JP2020028962A - Method for manufacture of microfluidic device with built-in membrane - Google Patents

Abstract

To provide a novel method for manufacture of a membrane built-in type microfluidic device by which the number of individual devices which cause liquid leakage is reduced and yielding percentage is increased.SOLUTION: A method for manufacture of a microfluidic device with a built-in membrane includes: a process in which a cut is formed in a silicone rubber substrate having a micro flow channel pattern; a process in which plasma treatment or corona discharge treatment is performed for the substrate while holding the substrate in a bent state so that a notch port of the cut is opened; and a process in which a porous membrane is inserted into the cut, and a cut surface is crimped.SELECTED DRAWING: Figure 1

Description

  The present invention relates to microfluidic devices.

  In order to deliver a drug from a bloodstream to a target tissue, a drug delivery system using nanoparticles or the like is widely used. In order to develop an effective drug delivery system, it is important to elucidate the relationship between experimentally observed drug vascular permeability and various physical parameters.

  Nanoparticles can penetrate the pores of the vessel wall in vivo, but the size and shape of the pores in the vessel wall and the composition of the extravascular stroma are non-uniform and difficult to grasp accurately. Did not accurately analyze the effects and interactions of various physical parameters (eg, pore size and nanoparticle size) on permeation. Ethical and efficiency issues can also accompany animal testing.

  On the other hand, in vitro assays based on cultured cells, for example using cell culture inserts, have the advantage of at least avoiding ethical issues and of being able to test under defined (pseudo) stromal compositions. However, such an assay would require experiments to be performed on large-sized instruments far from the actual blood vessels, would not be able to reproduce the presence of blood flow and the associated fluid pressure, and would result in non-uniform cell cultures on the insert. In this case, there is a disadvantage that data reliability is lost (for example, the cell density tends to be low near the outer edge of the insert). In addition, the problem that even in vitro assays based on cultured cells cannot accurately analyze the effects and interactions of various physical parameters remains.

  The present inventors have developed a microfluidic device in which an artificial porous membrane is incorporated into a silicone rubber substrate, and by measuring the permeation behavior of nanoparticles through the micropores of this porous membrane, animal experiments and cell experiments can be performed. It has been reported that the observable permeability can be related to specific physical parameters, and it has been proposed to rationally design nanopharmaceuticals by such an approach (Non-Patent Document 1).

  A microfluidic device incorporating a porous membrane can be used to culture a layer of vascular endothelial cells on the membrane to build a simulated vascular wall and evaluate the material permeability of the cell layer. Alternatively, the porous membrane itself can be regarded as a blood vessel wall and applied to permeability evaluation (Non-Patent Document 1). Such a membrane-embedded microfluidic device has made it possible to precisely evaluate the permeability while imitating the size and liquid flow equivalent to those of a real blood vessel.

  Early film-incorporating devices incorporated a film parallel to the observation plane (for example, Non-Patent Documents 1 and 2). Therefore, the sample (particles) moves perpendicularly to the observation surface during transmission. In other words, transmission in such a device means that particles cross the membrane in a direction toward or away from the microscope lens, and there is a problem that it is difficult to quantitatively evaluate the amount of transmission during microscopic observation. Was. The inventors have developed a method of incorporating the membrane perpendicularly to the observation surface and solved the problem (Non-Patent Document 3), and applied it to the evaluation of the vascular leakage of nanoparticles in a simulated blood environment. (Non-Patent Document 4).

Anal.Sci. 2016, 32, 1307-1314 Anal. Chem. 2001, 73, 5207-5213. 20th International Conference on Miniaturized Systems for Chemistry and Life Sciences, October 9-13, 2016, Dublin, Ireland, pp. 1023-1024 21st International Conference on Miniaturized Systems for Chemistry and Life Sciences, October 22-26, 2017, Savannah, Georgia, USA, pp. 1211-1212

  The above-described microfluidic device mainly includes a silicone rubber substrate having a microchannel pattern. This substrate can be prepared by a method including mixing a raw material for a reaction for forming a silicone rubber and a polymerization initiator, pouring the mixture into a mold, and heating. Conventionally, a device in which a film is incorporated perpendicularly to an observation surface (hereinafter, referred to as a “vertical film-incorporated device”) is conventionally provided with a cut in a substrate after the above-mentioned heating and curing. After the porous film was sandwiched in the cut, the cut surface was joined by heating again (Non-Patent Document 3).

  After making a cut in the substrate as described above, the present inventors perform plasma processing on the substrate while holding the substrate in a bent state so that the cutout is opened, and then press the cut surface by sandwiching the porous film When a membrane-incorporated device is manufactured by the step of performing the process, it has been found that the number of individual devices that cause liquid leakage decreases and the yield increases, and the present invention has been completed.

The present invention includes the following embodiments.
[1]
A step of making a cut in a silicone rubber substrate having a microchannel pattern,
Performing a plasma treatment or a corona discharge treatment on the substrate while holding the substrate in a bent state so that the slit of the slit is opened;
A method of sandwiching a porous membrane in the cut and pressing the cut surface in pressure.
[2]
The method according to [1], wherein the silicone rubber includes polydimethylsiloxane.
[3]
The method according to [1] or [2], wherein no additional silicone or a prepolymer thereof is included in the cut during the crimping.
[4]
Any one of [1] to [3], wherein the porous membrane is sandwiched between the cuts so that only the membrane surface of the porous membrane is exposed to the space in the flow path and the membrane edge is not exposed to the space in the flow path; The method described in.
[5]
The method according to any one of [1] to [4], further comprising a step of preparing a silicone rubber substrate having the microchannel pattern before the step of cutting.
[6]
The step of fabricating the silicone rubber substrate having the microchannel pattern includes superposing two silicone rubber substrate materials and joining them by heating, and at least one of the two silicone rubber substrate materials. Has a flow path molded concavely on the surface, and the surface having the flow path faces the surface of the other silicone rubber substrate material to form a micro flow path pattern, [5]. Method.

  According to the embodiments of the present invention, in the production of a membrane-embedded microfluidic device, particularly a vertical membrane-embedded device, it is possible to reduce the number of individual devices that cause liquid leakage and to improve the yield. Since the conventional manufacturing method was essentially the same in that the contacted siloxane skeletons were crosslinked, it is not necessarily the case that the liquid leakage is reduced by performing the crosslinking by the plasma treatment as in the embodiment of the present invention. I could not predict. Non-Patent Document 2 discloses a microchannel device in which two silicon substrate members each having a flow path are vertically stacked and a porous film is sandwiched between the two silicon substrate materials to separate the upper and lower flow paths by a porous film. Describes that oxygen plasma treatment was used to join the upper and lower substrate materials, but there was a problem that liquid leakage occurred. Non-Patent Document 2 discloses a problem of liquid leakage by forming a sandwich as described above after adding a silicone prepolymer to the edge of a porous film after oxygen plasma treatment of a substrate material and heating at 65 ° C. for 1 hour. Has been resolved. In view of the teachings of Non-Patent Document 2, it was further unexpected that embodiments of the present invention achieved a reduction in liquid leakage without the addition of a prepolymer.

  Further, according to the embodiment of the present invention, it is possible to manufacture a microfluidic device with a built-in membrane, particularly a device with a built-in vertical membrane, in a significantly shorter time than before, and it is possible to reduce energy consumption and cost. Furthermore, embodiments of the present invention allow for the incorporation of porous membranes containing components that may be denatured or destroyed by heat, such as, for example, Matrigel-coated porous membranes, into the device. For example, a membrane can be coated with various scaffold molecules used for cell culture, and can be directly incorporated into a device. In the conventional method that required heating for membrane incorporation, coating had to be performed after membrane incorporation, so such scaffold molecules were to be distributed over the entire inner surface of the flow channel. In this method, scaffold molecules can be localized only on the membrane surface. By incorporating a plurality of membranes, it becomes possible to arrange different types of scaffold molecules at arbitrary positions in the same channel. As described above, the degree of freedom in designing a microfluidic device incorporating a membrane is remarkably improved.

In FIG. 1, a razor is cut into a silicone rubber substrate having a micro-channel pattern (a), the substrate is held in a bent state so that the slit is opened (b), and the substrate is subjected to plasma treatment ( c) shows a manufacturing process in one embodiment in which a porous membrane is sandwiched between cuts (d), and the silicone rubber substrates sandwiching the porous membrane are pressed together (e) to obtain a vertical membrane embedded microfluidic device. FIG. 2A shows an overview (top) of a vertical membrane-embedded microfluidic device manufactured by the method of one embodiment, and an enlarged view (bottom) of a porous membrane-embedded portion. Fig. 2 (b) shows an experiment where the lower channel was filled with genipin-crosslinked collagen gel (looking black) and the suspension of fluorescently labeled nanoparticles (appearing white) was flowed into the upper channel. Show. FIG. 2 (c) shows the result of quantifying the cumulative amount of fluorescently labeled nanoparticles that have passed through the porous membrane in the experiment of (b).

  In the present disclosure, terms such as “up”, “down”, “first”, and “second” are used for convenience in distinguishing a plurality of elements or steps, and are not necessarily absolute positional relations or orders. Is not intended to be specified. For example, two flow paths located on the same horizontal plane may be referred to as an upper flow path and a lower flow path. In this case, even if the names of the upper flow path and the lower flow path are interchanged, there is no change in structure. It is possible.

  The present disclosure, in one embodiment, a step of making a cut in a silicone rubber substrate having a microchannel pattern, and holding the substrate in a bent state so that the cut opening of the cut is opened, while plasma is applied to the substrate. Provided is a method for manufacturing a membrane-embedded microfluidic device, which includes a step of performing a treatment and a step of pressing a cut surface by sandwiching a porous membrane in the cut. The microfluidic device according to the present disclosure uses silicone rubber as a device body, that is, a substrate. The silicone rubber in the present disclosure typically contains polydimethylsiloxane, and may be made of polydimethylsiloxane. At least a part of the methyl group may be replaced with another substituent such as a vinyl group or a phenyl group. It is considered that these hydrocarbon groups are removed by plasma treatment or corona discharge treatment to form silanol groups, and the silanol groups are cross-linked to cause bonding between the silicone rubber substrates.

  The substrate usually has a substantially flat outer shape. The flat plate preferably has a size of not more than 25 mm in length and not more than 75 mm in width, and more preferably not more than 20 mm in length and not more than 30 mm in width. The terms "vertical" and "horizontal" are not intended to limit the flat plate to a rectangular flat plate. The thickness of the flat plate is preferably within 20 mm, more preferably within 10 mm. With these dimensions, observation with a microscope becomes easy.

  Incising a silicone rubber substrate is typically accomplished by inserting a sharp blade halfway into the silicone rubber substrate (FIG. 1a). The notch does not mean that the blade penetrates and the silicone rubber substrate is completely cut into two blocks, but that the substrate block on both sides of the blade is connected by leaving the uncut part without the blade penetrating Means that. A razor is suitable, but not limited to, a cutting means that can be used for this purpose.

  The cut surface is usually perpendicular to the surface of the substrate, ie, 90 °, as shown on the right of FIG. 1a, but the angle of the cut surface with respect to the substrate surface may be varied, for example, in the range of 45 ° to 90 °. It is understood that the angle of the cut surface corresponds to the angle of the porous membrane after being incorporated. If the flow path in the substrate evolves in a substantially horizontal plane direction and the porous membrane is incorporated into the substrate almost vertically and separates the flow path, the microscope lens should be directed in the vertical direction, that is, in the thickness direction of the substrate. Thereby, it is possible to observe particles passing through the porous membrane in real time.

  As is commonly understood by those skilled in the art, a channel in a microfluidic device is a space formed inside a substrate that can be filled with a microliter volume of liquid from outside (usually through a port). is there. The liquid in the flow path can be kept flowing or can be retained. The channel may be filled with a gel-like substance instead of a liquid.

  The cut substrate is then held in a bent state so that the cut is opened (FIG. 1b). In this state, for example, two protrusions (for example, acrylic rods) fixed at a shorter interval than the entire length of the substrate are prepared, one end of the substrate is fixed to one protrusion, and the other end of the substrate is fixed to the other protrusion. This can be achieved by holding the substrate by warping the substrate so that the cut side is a convex surface. On the right side of FIG. 1b, the sample stage and the two projections fixed thereto are shown in black. In this state, the substrate is subjected to a plasma treatment or a corona discharge treatment (FIG. 1c).

  As known to those skilled in the art, the plasma processing includes a vacuum (low pressure) plasma processing performed after reducing the pressure in a closed sample chamber and an atmospheric pressure plasma processing performed while maintaining the atmospheric pressure in the sample chamber. Although both can be used in the present embodiment, vacuum plasma processing is particularly preferable. Source gases for the plasma include, but are not limited to, air, nitrogen, helium, argon, and mixtures of any of these. Air is a particularly preferred source gas.

  The corona discharge treatment is different from the plasma treatment in which a parallel plate type electrode is generally used, in which a discharge is caused in the atmosphere from a sharp electrode to irradiate a sample to perform surface modification. Corona discharge treatment also utilizes the generation of plasma, and can be used instead of plasma treatment. The time for performing the plasma treatment or the corona discharge treatment is usually 10 seconds to 5 minutes, preferably 20 seconds to 1 minute.

  An example of preferable plasma processing conditions is to perform the process at a pressure of 0.5 torr, a gas flow rate of 30 sccm, a power of 80 W, and a frequency of 50 kHz for 30 seconds using air as a source gas. One skilled in the art can adjust these parameters based on common knowledge. For example, a pressure setting of 0.1-100 torr, more preferably 0.3-10 torr, a gas flow of 1-100 sccm, more preferably 5-50 sccm, and / or a power of 10-300 W, more preferably 50-100 W. You may adjust suitably within a range.

  After the plasma treatment or the corona discharge treatment, the cut surfaces are pressed against each other with the porous film sandwiched between the cuts (FIG. 1d) (FIG. 1e). This step is preferably performed within 2 hours after the plasma treatment or corona discharge treatment, more preferably within 1 hour, further preferably within 20 minutes, particularly preferably within 5 minutes. Crimping may be achieved by the elasticity of the silicone rubber substrate itself adjacent to the cut, but pressure may be applied, for example, with a finger. Crimping can usually be completed within 1 minute, for example, within 30 seconds.

  Since the porous membrane itself is passively sandwiched and may or may not actively form a crosslink with the silicone rubber, the material of the porous membrane is not essentially limited. The porous membrane is typically made of a synthetic resin. Examples of synthetic resins include polyester, polycarbonate, polyacrylonitrile, ethylene-vinyl alcohol copolymer, polyolefin, cyclic polyolefin, polyvinyl chloride, polystyrene, polyvinyl alcohol, polyamide and the like, and mixtures thereof. Not limited. It is particularly preferred that the porous membrane comprises polyester or polycarbonate, or that the porous membrane comprises polyester or polycarbonate. The polyester is preferably polyethylene terephthalate (PET).

  In the present disclosure, the liquid leakage means that the liquid put in the channel of the device leaks to a region other than the channel (for example, an interface between the silicone rubber substrate and the porous membrane). Leakage of liquid from one flow path to another through a porous membrane is not included in liquid leakage.

  As described above, since the conventional manufacturing method of joining the cut surfaces by heating is essentially the same in that the contacted siloxane skeletons are crosslinked, the crosslinking is performed by plasma treatment as in the present embodiment. It was not always possible to predict that the liquid leakage would decrease and the yield would increase. While not being bound by any particular theory, when performing plasma treatment or corona discharge treatment by opening the cut, not only the cut cross section, but also the inner wall of the flow channel in the vicinity is exposed to plasma to acquire hydrophilicity. It is conceivable that the newly imparted hydrophilicity to the inner wall of the flow channel may make it difficult for the finished device to leak liquid from the flow channel. Further, in the present embodiment in which the film is inserted into the cut of one substrate instead of sandwiching the film between the two substrate materials, the cut surface itself attempts to restore the original state due to the elasticity of the silicone rubber substrate. May work, which may result in a decrease in liquid leakage. Cross-linking by heating takes a long time, but cross-linking after surface activation by plasma treatment is completed in a short time, so the process of self-repairing the opened incision by elasticity and the process of molecular cross-linking are well synchronized. It is possible.

  It is understood that, typically, the porous membrane is inserted in at least a part of the microchannel so as to separate one space into two. It will also be appreciated that the cuts described above are also placed in a position that allows such insertion of the porous membrane. For example, depending on what experiment is intended to be performed with the device, for example, a porous membrane can be inserted to separate a relatively thick channel into two smaller channels. Alternatively, a configuration may be adopted in which a porous membrane is inserted in such a manner that one flow path is blocked by the porous membrane, and the liquid passing through the flow path is forced to pass through the porous membrane.

  The porous membrane preferably has an area smaller than the cross-sectional area of the cut. It is preferable that the porous membrane has a size that does not protrude from the silicone rubber substrate when sandwiched between the cuts. It is preferable that the porous membrane is incorporated into the device in a state where the outer edge is not exposed to the space in the flow channel. That is, it is preferable that the porous membrane is sandwiched between the cuts so that only the membrane surface of the porous membrane is exposed to the space in the flow path, and the membrane edge is not exposed to the space in the flow path.

  It is not necessary to include additional silicone or its prepolymer in the cut when pressing the cut surface with the porous membrane sandwiched between the cuts. Rather, it is preferable to perform pressure bonding without adding silicone or its prepolymer into the cut. It is preferable that only the porous membrane is included in the cut during the pressure bonding.

  The thickness of the porous membrane is preferably 200 μm or less. If the porous membrane is too thick, its significance as a biological tissue model may be diminished. The thickness of the porous membrane is more preferably 100 μm or less, further preferably 50 μm or less, and particularly preferably 20 μm or less. The thickness of the porous membrane has no particular lower limit, but is usually 0.5 μm or more, for example, 1 μm or more or 5 μm or more.

  The pore size and pore density of the pores of the porous membrane may vary depending on the purpose of the experiment intended by the user, and are not particularly limited. For example, the pore size can be 0.05-10 μm, with 0.1-1 μm being preferred. A wide variety of porous membranes are commercially available and can be used in this embodiment. For example, the bottom of a commercially available cell culture insert is porous, and a porous membrane obtained by cutting out the bottom can be incorporated into the microfluidic device of the present embodiment. Alternatively, the porous film may be prepared using a technique such as a track etching method.

  Methods of forming a microchannel pattern inside a silicone rubber substrate are known to those skilled in the art. For example, a flow channel mold made of an appropriate material is combined with a mold if necessary, a silicone prepolymer is poured into the mold, and the silicone is cured according to the shape of the mold, so that the surface is concave. A substrate having a molded microchannel pattern can be obtained. By joining the flow path side surface of this substrate material to the flat surface of another substrate material and joining, it is possible to obtain a substrate having a flow path space inside the substrate instead of the substrate surface. This bonding can be preferably performed by heating at about 100 ° C. for about 30 minutes, for example, but the bonding method is not limited to this. The material of the mold can be appropriately selected by a person skilled in the art based on ordinary knowledge, but acrylic is suitable for precisely processing a micrometer-scale flow path by laser processing technology combined with drawing software. Resins are particularly preferred. By penetrating the holes from the substrate surface to the respective flow paths, it is possible to form a port capable of filling the flow paths and generating a liquid flow in the flow paths.

  Therefore, one embodiment of the present method further includes a step of preparing a silicone rubber substrate itself having a microchannel pattern prior to the step of cutting. In one example, this step includes superimposing and bonding two silicone rubber substrate materials by heating, wherein at least one of the two silicone rubber substrate materials has a channel formed in a concave surface. The surface having the flow path faces the surface of the other silicone rubber substrate material to form a micro flow path pattern. The heating temperature for the bonding is usually 60 ° C. or higher, preferably 65 ° C. or higher, more preferably 90 ° C. or higher, for example, 100 ° C. or higher.

  It is preferable that the method according to the embodiment of the present invention does not include heating the substrate and the porous film at a temperature of 60 ° C. or more after the time when the porous film is sandwiched between the cuts and pressed. More preferably, the method according to the embodiment of the present invention does not include heating the substrate and the porous membrane at a temperature of 60 ° C. or more after the time of cutting the silicone rubber substrate having the microchannel pattern. It is preferable that all the steps after these points are performed at room temperature. Room temperature is usually 4-36 ° C, preferably 10-30 ° C.

  Hereinafter, embodiments of the present invention will be described more specifically with reference to Examples, but the present invention is not limited to these Examples. Unless otherwise specified, experimental operations in Examples were performed at room temperature.

[Production of microfluidic device with embedded membrane]
Preparation of Mold First, an acrylic plate (Kuralex, Nitto Jushi Kogyo) having a thickness of 1 mm was processed into a rectangular shape having a length of 76 mm and a width of 26 mm using a laser processing machine (HAJIME, O-Laser). The processing parameters were set such that the output was 16%, the cutting speed was 6 mm / s, and the number of cuts was one. The processed rectangular acrylic plate was used as a mold for a lower substrate material having no flow path pattern.

  Next, an acrylic plate (Clarex, Nitto Jushi Kogyo) having a thickness of 0.2 mm was processed into an H-shaped channel pattern. The processing parameters were set to 8% output, 6 mm / s cutting speed, and one cutting. The height of the H-shape was 10 mm, the width of the center horizontal line was 3.0 mm, the width of the left and right vertical lines was 0.6 mm, and the distance between the left and right vertical lines was 4.0 mm. In order to eliminate the warpage of the H-shaped acrylic plate, it was sandwiched between glass slides, placed with a weight of 1 kg, and baked in a table-type blow dryer (MD-100, Yonezawa) (80 ° C., 1 hour). The baked H-shaped acrylic plate was attached to the same rectangular acrylic plate (1 mm thick) as described above using an acrylic adhesive (Acrysandy) and a thermosyringe (1 mL). At that time, first, one drop of the adhesive was dropped on a rectangular acrylic plate using a Nipro brand needle (27G, 02-165, Nipro), and an H-shape was put thereon and fixed. Thereafter, using a Terumo injection needle (27 G, with a blade edge), an adhesive was injected little by little from the tip of the H-shape to completely adhere. Two H-shaped acrylic plates were attached side by side to one rectangular acrylic plate ([H H]). After pasting, it was baked (65 ° C., 30 minutes) in a circulating constant temperature dryer (MOV-102F, Sanyo Electric). This plywood in which an H-shaped acrylic plate was bonded to a rectangular acrylic plate was used as a mold for an upper substrate material having a flow path pattern.

Preparation of Substrate Material The base material and the curing agent of polydimethylsiloxane PDMS (SILPOT 184, Dow Corning Toray) and a curing agent are put into a balance tray (1-52333-01, As One) at a weight ratio of 10: 1, and despet (6-760-). 02, As One). Thereafter, defoaming was performed using a vacuum desiccator (VS, Asone) and a vacuum pump (DAP-6D, ULVAC) for about 20 minutes to prepare a prepolymer. Prepare a 2mm thick acrylic plate with the same dimensions as the mold, and place Bencot (non-woven fabric cut to an appropriate size), the mold made above, and the mold in this order, and clip on all sides. I stopped. A mold having a thickness of 2 mm was laminated on the mold of the upper substrate material, and a mold having a thickness of 4 mm was laminated on the mold of the lower substrate material.

  About 3.2 g of PDMS prepolymer was poured into the mold of the upper substrate material, and about 6.4 g of PDMS prepolymer was poured into the mold of the lower substrate material so as to fill the inside of the mold. Thereafter, degassing was performed in a vacuum desiccator for about 40 minutes. After defoaming, it was placed in a circulating thermostatic dryer and subjected to first baking (65 ° C., 1 hour). An upper substrate material and a lower substrate material each having a length of 74 mm and a width of 24 mm were obtained. Two H-shaped channels are concavely formed on the surface of the former.

Preparation of Substrate (Device Body) The PDMS substrate material after the first baking was removed from the mold / mold, and placed on a slide glass washed with ethanol. From the PDMS substrate material, two 18 × 23 mm rectangles were cut out with a cutter. That is, as for the upper substrate material, two upper substrate materials each having one H-shaped flow path were cut out from the 74 mm × 24 mm substrate material obtained above. Further, as for the upper substrate material, a hole was made to pass through a hole from an upper surface of the substrate material to an end portion of the H-shaped flow path using eyelets, thereby producing a port. The upper substrate and the lower substrate having the dimensions of 18 × 23 mm were overlapped while preventing air from entering, put into a table-type blow dryer, and were bonded by performing a second bake (100 ° C., 30 minutes). . The lower surface (the flow path side) of the upper substrate material and the flat upper surface of the lower substrate material were bonded together to obtain a device body in which an H-shaped flow path was formed. Hereinafter, this device body is referred to as a substrate.

Incorporation of a porous membrane While observing the substrate after the second bake under a microscope, the razor blade is pushed down vertically from the upper surface of the substrate, and the channel corresponding to the horizontal line of the H shape is divided into two channels. Then, a cut having a depth of 3 mm was made. The razor blade separates the flow path corresponding to the H-shaped horizontal line into a flow path having a width of about 150 μm (referred to as an upper flow path) and a flow path having a width of about 2850 μm (referred to as a lower flow path). In the PDMS substrate on the extension of the horizontal line (see FIG. 1a).

  After pulling out the razor blade, the substrate was held in a bent state so that the slit was opened. This was achieved by attaching two acrylic rods to the sample stage and holding the substrate in a state where the cutout was opened, as shown on the right side of FIG. 1b. That is, since the distance between the two acrylic rods is shorter than the length of the substrate, the substrate was fixed in a bridge-like state. With the cut opening thus opened, the substrate together with the sample table was placed in a sample chamber of a vacuum plasma apparatus (CUTE-1MPR, FEMTO SCIENCE) and plasma-treated for 30 seconds (sample chamber capacity: 3.6 L (W150 mm × D200 mm) × H120 mm), source gas: air, pressure setting: 0.5 torr (actual measured value is about 0.65 torr due to inflow of source gas), gas flow rate: 30 sccm, power: 80 W) (FIG. 1c). Separately, a PET porous membrane (pore diameter 3 μm, bottom of cell culture insert (Corning Corp. 353091)) was cut into a size of 2 mm × 7 mm. This porous film is inserted into the cutout of the substrate within 1 minute after the plasma treatment (FIG. 1d). After the cutout is closed, the cut surface is pressed by hand while holding the substrate from the left and right for 10 seconds to sandwich the film. The cut sections of the flexible substrates were joined (FIG. 1e). Thus, a microfluidic device incorporating a membrane was obtained.

[Evaluation of nanoparticle permeability]
200 μL of a 1% collagen I solution (307-31611, Nippon Ham) was added to 200 μL of a 16 mM genipin solution in 2 × phosphate buffered saline (PBS), mixed with a vortex, and allowed to stand in an ice bath. In order to perform a permeation test, this mixed solution was filled in the lower channel of the membrane-incorporated microfluidic device to form a pseudo stroma. The filling was performed according to the following procedure. First, the device was evacuated for over an hour. The temperature control plate (TP-CH110R, Tokai Hit) attached to the stage part of the microscope was set at 4 ° C., and the device was placed thereon. Using a micropipette, 30 μL of the genipin / collagen solution was taken and injected into the device from a port in the lower channel. Thereafter, the device was transferred to an incubator (SCA-80DS, Astec) and warmed (37 ° C., 1 hour) to solidify the genipin-crosslinked collagen gel.

  A permeation test was performed by connecting a syringe pump (LEGATO111, KD Scientific) to the upper channel of the membrane-embedded microfluidic device and flowing a suspension of nanoparticles. In one example, fluorescently labeled dextran (average molecular weight 70000) suspended in PBS was used for testing as model nanoparticles. The mode of the syringe pump was set to “Infuse Only”, the target was set to “No target”, and a micro syringe (1750 TLL, Hamilton) was installed. The flow rate used in the experiment was calculated based on the linear velocity of the capillary. Observed with an inverted microscope (IX71, Olympus) and a CMOS camera (ORCA-Flash 4.0V2, Hamamatsu Photonics), and took a fluorescence image every 10 minutes for one hour after the nanoparticle suspension was filled in the upper channel did. The microscope used a 10 × objective lens, the camera was set to 4 binning, and the exposure time was set to 100 ms.

  FIG. 2 shows an overview of an example of the fabricated membrane-embedded microfluidic device, an enlarged view of the flow channel and the porous membrane portion, and an example of the results of quantifying the membrane permeation of the fluorescently labeled nanoparticles with time. Compared with the conventional manufacturing method in which the porous film is interposed between the cuts of the substrate and baked at about 100 ° C. for about 1 hour, the plasma processing is performed on the substrate while holding the substrate in a bent state so that the cutout is opened. In the manufacturing method of this example, it was recognized that the number of individual devices causing liquid leakage was reduced, that is, the yield was significantly improved. 2 (a) and 2 (b), black arrows indicate insertion positions of the porous membrane, and * and ** indicate two flow paths separated by the porous membrane (upper flow path and lower flow path, respectively). , White arrows indicate fluorescently labeled nanoparticles that have permeated the collagen gel side of the porous membrane. As shown in FIG. 2C, the amount of permeation over time could be accurately measured. Using this system, it is necessary to accurately analyze the effects of various parameters on transmittance, such as the particle size of nanoparticles, the pore size of the porous membrane, the composition of the material that fills each flow path, the composition of the porous membrane surface coating, and the flow rate. Can be.

[Preparation and evaluation of microfluidic device incorporating Matrigel coating film]
In this example, a porous membrane coated with Matrigel was incorporated into a microfluidic device. The production of the mold, the substrate material, and the substrate was performed in substantially the same manner as in the above-described example. However, the bonding of the upper substrate material and the lower substrate material was not performed by the above-described heat treatment, but by performing vacuum plasma processing on the surface to be bonded, and then overlaying the upper and lower substrate materials and pressing them by hand for 10 seconds.

  The porous membrane coated with Matrigel was cut from the bottom of a Matrigel invasion chamber (354481, Corning) with a scalpel and scissors. The formation of cuts in the substrate, the plasma treatment, the insertion of the film, and the like were performed in substantially the same manner as in the above example, to obtain a microfluidic device incorporating a Matrigel-coated film. All of the above processes are performed at room temperature, and since this Matrigel is not exposed to high heat of 60 ° C. or more, the biomolecules in Matrigel are intact.

  The following experiment was performed to confirm the practicality. That is, PBS was introduced into one of the two channels separated by the membrane. A solution of fluorescently labeled dextran (average molecular weight 10,000) was sent to the other flow path at a flow rate of 1 μL / min using a syringe pump. Observation with a fluorescent microscope over time confirmed that dextran permeated through the membrane and diffused into the other flow path. It was shown that there was no liquid leakage from the channel and the permeability of the nanoparticles could be evaluated with this device.

  Embodiments of the present invention can be used in the fields of medical / biological / pharmaceutical research devices, microreactors, and drug delivery system development. Embodiments of the present invention can be utilized for the construction of various other biological model systems as well as vascular permeability.

Claims (6)

A step of making a cut in a silicone rubber substrate having a microchannel pattern,
Performing a plasma treatment or a corona discharge treatment on the substrate while holding the substrate in a bent state so that the slit of the slit is opened;
A method of sandwiching a porous membrane in the cut and pressing the cut surface in pressure.   The method of claim 1, wherein the silicone rubber comprises polydimethylsiloxane.   3. The method according to claim 1 or 2, wherein no additional silicone or prepolymer thereof is included in the cut during the crimping.   4. The porous membrane according to any one of claims 1 to 3, wherein only the membrane surface of the porous membrane is exposed to the space in the flow path, and the membrane edge is not exposed to the space in the flow path, and the porous membrane is sandwiched between the cuts. The method described in.   The method according to any one of claims 1 to 4, further comprising a step of preparing a silicone rubber substrate having the microchannel pattern before the step of cutting.   The step of fabricating the silicone rubber substrate having the micro-channel pattern includes overlapping two silicone rubber substrate materials and joining them by heating, and at least one of the two silicone rubber substrate materials. Has a flow path molded concavely on the surface, and the surface having the flow path faces the surface of the other silicone rubber substrate material to form a micro flow path pattern. Method.