CN115044472A

CN115044472A - Multi-stage filtration exosome separation enrichment device

Info

Publication number: CN115044472A
Application number: CN202210900343.8A
Authority: CN
Inventors: 薛梅; 向安; 李旭; 卢兹凡
Original assignee: First Affiliated Hospital of Medical College of Xian Jiaotong University
Current assignee: First Affiliated Hospital of Medical College of Xian Jiaotong University
Priority date: 2022-07-28
Filing date: 2022-07-28
Publication date: 2022-09-13

Abstract

The invention discloses a multi-stage filtration exosome separation and enrichment device, which comprises: the heat-conducting module comprises a multi-stage cavity, a heat-conducting block, a refrigerating semiconductor, a heat radiating fin and a heat radiating fan; one side of the heat conducting block is tightly attached to the periphery of the multistage cavity, and the other side of the heat conducting block is tightly attached to the refrigerating surface of the refrigerating semiconductor; the radiating fins are tightly attached to the heat conducting surface of the refrigeration semiconductor, and the radiating fan is embedded between the radiating fins; and screening exosomes in the sample based on the multistage cavity to complete filtration, separation and enrichment. The invention solves the technical barrier that the existing exosome separation technology is difficult to realize the rapid and efficient acquisition of high-purity exosomes from various types of body fluids, secretions, excretions, cell culture solution supernatant and other large samples of dozens of or even hundreds of milliliters.

Description

Multi-stage filtration exosome separation enrichment device

Technical Field

The invention belongs to the field of exosome separation, and particularly relates to a multi-stage filtration exosome separation and enrichment device.

Background

Extracellular Vesicles (EVs) are active nanoscale membrane-bound vesicles that all cells have a secretory function. EVs can be classified into apoptotic bodies (mainly 1000-5000 nm), microbubbles (mainly 100-1000 nm), and exosomes (mainly 15-150 nm) according to their size. Wherein the exosome is derived from a lipid bilayer membrane vesicle formed by fusing an intracellular vesicle-restricted membrane with a cytoplasmic membrane. Research finds that exosomes exist in various body fluids (blood, cerebrospinal fluid and the like), secretions (saliva, tears, milk, sweat, semen and the like) and excretions (urine and the like) and various effusion under pathological conditions, and are a special intercellular information transfer or pathological characterization, so that the contained components such as nucleic acid, protein, lipid and the like have important research values and application potentials in prevention, diagnosis, treatment and the like of important diseases such as cardiovascular diseases, metabolism, nerves, tumors and the like. Meanwhile, the artificial modification research of the exosome for drug delivery is developed rapidly, and the product enters the clinical application stage.

How to separate exosomes from the various types of body fluids, secretions, excretions and cell culture supernatant efficiently and stably is a precondition for the basic research and clinical application of biomedicine. The existing exosome separation technology comprises ultracentrifugation (relying on ultra-high speed centrifugation equipment, long time consumption, exosome damage caused by ultra-high speed centrifugation force), precipitation (coagulant such as polyethylene glycol influences purity, more foreign proteins limit subsequent identification and application), an ultrafiltration system (protein and lipid pollution, easy adhesion and membrane blockage), immunoaffinity (relying on specific antigen-antibody recognition but exosome is still not clear of all surface markers, high reagent cost and low efficiency), microfluidic separation (relying on a professional instrument, long time consumption, low sample capacity, lack of standardization and method verification) and other technologies, and has certain challenges and technical difficulties for quickly and efficiently obtaining high-purity exosomes from a large sample of several milliliters to hundreds of milliliters.

The membrane separation is a mixture pore membrane screening technology driven by fluid pressure difference, a pore membrane is a selective passing medium between two phases, and particles or small molecular solutes which can be intercepted by the membrane pores are intercepted by utilizing the size selectivity of the membrane pores to the separated components, so that the particles or the small molecular solutes which can not be intercepted by the membrane pores permeate the membrane, and further the separation, concentration and purification effects are realized. Except the ultrafiltration (the operating pressure is about 0.1-0.9 MPa, the separation scale is about 1-dozen nanometers), the microfiltration (about less than 0.1 MPa, about dozens nanometers to dozens of micrometers) and the reverse osmosis (about 1-9 MPa, about less than 1 nanometer) belong to membrane separation technologies driven by pressure. Thus, the exosome particle size is near the separation scale boundary of ultrafiltration and microfiltration. Most microfiltration employs a vertical flow filtration strategy in which the direction of liquid flow coincides with the direction of filtration. The mode is convenient to operate, the device is simple, high-power-ratio concentration of a target object is easy to realize, and the device is suitable for concentrating and enriching particulate matters such as exosomes. However, when the aperture of the filter membrane is small or the particulate matters in the feed liquid are many, the particles larger than the aperture of the membrane are intercepted on the membrane in the early stage of filtration, small particles enter the pores of the membrane, and some particles are adsorbed in the pores of the membrane due to various forces, so that the effective pores of the membrane are reduced. After the filtration process, the particles begin to form a filter cake layer on the surface of the membrane, and the adsorption inside the membrane pores gradually tends to saturation. As more particles are trapped on the membrane surface, the adsorption inside the membrane pores also tends to be saturated, and the particles begin to block the membrane pores to form a gradually thickened filter cake layer/gel layer and enter the filtration termination stage. Thus, vertical allows conventional vertical flow filtration to handle only small volumes of feed solution. And when the liquid flow is vertical to the filtering direction, namely the liquid flows along the direction parallel to the membrane (namely tangential flow filtration), the liquid flow washes the filter cake/gel layer on the surface of the membrane to reduce the accumulation of the filter cake/gel layer, so that the stable filtering speed is ensured, and the high-efficiency filtration of the feed liquid with larger scale is realized. But this approach must have sufficient flow volume to ensure continuous scouring of the membrane, otherwise membrane plugging occurs. Thus, it is difficult to achieve high-power concentration of exosomes, for example.

Disclosure of Invention

In order to solve the above problems, the present invention provides the following solutions: a multi-stage filtration exosome separation enrichment device comprises:

the heat-conducting module comprises a multi-stage cavity, a heat-conducting block, a refrigerating semiconductor, a heat radiating fin and a heat radiating fan;

one side of the heat conducting block is tightly attached to the periphery of the multistage cavity, and the other side of the heat conducting block is tightly attached to the refrigerating surface of the refrigerating semiconductor;

the radiating fins are tightly attached to the heat conducting surfaces of the refrigeration semiconductors, and the radiating fan is embedded between the radiating fins;

and screening exosomes in the sample based on the multistage cavity to complete filtering, separating and enriching.

Preferably, the multistage cavity adopts multistage continuous vertical filtration, and particulate matters with different apertures are sequentially and continuously screened from top to bottom;

the multi-stage cavity comprises a sample input port, a plurality of separation cavities and a liquid outlet;

and a sample to be separated is input into the sample input port, and after being filtered, separated and enriched by the separation cavity, a filtrate flows out from the liquid outlet.

Preferably, the separation cavity comprises a cavity outer wall, an electromagnetic stator and a filtering and screening membrane;

the electromagnetic stator is tightly attached to the outer wall surface of the outer wall of the cavity;

the filtering and screening membrane is arranged at the bottom of the separation cavity and is supported by a porous supporting net.

Preferably, the electromagnetic stator comprises a fixed winding and a reversing element which are arranged on the periphery of the filter cavity body.

Preferably, the separation cavity further comprises a stirrer, and the stirrer is arranged in the center of the separation cavity and on the upper surface of the filtering and screening membrane.

Preferably, the stirrer is internally provided with a permanent magnet rotor consisting of Ru Fe B permanent magnets, and the permanent magnet rotor and the stirrer are arranged in a concentric circle.

Preferably, the separation cavity comprises a first separation cavity, a second separation cavity, a third separation cavity, a fourth separation cavity and a fifth separation cavity;

the bottom end filtering and screening membrane of the first separation cavity is a porous membrane with the aperture of 5 mu m and is used for intercepting cells with large particle size;

the bottom end filtering and screening membrane of the second separation cavity is a porous membrane with the aperture of 1um and is used for intercepting apoptotic bodies with the particle size of 1-5 um;

the bottom end of the third separation cavity is provided with a filtering and screening membrane which is a porous membrane with the aperture of 220nm and is used for intercepting microbubbles and virus particles with the particle size of 220nm-1 um;

the bottom end of the fourth separation cavity is provided with a filtering and screening membrane which is a porous membrane with the aperture of 150nm and is used for intercepting microbubbles with the particle size of 150nm-1 um;

the fifth separation cavity is an exosome separation-concentration cavity, and the bottom end filtering and screening membrane is a porous membrane with the aperture of 15nm and is used for intercepting and concentrating exosomes with the particle size of 15nm-150 nm.

Preferably, the porous membranes of the first separation cavity, the second separation cavity and the third separation cavity are laser etching hole arrays made of transparent polycarbonate;

and the bottom end filtering and screening membranes of the fourth separation cavity and the fifth separation cavity are nano through hole array membranes made of semitransparent anodized aluminum materials with the pore diameters being in regular V-shaped characteristics and gradually enlarged.

Preferably, the upper surface of a small-aperture filter membrane of the filtering and screening membrane at the bottom end of the fourth separation cavity is a filtering inlet end, and the aperture is 150 nm; the lower surface of the large-aperture filter membrane is a filtering outlet end, and the aperture is 400 nm;

the upper surface of a small-aperture filter membrane of the filtering and screening membrane at the bottom end of the fifth separation cavity is a filtering inlet end, and the aperture is 15 nm; the lower surface of the large-aperture filter membrane is provided with a filtering opening end, and the aperture is 30 nm.

The invention discloses the following technical effects:

according to the multi-stage filtration exosome separation and enrichment device provided by the invention, a V-shaped through hole membrane is introduced into vertical microporous filtration to prevent membrane pores from being blocked; and driving liquid flow to scour the filter membrane by electromagnetic induction force to prevent a filter cake/gel layer from forming, simultaneously making particles impact the filter membrane to improve the screening efficiency of the filter membrane and other strategies, so as to give full play to the proper high-power ratio concentration advantage of vertical microporous filtration and make up the defect that the filter cake cannot be used for large-volume feed liquid by using the characteristic that the liquid flow scour avoids the filter cake from blocking the membrane in tangential flow microporous filtration. And further solves the technical barrier that the existing exosome separation technology is difficult to realize the rapid and efficient acquisition of high-purity exosomes from various types of body fluids, secretions, excretions, cell culture solution supernatants and other large samples of dozens of or even hundreds of milliliters.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is a schematic cross-sectional view of the overall structure of an apparatus according to an embodiment of the present invention;

FIG. 2 is a schematic cross-sectional view of the overall structure of an apparatus according to an embodiment of the present invention;

FIG. 3 is a schematic cross-sectional view of a multi-stage chamber in an apparatus according to an embodiment of the present invention;

FIG. 4 is a schematic cross-sectional view of an apparatus according to an embodiment of the present invention;

FIG. 5 is a schematic cross-sectional view of a multi-stage chamber at one location in an apparatus according to an embodiment of the invention;

FIG. 6 is a schematic view of a filtration and sieving membrane of a fourth stage chamber of an embodiment of the present invention;

FIG. 7 is a scanning electron micrograph of a filtration and sieving membrane of a fourth stage chamber of an embodiment of the present invention;

FIG. 8 is a schematic view of a filtration and sieving membrane of a fifth stage chamber of an embodiment of the present invention;

FIG. 9 is a scanning electron micrograph of a filtration and sieving membrane of a fifth stage chamber according to an embodiment of the present invention;

FIG. 10 is a schematic diagram of the filtering of large and small particles in a chamber with and without a stir head according to an embodiment of the present invention;

FIG. 11 is a schematic view of a V-shaped via and II-shaped hole loaded at the bottom of the fourth and fifth stage chambers according to an embodiment of the present invention;

wherein, 1-a multi-stage cavity, 2-a sample enters an inlet of the multi-stage cavity, 3-a liquid outlet of the sample after being filtered, separated and enriched by the multi-stage cavity, 4-U-shaped aluminum heat conducting block, 5-a refrigerating semiconductor, 6-a copper radiating fin, 7-a radiating fan, 8-a cavity outer wall, 9-an electromagnetic stator, 10-a stirrer, 11-a filtering and screening membrane, 12-a first-stage cavity, 13-a second-stage cavity, 14-a filtering and screening membrane, 15-a third-stage cavity, 16-a filtering and screening membrane, 17-a fourth-stage cavity, 18-V-shaped through hole alumina semipermeable membrane, 19-a fifth-stage cavity, 20-V-shaped through hole alumina semipermeable membrane, 21-a filtrate collecting cavity, 22-Ru permanent magnet iron boron ring and 23-an electromagnetic stator, 24-150 nm sieve pore size, 25-400 nm membrane pore size, 26-15 nm sieve pore size, 27-30 nm membrane pore size, 28-particle, 29-stirring form, 30-particle, 31-filter cake or gel layer.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in further detail below.

As shown in fig. 1, the present invention provides a multi-stage filtration exosome separation and enrichment device, comprising:

the device comprises a multistage cavity 1, a sample entering an inlet 2 of the multistage cavity, a liquid outlet 3 for the sample after being filtered, separated and enriched by the multistage cavity, a U-shaped aluminum heat conduction block 4, a refrigeration semiconductor 5, a copper radiating fin 6 and a radiating fan 7.

Wherein, the multistage cavity adopts multistage continuous vertical filtration, and the particles with different apertures are continuously screened from top to bottom in sequence; the multi-stage cavity 1 comprises an inlet 2 for a sample to enter the multi-stage cavity, a plurality of separation cavities and a liquid outlet 3; a sample to be separated is input into the sample input port 2, and after being filtered, separated and enriched by the separation cavity, filtrate flows out from the liquid outlet 3.

One side of the U-shaped aluminum heat conduction block 4 is tightly attached to the periphery of the multistage cavity 1, and the other side is tightly attached to the refrigerating surface of the refrigerating semiconductor 5; the copper radiating fins 6 are tightly attached to the heat conducting surface of the refrigeration semiconductor 5, and the radiating fan 7 is embedded between the copper radiating fins 6.

As shown in fig. 2, the separation cavity comprises a cavity outer wall 8, an electromagnetic stator 9 and a filtering and screening membrane 11; the electromagnetic stators 9 are tightly attached to the outer wall surface of the outer wall 8 of the cavity, and the number of the outer wall 8 of the cavity and the number of the electromagnetic stators 9 are 5; the filtering and screening membranes 11 are arranged at the bottom of the separation cavity and supported by a porous supporting net, and the whole device comprises 5 groups of filtering and screening membranes with different shapes and apertures. The separation cavity further comprises 5 stirrers 10, and the stirrers 10 are arranged in the center of the separation cavity and on the upper surface of the filtering and screening membrane 11.

And (3) adopting multistage continuous vertical filtration to continuously screen particles with different sizes from top to bottom in sequence. The first, second and third stages are all pre-separation chambers, wherein the bottom end of the first stage chamber is loaded with a porous membrane with the screening pore size of 5um, and a great number of cells are intercepted. The particles passing through the first-stage sieving porous membrane directly enter the second-stage separation cavity along with liquid flow, a porous membrane with the sieving pore diameter of 1um is loaded at the bottom end of the second-stage cavity, and the particles with the particle diameter of 1-5um, namely apoptotic bodies, are intercepted by the second-stage cavity. Particles passing through the second-stage sieving porous membrane directly enter a third-stage separation cavity along with liquid flow, a porous membrane with the sieving pore size of 220nm is loaded at the bottom end of the third-stage cavity, and the third-stage cavity intercepts particles with the particle size of 220nm-1um, namely most microbubbles and virus particles. The bottom end of the fourth-stage chamber is loaded with a porous membrane with the sieving aperture of 150nm, and the fourth-stage chamber intercepts particles with the particle size of 150nm-1um, namely most of microbubbles. The particles of the fourth-stage sieving porous membrane directly enter a fifth-stage separation cavity along with liquid flow, the fifth-stage cavity is an exosome separation-concentration cavity, and a porous membrane with the sieving pore diameter of 15nm is loaded at the bottom end of the fifth-stage separation-concentration cavity, so that the purpose of intercepting and concentrating the particles with the particle diameter of 15nm-150nm, namely exosomes, is achieved.

The screening porous membranes loaded at the bottom ends of the fourth-stage chamber and the fifth-stage chamber in the multistage continuous vertical filtration are nano through hole array membranes made of semitransparent anodized aluminum materials with the gradually enlarged regular V-shaped characteristic pore diameter, and the porous membranes loaded at the bottom ends of the first-stage chamber, the second-stage chamber and the third-stage chamber and used for screening are laser etching hole arrays made of transparent polycarbonate materials. One surface of the porous membrane loaded in the fourth stage chamber has a small pore diameter D3 (150 nm) and is arranged as a filtration inlet end (upper surface of the filter membrane), and the other surface of the porous membrane has a large pore diameter D3 (400 nm) and is arranged as a filtration outlet end (lower surface of the filter membrane). The screening porous membrane loaded at the bottom end of the fifth-stage chamber is also a nano-pore array membrane made of anodic alumina, and the aperture of the nano-through holes distributed in an array on the semitransparent membrane is characterized by regular V-shaped gradual expansion. One side of the porous membrane was set to be a filtration inlet port (upper surface of the filter) with a small pore diameter D4 of 15nm, and the other side was set to be a filtration outlet port (lower surface of the filter) with a large pore diameter D4 of 30 nm. The small pore diameter on one surface of the alumina array pore membrane loaded in the fourth and fifth-stage chambers is the particle size screening size of the filter membrane, and particles smaller than the size can enter the membrane pores through the small pore diameter filtration inlet positioned on the upper surface of the filter membrane, then enter the gradually enlarged pore channels and finally flow out from the filtration outlet on the other surface of the pore membrane with the large pore diameter. The V-shaped straight through hole screening hole can achieve the purpose of effectively preventing and reducing the effective membrane holes caused by the blocking of the membrane holes by particles.

A mechanical stirrer is arranged in the center of each chamber of the continuous vertical filtration, which is close to the upper surface of the filter membrane. The stirring blade is in a two-blade or three-blade propeller type and can rotate at a high speed under the action of external force, so that liquid flow with the direction pointing to the filter membrane is generated in respective chambers, and the phenomenon of membrane blockage caused by the formation and accumulation of a filter cake layer/gel layer on the surface of the filter membrane is prevented and avoided through the scouring of the liquid flow to the surface of the filter membrane. Meanwhile, along with the continuous impact of the liquid flow on the upper surface of the filter membrane, the particles with different particle sizes wrapped in the filter membrane collide with the filter membrane continuously, so that the probability that the small particles pass through the small-aperture filter inlet is increased, and the purpose of improving the particle screening efficiency of the filter membrane is finally achieved.

The periphery of each stage of filtering chamber is provided with an electromagnetic stator formed by fixed winding, reversing and other elements, and a permanent magnet rotor formed by Ru-Fe-B permanent magnets is arranged in the propeller-type stirrer in the chamber. When the stator coil is energized, it becomes an electromagnet and begins to produce a magnetic field. Trapezoidal waveform alternating current voltage generated through direct current conversion. As the windings are switched to high and low signals, the respective windings are energized as north and south poles. The permanent magnet rotor with the north pole and the south pole is aligned with the stator pole and receives the interaction force between the electromagnetic stator and the permanent magnet rotor, and finally the purpose that the permanent magnet rotor in the cavity continuously rotates with the stirrer to drive liquid flow in the cavity is achieved.

Further, as shown in fig. 3, the bottom end of the first-stage separation chamber supported by the porous support net filters the sieving membrane 11 with a sieving pore size of 5 μm. The first-stage chamber 12 is formed by sealing the outer wall 8 of the chamber body, the electromagnetic stator 9 and the filtering and screening membrane 11, the stirrer 10 is arranged in the chamber, and the other second, third, fourth and fifth chambers are also arranged in the chamber. And a second-stage chamber 13, the bottom end of which is provided with a filtering and screening membrane 14 with the screening pore size of 1 micron. And a third stage chamber 15, the bottom end of which is provided with a filtering and screening membrane 14 with the screening pore size of 0.22 micron. And the bottom end of the fourth-stage chamber 17 is provided with a filtering and screening membrane 18 which is a V-shaped through-hole alumina semipermeable membrane, the aperture of the upper surface filtering inlet is 150 nanometers, namely, the screening aperture, and the aperture of the lower surface filtering outlet is 400 nanometers. In the fifth-stage chamber 19, the bottom end filtering and screening membrane 20 is also a V-shaped through-hole alumina semipermeable membrane, the upper surface filtering inlet aperture is 15 nanometers, and the lower surface filtering outlet aperture is 30 nanometers. The filtrate collecting chamber 21 is disposed below the fifth stage chamber 19 and has a liquid outlet 3 at the bottom.

Further, as shown in fig. 4-5, 11/14/16/18/20 are first through fifth stages of filtration and screening membranes, respectively, supported by a porous support mesh. The Ru ferroboron permanent magnet ring 22 and the propeller type stirrer are arranged in a concentric circle. The electromagnetic stator 23 is formed by a plurality of circles of painted copper wires wound on an iron core, the iron core, a ring and other structures.

Further, as shown in fig. 6-8, the fourth stage chamber bottom loaded V-shaped through-hole alumina translucent sieving pore array membrane 18 is composed of a membrane upper surface 24 of 150nm sieving pore size and a membrane lower surface 25 of 400nm through sieving pore size.

The V-shaped through-hole alumina translucent sieving hole array membrane 20 loaded at the bottom of the fifth-stage chamber is composed of a membrane upper surface 26 with 15nm sieving hole diameter and a membrane lower surface 27 with 30nm passing through sieving hole diameter.

Further, the filtration of the large and small particles 28 in the first to fifth stages of the chamber by the propeller stirrer 10 with and without loading is shown in fig. 10. The left panel shows the membrane with a propeller stirrer on the upper surface. The agitator is turned on as shown at 29 so that the particles on the membrane surface cannot form a filter cake or gel layer 31 on the membrane surface when the propeller-free agitator is started as shown in the right figure due to the scouring effect of the liquid flow on the membrane, and the contact of the particles with the screening membrane holes is increased so as to increase the screening efficiency.

Further, a schematic view of V-shaped through holes and II-shaped holes for particles loading through the bottom of the fourth and fifth stage chambers is shown in fig. 11. In the left figure 24, 26 are the fourth or fifth chamber upper surface screening apertures and 25, 27 are the fourth or fifth chamber lower surface exit apertures. 31 are II-shaped apertures and correspond to 24, 26. After the particulate matter 30 matched with the screening aperture of the upper surface of the V-shaped hole passes through the screening aperture of the upper surface of the V-shaped hole, the passing holes inside the filtering membrane are gradually enlarged, blocking or adsorption of the particulate matter 30 is difficult to occur, and retention and blockage of the particulate matter in the holes can be effectively reduced. After the particles enter the II-shaped holes in the right drawing, the particle size of the particles is always the same as the size of the holes, the particles pass through the holes slowly and are easy to block or adsorb, so that the effective screening holes are reduced, and the filtering and screening efficiency is influenced.

In a further optimized scheme, the process of separating exosomes in cell culture solution supernatant based on the device specifically comprises the following steps:

I. device preparation and balancing

Collecting 100 ml of the culture supernatant of the bladder cancer cell line, placing the collected supernatant into a centrifuge tube, and taking 50 ml of filtered phosphate buffer solution to place the supernatant into another centrifuge tube

Assembling a multi-stage continuous vertical filtering device, and loading a filtering and screening membrane in each stage of cavity, a stirrer in the cavity and a permanent magnet ring;

a peristaltic pump is adopted to suck a phosphate buffer solution through a silicon rubber tube at the speed of 2-10 ml per minute, and the phosphate buffer solution enters a first-stage separation chamber through an inlet 2 in the device;

meanwhile, the outlet 3 of the device is connected with a negative pressure pump and is opened, and pressure difference is formed in the multistage separation chamber;

starting an electromagnetic stator power supply to drive the permanent magnet rotor in the cavity to rotate;

starting a refrigeration semiconductor to cool the electromagnetic stator, and starting a cooling fan to dissipate heat of a refrigeration semiconductor cooling surface;

starting to stably flow out liquid in a liquid collecting bottle connected with the negative pressure pump;

separation and concentration

1-10 ml of the culture supernatant of the bladder cancer cell line is pumped by a peristaltic pump and enters the inlet of the device at a speed of every minute;

keeping the outlet end negative pressure pump, the electromagnetic rotor, the refrigeration semiconductor and the cooling fan to be started;

when the cell supernatant is completely pumped by the peristaltic pump, continuously pumping the phosphate buffer solution to the inlet of the device for 20 milliliters;

stopping the inlet peristaltic pump to feed liquid, and normally starting other elements;

stopping the negative pressure pump, the electromagnetic rotor, the refrigeration semiconductor and the fan when no liquid flows out from the outlet of the device;

unloading a fifth-stage separation chamber in the device body, and taking 1 ml of phosphate buffer solution by a pipettor to wash the spiral stirrer and remove the stirrer from the chamber;

sucking the phosphate buffer solution surfing bottom end filter screening hole array membrane by a liquid shifter, and transferring the phosphate buffer solution into an EP pipe;

the nano-particle size and concentration were analyzed.

The V-shaped through hole membrane is introduced into the multistage continuous vertical filtration to prevent the membrane pores from being blocked; and driving liquid flow to scour the filter membrane by electromagnetic induction force to prevent a filter cake/gel layer from forming, simultaneously making particles impact the filter membrane to improve the screening efficiency of the filter membrane and other strategies, so as to give full play to the proper high-power ratio concentration advantage of vertical microporous filtration and make up the defect that the filter cake cannot be used for large-volume feed liquid by using the characteristic that the liquid flow scour avoids the filter cake from blocking the membrane in tangential flow microporous filtration. And further solves the technical barrier that the existing exosome separation technology is difficult to realize the rapid and efficient acquisition of high-purity exosomes from various types of body fluids, secretions, excretions, cell culture solution supernatants and other large samples of dozens of or even hundreds of milliliters.

The present embodiment employs a multi-stage continuous vertical filtration device design. A porous membrane with the screening pore diameter of 7um is loaded at the bottom end of the first-stage chamber, and can retain most cells; a porous membrane with the screening pore diameter of 5um is loaded at the bottom end of the first-stage chamber, and can intercept most cells; a porous membrane with the screening pore diameter of 1um is loaded at the bottom end of the second-stage chamber, and particles with the particle diameter of 1-5um, namely apoptotic bodies, can be intercepted; a porous membrane with the sieving aperture of 150um is loaded at the bottom end of the third-stage chamber, and particles with the particle size of 150nm-1um, namely most microbubbles, can be intercepted; the bottom end of the fourth stage chamber is loaded with a porous membrane with the sieving pore diameter of 15nm, and can intercept and concentrate particles with the particle diameter of 15nm-150um, namely exosomes. Therefore, continuous screening of particles with different particle sizes, such as apoptotic bodies, microbubbles, particularly exosomes, and the like in the same sample can be realized.

In the multistage continuous vertical filtration adopted in this embodiment, the sieving porous membrane loaded at the bottom ends of the third and fourth stage chambers is a nano through-hole array membrane made of semitransparent anodized aluminum with a gradually enlarged pore diameter and a regular V-shaped characteristic, wherein one end of the membrane with a small pore diameter is a filtration inlet, and the other end with a large pore diameter is a filtration outlet. The small pore diameter is the particle size screening size of the filter membrane, and particles smaller than the size can enter the membrane pores through the small pore diameter filtration inlet positioned on the upper surface of the filter membrane, then enter the gradually enlarged pore channels and finally flow out from the filtration outlet on the other surface of the porous membrane with the large pore diameter. Therefore, the V-shaped straight-through hole screening hole can achieve the purpose of effectively preventing and reducing the effective membrane holes caused by the blockage of the membrane holes by nano-scale particles;

in this example, the small pore diameter (filtration inlet) D3 of the anodized aluminum sieving porous membrane loaded at the bottom end of the third-stage chamber of the multistage continuous vertical filtration is 150nm, and the large pore diameter (filtration outlet) D3 is 400 nm. The bottom end of the fourth stage chamber is loaded with a sieving porous membrane made of anodic alumina, the small pore diameter (filtration inlet) D4 is 15nm, and the large pore diameter (filtration outlet) D4 is 30 nm. Thus, sieving and concentration of exosomes having diameters in the range of 15-150 nm may be achieved in the fourth chamber.

Each chamber of the continuous vertical filtration described in this example was provided with a mechanical stirrer near the center of the upper surface of the membrane. The stirring blade is in a two-blade or three-blade propeller type and can rotate at a high speed under the action of external force, so that liquid flow with the direction pointing to the filter membrane is generated in respective chambers, and the phenomenon of membrane blockage caused by the formation and accumulation of a filter cake layer/gel layer on the surface of the filter membrane is prevented and avoided through the scouring of the liquid flow to the surface of the filter membrane. Meanwhile, along with the continuous impact of the liquid flow on the upper surface of the filter membrane, the particles with different particle sizes wrapped in the filter membrane collide with the filter membrane continuously, so that the probability that the small particles pass through the small-aperture filter inlet is increased, and the purpose of improving the particle screening efficiency of the filter membrane is finally achieved.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims

1. A multi-stage filtration exosome-separating-and-enriching device, comprising:

2. The multi-stage filtration exosome-separating-and-enriching device according to claim 1,

the multistage cavity adopts multistage continuous vertical filtration, and the particles with different apertures are sequentially and continuously screened from top to bottom;

3. The multi-stage filtration exosome-separating-and-enriching device according to claim 1,

the separation cavity comprises a cavity outer wall, an electromagnetic stator and a filtering and screening membrane;

4. The multi-stage filtration exosome-separating-and-enriching device according to claim 3,

the electromagnetic stator comprises a fixed winding and a reversing element which are arranged on the periphery of the filter cavity.

5. The multi-stage filtration exosome-separating-and-enriching device according to claim 3,

the separation cavity further comprises a stirrer, and the stirrer is arranged in the center of the separation cavity and on the upper surface of the filtering and screening membrane.

6. The multi-stage filtration exosome-separating-and-enriching device according to claim 5,

the stirring rod is internally provided with a permanent magnet rotor consisting of Ru-Fe-B permanent magnets, and the permanent magnet rotor and the stirring rod are arranged in a concentric circle.

7. The multi-stage filtration exosome-separating-and-enriching device according to claim 2,

the separation cavity comprises a first separation cavity, a second separation cavity, a third separation cavity, a fourth separation cavity and a fifth separation cavity;

8. The multi-stage filtration exosome-separating-and-enriching device according to claim 7,

the porous membranes of the first separation cavity, the second separation cavity and the third separation cavity are laser etching hole arrays made of transparent polycarbonate materials;

9. The multi-stage filtration exosome-separating-and-enriching device according to claim 7,

the upper surface of a small-aperture filter membrane of the filtering and screening membrane at the bottom end of the fourth separation cavity is a filtering inlet end, and the aperture is 150 nm; the lower surface of the large-aperture filter membrane is a filtering outlet end, and the aperture is 400 nm;