A microfluidic pulse filtration system, a preparation method, a filtration method and application thereof

Abstract

The present application provides a microfluidic pulse circulation filtration system, its preparation and filtration method and application. The microfluidic pulse circulation filtration system comprises a filter containing a filter membrane and a microfluidic chip. The present application uses microfluidic pulse filtration to prevent filter membrane blockage and filter membrane pollution, and gently filters and recovers micro-nanoparticles based on size. By using different pore sizes of filter membranes, micro-nanoparticles of different sizes can be separated. For example, large particles can be removed to recover small particles, or small particles can be removed to recover large particles. Microfluidic pulse filtration can minimize mechanical damage to particles during filtration and completely recover particles.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of materials, and particularly relates to a microfluidic pulse circulation filtration system, a preparation method and application thereof, and a filtration method. BACKGROUND

[0002] There are various applications of filtration using filter membranes, such as removing red blood cells (6 micrometer particles) in whole blood to separate plasma, removing free proteins (less than 3 nanometers) in plasma to recover extracellular vesicles (greater than 30 nanometers) in plasma, and the like. The traditional filtration method using filter membranes has the inherent problem of filter membrane clogging. In order to solve the problem of filter membrane clogging, a microfluidic pulse circulation filtration system is developed. Pulse filtration is to use repeated pulse flow to prevent filter membranes from being clogged by the accumulation of micro-nano particles. However, the traditional pulse filtration requires the joint driving of a large number of auxiliary equipment, such as a syringe pump, a programmable computer, a single-chip microcomputer, a valve, and related professional knowledge. Due to the use of a large number of auxiliary equipment, it is difficult to achieve microfluid pulse filtration or greatly increases the cost of achieving microfluid pulse filtration.

[0003] In the current prior art, only the filtration system of pulse filtration can remove red blood cells in whole blood samples to separate plasma, and cannot concentrate cell vesicles in cell culture medium, remove free proteins in plasma to recover extracellular vesicles, and the recovery rate is not ideal. The pollution and clogging rate of filter membranes need to be improved. Moreover, the template of the chip is made by traditional photolithography technology, which has the disadvantages of complex process and long production cycle. At the same time, the filtration system of pulse filtration does not have a circulation washing function, so that the sample on the filter membrane will continuously concentrate during pulse filtration, resulting in the aggregation of the sample on the filter membrane. SUMMARY

[0004] Therefore, the purpose of the present application is to overcome the defects in the prior art, and to provide a microfluidic pulse circulation filtration system, a preparation method and application thereof, and a filtration method. The present application uses the ingenious arrangement of filter membranes, elastic membranes, microchannels, one-way valves and other microfluidic elements to form a microfluidic pulse circulation filtration system, which can gently filter samples and reduce the damage of micro-nano particles during filtration. The present application realizes microfluid pulse filtration, reduces the cost, size and sample processing capacity of the microfluid pulse filtration system.

[0005] Before the summary of the present application, the terms used in the present application are defined as follows:

[0006] The term "PDMS" refers to polydimethylsiloxane.

[0007] The term "Teflon" refers to polytetrafluoroethylene.

[0008] To achieve the above object, the first aspect of the present application provides a microfluidic pulse circulation filtration system, comprising a circulation washing system, a pulse blow system, a filter containing a filter membrane and a microfluidic chip, wherein,

[0009] The circulation washing system, the pulse blow system, the filter containing a filter membrane and the microfluidic chip are connected by a needle or a pipeline; and / or

[0010] The microfluidic chip comprises an elastic membrane chamber, a microfluidic one-way valve and a microchannel.

[0011] The microfluidic pulse circulation filtration system according to the first aspect of the present application, wherein,

[0012] The microfluidic pulse circulation filtration system further comprises a pulse pressure inlet, a pulse pressure outlet and a filtered sample outlet;

[0013] The circulation washing system comprises a waste liquid bottle and a washing bottle, and the filtered sample outflow outlet is connected to the waste liquid bottle and the washing bottle in series;

[0014] The pulse blow system comprises a blow elastic membrane and a blow channel;

[0015] The elastic membrane chamber comprises an upper chamber above the elastic membrane, a lower chamber below the elastic membrane and an intermediate layer of the elastic membrane, and the upper chamber above the elastic membrane and the lower chamber below the elastic membrane are separated by the intermediate layer of the elastic membrane; and / or

[0016] The microchannel comprises a first microchannel and a second microchannel;

[0017] Preferably, the lower part of the blow elastic membrane is connected to the pulse pressure inlet, and the upper part of the blow elastic membrane is connected to the upper part of the filter containing a filter membrane through the blow channel;

[0018] Preferably, the waste liquid bottle and the washing bottle of the circulation washing system are connected to the filtered sample outflow outlet in series through a pipeline, the lower part of the washing bottle is provided with an opening, so that liquid forms droplets, and the droplets are aligned to the upper part of the filter containing a filter membrane;

[0019] Preferably, the filter containing a filter membrane, the upper chamber above the elastic membrane, the microfluidic one-way valve and the first microchannel are connected in series to form a microfluidic flow path;

[0020] Preferably, the lower chamber below the elastic membrane is connected to the second microchannel, the pulse pressure inlet and the pulse pressure outlet; and / or

[0021] Preferably, the microfluidic one-way valve is located between the upper chamber above the elastic membrane and the first microchannel, and the first microchannel is connected to the filtered sample outflow outlet.

[0022] According to the microfluidic pulse circulation filtration system of the first aspect of the present application, wherein,

[0023] The flow resistance of the micro-nanopore is obtained by formula (1):

[0024] The flow resistance R of the filter membrane Filter = the pressure difference passing through the filter membrane / the flow rate passing through the filter membrane formula (1);

[0025] The mechanical capacitance of the elastic membrane is obtained by formula (2):

[0026] Wherein,

[0027] C Membrane is the elastic membrane, r is the radius of the elastic membrane, T is the thickness of the elastic membrane, E is the Young's modulus of the elastic membrane, and μ is the Poisson's ratio of the elastic membrane; and / or

[0028] The relationship between the flow resistance of the micro-channel and the size of the micro-channel is obtained by formula (3)-(5):

[0029]

[0030] Wherein, R Channel is the flow resistance of the micro-channel, w, h, and l are the width, height, and length of the micro-channel, respectively, and ν is the dynamic viscosity of the fluid.

[0031] According to the microfluidic pulse circulation filtration system of the first aspect of the present application, wherein,

[0032] The material of the micro-channel is PDMS or Teflon, and most preferably PDMS;

[0033] The material of the elastic membrane of the elastic membrane chamber and / or the material of the elastic membrane of the blow and beat is PDMS or Teflon, and most preferably PDMS; and / or

[0034] The material of the blow and beat channel is a polyethylene tube;

[0035] Preferably, the diameter of the micro-nanopore is 20-600 nanometers, more preferably 20-220 nanometers, and further preferably 20-100 nanometers.

[0036] The second aspect of the present application provides a method for preparing the microfluidic pulse circulation filtration system of the first aspect, which comprises: preparing a microfluidic chip, connecting a circulation washing system, a pulse blow and beat system, and a filter containing a filter membrane to the microfluidic chip through a needle or a channel;

[0037] Preferably, the method for preparing the microfluidic pulse circulation filtration system comprises the following steps:

[0038] (A) preparing a microfluidic chip;

[0039] (B) connecting one side of the microfluidic chip with a waste liquid bottle and a washing bottle in a circulating washing system, and connecting the other side with a pulse blowing system and a filter containing a filter membrane, thereby obtaining the microfluidic pulse circulating filtration system;

[0040] More preferably, the step (B) further comprises: punching the waste liquid bottle and the washing bottle at the top and the bottom, connecting the hole at the top of the waste liquid bottle with the hole at the top of the washing bottle through a pipeline, connecting the hole at the bottom of the waste liquid bottle with the sample outflow outlet, and locating the hole at the bottom of the washing bottle above the filter containing the filter membrane; and / or the pulse blowing system is prepared by soft lithography.

[0041] According to the preparation method of the second aspect of the present application, in the step (A), the operation of preparing the microfluidic chip comprises the following steps:

[0042] (1) duplicating the upper layer material and the lower layer material containing the chamber and the microchannel through the mold printed by the 3D printer;

[0043] (2) heating and bonding the lower layer material containing the chamber and the microchannel prepared in step (1) with the elastic film of the intermediate layer;

[0044] (3) heating and bonding the elastic film with the lower layer material obtained in step (2) with the upper layer material containing the chamber and the microchannel, thereby forming the microfluidic chip with the upper layer material containing the chamber and the microchannel, the lower layer material and the intermediate layer being the elastic film;

[0045] Preferably, the upper layer material containing the chamber and the microchannel forms the chamber above the elastic film, the one-way valve and the first microchannel; and / or

[0046] Preferably, the lower layer material containing the chamber and the microchannel forms the chamber below the elastic film and the second microchannel.

[0047] According to the preparation method of the second aspect of the present application, in the step (A),

[0048] The step (1) further comprises: mixing the upper layer material and the lower layer material with the curing agent on the mold containing the chamber and the microchannel, heating, and then peeling off after the material is cured, thereby obtaining the upper layer material containing the chamber and the microchannel, and the lower layer material; and / or

[0049] In the steps (2) and (3), the bonding method is plasma or vacuum thermal compression bonding, and the most preferred method is plasma; the bonding temperature is 100-150℃, preferably 110-130℃, and most preferably 120℃; the bonding time is 2-30min, preferably 5-20min, and most preferably 15min.

[0050] According to the preparation method of the second aspect of the present invention, in step (1): the mass ratio of the upper layer material and the lower layer material to the curing agent is 5-20:1, preferably 5-15:1, and most preferably 10:1; the heating time is 10-36 h, preferably 10-24 h, and most preferably 12 h; the heating temperature is 60-100 °C, preferably 70-90 °C, and most preferably 80 °C; and / or

[0051] In step (3), when bonding the check valve, a pad is used to cover the check valve diaphragm and valve seat.

[0052] Preferably, the curing agent is a polydimethylsiloxane curing agent.

[0053] A third aspect of the present invention provides a pulse filtering method, wherein the method uses the microfluidic pulse cyclic filtering system described in the first aspect or the microfluidic pulse cyclic filtering system prepared by the method described in the second aspect to perform pulse cyclic filtering;

[0054] Preferably, the pulse filtration method includes: when a pulse pressure is provided to the microfluidic pulse circulation, the microfluidic pulse circulation filtration system automatically filters the sample on the filter membrane; the filtered sample flows out of the filter sample outlet through the filter membrane, the upper layer of the elastic membrane chamber, the microfluidic one-way valve, and the microchannel, and is prevented from clogging by the back-and-forth pulse flow of the filter membrane; during pulse filtration, the circulating washing system continuously replenishes washing liquid on the filter containing the filter membrane to achieve circulating washing; the pulse blowing system generates pulse flow by blowing the deformation of the elastic membrane, continuously blowing the sample above the filter containing the filter membrane to reduce filter membrane clogging and contamination.

[0055] The fourth aspect of the present invention provides the application of the microfluidic pulsed circulating filtration system described in the first aspect or the microfluidic pulsed circulating filtration system prepared by the method described in the second aspect in the preparation of micro / nano filter devices.

[0056] According to a specific embodiment of this application, the microfluidic pulse circulation filtration system comprises a filter with a filter membrane and a microfluidic chip. The microfluidic chip includes a chamber with an elastic membrane, a microfluidic one-way valve, and a microchannel. The filter containing the filter membrane, the upper layer of the chamber with the elastic membrane, the one-way valve, and the microchannel are connected in series to form a microfluidic flow path. The lower layer of the chamber with the elastic membrane is connected to a microchannel, which is connected to the pulse pressure-driven pulse filtration microfluidic chip.

[0057] The filter membrane has micro- and nanopores, and its flow resistance can be obtained by measuring the ratio of the pressure difference to the flow rate through the membrane. Therefore,

[0058] Flow resistance R of the filter membrane Filter = Pressure difference through the filter membrane / Flow rate through the filter membrane Formula (1).

[0059] Mechanical capacitance (C Membrane ) of the elastic membrane can be obtained by using the theory of the plate:

[0060]

[0061] where r and T are the radius and thickness of the membrane, respectively, and E and μ are the Young's modulus and Poisson's ratio of the elastic membrane, respectively.

[0062] Flow resistance (R Channel ) of the microchannel is controlled by changing the size of the channel.

[0063]

[0064] where w, h, and l are the width, height, and length of the microchannel, respectively, and v is the dynamic viscosity of the fluid.

[0065] The microfluidic chip of the microfluidic pulse circulation filtration system is composed of polydimethylsiloxane (PDMS) and is manufactured using soft lithography technology. The chip is composed of 3 layers of PDMS, the upper and lower layers being microchannels and the middle layer being an elastic membrane. The upper and lower layers of microchannels are obtained by copying PDMS from a 3D mold. After mixing the curing agent and PDMS at a ratio of 1:10 and placing them on the 3D mold, heating at 80 degrees Celsius for 12 hours, the liquid PDMS becomes an elastic solid and is peeled off from the 3D mold to obtain the upper and lower layers of microchannels. The bonding of each layer of PDMS is performed by plasma treatment, after which the treated PDMS is tightly attached together and is bonded by heating at 120 degrees Celsius for 15 minutes. When bonding the one-way valve, a PDMS cushion is used to cover the membrane and valve seat part of the valve to prevent this part from being treated by plasma, and the one-way valve can be opened after the heating process. The microfluidic chip and the pulse pressure are connected using a 90-degree bent needle and a polyethylene hose.

[0066] According to another specific embodiment of the present application, the preparation of the circulation filtration system comprises the following steps:

[0067] (1) punching the upper and lower parts of the waste liquid bottle and the washing bottle in the circulation washing system;

[0068] (2) connecting the upper holes of the waste liquid bottle and the washing bottle by a pipeline;

[0069] (3) connecting the lower hole of the waste liquid bottle with the sample outflow outlet;

[0070] (4) locating the lower hole of the washing bottle above the filter containing the filter membrane;

[0071] Preferably, the operation of the preparation of the circulation washing system comprises the following steps:

[0072] (5) copying the lower layer material by a 3D printer.

[0073] (6) Heating and bonding the lower material prepared in step (5) with the elastic film of the intermediate layer;

[0074] (7) The upper part of the elastic film is connected to the upper part of the filter containing the filter membrane through a polyethylene tube.

[0075] The pulse filtration system structure of the present application: the pulse filtration system is composed of a filter containing a filter membrane and a microfluidic chip. The microfluidic chip contains an elastic film, an upper chamber above the elastic film, a lower chamber below the elastic film, a one-way valve, a first microchannel, and a second microchannel. The middle membrane of the one-way valve contains a hole. The filter membrane containing the filter is connected to the upper chamber of the elastic film, the one-way valve, and the first microchannel, and is connected to the filtered sample outflow outlet. The pulse pressure inlet is connected to the lower chamber of the elastic film and the second microchannel, and is connected to the pulse pressure outlet.

[0076] Circulating filtration system: the waste liquid bottle and the washing bottle are connected in sequence to the filtered sample outflow outlet through a pipeline. The lower part of the washing bottle has a hole to form liquid droplets, which are aligned above the filter containing the filter membrane. The washing liquid in the washing bottle and the waste liquid in the waste liquid bottle are separated by air to prevent contamination of the washing liquid and the waste liquid.

[0077] Pulse blowing system: the pulse blowing system is composed of a blowing elastic film and a blowing channel. The lower part of the blowing elastic film is connected to the pulse pressure inlet, and the upper part of the blowing elastic film is connected to the upper part of the filter containing the filter membrane through the blowing channel.

[0078] According to another specific embodiment of the present application, the pulse filtration method comprises: when the microfluidic pulse circulation is provided with a pulse pressure, the microfluidic pulse circulation filtration system automatically filters the sample on the filter membrane, the filtered sample passes through the filter membrane, the upper layer of the elastic film chamber, the microfluidic one-way valve, the microchannel, and flows out of the filtered sample outflow outlet, and the back-and-forth pulse flow of the filter membrane prevents clogging of the filter; the continuous washing system continuously supplements the washing liquid on the filter containing the filter membrane to achieve continuous washing during pulse filtration; the pulse blowing system generates a pulse flow by deforming the blowing elastic film, continuously blows the sample above the filter containing the filter membrane, and reduces clogging and contamination of the filter membrane.

[0079] The working principle of the pulse filtration system is as follows: under the driving of the pulse pressure, the elastic membrane repeatedly deviates upward and downward, and repeatedly changes between state 1 and state 2. When the elastic membrane deviates upward (state 1), the liquid in the cavity above the elastic membrane flows out of the filter sample outlet through the one-way valve and the first microchannel. At the same time, the liquid in the cavity above the elastic membrane flows to the upper side of the filter membrane. When the elastic membrane deviates downward (state 2), the liquid above the filter membrane flows to the cavity above the elastic membrane through the filter membrane. At this time, the one-way valve is closed, and the liquid flowing out of the filter sample outlet flows into the cavity above the elastic membrane. When repeatedly changing between state 1 and state 2, the pulse filtration system can generate a pulse flow through the filter membrane, prevent the filter membrane from being blocked and contaminated, and effectively filter the sample to the filter sample outlet.

[0080] The working principle of the cycle filtration system is as follows: when the pulse filtration system is filtering, the sample is continuously fed into the waste liquid bottle through the filter sample outlet. At this time, the waste liquid bottle is filled with the filtered sample, and the same volume of the washing liquid in the washing bottle is transported in the form of droplets to the upper side of the filter containing the filter membrane, so that the volume of the sample above the filter membrane remains unchanged, and the cycle filtration of the automatically washed sample is realized.

[0081] The working principle of the pulse blow system is as follows: under the driving of the pulse pressure, the blow elastic membrane repeatedly deviates upward and downward (state 1 and state 2). When the blow elastic membrane deviates upward (state 1), the liquid above the filter containing the filter membrane is blown once through the blow channel. When the blow elastic membrane deviates downward (state 2), the liquid above the filter containing the filter membrane is sucked once through the blow channel. When repeatedly changing between state 1 and state 2 by using the pulse pressure, the liquid above the filter containing the filter membrane can be continuously blown and sucked, the liquid above the filter membrane is mixed, and the blocking and contamination of the filter membrane are further prevented.

[0082] When the pulse pressure is provided to the microfluidic pulse cycle filtration system, the system can automatically filter the sample on the filter membrane, and the filtered sample flows out of the filter sample outlet through the filter membrane, the upper layer of the elastic membrane cavity, the one-way valve and the microchannel. At the same time, the back-and-forth pulse flow through the filter membrane is generated to prevent the filtration from being blocked.

[0083] The application can be used for removing red blood cells from complex biological samples such as whole blood to recover plasma, removing free proteins from plasma to recover extracellular vesicles, sample pretreatment for disease detection, and micro-nanoparticle filtration and enrichment.

[0084] The filter membrane of the application has a nanometer-pore thin film, and the microfluidic chip can generate a pulse flow of microfluid. The pulse filtration of microfluid can be realized by combining the filter membrane and the microfluidic chip.

[0085] Compared with the prior art, the difference lies in that the circulating filtration can automatically clean the filtered sample without an external control system, and automatic and quantitative injection of washing liquid can be realized. The system of the application can ensure that the volume of the solution above the filter membrane is constant, effectively prevent the filter membrane from being blocked due to the rapid increase of the concentration of the substances in the sample when the solution above the membrane is filtered dry during the filtration process, and also prevent damage to the sample caused by drying of the solution.

[0086] Currently, there is no method for pulse filtration in the prior art. Pulse filtration can prevent blockage and reduce damage to micro-nano particles, and achieve rapid and effective separation. The pulse filtration system of the application can be applied to various filtrations by changing the size of the filter membrane pore size (600 nanometers, 20 nanometers), for example, red blood cells can be removed from whole blood to separate plasma, and extracellular vesicles can be concentrated in cell culture solution.

[0087] The pulse filtration system of the application can add a waste liquid and a washing bottle to realize pulse circulation washing, continuously add washing liquid to the sample during filtration, prevent the concentration and drying of the sample on the filter membrane during filtration, and further reduce the blockage and pollution of the filter membrane by using the pulse deformation of the elastic film to pulse the sample on the sample filter membrane through the micro-pipe.

[0088] The application can concentrate cell vesicles in cell culture solution, remove free proteins in plasma to recover extracellular vesicles, gently filter extracellular vesicles and prevent the concentration of extracellular vesicles, and improve the recovery rate of extracellular vesicles by using the circulating washing system. However, the current prior art pulse filtration system cannot achieve this.

[0089] Compared with the pulse filtration alone, the pulse filtration system of the application can further improve the anti-blocking and anti-pollution of the filter membrane, and improve the purity and recovery rate of particles by combining the pulse filtration with the circulating washing and pulse blowing.

[0090] The template of the chip in the pulse filtration system alone is made by traditional photolithography technology, which has the disadvantages of complex process and long production cycle. The template in the application is made by 3D printing technology, which can make the template simply and quickly.

[0091] The pulse filtration system alone does not have the function of circulating washing, so the sample on the filter membrane will be concentrated during pulse filtration, resulting in aggregation of the sample on the filter membrane. The application can prevent the concentration and aggregation of the sample on the filter membrane during pulse filtration by using circulating washing.

[0092] The microfluidic pulse circulating filtration system of the application can have the following beneficial effects, but is not limited to:

[0093] 1. The microfluidic pulse circulating filtration system of the application uses microfluidic pulse filtration to prevent filter membrane blockage and filter membrane pollution, and based on size, gently filters and recovers micro-nano particles.

[0094] 2. Different pore sizes of filter membranes can be used to separate micro- and nano-particles of different sizes. For example, large particles can be removed to recover small particles, or small particles can be removed to recover large particles.

[0095] 3. Microfluidic pulse filtration can minimize mechanical damage to particles during filtration and recover particles intact. For example, red blood cells in whole blood can be filtered to recover plasma, and free proteins in plasma can be filtered to recover extracellular vesicles. By filtering through a gentle microfluidic pulse flow, problems such as rupture of red blood cells caused by extrusion and low recovery rate of extracellular vesicles caused by contact with the filter membrane can be prevented.

[0096] 4. Compared with traditional constant pressure (or constant flow) filtration, pulse flow can effectively reduce filter membrane clogging and contamination during filtration, and improve separation efficiency depending on filter membrane pore size. Compared with traditional pulse filtration systems, the microfluidic pulse circulation filtration system can precisely control microfluids, reduce sample processing volume, reduce cost, and reduce the size of the device, achieving high parallel separation.

[0097] 5. The present application uses the ingenious arrangement of filter membranes, elastic membranes, microchannels, and one-way valves to form a microfluidic pulse circulation filtration system, which realizes pulse filtration of microfluids and reduces the cost, size, and sample processing volume of the pulse filtration system. BRIEF DESCRIPTION OF DRAWINGS

[0098] The embodiments of the present application will be described in detail below with reference to the accompanying drawings, in which:

[0099] Figure 1 A structure diagram of the microfluidic pulse circulation filtration system prepared in Example 1 is shown. In the diagram, Figure 1 A shows the structure diagram of the microfluidic pulse circulation filtration system in repeated state 1 when the elastic membrane repeatedly shifts upward; Figure 1 B shows the structure diagram of the microfluidic pulse circulation filtration system in repeated state 2 when the elastic membrane repeatedly shifts downward.

[0100] Figure 2 The results of washing free proteins by the microfluidic pulse circulation filtration system of the present application in Example 2 are shown. In the diagram, Figure 2 A shows that the removal rate of free proteins increases with the increase of the volume of the circulating washing liquid. When 1000 microliters of washing liquid is used for circulation washing, the protein removal rate can be more than 99%; Figure 2 B shows that the amount of residual free proteins decreases with the increase of the volume of the circulating washing liquid. When 1000 microliters of washing liquid is used for circulation washing, the concentration of free proteins decreases from 1.5 milligrams per milliliter to 5 micrograms per milliliter.

[0101] Figure 3Comparative Example 1 shows the comparison of pulse filtration of whole blood and constant flow filtration of whole blood; wherein, Figure 3 A shows the flow rate when pulse filtration of whole blood, pulse filtration can continuously remove blood cells from whole blood to separate plasma, and prevent the filter membrane from being blocked and contaminated by blood cells; Figure 3 B shows the flow rate when constant flow filtration of whole blood, the filter membrane is blocked by blood cells in whole blood, and the flow rate of constant flow filtration decreases from 16 microliters per second to almost 0 microliters per second within 100 seconds; Figure 3 C shows the plasma separated from whole blood by pulse filtration, the plasma separated by pulse filtration is clear and free of blood cell rupture; Figure 3 D shows the plasma separated from whole blood by constant flow filtration, the separated plasma contains blood cell rupture, resulting in hemolysis.

[0102] Figure 4 Comparative Example 2 shows the comparison of the results of concentrating extracellular vesicles in cell culture medium by pulse filtration and constant flow filtration; wherein, Figure 4 A shows the concentration of extracellular vesicles before filtration, the concentration of extracellular vesicles after concentration by pulse filtration, and the concentration of extracellular vesicles after concentration by constant flow filtration; the concentration of extracellular vesicles after concentration by pulse filtration is higher than that of extracellular vesicles after concentration by constant flow filtration, which can prove that pulse filtration can reduce damage to extracellular vesicles during filtration, while constant flow filtration causes damage to extracellular vesicles, so the concentration of extracellular vesicles after concentration by pulse filtration is higher than that of extracellular vesicles after concentration by constant flow filtration; Figure 4 B shows the recovery rate of extracellular vesicles concentrated by pulse filtration and constant flow filtration, the recovery rate of extracellular vesicles concentrated by pulse filtration is 75%, which is much higher than the recovery rate of extracellular vesicles concentrated by constant flow filtration.

[0103] Figure 5 Comparative Example 3 shows the comparison of the recovery rate of extracellular vesicles after removing free proteins in plasma by pulse circulation filtration and constant flow filtration; after removing free proteins and other impurities with 1000 microliters of washing solution, the recovery rate of extracellular vesicles by pulse circulation filtration is 60%, while the recovery rate of extracellular vesicles by constant flow filtration is only 15%.

[0104] Figure 6 Comparative Example 3 shows that pulse pressure is used to realize the deformation of the beating elastic membrane to generate pulse flow, and the pulse flow is used to beat the liquid above the filter membrane to further prevent the filter membrane from being blocked and contaminated; wherein, Figure 6 A shows that the upward deflection of the beating elastic membrane caused by pulse high pressure can beat the sample above the filter membrane; Figure 6 B shows that the downward deflection of the beating elastic membrane caused by pulse low pressure can pump the sample above the filter membrane, and by repeatedly beating and pumping, the sample above the filter containing the filter membrane is mixed, the blockage and contamination of the filter membrane are reduced, and the filtration speed is improved. Detailed Implementation

[0105] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for more detailed and specific illustration and should not be construed as limiting the present invention in any way.

[0106] This section provides a general description of the materials and testing methods used in the experiments of this invention. While many of the materials and methods of operation used to achieve the objectives of this invention are well known in the art, the invention is still described in as much detail as possible herein. It will be apparent to those skilled in the art that, unless otherwise stated in the context, the materials and methods of operation used in this invention are well known in the art.

[0107] The reagents and instruments used in the following examples are as follows:

[0108] Reagents:

[0109] PDMS, purchased from SYLGARD.

[0110] instrument:

[0111] Plasma bonding instrument, PDC-002, purchased from Harrick Plasma.

[0112] Example 1

[0113] This embodiment illustrates the preparation method of the microfluidic pulsed circulating filtration system of the present invention.

[0114] The microfluidic pulsed circulation filtration system is composed of polydimethylsiloxane (PDMS) and is manufactured using soft photolithography, specifically including the following steps:

[0115] (1) Fabrication of the microfluidic chip: The microfluidic chip consists of three layers of PDMS, with the upper and lower layers containing structures and the middle layer being a PDMS thin film. First, a mold is printed on glass using a 3D printer. The upper and lower PDMS layers containing structures are obtained by replicating from the mold. Each PDMS layer is bonded through plasma treatment and heating to form a PDMS microfluidic chip with microchannels and chambers in the upper and lower layers and an elastic thin film in the middle layer. The microfluidic chip after three-layer bonding contains microfluidic components such as elastic films in chambers, one-way valves, and microchannels.

[0116] (2) Preparation of a filter containing a filter membrane: The commercial filter membrane is installed in the commercial filter to form a filter containing a filter membrane.

[0117] (3) Preparation of a circulating filtration system:

[0118] (a) Drill holes above and below the waste liquid bottle and the washing bottle in the circulating washing system;

[0119] (b) the hole on the top of the waste liquid bottle and the washing bottle is connected by a pipe;

[0120] (c) the hole on the bottom of the waste liquid bottle is connected with the sample outflow outlet;

[0121] (d) the hole on the bottom of the washing bottle is above the filter containing the filter membrane.

[0122] (4) preparation of the pulse filtration system: connecting the side of the microfluidic chip with the waste liquid bottle and the washing bottle in the circulating washing system, and connecting the other side with the pulse blowing system and the filter containing the filter membrane, thus obtaining the microfluidic pulse circulating filtration system. In this embodiment, the microfluidic chip and the pulse pressure are connected by using a 90-degree bending needle and a polyethylene hose to form the pulse filtration system.

[0123] Example 2

[0124] This embodiment is used to illustrate the microfluidic pulse circulating filtration system of the present application.

[0125] Figure 1 The structural diagram of the microfluidic pulse circulating filtration system prepared in Example 1 is shown; wherein, Figure 1 A shows the structural diagram of the microfluidic pulse circulating filtration system in repeated state 1 when the elastic film repeatedly deviates upward; Figure 1 B shows the structural diagram of the microfluidic pulse circulating filtration system in repeated state 2 when the elastic film repeatedly deviates downward. As Figure 1 shown, when the waste liquid and washing liquid bottles are connected in series at the filter sample outflow outlet, the pulse filtration microfluidic chip can automatically wash the sample on the filter membrane in a circulating manner. The pulse filtration method of the present application is suitable for cleaning small particles in the sample and recovering particles larger than the pore size of the filter membrane. When the pulse filtration microfluidic chip is working, the waste liquid bottle flows in the filtered waste liquid, and the washing liquid in the washing bottle is dropped on the filter membrane in the form of droplets. The waste liquid and the washing liquid are separated by air to prevent the washing liquid from being contaminated by the waste liquid. At this time, the sample volume above the filter membrane is almost unchanged. Each time the droplet volume of the waste liquid is filtered, the droplet of the washing liquid is automatically supplemented into the sample on the filter membrane, which can prevent the concentration and drying of particles larger than the pore size of the filter membrane. The removal efficiency (η) of particles smaller than the pore size of the filter membrane is

[0126]

[0127] V Loading and V Droplet are the sample volume above the filter membrane and the droplet volume of the washing liquid, respectively, and N is the number of droplets dropped. Therefore, when the sample volume is 200 microliters and washed with 1 milliliter of washing liquid (37 droplets), the removal rate of particles smaller than the pore size of the filter membrane is 99.9%.

[0128] To verify the effectiveness of the cyclic washing method, 200 μL of free protein was washed with 1 mL of PBS, and the protein removal rate was as high as 99.9%. Figure 2 )

[0129] Figure 2 The results of washing free proteins using the microfluidic pulsed cyclic filtration system of the present invention in Example 2 are shown; wherein, Figure 2 A shows that the free protein removal rate increases with the increase of the circulating wash volume, and more than 99% of the protein can be removed when circulating wash with 1000 μL of washing solution. Figure 2 B shows that the amount of residual free protein decreases with increasing washing volume, decreasing from 1.5 mg / mL to 5 μg / mL when washing with 1000 μL of washing solution.

[0130] After washing with 1 ml of washing solution, the removal rate of free protein is greater than 99.9%. As the washing volume increases, the amount of residual protein decreases. This demonstrates that the microfluidic pulsed circulation filtration system of the present invention can effectively filter particles smaller than the pore size of the filter membrane.

[0131] Example 3

[0132] This embodiment is used to illustrate the preparation method of the pulse blowing system of the present invention.

[0133] The pulse blowing system of the present invention is composed of polydimethylsiloxane (PDMS) and is manufactured using a soft photolithography method, specifically including the following steps:

[0134] (1) The lower layer material is obtained by replicating the mold printed by a 3D printer.

[0135] (2) Heat and bond the lower layer material prepared in step (2) to the elastic film of the middle layer.

[0136] (3) The upper part of the elastic membrane is connected to the upper part of the filter containing the filter membrane through a polyethylene pipe.

[0137] Example 4

[0138] This embodiment illustrates the pulse filtering method of the microfluidic pulse circulating filtration system of the present invention.

[0139] The pulse pressure is provided to the pulse filtration microfluidic chip, and the pulse pressure can be provided by a pulse pressure generator, opening and closing of a solenoid valve, and constant pressure provided by a constant pressure pump. In this embodiment, the pulse pressure is provided by connecting the solenoid valve inlet to the constant pressure pump, and the pulse pressure is provided at the solenoid valve outlet when the solenoid valve is opened and closed. When the pulse pressure is connected to the chip, the chip can automatically filter the sample on the filter membrane. The filtered sample flows through the filter membrane, the upper layer of the elastic membrane chamber, the one-way valve, and the microchannel, and then flows out of the filter sample outlet. At the same time, the back-and-forth pulse flow through the filter membrane is generated to prevent the filter from being blocked. Specifically,

[0140] The pulse filtration system is driven by the pulse pressure, and the elastic membrane repeatedly deviates upward and downward, and repeatedly changes between state 1 and state 2. When the elastic membrane deviates upward (state 1), the liquid in the chamber above the elastic membrane flows out of the filter sample outlet through the one-way valve and the first microchannel. At the same time, the liquid in the chamber above the elastic membrane flows to the upper side of the filter membrane through the filter membrane. When the elastic membrane deviates downward (state 2), the liquid above the filter membrane flows to the chamber above the elastic membrane through the filter membrane. At this time, the one-way valve is closed, and the liquid flowing out of the filter sample outlet flows to the chamber above the elastic membrane. When the elastic membrane repeatedly changes between state 1 and state 2, the pulse filtration system can generate a pulse flow through the filter membrane, prevent the filter from being blocked and contaminated, and effectively filter the sample to the filter sample outlet.

[0141] The pulse filtration system filters the sample, and continuously flows the sample into the waste bottle through the filter sample outlet. At this time, the waste bottle is filled with the filtered sample, and an equal volume of the washing liquid in the washing bottle is transported in the form of droplets to the upper side of the filter containing the filter membrane, so that the volume of the sample above the filter membrane remains unchanged, and the cycle filtration of the automatically washed sample is realized.

[0142] The pulse blow system is driven by the pulse pressure, and the blow elastic membrane repeatedly deviates upward and downward (state 1 and state 2). When the blow elastic membrane deviates upward (state 1), the liquid above the filter containing the filter membrane is blown once through the blow channel. When the blow elastic membrane deviates downward (state 2), the liquid above the filter containing the filter membrane is sucked once through the blow channel. When the pulse pressure repeatedly changes between state 1 and state 2, the liquid above the filter containing the filter membrane can be continuously blown and sucked, the liquid above the filter membrane is mixed, and the blockage and contamination of the filter membrane are further prevented.

[0143] Example 5

[0144] This embodiment is used to illustrate the technical effects of the microfluidic pulse cycle filtration system of the present application.

[0145] The microfluidic pulse filtration realizes the pulse flow of microfluid, reduces the blockage and pollution of the filter membrane, and gently filters the sample. When filtering the whole blood sample, the collision and blockage of blood cells with the filter membrane are reduced, and the rupture of blood cells is prevented. When filtering the extracellular vesicles, the loss caused by the collision of extracellular vesicles with the filter membrane is reduced, and the particle recovery rate of extracellular vesicles is improved.

[0146] Example 6

[0147] The present embodiment is used to illustrate the technical effect of the microfluidic pulse circulation filtration system of the present application.

[0148] The microfluidic pulse filtration realizes the pulse flow of precise microfluid, can reduce the sample processing amount, and reduce the cost and size of the pulse filtration system. Traditional pulse filtration requires the joint driving of large and expensive injection pumps, motors and other auxiliary equipment, and it is difficult to realize the pulse flow of microfluid, or the cost of realizing the pulse filtration of microfluid is greatly increased. The microfluidic pulse flow can realize the fluid control of microfluid only by using one pulse pressure, which has a significant advantage compared with the traditional method.

[0149] Example 7

[0150] The present embodiment is used to illustrate the technical effect of the pulse blow system of the present application.

[0151] The pulse blow system realizes repeated blow mixing of the sample containing the filter membrane above, further reduces the blockage and pollution of the filter membrane during filtration, and improves the speed and separation efficiency of the pulse circulation filtration.

[0152] Comparative Example 1

[0153] The present comparative example is used to compare the effect of the microfluidic pulse circulation filtration system and the constant flow filtration system of the present application on the recovery of plasma from whole blood.

[0154] The whole blood is filtered to recover the plasma by using a filter membrane with a pore size of 600 nanometers.

[0155] Figure 3 The comparative example 1 shows the comparison of pulse filtration of whole blood and constant flow filtration of whole blood; wherein, Figure 3 A shows the flow rate when the whole blood is pulse filtered, the pulse filtration can continuously remove blood cells from the whole blood to separate the plasma, and prevent the filter membrane from being blocked and polluted by the blood cells; Figure 3 B shows the flow rate when the whole blood is constant flow filtered, the filter membrane is blocked by the blood cells in the whole blood when the constant flow is filtered, and the flow rate of the constant flow filtration decreases from 16 microliters per second to almost 0 microliters per second within 100 seconds; Figure 3 C shows the plasma separated from the whole blood by pulse filtration, the plasma separated by pulse filtration is clear and has no rupture of blood cells; Figure 3D shows the plasma separated from whole blood by constant flow filtration, the separated plasma contains the rupture of blood cells, resulting in hemolysis.

[0156] When pulse filtration, the plasma can be continuously separated from the whole blood, the pulse filtration flow is effectively prevented from being blocked by blood cells due to the anti-blocking effect of the pulse flow, so that the flow rate slowly decreases.

[0157] When constant flow filtration, blood cells quickly block the filter membrane, causing the constant flow filtration flow to rapidly decrease in a short time. Pulse filtration gently filters the whole blood sample, and the obtained sample has no hemolysis. Constant flow filtration continuously extrudes blood cells and filter membranes, causing the blood cells to rupture, and the obtained sample is red and hemolyzed.

[0158] The filter membrane of the present application has a nanometer-pore thin film, and the microfluidic chip can generate a pulse flow of microfluid. The present application can realize pulse filtration of microfluid by combining the filter membrane and the microfluidic chip.

[0159] Comparative Example 2

[0160] The present comparative example is used to compare the effects of the microfluidic pulse circulation filtration system and the constant flow filtration system of the present application on the filtration of concentrated extracellular vesicles.

[0161] A 20-nanometer-pore filter membrane is used to filter concentrated extracellular vesicles from a breast epithelial cell (MCF-10A) culture solution.

[0162] Figure 4 The results of comparing the pulse filtration and constant flow filtration of concentrated extracellular vesicles in a cell culture solution in Comparative Example 2 are shown; wherein, Figure 4 A shows the concentration of extracellular vesicles before filtration, the concentration of extracellular vesicles concentrated by pulse filtration, and the concentration of extracellular vesicles concentrated by constant flow filtration; the concentration of extracellular vesicles concentrated by pulse filtration is higher than the concentration of extracellular vesicles concentrated by constant flow filtration, which can prove that pulse filtration can reduce the damage to extracellular vesicles during filtration, while constant flow filtration causes damage to extracellular vesicles, so the concentration of extracellular vesicles concentrated by pulse filtration is higher than the concentration of extracellular vesicles concentrated by constant flow filtration; Figure 4 B shows the recovery rate of extracellular vesicles concentrated by pulse filtration and constant flow filtration, the recovery rate of extracellular vesicles concentrated by pulse filtration is 75%, which is much higher than the recovery rate of extracellular vesicles concentrated by constant flow filtration.

[0163] Extracellular vesicles can be concentrated by pulse filtration and constant flow filtration. The concentration after pulse filtration is greater than that after constant flow filtration. Therefore, pulse filtration achieves gentle filtration, reduces damage to extracellular vesicles during filtration, and the recovery rate of extracellular vesicles by pulse filtration is about 75%. During constant flow filtration, continuous extrusion of extracellular vesicles and filter membranes leads to loss of extracellular vesicles, and the recovery rate of extracellular vesicles by constant flow filtration is about 20%.

[0164] Pulse filtration can gently filter particles, reduce clogging and contamination of the filter membrane, reduce friction, damage, and loss between particles and filter membrane pores, and can improve the recovery rate of particles.

[0165] Comparative Example 3

[0166] The comparative example is used to compare the microfluidic pulse circulation filtration system of the present application with the filtration system of only pulse filtration.

[0167] 1. Difference one:

[0168] (1) The microfluidic pulse circulation filtration system of the present application: The microfluidic pulse circulation filtration system of the present application comprises a circulation washing system, a pulse blow-off system, a filter containing a filter membrane, and a microfluidic chip. The circulation washing system comprises a waste liquid bottle and a washing bottle, and the filter sample outlet is connected in series with the waste liquid bottle and the washing bottle; the pulse blow-off system comprises a blow-off elastic membrane and a blow-off channel. During filtration of the sample, the waste liquid automatically flows into the waste liquid bottle, and the washing liquid automatically drops in the form of droplets into the filtered sample, thereby washing small particles such as free proteins in the filtered sample. The pulse blow-off mixing system automatically cleans the micro-nano particles above the filter membrane during filtration, thereby preventing clogging and contamination of the filter membrane.

[0169] (2) Filtration system of only pulse filtration: does not comprise a waste liquid bottle, a washing bottle, droplets, a pulse pressure inlet, a pulse pressure outlet, a second microchannel, a blow-off elastic membrane, and a blow-off channel.

[0170] 2. Difference two:

[0171] (1) The microfluidic pulse circulation filtration system of the present application: Because the circulation washing system and the pulse blow-off system are used, extracellular vesicles can be concentrated from cell culture liquid, free proteins can be removed from plasma to recover extracellular vesicles, further reducing contamination and clogging of the filter membrane, and improving the recovery rate of extracellular vesicles.

[0172] (2) Filtration system of only pulse filtration: It is difficult to concentrate extracellular vesicles from a large amount of cell culture liquid, to effectively remove free proteins from plasma to recover extracellular vesicles, and to slow the filtration speed; contamination and clogging of the filter membrane cannot be solved; and the recovery rate of extracellular vesicles needs to be improved.

[0173] 3. Difference three:

[0174] (1) The microfluidic pulse circulation filtration system of the present application: The circulation washing system of the present application can automatically add washing liquid without manual operation to automatically clean the free proteins in the filtered plasma and recover extracellular vesicles. The pulse blow-mixing system can automatically blow and mix the sample, further preventing the clogging and pollution of the filter membrane during filtration. The separation effect and speed based on the pore size of the filter membrane are improved, and the extracellular vesicles are effectively filtered and separated with high recovery rate.

[0175] (2) The filtration system of only pulse filtration: The above technical effects cannot be achieved in the filtration system of only pulse filtration.

[0176] In summary, compared with the filtration system of only pulse filtration, the microfluidic pulse circulation filtration system of the present application can automatically clean the filtered sample without an external control system, and can automatically and quantitatively add washing liquid. The system of the present application can ensure that the volume of the solution above the filter membrane remains unchanged, effectively preventing the clogging of the filter membrane caused by the rapid increase in the concentration of substances in the sample when the solution above the membrane is filtered dry, and also preventing damage to the sample caused by drying of the solution.

[0177] Figure 5 As shown in Comparative Example 3, the extracellular vesicle recovery rate after removing free proteins in plasma by pulse circulation filtration and constant flow filtration was compared; after removing free proteins and other impurities by using 1000 microliters of washing liquid, the extracellular vesicle recovery rate by pulse circulation filtration was 60%, while the extracellular vesicle recovery rate by constant flow filtration was only 15%. As shown in FIG. 1, when the pulse circulation filtration of the present application is used, the concentration and drying of extracellular vesicles are prevented, and the extracellular vesicle recovery rate after washing with 1 mL of washing liquid is 60%. However, if only the pulse filtration of the prior art is used, due to the concentration and drying of extracellular vesicles during filtration, the extracellular vesicle recovery rate after washing with 1 mL of washing liquid is only 15%. Therefore, pulse circulation filtration can improve the recovery rate of extracellular vesicles after washing. Figure 5

[0178] Figure 6 As shown in Comparative Example 3, the use of pulse pressure to achieve the deformation of the elastic membrane generates a pulse flow, and the use of pulse flow to blow the sample above the filter membrane further prevents the clogging and pollution of the filter membrane; wherein, Figure 6 A shows that the upward deviation of the blow elastic membrane caused by pulse high pressure can blow the sample above the filter membrane; Figure 6 B shows that the downward deviation of the blow elastic membrane caused by pulse low pressure can draw the sample above the filter membrane, and the sample above the filter containing the filter is realized by repeated blow and draw, reducing the clogging and pollution of the filter membrane and improving the filtration speed. As shown in FIG. 2, Figure 6 ​As shown, when the pulse cycle filtration of the present application is used, further by blowing the pulse deformation of the membrane to blow the sample above the filter membrane, the clogging and pollution of the filter membrane can be reduced. The separation effect and separation speed based on the pore size of the filter membrane are improved, and the free protein is effectively filtered to separate the extracellular vesicles with high recovery rate.

[0179] Although the present application has been described to a certain degree of description, it is obvious that appropriate changes can be made to each condition without departing from the spirit and scope of the present application. It can be understood that the present application is not limited to the described embodiments, but is subject to the scope of the claims, which includes equivalent substitutions of each factor described.

Claims

1. A microfluidic pulsed circulating filtration system, characterized in that, The microfluidic pulse circulation filtration system includes: a circulation washing system, a pulse blowing system, a filter containing a filter membrane, and a microfluidic chip; wherein, The circulating washing system, pulse blowing system, filter containing filter membrane, and microfluidic chip are connected by needles or tubes. The microfluidic chip includes an elastic thin-film chamber, a microfluidic one-way valve, and a microchannel; The microfluidic pulse circulation filtration system further includes: a pulse pressure inlet, a pulse pressure outlet, and a filtered sample outlet; The circulating washing system includes a waste liquid bottle and a washing bottle, and the filtered sample outlet is connected in series with the waste liquid bottle and the washing liquid bottle. The pulse blowing system includes blowing an elastic film and a blowing channel; The elastic film chamber includes an upper elastic film chamber, a lower elastic film chamber, and an intermediate elastic film layer. The upper elastic film chamber and the lower elastic film chamber are separated by the intermediate elastic film layer. The microchannel includes a first microchannel and a second microchannel; The lower part of the blow-blow elastic film is connected to the pulse pressure inlet, and the upper part of the blow-blow elastic film is connected to the upper part of the filter containing the filter membrane through the blow-blow channel; The waste liquid bottle of the circulating washing system is connected to the washing bottle and the filtered sample outlet through pipes. The bottom of the washing bottle has an opening to allow liquid to pass through and form droplets, which are aligned with the filter containing the filter membrane. The filter containing the filter membrane, the upper chamber of the elastic membrane, the microfluidic one-way valve, and the first microchannel are sequentially connected to form a microfluidic flow path.

2. The microfluidic pulse circulating filtration system according to claim 1, characterized in that: The chamber beneath the elastic film is connected to the second microchannel, the pulse pressure inlet, and the pulse pressure outlet; and / or The microfluidic one-way valve is located between the upper chamber of the elastic membrane and the first microchannel, which is connected to the outlet of the filtered sample.

3. The microfluidic pulsed circulating filtration system according to claim 1 or 2, characterized in that: The flow resistance of the micro- and nanopores of the filter membrane is obtained by formula (1): Flow resistance R of the filter membrane Filter = Pressure difference through the filter membrane / Flow rate through the filter membrane (1); The mechanical capacitance of the elastic film is obtained by formula (2): C Membrane Let r be the mechanical capacitance of the elastic film, r be the radius of the elastic film, T be the thickness of the elastic film, E be the Young's modulus of the elastic film, and μ be the Poisson's coefficient of the elastic film; and / or The relationship between the flow resistance and the size of the microchannel is obtained by formulas (3)-(5): Among them, R Channel ν is the flow resistance of the microchannel, w, h, l are the width, height, and length of the microchannel, respectively, and ν is the dynamic viscosity of the fluid.

4. The microfluidic pulse circulating filtration system according to claim 1, characterized in that: The microchannel material is polydimethylsiloxane or polytetrafluoroethylene; The elastic film of the elastic film chamber and / or the material of the blown elastic film is polydimethylsiloxane or polytetrafluoroethylene; and / or The material of the blowing channel is polyethylene pipe.

5. The microfluidic pulse circulating filtration system according to claim 4, characterized in that: The diameter of the micro- and nanopores in the filter membrane is 20 to 600 nanometers.

6. The microfluidic pulse circulation filtration system according to claim 5, characterized in that, The diameter of the micro- and nanopores in the filter membrane is 20 to 220 nanometers.

7. The microfluidic pulse circulation filtration system according to claim 6, characterized in that, The diameter of the micro- and nanopores in the filter membrane is 20 to 100 nanometers.

8. A method for preparing the microfluidic pulsed circulating filtration system according to any one of claims 1 to 7, characterized in that, The method includes: preparing a microfluidic chip, and connecting a circulating washing system, a pulse blowing system, a filter containing a filter membrane, and the microfluidic chip together via a needle or channel.

9. The method according to claim 8, characterized in that, The method for preparing the microfluidic pulsed circulating filtration system includes the following steps: (A) Fabrication of microfluidic chips; (B) Connect one side of the microfluidic chip to the waste liquid bottle and washing bottle in the circulating washing system, and connect the other side to the pulse blowing system and the filter containing the filter membrane to obtain the microfluidic pulse circulating filtration system.

10. The method according to claim 9, characterized in that, Step (B) further includes: punching holes above and below the waste liquid bottle and the washing bottle, connecting the holes above the waste liquid bottle and the washing bottle through pipes, connecting the hole below the waste liquid bottle to the sample outlet, and placing the hole below the washing bottle above the filter containing the filter membrane; and / or the pulse blow system is fabricated by soft photolithography.

11. The method according to claim 9 or 10, characterized in that, In step (A), the microfluidic chip fabrication operation includes the following steps: (1) The upper and lower materials containing chambers and microchannels are replicated by a mold printed by a 3D printer; (2) The lower layer material containing chambers and microchannels prepared in step (1) is heated and bonded together with the elastic film of the middle layer; (3) The elastic film with the lower layer material obtained in step (2) is heated and bonded together with the upper layer material containing the chamber and microchannel to form a microfluidic chip with the upper layer material containing the chamber and microchannel, the lower layer material and the middle layer being an elastic film.

12. The method according to claim 11, characterized in that, An upper layer material containing chambers and microchannels forms an upper chamber above the elastic membrane, a one-way valve, and a first microchannel; and / or The lower layer material containing chambers and microchannels forms the lower chamber and second microchannel beneath the elastic membrane.

13. The method according to claim 11, characterized in that: Step (1) further includes: mixing the upper and lower layer materials with a curing agent on a mold containing chambers and microchannels, heating the mixture, and peeling it off after curing to obtain the upper and lower layer materials containing chambers and microchannels; and / or In steps (2) and (3), the bonding method is plasma bonding or vacuum hot pressing bonding, the bonding temperature is 100-150℃, and the bonding time is 2-30 min.

14. The method according to claim 13, characterized in that: In steps (2) and (3), the bonding method is plasma bonding, the bonding temperature is 110-130°C, and the bonding time is 5-20 min.

15. The method according to claim 14, characterized in that: In steps (2) and (3), the bonding temperature is 120°C and the bonding time is 15 min.

16. The method according to claim 11, characterized in that: In step (1): the mass ratio of the upper layer material and the lower layer material to the curing agent is 5-20:1, the heating time is 10-36 hours, and the heating temperature is 60-100°C; and / or In step (3), when bonding the check valve, a pad is used to cover the check valve diaphragm and valve seat.

17. The method according to claim 16, characterized in that: In step (1): the mass ratio of the upper layer material and the lower layer material to the curing agent is 5 to 15:1, the heating time is 10 to 24 hours, and the heating temperature is 70 to 90°C.

18. The method according to claim 17, characterized in that: In step (1): the mass ratio of the upper layer material and the lower layer material to the curing agent is 10:1, the heating time is 12h, and the heating temperature is 80℃.

19. The method according to claim 16, characterized in that, The curing agent is a polydimethylsiloxane curing agent.

20. A pulse filtering method, said method using the microfluidic pulse circulating filtration system according to any one of claims 1 to 7 or the microfluidic pulse circulating filtration system prepared by the method according to any one of claims 8 to 19.

21. The pulse filtering method according to claim 20, characterized in that, The pulse filtration method includes: when a pulse pressure is provided to the microfluidic pulse circulation filtration system, the microfluidic pulse circulation filtration system automatically filters the sample on the filter membrane. The filtered sample flows out of the filter sample outlet through the filter membrane, the upper layer of the elastic membrane chamber, the microfluidic one-way valve, and the microchannel, and is prevented from clogging by the back-and-forth pulse flow of the filter membrane; during pulse filtration, the circulating washing system continuously replenishes the filter containing the filter membrane with washing liquid to achieve circulating washing; the pulse blowing system generates a pulse flow by blowing the deformation of the elastic membrane, continuously blowing the sample above the filter containing the filter membrane to reduce filter membrane clogging and contamination.

22. The application of the microfluidic pulsed circulating filtration system according to any one of claims 1 to 7 or the microfluidic pulsed circulating filtration system prepared by the method according to any one of claims 8 to 19 in the preparation of micro / nano filter devices.

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