CN114713302A - Microfluidic chip and application thereof - Google Patents
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- CN114713302A CN114713302A CN202210404604.7A CN202210404604A CN114713302A CN 114713302 A CN114713302 A CN 114713302A CN 202210404604 A CN202210404604 A CN 202210404604A CN 114713302 A CN114713302 A CN 114713302A
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Abstract
The invention relates to the field of microfluidic chips, in particular to a microfluidic chip and application thereof. The present invention provides a microfluidic chip comprising: a first chip and a second chip; the first chip comprises a first upper surface and a first lower surface; the second chip comprises a second upper surface and a second lower surface; the first chip is provided with a first inlet and a first outlet; the second chip is provided with a second inlet and a second outlet; the first lower surface is provided with a first channel; the second upper surface is provided with a second channel; the first channel and the second channel are respectively provided with a groove array; the first channel and the second channel are attached oppositely; a porous membrane is arranged between the first channel and the second channel; the first chip, the porous film, and the second chip are arranged in this order from top to bottom. The micro-fluidic chip provided by the invention is realized based on a flow focusing-membrane separation technology, can continuously, efficiently and safely remove the cell cryoprotectant, and can be applied to removing cytotoxins or magnetic nanoparticles in extracellular fluid.
Description
Technical Field
The invention relates to the field of microfluidic chips, in particular to a microfluidic chip and application thereof.
Background
Cryopreservation technology refers to a technology for cryopreserving organisms, can remarkably prolong the preservation time of biological samples, and is widely applied to long-term preservation of various cell products (such as blood cells, stem cells, strains and the like). During the process of cell cryopreservation, the precipitated ice crystals can cause damage to cells. To reduce cell damage during cryopreservation, a certain amount of cryoprotectant (e.g., dimethyl sulfoxide (DMSO), glycerol, polyethylene glycol, etc.) needs to be added to the cell suspension prior to cryopreservation of the cells. Cryoprotectants reduce cell damage during cryopreservation, however cryoprotectants are generally somewhat cytotoxic and can also lead to cell permeability damage. Thus, prior to using cryopreserved cells, the cryoprotectant needs to be removed.
In recent years, with the development of microfluidic technology and soft lithography technology, microfluidic chips capable of accurately controlling micro-amount of liquid gradually emerge. The microfluidic chip has the advantages of integration, high flux, less sample consumption and the like, and is expected to become a new platform for efficiently removing the cell cryoprotectant.
However, the existing methods for removing cryoprotectants (such as one-step or multi-step centrifugation, dialysis, diffusion, etc.) have weak removing ability, low cell recovery rate, and no means for removing cryoprotectants from trace cells.
Disclosure of Invention
In view of the above, the present invention provides a microfluidic chip and applications thereof. The microfluidic chip provided by the invention is realized based on a flow focusing-membrane separation technology and can be used for removing a low-temperature protective agent in cells. The microfluidic chip provided by the invention can continuously remove and remove the cell cryoprotectant, so that cell pollution caused by open operation is avoided; the cell cryoprotectant can be efficiently removed, and the removal rate can reach more than 95%; can safely remove the cell cryoprotectant, not only greatly reduces osmotic pressure damage in the removal process, but also avoids mechanical damage of the device. The invention can realize high-flux cell washing by expanding in a parallel or serial mode, and can also be applied to removing cytotoxin or magnetic nanoparticles in extracellular fluid.
In order to achieve the above object, the present invention provides the following technical solutions:
the present invention provides a microfluidic chip comprising: a first chip 1 and a second chip 4; the first chip 1 includes a first upper surface and a first lower surface; the second chip 4 includes a second upper surface and a second lower surface; the first chip 1 is provided with a first inlet 8 and a first outlet 7; the second chip 4 is provided with a second inlet 6 and a second outlet 9; a first channel 2 is provided on the first lower surface of the first chip 1; a second channel 5 is formed on the second upper surface of the second chip 4; the first channel 2 and the second channel 5 are respectively provided with an array of grooves 13; the first channel 2 and the second channel 5 are oppositely attached; a porous membrane 3 is provided between the first channel 2 and the second channel 5; the first chip 1, the porous membrane 3, and the second chip 4 are arranged in this order from top to bottom.
In some embodiments of the invention, the first inlet 8, the first outlet 7, the second inlet 6 and the second outlet 9 are independent of each other.
In some embodiments of the invention, the first inlet 8 is a wash solution inlet, the first outlet 7 is a wash solution outlet, the second inlet 6 is a cell suspension inlet, and the second outlet is a cell suspension after washing, i.e. a product outlet.
In some embodiments of the present invention, the first chip 1 and the second chip 4 have a shape including a square and/or a polygon, the material includes one or more of polymethyl methacrylate, polycarbonate, polydimethylsiloxane, and/or glass, and the first chip 1 and/or the second chip 4 have an area of 1cm2～200cm2And the total thickness is 8 mm-20 mm.
In some embodiments of the present invention, the first chip 1 and the second chip 4 are rectangular in shape, and the material is polydimethylsiloxane, and the length, width and height thereof are 4cm, 2cm and 0.5cm, respectively.
In some embodiments of the present invention, the depth of the first channel 2 in the above microfluidic chip is smaller than the thickness of the first chip 1, and the depth of the second channel 5 is smaller than the thickness of the second chip 4.
In some embodiments of the present invention, the depth of the first channel 2 and/or the second channel 5 is 10 to 80 μm.
In some embodiments of the invention, the depth of the first channels 2 and/or the second channels 5 is 40 μm.
In some embodiments of the present invention, the area of the facing surfaces of the microfluidic chip is smaller than the surface area of the first channel 2 and/or the second channel 5.
In some embodiments of the present invention, the grooves 13 in the array of grooves 13 in the microfluidic chip are parallel to each other and are equally spaced.
In some embodiments of the present invention, the angle between the groove 13 in the array of grooves 13 on the surface of the first channel 2 in the above-mentioned microfluidic chip and the long side wall of the first channel 2 is 20 to 70 °, and the angle between the groove 13 in the array of grooves 13 of the second channel 5 in the above-mentioned microfluidic chip and the long side wall of the second channel 5 is 20 to 70 °.
In some embodiments of the present invention, the groove 13 in the array of grooves 13 of the first channel 2 in the microfluidic chip is at an angle of 30 ° to the long side wall of the first channel 2, and the groove 13 in the array of grooves 13 of the second channel 5 in the microfluidic chip is at an angle of 30 ° to the long side wall of the second channel 5.
In some embodiments of the present invention, the array of grooves 13 is configured to create a swirling flow field in the channel, which accelerates the mixing and mass transfer efficiency of the liquid in the channel.
In some embodiments of the present invention, the width of the groove 13 array in the microfluidic chip is 10 to 40 μm, the depth is 10 to 40 μm, and the pitch is 10 to 40 μm.
In some embodiments of the present invention, the width of the groove 13 array in the microfluidic chip is 25 μm, the depth is 25 μm, and the pitch is 25 μm.
In some embodiments of the present invention, the array of grooves 13 in the first channel 2 and the array of grooves 13 in the second channel 5 in the microfluidic chip are independent in size.
In some embodiments of the present invention, the porous membrane 3 in the microfluidic chip covers the first channel 2 and the second channel 5, and does not cover the first inlet 8, the first outlet 7, the second inlet 6, and the second outlet 9.
In some embodiments of the present invention, the porous membrane 3 in the microfluidic chip covers the first channel 2 and the second channel 5 to prevent liquid leakage.
In some embodiments of the present invention, the material of the porous membrane 3 includes a polycarbonate blotting-etched membrane and/or a polydimethylsiloxane porous membrane, and the thickness of the porous membrane 3 is 5 to 25 μm, and the pore diameter is 0.1 to 5 μm.
In some embodiments of the present invention, the porous membrane 3 has a pore size of 0.8 μm and is made of a polycarbonate imprinted etched membrane.
In some embodiments of the present invention, the overlapping portion of the first channel 2 and the second channel 5 in the microfluidic chip is a dialysis area 10;
a focus holding area 11 is formed between the dialysis area 10 and a long side wall of the second channel 5 with respect to the dialysis area 10;
the portion of the second channel 5 with the array of grooves 13 is a focusing area 12 excluding the dialysis area 10 and the focus holding area 11.
In some embodiments of the present invention, the first chip 1, the porous membrane 3, and the second chip 4 in the microfluidic chip are bonded by polydimethylsiloxane prepolymer and toluene, and the mass ratio of the polydimethylsiloxane prepolymer to the toluene is 2: 1.
In some embodiments of the present invention, the first chip 1 and the second chip 4 are manufactured by a soft lithography method, and the porous film 3 is manufactured by an electron beam etching method.
In some embodiments of the invention, a plurality of the microfluidic chips described above are connected in series and/or in parallel.
In some embodiments of the invention, the microfluidic chip is used for removing one or more of a cell cryoprotectant, a cytotoxin and/or a magnetic nanoparticle in a cell suspension.
The present invention provides a microfluidic chip comprising: a first chip 1 and a second chip 4; the first chip 1 includes a first upper surface and a first lower surface; the second chip 4 includes a second upper surface and a second lower surface; the first chip 1 is provided with a first inlet 8 and a first outlet 7; the second chip 4 is provided with a second inlet 6 and a second outlet 9; a first channel 2 is provided on the first lower surface of the first chip 1; a second channel 5 is formed on the second upper surface of the second chip 4; the first channel 2 and the second channel 5 are respectively provided with an array of grooves 13; the first channel 2 and the second channel 5 are oppositely attached; a porous membrane 3 is provided between the first channel 2 and the second channel 5; the first chip 1, the porous membrane 3, and the second chip 4 are arranged in this order from top to bottom.
The micro-fluidic chip provided by the invention removes the cell low-temperature protective agent by using a flow focusing-membrane separation technology, a washing solution is injected into the first channel 2 according to a certain flow rate, a cell suspension is injected into the second channel 5 in the opposite direction, cells in the cell suspension are focused on one side far away from the dialysis area 10 when passing through the focusing area 12, then enter the focusing holding area 11, and maintain the focusing state in the focusing holding area 11 until the cells flow out of the chip. Meanwhile, the washing solution dialyzes the extracellular fluid in the dialysis area 10, so that the low-temperature protective agent in the extracellular fluid is diffused into the washing solution and continuously flows out of the chip along with the washing solution, thereby completing the clearing process of the low-temperature protective agent. This allows the osmotic damage experienced by the cells to be greatly reduced, since the cells are focused on the side away from the dialysis zone 10, while avoiding cell loss due to capture by the porous membrane 3, thereby improving cell recovery. In addition, the micro-fluidic chip for removing the low-temperature protective agent in the cell suspension can also be used for removing cytotoxin, washing residual magnetic nanoparticles in extracellular fluid and the like, and has better universality and convenience.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below.
Fig. 1 shows a schematic three-dimensional structure of a microfluidic chip provided in an embodiment of the present invention;
fig. 2 shows a schematic plan view of a first chip 1, a second chip 4 and a combined plan structure of the microfluidic chip provided in the embodiment of the present invention, where the schematic plan view of the first chip 1 corresponds to (i), the schematic plan view of the second chip 4 corresponds to (ii), and the schematic plan structure after combination corresponds to (iii);
fig. 3 is a schematic diagram showing a cross section of a first chip 1 and a structure of a groove 13 of a microfluidic chip according to an embodiment of the present invention, wherein (i) is a schematic plan view of the structure of the first chip 1, wherein a broken line represents a cutting line, an arrow represents a projection direction, and (ii) is a cross-sectional view and a partially detailed enlarged view;
fig. 4 is a schematic cross-sectional structure diagram of a microfluidic chip according to an embodiment of the present invention, in which (i) is a plan view, (ii) is a schematic cross-sectional view and a detailed enlarged view of a part thereof, and (iii) is a schematic cross-sectional view and a detailed enlarged view of a part thereof;
1 denotes a first chip, 2 denotes a first channel, 3 denotes a porous membrane, 4 denotes a second chip, 5 denotes a second channel, 6 denotes a second inlet, 7 denotes a first outlet, 8 denotes a first inlet, 9 denotes a second outlet, 10 denotes a dialysis zone, 11 denotes a focus holding zone, 12 denotes a focusing zone, and 13 denotes a groove.
Detailed Description
The invention discloses a micro-fluidic chip and application thereof.
It should be understood that one or more of the expressions "… …" individually includes each of the stated objects after the expression and various different combinations of two or more of the stated objects, unless otherwise understood from the context and usage. The expression "and/or" in connection with three or more of the stated objects shall be understood to have the same meaning unless otherwise understood from the context.
The use of the terms "comprising," "having," or "containing," including grammatical equivalents thereof, is generally to be construed as open and non-limiting, e.g., without excluding other unstated elements or steps unless specifically stated otherwise or otherwise understood from the context.
It should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Further, two or more steps or actions may be performed simultaneously.
The use of any and all examples, or exemplary language such as "for example" or "including" herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Moreover, the numerical ranges and parameters setting forth the invention are approximations that may have numerical values that are within the numerical ranges specified in the specific examples. Any numerical value, however, inherently contains certain standard deviations found in their respective testing measurements. Accordingly, unless expressly stated otherwise, it is understood that all ranges, amounts, values and percentages used in this disclosure are by weight modified by "about". As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.
In examples 1 to 6 of the present invention, the raw materials and reagents used were all commercially available.
The invention is further illustrated by the following examples:
example 1
A microfluidic chip having the structure shown in fig. 1 includes:
the length, width and height of the microfluidic chip are respectively 4cm, 2cm and 0.5cm of rectangular first chip 1 and second chip 4; the first chip 1 and the second chip 4 are made of polydimethylsiloxane. The first lower surface of the first chip 1 comprises first channels 2 having a depth of 40 μm, a body portion (i.e. the portion of the constriction not comprising the access opening) having a width of 1mm and a length of 1.5 cm. An array of slanted grooves 13 is formed in the first channel 2 at the first lower surface of the first chip 1, the grooves 13 have a width of 25 μm and a depth of 25 μm, the axial direction of the grooves 13 forms an angle of 60 ° with the long side walls of the first channel 2, the distance between the grooves 13 is 25 μm, and the grooves 13 are arranged in an array over the straight channel portion.
As shown in FIG. 2, the first chip 1 comprises four through holes, wherein the first inlet 8 and the first outlet 7 are an inlet for the washing solution and an outlet for the waste solution, respectively, and the second inlet 6 and the second outlet 9 are an inlet for the cell suspension and an outlet for the washed products.
The second channel 5 on the second upper surface of the second chip 4 of the microfluidic chip had a depth of 40 μm, a main body portion (i.e., the portion of the constriction not including the inlet/outlet) having a width of 1mm and a length of 1.5 cm. The lower surface of the second channel 5 at the second upper surface of the second chip 4 is provided with an array of grooves 13, the grooves 13 having a width of 25 μm and a depth of 25 μm, and having a structure similar to the structure of the grooves 13 in the first channel 2, but in the opposite direction. As shown in fig. 2, the cell suspension is introduced through the second inlet 6, enters the second channel 5, and exits through the second outlet 9 after being washed.
The overlapping part of the first channel 2 and the second channel 5, i.e. the dialysis area 10, has a width of 0.5mm and a length of 1 cm.
The porous membrane 3 disposed between the first chip 1 and the second chip 4 is a polycarbonate print-etched membrane having a pore size of 0.8 μm, and has a rectangular shape and a size covering the entire dialysis area 10.
The first chip 1, the second chip 4, and the porous membrane 3 were bonded to each other after alignment using toluene and PDMS prepolymer mixed in a ratio of 1: 2 as an adhesive and a filler.
Example 2
In an embodiment of the invention, the first channels 2 are flow cavities at a first lower surface of the first chip 1 and the second channels 5 are flow cavities at a second upper surface of the second chip 4. The first and second channels 2, 5 are similarly shaped and the flow chamber is divided into an inlet and outlet section at both ends and a body chamber section in the middle. A porous membrane 3 is sandwiched between the first chip 1 and the second chip 4. The surfaces of the first chip 1 and the second chip 4 with the channels are attached to each other. The first chip 1 and the second chip 4 are arranged in parallel, and are staggered by a certain distance along the transverse direction and the longitudinal direction after the main body cavity parts are completely aligned and overlapped, so that the effect of overlapping the main body cavity parts is realized. The body chamber overlapping portion of the second channel 5 constitutes a dialysis zone 10, the flow chamber upstream of the dialysis zone 10 constitutes a focusing zone 12, and the flow chamber adjacent to the dialysis zone 10 (i.e., the remaining portion of the body chamber of the second channel 5 excluding the dialysis zone 10 and the focusing zone 12) constitutes a focus-holding zone 11, as shown in fig. 2.
Example 3
In the embodiment of the present invention, the depth of the first channel 2 is smaller than the thickness of the first chip 1, and the depth of the second channel 5 is smaller than the thickness of the second chip 4. The overlapping part of the first channel 2 and the second channel 5 is a dialysis area 10, the position and shape of which is shown in fig. 2.
In fig. 2, the first inlet 8 is a cleaning liquid inlet, and the first outlet 7 is a cleaning liquid outlet. In the embodiment of the present invention, the first channel 2 is a cleaning solution channel, and the isotonic cleaning solution without cryoprotectant enters the chip from the first inlet 8 at a constant flow rate, and the dialysis effect occurs between the dialysis zone 10 and the liquid in the second channel 5 of the second chip 4. In the first channel 2 of the first chip 1, parallel, elongated (rectangular in cross section) bumps are configured, a groove 13 is formed between adjacent bumps, and a plurality of equally spaced, parallel grooves 13 constitute a groove array, the structure of which is shown in fig. 3 and 4.
In the second channel 5 of the second chip 4, parallel, elongated (rectangular in cross section) bumps are also formed, the grooves 13 are formed between adjacent bumps, and a plurality of equally spaced, parallel grooves 13 form a groove array, the structure of which is shown in fig. 3 and 4.
Example 4
The microfluidic chip provided by the invention comprises a second channel 5 arranged on the second upper surface of the second chip 4, and the depth of the second channel 5 is smaller than the thickness of the second chip 4. The second channel 5 is a cell suspension channel. As shown in fig. 2, the second inlet 6 is a cell suspension inlet, and the second outlet 9 is a cell suspension (i.e., product) outlet after washing. The definition of the focusing region 12, the dialysis region 10 and the focus-and-hold region 11 is shown in FIG. 2.
In practical operation of the embodiment of the present invention, the cell suspension is injected from the second inlet 6 at a certain flow rate, focused under the action of the array structure of the grooves 13 in the focusing region 12, and then enters the focusing and maintaining region 11. Cell focusing refers to the gathering of randomly distributed cells in a cell suspension to a narrow range of positions, i.e., one side of a channel. The principle is as follows: taking the second channel 5 (cell suspension channel) as an example, when the liquid flows, the inclined groove 13 structure in the second channel 5 firstly constructs a flow field (flow mode) spirally advancing along the channel in the second channel 5; in addition, the width and depth of the channel 13 are designed to approximate the diameter of the cell, so that the cell is constrained to move within the channel body (without entering the channel) due to steric effects, and thus lateral migration occurs by fluid drag until it is collected on one side of the channel.
The extracellular fluid is dialyzed against the cleaning fluid in the dialysis zone 10, thereby achieving the removal of the cryoprotectant.
In the existing cryoprotectant removing device using the dialysis membrane, cells are easily adsorbed by the porous membrane because the cells are directly contacted with the porous membrane; in addition, the cells are randomly distributed in the cell suspension, and if the cells are directly contacted with a low-permeability washing solution, the drastic osmotic pressure change can cause the cells to crack and die due to excessive swelling caused by water absorption. In an embodiment of the invention, the cells in the cell suspension have been collected on the side remote from the dialysis zone 10 and free of transmembrane flow before being subjected to washing, and therefore there is no risk of adsorption by the porous membrane 3. At the same time, by controlling the flow of the cleaning fluid in the first channel 2, it is possible to control the osmotic pressure variation in the dialysis zone 10 of the second channel 5 along the flow direction, thereby controlling the osmotic pressure damage to which the cells are subjected during the cryoprotectant removal process.
The array of grooves 13 of the focus holding section 11 is to maintain such a flow field to keep the state of cell aggregation unchanged. In the first channel 2 (wash solution channel) there is also an array of grooves 13, which is not used for cell focusing, but for faster mixing. The invention is designed into a mode that the flow rate of the cleaning solution and the flow rate of the cell suspension can be adjusted, so that the osmotic pressure change can be accurately controlled by adjusting the flow of the cleaning solution, and the osmotic damage of cells can be reduced hopefully.
Example 5
The micro-fluidic chip provided in example 1 is used to remove the RAW264.7 cell cryoprotectant, and the specific process is as follows:
adding phosphate buffer solution containing 20% (v/v) of cell cryopreservation grade DMSO dropwise into equal amount of RAW264.7 cell suspension, and mixing uniformly to obtain RAW264.7 cell suspension (cell density 10) with final DMSO content of 10% (v/v)6mL) as a test sample. Phosphate buffer was used as the wash.
The test sample was injected at a flow rate of 5. mu.L/min from the second inlet 6 of the second channel 5 of the microfluidic chip of example 1, the phosphate buffer solution without DMSO was injected at a flow rate of 15. mu.L/min from the first inlet 8 of the first channel 2 of the microfluidic chip of example 1, and the waste liquid was withdrawn at a flow rate of 15. mu.L/min at the first outlet 7 of the first channel 2. After the flow has stabilized, a sample is collected from the second outlet 9 of the second channel 5. The collected cell samples were then subjected to dead-live staining to analyze cell viability and recovery, and DMSO content was determined to analyze DMSO clearance.
Example 6
The removal of the NE-4C cell cryoprotectant by using the microfluidic chip provided in the embodiment 1 comprises the following specific steps:
adding phosphate buffer solution containing 20% (v/v) of cell freezing grade DMSO dropwise into the same amount of NE-4C cell suspension, and mixing uniformly to obtain NE-4C cell suspension (cell density of 10%) with final DMSO content of 10% (v/v)6mL), as a test sample. Phosphate buffer was used as the wash.
The test sample was injected from the second inlet 6 of the second channel 5 of the microfluidic chip of example 1 at a constant flow rate, denoted as QCPhosphate buffer was injected at a constant flow rate, denoted as Q, from the first inlet 8 of the first channel 2 of the microfluidic chip of example 1WAt the first outlet 7 of the first channel 2 at the same flow rate QWAnd pumping the cleaning waste liquid.
After the flow was stabilized, about 40. mu.L of the product was collected from the second outlet 9 of the second channel 5, and the sample cells were subjected to dead-live staining using calcein AM and propidium iodide to measure the cell viability. The entrance and exit cell densities were counted using a hemocytometer. DMSO content was determined using uv spectrophotometer to analyze DMSO clearance.
Note that DMSO content in the untreated sample is CIThe cell density of the treated product is CO. The DMSO clearance λ is calculated as follows:
λ＝(1-CO/CI)×100％
recording the cell density in the untreated sample as DIThe cell density in the treated product is DOThe cell viability of the untreated cell sample was SIThe cell survival rate of the treated product is SOThe cell recovery R is calculated as follows:
R＝(SO×DO)/(SI×DI)×100％
tables 1-2 below show the recovery and cryoprotectant clearance data for three different flow rate configurations using the microfluidic chip provided in example 1 of the present invention for clearance of DMSO-loaded NE-4C cell suspensions. As described above, QC、QWRespectively representing the flow rate of the cell suspension and the flow rate of a cleaning solution (phosphate buffer solution), wherein the unit is mu L/min, and N represents the test times.
TABLE 1 example 1 of the present invention clearance of DMSO in NE-4C cells
TABLE 2 recovery of DMSO from NE-4C cells in inventive example 1
The experimental result shows that when the microfluidic chip provided by the embodiment 1 of the invention is used for removing the cryoprotectant of the NE-4C cell, the clearance rate of the cryoprotectant under the given condition can reach more than 95%, and the cell recovery rate can reach more than 90%
From the above embodiments, the present invention provides a microfluidic chip for continuously removing a cryoprotectant from a cell suspension, comprising: a first chip 1 and a second chip 4; the first chip 1 comprises a first upper surface and a first lower surface; the second chip 4 comprises a second upper surface and a second lower surface; the first chip 1 is provided with a first inlet 8 and a first outlet 7; the second chip 4 is provided with a second inlet 6 and a second outlet 9; a first channel 2 is arranged on the first lower surface of the first chip 1; a second channel 5 is arranged on the second upper surface of the second chip 4; the first channel 2 and the second channel 5 are respectively provided with a groove 13 array, and the groove arrays can focus cells and accelerate the diffusion of the low-temperature protective agent, so that the cell loss is reduced, and the cell permeability damage is relieved; the first channel 2 and the second channel 5 are oppositely attached; a porous membrane 3 is arranged between the first channel 2 and the second channel 5, one surface of the porous membrane covers the first channel 2, and the other surface of the porous membrane covers the second channel 5; the first chip 1, the porous membrane 3, and the second chip 4 are arranged in this order from top to bottom; the fluid in the dialysis zone 10 in the first channel 2 and the second channel 5 is exchanged through the porous membrane 3 to complete the cryoprotectant removal process. Based on the principle of the invention, the micro-fluidic chip provided by the invention not only can continuously, efficiently and safely remove the cell cryoprotectant, but also can remove substances such as cytotoxin or magnetic nanoparticles in extracellular fluid, and has better universality and convenience.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.
Claims (10)
1. A microfluidic chip, comprising:
a first chip (1) and a second chip (4);
the first chip (1) comprises a first upper surface and a first lower surface;
the second chip (4) comprises a second upper surface and a second lower surface;
the first chip (1) is provided with a first inlet (8) and a first outlet (7);
the second chip (4) is provided with a second inlet (6) and a second outlet (9);
the first lower surface of the first chip (1) is provided with a first channel (2);
the second upper surface of the second chip (4) is provided with a second channel (5);
the first channel (2) and the second channel (5) are respectively provided with an array of grooves (13);
the first channel (2) and the second channel (5) are attached oppositely;
a porous membrane (3) is arranged between the first channel (2) and the second channel (5);
the first chip (1), the porous membrane (3), and the second chip (4) are arranged in this order from top to bottom.
2. The microfluidic chip according to claim 1, wherein the depth of the first channel (2) is less than the thickness of the first chip (1) and the depth of the second channel (5) is less than the thickness of the second chip (4).
3. The microfluidic chip according to claim 1 or claim 2, wherein the area of the facing abutment is smaller than the surface area of the first channel (2) and/or the second channel (5).
4. Microfluidic chip according to anyone of claims 1 to 3, characterized in that the grooves (13) of the array of grooves (13) are parallel to each other and equally spaced.
5. The microfluidic chip according to any of claims 1 to 4, wherein the grooves (13) of the array of grooves (13) are angled 20 to 70 ° with respect to the long side walls of the first channel (2) and the second channel (5), respectively.
6. The microfluidic chip according to any of claims 1 to 5, wherein the grooves (13) of the array of grooves (13) have a width of 10 to 40 μm, a depth of 10 to 40 μm, and a pitch of 10 to 40 μm.
7. The microfluidic chip according to any of claims 1 to 6, wherein the porous membrane (3) covers the first channel (2) and the second channel (5) and does not cover the first inlet (8), the first outlet (7), the second inlet (6) and the second outlet (9).
8. The microfluidic chip according to any of claims 1 to 7, wherein the overlapping part of the array of grooves (13) of the first channel (2) and the array of grooves (13) of the second channel (5) is a dialysis zone (10);
the dialysis zone (10) and the array of grooves (13) between the second channel (5) with respect to the long side walls of the dialysis zone (10) are focus-holding zones (11);
the array of grooves (13) of the second channel (5) is a focal region (12) except for the dialysis region (10) and the focus-holding region (11).
9. The microfluidic chip according to any of claims 1 to 8, wherein the first chip (1), the porous membrane (3), and the second chip (4) are bonded with a polydimethylsiloxane prepolymer and toluene at a mass ratio of 2: 1.
10. Use of a microfluidic chip according to any one of claims 1 to 9 for the removal of one or more of cytoprotective agents, cytotoxins and/or magnetic nanoparticles from a cell suspension.
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