CN113005033B

CN113005033B - Device and method for capturing and separating exosomes and cells in biological sample

Info

Publication number: CN113005033B
Application number: CN202110203960.8A
Authority: CN
Inventors: 王晗; 刘君元
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2021-02-24
Filing date: 2021-02-24
Publication date: 2022-08-26
Anticipated expiration: 2041-02-24
Also published as: CN113005033A

Abstract

The invention discloses a device for capturing and separating exosomes and cells in a biological sample, which comprises: integrated reaction chip, integrated reaction chip is including integrated reaction chamber, integrated reaction chamber has the entry, specific cell that communicate in proper order and catches chamber, cell interception chamber, exosome and catches chamber and export, specific cell catches the structure that the intracavity has and can decorate specific cell binding element, the cell interception intracavity is equipped with the interception array that is used for intercepting the cell, be equipped with the hole on the interception array, the size in hole can make exosome pass through and can not make the cell pass through, exosome catches the structure that the intracavity has the exosome binding element that can decorate. The invention also discloses a method for capturing and separating exosomes and cells in a biological sample by adopting the device for capturing and separating exosomes and cells in the biological sample.

Description

Device and method for capturing and separating exosomes and cells in biological sample

Technical Field

The invention relates to the technical field of biomedical engineering, in particular to a capturing and separating device and a capturing and separating method for exosomes and cells in a biological sample.

Background

Exosomes are vesicle-like bodies having polymorphism, originate from multivesicular bodies in endocytic systems, and include many substances such as proteins, lipids, mRNA, miRNA, lncRNA, and DNA in cytoplasm. Exosomes are membrane vesicles with a diameter of 30-120nm that are secreted intracellularly to the exterior of the cell. Exosomes were originally found as waste thought to be excreted by cells. Until 2007, it was discovered that exosomes could serve as a mechanism of intercellular gene communication, and exosomes were of interest to scientists. Almost all cells in humans produce and secrete exosomes, which are detectable in biological samples such as blood, urine, saliva, cerebrospinal fluid, and the like. Taking a biological sample as an example, the protein contained in the exosome of the biological sample is derived from glomerulus, renal tubule, prostate, bladder cell, etc., and can be used as a biological marker of urinary system diseases. Therefore, the proteomics method for developing the exosomes of the biological sample plays an important role in developing proteomics analysis on the exosomes of the biological sample, and searching potential novel urinary system disease biomarkers and in applications such as disease diagnosis and treatment. Based on this, how to capture target exosomes in biological samples efficiently is the key to implementing this method.

The components in biological samples are very complex and contain not only exosomes but also cells, microvesicles, proteins, etc. Different from samples such as plasma and cell culture supernatant, biological sample samples are relatively more concentrated, and components are more complex, so that how to avoid the influence of high-concentration contaminating proteins and other impurities on the extraction of exosomes of the biological sample needs to be considered. Currently, the common methods for separating exosomes from biological samples include ultracentrifugation, filtration, chemical precipitation, and affinity precipitation. Ultracentrifugation exploits the differences in particle size/density, particle size, of exosomes from other components, but because exosomes are very small in volume (particle size 40-120nm) and overlap volumetrically with other components in the sample, such as microbubbles, their isolation and purification is difficult and the exosomes are damaged. The filtration method separates exosomes and proteins through a mesh-like structure, which is easily clogged and causes physical damage to exosomes during the separation process. Chemical precipitation methods that precipitate exosomes based on changing the solubility of exosomes based on changing the properties of the fluid; the affinity precipitation method realizes separation based on the affinity of exosomes for different substrates, and both have the defects of low enrichment efficiency, loss of part of exosomes and difficulty in obtaining sufficient amount of exosomes for subsequent analysis experiments. In addition, the above method for separating exosomes cannot simultaneously separate and analyze indices other than exosomes in a biological sample, such as specific epithelial cells.

Disclosure of Invention

Therefore, it is necessary to provide a device and a method for capturing and separating exosomes and cells in a biological sample, which do not destroy exosomes, have high exosome enrichment efficiency, and can simultaneously separate multiple indexes in the biological sample, in order to overcome the defects of the conventional exosome separation method.

A device for trapping and separating exosomes and cells in a biological sample, comprising:

integrated reaction chip, integrated reaction chip includes integrated reaction chamber, integrated reaction chamber has entry, specific cell that communicate in proper order and catches chamber, cell interception chamber, exosome and catches chamber and export, the cell interception intracavity is equipped with the interception array that is used for intercepting the cell, be equipped with the hole on the interception array, the size in hole can make exosome pass through and can not make the cell pass through.

In some of these embodiments, a stepped configuration of turbulence plate bodies is provided within the exosome-capturing chamber.

In some of these embodiments, the turbulence plate body is provided in plurality, and a plurality of turbulence plate bodies are arranged in parallel with respect to each other.

In some of these embodiments, the turbulence plate extends horizontally and forms a vertical protrusion.

In some of these embodiments, the height of the protrusions is from 5 μm to 100 μm.

In some embodiments, the exosome-binding unit is conjugated to the turbulence plate body.

In some of these embodiments, the pores on the intercepting array have a pore size of 3 μm or less.

In some of these embodiments, the specific cell-capture chamber has a specific cell-binding element conjugated to the inner wall of the chamber.

In some embodiments, a liquid adding cavity is communicated between the cell intercepting cavity and the exosome capturing cavity, and a liquid adding inlet is arranged on the liquid adding cavity.

In some of these embodiments, the feeding chamber consists of an elongated chamber connected to the cell intercepting chamber, a protruding chamber located between the elongated chamber and the exosome-capturing chamber and protruding to both sides of the integrated reaction chamber.

In some embodiments, the integrated reaction chamber has a specific cell capturing chamber inlet, a specific cell capturing chamber outlet, a cell intercepting chamber inlet, a cell intercepting chamber outlet, an exosome capturing chamber inlet and an exosome capturing chamber outlet in sequence from the inlet to the outlet of the integrated reaction chamber, the specific cell capturing chamber has a width that increases and then decreases from the specific cell capturing chamber inlet to the specific cell capturing chamber outlet, the cell intercepting chamber has a width that increases and then decreases from the cell intercepting chamber inlet to the cell intercepting chamber outlet, and the exosome capturing chamber has a width that increases and then decreases from the exosome capturing chamber inlet to the exosome capturing chamber outlet.

In some of these embodiments, the separation device comprises a pressure pump for providing a pressure to flow the biological sample from the inlet of the integrated reaction chamber to the outlet of the integrated reaction chamber.

A method for capturing and separating exosomes and cells in a biological sample adopts a device for capturing and separating exosomes and cells in the biological sample, and comprises the following steps:

and injecting a biological sample into the integrated reaction chamber from an inlet of the integrated reaction chamber, capturing specific cells by a specific cell capture chamber pre-conjugated with a specific cell binding unit, capturing non-specific cells at the front end of the cell interception chamber, and capturing exosomes by an exosome capture chamber pre-conjugated with an exosome binding unit.

In some of these embodiments, further comprising:

and adding magnetic beads which are provided with fluorescent groups and conjugated with a second exosome binding unit into the exosome capturing cavity, binding exosomes captured by the exosome binding unit, introducing a cleaning solution, washing away unbound magnetic beads, and detecting the fluorescence value in the exosome capturing cavity.

In some of these embodiments, the exosome-binding unit is conjugated within the exosome-capture chamber by a disulfide bond.

In some of these embodiments, further comprising:

adding a disulfide bond cleaving reagent to the exosome-exosome binding unit complex to detach the exosome-exosome binding unit complex from the exosome-capture chamber, washing the exosome-capture chamber with a buffer to collect the exosome-exosome binding unit complex.

The invention provides a high-efficiency capturing and separating method and a device without damaging exosomes, wherein a biological sample to be separated firstly passes through a specific cell capturing cavity, and a target specific cell is captured in the specific cell capturing cavity; then the biological sample passes through the cell interception cavity, the interception array is provided with pores, and the cells are totally intercepted at the front end of the cell interception cavity, so that exosomes enter the rear end of the cell interception cavity; the acellular biological sample passes through the exosome capturing cavity, and the exosome is captured, so that the capturing, separation and collection of the cells and the exosome are realized. According to the selection of the specific cell capturing cavity for the specific cell binding unit and the selection of the exosome capturing cavity for the exosome binding unit, the capturing, separating and collecting of the specific cell or the specific exosome can be realized. The invention can realize efficient capture and enrichment of the exosome without damaging the exosome, and can synchronously capture and separate multiple indexes in the biological sample.

Drawings

Fig. 1 is a schematic structural diagram of a microfluidic chip according to an embodiment of the present invention;

FIG. 2 is a schematic diagram of a reaction of a specific cell-capturing chamber according to an embodiment of the present invention;

FIG. 3 is a schematic diagram of a cell-intercepting chamber according to an embodiment of the present invention;

FIG. 4 is a schematic diagram of an intercepting array structure according to an embodiment of the present invention;

FIG. 5 is a schematic diagram of the reaction of a turbulence plate according to an embodiment of the present invention;

FIG. 6 is a schematic diagram of an implementation of exosome analysis using fluorescent specific binding member modified magnetic beads according to an embodiment of the present invention;

FIG. 7 is a schematic diagram of elution of captured exosomes intact and enrichment by magnetic force in one embodiment of the present invention;

FIG. 8 is a pictorial view of a specific cell capture chamber capturing cells for nuclear labeling using Hochest fluorochrome in an experiment in accordance with an embodiment of the present invention;

FIG. 9 is a diagram of a cell-intercepting array intercepting blood cells according to an embodiment of the present invention;

FIG. 10 is a diagram of an exosome analysis performed by an exosome capture region through fluorescent magnetic beads in an embodiment of the present invention;

FIG. 11 is a graph showing a particle size distribution of a sample exosome according to an embodiment of the present invention;

wherein,

inlet 100, outlet 200, specific cell capture chamber 300, cell interception chamber 400, interception array 410, aperture 411, exosome capture chamber 500, turbulence plate 510, protrusion 511, priming chamber 600, extension chamber 610, protrusion chamber 620, exosome 710, target exosome 711, non-target exosome 712, specific cell 720, non-specific cell 730, exosome binding unit 740, specific cell binding unit 750, magnetic bead 760, magnet 770.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Referring to fig. 1 to 5, an embodiment of the invention provides a device for capturing and separating exosomes and cells in a biological sample, including:

an integrated reaction chip, integrated reaction chip includes integrated reaction chamber, integrated reaction chamber has entry 100, specific cell that communicate in proper order and catches chamber 300, cell interception chamber 400, exosome and catches chamber 500 and export 200, be equipped with the interception array 410 that is used for intercepting the cell in the cell interception chamber 400, be equipped with the hole 411 on the interception array 410, the size of hole 411 can make exosome 710 pass through and can not make the cell pass through.

The embodiment of the invention also provides a method for capturing and separating exosomes and cells in a biological sample, which adopts the device for capturing and separating exosomes and cells in the biological sample and comprises the following steps:

a biological sample is injected into the integrated reaction chamber through the inlet 100 of the integrated reaction chamber, specific cells are captured by the specific cell capture chamber 300 pre-conjugated with the specific cell binding unit 750, non-specific cells 730 are intercepted at the front end of the cell interception chamber 400, and exosomes are captured by the exosome capture chamber 500 pre-conjugated with the exosome binding unit 740.

The invention provides a high-efficiency capturing and separating method and a device without damaging exosomes, wherein a biological sample to be separated firstly passes through a specific cell capturing cavity 300, and a target specific cell 720 is captured in the specific cell capturing cavity 300; then the biological sample passes through the cell interception cavity 400, the interception array 410 is provided with pores 411, and all cells are intercepted at the front end of the cell interception cavity 400, so that the exosome 710 enters the rear end of the cell interception cavity 400; the decellularized biological sample passes through the exosome capturing chamber 500, and the exosomes are captured by the exosome capturing chamber 500, thereby realizing the capture, separation and collection of the cells and the exosomes 710. The capture separation and collection of specific cells or specific exosomes can be achieved according to the selection of specific cell-capturing chamber for specific cell-binding unit 750 and the selection of exosome-capturing chamber for exosome-binding unit 740. The invention can realize efficient capture and enrichment of the exosome without damaging the exosome, and can synchronously capture and separate multiple indexes in the biological sample.

The present invention is applicable to any biological sample containing exosomes, including but not limited to, blood, urine, saliva, cerebrospinal fluid, and the like.

In some embodiments, the microfluidic chip can be an integral injection molding structure or a split structure. The split structure can include an upper cover sheet and a lower cover sheet, the upper and lower cover sheets forming the integrated reaction chamber therebetween.

The material of the micro-fluidic chip is any one or more of polystyrene, plastic, cellulose, polyacrylamide, polyethylene polypropylene, cross-linked dextran, glass, silica gel, silicon wafer and agarose gel.

In some embodiments, the capture and separation device may not include a pressure device, and the microfluidic chip may be vertically placed, and the capture and separation of the exosome 710 and the cell may be achieved by the gravity of the biological sample fluid itself, i.e. by the flow of the fluid in the direction from the inlet 100 to the outlet 200.

In some embodiments, the capture and separation device comprises a pressure pump for providing a pressure for flowing the biological sample from the inlet 100 of the integrated reaction chamber to the outlet 200 of the integrated reaction chamber.

In some embodiments, a stepped configuration of turbulence plates 510 is provided within the exosome capture chamber 500.

In some embodiments, exosome-binding cells 740 are conjugated to the turbulence plate body 510.

In some embodiments, there are a plurality of turbulence plates 510, and a plurality of turbulence plates 510 are disposed in parallel with respect to each other.

In some embodiments, the turbulence plate 510 extends horizontally, and the turbulence plate 510 has a protrusion 511 formed thereon in the vertical direction. The plurality of turbulence plates 510 extend in a horizontal direction and are arranged in a vertical direction. The biological sample flows from the inlet 100 to the outlet 200 of the microfluidic chip, enters the exosome-capturing chamber 500, flows in the horizontal direction, meets the structure of the protrusions 511 and is blocked in the flow direction, so that turbulent flow is formed. The turbulent flow pattern facilitates sufficient contact of the biological sample with the turbulence plate body 510 such that exosomes 710 in the biological sample are more easily bound to the turbulence plate body 510.

In some embodiments, the height of the protrusions 511 may be 5 μm to 100 μm. Specifically, the particle diameter may be 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm or 100 μm.

Referring to fig. 4, in some embodiments, the aperture of the aperture 411 of the intercepting array 410 is less than or equal to 3 μm. In some embodiments, the pore size of the pores 411 should be larger than the diameter of the exosomes, e.g. larger than 500nm, 600nm, 700nm, 800nm, 900nm or 10000 nm. Preferably, the compactness and number of the interception arrays 410 are such that almost all cells are intercepted in the cell interception cavities 400.

The shape of the intercepting array 410 may be, and is not limited to, a triangular array, a circular array, a rectangular array, and other shaped arrays.

In some embodiments, the specific cell binding unit 750 is conjugated on the inner wall of the specific cell capture chamber 300.

In some embodiments, a liquid adding chamber 600 is communicated between the cell intercepting chamber 400 and the exosome capturing chamber 500, and a liquid adding inlet is arranged on the liquid adding chamber 600 and is used for adding a cleaning solution, a buffer solution and the like into the integrated reaction chamber. The liquid feeding cavity 600 is arranged between the cell intercepting cavity 400 and the exosome capturing cavity 500, liquid enters the integrated reaction chamber from the inlet of the liquid feeding cavity 600, then is respectively distributed into the cell intercepting cavity 400 and the exosome capturing cavity 500 towards two sides, the cell intercepting cavity 400 and the exosome capturing cavity 500 are respectively washed or collected, and then respectively flow out from the inlet 100 and the outlet 200 of the integrated reaction chamber, so that the treatment of the cells and the exosomes 710 is respectively carried out without mutual interference.

In some embodiments, the feeding chamber 600 is composed of an extension chamber 610 connected to the cell intercepting chamber 400, and a protruding chamber 620 located between the extension chamber 610 and the exosome capturing chamber 500 and protruding to both sides of the integrated reaction chamber. The structural design increases the liquid pressure entering the integrated reaction chamber in the aspect of fluid flow, improves the impact force of liquid entering the chambers on the two sides, and has better cleaning effect.

In some embodiments, from the inlet 100 to the outlet 200, the integrated reaction chamber has a specific cell capturing chamber inlet, a specific cell capturing chamber outlet, a cell intercepting chamber inlet, a cell intercepting chamber outlet, an exosome capturing chamber inlet and an exosome capturing chamber outlet in sequence, the width of the specific cell capturing chamber 300 increases and then decreases from the specific cell capturing chamber inlet to the specific cell capturing chamber outlet, the width of the cell intercepting chamber 400 increases and then decreases from the cell intercepting chamber inlet to the cell intercepting chamber outlet, and the width of the exosome capturing chamber 500 increases and then decreases from the exosome capturing chamber inlet to the exosome capturing chamber outlet. The end tightening structure with the diameter increased and then reduced is adopted in each chamber, so that the pressure of liquid entering each chamber can be improved, and the combination or separation effect of the biological sample in each chamber is improved.

Specific cell binding members 750 include, but are not limited to, nucleic acids, antibodies, receptors, aptamers, and the like.

In some embodiments, the specific cell binding unit 750 may be immobilized on the specific cell capture chamber 300 by covalent bonding or physical adsorption; the covalent bonding means includes, but is not limited to, the fixation of the specific cell binding unit 750 on the surface of the specific cell capturing chamber 300 through the formation of covalent bonds by functional groups such as-SH, -OH, -COOH, etc.; or streptavidin is fixed on the surface of the specific cell capture chamber 300 through covalent bonds formed by functional groups such as-SH, -OH, -COOH, etc., and then the specific cell binding unit 750 modified with biotin is connected to the avidin. The physical adsorption may be performed by attaching the specific cell binding unit 750 to BSA, and then incubating the BSA through the tube and adsorbing the BSA to the specific cell capturing chamber 300, thereby fixing the specific cell binding unit 750 to the specific cell capturing chamber 300.

Exosome binding unit 740 includes, and is not limited to, nucleic acids, antibodies, receptors, aptamers, and the like.

The exosome binding unit 740 may bind to a marker of an exosome selected from AT least one of CD63, CD9, CD81, HSP70, Tsg101, EpCam, flotillin, Syntenin, Alix, HSP90, LAMP2B, LMP1, ADAM10, nicastrin, AChE, AQP2, RPL5, and a-1AT to achieve exosome capture.

In some embodiments, the specific cell capturing chamber 300 and the surface of the turbulence plate 510 are further modified with epoxy, amino, aldehyde, hydroxyl, carboxyl, oxo, thiol, etc. groups capable of immobilizing the groups coating the exosome-binding unit 740 and the specific cell-binding unit 750. In some embodiments, exosome binding unit 740 may be immobilized on exosome capture chamber 500 by means of covalent bond binding or physisorption; the covalent bonding means includes, but is not limited to, immobilization of the exosome-binding unit 740 on the exosome-capturing chamber 500 surface through covalent bonds formed by functional groups such as-SH, -OH, -COOH; or streptavidin is immobilized on the surface of the exosome capture chamber 500 through a covalent bond formed by functional groups such as-SH, -OH, -COOH, etc., and then the biotin-modified exosome binding unit 740 is connected to avidin. And the physical adsorption may be performed by attaching the exosome-binding element 740 to BSA, and then incubating and adsorbing the BSA to the exosome-capturing chamber 500 through a channel, thereby immobilizing the exosome-binding element 740 to the exosome-capturing chamber 500.

In some embodiments, the exosome binding unit 740 is conjugated within the exosome capture chamber 500 through a highly cleavable chemical bond. The "chemical bond highly cleavable" herein means a chemical bond capable of withstanding the shearing force of a fluid without breaking the bond. Preferably, the chemical bond can be cleaved under certain conditions (non-shearing force), for example, a disulfide bond is a chemical bond that can be efficiently cleaved.

In some embodiments, the separation method further comprises:

disulfide bond cleaving reagent is added to the exosome-capturing chamber 500 chamber to detach exosome 710-exosome binding unit 740 complex from the exosome-capturing chamber 500, and exosome 710-exosome binding unit 740 complex is collected by washing the exosome-capturing chamber 500 with buffer.

The disulfide bond cleaving agent may be selected from DTT, 2-MEA, and the like.

Referring to fig. 6, in some embodiments, the separation method further includes:

and adding magnetic beads 760 which are provided with fluorescent groups and conjugated with a second exosome-binding unit into the exosome-capturing chamber 500, binding the magnetic beads with the exosomes 710 captured by the exosome-binding unit 740, introducing a washing solution, washing away the unbound magnetic beads 760, and detecting the fluorescence value in the exosome-capturing chamber 500.

Referring to fig. 7, in some embodiments, the separation method further includes:

adding a disulfide bond cleaving reagent to the exosome capture chamber 500 to detach exosome 710-exosome binding unit 740-magnetic bead 760 complexes from the exosome capture chamber 500;

the exosome 710-exosome binding unit 740-magnetic bead 760 complexes are enriched by magnetic force.

The magnetic enrichment method can be that magnetic beads 760 are adsorbed by a magnet 770.

In the embodiment of the present invention, the specific capture and analysis of the target exosomes 711 in the biological sample are realized by combining the microfluidic technology with the immunofluorescence technology. The separation and analysis of the target exosomes 711 in the biological sample are integrated and automated through the disulfide bonds, the disulfide bonds can be cut off for subsequent biochemical analysis after immunocapture and fluorescent labeling, separation, detection and analysis equipment for the target exosomes 711 in the biological sample which can be clinically used is constructed, the system integration level is high, the operation is simple and convenient, and the flow time is short. The cell capture and analysis in the biological sample and the capture and analysis of the exosome 710 are integrated into one chip, so that the multi-parameter analysis of different biological substances in the biological sample is realized. The specific cells 720 (such as epithelial cells) and other cells (such as blood cells) in the biological sample are respectively captured and analyzed, and the specific cells 720 are subjected to in-situ immunofluorescent labeling or staining, so that the biological sample can be analyzed on a chip, and the separation and analysis integration can be realized.

Preferably, the invention is realized by integrating the cell in-situ capture, analysis and target exosome 711 capture and analysis in the device, and the device consists of a specific cell capture chamber 300, a cell interception chamber 400, a charging chamber 600 and an exosome capture chamber 500. The inner surface of the specific cell capturing cavity 300 is modified with an antibody (specific cell binding unit 750) corresponding to a specific antigen of the specific cell 720, and the specific cell 720 specific antibody can capture the specific cell 720 (such as epithelial cells in urine) which is exfoliated from a biological sample. The cell intercepting chamber 400 is provided with an intercepting array 410 having apertures 411, the apertures 411 having a size not larger than 3 microns and a size smaller than the size of the cells in the biological sample, and being used for separating the cells in the biological sample. A stepped turbulence plate body 510 is arranged in the exosome capturing cavity 500, a specific exosome binding unit 740 is modified on the surface of the turbulence plate body 510, and the specific exosome binding unit 740 is a biomolecule capable of specifically binding to a target exosome 711, and includes but is not limited to nucleic acid, antibody, receptor, aptamer and the like, and is used for specifically capturing the target exosome 711 in a biological sample. The surfaces of turbulence plate 510 and exosome-binding cells 740 are connected by highly cleavable chemical bonds, such as disulfide bonds. The step height is 5-100 microns and turbulence may be created to achieve sufficient contact of the target exosomes 711 with the exosome-binding units 740 surface-modified turbulence plate body 510.

The biological sample first passes through the specific cell-capturing chamber 300, the specific cells 720 exfoliated from the biological sample are captured in the specific cell-capturing chamber 300, and the non-specific cells 730 (such as blood cells, etc.) in the biological sample enter the downstream conduit. Next, the biological sample passes through the cell intercepting chamber 400, the cells in the biological sample are left by the cell intercepting chamber 400, and the substances with smaller size in the biological sample, including the target exosomes 711, flow to the exosome capturing chamber 500 through the pores 411 of the intercepting array 410, and then the target exosomes 711 are in sufficient contact with the exosome binding unit 740 surface-modified in the exosome capturing chamber 500, and are specifically adsorbed in the exosome capturing chamber 500, and other substances such as the non-target exosomes 712 are washed away by the washing liquid added in the charging chamber 600, thereby completing the capturing and separating of the target exosomes 711 and the specific cells 720 in the biological sample.

Then, an antibody with a fluorescent label or a staining reagent can be added to carry out immunofluorescence labeling or staining on the specific cell 720 captured by the specific cell capture cavity 300, and the in-situ capture and analysis of the cells in the biological sample can be realized by detecting the fluorescence value or staining result of the specific cell capture cavity 300; and a magnetic bead 760 modified by a specific binding unit with a fluorescent label is added to be bound with the target exosome 711, after the unbound magnetic bead 760 is washed away, the exosome 710 is analyzed by detecting the fluorescent value in the exosome capturing cavity 500, and a solution is provided for rapid detection of related diseases. After the connection between the target exosomes 711 and the turbulence plate body 510 of the exosome capture chamber 500 is cut off at a highly cleavable chemical bond (e.g. a disulfide bond), the complexes of magnetic beads 760 and exosomes 710 are enriched by magnetic force for downstream detection.

The following are specific examples.

As shown in FIG. 1, a urine sample is injected from an inlet 100 at a flow rate of 0.1-6 ml/hr, and first passes through a specific cell-capturing chamber 300, and exfoliated epithelial cells in the urine are captured in the specific cell-capturing chamber 300. And then passes through the cell intercepting chamber 400, the cell intercepting chamber 400 having 30-500 intercepting arrays 410 shown in fig. 3 and 4. Intercepting array 410 aperture 411 is no more than 3 microns, cells in urine are left by intercepting array 410, and the smaller-sized substances in urine containing exosomes 710 flow to exosome capturing chamber 500 through cell intercepting chamber 400, and exosome capturing chamber 500 is provided with ladder-type structure turbulence plate body 510, the height of the ladder is 5-100 microns, and turbulence can be formed. Exosome 710 is then contacted with modified exosome-binding element 740 in exosome-capturing chamber 500, specifically adsorbed in exosome-capturing chamber 500. And (3) introducing a cleaning solution through the inlet of the liquid adding cavity 600 at the flow rate of 0.1-6 ml/h, so that substances such as the non-target exosomes 712 are washed away by the cleaning solution, and the target exosomes 711 in the urine are captured.

Next, an antibody or a staining reagent with a fluorescent label may be added to immunofluorescent label or stain the captured epithelial cells, and the in situ capture and analysis of epithelial cells in urine may be achieved by detecting the fluorescence value or staining result at the specific cell capture chamber 300.

Next, as shown in fig. 6, magnetic beads 760 having a fluorescent group and conjugated with a second exosome binding unit may be added to bind to the captured exosomes 710, the binding reaction time is 5-60 minutes, then a washing solution is introduced through the inlet of the loading chamber 600 at a flow rate of 0.1-6 ml/hour, the unbound magnetic beads 760 are washed away, and the target exosomes 711 analysis is realized by detecting the fluorescence value in the device.

Next, as shown in fig. 7, the target exosomes 711 are cleaved from the exosome capture chamber 500 at a chemical bond (disulfide bond) that can be efficiently cleaved, and complexes of magnetic beads 760 and exosomes 710 are enriched by magnetic force for downstream detection. The content of the target exosome 711 can be analyzed by western blotting, enzyme-linked immunosorbent, or the like.

The results of actually applying the trapping and separating device to separate exosomes in the sample are shown in fig. 8-11.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A device for trapping and separating exosomes and cells in a biological sample, comprising:

the integrated reaction chip comprises an integrated reaction chamber, the integrated reaction chamber is provided with an inlet, a specific cell capturing cavity, a cell intercepting cavity, an exosome capturing cavity and an outlet which are sequentially communicated, an intercepting array for intercepting cells is arranged in the cell intercepting cavity, and pores are arranged on the intercepting array, and the size of the pores can enable exosomes to pass through and cannot enable the cells to pass through; a turbulence plate body with a stepped structure is arranged in the exosome capturing cavity, and an exosome combining unit is conjugated on the turbulence plate body;

a liquid adding cavity is communicated between the cell intercepting cavity and the exosome capturing cavity, and a liquid adding inlet is formed in the liquid adding cavity; the liquid feeding cavity consists of an extension cavity connected with the cell intercepting cavity and a protruding cavity which is positioned between the extension cavity and the exosome capturing cavity and protrudes towards the two sides of the integrated reaction cavity.

2. The apparatus for capturing and separating exosomes and cells according to claim 1, wherein said turbulence plate body is provided in plurality, and a plurality of turbulence plate bodies are arranged in parallel with each other;

and/or the turbulence plate body extends along the horizontal direction, and a protrusion in the vertical direction is formed on the turbulence plate body.

3. The apparatus for capturing and separating exosomes and cells according to claim 2, wherein the height of the projections is 5 μm to 100 μm.

4. The device for trapping and separating exosomes and cells according to claim 1, wherein the pore size of the pores on the intercepting array is less than or equal to 3 μm.

5. The device for capturing and separating exosomes and cells according to claim 1, wherein the specific cell-binding unit is conjugated on the inner wall of the specific cell-capturing chamber.

6. The device for capturing and separating exosomes and cells in a biological sample according to any one of claims 1 to 5, wherein the integrated reaction chamber is provided with a specific cell capturing chamber inlet, a specific cell capturing chamber outlet, a cell intercepting chamber inlet, a cell intercepting chamber outlet, an exosome capturing chamber inlet and an exosome capturing chamber outlet in sequence from the inlet to the outlet, the width of the specific cell capturing chamber is increased and then decreased from the specific cell capturing chamber inlet to the specific cell capturing chamber outlet, the width of the cell intercepting chamber is increased and then decreased from the cell intercepting chamber inlet to the cell intercepting chamber outlet, and the width of the exosome capturing chamber is increased and then decreased from the exosome capturing chamber inlet to the exosome capturing chamber outlet.

7. The apparatus for capturing and separating exosomes and cells according to any one of claims 1 to 5, wherein the separation apparatus comprises a pressure pump for providing pressure to flow the biological sample from the inlet of the integrated reaction chamber to the outlet of the integrated reaction chamber.

8. A method for capturing and separating exosomes and cells in a biological sample, which adopts the device for capturing and separating exosomes and cells in the biological sample, and comprises the following steps:

injecting a biological sample into the integrated reaction chamber from an inlet of the integrated reaction chamber, capturing specific cells by a specific cell capture chamber pre-conjugated with a specific cell binding unit, capturing non-specific cells at the front end of the cell interception chamber, and capturing exosomes by an exosome capture chamber pre-conjugated with an exosome binding unit.

9. The method for capturing and separating exosomes and cells according to claim 8, further comprising:

and adding magnetic beads which are provided with fluorescent groups and conjugated with a second exosome binding unit into the exosome capturing cavity, binding the magnetic beads with the exosomes captured by the exosome binding unit, introducing a cleaning solution, flushing away the unbound magnetic beads, and detecting the fluorescence value in the exosome capturing cavity.

10. The method for capturing and separating exosomes and cells in a biological sample according to claim 8 or 9, wherein the exosome-binding unit is conjugated in the exosome-capturing chamber through a disulfide bond.

11. The method of claim 10, further comprising: