JP2018130676A - Microfluidic transportation structure where through hole and flow channel are integrated, and method for manufacturing the same - Google Patents

Abstract

A microfluidic transport structure including a flow path for efficiently transporting a liquid containing solid particles such as a single cell, a through-hole having a diameter and a depth suitable for dropping them, and a method for manufacturing the same I will provide a. A flow path bottom made of a highly rigid material is provided with a plurality of through holes 31 having a ratio of the depth dimension to the diameter dimension (aspect ratio) of 1.0 or less. The part is provided with a movable film in the vicinity of the through hole at the lower part of the flow path, and has a valve function for opening and closing the through hole when the movable film is expanded by air pressure. [Selection] Figure 2

Description

  The present invention relates to a transport structure having a through hole for transporting a liquid containing solid particles such as cells and dropping it at a predetermined position, and a method for manufacturing the transport structure.

  In the life science and chemistry fields, research and development using micro-TAS (micro total analysis system) and a device incorporating a minute flow path and a reactor are actively performed. In research and development using μTAS, an attempt is made to create a structure in which microchannels and reactors are combined in a complex manner, and to mix, synthesize, and react a solution containing solid particles such as liquid and cells. Particularly in the biochip research field, recently, MEMS (Micro Electro Mechanical Systems) micro cell sorting and cell function evaluation technologies have been actively developed as methods for isolating rare stem cells in regenerative medical technology using stem cells. Has been.

  Cells are fundamental components of living organisms, and elucidation of their functions is an issue. A tissue in a multicellular organism has a three-dimensional structure in which multiple types of cells are assembled, and each cell changes its function by interacting with surrounding cells. In a living body, a plurality of types of cells take a specific arrangement state, and individual cell functions are regulated by interaction. The cell-cell interaction refers to physical / physiological communication that occurs between two or more cells, and includes a mechanism through a signaling substance between cells and a mechanism in which cells directly contact each other. Reconstructing a specific arrangement of cells found in vivo in an in vitro experimental environment and analyzing cell functions greatly advance medical and biological research. If the 3D tissue can be reconstructed outside the body, the phenomenon can be analyzed with high efficiency. Reconstruction at the single cell level requires a system that assembles single cells precisely in three dimensions.

  In addition, when performing drug tests using cells, toxicity tests, gene injections, and the like, it is desired to arrange a large number of cells and continuously observe them while identifying each individual. Currently, micromachining technology is widely applied to elucidation of biological mechanisms, measurement and treatment for medical treatment, and manipulation at the cell and gene level.

  μTAS performs high-precision microfabrication on a substrate such as silicon or glass, and microfabricates various elements such as mixing, reaction, detection and separation / recovery that are unit operations of chemical synthesis and chemical analysis on the substrate. An integrated one is common.

  As an example of μTAS, Patent Document 1 discloses a plurality of sample inflow portions into which a sample fluid flows, a sample outflow portion from which the sample fluid flows out, and a plurality of sample flow paths that connect the sample inflow portion and the sample outflow portion. A lab-on-a-chip technique is disclosed that includes a belt-like sheet in which a flow-through cell is disposed.

  In the case of such μTAS, a silicon wafer, glass, quartz or the like is often used as a chemically stable substrate. However, in order to form a flow path or the like on these substrates, a rigid and hard substrate surface is highly accurate. Therefore, it is difficult to manufacture. For this reason, Patent Document 1 describes that a resin that is easily processed is preferable as the substrate. Furthermore, there is a description that when an organic solvent is used in the flow path, it is desirable to use a highly rigid material crosslinked with an inorganic material or an organic material. In addition, a method for forming a hydroxy group or the like by plasma treatment or the like is disclosed for a portion to be given lipophilicity or hydrophilicity.

In Patent Document 2, a groove is provided for the purpose of providing a practical μTAS that is easy to manufacture, inexpensive, and suitable for mass production as compared to a conventional μTAS having a substrate such as silicon wafer, glass, or quartz. Discloses that a groove can be easily formed by stamping or injection molding of a plastic substrate having a surface having a channel-like shape. The plastic substrate uses PDMS (Polydimethylsiloxane), which is one of the photo-curing resins, and can transfer patterns on the sub-micron order onto the plastic substrate by embossing, etc. It is said that an excellent μTAS can be realized. When such a plastic substrate is made of PMMA, a pattern can be easily formed on the surface of the plastic substrate with photolithography. In these Patent Documents 1 and 2, a technique for integrally forming the flow path and the hole is not disclosed, and the flow path is made of a highly rigid material. In the processing of Pyrex (registered trademark) glass, holes can be formed by anisotropic etching using SF ₆ plasma. (Non-Patent Document 1)

  The μTAS realizes the function of transporting the liquid to the reactor or dropping the liquid at a predetermined position. However, the diameter of the joint between the hose for actually flowing the liquid into the μTAS and the inlet of the μTAS is several. It is very small, about 100 μm to several mm, and is extremely difficult and has poor reliability. Normally, when connecting the liquid inlet of the equipment and the hose, there is a wide range of methods in which a bamboo shoot-like joint is provided on the equipment side, the hose is inserted so that the inside of the hose is in contact with the joint, and the hose is tightened with a band fitting. Yes. Such a connection method has not been established.

  As a method for transporting a liquid containing cells by μTAS, a structure for arranging a cell trap in a flow path and arranging cells is tried, and it can be arranged in a relatively short time. Since density is required, there is a drawback that it is impossible to evaluate the essential behavior of single cells. In addition, attempts have been made to arrange cells using dielectrophoresis, but there is a drawback that denaturation occurs when a cell suspension touches an electrode, resulting in damage to the cells. (Non-Patent Document 2)

  Until now, cell patterning by an inkjet method (Non-patent Document 3) in which single cells are discharged from a nozzle has been reported. In this method, the inside of a single nozzle is optically observed, and each nozzle is provided with a detection system and a signal processing unit, so that the structure is complicated. Since the number of detection systems and the number of signal processing units are proportional to the number of nozzles, it is difficult to improve throughput. In order to increase the number of cells and build a tissue level at a high speed, a plurality of nozzles are connected in an array. However, it is necessary to prepare a detection system and a signal processing unit in proportion to the number of nozzles. As a simple method of dropping cells at a predetermined position, a nozzle that projects the flow channel and cells is designed using a hydrodynamic design method based on flow channel resistance without detecting the presence of the cell. However, it is not possible to manufacture a microfluidic transport structure using a manufacturing method that faithfully realizes the above and to automatically arrange cells.

JP 2004-156925 A JP 2006-153823 A

Xinghua Li, Takashi Abe, Yongxun Liu, and Masayoshi Esashi, Fabrication of High-Density Electrical Feed-Throughs by Deep-Reactive-Ion Etching of Pyrex Glass, Journal of Microelectromechanical Systems Volume: 11, Issue: 6, (2002) 625- 630. Chih-Hao Wang, Gwo-Bin Lee, Pneumatically driven peristaltic micropumps utilizing serpentine-shape channels, J. Micromech. Microeng, 16 (2006) 341-348. Azmi Yusof, Helen Keegan, Cathy D. Spillane, Orla M. Sheils, Cara M. Martin, John J. O'Leary, Roland Zengerlead and Peter Koltay, Inkjet-like printing of single-cells, Lab Chip, 11, (2011 ) 2447-2454. Shinji Sugiura, Tatsuya Oda, Yasuyuki Aoyagi, Mitsuo Satake, Nobuhiro ohkohchi, mitsutoshi Nakajima, Tubular gel fabrication and cell encapsulation in laminar flow stream formed by microfabricated nozzle arrey, The Royal Society of Chemistry 2008

  However, the transparent substrate used for such μTAS is generally a silicon substrate, and the substrate thickness is about 400 μm and the through-hole diameter is only about 60 μm (Non-Patent Document 4). It was difficult to form a flow path and a through-hole suitable for the above. In addition, the limit of the ratio of the depth dimension to the diameter dimension of the through hole for dropping cells (aspect ratio) can only be realized from 5 to 7. If you try to reduce the depth of the through hole by reducing the thickness of the substrate to lower the fluid resistance, the rigidity of the substrate will decrease, so it will be easily destroyed at the gas-liquid interface during handling and wet etching. It becomes easy. When glass is melted by wet etching, it becomes isotropic etching, so it is difficult to make the aspect ratio smaller than 1. Further, it takes a long time of 10 hours to form a hole having a hole diameter of 50 μm and a depth of 150 μm, and the productivity is extremely poor and the practicality is poor.

  In addition to such poor productivity, a nozzle with a small channel resistance and an optimum hole diameter for dropping single cells has not been realized, and a microfluidic transport structure that handles single cells has not yet been provided. .

  In addition, the structure including the flow path constituting the μTAS is made of a highly rigid material, and a complicated mechanism is required to control the protrusion of the droplet from the nozzle, and the miniaturization cannot be realized. .

  The present invention has been made in view of the above-mentioned problems, and the object of the present invention is to provide a channel for efficiently transporting a liquid containing a microfluid, such as a single cell, and a diameter suitable for dropping. It is an object of the present invention to provide a microfluidic transport structure including through holes having a depth and a method for manufacturing the same.

  In order to achieve this object, a microfluidic transport structure obtained by integrating the lower structure and the upper structure according to claim 1, wherein the lower structure has a side wall for forming a liquid transport channel. A plurality of through-holes having a ratio of the depth dimension to the diameter dimension (aspect ratio) of 1.0 or less, and the upper structure is stacked on the lower structure. A microfluidic transport structure having an upper surface portion for forming the liquid transport channel.

  3. The lower structure according to claim 2, wherein the lower structure is made of a photocurable resin, is formed on a surface of a sacrificial layer formed on a substrate, and is formed by a remaining part from which the sacrificial layer is removed. The microfluidic transport structure according to claim 1, wherein:

  The upper structure according to claim 3 includes a partition wall that is appropriately spaced from an upper surface portion, and a liquid transport channel is formed between the partition wall and the lower structure, and the partition wall and the upper surface portion An air pressure flow channel for allowing pressurized air to flow in between is formed, the partition wall is a movable film formed of an elastic material, and the movable film is divided into a movable area and a fixed area, and is movable. The region is deformable in an appropriate range facing each of the plurality of through holes of the lower structure, and the fixed region is supported by the upper surface portion in a range excluding the movable region. A microfluidic transport structure according to claim 1 or 2.

  The movable region according to claim 4 is a circular region formed in a circular range, and the circular region has a circular shape centering around an extension line of a central axis of a plurality of through holes of the lower structure. The macrofluid transport structure according to claim 3, wherein the macrofluid transport structure is formed.

  5. The microfluidic transport structure according to claim 4, wherein the circular region according to claim 5 is formed in a circular shape centering on a position deviated from an extension line of the central axis of the through hole.

  The manufacturing method according to claim 6 is the manufacturing method of the microfluidic transport structure according to claim 1 or 2, and includes an upper structure forming step, a lower structure forming step, and an integration step, The upper structure forming step includes a mold forming step of forming a mold die having a shape that matches a liquid transport channel planned area on the substrate, and an upper constituent material filling that fills the mold die with the upper constituent material. A step of releasing the mold from the mold after curing the upper constituent material, and a step of punching a part of the molded product that has been released, and the lower structure forming step includes: A sacrificial layer forming step of forming a sacrificial layer on the substrate, and a channel bottom surface forming step of patterning a lower constituent material in the liquid transport channel forming region except for a portion corresponding to the through hole on the surface of the sacrificial film And removing the sacrificial layer A separation step of separating the substrate and the lower constituent material, and the integration step includes a structure formed by the upper structure forming step, and a structure formed by the lower structure forming step. Is a method of manufacturing a microfluidic transport structure, which includes a pasting step in which the respective liquid transport flow path scheduled regions and the liquid transport flow path formation regions are bonded together.

  A manufacturing method according to claim 7 is the manufacturing method of the microfluidic transport structure according to any one of claims 3 to 5, wherein the upper structure forming step, the lower structure forming step, and the integrating step And the upper structure forming step includes an air pressure channel forming step, a movable film forming step, a laminating step, and a liquid supply hole forming step, and the air pressure channel forming step is performed on the substrate. A mold forming step for forming a mold having a shape that matches the planned flow area for air pressure, an upper constituent material filling step for filling the mold with the upper constituent material, and curing the upper constituent material A mold release step for releasing the mold from the mold, and a first punching step for punching a part of the molded product that has been released, wherein the movable film forming step is performed by using an elastic material on the substrate. Includes an elastic material film forming process to form a predetermined layer The laminating step includes a first laminating step of laminating the structure formed by the pneumatic flow path forming step on the surface of the movable film scheduled layer formed by the elastic material film forming step, and the liquid The supply hole forming step includes a second punching step of punching a part of a region where the structure formed by the pneumatic flow path forming step and the movable film planned layer are laminated, and forming the lower structure The process includes a sacrificial layer forming process for forming a sacrificial layer on the substrate, and a flow path for patterning the lower constituent material in the liquid transport flow path forming region except for a portion corresponding to the through hole on the surface of the sacrificial layer. And a separation step of separating the substrate and the lower constituent material by removing the sacrificial layer, and the integration step includes a structure formed by the upper structure forming step, and the lower portion Structure formation A microfluidic transport characterized in that it includes a second laminating step in which the structure formed by the process is pasted in a state where the respective liquid transport channel planned region and the liquid transport channel forming region match. It is a manufacturing method of a structure.

  The microfluidic transport structure of the present invention has a flow path capable of transporting a microfluid, for example, a liquid containing solid particles such as a single cell, and a penetration having an aspect ratio of 1.0 or less indicating the ratio of the diameter dimension to the depth dimension. Since it is comprised with a hole, there exists an effect that a micro liquid can be dripped at a predetermined position accurately.

These are basic cross-sectional schematic diagrams of a microfluidic transport structure. FIG. 5 is a schematic cross-sectional view of a microfluidic transport structure having a pneumatic flow path and a valve added thereto. These are the cross-sectional schematic diagrams which show the principle of the cell protrusion of the single cell inkjet. These are the schematic diagrams of the nozzle array provided with 16 holes and nozzles as an example as a model for demonstrating the design method of a microfluidic transport structure. FIG. 5 is a perspective view of the nozzle array of FIG. 4. Fig. 4 is an equivalent circuit of a nozzle array configured by connecting four nozzles in series in Fig. 4. These are the equivalent circuits of the nozzle part of a microfluidic transport structure. These are the equivalent circuits in the bubble cross section of a microfluidic transport structure. Is a manufacturing process of the microfluidic transport structure. Is the relationship between the spin coat rotation speed and the film thickness in the step of producing the movable film at the bottom of the flow path. These are photographs observed from the upper surface of the nozzle array produced in the experiment. FIG. 12 is a cross-sectional observation photograph of the nozzle array indicated by A-A ′ shown in FIG. 11. These are observation photographs from the upper surface of the nozzle array before and after the valve operation experiment.

  Hereinafter, embodiments of the present invention will be described in detail. FIG. 1 is a basic cross-sectional schematic diagram of a microfluidic transport structure 1. Although three through holes 31 are provided in a part of the flow path in this figure, it is only an example, and it is needless to say that it can be appropriately changed depending on the application.

  The microfluidic transport structure 1 of this embodiment is a structure in which a lower structure 3 made of a highly rigid material and an upper structure 2 having an upper surface portion are joined. A groove 32 is provided at a predetermined position in the lower structure, and a plurality of through holes 31 for dropping liquid and cells are provided at the bottom of the groove of the lower structure. By joining the upper structure and the lower structure, it becomes a cover of the groove of the lower structure, and a flow path having both ends opened can be formed, and both ends of the formed flow path 32 are liquid inlets / outlets 21. The entrance / exit 21 made of an elastic body allows a liquid to flow in by inserting a bamboo shoot-like joint attached to the tip of the hose.

  FIG. 2 shows a form in which the upper structure 2 of the microfluidic transport structure 1 of FIG. 1 is partially deformed. In this embodiment, a portion corresponding to the vicinity of the upper portion of the through hole 31 of the lower structure 3 is shown. A partition wall 24 that exhibits a valve function and an air pressure channel 25 having a space 23 disposed above the partition wall 24 are provided. Since the partition wall is partially or entirely made of an elastic body, it acts as a movable film. In FIG. 2, the partial region of the partition wall 24 functioning as a movable film is shown as an example of a circular shape centered on the center line with respect to the through hole (circular hole) 31. The range or shape can be changed as appropriate. The air flow path provided in the upper structure is provided with a pressurized air inlet 22 at one end. In this figure, there is a structure in which three through holes are provided in a part of the flow path, and a pneumatic film having a movable membrane and a space arranged near the upper part of the through hole is provided. Needless to say, it can be appropriately changed depending on the application.

  Although FIG. 1 and FIG. 2 differ in the structure of an upper structure, since others are the same structures, the common part is demonstrated in detail first.

  The elastic body of the upper structure 2 is an organic material. Specifically, examples of the organic material having elasticity include silicone elastomer, PDMS (polydimethylsiloxane), polyurethane, thermosetting polyester, and silicone hydrogel. When observing the movement of droplets and cells, PDMS which is a transparent material is suitable.

  The lower structure 3 is a photo-curing resin, and both a negative-type photo-curing resin capable of patterning on a light-irradiated portion and a positive-type photo-curing resin capable of patterning on an unexposed portion can be used. It can be formed on a thick film of the order, and has the property of forming a multilayer structure on the top. Specifically, examples of the photo-curable resin include epoxy-type chemically sensitized and thick-film negative resists, polyimide resists, acrylic resists, and novolak resists.

  The cross-sectional shape of the flow path formed by joining the upper structure and the lower structure may be a rectangle, a triangle, a trapezoid, or a parallelogram. The length, width, and height of the channel can be arbitrarily determined, and the channel resistance when a single cell flows is determined by these design parameters.

  The dimensions of the flow path of the microfluidic transport structure according to the present embodiment are designed according to the type of solid particles such as cells contained in the microfluid flowing in the flow path, but the range is 5 to 300 μm in height and 10 to 1,000 μm in width. It is. In the case of flowing a solution containing a single cell coated with a monomer, the height is preferably 30 μm and the width is preferably 50 μm in consideration of the size of the single cell. In order to allow cells to flow efficiently, it is necessary to design the flow path of the nozzle array, which will be described later, based on the flow resistance of each part, and in order to allow cells to protrude from the nozzle, the flow resistance of the nozzle part is It is necessary to make it lower than the resistance of other flow paths. The flow path resistance of the through holes constituting the nozzle is determined by the hole diameter and the thickness of the lower structure of the hole. The flow path for transporting cells is desirably large to prevent blockage by the cells, but on the other hand, in order to improve controllability of cell transport, it is desirable to make the flow path close to the size of the cells and coat with monomer. For transporting single cells, the width is about 50 μm and the height is about 30 μm. The nozzle's through-hole minimizes dead volume (space not related to the transport function) with a diameter suitable for the size of the coated cell in order to accurately drop the coated single cell at a predetermined position. That is good. In order to make the flow resistance of the nozzle lower than the flow resistance of the other flow paths and drop the cells to a predetermined position with high precision, the aspect ratio of the through hole of the nozzle is 1.0 or less, and the diameter of the hole is It is preferable that the particle size of the cell to be handled is slightly larger than the particle size coated with the monomer. Preferably, the aspect ratio is 0.5, the diameter of the through hole is 40 μm, and the hole depth is 20 μm. If the aspect ratio is around 1.0, it is within the range of the present embodiment.

  A structure having a valve function provided in a part of the upper structure will be described with reference to FIG. A space 23 is provided in the vicinity of the partition wall 24 in the bottom portion of the upper structure near the upper portion of the through hole 31 provided in the lower structure, and an air pressure channel 25 and an air inlet 22 for sending pressurized air into the space are provided. Provide. The partition wall 24 at the bottom of the space 23 provided in the upper structure is an elastic body, functions as a movable film, and has a thickness that can be deformed so that a valve function can be exhibited by predetermined pressurized air. The thickness of the movable film 24 may be the same as the thickness of the pneumatic flow path portion of the upper structure, but it is preferably made thinner to increase the movable response and to obtain a predetermined bulge with low pressure. Is preferable. Since the movable film 24 is deformed from other portions supported by the wall when pressurized with air from the inlet 22, it can function as a valve for the through hole. The elastic modulus of the upper structure is appropriately selected according to the application depending on the structure holding strength as the structure and the operation performance as the film for the valve operation. When dripping cells, in order to move the cells along the flow path 4 along with the liquid toward the through hole, the liquid containing the cells needs to flow, so the valve is not completely closed. The opening of the movable membrane is controlled so as not to pass the cells, and the swelling of the movable membrane is controlled so that the liquid flows through the through hole 31 to the outside. The lowest point of deformation of the movable film 24 when operated as a valve may coincide with the center line of the through-hole 31 of the lower structure, but preferably prevents the cell from dripping and allows only liquid to flow. The lowermost point of the bulge of the movable film 24 is displaced from the center of the through hole so that the bulge of the movable film 24 comes into contact with the upper part of the through hole. Because it hits the lower structure, it is easy to control.

  The operation of the single cell printer using the microfluidic transport structure of this embodiment will be described in detail.

  FIG. 3 is a conceptual diagram of a protruding operation of a single cell covered by a single cell printer using a microfluidic transport structure. The single cell printer has a structure part for coating a single cell with a monomer, a valve part having a movable membrane 24 having a valve function for moving the coated single cell to a nozzle part at a predetermined time, and one cell at a time. A unit structure constituted by a nozzle portion provided with a through-hole 31 for projecting is formed by a nozzle array arranged in an array. However, FIG. 3 does not show a structure for coating a single cell with a monomer.

  First, a liquid in which a single cell and a photocurable monomer are mixed is poured into the channel to cover the single cell. Next, as shown in the valve closed state in the figure, the coated cells are blocked to the extent that the cells are trapped by the expansion of the movable membrane 24 of the upper structure as indicated by the white arrow shown at the left end in the figure. It moves according to the flow of the liquid to the valve, and the cell is trapped. In the figure, the flow of liquid is indicated by white arrows. Thereafter, the pressure in the space 23 is reduced at a predetermined timing, and the movable membrane 24 returns to its original position, so that the coated cells protrude outside through the through holes 31 that are opened as valves and provided in the flow paths. As shown in the drawing, the center of the movable film 24 is provided at a position deviated from the center line of the through hole 31. As a result, the pressure of the space 23 is increased to enter the flow path (to the through hole 31). In a bulged state (valve “closed” state), even when a part of the movable film 24 is in contact with a part of the edge of the through hole 31, a predetermined gap is formed between the edge of the opposite side. A range of clearances can be formed. Thereby, even if the valve is in the “closed” state, only the liquid can flow downward from the through hole 31. When the center of the movable film 24 is provided on the center line of the through hole 31, the pressure of the space 23 is controlled, and a predetermined clearance is formed between the edge of the through hole 31. May be.

  Next, a method for designing the flow path of the nozzle array will be described in detail. In FIG. 4, four through-holes 31 of the microfluidic transport structure of this embodiment are connected in series by a bypass channel 5 to form one set, and four sets are connected in parallel and arranged in an array. Nozzle array 6 has a liquid inlet and the other end 7 has an outlet. The liquid flows from the inlet 6 toward the lower side of the paper surface. FIG. 5 is a perspective view of this array. A portion surrounded by a broken line in the plan view of FIG. 4 is a range in which the flow channel model of FIG. 6 is replaced with an equivalent circuit. However, the flow path leading to the outlet 7 was omitted. The array structures in FIGS. 4 and 5 are merely examples, and it goes without saying that they can be changed as appropriate according to the application.

The flow rate Q [m ³ / s] in the microchannel is obtained by Equation 1.

Δp [Pa] is a pressure difference, and R [Pa · s · m ⁻³ ] is a channel resistance. Also, according to Hagen-Poiseuille's law, Equation 2 is established.

Here, η [Pa · s] is the viscosity, L [m] is the channel length, and γ _H [m] is the hydraulic radius of the channel. In addition, since the design flow path of a present Example has a rectangular flow path shape, hydraulic radius (gamma) _H was computed from several.

Here, De [m] is an equivalent diameter, A _f [m] is a channel cross-sectional area, and W _p [m] is a channel cross-sectional perimeter. From Equations 1 and 2, the channel resistance can be calculated by Equation 4.

The flow path resistance of the nozzle array can be calculated as an equivalent circuit using the fluid resistance of each flow path. The equivalent channel resistance when N channels are arranged in series is R _{H, eq} = R _{H, 1} + R _{H, 2} +... + R _{H, N.} When arranged in parallel, 1 / R _{H, eq} = 1 / R _{H, 1} + 1 / R _{H, 2} +... + 1 / R _{H, N.} Even in the case of a channel network combining series and parallel, the combined channel resistance can be calculated using the above formula. Therefore, since the flow rate ratio is determined from the channel resistance determined only by the channel size, each channel resistance can be obtained to determine the channel size.

  FIG. 6 is a plan view of a flow path model equivalent circuit used for explaining the nozzle array design method. The equivalent circuit channel model of FIG. 6 is an equivalent circuit of a portion 8 surrounded by a broken line on the plan view of FIG. In the figure, R is the channel resistance, and the subscript numerals attached to each indicate the channel number. When pressure is applied to the diaphragm forming the valve and the channel is closed due to the blockage, the flow rate in the direction of the outlet 7 is faster than the bypass channel 5 connected to the valve. A branched flow path reaching the through hole 31 is designed. As a method of capturing cells, a method of trapping by controlling the flow rate ratio was adopted. The flow rate ratio is designed from the ratio of flow path resistance determined only by the flow path dimensions. The flow rate to the nozzle through-hole is designed to be larger than the flow rate toward the outlet, in other words, the fluid resistance to the nozzle hole is smaller than the fluid resistance to the outlet.

Next, the same flow path model was replaced with an equivalent circuit for calculation. R shown in FIG. 6 is a flow path resistance, which is a normal resistance in the flow path of the equivalent circuit of the flow path model. In addition, it is assumed that R _t1 = R _t2 = R _t3 = 0 in an equivalent circuit when cells are trapped. FIG. 7 is a flow path equivalent circuit near the nozzle. The white arrow in the figure is the flow rate. Design the flow path so that the flow resistance R _t4 going to the nozzle hole is smaller than the bypass flow resistance R _Bypass so that the flow _quantity Q _t4 going to the nozzle hole is larger than the flow _quantity Q _Bypass of the bypass flow path. I do.

From this, if subscripts are added to L and r, and the flow path length and hydraulic radius of each flow path are given, they can be rewritten as shown in Equation 5.

FIG. 8 is an equivalent circuit obtained by subdividing the resistance _Rt4 from the bypass flow path to the through hole 31 of the valve, and the entire flow path resistance can be expressed by Equation 6. Finally, the flow path is designed to satisfy Equation 7. In addition, the flow path length and radius are expressed as shown in Equation 8.

Next, the manufacturing method of the microfluidic transport structure according to this embodiment will be described in detail.

  FIG. 9 is a schematic view showing a nozzle array manufacturing process. The process will be described in detail with reference to FIG. In addition, the following embodiment is only an example which actualized this invention, and it cannot be overemphasized that this embodiment can be changed suitably in the range which does not change the main point of this invention.

  Step 1 / Upper Structure Manufacturing Process: The surface of the substrate 10 for forming the upper structure 2 of the microfluidic transport structure according to the present embodiment is washed with acetone, and subsequently replaced and rinsed with isopropanol (IPA). . The substrate is flat and any material can be used as long as no deformation or distortion occurs during the process. Specifically, such substrates include Si wafers, glass wafers, quartz wafers, SiC (silicon carbide) wafers, sapphire wafers, compound semiconductor wafers such as GaP (gallium phosphide) wafers, GaAs (gallium arsenide) wafers, and InP (phosphorization). Indium) wafers and GaN (gallium nitride) wafers can be used. Further, a film substrate can be used for continuous production, and specifically, a heat-resistant film such as polyimide or PET can be used. Further, a substrate having a metal or organic material thin film on its surface can be used as well.

  Next, a photoresist is applied to the substrate surface with a uniform film thickness by a technique such as spin coating. Thereafter, predetermined flow paths and structures are formed on a mask used in the semiconductor manufacturing process, and a photoresist film is patterned into a necessary shape by photolithography to form a mold 12 for the upper structure. In addition to patterning the photoresist film by photolithography, a mold can be produced by machining.

  Next, in order to make an upper structure, the main component and the curing agent of the organic material are weighed at a predetermined ratio, and stirred and mixed while removing bubbles in the liquid with a vacuum stirring and defoaming mixer. The ratio of base agent to curing agent can be selected to change the rate of crosslinking. The stirred organic material is poured into a flow path mold, and further put into a vacuum chamber to remove bubbles in the resin material. Then, it hardens | cures at the temperature and time matched with the hardening conditions of the organic material. After curing, the upper structure is removed from the mold and, if necessary, a predetermined hole is made at a predetermined position corresponding to the inlet using a punch, thereby completing one part 2a of the upper structure.

  Process 2 / Valve Manufacturing Process: Process 2 of the present embodiment is a process of manufacturing an upper structure provided with a pneumatic flow path and a valve. This step is omitted when the upper structure of the present embodiment has no valve. As in the production of step 1, the same organic material main component and curing agent are mixed at a predetermined ratio while vacuum degassing, and then coated on the substrate 10 with a spin coater. The substrate used here is one that can be easily released from the organic material used for the upper structure, and preferably a Si wafer substrate. Thereafter, the film 2b is obtained by curing under predetermined curing conditions. FIG. 10 shows, as an example, changes in the PDMS film thickness when the rotational speed is changed when a spin coater is applied for 60 seconds when PDMS is used as the organic material. Moving performance changes by changing the film thickness, and the pressure and responsiveness of valve operation can be changed.

  Next, a film is formed by spin coating on the part 2a and the substrate of the upper structure produced in step 1, and then the cured film 2b is placed in a plasma processing apparatus and subjected to plasma processing in an air atmosphere. A silicone resin material can be used for the upper structure, and the surface can be excited even by an atmospheric pressure plasma or vacuum ultraviolet light irradiation apparatus. Then, both structures are bonded together and joined by heating. Then, the upper structure 2 provided with the air flow path and the movable film is obtained by mechanically removing the substrate. For the bonding, other bonding methods such as an adhesive can be used as appropriate.

  Step 3 / Process for Producing Lower Structure: Similar to the upper structure, the same substrate 10 is used when producing the lower structure. In order to release the fabricated lower structure from the substrate without applying any mechanical stress, the sacrificial layer 11 is formed on the substrate in advance. If the lower structure is fabricated directly on the substrate and mechanically peeled off, the bottom having a thickness of only about 20 μm is damaged, so that the bottom needs mechanical strength and has a limit on the thickness. In this embodiment, in order to separate the lower structure from the substrate without applying mechanical stress, a sacrificial layer that dissolves in a solvent is formed on the substrate in advance. The solvent dissolves only the sacrificial layer without chemically reacting with the lower structure. In the selection of the material of the sacrificial layer corresponding to the organic material of the substructure of the present embodiment, selected from photosensitive resin, polyvinyl alcohol, starch, dextrin, amylose, gelatin, agar, carrageenan, pectin, or roast bean gum. One or more organic materials can be used. As such a sacrificial layer, a lift-off resist made of polyaliphatic imide copolymer or polydimethylglutarimide which can be dissolved in an alkaline solution can also be used. Such a sacrificial layer can be formed by spin coating on a substrate surface obtained by plasma treatment of these solutions. As the sacrificial layer, a thin film of silicon dioxide, spin-on-glass, or metal (aluminum, chromium, gold, silver, etc.) formed on the substrate can also be used. The film forming method at this time may be performed by a conventionally known method such as coating by spin coater, slit coater, spraying or the like, or vapor deposition by physical vapor deposition (PVD) or chemical vapor deposition (CVD) depending on the material of the thin film. it can. The film thickness of the thin film is appropriately set depending on the shape of the flow path to be produced.

  A photoresist is applied onto the deposited sacrificial layer 11 by a spin coat method, and through holes are formed in the photoresist layer by photolithography using a mask having a pattern of through holes. Next, the same process is repeated on this layer, and the bottom and walls of the flow path are formed in the photoresist layer by photolithography using a mask having a patterned flow path shape. The depth and structure of the flow path can be controlled by changing the photoresist application conditions and the number of times.

Step 4: Joining of the upper structure and the lower structure: The prepared upper structure 2 and lower structure 3 are placed in a plasma processing apparatus and subjected to N ₂ plasma treatment. Similar results can be obtained with the plasma processing equipment in three types of discharge: direct current, alternating current, and high frequency. In addition, when PDMS is used as the material of the upper structure, the same effect can be obtained even if a silane coupling agent modified with an amino group is used for the surface treatment. When the photo-curable material is changed, a strong bonding with the silicone resin material can be obtained by using a silane coupling agent that is modified with an amino group. Thereafter, the joining positions of the components are aligned using a stereomicroscope, the upper structure and the lower structure are bonded together, and then joined by heating. Heating is used to shorten the time, but bonding can be realized at room temperature to 300 ° C., which is a temperature range in which the material does not soften. It goes without saying that the method of joining the upper structure and the lower structure differs depending on the selection of the material.

  Step 5 / Removal of Substrate: The microfluid transport structure 1 is obtained by immersing in the solvent of the sacrificial layer 11 and dissolving the sacrificial layer to separate the joined body from the substrate. When the above-described sacrificial layer is formed using an organic material, the microfluidic transport structure 1 is obtained by dissolving using a solvent having a re-dissolvable temperature and peeling from the substrate in this peeling step. Except for this embodiment, the solvent can be selected and dissolved in accordance with the material of the sacrificial layer to peel the structure from the substrate. In addition, by performing ultrasonic waves or solution agitation, the contact amount of the solution can be increased, dissolution of the sacrificial layer can be promoted, and peeling can be performed in a short time.

  The manufacturing method of the microfluidic transport structure is also within the scope of the present invention.

  Hereinafter, experimental examples for verifying the effects of the manufacturing method of the present invention will be described.

The design values of the nozzle array produced in the experiment were set as shown in Table 1. As a result of calculating the right side of Equation 7, it was 0.55, and a design satisfying Equation 7 could be performed.

  The manufacturing conditions of the nozzle array manufactured based on the manufacturing method of this embodiment will be described for each process.

Step 1 / Manufacturing process of superstructure: The surface of a Si wafer (3 inches) used as a substrate was washed with acetone, and subsequently replaced and rinsed with isopropanol (IPA). Next, photoresist SU-8 3050 was applied to the substrate surface with a uniform film thickness by spin coating. Thereafter, a predetermined flow path was formed in the photomask, and the photoresist film was etched into a flow path shape by photolithography to produce a flow path mold. Table 2 shows the detailed process conditions.

  Next, PDMS (Silpot 184, manufactured by Toray Dow Goning Co., Ltd.) is used as the material of the upper structure, the main agent and the curing agent are weighed at a ratio of 10: 1, and bubbles in the liquid are removed by a vacuum stirring deaerator mixer. While stirring, the mixture was mixed. After stirring, PDMS was poured into a flow path mold, and further put in a vacuum chamber to remove bubbles in PDMS. Thereafter, it is allowed to stand at room temperature for 24 hours to be cured. After curing, the PDMS was removed from the mold of the flow path, and a hole with a diameter of 0.5 mm was formed using a punch at a predetermined position corresponding to the inlet.

  Step 2 / Manufacturing process of the valve: As in the manufacture of Step 1, the main component of PDMS and the curing agent are mixed at a ratio of 10: 1 while vacuum degassing, and then PDMS is coated on the Si wafer with a spin coater. . The operating condition of the spin coater was set to slope (5 s) → 500 rpm (20 s) → slope (5 s) → 3000 rpm (60 s) → slope (10 s)). Thereafter, it was cured by heating at 80 ° C. for 40 min on a hot plate.

  The parts produced in step 1 are joined to the PDMS film on the Si wafer. These are placed in a plasma processing apparatus and treated with an air plasma containing oxygen for 45 seconds to activate the surface. Then, both were bonded together and joined by heating at 80 ° C. for 15 minutes on a hot plate.

  Step 3 / Substructure manufacturing step: Dextran (Dextran molecular weight 60,000 manufactured by Wako Pure Chemical Industries, Ltd.) is selected as the material for the sacrificial layer, and this is mixed in pure water at a rate of 10 w / v%. A dextran aqueous solution was prepared. This solution was applied to the surface of a silicon wafer subjected to plasma treatment by a spin coat method.

A photoresist (SU-8 3050) was applied onto the formed dextran film by spin coating, and through holes and flow channel shapes were formed by photolithography using a mask in which the through holes and flow channel shapes were respectively patterned. Table 3 shows the photolithography process conditions.

Step 4 / junction of the upper structure and the lower structure: the PDMS movable membrane of the upper structure by a plasma processing apparatus (manufactured by Vacuum Device PIB-20B), N ₂ plasma treatment (gas flow 10 sccm, the ambient pressure 50 Pa , Current value 20 mA, treatment time 2 min30 s). Thereafter, the joining positions of the upper structure and the lower structure were aligned using a stereomicroscope and bonded together, and then joined by heating at 120 ° C. for 15 minutes on a hot plate. Heating is used to shorten the time, but bonding can be realized at room temperature to 300 ° C., which is a temperature range in which the material does not soften.

  Step 5 / Removal of Substrate: The microfluidic transport structure device was separated from the silicon substrate by immersing in pure water at 50 ° C. to dissolve the sacrificial layer of dextran.

  As described above, the microfluidic transport structure can be manufactured by the embodiment of the manufacturing method.

  FIG. 11 is an image obtained by observing the nozzle array produced in the example from the bottom with an inverted microscope (Nikon Corporation, ECLIPSE Ti). In the observed image, it can be seen that a “cell channel” for flowing a fluid, an “opening” serving as a through hole, and a pneumatic channel for expressing a valve function are formed. The misalignment of the upper structure (SU-8 layer) and lower structure (PDMS layer) interface was within 5 μm. From this, the movable film could be installed on the upper part of the predetermined through hole, and the movable film had the accuracy necessary to be deformed so as to exhibit the valve function.

  FIG. 12 is a microscopic image observed by cutting along the A-A ′ cross section of FIG. 11. The flow path height of the pneumatic flow path portion is 51.4 ± 0.4 μm (n = 4). In addition, the height of the opening (SU-8 1st) of the cell capture channel is 18.4 ± 0.4 μm (n = 4), and the height of the channel (SU-8 2nd) is 27.4 ±. It was 0.5 μm (n = 4). The opening diameter of the nozzle through-hole was 39.0 ± 1.4 μm (n = 16), and the aspect ratio was about 0.46. The n number here represents the measured number.

  The PDMS valve part at the time of cell capture has a diaphragm structure and can be operated by changing the air pressure. A device in which the film thickness of the PDMS superstructure forming the diaphragm structure was changed was prepared, and an air pressure up to a gauge pressure of 190 kPa was applied with a compressor. When air pressure was applied up to 190 kPa in each microfluidic transport structure with the film thickness changed under three conditions, the operation of the valve could be confirmed. No damage to the valve was observed after the experiment. At this time, the valve is deformed to a maximum of about 20 μm by calculation by applying air pressure, and is a deformation that can capture cells. The air pressure can be applied from a vacuum pressure to a gauge pressure of 300 kPa, which is not damaged, and the valve can be deformed.

  FIG. 13 is a microscopic image comparing a before and after application of air pressure in a valve array having a PDMS movable film thickness of 17.3 μm. Although the pneumatic flow path produced by PDMS was deformed and expanded, it withstood air pressure for about 30 seconds without any problem.

DESCRIPTION OF SYMBOLS 1 Microfluid transport structure 2 Upper structure 2a One part 2b of upper structure Film formed and hardened film 3 Lower structure 4 Groove 5 Bypass flow path 6 One end (inlet) of flow path
7 The other end (outlet) of the flow path
8 Range set by equivalent circuit 10 Substrate 11 Sacrificial layer 12 Mold 21 Entrance / exit 22 Inlet 23 Space 24 Partition 31 Through-hole 32 Channel

Claims (7)

  A microfluidic transport structure in which a lower structure and an upper structure are integrated, and the lower structure includes a side wall portion and a bottom surface portion for forming a liquid transport channel, and has a diameter on the bottom surface portion. A plurality of through-holes having a ratio of the depth dimension to the dimension (aspect ratio) of 1.0 or less are provided, and the upper structure is stacked on the lower structure to form the liquid transport channel. A microfluidic transport structure characterized by having a portion.   The lower structure is composed of a photocurable resin, and is formed on the surface of a sacrificial layer formed on a substrate and is composed of a remaining part from which the sacrificial layer is removed. The microfluidic transport structure according to claim 1.   The upper structure includes a partition that is appropriately spaced from the upper surface, and a liquid transport channel is formed between the partition and the lower structure, and a pressure is applied between the partition and the upper surface. A pneumatic flow path through which air flows is formed, the partition wall is a movable film formed of an elastic material, and the movable film is divided into a movable area and a fixed area, and the movable area is the lower part 3. The structure according to claim 1, wherein the structure is deformable in an appropriate range facing each of the plurality of through holes of the structure, and the fixed region is supported by the upper surface portion in a range excluding the movable region. 2. A microfluidic transport structure according to 1.   The movable region is a circular region formed in a circular range, and the circular region is formed in a circular shape centering around an extension line of a central axis of a plurality of through holes of the lower structure. The macrofluid transport structure according to claim 3, wherein the macrofluid transport structure is provided.   5. The microfluidic transport structure according to claim 4, wherein the circular region is formed in a circular shape centered at a position deviated from an extension line of a central axis of the through hole.   3. The method of manufacturing a microfluidic transport structure according to claim 1, comprising an upper structure forming step, a lower structure forming step, and an integration step, wherein the upper structure forming step includes: A mold forming process for forming a mold having a shape that matches a liquid transport channel planned area on the upper side, an upper constituent material filling process for filling the mold with the upper constituent material, and curing the upper constituent material A mold release step of releasing the mold from the mold and a punching step of punching a part of the released molded product, wherein the lower structure forming step forms a sacrificial layer on the substrate A sacrificial layer forming step, a flow path bottom surface forming step of patterning a lower constituent material in a liquid transport flow path forming region except for a portion corresponding to the through hole on the surface of the sacrificial film, and removing the sacrificial layer By the board and lower structure A separation step of separating the material, and the integration step includes transporting the structure formed by the upper structure formation step and the structure formed by the lower structure formation step to each of the liquid transports. A method of manufacturing a microfluidic transport structure, comprising a pasting step in which the planned flow path region and the liquid transport flow path forming region are bonded together. A method for manufacturing a microfluidic transport structure according to any one of claims 3 to 5, comprising an upper structure formation step, a lower structure formation step, and an integration step, and further comprising an upper structure formation The process includes an air pressure flow path forming process, a movable film forming process, a stacking process, and a liquid supply hole forming process, and the air pressure flow path forming process matches the planned air pressure flow path area on the substrate. A mold forming step for forming a mold having a protruding shape, an upper constituent material filling step for filling the mold with the upper constituent material, and releasing the mold from the mold after the upper constituent material is cured. An elastic material film forming step including forming a movable film pre-determined layer with an elastic material on a substrate, including a mold releasing step and a first hole punching step of punching a part of the mold that has been released; Including the step, the laminating step, the elasticity Including a first laminating step of laminating the structure formed by the pneumatic flow path forming step to the surface of the movable film planned layer formed by the material film forming step, the liquid supply hole forming step, Including a second perforating step for perforating a part of a region where the structure formed by the pneumatic flow path forming step and the movable film planned layer is laminated, and the lower structure forming step is sacrificed on the substrate A sacrificial layer forming step of forming a layer, a flow path bottom surface forming step of patterning a lower constituent material in a liquid transport flow path forming region except for a portion corresponding to the through hole on the surface of the sacrificial layer, and the sacrificial layer A separation step of separating the substrate and the lower constituent material by removing the layer, and the integration step is formed by the structure formed by the upper structure formation step and the lower structure formation step. Structure Preparative method of the microfluidic transport structure, characterized in that in which each of the liquid transport channel scheduled region and the liquid transport channel formation region comprises a second lamination step of laminating a state matching.