KR102043027B1 - Microchip Containing Perpendicularly Bonded Functional Nano-porous Membrane and Manufacturing Method Thereof - Google Patents

Abstract

The present invention relates to a PDMS-based or PMMA-based microchip in which a functional nanopermeable membrane is vertically and permanently fixed and a method of manufacturing the same.
The PDMS or PMMA-based substrate of the present invention has excellent optical properties, has the advantage of easily forming microfluidic channels through lithography and laser processing, and the functional nano-permeable membrane of the present invention has high porosity. It has high mass transfer efficiency, selective mass transfer through control of pore size, and low resistance and reactivity to the injected fluid and solution, so it can be used to manufacture selective microfluidic reactor. There is an advantage. In addition, the semi-cured PDMS solution of the present invention is cured between the functional nano-permeable membrane and the wall or pillar of the PDMS-based or PMMA-based substrate to fix the functional nano-permeable membrane vertically and permanently, thereby providing selective permeation to the microfluidic channel. There is an advantage to grant.
Therefore, the microfluidic reaction based on the PDMS or PMMA-based microchip in which the nano-permeable membrane of the present invention is vertically fixed is based on the principle of physical and chemical reactions caused by selective mass transfer between samples separated spatially by the nano-permeable membrane. It can be applied to a microfluidic reaction system.

Description

Microchip Containing Perpendicularly Bonded Functional Nano-porous Membrane and Manufacturing Method Thereof}

The present invention relates to a microchip in which a functional nanopermeable membrane is vertically and permanently fixed and a method of manufacturing the same.

Microchips to which a microfluidic reaction system is applied have an advantage of detecting the presence of a target substance qualitatively and quantitatively with only a small amount of a sample. The microfluidic reaction system overcomes the disadvantages of the conventional bulk process, uses a small amount of reagents, shortens the reaction time, improves material transfer and heat transfer rates, and improves diffusion distance and side reaction. There is an advantage to minimize. Microchips used as biosensors are commonly used to measure color changes or luminescence due to binding between biomolecules. Therefore, it is important that microchips for use as biosensors have excellent optical properties. Substrates based on polydimethylsiloxane (PDMS) or polymethylmetacrylate (PMMA) have excellent optical transmission and easily form microfluidic channels through various lithography methods and laser processing methods. There is an advantage to this. However, PDMS-based or PMMA-based substrates can form a microfluidic channel with a planar structure that can control depth and width through the microfluidic channel formation method, but attach a functional material or change the structure of the microfluidic channel in three dimensions. There is a limitation in providing functionality to the microfluidic channel through the method. In particular, the copolymer (siloxane) containing a siloxane (siloxane) group (polymer) due to the low chemical bonding properties are difficult to chemically bond with other materials. This attempted to immobilize the functional separator using the plasma technique, but there was a disadvantage that the fixing is not permanent and only copolymers of the same functional group can be used. Therefore, the conventional microfluidic reaction system using a microfluidic channel has been mainly used for synthesizing nanomaterials as a microfluidic reactor that induces a chemical reaction by continuously flowing two or more raw materials. To detect the response of the target biomolecules, it is important to exclude the binding of other biomolecules present in the sample. In particular, in the microfluidic reaction system, the detection sensitivity of the biomolecule reaction is very high. Therefore, the reaction of the target biomolecule cannot be detected without removing the side reaction with other biomolecules. Since biomolecules have various sizes, water solubility, and excellent diffusivity, the detection of microfluidic reaction system can be dramatically improved by screening according to the size of biomass. Therefore, if surface modification method is developed to overcome this problem for copolymers having low chemical bonding due to difficult surface modification, it is possible to manufacture a microfluidic reaction system having improved detection level by bonding various functional separators. It is expected.

The patents and references mentioned herein are incorporated herein by reference to the same extent as if each document was individually and clearly specified by reference.

Korean Patent Registration No. 1446687

The inventors of the present invention have excellent optical properties, but are difficult to modify the surface, so that the PDMS solution of a polydimethylsiloxane (PDMS) or polymethylmetacrylate (PMMA) -based substrate having a low chemical bonding and a functional nanopermeable membrane is semi-cured. The present invention has been completed by manufacturing a microfluidic channel having selective permeability and manufacturing a microchip including the same by vertically and permanently bonding using the same. Accordingly, an object of the present invention is a PDMS or PMMA based substrate; A microfluidic channel formed on the substrate; And it provides a microchip comprising a functional nano-permeable membrane (nonporous-membrane) vertically fixed to the wall or pillar of the microfluidic channel.

Another object of the present invention is to provide a method for manufacturing the microchip.

Other objects and technical features of the present invention are more specifically shown by the following detailed description, claims and drawings.

According to one aspect of the invention, the present invention is a polydimethylsiloxane (polydimethylsiloxane, PDMS) or polymethyl methacrylate (polymethylmetacrylate, PMMA) based substrate; A microfluidic channel formed on the substrate; And it provides a microchip comprising a functional non-permeable membrane (perforated vertically and permanently perpendicular to the wall or pillar of the microfluidic channel). According to one embodiment of the present invention, the functional nano-permeable membrane of the present invention is prepared using electrospinning and gives hydrophilicity or hydrophobicity by performing plasma surface treatment. According to another embodiment of the present invention, the functional nanopermeable membrane of the present invention is vertically and permanently fixed to the wall or pillar of the microfluidic channel using a half-cured PDMS solution.

According to another aspect of the present invention, the present invention provides a method for manufacturing a microchip in which a functional nanopermeable membrane is vertically fixed, comprising the following steps:

Step (a): preparing a semi-cured polydimethylsiloxane (PDMS) solution;

Step (b): forming a microfluidic channel by performing soft lithography or laser processing on PDMS or polymethylmethacrylate (PMMA) -based substrate;

Step (c): preparing a functional nanopermeable membrane using electrospinning and performing plasma surface treatment to have hydrophilicity or hydrophobicity;

Step (d): cutting the hydrophilic or hydrophobic functional nanopermeable membrane to fit the size of the microfluidic channel and compressing it vertically to the wall or pillar of the microfluidic channel using static electricity;

Step (e): supplying the semi-cured PDMS solution to the hydrophilic or hydrophobic functional nanopermeable membrane and the compressed surface of the wall or the pillar of the microfluidic channel; And

Step (f): The substrate of step (e) is heated at a temperature in the range of 70-90 ° C. for 0.5-1.5 hours, thereby compressing the hydrophilic or hydrophobic functional nanopermeable membrane and the wall or pillar of the microfluidic channel. Curing the semi-cured PDMS solution supplied to the vertically and permanently fixing the functional nanopermeable membrane to the wall or pillar of the microfluidic channel.

The present invention relates to a PDMS-based or PMMA-based microchip in which a functional nanopermeable membrane is vertically and permanently fixed and a method of manufacturing the same. PDMS or PMMA-based substrates of the present invention have excellent optical properties and have the advantage of easily forming microfluidic channels through lithography and laser processing. Functional nano-permeable membrane of the present invention has a pore has the advantage of high material transfer efficiency, by controlling the pore size and porosity to selectively transfer the material according to the size and surface properties of the material and low for the injected fluid and solution There is an advantage that can be used to prepare a microfluidic reactor (resistance and reactivity) excellent selectivity. The present invention uses a semi-cured PDMS solution to vertically and permanently fix the functional nanopermeable membrane between the walls or pillars of the PDMS or PMMA-based substrate, so that the microfluidic channel is selectively partitioned by the functional nanopermeable membrane. There is an advantage that can form a microfluidic channel that is selectively transmitted.

Therefore, the microfluidic reaction based on the PDMS or PMMA-based microchip in which the nano-permeable membrane of the present invention is vertically fixed is based on the principle of physical and chemical reactions caused by selective mass transfer between samples separated spatially by the nano-permeable membrane. It can be applied to a microfluidic reaction system.

Figure 1 shows the degree of improvement of the microfluidic channel to which the functional nanopermeable membrane is vertically and permanently bonded. Panel A shows a conceptual diagram of a channel portion to which a functional nanopermeable membrane is attached and a channel portion to which a functional nanopermeable membrane is not attached. Panel B shows a conceptual diagram of culturing cells in a space partitioned by the functional nanopermeable membrane.
Figure 2 shows the vertical and permanent fixation of the functional nano-permeable membrane in the microfluidic channels having various heights and widths of the microchip.
Panel A of FIG. 3 shows the result of capturing the vertically and permanently fixed pillars of the functional nano-permeable membrane and the microfluidic channel, and the panel B shows that the pillars of the functional nano-permeable membrane and the microfluidic channel are vertical. Laser scanning of the permanently fixed microfluidic channel shows that the functional permeable membrane is attached at the same height as the channel.
FIG. 4 shows the green dye passed through chemical diffusion through a vertical, permanently fixed functional nanopermeable membrane and silica microparticles (red) controlled permeation.
5 is a cell culture in a microfluidic channel separated by a vertical and permanently fixed functional nanopermeable membrane, the contact between the cells is physically controlled by the functional nanopermeable membrane, and in the cell through the fixed functional nanopermeable membrane The secreted chemical signal is shown to be transmitted to each other cells. Panels A and B show the results of the cultured MDA-MB-23 cells injected into the channel and compartmentalized by functional nanopermeable membranes. Panel C shows MDA-MB-23 cells and hMVECs cells by functional nanopermeable membranes, respectively. The cell movement was limited as a result of injecting into the partitioned channel and cultured, but the cell culture medium containing the signal transducing agent was freely moved.

According to one aspect of the invention, the present invention is a polydimethylsiloxane (polydimethylsiloxane, PDMS) or polymethyl methacrylate (polymethylmetacrylate, PMMA) based substrate; A microfluidic channel formed on the substrate; And it provides a microchip comprising a functional non-permeable membrane (perforated vertically and permanently perpendicular to the wall or pillar of the microfluidic channel).

The polydimethylsiloxane (PDMS) is a kind of polymeric organosillicon compound, commonly called silicon.

The PDMS has excellent optical transmittance but has a disadvantage in that it is difficult to chemically bond with other materials due to difficult surface modification.

The polymethylmethacrylate (PMMA) is a resin that has excellent transmittance, chemical stability and toughness of up to 92% of light transmittance. The PMMA is used as an organic glass having excellent strength and good workability and permeability compared to glass. Therefore, the PDMS and PMMA are frequently used in a detection sensor that detects a target material by measuring color change or luminescence due to excellent optical properties, but surface modification is difficult, and thus functional addition is difficult in addition to the formation of planar channels.

The microfluidic channel refers to a microchannel having a width and depth of several hundred micrometers and through which a fluid can flow.

According to one embodiment of the invention, the microfluidic channel is formed using soft lithography or laser processing. The soft lithography means a transfer method using alkane having a functional group and a polymer material to make a pattern or a structure without using a complicated device used in conventional photolithography. The soft lithography has an advantage that it is easy to fabricate shapes and structures in nano or micrometer units quickly and inexpensively. The soft lithography is a micro-contact printing (μCP) method, decal transfer microlithography method, light stamp printing method, replica molding method, capillary force lithography method, Capillary-micromolding in capillaries, microtransfer molding (μTM), nanoimprinting or liquid-bridgemediated nanotransfer molding (LB-nTM) can be used. Preferably, liquid-bridgemediated nanotransfer molding (LBN) method is used.

According to one embodiment of the invention, the functional nano-permeable membrane is a polymer, for example, elastin, hyaluronic acid, alginate, gelatin, collagen, cellulose, polyethylene glycol (PEG), polyethylene oxide (PEO), polycaprolactone (PCL) ), Polylactic acid (PLA), polyglycolic acid (PGA), polyethersulfone (PES), polyurethane (PU), poly [(lactic-co- (glycolic acid)) (PLGA), poly [(3-hydro Oxybutyrate) -co- (3-hydroxyvalorate) (PHBV), polydioxanone (PDO), poly [(L-lactide) -co- (caprolactone)], poly (esterurethane) (PEUU) , Poly [(L-lactide) -co- (D-lactide)], poly [ethylene-co- (vinyl alcohol)] (PVOH), polyacrylic acid (PAA), polyvinyl alcohol (PVA), polyvinyl Formed of one or more polymers or copolymers thereof or mixtures thereof selected from the group consisting of pyrrolidone (PVP), polystyrene (PS) and polyaniline (PAN) The not.

Preferably, the functional natto permeable membrane is formed of, but is not limited to, one or more polymers or copolymers thereof or mixtures thereof selected from the group consisting of polyurethane, polyethersulfone, polyvinyl alcohol, polycaprolactone and silicone.

The functional nanopermeable membrane is prepared by electrospining. The electrospinning means a method of realizing a continuous fiber having a diameter of several μm to several nm scale using an electric field. The electrospinning method is simpler than the self-assembly, phase separation, and mold synthesis method, and there is no limitation of material selection, and it is easy to control specific surface area, porosity, structure, and size due to shape. The electrospinning methods include coaxial electrospining, mixed electrospining, multilayer and mixed electrospining, forced air electropspining or air gap electrospinning. Air gap electrospining may be used, but is not limited thereto. Preferably, coaxial electrospinning is used.

Functional nano-permeable membrane of the present invention can be produced by varying the process conditions of the electrospinning functional nano-permeable membrane having a variety of surface area characteristics and varying the degree of separation or permeation depending on the size. Morphological characteristics of the nanofibers by the electrospinning depends on the process conditions of electrospinning. The process conditions during the electrospinning include physical properties of the polymer solution such as molecular weight of the polymer, viscosity of the polymer solution, electrical conductivity and surface tension, applied voltage, tip to collector distance (TCD), radiation velocity and It is influenced by the same fair factor.

According to an embodiment of the present invention, the functional nano-permeable membrane has a voltage of 10-20kV, a spinning speed of 0.05-0.2ml / h, a diameter of 20-30G of a needle, a spinning distance of 25-45cm, and a contact angle of 20-150 °. 8-12 ml of the electrospinning solution containing the biocompatible polymer is electrospun for 60-80 hours.

When the electrospinning is performed outside the above conditions, the nanofibers may be excessively stacked or insufficiently stacked to prepare nanofibers of desired physical properties. The contact angle is a factor that determines the porosity of the nano-permeable membrane, the nano-permeable membrane of the present invention is preferably prepared to have an average porosity of 85% or more. When the nano-permeable membrane is manufactured with the contact angle of less than 20 °, the porosity of the nano-permeable membrane is prepared to be less than 85% on the average, thereby decreasing the selectivity of the permeable material. When the nano-permeable membrane is manufactured with the contact angle exceeding 150 °, The porosity is so excessive that it is impossible to permeate the material.

Functional nano-permeable membrane of the present invention can be integrated to the nano-permeable membrane up to 100㎛ depending on the conditions of the fluid flowing into the injected microchip and the state of the material to be detected and analyzed. The electrospinning time is a factor that determines the thickness of the nano-permeable membrane and the pore size. According to one specific embodiment of the present invention, the functional nano-permeable membrane of the present invention may have a thickness of 15-100 μm and may be prepared as a nano-permeable membrane having a pore size of 1-20 μm. Preferably, the functional nanopermeable membrane of the present invention may have a thickness of 20-60 μm and may be prepared as a nanopermeable membrane having a pore size of 2-10 μm. More preferably, the functional nanopermeable membrane of the present invention may have a thickness of 30 μm and may be prepared as a nanopermeable membrane having a pore size of 3-5 μm.

The electrospinning time is less than 60 hours, the thickness of the nano-permeable membrane is less than 15㎛ and the pore size may be prepared as a nano-permeable membrane exceeding 5㎛. If the thickness is less than 15 μm, the physical strength of the nano-permeable membrane may be degraded, and if the pore size exceeds 20 μm, the selectivity to the permeable material may be reduced. In addition, when the electrospinning time exceeds 80 hours, the nano-permeable membrane may have a thickness of more than 100 μm and a pore size of less than 1 μm. If the thickness exceeds 100㎛ is not suitable for installation in the microchip and if the pore size is less than 1㎛ there is a disadvantage that the material transmission efficiency is lowered.

According to one embodiment of the present invention, the functional nano-permeable membrane of the present invention is subjected to plasma surface treatment to have hydrophilicity or hydrophobicity.

The plasma refers to a gas state separated from electrons having negative charges and positively charged ions at very high temperatures. The plasma is a gas composed of electrons, ions, and neutral particles generated by an arc discharge, and thermal plasma or low pressure or atmospheric pressure in the form of a high-speed jet flame having a constituent particle of 1,000-20,000 ° C. and 100-2,000 m / s. ) May be a low-temperature plasma (atmospheric pressure plasma) generated by a pulse corona discharge or a dielectric barrier discharge method, but is not limited thereto.

The plasma surface treatment of the present invention may use a plate-type reactor, a vertical cylinder reactor, or a needle-to-cylinder reactor. It is not limited. When the properties of the functional nanopermeable membrane are changed to hydrophilic or hydrophobic through the plasma surface treatment, the selectivity of materials that can pass through the nanopermeable membrane is improved.

According to one embodiment of the invention, the functional nano-permeable membrane of the present invention is fixed vertically to the wall or pillar of the microfluidic channel using a half-cured PDMS solution.

The process of vertically fixing the functional nanopermeable membrane on the wall or pillar of the microfluidic channel may be performed by the following steps:

Step (d): cutting the hydrophilic or hydrophobic functional nanopermeable membrane to fit the size of the microfluidic channel and compressing it vertically to the wall or pillar of the microfluidic channel using static electricity;

Step (e): supplying the semi-cured PDMS solution to the hydrophilic or hydrophobic functional nanopermeable membrane and the compressed surface of the wall or the pillar of the microfluidic channel; And

Step (f): The substrate of step (e) is heated at a temperature in the range of 70-90 ° C. for 0.5-1.5 hours, thereby compressing the hydrophilic or hydrophobic functional nanopermeable membrane and the wall or pillar of the microfluidic channel. Curing the semi-cured PDMS solution supplied to the vertically and permanently fixing the functional nanopermeable membrane to the wall or pillar of the microfluidic channel.

Hereinafter, a process of vertically and permanently fixing the functional nanopermeable membrane to the wall surface or the pillar of the microfluidic channel will be described in detail.

Step (d): functional with said hydrophilicity or hydrophobicity Nano-permeable membrane  Cutting to fit the size of the microfluidic channel and compressing vertically to the wall or pillar of the microfluidic channel by using static electricity.

The functional nanopermeable membrane is prepared by cutting to the depth of the microfluidic channel and the width of the place where the functional nanopermeable membrane is attached. The cutting of the functional nanopermeable membrane is cut by using a scale of a microscope lens in a microscope.

According to a specific embodiment of the present invention, the functional nano-permeable membrane is manufactured to be used in a microfluidic channel having a depth of 150-300 μm by cutting to a width of 150-300 μm. Preferably, the functional nano-permeable membrane is cut to a width of 175-270 μm to prepare a microfluidic channel having a depth of 175-270 μm. More preferably, the functional nano-permeable membrane is cut to a width of 200-240 μm to prepare a microfluidic channel having a depth of 200-240 μm.

The cut functional nano-permeable membrane is pressed vertically to the adhesive portion of the microfluidic channel using tweezers. The compression is maintained by electrostatic attraction so that a separate adhesive material is not needed.

Step (e): functional with said hydrophilicity or hydrophobicity Nano-permeable membrane  Supplying the semi-cured PDMS solution to the wall surface of the microfluidic channel or the pressing surface of the pillar.

According to one embodiment of the present invention, the semi-cured PDMS solution is 0-10 after mixing the PDMS solution and the curing agent in a molar ratio of 8: 1 to 12: 1 (PDMS solution: curing agent) and removing bubbles Store in the temperature range of ℃. Preferably, the semi-cured PDMS solution is mixed at a molar ratio of the PDMS solution and the curing agent at a molar ratio of 9: 1 to 11: 1 (PDMS solution: curing agent) and removed from air bubbles, and then stored at a temperature range of 0-10 ° C. do. More preferably, the semi-cured PDMS solution is mixed at a molar ratio of the PDMS solution and the curing agent at a molar ratio of 10: 1 (PDMS solution: curing agent), and the bubble is removed and stored at a temperature range of 0-10 ° C.

According to a specific embodiment of the present invention, the curing agent may use a silicone curing agent (sillicone curing agent). The silicone curing agent is di- (2,4-dichlorobenzoyl) -peroxide (Di- (2,4-dichlorobenzoyl) -peroxide), dibenzoyl peroxide, tert-butyl cumyl peroxide (tert- Butylcumylperoxide, Di-tert-butylperoxide, 2,5-dimethyl-2,5 di (thi-butylperoxy) -hexane (2,5-Dimethyl-2,5 di ( t-butylperoxy) -hexane), di- (2,4-dichlorobenzoyl) -peroxide (Di- (2,4-dichlorobenzoyl) -peroxide), dicumylperoxide, di-ty-butylperoxide (Di-t-butylperoxide) and one or more curing agents selected from the group consisting of t-butylcumylperoxide or mixtures thereof, but is not limited thereto. The bubbles may be mixed and removed in a low pressure atmosphere using a vacuum chamber, and may be removed by passing through a filter paper having micro-sized pores. Preferably the bubbles are removed by mixing in a low pressure atmosphere using a vacuum chamber. If the semi-cured PDMS solution does not store oxygen, nitrogen, and bubbles present therein and is not stored at a temperature range of 0-10 ° C., bubbles may be generated from oxygen and nitrogen dissolved in the solution according to temperature change. When the semi-cured PDMS solution in which the bubbles are not removed is used to combine the functional nano-permeable membrane and the wall or pillar of the microfluidic channel, bubbles are generated during heating for curing of the semi-cured PDMS solution, thereby providing a fixed effect. It will fall significantly.

According to one embodiment of the invention, the semi-cured PDMS solution is stored for 10-30 minutes in the temperature range of 20-30 ℃ before being supplied to the functional nano-permeable membrane and the pressing surface of the wall or pillar of the microfluidic channel Use it after. Since the semi-cured PDMS solution was stored at a temperature range of 0-10 ° C., bubbles may occur when the temperature is rapidly changed through heatings. Therefore, in order to minimize the occurrence of bubbles due to the temperature change it is preferable to use after storing for 10-30 minutes in the temperature range of 20-30 ℃. The semi-cured PDMS solution may be supplied by applying a thinly processed spatula on the functional nano-permeable membrane compressed on the wall or pillar of the microfluidic channel. Since the functional nanopermeable membrane has pores, when the semi-cured PDMS solution is coated on the functional nanopermeable membrane, the functional nanopermeable membrane penetrates into the functional nanopermeable membrane by osmotic pressure, so that the functional nanopermeable membrane and the wall or pillar of the microfluidic channel are formed. It is located on the pressing surface.

Step (f): The substrate of step (e) is 0.5- at a temperature range of 70-90 ° C. For 1.5 hours  Functionality having the hydrophilicity or hydrophobicity by heating Nano-permeable membrane  Wall or column of the microfluidic channel On the pressing surface  The semi-cured PDMS solution fed to cure the functional Nano-permeable membrane  Vertically and permanently fixing the wall or pillar of the microfluidic channel

The semi-cured PDMS solution is cured to a copolymer when heated for 0.5-1.5 hours in the temperature range of 70-90 ℃. Therefore, when the substrate including the functional nano-permeable membrane, the wall or pillar of the microfluidic channel, and the semi-cured PDMS solution positioned therebetween is cured, the semi-cured PDMS solution is cured while the functional nano-permeable membrane and the microfluid are The walls or columns of the channels are permanently fixed.

The heating may be used without limitation as long as it is a heater capable of sufficiently heating the hydrophilic or hydrophobic functional nanopermeable membrane and the semi-cured PDMS solution supplied to the pressing surface of the wall or the pillar of the microfluidic channel. Preferably it may be carried out in a hot-plate or oven, but is not limited thereto.

According to one embodiment of the present invention, the process for curing the semi-cured PDMS solution is supplied with the semi-cured PDMS solution to the functional nano-permeable membrane having the hydrophilic or hydrophobic and the pressing surface of the wall or pillar of the microfluidic channel The prepared substrates are heated by heating at a temperature in the range of 70-90 ° C. for 0.5-1.5 hours. Preferably, the substrate having the semi-cured PDMS solution supplied to the hydrophilic or hydrophobic functional nanopermeable membrane and the pressing surface of the wall or the pillar of the microfluidic channel is heated by heating at a temperature range of 80 ° C. for 1 hour. When the heat treatment is performed in the curing of the PDMS curing solution, there is an advantage in that the hardness of the PDMS can be improved in preparation for a method of naturally curing at room temperature (free standing). If the temperature of the oven is less than 70 ℃ heat treatment takes more time, even if the temperature of the oven exceeds 90 ℃ heat treatment effect is the same as the heat treatment in 70-90 ℃ oven.

According to another aspect of the present invention, the present invention provides a method for manufacturing a microchip in which a functional nanopermeable membrane is vertically fixed, comprising the following steps:

Step (a): preparing a semi-cured polydimethylsiloxane (PDMS) solution;

Step (b): forming a microfluidic channel by performing soft lithography or laser processing on PDMS or polymethylmethacrylate (PMMA) -based substrate;

Step (c): preparing a functional nanopermeable membrane using electrospinning and performing plasma surface treatment to have hydrophilicity or hydrophobicity;

Step (d): cutting the hydrophilic or hydrophobic functional nanopermeable membrane to fit the size of the microfluidic channel and compressing it vertically to the wall or pillar of the microfluidic channel using static electricity;

Step (e): supplying the semi-cured PDMS solution to the hydrophilic or hydrophobic functional nanopermeable membrane and the compressed surface of the wall or the pillar of the microfluidic channel; And

Step (f): The substrate of step (e) is heated at a temperature in the range of 70-90 ° C. for 0.5-1.5 hours, thereby compressing the hydrophilic or hydrophobic functional nanopermeable membrane and the wall or pillar of the microfluidic channel. Curing the semi-cured PDMS solution supplied to the vertically and permanently fixing the functional nanopermeable membrane to the wall or pillar of the microfluidic channel.

EXAMPLE

Example 1 Preparation of PDMS or PMMA Microchips

1) PDMS or PMMA microchips with vertically fixed functional nanopermeable membranes

1 shows a conceptual diagram of a PDMS or PMMA microchip in which a functional nano-permeable membrane is vertically and permanently fixed to a microfluidic channel. According to the conceptual diagram, a nano-permeable membrane vertically fixed to a channel and a pillar of the channel is positioned, a space partitioned by the nano-permeable membrane and an open channel space are located, and the open space (mixed channel) is a cell-matrix. It is shown that gels can be located that can create an environment similar to that of Since the PDMS or PMMA has excellent optical properties, it is possible to confirm both the structure of the microfluidic channel and the reaction in the microfluidic channel. The microfluidic channel has a plurality of channels formed to allow various kinds of samples to flow, and there is a mixing channel through which the samples flowing through the microfluidic channel can be mixed. According to panel A of FIG. 2, two microchannels having a depth of 200 μm are formed in the microchip, and a mixed channel having two separate microchannels meets. In the mixed channel, the wall surface of each channel is composed of a plurality of pillars, and a functional nano-permeable membrane is vertically and permanently fixed between any one channel pillar. The functional nanopermeable membrane has a thickness of 30 μm. According to panel B of FIG. 2, four microfluidic channels having a depth of 240 μm are formed on the microchip, and a mixed channel is formed where the four microfluidic channels meet. In the mixed channel, a wall of each channel is composed of one pillar and two passages, and a functional nano-permeable membrane is vertically and permanently fixed between any one passage. The functional nanopermeable membrane has a thickness of 30 μm.

2) Vertical fixation of functional nanopermeable membrane using electron microscope

Figure 3 is a result of photographing the functional nano-permeable membrane perpendicular to the wall or pillar of the microfluidic channel and permanently fixed using JEOL's JSM-5410 electron microscope and the result of the nano-permeable membrane bonded to the same height as the microfluidic channel. Shows. According to panel A of FIG. 3, it is confirmed that the pillar of the microchannel channel and the functional nanopermeable membrane are permanently bonded by the semi-cured PDMS solution. The semi-cured PDMS solution forms a nano-permeable membrane and a copolymer in the process of curing by heating to permanently fix the functional nano-permeable membrane and the microfluidic channel. According to panel B of FIG. 3, it is confirmed that the nano-permeable membrane is bonded to the same height as the microfluidic channel. MDA-MB-23 cells were injected into one channel partitioned by the nanopermeable membrane and hMVECs cells were injected into the other channel partitioned by the nanopermeable membrane, followed by laser surface scanning. As a result, it was confirmed that each injected cell was located only at each compartment, and each compartment was clearly performed by a nanopermeable membrane (blue).

Experimental Example 1: Selective Permeation by Vertically Fixed Functional Nanopermeable Membranes

Figure 4 shows the selective permeability by the vertically and permanently fixed functional nanomembrane. In the microchip of the present invention, two microfluidic channels having a depth of 200 μm are formed and a functional nano-permeable membrane manufactured to a thickness of 30 μm is vertically fixed. Through the microchip's input port, BSA-FITC (about 37-45kDa) fluorescent dye, which is fluorescently expressed in liquid green color, and fluorescent silica particles having a size of 5 μm and 10 μm, which are expressed in red fluorescence, are injected together. In the fluorescent dye (green) and fluorescent silica particles (red) it was confirmed that the material movement. As a result of the experiment, the fluorescent silica particles having a size of 5 to 10 μm do not penetrate the functional nano-permeable membrane and are divided into a channel in which the fluorescent silica particles are present and a channel in which the fluorescent silica particles do not exist, based on the functional nano-permeable membrane. It was confirmed. In addition, in the case of the liquid BSA-FITC fluorescent dye having a size of about 37 ~ 45kDa permeate the functional nano-permeable membrane freely, it was observed that the fluorescence was observed in all spaces regardless of the channel partitioned by the nano-permeable membrane. Therefore, it was confirmed that the functional nanopermeable membrane of the present invention can transmit only particles smaller than 5 μm.

Experimental Example 2 Results of Cell Culture on Vertically Fixed Functional Nanopermeable Membranes

5 is a cell culture in a microfluidic channel separated by a vertically fixed functional nanopermeable membrane (membrane), the contact between the cells is physically controlled by the nanopermeable membrane, secreted from the cell through the fixed functional nanopermeable membrane Chemical signals are shown to be delivered to each other's cells. To this end, MDA-MB-23 cells were cultured in a cell-culture dish, and then injected into the microchip through a fluid input port secured to the microchip together with the cell culture solution (Panels A and B of FIG. 5). . As a result of the injection, it was confirmed that the MDA-MB-23 cells exist only in the injected channel, but did not pass through the functional nanopermeable membrane, and thus did not exist in other channels partitioned by the functional nanopermeable membrane. Cell culture is considered to be able to use the cells cultured according to the general cell culture method, it is determined that the amount of cells and the cell culture fluid injected may vary depending on the microchip to be produced. Therefore, the cell is considered to be a normal cell-line cell and stem cell. Microchips have a built-in nano-permeable membrane between them and one channel can place cells and the other can be filled with media, collagen or hydrogels into which the cell signaling material can diffuse, optionally cell culture, other cells, bioactivity Various materials such as materials can be added between nanopermeable membranes.

Based on the results, the cultured MDA-MB-23 cells and hMVECs cells were injected into the respective channels partitioned by the functional nano-permeable membrane, incubated for a predetermined time, and then polarized using confocal laser microscopy. Microscopy was used to confirm the movement of cells and the movement of signal transduction materials (Panel B of FIG. 5). Experimental results showed that cell migration was not confirmed, while cell culture was free to migrate.

Specific embodiments described herein are meant to represent preferred embodiments or examples of the present invention, whereby the scope of the present invention is not limited. It will be apparent to those skilled in the art that variations and other uses of the invention do not depart from the scope of the invention described in the claims.

Claims (11)

delete delete delete delete delete delete delete (a) preparing a semi-cured polydimethylsiloxane (PDMS) solution;
(b) forming a microfluidic channel by performing soft lithography or laser processing on a PDMS or polymethylmetacrylate (PMMA) -based substrate;
(c) preparing a functional nanopermeable membrane using electrospinning and performing plasma surface treatment to have hydrophilicity or hydrophobicity;
(d) cutting the hydrophilic or hydrophobic functional nanopermeable membrane to fit the size of the microfluidic channel, and compressing the hydrophilic or hydrophobic layer to the wall or pillar of the microfluidic channel by using static electricity;
(e) supplying the semi-cured PDMS solution to the hydrophilic or hydrophobic functional nanopermeable membrane and the compressed surface of the wall or the pillar of the microfluidic channel; And
(f) heating the substrate of step (e) for 0.5-1.5 hours in the temperature range of 70-90 ° C. and supplying the hydrophilic or hydrophobic functional nanopermeable membrane and the compressed surface of the wall or pillar of the microfluidic channel. And curing the semi-cured PDMS solution to fix the functional nanopermeable membrane vertically and permanently to the wall or pillar of the microfluidic channel.
The method of claim 8, wherein the semi-cured PDMS solution is a mixture of the PDMS solution and the curing agent in a molar ratio of 8: 1 to 12: 1 (PDMS solution: curing agent) in a molar ratio (molar ratio) and after removing the bubble temperature of 0-10 ℃ A method of manufacturing a microchip in which a functional nano-permeable membrane is vertically and permanently fixed, characterized in that it is stored in a range.
The method of claim 8, wherein the semi-cured PDMS solution is used after storing for 10-30 minutes in the temperature range of 20-30 ℃ before being supplied to the functional nano-permeable membrane and the pressing surface of the wall or pillar of the microfluidic channel A method of manufacturing a microchip in which a functional nano-permeable membrane is vertically and permanently fixed.
The method of claim 8, wherein the functional nanopermeable membrane is one or more polymers selected from the group consisting of polyurethane, polyethersulfone, polyvinylalchol, polycarprolactone and silicone Or a method of manufacturing a microchip in which a functional nanopermeable membrane is vertically and permanently fixed, characterized in that it is prepared using a copolymer or a mixture thereof.

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