Exosome pneumatic centrifugal separation chip

Abstract

The application discloses an exosome pneumatic centrifugal separation chip, and belongs to the field of microfluidic chips, which comprises one or more separation mechanisms; the separation mechanism comprises N-stage separation modules; the separation module comprises a sample adding chamber, a filtering unit in communication with the sample adding chamber, a nanofiltration membrane arranged in the filtering unit, a pneumatic chamber in communication with the filtering unit, a waste liquid chamber in communication with the filtering unit, and a sample collection pipeline with one end connected with the bottom end of the filtering unit and the other end serving as a sample collection opening and being higher than the top end of the sample adding chamber; when N>1, the pore diameter of the nanofiltration membrane decreases from the first-stage separation module to the N-stage separation module, and in the two adjacent separation modules, the sample adding chamber of the next-stage separation module is arranged downstream of the waste liquid chamber of the previous-stage separation module and the two chambers are connected. The application can effectively overcome the problems of long time consumption and low efficiency caused by the blockage commonly existing in the current nanometer pore membrane separation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microfluidic chips, and more particularly relates to an exosome pneumatic centrifugal separation chip. BACKGROUND

[0002] Exosomes are nanoscale extracellular vesicles (30-200 nm in diameter) secreted by cells, mediate numerous key life activities such as cell communication, and are crucial in the fields of painless early screening of diseases, precision treatment, prognosis monitoring, and integrated diagnosis and treatment. On the one hand, the rich markers on the surface and inside of the exosomes are considered as a new generation of non-invasive disease diagnosis and detection targets. On the other hand, due to the excellent biocompatibility, extremely low immunogenicity, and easy functionalization characteristics, the exosomes are also increasingly becoming a new type of biological nanodiagnosis and treatment integrated carrier. At present, the extraction of exosomes mainly comes from body fluids such as blood, saliva, urine, cerebrospinal fluid, semen, saliva, pleural effusion, and milk.

[0003] Exosomes are 100 times smaller in diameter and 1 million times smaller in volume than cells, and the separation thereof faces great challenges. The ultracentrifugation method is the main technology for separating exosomes at present, but this technology also has obvious defects such as long time consumption, low yield and purity of separated exosomes. Other methods, such as polyethylene glycol precipitation method, phosphatidylserine affinity capture method, size exclusion chromatography method, and magnetic bead immunization method, have been used for exosome separation, but the above methods for extracting exosomes usually need multiple sample addition and replacement, and have the disadvantages of long time consumption and complicated operation process.

[0004] Microfluidic chip integrated nanofiltration membrane is a promising alternative. Liu et al. reported a nanomembrane filtration-based platform for exosome total isolation chip (ExoTIC) with the advantages of simplicity, ease of use, and modularity, which is convenient for rapid isolation of high-purity exosomes from body fluids, including plasma, urine, and lavage fluid. Compared with traditional ultracentrifugation, the platform obtains up to 1000 times higher yield of exosomes (ACS Nano 201711(11), 10712-10723). However, when separating a large number of samples, the continuous separation process can form a "filter cake", which can cause filter membrane blockage, reduce efficiency, and even cause filter membrane rupture. Woo et al. integrated two different pore size filters (20 nm and 600 nm in size) and proposed a rapid, label-free, and highly sensitive centrifugal microfluidic chip platform for exosome separation and quantification (Exodisc), which can automatically enrich 20-600 nm vesicles from urine samples within 30 minutes with simple centrifugation. Compared with the gold standard ultracentrifugation method, the Exodisc isolated exosome mRNA up to 100 times (ACS Nano 2017 11(2), 1360-1370). However, this method also has the same problems as the above-mentioned methods, such as the formation of "filter cake" and filter membrane blockage, which reduces the separation efficiency. In the patent document with the application publication number CN115672423A, a disc-type chip is provided, which can automatically complete the filtration process, sample separation and enrichment, and detection process without manual intervention on the centrifugal chip. However, when separating a large number of samples, the same problems exist, such as filter membrane blockage and low separation efficiency. SUMMARY

[0005] In view of the defects of the prior art and the need for improvement, the present application provides an exosome pneumatic centrifugal separation chip, which aims to effectively overcome the problems of long time consumption and low efficiency caused by blockage in current nanometer pore membrane separation.

[0006] To achieve the above-mentioned purpose, according to one aspect of the present application, an exosome pneumatic centrifugal separation chip is provided, comprising: one or more separation mechanisms; the separation mechanism comprises N-stage separation modules; the separation module comprises:

[0007] a sample adding chamber, which is provided with a sample adding port;

[0008] a filtration unit, which is arranged downstream of the sample adding chamber and communicates with the sample adding chamber, and a nanofiltration membrane is arranged in the filtration unit;

[0009] a pneumatic chamber, which is arranged downstream of the filtration unit and communicates with the filtration unit;

[0010] a waste liquid chamber arranged downstream of the filtration unit and in communication with the filtration unit;

[0011] and a sample collection pipe, one end of which is connected to the bottom end of the filtration unit, and the other end of which serves as a sample collection port and is higher than the top end of the sample addition chamber;

[0012] wherein N is a positive integer; when N>1, the pore size of the nanofiltration membrane contained in the first-stage separation module to the N-stage separation module decreases in turn, and in the adjacent two separation modules, the sample addition chamber of the lower-stage separation module is arranged downstream of the waste liquid chamber of the upper-stage separation module and the two chambers are in communication.

[0013] In some optional embodiments, in the adjacent two separation modules, the sample addition chamber of the lower-stage separation module is arranged outside the waste liquid chamber of the upper-stage separation module, and the two chambers are in communication through a microchannel.

[0014] In some optional embodiments, in the adjacent two separation modules, the sample addition chamber of the lower-stage separation module is in communication with the waste liquid chamber of the upper-stage separation module through a pumping unit, and the pumping unit comprises:

[0015] an inlet channel in communication with the waste liquid chamber of the upper-stage separation module;

[0016] an outlet channel in communication with the sample addition chamber of the lower-stage separation module;

[0017] and an inward pumping pneumatic chamber arranged downstream of the waste liquid chamber of the upper-stage separation module and in communication with the inlet channel and the outlet channel, respectively.

[0018] Further, in the same pumping unit, the outlet channel is thicker than the inlet channel.

[0019] Further, the pneumatic chamber is connected to the filtration unit through a first microchannel, and the bottom edge of the pneumatic chamber is arc-shaped; one end of the arc-shaped edge is lower, which is the connection point of the pneumatic chamber and the first microchannel; the other end of the arc-shaped edge is higher.

[0020] Further, the end of the first microchannel connected to the pneumatic chamber is in a swollen structure.

[0021] Further, the filtration unit comprises, from top to bottom, a filtration chamber, a nanofiltration membrane, and a filter membrane support layer; the filter membrane support layer is provided with a plurality of openings, and the pore size of the openings is larger than that of the corresponding nanofiltration membrane.

[0022] Overall, the above technical solutions conceived by the present application can achieve the following beneficial effects:

[0023] (1) The exosome pneumatic centrifugal separation chip provided by the application is provided with a separation module, can effectively realize separation of exosome subgroups, and is provided with a pneumatic chamber in communication with a filtering unit in which a nanofiltration membrane is located in each separation module. In a centrifugal acceleration process, liquid flows through the surface of the filtering membrane to flush the filtering membrane, and at the same time, pneumatic energy is stored. In a centrifugal deceleration process, the pneumatic energy stored in the pneumatic chamber is released to push the liquid to flow reversely through the surface of the filtering membrane to flush the filtering membrane again. In this way, the liquid continuously flows bi-tangentially through the surface of the nanomembrane, so that the accumulated nanoparticles intermittently move out of the surface of the filtering membrane, and synchronous reciprocating radial and tangential flow separation is realized on the surface of the nanomembrane, so that the nanomembrane is prevented from being blocked by nanoparticles, and the separation efficiency is ensured.

[0024] (2) When the exosome pneumatic centrifugal separation chip provided by the application comprises a plurality of separation modules, the pore diameters of the nanofiltration membranes in the plurality of separation modules decrease in turn, the sample adding chamber of a lower separation module is arranged downstream of the waste liquid chamber of an upper separation module, and the two chambers are in communication, so that multi-stage separation of exosome subgroups can be realized.

[0025] (3) When the exosome pneumatic centrifugal separation chip provided by the application comprises a plurality of separation modules, the adjacent two separation modules are connected through a pumping unit comprising an inward pumping pneumatic chamber, liquid can be pumped inward, so that the length of the same separation structure distributed along the radial direction can be reduced, and the length is not limited by the radius of the centrifugal disc. A plurality of nanofiltration membranes are connected in series through radial inward pumping of liquid, and more exosome subgroups of different sizes are separated. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The exosome pneumatic centrifugal separation chip provided for the embodiment 1 of the application is shown in the schematic diagram.

[0027] Figure 2 The separation module provided for the embodiment 1 of the application is shown in the schematic diagram.

[0028] Figure 3 The exosome sub-group separation principle provided for the embodiment 1 of the application is shown in the schematic diagram.

[0029] Figure 4 The multi-layer structure of the chip provided for the embodiment 1 of the application is shown in the schematic diagram.

[0030] Figure 5 The exosome pneumatic centrifugal separation chip provided for the embodiment 2 of the application is shown in the schematic diagram.

[0031] In all the drawings, the same reference signs are used to represent the same elements or structures, in which:

[0032] 1-sample adding chamber; 11-sample adding port

[0033] 2 - filtration unit; 21 - filtration chamber, 22 - nanofiltration membrane, 23 - support layer of the filtration membrane;

[0034] 3 - waste liquid chamber;

[0035] 4 - sample collection conduit; 41 - sample collection port;

[0036] 5 - first microchannel;

[0037] 6 - second microchannel;

[0038] 7 - pumping unit; 71 - inlet channel, 72 - outlet channel, 73 - inward pumping pneumatic chamber;

[0039] 8 - pneumatic chamber. DETAILED DESCRIPTION

[0040] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and should not be used to limit the present application. In addition, the technical features involved in the various embodiments of the present application described below can be combined with each other as long as they do not conflict with each other.

[0041] In the present application, the terms "first", "second", etc. (if any) in the present application and the accompanying drawings are used to distinguish similar objects, and do not necessarily describe a specific order or sequence.

[0042] In order to effectively overcome the problems of long time consumption and low efficiency caused by blockage in the process of separating exosomes by existing nanometer pore membranes, the present application provides an exosome pneumatic centrifugal separation chip, the overall idea of which is to design a pneumatic chamber in communication with the filtration unit in the chip, so that the liquid flows through the surface of the filtration membrane to flush the filtration membrane during the centrifugal acceleration process, and the pneumatic energy is stored at the same time, during the centrifugal deceleration process, the pneumatic energy stored in the pneumatic chamber is released to push the liquid to flow through the surface of the filtration membrane in the opposite direction, flushing the filtration membrane again, so that the liquid continuously flows through the surface of the nanometer membrane in the double tangential direction, so that the accumulated nanometer particles are intermittently removed from the surface of the filtration membrane, and the synchronous reciprocating radial and tangential flow separation is realized on the surface of the nanometer membrane, avoiding the blockage of the nanometer filtration membrane by nanometer particles.

[0043] It should be noted that the all-integrated cell exosome separation and analysis microfluidic chip provided by the application is a centrifugal microfluidic chip, which has a rotation center. It is easy to understand that in this type of chip, "inner" and "outer" respectively refer to the side closer to the rotation center and the side farther from the rotation center; "closer" and "farther" respectively refer to closer to the rotation center and farther from the rotation center; "higher" and "lower" respectively refer to closer to the rotation center and farther from the rotation center; "top end" and "bottom end" respectively refer to the end closest to the rotation center and the end farthest from the rotation center; "start", "head", "end", "upstream" and "downstream" are determined with reference to the flow direction of the liquid during centrifugation, for example, in a section of the channel, the end through which the liquid flows first is the head end, and the end through which the liquid flows last is the end; for example, the position through which the liquid flows first is the upstream position, and the position through which the liquid flows later is the downstream position. In the following examples, unless otherwise specified, the meaning of the terms is as described herein.

[0044] Considering that exosomes of different sizes have different characteristics, the all-integrated cell exosome separation and analysis microfluidic chip provided by the application can realize the separation of exosomes of different sizes by cascading filtration units with nanofiltration membranes of different pore sizes.

[0045] The following is an example.

[0046] Example 1:

[0047] An exosome pneumatic centrifugal separation chip, as shown in Figure 1 , comprising: two separation mechanisms; the separation mechanism comprises a three-stage separation module; the three-stage separation module is respectively marked as a first-stage separation module, a second-stage separation module and a third-stage separation module;

[0048] As shown in Figure 1 and Figure 2 , the separation module comprises:

[0049] a sample adding chamber 1, which is provided with a sample adding port 11;

[0050] a filtration unit 2, which is arranged downstream of the sample adding chamber 1 and communicates with the sample adding chamber 1, and a nanofiltration membrane 22 is arranged in the filtration unit 2;

[0051] a pneumatic chamber 8, which is arranged downstream of the filtration unit 2 and communicates with the filtration unit 2;

[0052] a waste liquid chamber 3, which is arranged downstream of the filtration unit 2 and communicates with the filtration unit 2;

[0053] and a sample collection pipeline 4, one end of which is connected to the bottom end of the filtration unit 2, and the other end serves as a sample collection port 41 and is higher than the top end of the sample adding chamber;

[0054] The pore size of the nanofiltration membrane contained therein decreases successively from the first separation module to the third separation module, and in the two adjacent separation modules, the sample adding chamber of the next stage separation module is arranged downstream of the waste liquid chamber of the previous stage separation module and the two chambers are in communication; optionally, in the embodiment, the pore size of the nanofiltration membrane successively is 200 nm, 100 nm and 20 nm from the first separation module to the third separation module.

[0055] Specifically, as shown in Figure 1 and Figure 2 In the embodiment, in the two adjacent separation modules, the sample adding chamber of the next stage separation module is arranged outside the waste liquid chamber of the previous stage separation module, and the two chambers are in communication through a microchannel.

[0056] The exosome pneumatic centrifugal separation chip provided in the embodiment is used for exosome separation. For each separation module, after the cell culture supernatant sample with cells and cell debris removed by centrifugation is added from the sample adding port to the sample adding chamber, when the centrifuge is accelerated for centrifugation, the liquid in the sample adding chamber is affected by the centrifugal force and flows from the sample adding chamber to the filtration unit. Under the action of the centrifugal force, small molecules and impurity proteins smaller than the pore size of the nanofiltration membrane flow into the waste liquid chamber through the nanofiltration membrane, and exosomes larger than the pore size of the nanofiltration membrane are intercepted. Since the flow rate through the nanomembrane is less than the flow rate of the centrifugal force sample, a part of the sample will flow through the filtration unit from the surface of the nanomembrane and flow into the pneumatic chamber, so that the air in the pneumatic chamber is compressed, thereby increasing the internal pressure of the pneumatic chamber. Next, the centrifugal speed is reduced, and the previously compressed gas will push the sample in the pneumatic chamber to flow through the surface of the filter membrane in the filtration unit and return to the sample adding chamber. In this way, under the control of the automatic program, the liquid continuously flows bidirectionally to flush the surface of the nanomembrane, so that the accumulated nanoparticles intermittently move away from the surface of the filter membrane, restoring the filtering function of the nanofiltration membrane, thereby realizing anti-blocking ultrafast nanoparticle separation. After the separation is completed, the exosomes are on the surface of the filter membrane in the filtration chamber, and the sample can be directly sucked out through the sample collection tube by using a pipette gun or the like at the sample collection port, thereby realizing exosome pneumatic centrifugal separation.

[0057] Figure 3Figure 9 shows a process schematic diagram of separating different size exosome subgroups by the exosome pneumatic centrifugal separation chip provided in the embodiment; the cell culture supernatant with cells and cell debris removed by centrifugation is added into the sample chamber of the first separation module, the centrifuge is driven to rotate the chip according to the above operation process, and the sample flows through the three nanofiltration membranes in turn. Due to the existence of the pneumatic chamber, the sample flows back and forth in the double tangential direction to flush the membrane surface. The vesicles with a size greater than 200 nm are enriched on the first nanofiltration membrane, the vesicles with a size less than 200 nm and some small molecule substances pass through the first nanofiltration membrane and flow into the sample chamber of the second separation module, the vesicles with a size between 100 nm and 200 nm are enriched on the second nanofiltration membrane, the vesicles with a size less than 100 nm and small molecules pass through the second nanofiltration membrane and flow into the sample chamber of the third separation module, the vesicles with a size between 20 nm and 100 nm are enriched on the third nanofiltration membrane, and the small molecules and impurities finally flow into the waste chamber. After the separation is completed, PBS is added for washing, and finally the vesicles on the membrane surface are directly sucked out from the three sample collection ports by a pipette, that is, the exosome subgroups are separated. In other embodiments, sample collection can also be achieved by setting a sample collection chamber and a siphon valve.

[0058] As shown in Figure 1, in the embodiment, the pneumatic chamber 8 and the filtration unit 2 are connected through the first microchannel 5, and the bottom edge of the pneumatic chamber 8 is arc-shaped. One end of the arc-shaped edge is lower, which is the connection point of the pneumatic chamber and the first microchannel 5, and the other end of the arc-shaped edge is higher. Based on the structural design of the pneumatic chamber, the liquid flowing through the filtration unit 2 can smoothly enter the pneumatic chamber 8 during high-speed centrifugation, and the liquid in the pneumatic chamber 8 can flow back to the sample chamber 1 during low-speed centrifugation. Figure 1 As shown in Figure 1, in the embodiment, the pneumatic chamber 8 and the filtration unit 2 are connected through the first microchannel 5, and the bottom edge of the pneumatic chamber 8 is arc-shaped. One end of the arc-shaped edge is lower, which is the connection point of the pneumatic chamber and the first microchannel 5, and the other end of the arc-shaped edge is higher. Based on the structural design of the pneumatic chamber, the liquid flowing through the filtration unit 2 can smoothly enter the pneumatic chamber 8 during high-speed centrifugation, and the liquid in the pneumatic chamber 8 can flow back to the sample chamber 1 during low-speed centrifugation.

[0059] Figure 1 As shown in Figure 1, in the embodiment, the sample chamber 1 and the filtration unit 2 are connected through the second microchannel 6, and each sample chamber is provided with two ports. In actual application, one port is used as a sample addition port for adding reaction reagents, and the other port is used as an air hole for balancing air pressure to prevent the liquid in the sample chamber from failing to flow into the filtration unit. Figure 2 The exosome pneumatic centrifugal separation chip provided in the embodiment is a multilayer chip,

[0060] As shown in Figure 1, in the embodiment, the sample chamber 1 and the filtration unit 2 are connected through the second microchannel 6, and each sample chamber is provided with two ports. In actual application, one port is used as a sample addition port for adding reaction reagents, and the other port is used as an air hole for balancing air pressure to prevent the liquid in the sample chamber from failing to flow into the filtration unit. Figure 4 Figure 4 As shown in Figure 1, in the embodiment, the filtration unit 2 includes a filtration chamber 21, a nanofiltration membrane 22 and a filter membrane support layer 23 arranged in sequence from top to bottom. The filter membrane support layer 23 is provided with a plurality of openings, and the pore size of the openings is greater than that of the corresponding nanofiltration membrane 22.

[0061] As shown in Figure 1, in the embodiment, the filtration unit 2 includes a filtration chamber 21, a nanofiltration membrane 22 and a filter membrane support layer 23 arranged in sequence from top to bottom. The filter membrane support layer 23 is provided with a plurality of openings, and the pore size of the openings is greater than that of the corresponding nanofiltration membrane 22. Figure 4 ​​As shown, the filter membrane support layer 23 in the filter unit is actually formed by setting openings in the corresponding positions of a chip layer, and the nanofiltration membrane 22 is covered on the filter membrane support layer 23; based on the filter unit structure, since the filter membrane is parallel to the chip packaging, the filtration area is large, and the filtration speed is fast, at the same time, the liquid flows from top to bottom, and the flow direction is perpendicular to the filtration direction, realizing tangential flow filtration.

[0062] With reference to the foregoing Figure 4 In this embodiment, the chip comprises, from top to bottom, a top cover, a first chamber layer, a channel layer, a second chamber layer, a support layer, a third chamber layer and a bottom cover; wherein:

[0063] The sample adding chamber and the pneumatic chamber are arranged in the first chamber layer;

[0064] The sample collection pipeline and other microchannels for connecting the chambers are arranged in the channel layer;

[0065] The filtration chambers of the filter units are arranged in the second chamber layer;

[0066] The filter membrane support layers of the filter units are arranged in the support layer;

[0067] The waste liquid chambers are arranged in the third chamber layer.

[0068] The nanofiltration membranes of the filter units are specifically anodic aluminum oxide membranes (AAO membranes), which are fixed on the corresponding filter membrane support layers by double-sided pressure sensitive adhesive.

[0069] In this embodiment, the material of the chip is PMMA, therefore, the chip specifically comprises 7 layers of PMMA, one layer of anodic aluminum oxide membrane (AAO membrane) and one layer of double-sided pressure sensitive adhesive; in some other embodiments of the present application, the material of the chip can also be PC or PP.

[0070] It should be noted that the layered design herein is only one optional implementation, and in actual application, the number of layers of the chip and the distribution manner of the structures in the chip layers can be flexibly adjusted according to actual needs. In addition, since centrifugal driving is a kind of fluid control technology without bias, multiple repeated units can be parallel on a single chip, and multiple chips can be synchronously stacked in the Z-axis, which is easy for subsequent high-throughput and industrialization.

[0071] Optionally, the processing mode of the centrifugal chip includes CNC, laser engraving, soft lithography technology, 3D printing technology, etc., but is not limited thereto.

[0072] Considering that there is a certain limitation in the processing precision in the actual processing of the chip; in order to prevent the first microchannel from being unable to be accurately connected with the pneumatic chamber due to insufficient processing precision, as an optional implementation, Figure 1 and Figure 2As shown, in this embodiment, the end of the first microchannel connected to the pneumatic chamber is an enlarged structure.

[0073] It should be noted that the number of stages of the separation module in the separation mechanism can be flexibly adjusted, and the upstream and downstream relationship of each separation module can be ensured through design.

[0074] Example 2:

[0075] An exosome pneumatic centrifugal separation chip, such as Figure 5 As shown, it includes one separation mechanism; the separation structure includes four separation modules, which are referred to as the first separation module, the second separation module, the third separation module and the fourth separation module in sequence.

[0076] The specific structure of each separate module is the same as that in Embodiment 1 above, except that, for example... Figure 5 As shown, in this embodiment, in two adjacent separation modules, the sample addition chamber of the next-level separation module and the waste liquid chamber of the previous-level separation module are connected through a pumping unit 7. The pumping unit 7 includes:

[0077] An inlet channel 71 that connects to the waste liquid chamber of the previous separation module;

[0078] An outlet channel 72 that connects to the sample loading chamber of the next-stage separation module;

[0079] And an inward pumping pneumatic chamber 73, which is connected to the inlet channel 71 and the outlet channel 72 respectively, and is located downstream of the waste liquid chamber of the previous separation module.

[0080] And, as Figure 5 As shown, in the same pumping unit 7, the outlet channel 72 is wider than the inlet channel 71.

[0081] The exosome pneumatic centrifugation separation chip provided in this embodiment achieves the following process for multi-stage separation of exosome subpopulations:

[0082] After the pneumatic centrifugal filtration of the first separation module is completed, the sample smaller than the pore size of the nanofiltration membrane flows into the waste liquid chamber of the first separation module, and a first-stage pneumatic inward pumping chamber is arranged below the first-stage waste liquid chamber. The sample is caused to flow from the waste liquid chamber of the first separation module into the inward pumping pneumatic chamber of the first separation module through the inlet channel by centrifugal acceleration rotation, the air in the inward pumping pneumatic chamber is compressed, thereby storing pneumatic energy. The inward pumping pneumatic chamber is connected to an outlet channel, which has a lower hydraulic resistance than the inlet channel. By rapidly reducing the rotation speed, the pneumatic energy stored in the inward pumping pneumatic chamber is rapidly released, in this way, the liquid is mainly pumped radially inward into the sample loading chamber of the second separation module through the outlet channel with a lower hydraulic resistance. After the separation is completed in the second separation module, the sample loading chamber of the third separation module is pumped again through the inward pumping pneumatic chamber of the second separation module, and the sample loading chamber of the fourth separation module is pumped again through the inward pumping pneumatic chamber of the third separation module. After the separation is completed, the sample is sucked from the sample collection port of each stage by a pipette, and the exosome subpopulation is obtained.

[0083] In other optional embodiments, in addition to radially inward pumping of liquid by storing pneumatic energy in the pneumatic chamber, heat radiation can also be used. The internal gas pressure of the waste liquid chamber is increased by heating, thereby pushing the liquid to be radially inward pumped to the next stage sample chamber.

[0084] In order to realize multi-stage separation of exosome subpopulations, the first embodiment needs to adopt a structure in which the separation modules are sequentially connected in the direction from the centrifugal center radially outward. Radially outwardly connecting multiple nanofiltration membranes in sequence is limited by the radius of the centrifugal disc. The first embodiment connects two adjacent separation modules by setting a pumping unit containing an inward pumping pneumatic chamber. The inward pumping pneumatic chamber can pump liquid inward, thereby reducing the length of the same separation structure distributed along the radial direction, and is not limited by the radius of the centrifugal disc. By radially inward pumping of liquid, multiple nanofiltration membranes are connected in series, and more exosome subpopulations of different sizes are separated.

[0085] It should also be noted that the number of separation modules in the separation mechanism can be flexibly adjusted, as long as the upstream and downstream relationships of the separation modules are ensured by design.

[0086] Those skilled in the art will readily understand that the above description is only the preferred embodiment of the present application, and is not intended to limit the present application. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. An exosome pneumatic centrifugal separation chip, characterized in that, The application relates to a separation device comprising: one or more separation mechanisms; the separation mechanism comprises an N-stage separation module; the separation module comprises: a sample loading chamber, which is provided with a sample loading port; a filter unit, which is arranged downstream of the sample loading chamber and communicates with the sample loading chamber, and a nanofiltration membrane is arranged in the filter unit; a pneumatic chamber, which is arranged downstream of the filter unit and communicates with the filter unit; the connecting point of the pneumatic chamber and the filter unit is located above the nanofiltration membrane; a waste liquid chamber, which is arranged downstream of the filter unit and communicates with the filter unit; the connecting point of the waste liquid chamber and the filter unit is located below the nanofiltration membrane; and a sample collection pipeline, one end of which is connected to the bottom end of the filter unit, and the other end serves as a sample collection port and is higher than the top end of the sample loading chamber; in, N It is a positive integer; N When >1, from the first-level separation module to the... N The separation modules consist of nanofiltration membranes with progressively smaller pore sizes. In adjacent separation modules, the sample loading chamber of the next separation module is located downstream of the waste liquid chamber of the previous separation module, and these two chambers are interconnected. During operation, for each separation module, the cell culture supernatant sample, after centrifugation to remove cells and cell debris, is added to the sample loading chamber through the sample inlet. When the centrifuge accelerates the centrifugation, the liquid in the sample loading chamber is subjected to centrifugal force and flows from the sample loading chamber to the filtration unit. Under the action of centrifugal force, small molecules and impurity proteins smaller than the pore size of the nanofiltration membrane pass through the nanofiltration membrane and flow into the waste liquid chamber. Exosomes larger than the pore size of the nanofiltration membrane are trapped. Since the flow rate through the nanopores is less than the flow rate of centrifugal injection, some samples will flow from the surface of the nanofiltration membrane through the filtration unit and into the pneumatic chamber, which compresses the air in the pneumatic chamber and increases the internal pressure. Next, the centrifugation speed is reduced, and the previously compressed gas will push the sample in the pneumatic chamber through the surface of the filter membrane in the filtration unit and return to the sample loading chamber. This process is repeated. Under the control of the automated program, the liquid continuously flows in two directions to flush the surface of the nanofiltration membrane, causing the accumulated nanoparticles to be intermittently removed from the surface of the filter membrane, restoring the filtration function of the nanofiltration membrane.

2. The exosome pneumatic centrifugation separation chip of claim 1, wherein, in adjacent two separation modules, the sample loading chamber of the lower-stage separation module is arranged outside the waste liquid chamber of the upper-stage separation module, and the two chambers communicate through a microchannel.

3. The exosome pneumatic centrifugation separation chip of claim 1, wherein, in adjacent two separation modules, the sample loading chamber of the lower-stage separation module communicates with the waste liquid chamber of the upper-stage separation module through a pumping unit, and the pumping unit comprises: an inlet channel, which communicates with the waste liquid chamber of the upper-stage separation module; an outlet channel, which communicates with the sample loading chamber of the lower-stage separation module; and an inward pumping pneumatic chamber, which communicates with the inlet channel and the outlet channel respectively and is arranged downstream of the waste liquid chamber of the upper-stage separation module.

4. The exosome pneumatic centrifugal separation chip of claim 3, wherein, in the same pumping unit, the outlet channel is thicker than the inlet channel.

5. The exosome pneumatic centrifugal separation chip according to any one of claims 1-4, wherein, the pneumatic chamber and the filter unit are connected through a first microchannel, and the bottom edge of the pneumatic chamber is arc-shaped; one end of the arc-shaped bottom edge is lower, which is the connecting point of the pneumatic chamber and the first microchannel; the other end of the arc-shaped bottom edge is higher.

6. The exosome pneumatic centrifugal separation chip of claim 5, wherein, the end of the first microchannel, which is connected to the pneumatic chamber, is in an expanded structure.

7. The exosome pneumatic centrifugation separation chip of any one of claims 1-4, wherein, the filter unit comprises a filter chamber, a nanofiltration membrane and a filter membrane support layer arranged in sequence from top to bottom; a plurality of openings are arranged on the filter membrane support layer, and the pore size of the openings is larger than that of the corresponding nanofiltration membrane.

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