Method for simultaneously detecting multiple membrane proteins on surfaces of extracellular vesicles

Abstract

The invention relates to the technical field of biological detection, in particular to a method for simultaneously detecting multiple membrane proteins on the surface of extracellular vesicles. On the basis of a biosensor, the invention provides an'electrical sandwich 'determination method, negative charges of the antibodies are increased by independently coupling the antibodies of the second to-be-detected membrane protein to the nth to-be-detected membrane protein with the single-chain DNA with negative charges, so that the electrical response of the antibodies when the antibodies are combined with the extracellular vesicle surface membrane protein is enhanced; the method can be used for detecting sEVs subtypes simultaneously containing various extracellular vesicle surface film proteins, and has important significance in the field of biological detection.

Description

Technical Field

[0001] The present invention 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) refer to heterogeneous tiny biological vesicles with a phospholipid bilayer structure that are released by cells into the extracellular body fluid environment. According to the size of EVs, they can be divided into small extracellular vesicles (sEVs, <200 nm) and large extracellular vesicles (lEVs, >200 nm). sEVs are widely present in cell culture supernatants and various body fluids (such as sweat, blood, urine, and cerebrospinal fluid). The membrane surface of sEVs contains a large number of membrane proteins. The types and relative abundance of these proteins are heterogeneous due to differences in parental cell types and microenvironments. The existence of this heterogeneity enables different sEVs subpopulations to provide important biological information on disease states and has the potential to be used in disease diagnosis and detection.

[0003] However, the heterogeneity of sEVs and the complexity of body fluids pose great challenges to the in-depth study of sEVs. Classification and analysis of specific protein-positive sEVs subpopulations are crucial for a more accurate understanding of the biological significance of sEVs. sEVs subpopulation detection methods can be divided into two categories. One is label-free methods, such as cryo-electron microscopy (cryo-EM), atomic force microscopy (AFM), nanoparticle tracking analysis (NTA), Raman tweezers microscopy (RTM), single particle interference reflection imaging sensor (SP-IRIS), etc. These methods are mainly used to characterize the morphology, concentration, size distribution and surface protein of sEVs, but the throughput is low, the information provided is limited, and the detection sensitivity is low; the other is fluorescent label-based labeling methods, such as high-resolution flow cytometry (hrFC), digital droplet PCR (ddPCR), super-resolution microscopy (SRM), fluorescence microscopy, etc. Compared with the above label-free methods, these methods have achieved higher sensitivity and specificity, but still have certain limitations. For example, fluorescence observation is easily interfered by background signals, and optical methods rely on complex optical systems. In addition, nanoflow detection technology usually relies on complex signal amplification strategies such as hybridization chain reaction (HCR) and rolling circle amplification (RCA) to improve sensitivity. Therefore, it is necessary to develop simpler and more direct detection methods to achieve more convenient and more sensitive sEVs subpopulation detection. 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 develops an "electrical sandwich" assay method 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, the method being performed using a biosensor, the biosensor being formed by bonding a silicon nanowire device to a polydimethylsiloxane microfluidic channel, the surface of the silicon nanowire device being provided with silicon nanowires, and the silicon nanowires being located in the polydimethylsiloxane microfluidic channel; The method comprises the following steps: (1) Surface modification of the silicon nanowire device in the biosensor is performed, 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 a control solution is passed into the polydimethylsiloxane microfluidic channel of the biosensor for electrical detection to obtain a basic threshold voltage. ; (2) Passing a solution containing extracellular vesicles into the polydimethylsiloxane microfluidic channel of the biosensor, allowing the extracellular vesicles expressing the first membrane protein to be detected to react with the antibody of the first membrane protein to be detected, and then performing electrical detection 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, wherein n is an integer greater than 1; The steps for determining the threshold voltage corresponding to each antibody of the membrane protein to be detected include: A solution containing an antibody to the mth membrane protein to be detected is passed into the polydimethylsiloxane microfluidic channel of the biosensor, so that the antibody to 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 of the mth membrane protein to be detected is coupled with single-stranded DNA; , , , It reflects the expression level of the mth membrane protein to be detected in extracellular vesicles.

[0006] The present disclosure provides an "electrical sandwich" assay method based on the biosensor, 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.

[0007] Among them, "electrical sandwich" refers to the structure of "antibody for the first membrane protein to be detected + extracellular vesicles + antibody for the second membrane protein to be detected to antibody for the nth membrane protein to be detected".

[0008] The following are preferred technical solutions of the present disclosure, but are not intended to be limitations of 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.

[0009] As a preferred technical solution of the present disclosure, the extracellular vesicles include small extracellular vesicles with a size less than 200 nm.

[0010] As a preferred technical solution of the present disclosure, the silicon nanowire device includes a P-type silicon nanowire device.

[0011] 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: The surface of the silicon nanowire device in the biosensor is silanized by using a silanization reagent to graft amino groups on the surface of the nanowire device; then, an aldehyde modification is performed by using an aldehyde modification reagent to combine the aldehyde group at any end of the aldehyde modification reagent with the amino group on the surface of the silicon nanowire device to complete the surface modification.

[0012] Preferably, the silanization agent comprises 3-aminopropyltriethoxysilane.

[0013] Preferably, the formaldehyde reagent comprises an aqueous solution of glutaraldehyde.

[0014] 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 comprises: A solution containing an antibody against a first membrane protein to be detected is introduced 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 agent, 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.

[0015] 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, but is not limited to the listed values, and other values ​​not listed within the range are also applicable.

[0016] Preferably, the blocking solution comprises BSA solution.

[0017] As a preferred technical solution of the present disclosure, the control solution in step (1) includes a PBS solution.

[0018] 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 above values, and other values ​​not listed in the above range are also applicable.

[0019] 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, but is not limited to the listed values. Other values ​​not listed in the range are also applicable, preferably 10 to 30, and more preferably 15.

[0020] In the present disclosure, single-stranded DNA carries negative charge, and 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.

[0021] It should be noted that there is no requirement for the nucleotide sequence of the single-stranded DNA, and any nucleotide sequence is acceptable.

[0022] 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, but is not limited to the listed values, and other values ​​not listed within the range are also applicable.

[0023] As a preferred technical solution of the present disclosure, 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.

[0024] Compared with the prior art, the technical solution provided by the embodiments of the present disclosure has the following advantages: The present disclosure provides an "electrical sandwich" assay method based on the biosensor, 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

[0025] 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.

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0027] Figure 1 It is a schematic diagram of the cross-sectional structure of the biosensor perpendicular to the microfluidic channel according to the specific embodiment of the present disclosure; Figure 2 A circuit diagram of a biosensor for measuring a threshold voltage corresponding to an extracellular vesicle membrane protein according to a specific embodiment of the present disclosure; Figure 3 For the embodiments 1-3 and comparative example 1 of the present disclosure as well as The result graph of Figure 4 For the embodiments 1-3 and comparative example 1 of the present disclosure Result graph.

[0028] Among them, 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 of the first membrane protein to be detected; 4. extracellular vesicles; 5. antibody of the second membrane protein to be detected; 5-1. single-stranded DNA; It should be noted that Figure 2 The cross-sectional view of the biosensor parallel to the fluid channel is shown. Figure 1 Cross-sectional view of plane AA, where S is the source and D is the drain. DETAILED DESCRIPTION

[0029] 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 in the embodiments can be combined with each other without conflict.

[0030] 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.

[0031] 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.

[0032] The silicon nanowire device 1 is a P-type silicon nanowire device.

[0033] The silicon nanowire device 1 includes, from bottom to top, a stacked Si layer 1-1 and a SiO2 layer 1-2, wherein the surface of the SiO2 layer 1-2 is provided with parallel and spaced silicon nanowires 1-3; Further, the surface of the silicon nanowire 1-3 is covered with a HfO2 layer 1-4; 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 nanowire 1-3 is located in the cavity, and the cavity is used to pass fluid.

[0034] In the biosensor, the HfO2 layers 1-4 serve as insulating dielectric layers, which can well isolate the liquid and the silicon nanowires 1-3, thereby achieving more stable electrical testing, and HfO2 is easier to achieve surface functionalization than silicon materials. In addition, compared with the commonly used dielectric layer silicon oxide, HfO2 has a higher relative dielectric constant and is more sensitive to charge changes caused by surface biomolecular interactions, thereby improving sensor sensitivity.

[0035] Further, 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 for the first membrane protein to be detected, an extracellular vesicle 4, an antibody 5 for the second membrane protein to be detected, and a single-stranded DNA 5-1.

[0036] Furthermore, the mechanism of using the biosensor disclosed in the present invention 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: The biosensor modifies the surface of the silicon nanowires 1-3 with antibodies to the 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 antibodies to the first membrane protein to be detected. The extracellular vesicles will change the carrier arrangement 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 circuit diagram of the test is shown in FIG. 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 on the device. 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 ).

[0037] Example 1 (1) Surface modification of silicon nanowire devices and modification of antibodies against the first membrane protein to be detected S1: (1) A mixed solution of isopropanol, 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 minutes, the surface of the silicon nanowire device 1 was grafted with amino groups. The device was then washed three times with deionized water to remove unbound APTES molecules. S2: Inject a 2.5wt% glutaraldehyde aqueous solution into the polydimethylsiloxane microfluidic channel 2 of the biosensor, and the aldehyde group at one end of the glutaraldehyde molecule combines with the amino group on the surface of the silicon nanowire device 1. After reacting for 30 minutes, wash it three times with deionized water; S3: Inject a solution containing CD81 antibody at a concentration of 50 μg / mL into the polydimethylsiloxane microfluidic channel 2 of the biosensor, and the amino group on the CD81 antibody reacts and combines with the aldehyde group at the other end of the glutaraldehyde molecule to achieve the fixation of the CD81 antibody on the surface of the silicon nanowire device 1. After reacting for 1 hour, wash three times with 0.01× PBS solution (obtained by diluting 1× PBS standard solution 100 times) to remove unbound CD81 antibody; S4: using a 0.5 wt % BSA solution to block the non-specific reaction sites on the surface of the silicon nanowire device 1 for 2 h to reduce the non-specific adsorption problem in the subsequent detection process, and then washing with a 0.01× PBS solution for 3 times; 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, and the threshold voltage was calculated. ; (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 for washing to remove the unbound 293F-sEVs. Then, electrical detection was performed to obtain a first threshold voltage. ; , It reflects the expression level of CD81 membrane protein in 293F-sEVs; (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 for washing to remove the unbound CD63 antibodies, and then electrical detection was performed to obtain a second threshold voltage. ; The CD63 antibody is coupled to a single-stranded DNA, the nucleotide sequence of the single-stranded DNA is AGT CT, and the number is 5; , , Reflects the expression level of CD63 membrane protein in 293F-sEVs.

[0038] Example 2 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 CTG GAT GTA GTC (SEQ ID NO: 1), and the number is 15.

[0039] Example 3 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.

[0040] Comparative Example 1 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.

[0041] The threshold voltage data results involved in Examples 1-3 and Comparative Example 1 are shown in Table 1.

[0042] Table 1

[0043] According to the results in Table 1, Figure 3 Demonstrated in Examples 1-3 and Comparative Example 1 as well as As a result, Figure 4 Shown result.

[0044] from Figure 3 and Figure 4 It can be seen that after step (2), the threshold voltage detected was 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 was 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. Figure 3It 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, and is helpful for the subpopulation analysis of extracellular vesicles based on membrane proteins.

[0045] The above description is only a specific embodiment of the present disclosure, so that those skilled in the art can understand or implement the present disclosure. Various modifications to these embodiments will be 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 will not be limited to the embodiments described herein, but will conform to the widest scope 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, wherein the biosensor is formed by bonding a silicon nanowire device to a polydimethylsiloxane microfluidic channel, wherein silicon nanowires are arranged on the surface of the silicon nanowire device, and the silicon nanowires are located in the polydimethylsiloxane microfluidic channel; 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 a control solution is passed into the polydimethylsiloxane microfluidic channel of the biosensor for electrical detection to obtain a basic threshold voltage ; (2) Passing a solution containing extracellular vesicles into the polydimethylsiloxane microfluidic channel of the biosensor, allowing the extracellular vesicles expressing the first membrane protein to be detected to react with the antibody of the first membrane protein to be detected, and then performing electrical detection 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, wherein n is an integer greater than 1; The steps for determining the threshold voltage corresponding to each antibody of the membrane protein to be detected include: A solution containing an antibody to the mth membrane protein to be detected is passed into the polydimethylsiloxane microfluidic channel of the biosensor, so that the antibody to 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 of the mth membrane protein to be detected is coupled with single-stranded DNA; , , , 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, characterized in that: In step (1), the step of surface modification of the silicon nanowire device in the biosensor comprises: The surface of the silicon nanowire device in the biosensor is silanized by using a silanization reagent to graft amino groups on the surface of the nanowire device; then, an aldehyde modification is performed by using an aldehyde modification reagent to combine the aldehyde group at any end of the aldehyde modification reagent with the amino group 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 comprises: A solution containing an antibody against a first membrane protein to be detected is introduced 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 agent, 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 reagent 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), the number of nucleotides in the single-stranded DNA is not less than 10.

9. 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.

10. 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.

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