An Exosome Sorting and Detection Chip, System and Method Based on Surface Acoustic Wave

Abstract

The present invention discloses a surface acoustic wave-based exosome sorting and detection chip, system and method, which includes a piezoelectric substrate. A microfluidic channel is bonded to the surface of the piezoelectric substrate. The microfluidic channel includes a main channel and a first buffer fluid channel, a first separation channel, a second buffer fluid channel and a second separation channel connected to one side of the main channel. An injection port is provided at one end of the main channel, and the other end is connected to a detection channel. The inner surface of the detection channel is modified with a fluorescence detection probe. A first sorting electrode for separating micron-sized particles in a body fluid sample from the first separation channel and a second sorting electrode for separating submicron-sized particles in the body fluid sample from the second separation channel are also provided on the piezoelectric substrate. The present invention can achieve rapid and efficient separation of exosomes and detect characteristic proteins on the surface of exosomes.

Description

Technical Field

[0001] The present invention relates to the technical fields of biological detection technology, microfluidic technology and exosome separation and detection technology, and particularly relates to a sorting and detection chip, system and method for exosomes based on surface acoustic waves. Background Art

[0002] Exosomes are polymorphic vesicle-like bodies actively secreted by cells, with diameters mostly between 30 and 100 nm. Exosomes carry and transmit a variety of important signaling molecules including proteins, mRNAs, miRNAs and lipid molecules, and are widely present in biological fluids such as cell culture supernatants, serum, plasma, saliva, urine, amniotic fluid, etc. In recent years, research has shown that exosomes not only play an important role in the material and information transfer between cells, but also have the advantages of small size, easy penetration of biological membranes, and low immunogenicity, and can be used as good carriers for drugs. The specific proteins and genetic materials carried by exosomes are more expected to become early diagnostic markers for various diseases such as cancer and coronary heart disease. For example, overexpressed protein markers such as tumor antigens and immunosuppressive proteins such as FasL, TRAIL and TGF-β are detected in exosomes produced by tumor cells, which makes exosomes of great significance in the diagnosis of tumors and related diseases; more than 60 miRNAs related to immune regulation are contained in exosomes extracted from human milk; exosomes of glioma cells contain a large number of mRNAs related to cell proliferation and migration, angiogenesis and immune response. Therefore, the efficient and rapid separation of exosomes from human body fluids is crucial for medical research and clinical diagnosis.

[0003] Although exosomes are abundant in body fluids, other types of extracellular vesicles (such as microvesicles, apoptotic bodies) and biological particles often interfere with their detection results. Therefore, the separation and purification of exosomes are the premise of exosome detection and an important link in disease diagnosis based on exosome detection. Currently, there is still no exosome separation method that can simultaneously ensure the content, purity and biological activity of exosomes.

[0004] Currently, common methods for exosome isolation include: centrifugation, filtration centrifugation, density gradient centrifugation, immunomagnetic beads, and chromatography. Centrifugation is the most commonly used method for exosome isolation. After collecting cell culture medium, centrifuge at 300g, 2000g, and 10000g in sequence to remove cell debris and macromolecular proteins, and finally centrifuge at 100000g to obtain exosomes. This method yields a large amount of exosomes, but the purity is insufficient, and exosomes are prone to aggregation. Filtration centrifugation uses ultrafiltration membranes with different molecular weight cut-offs (MWCO) to centrifuge and separate exosomes. Selecting ultrafiltration membranes of different sizes can separate exosomes from other macromolecules. This method is simple and time-saving, and does not affect the biological activity of exosomes, but there is also a problem of insufficient purity. Density gradient centrifugation is to ultracentrifuge the sample together with the gradient material, and different components in the sample sediment to their respective isopycnic zones. Prepare a continuous gradient system of two concentrations of sucrose solutions (such as 2.5M and 0.25M) in an ultracentrifuge tube in advance, spread the sample on the sucrose solution, and centrifuge at 100000g for 16h. Exosomes will sediment to the isopycnic zone (1.10 - 1.18 g / ml). The exosomes isolated by this method have high purity, but the preliminary preparation work is cumbersome, time-consuming, and the yield is small. Immunomagnetic beads are spherical magnetic particles coated with antibodies, which can specifically bind to the target substance. This method can ensure the integrity of exosome morphology, has high specificity, is simple to operate, and does not require expensive equipment, but this method is easily interfered by other biomolecules in body fluids and cannot be used for untreated body fluid samples. Chromatography separates solutes based on the relative relationship between the pore size of the gel pores and the size of the sample molecules. The purity of this method is relatively high compared to centrifugation, but the operation is complex and the yield is not high.

[0005] For exosomes isolated from body fluids, in addition to analyzing the size, morphology, etc. of exosomes through transmission electron microscopy, the proteins carried on their surface are also cell information that people are interested in. Currently, researchers most commonly use SDS-PAGE electrophoresis and Western-Blot to analyze the protein content and types in the obtained exosomes. The operations of these two methods are too complex, greatly reducing the timeliness of exosome detection.

[0006] Traditional methods for exosome detection and identification mainly include morphological characterization, surface protein detection, and internal nucleic acid detection, etc. Using SEM / TEM for morphological characterization is the most accurate, but it is not suitable for rapid measurement of a large number of samples; dynamic light scattering and nanoparticle analysis and tracking systems can quickly detect the size of exosomes, but it is difficult to distinguish protein precipitates with similar sizes from exosomes; surface proteins can be detected by immunoblotting, enzyme-linked immunosorbent assay, flow cytometry, etc., but there are deficiencies such as complex and time-consuming operations, poor repeatability, and many interfering factors; internal nucleic acids can be detected by PCR, which has strong specificity and low requirements for sample concentration, but the operation is complex and the equipment requirements are high.

[0007] In view of the disadvantages of traditional exosome separation and detection methods, such as large sample requirements, exosome damage, low recovery rate, complex and time-consuming operation, and high equipment requirements, there is an urgent need for a method to achieve rapid and efficient separation of exosomes and detect the characteristic proteins on the surface of exosomes. Summary of the Invention

[0008] The purpose of the present invention is to provide a surface acoustic wave-based exosome sorting and detection chip, system and method to overcome the defects of the existing technology. While rapidly and accurately purifying and separating exosomes, the present invention can also detect specific proteins carried on the surface of exosomes, achieving one-step exosome separation and detection.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] A surface acoustic wave-based exosome sorting and detection chip includes a piezoelectric substrate, on the surface of which a microchannel is bonded. The microchannel includes a main channel and a first buffer channel, a first separation channel, a second buffer channel, and a second separation channel connected to one side of the main channel. One end of the main channel is provided with a sample inlet, and the other end is connected to a detection channel. The inner surface of the detection channel is modified with a fluorescence detection probe. On the piezoelectric substrate, a first sorting electrode is also provided for separating micron-sized particles in the body fluid sample from the first separation channel, and a second sorting electrode is provided for separating submicron-sized particles in the body fluid sample from the second separation channel;

[0011] The free end of the first buffer channel is provided with a first buffer inlet, the free end of the first separation channel is provided with a first separation outlet, the free end of the second buffer channel is provided with a second buffer inlet, the free end of the second separation channel is provided with a second separation outlet, and the free end of the detection channel is provided with a fluorescence probe outlet.

[0012] Further, in the direction of the body fluid sample flow, the first buffer channel, the first separation channel, the second buffer channel, and the second separation channel are sequentially connected to one side of the main channel, and the first buffer channel and the second buffer channel are arranged in parallel, and the first separation channel and the second separation channel are arranged in parallel.

[0013] Further, the flow direction of the buffer liquid in the first buffer channel and the second buffer channel forms an acute angle with the flow direction of the body fluid sample, and the flow direction of the liquid in the first separation channel and the second separation channel forms an acute angle with the flow direction of the body fluid sample.

[0014] Further, the primary sorting electrode and the secondary sorting electrode are located on the other side of the main flow channel, and the primary sorting electrode is located between the primary buffer flow channel and the primary separation flow channel, while the secondary sorting electrode is located between the secondary buffer flow channel and the secondary separation flow channel.

[0015] Further, the detection flow channel is arranged in an S shape;

[0016] The fluorescence detection probe is a complex composed of a nucleic acid aptamer conjugated with heavy metal nanoparticles and a complementary sequence ssDNA conjugated with a fluorescent group;

[0017] The primary sorting electrode and the secondary sorting electrode are focused surface acoustic wave interdigital electrodes.

[0018] An exosome sorting and detection system based on surface acoustic waves includes a sampling unit, a surface acoustic wave control unit, a sample collector, a fluorescence signal detection unit, and a signal output terminal;

[0019] The sampling unit is connected to the sampling port, the primary buffer liquid inlet, and the secondary buffer liquid inlet. The surface acoustic wave control unit is connected to the primary sorting electrode and the secondary sorting electrode. The sample collector is connected to the primary separation outlet, the secondary separation outlet, and the fluorescence probe outlet. The fluorescence signal detection unit is aligned with the fluorescence probe outlet, and the signal output terminal is connected to the fluorescence signal detection unit.

[0020] Further, the sampling unit includes an air compression pump. The outlet end of the air compressor is connected to a sampling tube through a flow controller. The sampling tube includes a first sampling tube containing the sample to be separated and detected, a second sampling tube containing the primary buffer liquid, and a third sampling tube containing the secondary buffer liquid. The first sampling tube is connected to the sampling port, the second sampling tube is connected to the primary buffer liquid inlet, and the third sampling tube is connected to the secondary buffer liquid inlet.

[0021] Further, the surface acoustic wave control unit includes a signal generating circuit. The output end of the signal generating circuit is connected to a power amplification circuit, and the output end of the power amplification circuit is connected to the primary sorting electrode and the secondary sorting electrode;

[0022] The sample collector includes a primary separation liquid collection tube, a secondary separation liquid collection tube, and a detection sample collection tube. The primary separation liquid collection tube is connected to the primary separation outlet, the secondary separation liquid collection tube is connected to the secondary separation outlet, and the detection sample collection tube is connected to the fluorescence probe outlet.

[0023] Further, the fluorescence signal detection unit includes a laser diode, a dichroic mirror is disposed at the output end of the laser diode, a band-pass filter and a convex lens are sequentially disposed above the dichroic mirror, a photomultiplier tube is disposed above the convex lens, and the output end of the photomultiplier tube is connected to a signal output terminal through a signal detection circuit;

[0024] The signal output terminal includes signal analysis software for collecting, analyzing and recording fluorescence signals and a signal display device for displaying detection results.

[0025] A method for sorting and detecting exosomes based on surface acoustic waves. The sample to be separated and detected is injected through an injection unit. The sample to be separated and detected enters from the injection port, and two-stage buffer solutions enter from the primary buffer inlet and the secondary buffer inlet respectively. After passing through the primary sorting electrode and the secondary sorting electrode, biological particles that affect exosome detection are excluded, and the solution containing exosomes enters the detection flow channel. The exosomes are captured by fluorescence detection probes in the detection flow channel, and the complementary sequence solution connected with a fluorescent group is collected through the sample collector through the fluorescence probe outlet, and the fluorescence intensity is detected by the fluorescence signal detection unit. Further, the concentration of exosomes in the body fluid sample is obtained through the signal output terminal and displayed.

[0026] Compared with the prior art, the present invention has the following beneficial technical effects:

[0027] The present invention provides an integrated exosome separation and detection chip, which adopts a microfluidic chip and surface acoustic wave technology, and can complete the cumbersome separation, cleaning and detection steps adopted in traditional laboratories on the chip, greatly saving time and the workload of operators, and realizing one-step rapid sorting and detection of exosomes.

[0028] The exosome sorting technology proposed by the present invention based on surface acoustic wave technology utilizes the principle that particles of different sizes in a fluid are subjected to different acoustic radiation forces due to different sizes under the same acoustic field radiation. Two-stage separation of exosomes in a body fluid sample is performed on interfering particles of different sizes in human body fluids. In the first stage of separation, micron-sized particles are separated from the flow channel, and in the second stage of separation, sub-micron-sized particles are separated from the flow channel, ensuring high precision and high acquisition rate of sorting. Compared with traditional gradient density centrifugation, the exosome sorting technology proposed by the present invention has lower equipment requirements, and higher precision and acquisition rate.

[0029] The present invention uses nucleic acid aptamers modified on the inner surface of the flow channel as fluorescence detection probes. The highly purified exosomes obtained after separation of the body fluid sample directly enter the detection flow channel for detection without additional collection steps, simplifying the operation. The fluorescence probe has high specificity and low price, and can specifically capture specific proteins on the surface of exosomes, thereby causing changes in fluorescence signals and realizing precise detection of exosomes.

[0030] The system of the present invention can be used for rapid purification and separation of exosomes in a body fluid sample, and for detection of specific proteins carried on the surface of exosomes, achieving one-step separation and detection of exosomes. The body fluid sample and the buffer are respectively loaded into the sampling tubes and pumped into the exosome sorting and detection chip through the sampling system. The body fluid sample enters from the sampling port. After passing through the primary sorting electrode and the secondary sorting electrode, biological particles that will affect exosome detection are excluded, and the solution containing exosomes enters the detection flow channel. Since the nucleic acid aptamer of the specific protein on the surface of exosomes has a stronger affinity for the protein, the nucleic acid aptamer will bind to the protein to capture the exosomes, thereby releasing the complementary sequence linked with a fluorescent group. The solution of the complementary sequence linked with the fluorescent group is collected as a sample through the fluorescent probe outlet, and the fluorescence intensity is detected by the fluorescence detection unit. The signal analysis software collects and analyzes the fluorescence signal to obtain the concentration of exosomes in the body fluid sample, and the detection result is output through the signal output terminal. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings in the specification are used to provide a further understanding of the present invention, and constitute a part of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0032] Figure 1 It is a three-dimensional structure schematic diagram of the exosome sorting and detection chip of the present invention;

[0033] Figure 2 It is a schematic diagram of the principle of exosome sorting and detection based on surface acoustic wave of the present invention;

[0034] Figure 3 It is a planar structure schematic diagram of the exosome sorting and detection chip of the present invention;

[0035] Figure 4 It is a schematic diagram of the structure of the system of the present invention.

[0036] Among them, 1. Sampling unit; 1-1. Air compression pump; 1-2. Flow controller; 1-3. Sampling tube; 2. Surface acoustic wave control unit; 2-1. Signal generation circuit; 2-2. Power amplification circuit; 3. Exosome sorting and detection chip; 3-1. Piezoelectric substrate; 3-2. Microchannel; 3-3. Main channel; 3-4. Primary buffer flow channel; 3-5. Primary separation flow channel; 3-6. Secondary buffer flow channel; 3-7. Secondary separation flow channel; 3-8. Primary buffer liquid inlet; 3-9. Primary separation outlet; 3-10. Secondary buffer liquid inlet; 3-11. Secondary separation outlet; 3-12. Sampling port; 3-13. Detection flow channel; 3-14. Fluorescent probe outlet; 3-15. Primary sorting electrode; 3-16. Secondary sorting electrode; 4. Sample collector; 4-1. Primary separation liquid collection tube; 4-2. Secondary separation liquid collection tube; 4-3. Detection sample collection tube; 5. Fluorescent signal detection unit; 5-1. Laser diode; 5-2. Dichroic mirror; 5-3. Band-pass filter; 5-4. Convex lens; 5-5. Photomultiplier tube; 5-6. Signal detection circuit; 6. Signal output terminal; 6-1. Signal analysis software; 6-2. Signal display device; 7. Fluorescent detection probe; 8. Micron-sized particles; 9. Submicron-sized particles. Detailed implementation mode

[0037] The present invention will be described in detail below with reference to the accompanying drawings:

[0038] An exosome sorting and detection chip based on surface acoustic waves, as shown in the attached Figure 1 and attached Figure 2 and attached Figure 3As shown in the figure, it includes: a piezoelectric substrate 3-1, on one side of the piezoelectric substrate 3-1, there are a primary sorting electrode 3-15 and a secondary sorting electrode 3-16. A microchannel 3-2 is bonded to the upper surface of the piezoelectric substrate 3-1. The body fluid sample enters the microchannel 3-2 from the sample inlet 3-12, and the buffer solution enters from the primary buffer inlet 3-8. The primary sorting electrode 3-15 generates an acoustic radiation force-weak acoustic field to separate larger micron-sized particles 8 (including particles such as cell debris and platelets) from the primary separation outlet 3-9. Subsequently, the buffer solution is introduced from the secondary buffer inlet 3-10, and the secondary sorting electrode 3-16 generates an acoustic radiation force-strong acoustic field to separate submicron-sized particles 9 (including particles such as apoptotic bodies and microvesicles) larger than exosomes from the secondary separation outlet 3-11. Finally, the exosomes enter the detection channel 3-13, and the fluorescence detection probe 7 modified on the inner surface of the detection channel specifically captures and detects the exosomes expressing surface characteristic proteins; the microchannel 3-2 includes a sample inlet 3-12, and the sample inlet 3-12 is connected to the main channel 3-3; the primary buffer inlet 3-8 is connected to the primary buffer channel 3-4, and the primary separation outlet 3-9 is connected to the primary separation channel 3-5; the secondary buffer inlet 3-10 is connected to the secondary buffer channel 3-6, and the secondary separation outlet 3-11 is connected to the secondary separation channel 9-1; the main channel 3-3 is also connected to the detection channel 3-13, and the end of the detection channel 3-13 is connected to the fluorescence probe outlet 3-14.

[0039] The primary sorting electrode 3-15 and the secondary sorting electrode 3-16 are focused surface acoustic wave interdigital electrodes. When the primary sorting electrode 3-15 and the secondary sorting electrode 3-16 are turned on, they can respectively form acoustic fields with different acoustic radiation forces. The acoustic radiation force of the acoustic field formed by the primary sorting electrode 3-15 is weak and can drive larger micron-sized particles 8; the acoustic radiation force of the acoustic field formed by the secondary sorting electrode 3-16 is strong and can drive smaller submicron-sized particles 9 larger than exosomes.

[0040] The fluorescence detection probe 7 is a complex composed of a nucleic acid aptamer conjugated with heavy metal nanoparticles and a complementary sequence ssDNA conjugated with a fluorescent group based on the fluorescence resonance energy transfer effect between the nucleic acid aptamer and the fluorescent group. The nucleic acid aptamer can specifically capture protein markers expressed on the surface of exosomes, such as CD63, CD81, CD9, EpCAM, etc. The heavy metal nanoparticles have fluorescence quenching properties, such as gold nanoparticles. The fluorescent group includes but is not limited to carbon quantum dots, FAM, FITC, Cy3, Cy5, etc.

[0041] An exosome sorting and detection system based on surface acoustic waves, as shown in the appendix Figure 4As shown in the figure, it includes a sample injection unit 1, a surface acoustic wave control unit 2, a microfluidic chip 3, a sample collector 4, a fluorescence signal detection unit 5, and a signal output terminal 6.

[0042] The sample injection unit 1 includes an air compressor pump 1-1, a flow controller 1-2, and a sample injection tube 1-3. Samples to be separated and detected and buffer solution are respectively loaded in the sample injection tube 1-3. The air compressor pump 1-1 provides pressure to drive the liquid into the microfluidic chip 3, and the flow controller 1-2 can control the flow rate of the liquid in the microchannel system.

[0043] The surface acoustic wave control unit 2 includes a signal generation circuit 2-1 and a power amplification circuit 2-2. The signal generation circuit 2-1 can generate a high-frequency alternating current signal and input it into the interdigital electrodes in the microfluidic chip through the power amplification circuit 2-2. The piezoelectric substrate generates resonance under the excitation of the high-frequency voltage of the interdigital electrodes to form a surface acoustic wave. The parameters of the surface acoustic wave can be adjusted by controlling the frequency of the signal generator and the power amplification factor.

[0044] The microfluidic chip 3 is the above-mentioned exosome sorting and detection chip based on surface acoustic waves.

[0045] The sample collector 4 includes a primary separation liquid collection tube 4-1, a secondary separation liquid collection tube 4-2, and a detection sample collection tube 4-3. Among them, the primary separation liquid collection tube 4-1 is connected to the primary separation outlet, the secondary separation liquid collection tube 4-2 is connected to the secondary separation outlet, and the detection sample collection tube 4-3 is connected to the fluorescence probe outlet.

[0046] The fluorescence signal detection unit 5 includes a laser diode 5-1, a dichroic mirror 5-2, a band-pass filter 5-3, a convex lens 5-4, a photomultiplier tube 5-5, and a signal detection circuit 5-6. The laser generated by the laser diode 5-1 is reflected by the dichroic mirror 5-2 and then irradiated onto the microfluidic cell sorting chip 3. When the detection sample contains a fluorescence probe, the fluorescent label generates fluorescence under the excitation of the laser. The fluorescence then passes through the dichroic mirror 5-2, the band-pass filter 5-3, and the convex lens 5-4 in sequence and enters the photomultiplier tube 5-5. The photomultiplier tube 5-5 converts the fluorescence signal into an electrical signal, which is detected by the detection circuit 5-6 and sent to the signal output terminal 6.

[0047] The signal output terminal 6 includes a signal analysis software 6-1 and a signal display device 6-2. The signal analysis software 6-1 is software for collecting, analyzing, and recording fluorescence signals, and the signal display device 6-2 is a two-dimensional image display.

[0048] A method for sorting and detecting exosomes based on surface acoustic waves, wherein the sample to be separated and detected is injected through an injection unit 1, and the sample to be separated and detected enters from an injection port 3-12, and two-level buffer solutions enter from a primary buffer inlet 3-8 and a secondary buffer inlet 3-10 respectively, and after the sample to be separated and detected passes through a primary sorting electrode 3-15 and a secondary sorting electrode 3-16, after biological particles that may affect the detection of exosomes are eliminated, a solution containing exosomes enters a detection channel 3-13, and exosomes are captured by a fluorescent detection probe 7 in the detection channel 3-13, and a complementary sequence solution connected with a fluorescent group is collected through a sample collector 4 through a fluorescent probe outlet 3-14, and the fluorescence intensity is detected by a fluorescent signal detection unit 5, and further the concentration of exosomes in the body fluid sample is obtained and displayed through a signal output terminal 6.

[0049] The principle of the present invention is:

[0050] The surface acoustic wave generated by the interdigitated electrodes propagates along the surface of the piezoelectric substrate. Particles of different sizes are subjected to different acoustic radiation forces and viscous forces in the surface acoustic wave field, wherein the viscous force is proportional to the radius of the particle, and the acoustic radiation force is proportional to the volume of the particle. Under the combined action of the acoustic radiation force and the viscous force, the larger cell fragments, platelets and other micron-sized particles in the body fluid are subjected to the weaker acoustic field generated by the primary sorting electrode 3-15, and the flow path is deviated, thereby flowing to the primary separation channel 3-5, while the smaller submicron-sized particles larger than the exosomes will flow to the next-level sorting electrode, and under the stronger acoustic field generated by the secondary sorting electrode 3-16, the flow path is deviated and flows to the secondary separation channel 9-1; at this point, the cell fragments, platelets and submicron-sized particles in the body fluid that can interfere with the detection of exosomes are separated from the flow channel. For the separated exosomes, a fluorescent detection probe prepared based on the fluorescence resonance energy transfer (FRET) effect is used for detection, and the effect depends on the distance between the FRET donor-acceptor pair. In the present invention, the fluorescent group is used as a donor and the heavy metal nanoparticles are used as an acceptor. When the aptamer connected to the heavy metal nanoparticles binds to the complementary sequence connected to the fluorescent group, the donor-acceptor pair is close and the fluorescence of the fluorescent group is quenched; when the exosomes appear, due to the stronger affinity between the aptamer and the specific protein on the surface of the exosome, the aptamer binds to the specific protein on the surface of the exosome, releasing the complementary sequence connected to the fluorescent group, greatly increasing the distance between the donor-acceptor pair, resulting in the fluorescence recovery of the fluorescent group. By detecting the change in the fluorescence intensity of the fluorescent group, the concentration of exosomes in the body fluid sample can be inferred.

[0051] The present invention is described in further detail below in conjunction with embodiments:

[0052] Example 1

[0053] An acoustic surface wave-based exosome sorting and detection chip, comprising: a lithium niobate piezoelectric substrate, on one side of the lithium niobate piezoelectric substrate, there are a primary sorting electrode 3-15 and a secondary sorting electrode 3-16. A polydimethylsiloxane (PDMS) microchannel 3-2 is bonded to the upper surface of the piezoelectric substrate 3-1. A human whole blood sample enters the microchannel 3-2 from the sample inlet 3-12, and 0.1×PBS buffer (pH = 7.4) enters from the primary buffer inlet 3-8. The primary sorting electrode 3-15 generates a sound field with relatively weak acoustic radiation force to separate larger micron-sized particles 8 from the primary separation outlet 3-9. Subsequently, buffer is introduced from the secondary buffer inlet 3-10, and the secondary sorting electrode 3-16 generates a sound field with relatively strong acoustic radiation force to separate submicron-sized particles 9 larger than exosomes from the secondary separation outlet 3-11. Finally, the exosomes enter the detection channel 3-13, and the fluorescence detection probe 7 modified on the inner surface of the detection channel specifically captures and detects the exosomes expressing CD63 protein.

[0054] The primary sorting electrode 3-15 and the secondary sorting electrode 3-16 are focused surface acoustic wave interdigital electrodes, and when the primary sorting electrode 3-15 and the secondary sorting electrode 3-16 are turned on, they can respectively form sound fields with different magnitudes of acoustic radiation force. The sound field formed by the primary sorting electrode 3-15 has relatively weak acoustic radiation force and can drive larger micron-sized particles 8; the sound field formed by the secondary sorting electrode 3-16 has relatively strong acoustic radiation force and can drive smaller submicron-sized particles 9 larger than exosomes. The primary sorting electrode 1 is a focused arc-shaped interdigital electrode, with an electrode width of 10 μm, an electrode pitch of 10 μm, and 15 electrode pairs. The secondary sorting electrode 2 is a focused arc-shaped interdigital electrode, with an electrode width of 5 μm, an electrode pitch of 5 μm, and 15 electrode pairs.

[0055] The microchannel 3-2 includes a sample inlet 3-12, and the sample inlet 3-12 is connected to the main channel 3-3; the primary buffer inlet 3-8 is connected to the primary buffer channel 3-4, and the primary separation outlet 3-9 is connected to the primary separation channel 3-5; the secondary buffer inlet 3-10 is connected to the secondary buffer channel 3-6, and the secondary separation outlet 3-11 is connected to the secondary separation channel 9-1; the main channel 3-3 is also connected to the detection channel 3-13, and the end of the detection channel is connected to the fluorescence probe outlet 3-14.

[0056] The described fluorescence detection probe 7 is a complex composed of an aptamer conjugated with gold nanoparticles and a complementary sequence ssDNA conjugated with a fluorophore based on the fluorescence resonance energy transfer effect between the nucleic acid aptamer of CD63 protein on the surface of exosomes and the fluorophore. The nucleic acid aptamer can specifically capture the CD63 protein expressed on the surface of exosomes. The diameter of the gold nanoparticles is about 13 nm and they have the property of fluorescence quenching; the fluorophore is Cy5 fluorescent dye. The nucleic acid aptamer sequence of the CD63 protein is: 5’-COOH-CACCCCACCTCGCTCCCGTGACACTAATGCTA-SH-C6-3’; the complementary sequence ssDNA sequence is: 5’-TAGCATTAGTGTC-C6-Cy5-3’.

[0057] A method for integrated sorting and detection of exosomes based on a surface acoustic wave chip. The body fluid sample enters from the sample inlet 3-12. After passing through the primary sorting electrode 3-15 and the secondary sorting electrode 3-16, after excluding the biological particles that will affect the detection of exosomes, the solution containing exosomes enters the detection flow channel 3-13. Since the nucleic acid aptamer of the CD63 protein on the surface of exosomes has a stronger affinity for this protein, the nucleic acid aptamer will bind to the CD63 protein, capture the exosomes, and thus release the complementary sequence connected with the Cy5 fluorescent dye. The solution of the complementary sequence connected with the Cy5 fluorescent dye is collected through the fluorescence probe outlet 3-14 and the fluorescence intensity is detected at 670 nm under the excitation wavelength of 645 nm, and further the concentration of exosomes in the body fluid sample is obtained.

[0058] Example 2

[0059] A surface acoustic wave-based exosome sorting and detection chip includes: a lithium niobate piezoelectric substrate, and a primary sorting electrode 3-15 and a secondary sorting electrode 3-16 are provided on one side of the lithium niobate piezoelectric substrate. A PDMS microchannel 3-2 is bonded to the upper surface of the piezoelectric substrate 3-1. The whole blood sample of colon cancer patients enters the microchannel 3-2 from the sample inlet 3-12, and 0.1×PBS buffer solution (pH = 7.4) enters from the primary buffer inlet 3-8. The primary sorting electrode 3-15 generates a sound field with relatively weak acoustic radiation force to separate the micron-sized particles 8 with larger sizes from the primary separation outlet 3-9; then buffer solution is introduced from the secondary buffer inlet 3-10, and the secondary sorting electrode 3-16 generates a sound field with relatively strong acoustic radiation force to separate the submicron-sized particles 9 larger than exosomes from the secondary separation outlet 3-11. Finally, the exosomes enter the detection flow channel 3-13, and the fluorescence detection probe 7 modified on the inner surface of the detection flow channel specifically captures and detects the exosomes expressing CEA protein.

[0060] The first-stage sorting electrode 3-15 and the second-stage sorting electrode 3-16 are focused surface acoustic wave interdigital electrodes. When the first-stage sorting electrode 3-15 and the second-stage sorting electrode 3-16 are turned on, they can respectively form sound fields with different acoustic radiation forces. The sound field formed by the first-stage sorting electrode 3-15 has a relatively weak acoustic radiation force and can drive micron-sized particles 8 with larger sizes; the sound field formed by the second-stage sorting electrode 3-16 has a relatively strong acoustic radiation force and can drive submicron-sized particles 9 with smaller sizes that are larger than exosomes. The first-stage sorting electrode 1 is a focused arc-shaped interdigital electrode with an electrode width of 10 μm, an electrode pitch of 10 μm, and 15 electrode pairs. The second-stage sorting electrode 2 is a focused arc-shaped interdigital electrode with an electrode width of 5 μm, an electrode pitch of 5 μm, and 15 electrode pairs.

[0061] The microchannel 3-2 includes a sample inlet 3-12, and the sample inlet 3-12 is connected to the main channel 3-3; the first-stage buffer liquid inlet 3-8 is connected to the first-stage buffer liquid channel 3-4, and the first-stage separation outlet 3-9 is connected to the first-stage separation channel 3-5; the second-stage buffer liquid inlet 3-10 is connected to the second-stage buffer liquid channel 3-6, and the second-stage separation outlet 3-11 is connected to the second-stage separation channel 9-1; the main channel 3-3 is also connected to the detection channel 3-13, and the end of the detection channel is connected to the fluorescence probe outlet 3-14.

[0062] The fluorescence detection probe 7 is based on the fluorescence resonance energy transfer effect between the nucleic acid aptamer of the CEA protein on the surface of exosomes and the fluorescent group, and is a complex composed of a nucleic acid aptamer conjugated with gold nanoparticles and a complementary sequence ssDNA conjugated with a fluorescent group. The nucleic acid aptamer can specifically capture the CEA protein expressed on the surface of exosomes. The diameter of the gold nanoparticles is about 5 nm and has the property of fluorescence quenching; the fluorescent group is a FAM fluorescent dye. The nucleic acid aptamer sequence of the CEA protein is: 5’-COOH-TCGCGCGAGTCGTCTGGGGAACCATCGAGTTACACCGACCTTCTATGTGCGGCCCCCCGCATCGTCCTCCC-SH-C6-3’; the complementary sequence ssDNA sequence is: 5’-GGGAGGACGATGC-C6-FAM-3’.

[0063] An integrated exosome sorting and detection method based on a surface acoustic wave chip. The whole blood sample enters from the sample inlet 3-12. After passing through the primary sorting electrode 3-15 and the secondary sorting electrode 3-16, biological particles that will affect exosome detection are excluded. Then, the solution containing exosomes enters the detection flow channel 3-13. Since the nucleic acid aptamer of the CEA protein on the surface of exosomes has a stronger affinity for this protein, the nucleic acid aptamer will bind to the CEA protein, capture the exosomes, and thus release the complementary sequence linked to the FAM fluorescent dye. The solution of the complementary sequence linked to the FAM fluorescent dye is collected through the fluorescence probe outlet 3-14 and the fluorescence intensity is detected at 522 nm under an excitation wavelength of 494 nm, and further the concentration of exosomes expressing the CEA protein in the whole blood sample is obtained.

[0064] Example 3

[0065] An exosome sorting and detection chip based on surface acoustic waves, comprising: a lithium niobate piezoelectric substrate, on one side of which a primary sorting electrode 3-15 and a secondary sorting electrode 3-16 are provided. The upper surface of the piezoelectric substrate 3-1 is bonded with a polydimethylsiloxane (PDMS) microchannel 3-2. The whole blood sample of breast cancer patients enters the microchannel 3-2 from the sample inlet 3-12, and the 0.1×PBS buffer solution (pH = 7.4) enters from the primary buffer liquid inlet 3-8. The primary sorting electrode 3-15 generates a sound field with relatively weak acoustic radiation force to separate the larger micron-sized particles 8 from the primary separation outlet 3-9; then the buffer solution is introduced from the secondary buffer liquid inlet 3-10, and the secondary sorting electrode 3-16 generates a sound field with relatively strong acoustic radiation force to separate the submicron-sized particles 9 larger than exosomes from the secondary separation outlet 3-11. Finally, the exosomes enter the detection flow channel 3-13, and the fluorescent detection probe 7 modified on the inner surface of the detection flow channel specifically captures and detects the exosomes expressing the EpCAM protein.

[0066] The primary sorting electrode 3-15 and the secondary sorting electrode 3-16 are focused surface acoustic wave interdigital electrodes. When the primary sorting electrode 3-15 and the secondary sorting electrode 3-16 are turned on, they can respectively form sound fields with different magnitudes of acoustic radiation force. The sound field formed by the primary sorting electrode 3-15 has relatively weak acoustic radiation force and can drive the larger micron-sized particles 8; the sound field formed by the secondary sorting electrode 3-16 has relatively strong acoustic radiation force and can drive the smaller submicron-sized particles 9 larger than exosomes. The primary sorting electrode 1 is a focused arc-shaped interdigital electrode with an electrode width of 10 μm, an electrode pitch of 10 μm, and 15 electrode pairs. The secondary sorting electrode 2 is a focused arc-shaped interdigital electrode with an electrode width of 5 μm, an electrode pitch of 5 μm, and 15 electrode pairs.

[0067] The described microchannel 3-2 includes a sample inlet 3-12, and the sample inlet 3-12 is connected to the main channel 3-3; the primary buffer inlet 3-8 is connected to the primary buffer channel 3-4, and the primary separation outlet 3-9 is connected to the primary separation channel 3-5; the secondary buffer inlet 3-10 is connected to the secondary buffer channel 3-6, and the secondary separation outlet 3-11 is connected to the secondary separation channel 9-1; the main channel 3-3 is also connected to the detection channel 3-13, and the end of the detection channel is connected to the fluorescent probe outlet 3-14.

[0068] The described fluorescence detection probe 7 is based on the fluorescence resonance energy transfer effect between the nucleic acid aptamer of the EpCAM protein on the surface of exosomes and the fluorescent group, and is a complex composed of a nucleic acid aptamer conjugated with gold nanoparticles and a complementary sequence ssDNA conjugated with a fluorescent group. The nucleic acid aptamer can specifically capture the EpCAM protein expressed on the surface of exosomes. The diameter of the gold nanoparticles is about 20 nm and has the property of fluorescence quenching; the fluorescent group is graphene quantum dots (GQD). The nucleic acid aptamer sequence of the EpCAM protein is: 5’-COOH-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-SH-C6-3’; the complementary sequence ssDNA sequence is: 5’-CAGGCCAACCCCC-C6-GQD-3’.

[0069] Example 4

[0070] This example provides an exosome separation and detection system based on a microfluidic chip and surface acoustic wave technology, including a sample injection unit 1, a surface acoustic wave control unit 2, an exosome sorting and detection chip 3, a sample collector 4, a fluorescence signal detection unit 5, and a signal output terminal 6.

[0071] The sample injection unit 1 includes an air compressor pump 1-1, a flow controller 1-2, and a sample injection tube 1-3. The sample injection tube 1-3 contains the sample to be separated and detected and the buffer solution respectively. The air compressor pump 1-1 provides pressure to drive the liquid into the exosome sorting and detection chip 3, and the flow controller 1-2 can control the flow rate of the liquid in the microchannel system.

[0072] The surface acoustic wave control unit 2 includes a signal generator 2-1 and a power amplifier 2-2. The signal generator 2-1 can generate a high-frequency alternating current signal and then input it into the interdigital electrodes in the exosome sorting and detection chip 3 through the power amplifier 2-2. The lithium niobate piezoelectric substrate generates resonance under the excitation of the high-frequency voltage of the interdigital electrodes to form a surface acoustic wave. The parameters of the surface acoustic wave can be adjusted by controlling the frequency and power amplification factor of the signal generator 2-1.

[0073] The exosome sorting and detection chip 3 adopts the structure shown in Examples 1-3.

[0074] The sample collector 4 includes a primary separation liquid collection tube 4-1, a secondary separation liquid collection tube 4-2, and a detection sample collection tube 4-3. The primary separation liquid collection tube 4-1 is connected to the primary separation outlet 3-9, the secondary separation liquid collection tube 4-2 is connected to the secondary separation outlet 3-11, and the detection sample collection tube 4-3 is connected to the fluorescence probe outlet 3-14.

[0075] The fluorescence signal detection unit 5 includes a laser diode 5-1, a dichroic mirror 5-2, a band-pass filter 5-3, a convex lens 5-4, a photomultiplier tube 5-5, and a signal detection circuit 5-6. The laser generated by the laser diode 5-1 is reflected by the dichroic mirror 5-2 and then irradiated onto the exosome sorting and detection chip 3. When the detection sample contains a fluorescence probe, the fluorescent label generates fluorescence under the excitation of the laser. The fluorescence then passes through the dichroic mirror 5-2, the band-pass filter 5-3, and the convex lens 5-4 in sequence and enters the photomultiplier tube 5-5. The photomultiplier tube 5-5 converts the fluorescence signal into an electrical signal, and the signal detection circuit 5-6 detects and displays the fluorescence intensity.

[0076] The signal output terminal 6 is a tablet computer. The tablet computer is installed with signal analysis software 6-1, which can collect, analyze, and record the fluorescence signal, and also has a signal display device 6-2.

[0077] A method for integrated sorting and detection of exosomes based on a surface acoustic wave chip. The whole blood sample enters from the sample inlet 3-12. After passing through the primary sorting electrode 3-15 and the secondary sorting electrode 3-16, biological particles that will affect exosome detection are excluded. The solution containing exosomes enters the detection flow channel 3-13. Since the nucleic acid aptamer of the EpCAM protein on the surface of exosomes has a stronger affinity for this protein, the nucleic acid aptamer will bind to the EpCAM protein, capture the exosomes, and thus release the complementary sequence connected with GQD. The solution of the complementary sequence connected with GQD is collected through the fluorescence probe outlet 3-14, and the fluorescence intensity is detected at 445 nm under an excitation wavelength of 355 nm, so as to further obtain the concentration of exosomes expressing the EpCAM protein in the whole blood sample.

[0078] The above content is only to illustrate the technical idea of the present invention and cannot be used to limit the protection scope of the present invention. Any modification made on the basis of the technical solution according to the technical idea proposed by the present invention falls within the protection scope of the claims of the present invention.

Claims

1. A surface acoustic wave-based exosome sorting and detection chip, characterized in that, it includes a piezoelectric substrate (3-1), on the surface of the piezoelectric substrate (3-1) there is bonded a microfluidic channel (3-2), the microfluidic channel (3-2) includes a main channel (3-3) and a primary buffer fluid channel (3-4), a primary separation channel (3-5), a secondary buffer fluid channel (3-6) and a secondary separation channel (3-7) connected to one side of the main channel (3-3), one end of the main channel (3-3) is provided with a sample injection port (3-12), the other end is connected to a detection channel (3-13), the inner surface of the detection channel (3-13) is modified with a fluorescence detection probe (7), on the piezoelectric substrate (3-1) there is also provided a primary sorting electrode (3-15) for separating micron-sized particles (8) in the body fluid sample from the primary separation channel (3-5) and a secondary sorting electrode (3-16) for separating submicron-sized particles (9) in the body fluid sample from the secondary separation channel (3-7); the free end of the primary buffer fluid channel (3-4) is provided with a primary buffer fluid inlet (3-8), the free end of the primary separation channel (3-5) is provided with a primary separation outlet (3-9), the free end of the secondary buffer fluid channel (3-6) is provided with a secondary buffer fluid inlet (3-10), the free end of the secondary separation channel (3-7) is provided with a secondary separation outlet (3-11), the free end of the detection channel (3-13) is provided with a fluorescence probe outlet (3-14); According to the flow direction of the body fluid sample, the primary buffer fluid channel (3-4), the primary separation channel (3-5), the secondary buffer fluid channel (3-6) and the secondary separation channel (3-7) are sequentially connected to one side of the main channel (3-3), and the primary buffer fluid channel (3-4) and the secondary buffer fluid channel (3-6) are arranged in parallel, the primary separation channel (3-5) and the secondary separation channel (3-7) are arranged in parallel; the detection channel (3-13) is arranged in an S shape; the fluorescence detection probe (7) is a complex composed of a nucleic acid aptamer coupled with heavy metal nanoparticles and a complementary sequence ssDNA coupled with a fluorescent group; the primary sorting electrode (3-15) and the secondary sorting electrode (3-16) are focused surface acoustic wave interdigital electrodes.

2. The surface acoustic wave-based exosome sorting and detection chip according to claim 1, characterized in that, the flow direction of the buffer fluid in the primary buffer fluid channel (3-4) and the secondary buffer fluid channel (3-6) forms an acute angle with the flow direction of the body fluid sample, and the flow direction of the liquid in the primary separation channel (3-5) and the secondary separation channel (3-7) forms an acute angle with the flow direction of the body fluid sample.

3. The surface acoustic wave-based exosome sorting and detection chip according to claim 1, characterized in that, The primary sorting electrode (3-15) and the secondary sorting electrode (3-16) are located on the other side of the main flow channel (3-3), and the primary sorting electrode (3-15) is located between the primary buffer flow channel (3-4) and the primary separation flow channel (3-5), while the secondary sorting electrode (3-16) is located between the secondary buffer flow channel (3-6) and the secondary separation flow channel (3-7).

4. An exosome sorting and detection system based on surface acoustic wave, based on the exosome sorting and detection chip according to any one of claims 1-3, Characterized in that, it includes a sample injection unit (1), a surface acoustic wave control unit (2), a sample collector (4), a fluorescence signal detection unit (5) and a signal output terminal (6); The sample injection unit (1) is connected to the sample injection port (3-12), the primary buffer liquid inlet (3-8) and the secondary buffer liquid inlet (3-10), the surface acoustic wave control unit (2) is connected to the primary sorting electrode (3-15) and the secondary sorting electrode (3-16), the sample collector (4) is connected to the primary separation outlet (3-9), the secondary separation outlet (3-11) and the fluorescence probe outlet (3-14), the fluorescence signal detection unit (5) is aligned with the fluorescence probe outlet (3-14), and the signal output terminal (6) is connected to the fluorescence signal detection unit (5).

5. An exosome sorting and detection system based on surface acoustic wave according to claim 4, Characterized in that, the sample injection unit (1) includes an air compression pump (1-1), the outlet end of the air compressor (1-1) is connected to a sampling tube (1-3) through a flow controller (1-2), the sampling tube (1-3) includes a first sampling tube containing the sample to be separated and detected, a second sampling tube containing the primary buffer liquid and a third sampling tube containing the secondary buffer liquid, the first sampling tube is connected to the sample injection port (3-12), the second sampling tube is connected to the primary buffer liquid inlet (3-8), and the third sampling tube is connected to the secondary buffer liquid inlet (3-10).

6. An exosome sorting and detection system based on surface acoustic wave according to claim 4, Characterized in that, the surface acoustic wave control unit (2) includes a signal generation circuit (2-1), the output end of the signal generation circuit (2-1) is connected to a power amplification circuit (2-2), and the output end of the power amplification circuit (2-2) is connected to the primary sorting electrode (3-15) and the secondary sorting electrode (3-16); The sample collector (4) includes a primary separation liquid collection tube (4-1), a secondary separation liquid collection tube (4-2) and a detection sample collection tube (4-3), the primary separation liquid collection tube (4-1) is connected to the primary separation outlet (3-9), the secondary separation liquid collection tube (4-2) is connected to the secondary separation outlet (3-11), and the detection sample collection tube (4-3) is connected to the fluorescence probe outlet (3-14).

7. An exosome sorting and detection system based on surface acoustic wave according to claim 4, Characterized in that, The fluorescence signal detection unit (5) includes a laser diode (5-1). A dichroic mirror (5-2) is arranged at the output end of the laser diode (5-1). A band-pass filter (5-3) and a convex lens (5-4) are sequentially arranged above the dichroic mirror (5-2). A photomultiplier tube (5-5) is arranged above the convex lens (5-4). The output end of the photomultiplier tube (5-5) is connected to a signal output terminal (6) through a signal detection circuit (5-6); The signal output terminal (6) includes a signal analysis software (6-1) for collecting, analyzing, and recording fluorescence signals and a signal display device (6-2) for displaying detection results.

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