[0010] (3) sequentially measuring the threshold voltages corresponding to the antibodies for the second membrane protein to be detected to the antibodies for the nth membrane protein to be detected, where n is an integer greater than 1;

[0011] The steps for determining the threshold voltage corresponding to each antibody to be detected for each membrane protein include:

[0012] A solution containing an antibody against the mth membrane protein to be detected is passed into the polydimethylsiloxane microfluidic channel of the biosensor, so that the antibody against the mth membrane protein to be detected reacts with the extracellular vesicles expressing the mth membrane protein to be detected, and then electrical detection is performed to obtain the mth threshold voltage. ; Wherein, m is any integer from 2 to n;

[0013] The antibody for the mth membrane protein to be detected is coupled with single-stranded DNA;

[0014] , , , It reflects the expression level of the mth membrane protein to be detected in extracellular vesicles.

[0015] Based on the biosensor, the present disclosure provides an "electrical sandwich" assay method, and increases the negative charge of the antibody by independently coupling negatively charged single-stranded DNA to the antibody of the second membrane protein to be detected to the antibody of the nth membrane protein to be detected, thereby enhancing its electrical response when binding to the extracellular vesicle surface membrane protein. The method can realize the detection of sEVs subtypes containing multiple extracellular vesicle surface membrane proteins at the same time; in addition, compared with the two existing conventional detection methods, the operation is simple and direct, the sensitivity is high, and there is no need to rely on complex signal amplification strategies such as hybridization chain reaction (HCR) and rolling circle amplification (RCA), which is of great significance in the field of biological detection.

[0016] Among them, "electrical sandwich" refers to the structure of "antibody to the first membrane protein to be detected + extracellular vesicles + antibody to the second membrane protein to be detected to antibody to the nth membrane protein to be detected".

[0017] The following are preferred technical solutions of the present disclosure, but are not intended to limit the technical solutions provided by the present disclosure. Through the following technical solutions, the technical objectives and beneficial effects of the present disclosure can be better achieved and realized.

[0018] As a preferred technical solution of the present disclosure, the extracellular vesicles include small extracellular vesicles with a size of less than 200 nm.

[0019] As a preferred technical solution of the present disclosure, the silicon nanowire device includes a P-type silicon nanowire device.

[0020] As a preferred technical solution of the present disclosure, in step (1), the step of surface modification of the silicon nanowire device in the biosensor includes:

[0021] The surface of the silicon nanowire device in the biosensor is silanized using a silanization reagent to graft amino groups onto the surface of the nanowire device; then, an aldehyde group is used to formaldehyde the device so that the aldehyde group at any end of the aldehyde group combines with the amino groups on the surface of the silicon nanowire device to complete the surface modification.

[0022] Preferably, the silanization agent comprises 3-aminopropyltriethoxysilane.

[0023] Preferably, the formaldehyde-forming agent comprises an aqueous solution of glutaraldehyde.

[0024] As a preferred technical solution of the present disclosure, in step (1), the step of modifying the antibody of the first membrane protein to be detected onto the silicon nanowire of the silicon nanowire device includes:

[0025] A solution containing an antibody against a first membrane protein to be detected is passed into the polydimethylsiloxane microfluidic channel of the biosensor, so that the amino group on the surface of the antibody against the first membrane protein to be detected reacts with the aldehyde group at the other end of the aldehyde-forming reagent, thereby grafting the antibody against the first membrane protein to be detected onto the silicon nanowires of the silicon nanowire device; and then a blocking solution is used to block the nonspecific reaction sites on the surface of the silicon nanowire device.

[0026] Preferably, in the solution containing the antibody against the first membrane protein to be detected, the concentration of the antibody against the first membrane protein to be detected is 45-55 μg / mL, for example, 45 μg / mL, 47 μg / mL, 50 μg / mL, 53 μg / mL or 55 μg / mL, etc., but is not limited to the listed values, and other values ​​not listed within this range are also applicable.

[0027] Preferably, the blocking solution comprises BSA solution.

[0028] As a preferred technical solution of the present disclosure, the control solution in step (1) includes a PBS solution.

[0029] As a preferred technical solution of the present disclosure, in the solution containing extracellular vesicles in step (2), the concentration of extracellular vesicles is 1×10 9 ~ 3×10 9 / mL, for example 1×10 9 / mL, 2×10 9 / mL or 3×10 9 The above values ​​are not limited to the listed values, and other values ​​not listed in the above range are also applicable.

[0030] As a preferred technical solution of the present disclosure, in step (3), the number of nucleotides in the single-stranded DNA is not less than 10, for example, 10, 15, 20, 25, 30 or 35, etc., but is not limited to the listed values. Other values ​​not listed within this range are also applicable, preferably 10 to 30, and more preferably 15.

[0031] In the present disclosure, single-stranded DNA carries a negative charge. After coupling it to the antibody to be detected, the negative potential of the antibody to be detected increases, and the negative potential increases with the increase of the length of the DNA chain, thereby amplifying the electrical signal and enhancing the sensitivity.

[0032] It should be noted that there is no requirement for the nucleotide sequence of the single-stranded DNA, and any nucleotide sequence is acceptable.

[0033] As a preferred technical solution of the present disclosure, in step (3), in the solution containing the antibody against the mth membrane protein to be detected, the concentration of the antibody against the mth membrane protein to be detected is 45-55 μg / mL, for example, 45 μg / mL, 47 μg / mL, 50 μg / mL, 53 μg / mL or 55 μg / mL, etc., but is not limited to the listed values, and other values ​​not listed within the range are also applicable.

[0034] As a preferred technical solution of the present disclosure, in steps (2) and (3), after each antibody of the membrane protein to be detected reacts with the extracellular vesicles, the polydimethylsiloxane microfluidic channel of the biosensor is cleaned with a PBS solution to remove unreacted substances before electrical detection.

[0035] The technical solution provided by the embodiments of the present disclosure has the following advantages over the prior art:

[0036] Based on the biosensor, the present disclosure provides an "electrical sandwich" assay method, and increases the negative charge of the antibody by independently coupling negatively charged single-stranded DNA to the antibody of the second membrane protein to be detected to the antibody of the nth membrane protein to be detected, thereby enhancing its electrical response when binding to the extracellular vesicle surface membrane protein. The method can realize the detection of sEVs subtypes containing multiple extracellular vesicle surface membrane proteins at the same time; in addition, compared with the two existing conventional detection methods, the operation is simple and direct, the sensitivity is high, and there is no need to rely on complex signal amplification strategies such as hybridization chain reaction (HCR) and rolling circle amplification (RCA), which is of great significance in the field of biological detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present disclosure and, together with the description, serve to explain the principles of the present disclosure.

[0038] In order to more clearly illustrate the embodiments of the present disclosure or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0039] Figure 1 Schematic diagram of the cross-sectional structure of the biosensor perpendicular to the microfluidic channel according to a specific embodiment of the present disclosure;

[0040] Figure 2 This is a circuit diagram of the biosensor according to the specific embodiment of the present disclosure for measuring the threshold voltage corresponding to the extracellular vesicle membrane protein;

[0041] Figure 3For Examples 1-3 and Comparative Example 1 of the present disclosure as well as The result graph;

[0042] Figure 4 For Examples 1-3 and Comparative Example 1 of the present disclosure Result graph.

[0043] Wherein: 1. silicon nanowire device; 1-1. Si layer; 1-2. SiO2 layer; 1-3. silicon nanowire; 1-4. HfO2 layer; 2. polydimethylsiloxane microfluidic channel; 3. antibody to the first membrane protein to be detected; 4. extracellular vesicles; 5. antibody to the second membrane protein to be detected; 5-1. single-stranded DNA;

[0044] It should be noted that Figure 2 The biosensor is shown as a cross-sectional view parallel to the fluid channel, i.e., along Figure 1 Cross-sectional view of plane AA, where S is the source and D is the drain. DETAILED DESCRIPTION

[0045] In order to more clearly understand the above-mentioned objectives, features and advantages of the present disclosure, the scheme of the present disclosure will be further described below. It should be noted that the embodiments of the present disclosure and the features therein can be combined with each other in the absence of conflict.

[0046] In the following description, many specific details are set forth to facilitate a full understanding of the present disclosure, but the present disclosure may also be implemented in other ways different from those described herein; it is obvious that the embodiments in the specification are only part of the embodiments of the present disclosure, rather than all of the embodiments.

[0047] For example, the biosensor used in the present disclosure is formed by bonding a silicon nanowire device 1 to a polydimethylsiloxane microfluidic channel 2. The front view structural diagram of the biosensor is shown in FIG. Figure 1 shown.

[0048] The silicon nanowire device 1 is a P-type silicon nanowire device.

[0049] The silicon nanowire device 1 includes, from bottom to top, a stacked Si layer 1-1 and a SiO2 layer 1-2, wherein parallel and spaced silicon nanowires 1-3 are arranged on the surface of the SiO2 layer 1-2;

[0050] Furthermore, the surface of the silicon nanowire 1-3 is covered with a HfO2 layer 1-4;

[0051] The polydimethylsiloxane microfluidic channel 2 is bonded to the SiO2 layer 1-2 of the silicon nanowire device 1 to form a cavity, so that the silicon nanowires 1-3 are located in the cavity, and the cavity is used to pass fluid.

[0052] In the biosensor, HfO2 layers 1-4 act as insulating dielectric layers, effectively isolating the liquid from the silicon nanowires 1-3, enabling more stable electrical testing. Compared to silicon, HfO2 is easier to functionalize on the surface. Furthermore, compared to commonly used dielectric layers such as silicon oxide, HfO2 has a higher relative dielectric constant, making it more sensitive to charge changes caused by surface biomolecular interactions, thereby improving sensor sensitivity.

[0053] Furthermore, in order to demonstrate the working principle of the present disclosure when the biosensor is used for detection, Figure 1 Also shown are an antibody 3 against the first membrane protein to be detected, an extracellular vesicle 4, an antibody 5 against the second membrane protein to be detected, and a single-stranded DNA 5-1.

[0054] Furthermore, the mechanism of using the biosensor disclosed herein to measure the threshold voltage corresponding to the extracellular vesicle membrane protein is as follows, taking the first membrane protein to be detected as an example:

[0055] The biosensor modifies the surface of the silicon nanowires 1-3 with an antibody against a first membrane protein to be detected, and then passes a solution containing extracellular vesicles into the polydimethylsiloxane microfluidic channel 2 of the biosensor. If the extracellular vesicles express the first membrane protein to be detected, they will be captured by the antibody against the first membrane protein to be detected. The extracellular vesicles will change the carrier distribution inside the silicon nanowires 1-3, thereby further causing the electrical response of the biosensor (i.e., changes in source-drain current and threshold voltage). In order to obtain an electrical response signal, the biosensor needs to be electrically tested. The test circuit diagram is shown below. Figure 2 As shown, the source S is grounded. When a negative voltage is applied to the gate of the silicon nanowire device 1 of the biosensor, an inversion layer is formed on the surface of the P-type silicon nanowire, thereby forming a conductive channel, and at the drain voltage ( V D ) forms a current between the source and the drain, turning the device on. Set the appropriate drain voltage, gate voltage range and step size, and then scan all gate voltages ( V G ) corresponds to the drain current ( I D ), the transfer characteristic curve of the silicon nanowire device 1 can be obtained. Based on the obtained transfer characteristic curve, the threshold voltage ( V th ).

[0056] Example 1

[0057] (1) Surface modification of silicon nanowire devices and modification of antibodies against the first membrane protein to be detected

[0058] S1: (1) A mixed solution of isopropyl alcohol, deionized water, and 3-aminopropyltriethoxysilane (APTES) was prepared in a volume ratio of 100:100:1 and injected into the polydimethylsiloxane microfluidic channel 2 of the biosensor. After reacting for 20 min, amino groups were grafted onto the surface of the silicon nanowire device 1. The device was then washed three times with deionized water to remove unbound APTES molecules.

[0059] S2: A 2.5 wt% glutaraldehyde aqueous solution was injected into the polydimethylsiloxane microfluidic channel 2 of the biosensor. The aldehyde group at one end of the glutaraldehyde molecule combined with the amino group on the surface of the silicon nanowire device 1. After reacting for 30 minutes, the device was washed three times with deionized water.

[0060] S3: A solution containing CD81 antibodies at a concentration of 50 μg / mL was injected into the polydimethylsiloxane microfluidic channel 2 of the biosensor. The amino groups on the CD81 antibodies reacted with the aldehyde groups on the other end of the glutaraldehyde molecules to immobilize the CD81 antibodies on the surface of the silicon nanowire device 1. After reacting for 1 hour, the solution was washed three times with 0.01× PBS solution (obtained by diluting 1× PBS standard solution 100 times) to remove unbound CD81 antibodies.

[0061] S4: Block the nonspecific reaction sites on the surface of the silicon nanowire device 1 with a 0.5 wt % BSA solution for 2 h to reduce the nonspecific adsorption problem in the subsequent detection process, and then wash with a 0.01× PBS solution three times;

[0062] A 0.01× PBS solution was used as a control solution and injected into the polydimethylsiloxane microfluidic channel 2 of the biosensor. Electrical detection was performed after 30 minutes to calculate the threshold voltage. ;

[0063] (2) The concentration is 2×10 9 A solution containing 293F-sEVs at a concentration of 100 μg / mL was passed into the polydimethylsiloxane microfluidic channel 2 of the biosensor to allow the 293F-sEVs expressing the CD81 membrane protein to react with the CD81 antibody. After 30 minutes, a 0.01× PBS solution was passed through the channel for washing to remove unbound 293F-sEVs. Electrical detection was then performed to obtain a first threshold voltage. ;

[0064] , It reflects the expression level of CD81 membrane protein in 293F-sEVs;

[0065] (3) A solution containing CD63 antibodies at a concentration of 50 μg / mL was passed into the polydimethylsiloxane microfluidic channel 2 of the biosensor to allow the CD63 antibodies to react with the 293F-sEVs expressing the CD63 membrane protein. After 30 minutes, a 0.01× PBS solution was passed through the channel for washing to remove the unbound CD63 antibodies. Then, electrical detection was performed to obtain the second threshold voltage. ;

[0066] The CD63 antibody is coupled to a single-stranded DNA, wherein the nucleotide sequence of the single-stranded DNA is AGT CT and the number is 5;

[0067] , , Reflects the expression level of CD63 membrane protein in 293F-sEVs.

[0068] Example 2

[0069] This example refers to the method in Example 1, with the only difference being that the nucleotide sequence of the single-stranded DNA to which the CD63 antibody is coupled in step (3) is AGT CTG GAT GTA GTC (SEQ ID NO: 1), and the number is 15.

[0070] Example 3

[0071] This example refers to the method in Example 1, with the only difference being that the nucleotide sequence of the single-stranded DNA coupled to the CD63 antibody in step (3) is AGT CGT AGT TGA GTC AGT CTG GAT GTA GTC (SEQ ID NO: 2), and the number is 30.

[0072] Comparative Example 1

[0073] This comparative example refers to the method in Example 1, with the only difference being that the CD63 antibody in step (3) is not coupled to single-stranded DNA.

[0074] The threshold voltage data results involved in Examples 1-3 and Comparative Example 1 are shown in Table 1.

[0075] Table 1

[0076]

[0077] According to the results in Table 1, Figure 3 Demonstrated in Examples 1-3 and Comparative Example 1 as well as The result, Figure 4 Shown results.

[0078] from Figure 3 and Figure 4 It can be seen that after step (2), the threshold voltage detected is negatively biased, proving that the biosensor modified with CD81 antibody can effectively capture the 293F-sEVs subpopulation expressing CD81 membrane protein; and after step (3), the threshold voltage detected is positively biased, proving that the CD63 antibody further binds to the CD63 membrane protein on 293F-sEVs, thereby achieving dual analysis of CD81 membrane protein and CD63 membrane protein. And through Figure 3 It can be seen that the voltage change of single-stranded DNA with 15 CD63-coupled nucleotides is the largest, which effectively amplifies the electrical response after the detection antibody binds to the extracellular vesicle membrane protein, which is helpful for the subpopulation analysis of extracellular vesicles based on membrane proteins.

[0079] The foregoing description is intended only to provide specific embodiments of the present disclosure, intended to enable those skilled in the art to understand and implement the present disclosure. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure is not intended to be limited to the embodiments described herein, but rather to be construed in the broadest manner consistent with the principles and novel features disclosed herein.

Claims

1. A method for simultaneously detecting multiple membrane proteins on the surface of extracellular vesicles, characterized in that: The method is carried out using a biosensor, which is formed by bonding a silicon nanowire device to a polydimethylsiloxane microfluidic channel, wherein silicon nanowires are provided on the surface of the silicon nanowire device, and the silicon nanowires are located in the polydimethylsiloxane microfluidic channel; The surface of the silicon nanowire is covered with a HfO2 layer; The method comprises the following steps: (1) The silicon nanowire device in the biosensor is surface modified, and then the antibody of the first membrane protein to be detected is modified onto the silicon nanowire of the silicon nanowire device, and then the control solution is passed into the polydimethylsiloxane microfluidic channel of the biosensor for electrical detection to obtain the basic threshold voltage. ; (2) A solution containing extracellular vesicles is passed into the polydimethylsiloxane microfluidic channel of the biosensor, so that the extracellular vesicles expressing the first membrane protein to be detected react with the antibody of the first membrane protein to be detected, and then electrical detection is performed to obtain a first threshold voltage. ; , It reflects the expression level of the first membrane protein to be detected in extracellular vesicles; (3) sequentially measuring the threshold voltages corresponding to the antibodies for the second membrane protein to be detected to the antibodies for the nth membrane protein to be detected, where n is an integer greater than 1; The steps for determining the threshold voltage corresponding to each antibody to be detected for each membrane protein include: A solution containing an antibody against the mth membrane protein to be detected is passed into the polydimethylsiloxane microfluidic channel of the biosensor, so that the antibody against the mth membrane protein to be detected reacts with the extracellular vesicles expressing the mth membrane protein to be detected, and then electrical detection is performed to obtain the mth threshold voltage. ; Wherein, m is any integer from 2 to n; The antibody for the mth membrane protein to be detected is coupled with single-stranded DNA; The number of nucleotides in the single-stranded DNA is 10 to 30; , , , It reflects the expression level of the mth membrane protein to be detected in extracellular vesicles.

2. The method according to claim 1, characterized in that The extracellular vesicles include small extracellular vesicles with a size less than 200 nm.

3. The method according to claim 1 or 2, characterized in that The silicon nanowire device includes a P-type silicon nanowire device.

4. The method according to claim 1, wherein In step (1), the step of surface modification of the silicon nanowire device in the biosensor includes: The surface of the silicon nanowire device in the biosensor is silanized using a silanization reagent to graft amino groups onto the surface of the nanowire device; then, an aldehyde group is used to formaldehyde the device so that the aldehyde group at any end of the aldehyde group combines with the amino groups on the surface of the silicon nanowire device to complete the surface modification.

5. The method according to claim 4, characterized in that In step (1), the step of modifying the antibody of the first membrane protein to be detected onto the silicon nanowire of the silicon nanowire device includes: A solution containing an antibody against a first membrane protein to be detected is passed into the polydimethylsiloxane microfluidic channel of the biosensor, so that the amino group on the surface of the antibody against the first membrane protein to be detected reacts with the aldehyde group at the other end of the aldehyde-forming reagent, thereby grafting the antibody against the first membrane protein to be detected onto the silicon nanowires of the silicon nanowire device; and then a blocking solution is used to block the nonspecific reaction sites on the surface of the silicon nanowire device.

6. The method according to claim 5, characterized in that The silanization agent includes 3-aminopropyltriethoxysilane; and / or, the formaldehyde-forming agent comprises an aqueous solution of glutaraldehyde; and / or, in the solution containing the antibody against the first membrane protein to be detected, the concentration of the antibody against the first membrane protein to be detected is 45-55 μg / mL; And / or, the blocking solution includes BSA solution.

7. The method according to claim 1, characterized in that The control solution in step (1) includes a PBS solution; And / or, in the solution containing extracellular vesicles in step (2), the concentration of extracellular vesicles is 1×10 9 ~ 3×10 9 pieces / mL.

8. The method according to claim 1, characterized in that In step (3), in the solution containing the antibody against the mth membrane protein to be detected, the concentration of the antibody against the mth membrane protein to be detected is 45-55 μg / mL.

9. The method according to claim 1, characterized in that In step (2) and step (3), after each antibody of the membrane protein to be detected reacts with the extracellular vesicles, the polydimethylsiloxane microfluidic channel of the biosensor is cleaned with a PBS solution to remove unreacted substances before electrical detection.

Images