CN115739215A

CN115739215A - Micro-fluidic pulse filtering system, preparation method and filtering method thereof and application

Info

Publication number: CN115739215A
Application number: CN202211376562.7A
Authority: CN
Inventors: 孙佳姝; 李政霖; 田飞; 刘超; 邓瑾琦
Original assignee: National Center for Nanosccience and Technology China
Current assignee: National Center for Nanosccience and Technology China
Priority date: 2022-11-04
Filing date: 2022-11-04
Publication date: 2023-03-07

Abstract

The invention provides a micro-fluidic pulse circulating filtration system, a preparation method and a filtration method thereof and application. The microfluidic pulse circulation filtration system comprises a filter containing a filter membrane and a microfluidic chip. According to the invention, micro-fluidic pulse filtration is utilized to prevent the filter membrane from being blocked and the filter membrane from being polluted, and micro-nano particles based on the size are softly filtered and recovered. The micro-nano particles with different sizes can be separated by using the aperture of different filter membranes. For example, large particles can be removed to recover small particles, or small particles can be removed to recover large particles. The micro-fluidic pulse filtration can reduce the mechanical damage of particles during filtration to the maximum extent and completely recover the particles.

Description

Micro-fluidic pulse filtering system, preparation method and filtering method thereof and application

Technical Field

The invention belongs to the field of materials, and particularly relates to a micro-fluidic pulse circulating filtration system, a preparation method and a filtration method thereof and application thereof.

Background

Filtration using filtration membranes has a variety of applications, such as: red blood cells (6 micron particles) are removed from whole blood to separate plasma, free proteins (less than 3 nanometers) are removed from plasma, and extracellular vesicles (more than 30 nanometers) are recovered from plasma. The conventional method using a filter membrane has an inherent problem of clogging of the filter membrane. In order to solve the problem of blockage of the filter membrane, a micro-fluidic pulse circulation filtration system is developed. The pulse filtration is to use repeated pulse flow to prevent the filter membrane from being blocked by the accumulation of micro-nano particles. However, the traditional pulse filtering requires the combined driving of huge auxiliary devices, such as: syringe pump, programmable computer, single-chip computer, valve and need relevant professional knowledge. Due to the use of bulky auxiliary equipment, microfluidic pulse filtration is difficult to achieve or the cost of achieving microfluidic pulse filtration is greatly increased.

In the prior art, a filtration system only using pulse filtration can only remove red blood cells from a whole blood sample to separate plasma, cell vesicles in a cell culture solution cannot be concentrated, free proteins in the plasma are removed to recover extracellular vesicles, and the recovery rate is not ideal. The pollution and blockage rate of the filter membrane needs to be improved. And the template of the chip is manufactured by the traditional photoetching technology, and has the defects of complex process and long manufacturing period. Meanwhile, the filtration system only using pulse filtration has no circular washing function, so that samples on the filter membrane can be continuously concentrated during pulse filtration, and the samples on the filter membrane can be polymerized.

Disclosure of Invention

Therefore, the present invention aims to overcome the defects in the prior art and provide a microfluidic pulse circulation filtration system, a preparation method thereof, a filtration method thereof and an application thereof. The micro-fluidic pulse circulation filtering system is formed by skillfully arranging micro-fluidic elements such as the filter membrane, the elastic film, the micro-channel, the one-way valve and the like, so that a sample can be filtered softly, and the damage of micro-nano particles during filtering is reduced. The micro-flow pulse filtration is realized, and the cost, the system size and the sample processing capacity of the micro-flow pulse filtration system are reduced.

Before setting forth the context of the present invention, the terms used herein are defined as follows:

the term "PDMS" refers to: polydimethylsiloxane.

The term "Teflon" means: polytetrafluoroethylene.

To achieve the above object, a first aspect of the present invention provides a microfluidic pulse-cycle filtration system comprising: a circulating washing system, a pulse blowing system, a filter containing a filter membrane and a micro-fluidic chip; wherein the content of the first and second substances,

the circulating washing system, the pulse blowing system, the filter containing the filter membrane and the micro-fluidic chip are connected through a needle or a pipeline; and/or

The microfluidic chip comprises an elastic film chamber, a microfluidic one-way valve and a microchannel.

The microfluidic pulse cycle filtration system according to the first aspect of the present invention, wherein,

the microfluidic pulse circulation filtration system further comprises: a pulse pressure inlet, a pulse pressure outlet and a filtered sample outlet;

the circulating washing system comprises a waste liquid bottle and a washing bottle, and the filtering sample outflow outlet is connected with the waste liquid bottle and the washing liquid bottle in series;

the pulse blow-beating system comprises a blow-beating elastic film and a blow-beating channel;

the elastic film cavity comprises an upper cavity of the elastic film, a lower cavity of the elastic film and the elastic film of the middle layer, and the upper cavity of the elastic film and the lower cavity of the elastic film are separated by the elastic film of the middle layer; and/or

The microchannel comprises a first microchannel and a second microchannel;

preferably, the lower part of the blown elastic membrane is connected with a pulse pressure inlet, and the upper part of the blown elastic membrane is connected to the upper part of the filter containing the filter membrane through the blown channel;

preferably, the waste liquid bottle and the washing bottle of the circulation washing system are sequentially connected to the filtered sample outflow outlet through a pipeline, the lower part of the washing bottle is provided with an opening for allowing the liquid to pass through to form liquid drops, and the liquid drops are aligned above the filter containing the filter membrane;

preferably, 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;

preferably, the chamber below the elastic membrane is connected with the second microchannel, the pulse pressure inlet and the pulse pressure outlet; and/or

Preferably, the microfluidic one-way valve is located between the chamber above the elastic membrane and a first microchannel, and the first microchannel is connected with the filtered sample outflow port.

the flow resistance of the micro-nano hole is obtained through a formula (1):

flow resistance R of the Filter Membrane _Filter = pressure difference across the filter membrane/flow through the filter membrane equation (1);

the mechanical capacitance of the elastic membrane is obtained by equation (2):

C _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 coefficient of the elastic membrane; and/or

The relationship between the flow resistance of the microchannel and the size of the microchannel is obtained by the following equations (3) to (5):

wherein R is _Channel Is the flow resistance of the microchannel, w, h, l are the width, height, length of the microchannel, respectively, and ν is the dynamic viscosity of the fluid.

The microfluidic pulse-cycle filtration system according to the first aspect of the present invention, wherein,

the microchannel is made of PDMS or Teflon, and the PDMS is most preferably selected;

the elastic film of the elastic film chamber and/or the material of the blown elastic film is PDMS or Teflon, and the PDMS is the most preferable; and/or

The blow-beating channel is made of a polyethylene pipe;

preferably, the diameter of the micro-nano hole is 20-600 nm, more preferably 20-220 nm, and further preferably 20-100 nm.

A second aspect of the invention provides a method of making a microfluidic pulse-cycle filtration system according to the first aspect, the method comprising: preparing a micro-fluidic chip, and connecting a circulating washing system, a pulse blowing system, a filter containing a filter membrane and the micro-fluidic chip together through a needle or a channel;

preferably, the method of making the microfluidic pulse cycle filtration system comprises the steps of:

(A) Preparing a micro-fluidic chip;

(B) Connecting one side of a micro-fluidic chip with a waste liquid bottle and a washing bottle in the connection of a circulating washing system, and connecting the other side of the micro-fluidic chip with a pulse blowing system and a filter containing a filter membrane to obtain the micro-fluidic pulse circulating filtration system;

more preferably, the step (B) further comprises: punching holes above and below the waste liquid bottle and the washing bottle, wherein the hole above the waste liquid bottle is connected with the hole above the washing bottle through a pipeline, the hole below the waste liquid bottle is connected with a sample outflow outlet, and the hole below the washing bottle is positioned above a filter containing a filter membrane; and/or the pulse blow-beating system is prepared by a soft photoetching method.

The production method according to the second aspect of the present invention, wherein, in the step (a), the operation of producing the microfluidic chip comprises the steps of:

(1) Copying a mold printed by a 3D printer to obtain an upper layer material and a lower layer material containing a cavity and a micro-channel;

(2) Heating and bonding the lower layer material containing the cavity and the micro-channel prepared in the step (1) and the elastic film of the middle layer together;

(3) Heating and bonding the elastic film with the lower layer material obtained in the step (2) and the upper layer material containing the cavity and the micro-channel together to form a micro-fluidic chip with the upper layer material containing the cavity and the micro-channel, the lower layer material and the middle layer being the elastic film;

preferably, the upper layer of material comprising the chamber and the microchannel forms the chamber, the one-way valve and the first microchannel over the elastomeric film; and/or

Preferably, the lower layer of material containing the chambers and microchannels forms the chambers and second microchannels below the elastic membrane.

The production method according to the second aspect of the invention, wherein,

the step (1) further comprises: placing the upper layer material, the lower layer material and a curing agent on a mold containing a cavity and a micro-channel, mixing, heating, solidifying and peeling the materials to obtain the upper layer material and the lower layer material containing the cavity and the micro-channel; and/or

In the step (2) and the step (3), the bonding method is a plasma method or vacuum hot-pressing bonding, and most preferably a plasma method; the bonding temperature is 100-150 ℃, preferably 110-130 ℃, and most preferably 120 ℃; the bonding time is 2 to 30min, preferably 5 to 20min, and most preferably 15min.

The production method according to the second aspect of the present invention, wherein, in the step (1): the mass ratio of the upper layer material to the lower layer material to the curing agent is 5-20: 1, preferably 5 to 15:1, most preferably 10; the heating time is 10 to 36 hours, preferably 10 to 24 hours, and most preferably 12 hours; the heating temperature is 60-100 ℃, preferably 70-90 ℃, and most preferably 80 ℃; and/or

In the step (3), when the one-way valve is bonded, the cushion is used for covering the film of the one-way valve and the valve seat;

preferably, the curing agent is a polydimethylsiloxane curing agent.

A third aspect of the invention provides a pulse filtration method using a microfluidic pulse cycle filtration system according to the first aspect or a microfluidic pulse cycle filtration system prepared by the method according to the second aspect;

preferably, the pulse filtering method includes: when pulse pressure is provided for the micro-fluidic pulse circulation, the micro-fluidic pulse circulation filtering system automatically filters a sample on a filter membrane, the filtered sample flows out of a filtered sample outflow outlet through the filter membrane, the upper layer of the elastic membrane chamber, the micro-fluidic one-way valve and the micro-channel, and the filtered sample flows back and forth through the filter membrane to prevent blockage and filtration; the circulating washing system continuously supplements washing liquid on a filter containing a filter membrane during pulse filtration to realize circulating washing; the pulse blowing system generates pulse flow through the deformation of the blowing elastic film, continuously blows and blows a sample above the filter containing the filter membrane, and reduces the blockage and pollution of the filter membrane.

The fourth aspect of the invention provides an application of the microfluidic pulse circulation filtration system of the first aspect or the microfluidic pulse circulation filtration system prepared by the method of the second aspect in preparing a micro-nano filtration device.

According to a specific embodiment of the present application, the microfluidic pulse cycle filtration system of the present application comprises a filter of a filter membrane and a microfluidic chip. The micro-fluidic chip comprises a chamber with an elastic film, a micro-fluidic one-way valve and a micro-channel. The filter containing the filter membrane, the upper layer of the chamber of the elastic membrane, the one-way valve and the microchannel are connected in series to form the microfluidic flow path. The lower layer of the chamber of the elastic membrane is connected with a micro-channel which is connected with a pulse pressure driving pulse filtering micro-fluidic chip.

The filter membrane is provided with micro-nano holes, and the flow resistance of the filter membrane can be obtained by measuring the ratio of the pressure difference and the flow passing through the filter membrane. Therefore, the temperature of the molten metal is controlled,

flow resistance R of the Filter Membrane _Filter = pressure difference across the filter membrane/flow through the filter membrane equation (1).

Mechanical capacitance (C) of elastic film _Membrane ) Can be obtained by using a column plate theory:

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 coefficient, respectively, of the elastic membrane.

Flow resistance (R) of microchannels _Channel ) Controlled by varying the size of the channel.

Wherein, w, h and l are the width, height and length of the micro-channel respectively. ν is the dynamic viscosity of the fluid.

The micro-fluidic chip of the micro-fluidic pulse circulating filter system consists of Polydimethylsiloxane (PDMS) and is manufactured by a soft lithography technology. The chip is composed of 3 layers of PDMS, the upper layer and the lower layer are microchannels, and the middle layer is an elastic film. The upper and lower microchannels were made by replicating PDMS from a 3D mold. After mixing the curing agent and PDMS at a temperature of 1. Bonding of each PDMS layer was performed by plasma treatment, followed by heating the treated PDMS layers together at 120 degrees celsius for 15 minutes. When the check valve is bonded, the membrane and the valve seat of the valve are covered by the PDMS cushion to prevent the part from being treated by plasma, and the check valve can be opened after the heating process. The micro-fluidic chip and the pulse pressure are connected by a 90-degree bent needle and a polyethylene hose.

According to another specific embodiment of the present application, the preparative loop filtration system comprises the steps of:

(1) Punching holes at the upper part and the lower part of a waste liquid bottle and a washing bottle in the circulating washing system;

(2) The waste liquid bottle is connected with the hole above the washing bottle through a pipeline;

(3) The lower hole of the waste liquid bottle is connected with the sample outflow outlet;

(4) The lower hole of the washing bottle is positioned above the filter containing the filter membrane;

preferably, the preparation cycle washing system operation comprises the steps of:

(5) Copying a mold printed by a 3D printer to obtain a lower-layer material;

(6) Heating and bonding the lower layer material prepared in the step (5) and the elastic film of the middle layer together;

(7) The top of the elastic membrane is connected to the top of the filter containing the filter membrane by a polyethylene tube.

The pulse filtering system structure of the invention is as follows: the pulse filtering system consists of a filter containing a filter membrane and a microfluidic chip. And (4) a micro-fluidic chip. The micro-fluidic chip comprises an elastic film, an upper cavity of the elastic film, a lower cavity of the elastic film, a one-way valve, a first micro-channel and a second micro-channel. The intermediate film of the one-way valve contains holes. A filter membrane containing a filter is connected to the upper chamber of the elastic membrane, the check valve, and the first microchannel, and is connected to the filtered sample outflow port. The pulsating inlet is connected to the lower chamber of the elastic membrane, the second microchannel, and to the pulsating outlet.

A circulating filtering system: the waste liquid bottle and the washing bottle are sequentially connected to the filtered sample outflow outlet by pipelines. The holes in the lower part of the wash bottle allow the liquid to pass through forming droplets which are directed 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, so that the pollution of the washing liquid and the waste liquid is prevented.

Pulse blows and beats the system: the pulse blow-beating system consists of a blow-beating elastic film and a blow-beating channel. The lower part of the blowing elastic membrane is connected with the pulse pressing port, and the upper part of the blowing elastic membrane is connected to the upper part of a filter containing a filter membrane through a blowing channel.

According to yet another specific embodiment of the present application, the pulse filtering method comprises: when pulse pressure is provided for the micro-fluidic pulse circulation, the micro-fluidic pulse circulation filtering system automatically filters a sample on a filter membrane, the filtered sample flows out of a filtered sample outflow outlet through the filter membrane, the upper layer of an elastic membrane chamber, a micro-fluidic one-way valve and a micro-channel, and the filtered sample flows back and forth through the filter membrane to prevent blockage; the circulating washing system continuously replenishes washing liquid on a filter containing a filter membrane during pulse filtration to realize circulating washing; the pulse blowing system generates pulse flow through the deformation of the blowing elastic membrane, continuously blows and blows a sample above a filter containing the filter membrane, and reduces the blockage and pollution of the filter membrane.

The working principle of the pulse filtering system is as follows: the pulse filtering system is driven by pulse pressure, and the elastic membrane repeatedly deflects upwards and downwards to repeat states 1 and 2. When the elastic membrane deflects upwards (state 1), the liquid in the chamber above the elastic membrane flows out of the filtered sample outflow outlet through the one-way valve and the first microchannel. And simultaneously the liquid in the chamber above the elastic membrane flows to the upper part of the filter membrane through the filter membrane. When the elastic film deflects downwards (state 2), the liquid above the filter membrane flows to the chamber above the elastic film through the filter membrane, at the moment, the one-way valve is closed, and the liquid of the filtered sample flowing out of the outlet is prevented from flowing to the chamber above the elastic film. When the states 1 and 2 are repeated, the pulse filtering system can generate pulse flow passing through the filter membrane, so that the filter membrane is prevented from being blocked and polluted, and the sample is effectively filtered to the filtered sample outflow outlet.

The working principle of the circulating filtration system is as follows: when the pulse filtering system filters, the sample is continuously flowed into the waste liquid bottle through the filtering sample outflow outlet. At the moment, the waste liquid bottle is filled with the filtered sample, and the washing liquid in the washing bottle is conveyed to the upper part of the filter containing the filter membrane in a liquid drop mode in an equal volume, so that the volume of the sample above the filter membrane is kept unchanged, and the circulating filtration of automatically washing the sample is realized.

The working principle of the pulse blow-beating system is as follows: the pulse blowing system is driven by pulse pressure to repeatedly shift the elastic film upwards and downwards (state 1 and state 2). When the blown elastic membrane is deflected upward (state 1), the liquid above the filter containing the filter membrane is blown once through the blowing channel. When the blown elastic membrane is deflected downward (state 2), the liquid above the filter containing the filter membrane is pumped once through the blown channel. When the state 1 and the state 2 are repeated by using the pulse pressure, the liquid above the filter containing the filter membrane can be continuously blown and pumped, and the liquid above the filter membrane is uniformly mixed, so that the blockage and the pollution of the filter membrane are further prevented.

When pulse pressure is provided for the microfluidic pulse circulation filtration system, the system can automatically filter samples on the filter membrane, and the filtered samples flow out of the filtered sample outflow outlet through the filter membrane, the upper layer of the elastic membrane chamber, the one-way valve and the microchannel. Meanwhile, pulse flow back and forth through the filter membrane can be generated to realize anti-blocking filtration.

The invention can be used for sample pretreatment for detecting diseases, such as removing red blood cells from complex biological samples such as whole blood and recovering plasma, removing free protein from the plasma and recovering extracellular vesicles, and has application prospects in filtration and enrichment of micro-nano particles.

The filter membrane of the invention is provided with a film with nano holes, and the micro-fluidic chip can generate micro-fluidic pulse flow. The micro-flow pulse filtration can be realized by combining the filter membrane and the micro-flow control chip.

Compared with the prior art, the device has the difference that the sample can be automatically cleaned and filtered through circulating filtration, and the washing liquid can be automatically and quantitatively filled without an external control system. The utility model provides a system can guarantee that filter membrane top solution volume is unchangeable, causes the jam filter membrane that material concentration sharply improves and lead to in the sample when effectively preventing that membrane top solution from straining futilely in the filtration process, also can prevent that solution drying from causing the damage to the sample.

In the current prior art, there is no method for pulse filtering. The pulse filtration can prevent blockage and reduce the damage of micro-nano particles, and quick and effective separation is realized. The pulse filtration system of the invention can be used for a plurality of filtration applications only by changing the pore size of the filter membrane (600 nm, 20 nm), such as: can remove red blood cell separation plasma from whole blood, and concentrate extracellular vesicles in cell culture solution.

The pulse filtering system can be added with waste liquid and a washing bottle to realize pulse circulation washing, and washing liquid is continuously added to the sample during filtering to prevent the sample on the filter membrane from being concentrated and dried during filtering. The sample on the sample filter membrane can be blown by the impulse deformation of the elastic membrane through the micro-pipeline, and the blockage and pollution of the filter membrane are further reduced.

The invention can concentrate the cell vesicles in the cell culture solution through the circulating washing system, remove free protein in plasma and recover the extracellular vesicles, can filter the extracellular vesicles softly and prevent the concentration of the extracellular vesicles, and improves the recovery rate of the extracellular vesicles. Whereas the pulse-only filtering systems of the current prior art are not at all realizable.

The invention uses the mixture of circular washing and pulse blowing on the basis of pulse filtration, and compared with the pulse filtration, the invention can further improve the anti-blocking and pollution of the filter membrane and can improve the purity and recovery rate of particles.

The template of the chip in the filtering system only with pulse filtering is manufactured by the traditional photoetching technology, and has the defects of complex process and long manufacturing period. The template is manufactured by using a 3D printing technology, and the template can be simply and quickly manufactured.

The filtration system of the pulse filtration only has no circulation washing function, so the sample on the filter membrane is continuously concentrated during the pulse filtration, and the sample on the filter membrane is polymerized. The invention can prevent the sample on the filter membrane from concentrating and polymerizing during pulse filtration by using circulating washing.

The microfluidic pulse circulation filtration system of the present invention may have, but is not limited to, the following beneficial effects:

1. the micro-fluidic pulse circulation filtering system provided by the invention utilizes micro-fluidic pulse filtering to prevent the filter membrane from being blocked and prevent the filter membrane from being polluted, and softly filters and recovers micro-nano particles based on the size.

2. Micro-nano particles with different sizes can be separated by using different aperture of the filter membrane. For example, large particles can be removed to recover small particles, or small particles can be removed to recover large particles.

3. The micro-fluidic pulse filtration can reduce the mechanical damage of particles during filtration to the maximum extent and completely recover the particles. For example: the plasma can be recovered by filtering red blood cells from whole blood and the extracellular vesicles can be recovered by filtering free proteins from the plasma. When the soft micro-fluidic pulse flow filtration is carried out, the problems of rupture caused by extrusion of red blood cells and the filter membrane, low recovery rate caused by contact of extracellular vesicles and the filter membrane and the like can be prevented.

4. Compared with the traditional constant-pressure (or constant-flow) filtration, the pulse filtration can effectively reduce the blockage and pollution of the filter membrane during the filtration and improve the separation effect depending on the aperture size of the filter membrane. Compared with the traditional pulse filtering system, the micro-fluidic pulse circulating filtering system can accurately control micro-fluid, reduce the sample processing capacity, reduce the cost, reduce the volume of equipment and realize highly parallel separation.

5. The micro-fluidic pulse circulating filtration system is formed by skillfully arranging micro-fluidic elements such as the filter membrane, the elastic membrane, the micro-channel, the one-way valve and the like, so that the micro-fluidic pulse filtration is realized, and the cost, the system size and the sample processing capacity of the micro-fluidic pulse filtration system are reduced.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 shows a block diagram of a microfluidic pulse cycle filtration system prepared in example 1; wherein, fig. 1A shows a schematic diagram of a microfluidic pulse cycle filtration system in a repeated state 1 with repeated upward deflection of the elastic membrane; figure 1B shows a block diagram of a microfluidic pulse cycle filtration system in a repeated state 2 with repeated downward deflection of the elastic membrane.

Figure 2 shows the results of washing free protein by the microfluidic pulse cycle filtration system of the invention in example 2; wherein, fig. 2A shows that the removal rate of free protein increases with the increase of the volume of the circulating washing solution, and more than 99% of the removal rate of protein can be removed when the washing solution is circulated and washed by 1000 microliters; figure 2B shows that the amount of residual free protein decreased with increasing volume of the wash cycle, and that the concentration of free protein decreased from 1.5 milligrams per milliliter to 5 micrograms per milliliter with 1000 microliters of wash cycle.

FIG. 3 shows a comparison of pulse filtered whole blood and constant flow filtered whole blood in comparative example 1; wherein FIG. 3A illustrates the flow rate of whole blood through pulse filtration, which continuously removes blood cells from the whole blood to separate plasma and prevents the filter membrane from being clogged and contaminated by the blood cells; FIG. 3B shows the flow rate for constant flow filtration of whole blood, where the filter is clogged by blood cells in the whole blood, from 16 microliters per second to almost 0 microliters per second in 100 seconds; FIG. 3C shows plasma separated from whole blood by pulse filtration, which is clear with no rupture of blood cells; fig. 3D shows plasma separated from whole blood by constant flow filtration, the separated plasma containing rupture of blood cells, resulting in hemolysis.

FIG. 4 shows the results of comparing the concentration of extracellular vesicles in a cell culture solution by pulse filtration with constant-flow filtration in comparative example 2; wherein, fig. 4A 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-current filtration; the concentration of the extracellular vesicles after pulse filtration and concentration is higher than that of the extracellular vesicles after constant-current filtration and concentration, so that the pulse filtration can reduce the damage to the extracellular vesicles during filtration, and the constant-current filtration can damage the extracellular vesicles, so that the concentration of the extracellular vesicles after pulse filtration and concentration is high and the concentration of the extracellular vesicles after constant-current filtration and concentration; fig. 4B shows the recovery of pulse filtration and constant flow filtration concentrated extracellular vesicles, with a recovery of 75% for pulse filtration concentrated extracellular vesicles, which is far higher than the recovery of constant flow filtration concentrated extracellular vesicles.

FIG. 5 shows a comparison of extracellular vesicle recovery after removal of plasma free proteins using pulsed circulation filtration and constant flow filtration in comparative example 3; after removing impurities such as free protein by using 1000 microliters of washing solution, the recovery rate of the extracellular vesicles by pulse cycle filtration was 60%, while the recovery rate of the extracellular vesicles by constant flow filtration was only 15%.

FIG. 6 shows that the pulse flow generated by the pulse pressing to blow the elastic membrane in comparative example 3 can further prevent the filter membrane from being blocked and polluted by blowing the liquid above the filter membrane; wherein FIG. 6A shows the upward deflection of the blown elastic film caused by the pulsed high voltage, which blows the sample over the filter; fig. 6B shows that the downward deflection of the blown elastic membrane caused by the low pressure of the pulse can pump and filter the sample above the membrane, and the sample above the filter containing the filter membrane can be mixed by repeatedly blowing and pumping, so that the blockage and pollution of the filter membrane are reduced, and the filtering speed is increased.

Detailed Description

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

This section describes generally the materials used in the tests of the present invention, as well as the test methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible. It will be apparent to those skilled in the art that the materials and methods of operation used in the present invention are well within the skill of the art in this context, if not specifically mentioned.

The reagents and instrumentation used in the following examples are as follows:

reagent:

PDMS, available from SYLGARD.

The instrument comprises the following steps:

a Plasma bonding apparatus, PDC-002, was purchased from Harrick Plasma, inc.

Example 1

This example illustrates the method of making a microfluidic pulse-cycle filtration system according to the present invention.

The micro-fluidic pulse circulating filter system is composed of Polydimethylsiloxane (PDMS), is manufactured by a soft lithography method, and specifically comprises the following steps:

(1) Preparing a micro-fluidic chip: the microfluidic chip is composed of three layers of PDMS, the upper layer and the lower layer contain structures, and the middle layer is a PDMS film. A mold was first printed on glass using a 3D printer. The PDMS of the upper and lower layers containing the structure was obtained by replication from a mold. Each layer of PDMS is bonded by plasma treatment and heating to form a PDMS microfluidic chip with upper and lower layers of microchannels and chambers and an intermediate layer of an elastic film. The three layers of bonded microfluidic chips are provided with microfluidic elements such as an elastic film, a one-way valve, a microchannel and the like of a cavity.

(2) Preparation of filter containing filter membrane: commercial filter membranes are mounted in commercial filters to form filters containing the filter membranes.

(3) Preparing a circulating filtration system:

(a) Punching holes at the upper part and the lower part of a waste liquid bottle and a washing bottle in the circulating washing system;

(b) The waste liquid bottle is connected with the hole above the washing bottle through a pipeline;

(c) The lower hole of the waste liquid bottle is connected with the sample outflow outlet;

(d) The lower well of the wash bottle is located above the filter containing the filter membrane.

(4) Preparing a pulse filtering system: and connecting one side of the microfluidic chip with a waste liquid bottle and a washing bottle in the connection of the circulating washing system, and connecting the other side of the microfluidic chip with a pulse blowing system and a filter containing a filter membrane to obtain the microfluidic pulse circulating filtering system.

Example 2

This example illustrates the microfluidic pulse cycle filtration system of the present invention.

FIG. 1 shows a block diagram of a microfluidic pulse-cycle filtration system prepared in example 1; wherein, fig. 1A shows a schematic diagram of a microfluidic pulse cycle filtration system in a repeated state 1 with repeated upward deflection of the elastic membrane; figure 1B shows a block diagram of the microfluidic pulse cycle filtration system in repetitive state 2 with repeated downward deflection of the elastic membrane. As shown in fig. 1, when the outlet of the filtered sample is connected in series with the waste liquid and the washing liquid bottle, the pulse-filtering microfluidic chip can automatically wash the sample on the filter membrane in a circulating manner. The pulse filtering method is suitable for cleaning small particles in a sample and recovering particles larger than the aperture of the filter membrane. When the pulse filtration microfluidic chip works, filtered waste liquid flows into the waste liquid bottle, and washing liquid in the washing bottle drops above the filter membrane in a liquid drop mode. The waste liquid and the washing liquid are separated by air, so that the washing liquid is prevented from being polluted by the waste liquid. At this time, the volume of the sample above the filter is almost constant, and the droplets of the washing solution are automatically replenished into the sample on the filter each time the waste liquid of the droplet volume is filtered, thereby preventing the concentration and drying of particles larger than the pore diameter of the filter. The efficiency (eta) of particle removal smaller than the pore size of the filter membrane is

V _Loading And V _Droplet The sample loading volume above the filter membrane and the drop volume of the washing solution are respectively, and N is the number of drops falling. Thus, when the sample volume was 200. Mu.l and washed with 1ml of washing solution (37 droplets), the removal rate of particles smaller than the filter pore size was 99.9%.

To verify the effectiveness of the cyclic wash, 200. Mu.l of free protein was removed up to 99.9% after washing with 1ml PBS (FIG. 2)

Figure 2 shows the results of washing free protein by the microfluidic pulse-cycle filtration system of the invention in example 2; wherein, fig. 2A shows that the removal rate of free protein increases with the increase of the volume of the circulating washing, and the removal rate of protein of more than 99% can be removed when the washing is circulated by 1000 microliters of the washing liquid; figure 2B shows that the amount of residual free protein decreased with increasing volume of the wash cycle, and that the concentration of free protein decreased from 1.5 milligrams per milliliter to 5 micrograms per milliliter with 1000 microliters of wash cycle.

The removal of free protein was greater than 99.9% after washing with 1ml of wash solution. The volume of washing increased and the amount of residual protein decreased. The micro-fluidic pulse circulation filtering system can effectively filter particles with smaller pore sizes than the filter membrane.

Example 3

This example illustrates the preparation of the pulse blow-beating system of the present invention.

The pulse blow-beating system consists of Polydimethylsiloxane (PDMS) and is manufactured by a soft lithography method, and the pulse blow-beating system specifically comprises the following steps:

(1) And copying the mold printed by the 3D printer to obtain the lower-layer material.

(2) Heating and bonding the lower layer material prepared in the step (2) and the elastic film of the middle layer together.

(3) The top of the elastic membrane is connected to the top of the filter containing the filter membrane by a polyethylene tube.

Example 4

This example illustrates the pulse filtering method of the microfluidic pulse-cycle filtration system of the present invention.

The pulse pressure is provided for the pulse filtering micro-fluidic chip, the pulse pressure can be realized in a mode of combining a pulse pressure generator, the opening and closing of the electromagnetic valve and the constant pressure provided by the constant pressure pump, the pulse pressure of the embodiment is connected with an inlet of the electromagnetic valve through the constant pressure pump, the pulse pressure is realized at an outlet of the electromagnetic valve when the electromagnetic valve is opened and closed, the chip can automatically filter a sample on the filter membrane when the pulse pressure is connected to the chip, and the filtered sample flows out of an outlet of the filtered sample through the filter membrane, an upper layer of the elastic membrane chamber, the one-way valve and the microchannel. Meanwhile, pulse flow back and forth through the filter membrane can be generated to realize anti-blocking filtration. Specifically, the method comprises the following steps:

a pulse filtering system: the pulse filtering system is driven by pulse pressure, and the elastic membrane repeatedly deflects upwards and downwards to repeat the states 1 and 2. When the elastic membrane deflects upwards (state 1), the liquid in the chamber above the elastic membrane flows out of the filtered sample outflow outlet through the one-way valve and the first microchannel. And simultaneously, the liquid in the chamber above the elastic membrane flows to the position above the filter membrane through the filter membrane. When the elastic film deflects downwards (state 2), the liquid above the filter membrane flows to the chamber above the elastic film through the filter membrane, at the moment, the one-way valve is closed, and the liquid of the filtered sample flowing out of the outlet is prevented from flowing to the chamber above the elastic film. When the states 1 and 2 are repeated, the pulse filtering system can generate pulse flow passing through the filter membrane, so that the filter membrane is prevented from being blocked and polluted, and the sample is effectively filtered to the filtered sample outflow outlet.

A circulating filtering system: when the pulse filtering system filters, the sample is continuously discharged into the waste liquid bottle through the filtering sample outlet. At the moment, the waste liquid bottle is filled with the filtered sample, and the washing liquid in the washing bottle is conveyed to the upper part of a filter containing a filter membrane in a liquid drop mode in an equal volume mode, so that the volume of the sample above the filter membrane is kept unchanged, and the circulating filtration of automatically washing the sample is realized.

Pulse blows and beats the system: the pulse blowing system is driven by pulse pressure to repeatedly shift the elastic film upwards and downwards (state 1 and state 2). When the blown elastic membrane is deflected upward (state 1), the liquid above the filter containing the filter membrane is blown once through the blowing channel. When the blown elastic membrane is deflected downward (state 2), the liquid above the filter containing the filter membrane is pumped once through the blowing channel. When the state 1 and the state 2 are repeated by using the pulse pressure, the liquid above the filter containing the filter membrane can be continuously blown and pumped, and the liquid above the filter membrane is uniformly mixed, so that the blockage and the pollution of the filter membrane are further prevented.

Example 5

This example illustrates the technical effect of the microfluidic pulse-cycle filtration system of the present invention.

The micro-fluidic pulse filtration realizes micro-fluidic pulse flow, reduces the blockage and pollution of a filter membrane, and filters a sample softly. When the whole blood sample is filtered, the collision and blockage between blood cells and the filter membrane are reduced, and the rupture of the blood cells is prevented. When the extracellular vesicles are filtered, the loss caused by collision of the extracellular vesicles and the filter membrane is reduced, and the particle recovery rate of the extracellular vesicles is improved.

Example 6

The micro-fluidic pulse filtration realizes precise micro-fluidic pulse flow, can reduce the sample processing capacity and reduce the cost and size of a pulse filtration system. The traditional pulse filtration needs the combined drive of auxiliary equipment such as a huge and expensive injection pump, a motor and the like, and is difficult to realize the micro-flow pulse flow, or the cost for realizing the micro-flow pulse filtration is greatly increased. Microfluidic pulse flow enables microfluidic fluid manipulation with only one pulse pressure, and has significant advantages over conventional methods.

Example 7

This embodiment is provided to illustrate the technical effect of the pulse blow-beating system of the present invention.

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

Comparative example 1

The comparison example is used for comparing the effects of the microfluidic pulse circulation filtration system and the constant flow filtration system of the invention on whole blood filtration and plasma recovery.

Plasma was recovered by filtering whole blood using a 600 nm pore size filter.

FIG. 3 shows a comparison of pulse filtered whole blood and constant flow filtered whole blood in comparative example 1; wherein FIG. 3A illustrates the flow rate of whole blood through pulse filtration, which continuously removes blood cells from the whole blood to separate plasma and prevents the filter membrane from being clogged and contaminated by the blood cells; FIG. 3B shows the flow rate for constant flow filtration of whole blood, where the filter is clogged by blood cells in the whole blood, from 16 microliters per second to almost 0 microliters per second in 100 seconds; FIG. 3C shows plasma separated from whole blood by pulse filtration, which is clear with no rupture of blood cells; fig. 3D shows constant flow filtration of separated plasma from whole blood, which contains rupture of blood cells, resulting in hemolysis.

During pulse filtration, plasma can be continuously separated from whole blood, and the pulse filtration flow is effectively prevented from being blocked by blood cells due to the anti-blocking effect of pulse flow, so that the flow is slowly reduced.

During constant-flow filtration, the hemocyte quickly blocks the filter membrane, so that the constant-flow filtration flow is quickly reduced in a short time. Pulse filtration gently filters the whole blood sample and the sample obtained is free of hemolysis. Constant flow filtration continuously squeezes the blood cells and filter to rupture the blood cells and the sample obtained is red colored and hemolyzed.

The filter membrane of the invention is provided with a film with nano holes, and the microfluidic chip can generate micro-flow pulse flow. The invention can combine the filter membrane and the micro-fluidic chip to realize the micro-fluidic pulse filtration.

Comparative example 2

The comparative example is used for comparing the effects of the microfluidic pulse circulation filtration system and the constant-current filtration system of the invention on filtering and concentrating extracellular vesicles.

The concentrated extracellular vesicles were filtered from mammary epithelial cell (MCF-10A) culture using a 20 nm pore size filter.

FIG. 4 shows the results of comparing the concentration of extracellular vesicles in a cell culture solution by pulse filtration with constant-flow filtration in comparative example 2; wherein, fig. 4A 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-current filtration; the concentration of the extracellular vesicles after pulse filtration and concentration is higher than that of the extracellular vesicles after constant-current filtration and concentration, so that the pulse filtration can reduce the damage to the extracellular vesicles during filtration, and the constant-current filtration causes damage to the extracellular vesicles, so that the concentration of the extracellular vesicles after pulse filtration and concentration is higher than that of the extracellular vesicles after constant-current filtration and concentration; fig. 4B shows the recovery of pulse filtration and constant flow filtration concentrated extracellular vesicles, with a recovery of 75% for pulse filtration concentrated extracellular vesicles, which is far higher than the recovery of constant flow filtration concentrated extracellular vesicles.

Extracellular vesicles can be concentrated by pulse filtration and constant-current filtration, and the concentration after pulse filtration and concentration is greater than that after constant-current filtration and concentration. Therefore, the pulse filtration realizes soft filtration, reduces damage to the extracellular vesicles during filtration, and the recovery rate of the extracellular vesicles by pulse filtration is about 75%. During constant-flow filtration, the extracellular vesicles and the filter membrane are continuously squeezed to cause the loss of the extracellular vesicles, and the recovery rate of the extracellular vesicles subjected to constant-flow filtration is about 20%.

The pulse filtration can filter particles softly, reduce the blockage and pollution of the filter membrane, reduce the friction, damage and loss between the particles and the filter membrane holes, and can improve the recovery rate of the particles.

Comparative example 3

This comparative example serves to compare the microfluidic pulse-cycle filtration system of the present invention with a filtration system that is pulse-only filtered.

1. The first difference is as follows:

(1) The micro-fluidic pulse circulating filtration system comprises: the micro-fluidic pulse circulating filtration system comprises: a circulating washing system, a pulse blowing system, a filter containing a filter membrane and a microfluidic chip. The circulating washing system comprises a waste liquid bottle and a washing bottle, and the filtering sample outflow outlet is connected with the waste liquid bottle and the washing liquid bottle in series; the pulse blow-beating system comprises a blow-beating elastic film and a blow-beating channel. When the sample is filtered, the waste liquid automatically flows into a waste liquid bottle, the washing liquid automatically falls into the filtered sample in the form of liquid drops, and small particles such as free protein in the filtered sample are washed. The micro-nano particles above the filter membrane are automatically cleaned by the pulse blowing and beating mixing system during filtering, so that the filter membrane is prevented from being blocked and polluted.

(2) Pulse-only filtration system: the device does not comprise a waste liquid bottle, a washing bottle, liquid drops, a pulse pressing port, a pulse pressing outlet, a second micro-channel, a blowing elastic film and a blowing channel.

2. The difference is two:

(1) The micro-fluidic pulse circulating filtration system comprises: just because of using circulation washing system and pulse to blow and beat the system, can realize concentrating the extracellular vesicle in cell culture solution, get rid of the extracellular vesicle of recovery of free protein from the plasma, further reduce the pollution and the jam of filter membrane, improve the recovery rate of extracellular vesicle.

(2) Pulse-only filtration system: the extracellular vesicles are difficult to concentrate from a large amount of cell culture solution, free proteins are effectively removed from blood plasma, and the extracellular vesicles are recovered, so that the filtering speed is low; the pollution and blockage of the filter membrane cannot be solved; the recovery rate of extracellular vesicles is to be improved.

3. The difference is three:

(1) The micro-fluidic pulse circulating filtration system comprises: the circulating washing system can automatically add washing liquid, does not need manual operation, automatically cleans and filters free protein in plasma, and recovers extracellular vesicles. The pulse blowing and mixing system can automatically blow and mix the sample, and can further prevent the blockage and the pollution of a filter membrane during the filtration. Improves the separation effect and the separation speed based on the dependence of the aperture size of the filter membrane, and effectively filters the separated extracellular vesicles with high recovery rate of free protein.

(2) Pulse-only filtration system: the technical effects cannot be realized only by the pulse filtering system.

In conclusion, compared with the filtering system only adopting pulse filtration, the micro-fluidic pulse circulation filtering system can automatically clean the filtered sample, and can realize automatic and quantitative filling of the washing liquid without an external control system. The utility model provides a system can guarantee that filter membrane top solution volume is unchangeable, causes the jam filter membrane that material concentration sharply improves and lead to in the sample when effectively preventing that membrane top solution from straining futilely in the filtration process, also can prevent that solution drying from causing the damage to the sample.

FIG. 5 shows a comparison of extracellular vesicle recovery after removal of plasma free proteins using pulsed-circulation filtration and constant-flow filtration in comparative example 3; after removing impurities such as free protein by using 1000 microliters of washing solution, the recovery rate of the extracellular vesicles by pulse cycle filtration was 60%, while the recovery rate of the extracellular vesicles by constant flow filtration was only 15%. As shown in fig. 5, the present invention prevents concentration and drying of extracellular vesicles during pulse cycle filtration, and the recovery rate of extracellular vesicles after washing with 1mL of washing solution was 60%. However, in the case of pulse filtration according to the prior art, the recovery rate of extracellular vesicles after washing with 1mL of washing solution was only 15% due to concentration and drying of extracellular vesicles during filtration. Therefore, the pulse circulation filtration can improve the recovery rate of the extracellular vesicles after washing.

FIG. 6 shows that the pulse flow generated by the deformation of the elastic membrane in comparative example 3 is achieved by pulse pressing, and the sample above the filter membrane is blown by the pulse flow to further prevent the blockage and the pollution of the filter membrane; wherein figure 6A shows the upward deflection of the blown elastic membrane caused by the pulsed high voltage, which blows the sample above the filter membrane; FIG. 6B shows that the downward deflection of the blown elastic membrane is caused when the pulse is low-pressure, the sample above the membrane can be filtered, and the sample above the filter containing the filter membrane is mixed by repeatedly blowing and pumping, so that the blockage and pollution of the filter membrane are reduced, and the filtering speed is increased. As shown in FIG. 6, when the pulse circulation filtration is performed, the sample above the filter membrane is blown by the pulse deformation of the blown membrane, so that the blockage and the pollution of the filter membrane can be reduced. Improves the separation effect and the separation speed based on the pore size of the filter membrane, and effectively filters the separated extracellular vesicles with high recovery rate of free protein.

Although the present invention has been described to a certain extent, it is apparent that appropriate changes in the respective conditions may be made without departing from the spirit and scope of the present invention. It is to be understood that the invention is not limited to the described embodiments, but is to be accorded the scope consistent with the claims, including equivalents of each element described.

Claims

1. A microfluidic pulse cycle filtration system, comprising: a circulating washing system, a pulse blowing system, a filter containing a filter membrane and a micro-fluidic chip; wherein, the first and the second end of the pipe are connected with each other,

2. The microfluidic pulse cycle filtration system of claim 1, wherein:

the microfluidic pulse circulation filtration system further comprises: a pulse pressure inlet, a pulse extrusion port and a filtered sample outlet;

The microchannel comprises a first microchannel and a second microchannel;

preferably, the filter with 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;

3. The microfluidic pulse cycle filtration system of claim 1 or 2, wherein:

the flow resistance of the micro-nano hole is obtained through a formula (1):

the mechanical capacitance of the elastic membrane is obtained by equation (2):

wherein the content of the first and second substances,

4. The microfluidic pulse cycle filtration system of any one of claims 1 to 3, wherein:

The blow-beating channel is made of a polyethylene pipe;

5. A method of making a microfluidic pulse cycle filtration system according to any of claims 1 to 4, comprising: preparing a micro-fluidic chip, and connecting a circulating washing system, a pulse blowing system, a filter containing a filter membrane and the micro-fluidic chip together through a needle or a channel;

(A) Preparing a micro-fluidic chip;

(B) Connecting one side of a micro-fluidic chip with a waste liquid bottle and a washing bottle in the connection of a circulating washing system, and connecting the other side of the micro-fluidic chip with a pulse blowing system and a filter containing a filter membrane to obtain the micro-fluidic pulse circulating filtering system;

6. The method according to claim 5, wherein in the step (A), the operation of preparing the microfluidic chip comprises the following steps:

preferably, the upper layer of material comprising the chamber and the microchannel forms the chamber, the one-way valve and the first microchannel above the elastomeric film; and/or

7. The method according to claim 5 or 6, characterized in that:

8. The method according to any one of claims 5 to 7, characterized in that:

in the step (1): the mass ratio of the upper layer material to the lower layer material to the curing agent is 5-20: 1, preferably 5 to 15:1, most preferably 10; the heating time is 10-36 h, preferably 10-24 h, and most preferably 12h; the heating temperature is 60-100 ℃, preferably 70-90 ℃, and most preferably 80 ℃; and/or

preferably, the curing agent is a polydimethylsiloxane curing agent.

9. A pulse filtration method using the microfluidic pulse cycle filtration system of any one of claims 1 to 4 or the microfluidic pulse cycle filtration system prepared by the method of any one of claims 5 to 8;

preferably, the pulse filtering method comprises: when pulse pressure is provided for the micro-fluidic pulse circulation, the micro-fluidic pulse circulation filtering system automatically filters a sample on a filter membrane, the filtered sample flows out of a filtered sample outflow outlet through the filter membrane, the upper layer of an elastic membrane chamber, a micro-fluidic one-way valve and a micro-channel, and the filtered sample flows back and forth through the filter membrane to prevent blockage; the circulating washing system continuously supplements washing liquid on a filter containing a filter membrane during pulse filtration to realize circulating washing; the pulse blowing system generates pulse flow through the deformation of the blowing elastic film, continuously blows and blows a sample above the filter containing the filter membrane, and reduces the blockage and pollution of the filter membrane.

10. Use of the microfluidic pulse-cycle filtration system of any one of claims 1 to 4 or the microfluidic pulse-cycle filtration system prepared by the method of any one of claims 5 to 8 in the preparation of micro-nano filtration devices.