JP2009109249A - Microchip and master chip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To develop a technique capable of stably maintaining a desired concentration profile, even when liquid feed is temporarily halted and even when the speed of a liquid current of liquid feed is very slow on a microchip for adjusting the concentrations of solutions by multiple steps, by repeating the dispensation and mixture of solutions having different concentrations, which are introduced from a plurality of liquid inlets, in a fine channel network having a plurality of branch points and confluence points. <P>SOLUTION: Main channels 24 (mixture parts) for mixing liquids are connected to one another, by connecting channels 25 having a channel cross-sectional area which is smaller than those of the main channels 24, to form the microchip 1. A master chip is for resin molding of the microchip 1. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a microchip used for analyzing a liquid sample, and in particular, distributing and mixing solutions having different concentrations introduced from a plurality of liquid inlets in a microchannel network having a plurality of branch points and junction points. To a microchip that adjusts the concentration of the solution in multiple stages and a master chip for producing the microchip.

A microchip for performing a cell assay is expected as an analytical method capable of realizing a higher degree of integration than a conventional microwell plate. Non-Patent Document 1 published by the inventors and Non-Patent Document 2 published by an American group describe a microchip capable of testing the cytotoxicity of a plurality of drugs at once.
On the other hand, in general, in performing a cytotoxicity test, it is necessary to perform a test in a wide concentration range as described in Non-Patent Document 3. However, the liquid handled by the microchip is on the order of several nanoliters, and it is very difficult to accurately measure and dilute such a small amount of liquid.
In order to solve such a problem, a method of creating a plurality of liquid flows having different concentrations by using a micro flow channel network that repeats branching and merging is known (for example, Non-Patent Document 4).
Further, a method for adjusting solutions having various complex concentration profiles by devising the design of the channel network based on this principle has been reported (for example, Non-Patent Documents 5-8).

In general, in these methods, the flow resistance of each flow path forming the flow path network is set appropriately, and liquid is introduced at an appropriate flow rate using a syringe pump or the like from the liquid introduction port, whereby the concentration differs. It is possible to distribute the liquids at an appropriate ratio at the branching point and to mix the liquids at an appropriate ratio in the mixing portion, and to obtain a liquid having a desired concentration at the liquid discharge port existing at the most downstream side. However, since appropriate distribution at the branch point and appropriate mixing in the mixing unit are realized only in a state where the liquid is introduced from the liquid introduction port at a preset flow rate, the liquid in the syringe pump is insufficient. However, when the flow rate at the liquid inlet is significantly reduced, a liquid having a desired concentration cannot be obtained. This is a serious problem when a cell culture medium having a multi-stage concentration profile is adjusted by a micro flow channel network and applied to a cell culture chip for culturing cells downstream for a long time. In other words, in order to culture cells for a long period of time, it is necessary to replenish the culture solution appropriately to a syringe pump or the like. Therefore, there is a problem that the solute concentration in each flow path changes. This is a fatal problem in a cytotoxicity test chip in which the state of a cell is changed only by being exposed to a small amount of solute for a short time.
Further, although different from these methods, as a similar method, a method of creating a multi-stage concentration profile by performing branching and merging only one stage has been reported (for example, Non-Patent Document 9-11).

However, with these methods, it is difficult to produce concentration profiles having concentrations that differ by three orders of magnitude or more. In order to create a multistage concentration profile with a concentration range of several orders of magnitude for a microchip suitable for general cytotoxicity tests, it is desirable to use a structure that repeats branching and merging.
Furthermore, although different from these methods, as a similar method, a method of creating a concentration gradient using a diffusion phenomenon has been reported (for example, Non-Patent Document 12).
However, even when the concentration gradient is produced by the diffusion phenomenon, there is a drawback that the concentration profile changes greatly with the passage of time when the pump and the pressurizing device are once stopped.
Sugiura S., Edahiro J., Kikuchi K., Sumaru K., and Kanamori T.:Cell Culture Microchamber Array with Independent Perfusion Channel for Parallel Drug Toxicity Assay, The proceedings of microTAS 2007, Chemical and Biological Microsystems Society, pp1321-1323 (2007) Wang Z., Kim M.-C., Marquez M. and Thorsen T .: High-Density Microfluidic Arrays for Cell Cytotoxicity Analysis, Lab Chip, 7, 740-745 (2007) Takara K., Sakaeda T., Yagami T., Kobayashi H., Ohmoto N., Horinouchi M., Nishiguchi K. and Okumura K .: Cytotoxic Effects of 27 Anticancer Drugs in Hela and Mdr1-Overexpressing Derivative Cell Lines, Biol. Pharm. Bull., 25, 771-778 (2002) NL Jeon, SKW Dertinger, DT Chiu, IS Choi, AD Stroock and GM Whitesides: "Generation of Solution and Surface Gradients Using Microfluidic Systems", Langmuir, 16 (22), 8311-8316 (2000). SKW Dertinger, DT Chiu, NL Jeon, GM Whitesides, Generation of gradients having complex shapes using microfluidic networks, Anal. Chem. 73 (2001) 1240-1246. XY Jiang, JMK Ng, AD Stroock, SKW Dertinger and GM Whitesides: "A Miniaturized, Parallel, Serially Diluted Immunoassay for Analyzing Multiple Antigens", J. Am. Chem. Soc., 125 (18), 5294-5295 (2003) K. Campbell, A. Groisman, Generation of complex concentration profiles in microchannels in a logarithmically small number of steps, Lab Chip 7 (2007) 264-272. F. Lin, W. Saadi, SW Rhee, SJ Wang, S. Mittal and NL Jeon: "Generation of Dynamic Temporal and Spatial Concentration Gradients Using Microfluidic Devices", Lab Chip, 4 (3), 164-167 (2004) M. Yamada, T. Hirano, M. Yasuda and M. Seki: "A Microfluidic Flow Distributor Generating Stepwise Concentrations for High-Throughput Biochemical Processing", Lab Chip, 6 (2), 179-184 (2006) H. Bang, SH Lim, YK Lee, S. Chung, C. Chung, DC Han and JK Chang: "Serial Dilution Microchip for Cytotoxicity Test", J. Micromech. Microeng., 14 (8), 1165-1170 (2004 ) # 2286 GM Walker, N. Monteiro-Riviere, J. Rouse and AT O'Neill: "A Linear Dilution Microfluidic Device for Cytotoxicity Assays", Lab Chip, 7 (2), 226-232 (2007) BG Chung, F. Lin and NL Jeon: "A Microfluidic Multi-Injector for Gradient Generation", Lab Chip, 6 (6), 764-768 (2006)

  As described above, when performing a cytotoxicity test on a microchip, it is necessary to continue supplying the culture solution over a long period of time. Therefore, a configuration of a flow channel network that can supply a culture solution having an appropriate concentration even after the supply of the culture solution is temporarily stopped is required.

  In the present invention, a micro-fluid having a multi-stage concentration profile is prepared by repeatedly distributing and mixing solutions having different concentrations introduced from a plurality of liquid inlets in a microchannel network having a plurality of branch points and junction points. An object of the chip is to provide a microchip having a configuration capable of maintaining a concentration profile even if the introduction of liquid from the liquid inlet is temporarily stopped.

In order to solve the above problems, the present invention provides the following configuration.
The first invention includes a plurality of liquid inlets into which liquids having different concentrations are introduced, and four or more liquid outlets, and a micro guide for introducing the liquid introduced from the liquid inlet to the liquid outlet. A flow channel network is formed, the micro flow channel network has a plurality of branch points and a plurality of merge points, and the liquid introduced from the plurality of liquid inlets is repeatedly branched and merged twice or more. Accordingly, the microchip is configured to adjust solutions having different multi-stage concentrations for each liquid discharge port, and the microchannel network includes main channels for mixing liquids having different concentrations. Are connected via a connection channel having a narrow portion having a smaller channel cross-sectional area than the main channel,
The connection channel is a liquid between the main channel extending from the branch point to the upstream side in the liquid feeding direction and the main channel extending from the junction point to the downstream side in the liquid feeding direction in the micro channel network. Provided is a microchip characterized by constituting a flow path.
According to a second invention, there is provided the microchip according to the first invention, wherein the narrowed portion of the connection channel is a groove formed with a depth smaller than that of the main channel.
According to a third invention, in the microchip of the first or second invention, the microchannel network includes the main channel and a first resistance channel connected to an upstream end of the main channel in the liquid feeding direction, A second resistance channel connected to a downstream end of the main channel in the liquid feeding direction, and a plurality of liquid feeding lines extending from the liquid introduction port. The first resistance channel and the second resistance The flow channel is formed with a flow channel cross-sectional area smaller than that of the main flow channel, and each liquid feed line has its upstream end in the liquid feed direction communicated with the liquid inlet and the downstream end in the liquid feed direction with the liquid discharge port. The connection flow path is connected to the main flow path at the upstream end in the liquid feed direction at the branch point of the liquid flow direction downstream end of the main flow path of the liquid feed line, and downstream in the liquid feed direction. The end is communicated with the main flow path at the junction point at the upstream end in the liquid feed direction of the main flow path of another liquid feed line. Providing a microchip, characterized in that.
According to a fourth aspect of the present invention, in the microchip according to the third aspect, the first resistance channel and the second resistance channel, and the narrow portion of the connection channel have the same depth, and the mainstream Provided is a microchip characterized in that it is a groove formed with a depth smaller than that of a path.
A fifth invention provides the microchip according to any one of the first to fourth inventions, wherein a micro container is connected downstream of the liquid discharge port.
A sixth invention provides the microchip according to the fifth invention, wherein the micro container is a cell culture container.
According to a seventh invention, in the microchip of any one of the first to sixth inventions, the microchannel network can adjust a solution having a predetermined concentration by applying the same pressure to each liquid inlet. Provided is a microchip characterized by being designed.
An eighth invention is a master chip for producing the microchip of any one of the first to seventh inventions by replica molding, wherein the mainstream is formed by resin layers having different thicknesses formed on a base substrate. A main channel forming ridge for forming a path and a connection channel forming ridge for forming the connection channel are formed, and the connection channel forming ridge is used for forming the main channel A master chip is provided, which is formed of a resin layer having a thickness smaller than that of the ridge, and has a projection dimension from the base substrate that is smaller than that of the main channel forming ridge.
A ninth invention provides the master chip according to the eighth aspect, wherein the resin layer is a photoresist layer.
A tenth invention is the master chip of the eighth or ninth invention for producing the microchip of the fourth invention, and further, a first resistance channel for forming the first resistance channel The forming protrusion and the second resistance flow path forming protrusion for forming the second resistance flow path are formed by a resin layer having a smaller thickness on the base substrate than the main flow path forming protrusion. And a master chip characterized in that the projecting dimensions of the first and second resistance channel forming ridges from the base substrate are the same as the connection channel forming ridges. provide.

According to the present invention, liquid introduction from the liquid introduction port is temporarily performed by the configuration in which the main flow paths for liquid mixing are connected by the connection flow path having a narrow portion having a smaller flow path cross-sectional area than the main flow path. Even if stopped, the concentration profile in the microchannel network can be maintained.
Adjustment of a solution having a desired concentration in each main channel is based on the premise that the distribution of the solution at the branch point of the microchannel network and the mixing of the solution at the junction are performed by convection. The adoption of the connection flow path having the narrowed portion makes the mass movement between the main flow paths by diffusion through the connection flow path smaller than the mass transfer by convection. This makes it possible to maintain the concentration profile even if the introduction of liquid from the liquid inlet is temporarily stopped.
In addition to temporarily stopping the introduction of liquid from the liquid inlet, for example, for cell culture, the culture solution whose concentration is adjusted by the microchannel network is transferred from the liquid outlet to the culture vessel very slowly for a long period of time. Mass transfer by diffusion between the branch point and junction of the microchannel network even when the liquid is continuously flowing through the microchannel network at a very slow speed However, there is an advantage that a desired concentration profile can be maintained by being suppressed by the connection channel (specifically, the narrow portion).
In addition, the use of the connection flow path having the narrowed portion requires a short flow path length of the connection flow path, contributing to a reduction in chip size.
In addition, the configuration in which the depth of the connection channel is formed shallower than that of the main channel and the depth of the connection channel is aligned with the depth of the resistance channel is suitable for processing a channel network with a large channel cross-sectional area ratio. In addition to contributing to the convenience of processing, it is possible to predict the flow rate of the solution flowing through each flow channel when designing the flow channel network, so that it is possible to adjust a solution with a desired concentration in each main flow channel. .

Hereinafter, a microchip and a master chip embodying the present invention will be described with reference to the drawings.
FIG. 1 and FIG. 2 are schematic views of a microchannel network for adjusting a solution having a multistage concentration.
1 and 2, the microchip 1 has a structure in which a cover plate 3 is bonded to a chip substrate 2 (see FIG. 10G) on which a groove-shaped microchannel network 20 is formed. The microchannel network 20 is formed on one surface of the chip substrate 2 (hereinafter also referred to as a channel formation surface 2a), and the cover plate 3 covers the chip substrate 2 so as to cover the channel formation surface 2a. Are pasted together.
As the chip substrate 2 and the cover plate 3, for example, a material made of a well-known synthetic resin as a material of a microchip having a microchannel such as polydimethylsiloxysan (hereinafter also referred to as PDMS) can be adopted.

As shown in FIG. 1, the microchannel network 20 includes a plurality of (two in FIG. 1) liquid inlets 21 into which liquid is introduced and four or more (eight in FIG. 1) liquid outlets. 22, and the liquid introduced from the liquid inlet 21 is guided to the liquid outlet 22.
The microchannel network 20 adjusts the solutions introduced in a plurality of stages at different liquid outlets 22 by repeating the branching and merging of the liquids introduced from the plurality of liquid inlets 21 at least twice. It is configured.

The microchannel network 20 shown in FIGS. 1 and 2 will be specifically described.
1 and 2, reference numeral 23 is a liquid feed line, 24 is a main flow path (mixing section), 25 is a connection flow path connecting the main flow paths 24, 26 is a confluence, 27 is a branch point, and 28 is a first point. A resistance channel 29 is a second resistance channel.

The microchannel network 20 has one liquid supply line 23 (in the drawing, in the drawing) communicated with one of the two liquid introduction ports 21 (hereinafter also referred to as a first liquid introduction port, denoted by reference numeral 211 in the drawing). 231) and a liquid supply passage 21b extending from the other liquid introduction port 21 (hereinafter also referred to as a second liquid introduction port, denoted by reference numeral 212 in the figure), and through this liquid supply passage 21b. And a plurality of liquid feeding lines 23 (reference numerals 232 to 238 in the figure) communicated with the second liquid introduction port 212.
A liquid discharge port 22 is provided at an end of each liquid feed line 23 opposite to the liquid inlet 21 (downstream end in the liquid feed direction).

Each liquid feed line 23 includes a main flow path 24, a first resistance flow path 28 connected to the upstream end of the main flow path 24 in the liquid feed direction, and a second connected to the downstream end of the main flow path 24 in the liquid feed direction. A resistance channel 29 is provided.
The first resistance channel 28 and the second resistance channel 29 are formed to have a channel cross-sectional area smaller than that of the main channel 24, so that the liquid flowing through the microchannel network 20 has a channel resistance larger than that of the main channel 24. give.

The liquid supply line 231 communicated with the first liquid introduction port 211 has an upstream end in the liquid supply direction of the first resistance flow channel 28 communicated with a liquid supply flow channel 21a extending from the first liquid introduction port 211. It communicates with the first liquid inlet 211 via the flow path 21a. Further, in the liquid supply line 231, the downstream end of the second resistance channel 29 in the liquid supply direction is communicated with the liquid discharge port 22.
The liquid feed lines 232 to 238 communicating with the second liquid introduction port 212 are communicated with the liquid supply flow channel 21b extending from the second liquid introduction port 212 at the upstream end of the first resistance flow channel 28 in the liquid feed direction. The second liquid inlet 212 is communicated with the liquid supply channel 21b. In addition, in the liquid supply lines 232 to 238, the downstream end of the second resistance channel 29 in the liquid supply direction is communicated with the liquid discharge port 22.

In addition, the main flow paths 24 of the respective liquid feeding lines 23 have the same cross-sectional area.
In the microchannel network 20, channels other than the connection channel 25, the first and second resistance channels 28 and 29 (for example, the liquid supply channels 21 a and 21 b) They are aligned with the main flow path 24 of the liquid feed line 23.
However, in the present invention, there is a variation in the cross-sectional area of the main flow path 24 of each liquid supply line 23, or the main flow path 24, the connection flow path 25, the first and second resistance flows in the micro flow path network 20. The configuration in which the channel cross-sectional area of the channels other than the channels 28 and 29 is different from the channel cross-sectional area of the main channel 24 is not excluded.

In this microchannel network 20, a branch point denoted by reference numeral 27 is a downstream end of the main channel 24 of the liquid feeding line 23 in the liquid feeding direction, the second resistance channel 29, and the connection channel 25. It is a communication point.
Further, the confluence point denoted by reference numeral 26 is a communication point where the upstream end of the main flow path 24 of the liquid supply line 23, the first resistance flow path 28, and the connection flow path 25 communicate with each other.

  The liquid feed line 231 that communicates with the first liquid inlet 211 is provided with a branch point 27 that branches the downstream end of the main flow path 24 in the liquid feed direction into the second resistance flow path 29 and the connection flow path 25. Yes. At the branch point 27, the second resistance channel 29, the connection channel 25, and the main channel 24 are communicated with each other. The connection flow path 25 connected to the main flow path 24 of the liquid supply line 231 is one of the main flow paths 24 of the liquid supply lines 232 to 238 (liquid supply line 232) communicating with the second liquid introduction port 212. At the junction 26 at the upstream end in the liquid feeding direction, the first resistance flow path 28 and the main flow path 24 are communicated together, and the liquid is introduced from the main flow path 24 of the liquid feed line 231 to the main flow path 24 of the liquid feed line 232. It functions as a liquid flow path.

Among the liquid supply lines 232 to 238 communicating with the second liquid introduction port 212, the liquid supply line 23 denoted by reference numerals 232 to 236 is connected to the second resistance flow path 29 and the flow path at the downstream end of the main flow path 24 in the liquid supply direction. Branch point 27 branched to 25 (the two flow paths 29, 25 and the downstream end of the main flow path 24 in communication with each other), the first resistance flow path 28, the connection flow path 25, and the main flow path 24 The main flow path 24 of each liquid sending line 232-236 is connected in cascade through the connection flow path 25. Further, the liquid feed line 237 is provided with only the junction point 26 among the junction point 26 and the branch point 27. The connection flow path 25 communicated with the first resistance flow path 28 and the upstream end of the main flow path 24 in the liquid transfer direction at the junction 26 of the liquid supply line 237 is connected in cascade through the connection flow path 25. In the main flow path 24 of the liquid supply lines 232 to 236, the liquid is introduced from the first liquid introduction port 211 in the moving direction (the order in which the liquid introduced from the first liquid introduction port 211 flows in) is located on the most downstream side. The main flow path 24 (the main flow path 24 of the liquid supply line 236) communicates with the main flow path 24 at the branch point 27 at the downstream end in the liquid supply direction, and the main flow path 24 of the liquid supply line 236 communicates with the main flow path of the liquid supply line 237. It functions as a liquid flow path for introducing a liquid into 24.
Thereby, in the microchannel network 20, the liquid introduced into the first liquid introduction port 211 can flow into the main channel 24 of the liquid feeding lines 231 to 237.
In each connection channel 25, the end on the branch point 27 side is an upstream end in the liquid feeding direction, and the end on the confluence point 26 side is a downstream end in the liquid feeding direction.

  Of the liquid supply lines 232 to 238 communicating with the second liquid introduction port 212, the connection flow path 25 is not connected to the liquid supply line 23 denoted by reference numeral 238. The liquid feeding line 238 does not introduce liquid from other liquid feeding lines.

The first resistance flow path 28 of the liquid feed lines 232 to 237 communicating with the second liquid inlet 212 has a moving direction (first direction) of the liquid introduced from the first liquid inlet 211 among these liquid feed lines 232 to 237. The flow to be given to the liquid in the first resistance channel 28 sequentially from the liquid supply line 232 located at the most upstream to the liquid supply line located at the downstream side in the order in which the liquid introduced from the liquid introduction port 211 flows in). It is formed so as to increase the road resistance.
The difference in flow path resistance generated by the first resistance flow path 28 of the liquid supply lines 232 to 237 is realized by, for example, the difference in flow path cross-sectional area of the first resistance flow path 28 of the liquid supply lines 232 to 237. However, here, as the first resistance flow path 28 of each of the liquid feeding lines 232 to 237, those having the same flow path cross-sectional area are adopted, and the difference in flow path length of the first resistance flow path 28 is adopted. The difference in channel resistance is realized.
The first resistance flow paths 28 of the liquid feed lines 231 to 238 have the same flow path cross-sectional area, and each has a constant flow path cross-sectional area over the entire length of the flow path.

In addition, the second resistance flow path 29 of the liquid supply lines 231 to 237 is the movement direction of the liquid introduced from the first liquid introduction port 211 (introduced from the first liquid introduction port 211) among these liquid supply lines 231 to 237. The flow resistance given to the liquid of the second resistance flow path 29 is sequentially reduced from the liquid supply line 231 positioned at the most upstream in the order of the liquid flow) to the liquid supply line positioned downstream. Is formed.
The difference in flow path resistance generated by the second resistance flow path 29 of the liquid supply lines 231 to 236 is realized, for example, by the difference in flow path cross-sectional area of the second resistance flow path 29 of the liquid supply lines 231 to 236. However, here, as the second resistance flow paths 29 of the liquid feeding lines 231 to 236, those having the same flow path cross-sectional area are adopted, and the difference in flow path length of the second resistance flow paths 29 is adopted. The difference in channel resistance is realized.

However, with respect to the second resistance channel 29 of the liquid supply line 237, by increasing the channel cross-sectional area as compared with the second resistance channel 29 of the liquid supply line 236, the channel resistance can be reduced. Smaller than that.
However, the second resistance flow path 29 of the liquid supply line 237 also has the same flow cross-sectional area as the second resistance flow path 29 of the liquid supply lines 231 to 236, and the difference in flow path length (the liquid supply line 236 Needless to say, the flow path length is shorter than that of the second resistance flow path 29), and a flow path resistance smaller than that of the second resistance flow path 29 of the liquid feeding line 236 can be realized.
Each of the second resistance flow paths 29 of the liquid supply lines 231 to 238 has a constant flow path cross-sectional area over the entire length of the flow path.

The microchannel network 20 of the microchip 1 shown in the example is a multi-stage different for each liquid outlet 22 by repeating the branching and merging of the liquid introduced from the two liquid inlets 211 and 212 at least twice. It is configured to adjust a solution having a concentration of.
When the types of liquids introduced from the two liquid inlets 211 and 212 into the microchannel network 20 are different from each other, the main flow path 24 of the liquid feed lines 232 to 237 communicating with the second liquid inlet 212 is connected to the second liquid inlet. It functions as a mixing unit that mixes the liquid introduced into the liquid feed line from the port 212 and the liquid introduced from the connection flow path 25. The adjustment of the solution having a desired concentration in each main channel 24 is performed by diffusing the solution at the branch point 27 of the microchannel network 20 and mixing the solution at the junction 26 by diffusion in the main channel 24, respectively. Realized.
Further, a mechanism for promoting mixing may be provided in the main channel 24. For example, a stirring bar or the like may be introduced into the main flow path 24.

From the liquid discharge port 22 provided for each liquid feed line 23, a liquid in which the liquid introduced from the first liquid introduction port 211 and the liquid introduced from the second liquid introduction port 212 are mixed is discharged (supplied). The The solution discharged from the liquid discharge port 22 provided for each liquid feed line 23 is in the moving direction of the liquid introduced from the first liquid introduction port 211 (in the order in which the liquid introduced from the first liquid introduction port 211 flows). The concentration of the liquid introduced from the first liquid introduction port 211 sequentially decreases from the liquid delivery line 231 located on the most upstream side to the liquid delivery line located on the downstream side, and introduced from the second liquid introduction port 212. The liquid concentration increases.
In addition, the microchannel network 20 is designed so that a solution having a desired concentration individually set for each liquid discharge port 22 can be adjusted by applying the same pressure to each of the liquid introduction ports 211 and 212. Yes.

The connection channel 25 of the microchannel network 20 is formed to have a smaller channel cross-sectional area than the main channel 24 of each liquid feed line 23. The connection channel 25 having a smaller channel cross-sectional area than the main channel 24 functions as a narrow part (a narrow part having a smaller channel cross-sectional area than the main channel 24). As shown in FIG. 4, the connection channel 25 may have a configuration in which a narrow portion 25 a having a channel cross-sectional area smaller than that of the main channel 24 is provided in a part in the longitudinal direction.
In the microchannel network 20, the mass transfer between the main channels 24 due to diffusion through the connection channel 25 is smaller than the mass transfer due to convection. For this reason, the concentration profile in the microchannel network 20 can be maintained even if the introduction of the liquid from the liquid inlets 211 and 212 is temporarily stopped. The length of time during which the concentration profile can be maintained (hereinafter also referred to as profile maintenance time) can be adjusted by the channel cross-sectional area of the connection channel 25.
In order to lengthen the profile maintenance time, it is possible to increase the flow path length of the connection flow path 25, but it is possible to adjust the flow path cross-sectional area of the connection flow path 25 (reduce the flow path cross-sectional area). For example, the flow path length of the connection flow path 25 is short, which contributes to the miniaturization of the entire microchip 1.

  In addition, the use of the connection channel 25 whose entire length or part is a narrow portion having a smaller channel cross-sectional area than the main channel 24 is used when the flow velocity of the liquid flowing through the microchannel network 20 is very slow. The advantage that the desired concentration profile can be stably maintained by suppressing the mass transfer due to diffusion between the branch point 27 and the junction point 26 of the road network 20 by the connection channel 25 (specifically, the narrow portion). There is.

  Using the microchip shown in FIG. 1, pressurizing at 80 kPa from the liquid inlet and feeding two kinds of liquids having different concentrations to confirm the concentration profile, and then temporarily stopping the pressurization for 3 hours When the concentration profile was confirmed by pressurizing again at 80 kPa after being allowed to stand, no change in the concentration profile due to the stop of pressurization was confirmed.

Hereinafter, in order to obtain a culture solution for cell culture from each liquid discharge port 22, a culture solution having a high concentration of solute is introduced into the microchannel network 20 from the first liquid introduction port 211, and the second liquid introduction port 212. Taking the case of introducing a diluting liquid (hereinafter also referred to as a diluting liquid) as an example, the effect of the use of the connection flow path 25 that is a narrow part whose entire length or part is smaller than the main flow path 24 is smaller than the main flow path 24. consider.
Hereinafter, the main flow path 24 of the liquid feeding lines 232 to 237 is also referred to as a mixing unit 24.
Further, with respect to the liquid discharge port 22, the mixing unit 24, the connection channel 25, the branch point 27, the first resistance channel 28, and the second resistance channel 29 of the microchannel network 20, the branch point 27 is connected to the first liquid introduction port. The first stage, the second stage, etc. will be described in the order in which the liquid introduced from the port 211 flows, and the mixing unit 24 extending from the branch point 27 to the upstream side in the liquid feeding direction and the feeding of the mixing unit 24 The first resistance channel 28 communicating with the upstream end in the liquid direction, the second resistance channel 29 and the connection channel 25 extending from the branch point 27 downstream in the liquid feeding direction, and the downstream in the liquid feeding direction of the second resistance channel 29 The liquid discharge ports 22 at the ends will also be described as the first stage, the second stage, etc., corresponding to the branch points 27, respectively. Regarding the junction point 26, the junction point 26 at the upstream end of the mixing unit 24 extending from the second stage branch point 27 to the upstream side in the liquid feeding direction is referred to as the second stage junction point 26, and is hereinafter referred to as the first liquid inlet 211. In the order in which the liquid introduced from the flow-in, the third stage, the fourth stage, etc. will be described.
The liquid feed lines 231 to 238 are also described as the first stage, the second stage,... From the top in FIGS. 1 and 2, that is, the order in which the liquid introduced from the first liquid introduction port 211 flows. With respect to the liquid discharge port 22, the mixing unit 24, the junction 26, the branch point 27, the first resistance channel 28, the second resistance channel 29, the first stage, the second stage,. Called.

FIG. 3 is an enlarged view of the vicinity of the first-stage liquid feeding line 231 and the second-stage liquid feeding line 232.
As shown in FIG. 3, the liquid introduced from the first liquid inlet 211 branches at the first stage branch point 27 (indicated by reference numeral 271 in FIG. 3), and the first stage connection flow path 25 (in FIG. 3, It merges with the dilution liquid at the second-stage junction 26 (reference numeral 262 in FIG. 3) via the reference numeral 251).
In FIG. 3, reference numeral 241 denotes a first-stage main flow path, 242 denotes a second-stage main flow path (mixing unit), 252 denotes a second-stage connection flow path, 272 denotes a second-stage branch point, and 281 denotes a first-stage first resistance. The flow path, 291 is a first-stage second resistance flow path, 282 is a second-stage first resistance flow path, and 292 is a second-stage second resistance flow path.

  As shown in FIG. 3, the liquid introduced from the high-concentration liquid inlet (first liquid inlet 21) branches at the first stage branch point 271, partly flows to the first stage liquid outlet 221, and partly It flows to the first stage connection flow path 251. The liquid that has flowed into the first-stage connection channel 251 merges with the dilution liquid at the second-stage junction 262, and the liquid diluted at the junction 262 is mixed at the second-stage mixing unit 242. The liquid mixed in the second-stage mixing unit 242 branches at the second-stage branching point 272, partly flows to the second-stage liquid discharge port 222, and partly flows to the second-stage connection channel 252. At this time, the liquids flowing through the first-stage mixing unit 241 and the second-stage mixing unit 242 are different in concentration, and differ by culturing the cells by providing the micro container 31 downstream of each liquid discharge port 22. Cells can be cultured in a culture solution of a concentration.

  In general, when cell culture is performed, the culture solution must be continuously supplied for several days to several months. Therefore, when the culture medium is replenished to a pump or the like that supplies the liquid, it is necessary to temporarily stop the liquid. In the prior art, at this time, the liquid present in each mixing unit is mixed through the flow path between the mixing units, and the concentration of the solution differs from the desired concentration. This problem can be avoided by reducing the cross-sectional area of the road. In addition, by making the connection channel shallow, a connection channel having a small cross-sectional area and a small difference in depth and width can be easily obtained.

  In addition, in the flow channel network having the configuration shown in FIGS. 1 and 2, the culture solution is distributed at a predetermined ratio at the branch points of the respective stages, and mixed at a predetermined ratio at the junction and the mixing unit, so that a desired concentration can be obtained. Adjust the solution. The ratio of distribution and merging is determined by the flow rate of the solution flowing through each flow path. This is because the flow resistance of the first resistance flow path, the second resistance flow path, and the connection flow path section at each stage is appropriately set. It can be set freely by designing. At this time, if the channel cross-sectional area of the connection channel portion, the first resistance channel, and the second resistance channel is smaller than the channel cross-sectional area of the other channels, Since the calculation can be performed while ignoring the channel resistance of the channel, the channel network can be easily designed. Also, by making the resistance flow path shallower than the connection flow path, the first resistance flow path and the second resistance flow path, a connection flow path with a small cross-sectional area and a small difference in depth and width can be easily obtained. Can get to.

Hereinafter, the relationship between the channel cross-sectional area of the connection channel and the mass transfer due to diffusion will be examined in detail.
First, when the flow of the culture solution is stopped, the movement of the solute in the liquid in the mixing unit 24 at the Nth stage (N = 1, 2, 3,... Nmax) through the connection flow path 25 is considered. That is, the mass transfer by diffusion through the connecting flow path 25 between the N-th mixing unit 24 and the (N−1) -th and N + 1-th mixing units 24 is considered. The concentration of the solution in the N-th mixing unit 24 is CN [kg / m ³ ]. In general, the mass transfer flux J [kg / m ² / s] due to diffusion when the solution is stopped is expressed by the following equation.

Here, D [m ² / s] is a diffusion constant, c [kg / m ³ ] is a solute concentration, and x [m] is a coordinate in the length direction of the connection channel 25. Therefore, the average flux JN of the connection flow path 25 in the Nth stage can be estimated by the following expression, where the length of the connection flow path 25 is Lc [m].

Ｎ＞１、つまり二段目以降の混合部の濃度については以下の式である。

Ｎ＝Ｎｍａｘ、つまりＮｍａｘ段目の混合部の濃度については以下の式である。

Here, the cross-sectional area of the connection channel 25 is Ac [m ² ], and the width, depth, cross-sectional area and length of the mixing part are Wm [m], Hm [m], Am [m ² ], and Lm [m]. ]. Also, to simplify the discussion, it is assumed that a mixer is installed in the mixing section, and it is assumed that the solution is completely mixed in the mixing section after the solution is stopped. The concentration change in the N-th mixing portion is expressed by the following equation.
N = 1, that is, the concentration of the mixing portion in the first stage is the following equation.

N> 1, that is, the concentration of the mixing part after the second stage is the following expression.

N = Nmax, that is, the concentration of the mixing portion at the Nmax stage is the following equation.

When Nmax = 2, that is, when the flow path has two stages, these differential equations can be easily solved, and the concentration of the solution in the N-th mixing portion at the moment when the solution is stopped is expressed as C _{N, 0} [kg / m ³ ], the following solution can be obtained as a solution of the concentration change in the first and second mixing parts after the liquid feeding is stopped.

Here, it is assumed that the present invention tests the cellular response in a micro container in a wide concentration range of a low molecular solute, and the dilution rate of each stage is assumed to be 10 times, that is, the first stage and the second stage. The solute concentrations C _1,0 and C _2,0 in the mixing part are set to 1 [kg / m ³ ] and 0.1 [kg / m ³ ]. Further, assuming that the diffusion constant D is about 0.5 × 10 ⁻⁹ [m ² / s] in water of sucrose, the width Wm [m], the depth Hm [m], and the cross-sectional area Am [ m ² ] and length Lm [m] are 1 × 10 ⁻⁴ [m], 5 × 10 ⁻⁵ [m], 5 × 10 ⁻⁹ [m], and 1 × 10 ⁻³ [m], and the length Lc of the connection channel Set [m] to 1 × 10 ⁻⁴ [m].

FIG. 5 is a graph plotting the influence of the cross-sectional area ratio (mixing section / connecting flow path) between the connection flow path and the mixing section on the concentration change in the mixing section at each stage after the liquid supply is stopped.
When the cross-sectional area ratio between the connection flow path and the mixing part is 1, the concentration of the first and second mixing parts changes due to diffusion through the connection flow path within a few minutes after the liquid supply is stopped. It can be read that the cross-sectional area ratio of the mixing part is 10 or 100, that is, the change in the concentration of the mixing part in each stage is suppressed by making the connecting flow path thinner than the mixing part.

FIG. 6 shows the influence of the cross-sectional area ratio (mixing part / connecting flow path) between the connection flow path and the mixing part on the concentration of each stage 10 minutes after stopping the liquid feeding.
It can be seen that as the cross-sectional area ratio between the connection flow path and the mixing portion increases, that is, the concentration change in each stage can be suppressed 10 minutes after stopping the liquid feeding by making the connection flow path thinner than the mixing portion. From FIG. 6, it can be read that the change in the solute concentration at each stage is suppressed even after 10 minutes by setting the cross-sectional area of the connection channel to 1/10 or less of the cross-sectional area of the mixing portion.

When Nmax> 2, that is, when the flow path has three or more stages, it is not easy to solve these differential equations (3), (4), and (5). 5. It can be easily understood that the phenomenon shown in FIG. 6 occurs.
Further, when it is assumed that a multi-stage concentration solution is prepared in a wide concentration range by serial dilution, an experiment is performed in a wide concentration range of about 6 to 8 digits as in Non-Patent Document 3, for example. In some cases, it is necessary to adjust the solution at a relatively large dilution rate in each stage. In other words, since _CN is much larger than _{CN + 1} , it can be considered that C ₁ >> C ₂ >> C ₃ >>, ..., >> Cmax. 5 and almost the same graph as FIG. 6 are obtained.

Next, let us consider the mass transfer in the connection flow path at each stage when the solution is flowed at a certain flow rate.
The flow rates of the liquid flowing through the first resistance channel, the mixing unit, the second resistance channel, and the connection channel in the Nth stage shown in FIGS. 1 and 2 are respectively Q _{N, 1} , Q _{N, 2} , Q _{N, 3} , Q _{N, 4} . Further, downstream of the mixing section of the N-th stage, i.e., the mixing unit, a second resistor flow path, a concentration of the solution of the liquid outlet and C _N.
In the flow path network having the configuration shown in FIGS. 1 and 2, the solution is distributed and mixed at a predetermined ratio at the branching point and the merging point in order to adjust the solution. In order to adjust the solution having a desired concentration, this distribution is performed. The mass transfer needs to be performed by convection during mixing. Here, the flux J _{N, C} [kg / m ² / s] of mass transfer by convection in the _N-th connection channel is expressed by the following equation using the average flow velocity v _{N, 4} in the connection channel. The

In addition, the mass transfer flux due to diffusion is expressed by the following equation as in equation (1) _{, where} J _{N, D} [kg / m ² / s].

As described above, in order to adjust a solution having a desired concentration, it is necessary that the mass transfer by diffusion is dominant as compared with the mass transfer by convection, and in general, J _{N, C} / J _{N, D} > 10 is preferred. The following formulas are derived from formulas (8) and (9).

Here, as described above, the solute concentrations C _{1, 0} and C ₂ , ₀ of the first stage and the second stage of the mixing part are set to 1 [kg / m ³ ] and 0.1 [kg / m ³ ], and the connected flow The path length Lc [m] is set to 1 × 10 ⁻⁴ [m], and the diffusion constant D is assumed to be about 0.5 × 10 ⁻⁹ [m ² / s] in water.
On the other hand, in the present invention, it is assumed that cells are cultured by providing a micro container (reference numeral 31 in FIG. 2) downstream of the liquid discharge port.
Here, it is assumed that cells are cultured by providing a micro container having a diameter of 1.5 mm downstream of the liquid discharge port. This size is the size of the micro container used in Non-Patent Document 1. In general cell culture, about 10 mL of culture solution is put into a culture dish having a diameter of 10 cm, and the culture solution is changed every two days. This culture medium exchange rate corresponds to a rate of about 6 ml / mm ² / day. Therefore, the supply speed of the culture solution to be supplied to the micro container having a diameter of 1.5 mm is 1.4 × 10 ⁻¹⁴ [m ³ / s]. In the microchannel network having a dilution ratio of 10 times as described above, since the culture fluid having a flow rate of about 1/10 of the liquid outlet passes through the connection channel, Q _{N, 4} = 1.4 × 10 ⁻¹⁵ [M ³ / s]. FIG. 7 shows the relationship between J _{N, C} / J _{N, D} and Ac calculated by substituting the above conditions into equation (10).

From this figure, in order for J _{N, C} / J _{N, D to} be 10 or more, the connection flow path cross-sectional area Ac needs to be 3.1 × ^{10 −10} m ² or less. Assuming that the depth of the connection flow path is 5 × 10 ⁻⁵ [m] similarly to the depth of the mixing portion described above, the width of the connection flow path needs to be 6.2 × 10 ⁻⁶ [m] or less. A flow path having a depth / width ratio of about 8 is actually difficult to process.
In this case, assuming that the ratio of the width and depth of the connection flow path is 1, if the width and depth of the connection flow path are 1.8 × 10 ⁻⁵ [m] or less, J _{N, C} / J _{N, D} is 10 or more. Thus, by making the connection flow path shallower than the mixing section, a flow path configuration that can be processed practically is possible.
Making the connection channel shallower than the mixing unit facilitates the formation of a connection channel having a smaller channel cross-sectional area than the mixing unit.

In addition, in the case where J _{N, C} / J _{N, D} is 10 in FIG. 8, the cross-sectional area Ac [m ² ] of the connection channel derived from the equation (10) and the flow rate Q _{N of the} solution flowing through the connection channel _, The relationship of ₄ [m ³ / s] is shown.
From this figure, the range of the connection channel cross-sectional area Ac required for mass transfer in the connection channel controlled by convection can be read. Since J _{N, C} / J _{N, D} is preferably 10 or more so that the mass transfer in the connection channel is governed by convection, this is the flow rate Q _{N, 4} of the liquid flowing through a certain connection channel. It is sufficient that the value of Ac is smaller than Ac that can be read from the drawing.
Assuming that the culture solution is supplied to a micro container having a diameter of 1.5 mm as in the above discussion, the flow rate Q _{N, 4} of the liquid flowing through the connection channel is 1.4 × 10 ⁻¹⁵ [m ³ / s]. . In this case, it is preferable from FIG. 8 that the connection flow path cross-sectional area Ac is 3.1 × ^{10 −10} [m ² ] or less. Assuming that the depth of the connection flow path is 5 × 10 ⁻⁵ [m] similarly to the depth of the mixing portion, the width of the connection flow path needs to be 6.2 × 10 ⁻⁶ [m] or less. / A channel with a width ratio of about 8 is actually difficult to process. If the ratio of the width and depth of the connection flow path is 1, if the width and depth of the connection flow path are 1.8 × 10 ⁻⁵ [m] or less, J _{N, C} / J _{N, D} will be 10 or more, and connection By making the flow path shallower than the mixing section, a flow path configuration that can be processed practically becomes possible.
From this, it can be seen that by making the connection channel shallower than the mixing part, it is easy to form a connection channel having a smaller channel cross-sectional area than the mixing part.

  Next, a case where the microchip 1 having a connection channel that is a groove having a shallower depth than the mixing portion is produced (manufactured) by the replica molding method will be described.

In FIGS. 10 (e) and 10 (f), reference numeral 4 denotes a master chip used for producing the chip substrate 2 of the microchip 1. The master chip 4 has a configuration in which resin layers 42 and 43 having different thicknesses are provided on one surface of a base substrate 41.
The base substrate 41 is a plate-like member such as a silicon wafer or a chip (silicon chip) obtained by dicing the silicon wafer.

  The resin layers 42 and 43 are resin layers 42 (hereinafter referred to as shallow flow) for forming the connection flow path 25 and the resistance flow paths 28 and 29 which are shallow flow paths in the micro flow path network 20 of the chip substrate 2. And a resin layer 43 (hereinafter also referred to as a deep flow path forming resin layer) for forming the mixing portion 24 and the liquid supply flow path 21 which are deeply structured flow paths. Separated. The deep flow path forming resin layer 43 is thicker than the shallow flow path forming resin layer 42. In the master chip 4, when the surface of the surface forming base substrate 41 is the reference surface 4 </ b> S, a portion protruding from the reference surface 4 </ b> S is a flow path forming protrusion for forming the fine flow path network 20. Acts as an article.

As an example of a method for producing the master chip 4, a photoresist layer 44 (hereinafter also referred to as a first photoresist layer) is formed by heat-curing a resist applied to one surface (resin layer forming surface 41 a) of the base substrate 41. Then, as shown in FIG. 10B, the pattern of the shallow flow path forming resin layer 42 is formed on the first photoresist layer 44 by exposure and heating using the photomask 45. Bake.
Next, a resist is applied on the first photoresist layer 44 to form a second photoresist layer 46 (FIG. 10C). The second photoresist layer 46 is formed so as to cover the entire first photoresist layer 44 with a thickness greater than that of the first photoresist layer 44 (surface-forming resin layer 42).
Next, as shown in FIG. 10D, the resin layer 43 pattern is baked on the second photoresist layer 46 by exposure using the photomask 47 and heating.
Thereafter, in the development step shown in FIG. 10E, the first and second photoresist layers 44 and 46 are removed while leaving the baked portions of the resin layers 42 and 43.
Thereby, the master chip 4 having the resin layers 42 and 43 having a desired pattern is obtained.

In order to create (manufacture) the chip substrate 2 of the microchip 1 using the master chip 4 by the replica molding method, for example, the chip is used with PDMS or the like in a state where the master chip 4 is set in a mold. Resin molding of the substrate 2 is performed (FIG. 10F). Thereby, the chip substrate 2 having the fine channel network 20 is formed by the channel forming protrusions formed by the resin layer 42 (shallow channel forming resin layer) and the resin layer 43 (deep channel forming resin layer). can get.
Then, as shown in FIG. 10G, the microchip 1 can be assembled by attaching the cover plate 3 to the obtained chip substrate 2.

  According to the above-described method, the portion of the resin layer 42 and the resin layer 43 that functions as the flow path forming ridge is formed into a ridge having a rectangular cross section. , Formed in a groove shape having a rectangular cross section.

  Here, among the liquid channels constituting the fine channel network, the connection channel 25 having a smaller channel cross-sectional area than the main channel 24 (mixing unit) is shallower than the main channel 24 (mixing unit). In order to form the grooves, as shown in FIGS. 11A and 11B, resin layers 42 and 43 having different thicknesses are formed on the base substrate 41 as flow path forming resin layers, A main channel forming ridge 43a for forming the main channel 24 and a connection channel forming ridge 42a for forming the connection channel 25 are obtained. The connecting channel forming ridges 42a have a projecting dimension from the base substrate 41 and a projecting dimension from the reference surface 4S smaller than the main channel forming ridges 43a.

11A and 11B illustrate a configuration in which the second resistance flow path forming protrusion 42b for forming the second resistance flow path 29 is also formed by the resin layer 42. .
In this case, the second resistance flow path forming ridges 42b have the same projecting dimensions from the base substrate 41 (in other words, projecting dimensions from the reference surface 4S) as the connection channel forming ridges 42a. Will be.
When the microchannel network 20 has a configuration in which the connection channel 25 and the second resistance channel 29 have the same depth, as described above, the connection channel forming protrusion 42a and the second channel are formed by one resin layer 42. The two-resistance flow path forming protrusion 42b can be obtained.
The same applies to the relationship between the connection flow path 25, the first resistance flow path 28, the first resistance flow path forming ridge formed on the master chip 4, and the connection flow path forming ridge 42a.

11A and 11B, the width W1 of the main flow path forming ridge 43a, the width W2 of the connection flow path forming ridge 42a, and the width W3 of the second resistance flow path forming ridge 42b are as follows. A configuration that is the same is illustrated.
In this case, the microchannel network 20 of the chip substrate 2 is obtained in which the main channel 24, the connection channel 25, and the second resistance channel 29 have the same groove width. The channel cross-sectional area of each channel is adjusted by the depth of each channel.
The same applies to the relationship between the connection flow path 25, the first resistance flow path 28, the first resistance flow path forming ridge formed on the master chip 4, and the connection flow path forming ridge 42a.
The present invention includes a configuration in which one or more widths of the connection channel 25, the first resistance channel 28, and the second resistance channel 29 are aligned with the width of the main channel 24.

FIG. 9 shows the results of evaluating the concentration gradient generation mechanism of the prototyped microchip.
A microchip was prototyped using PDMS.
The connection flow path, the first resistance flow path, and the second resistance flow path are grooves having a rectangular cross section (square grooves) having a width of 20 μm and a depth of 10 μm, and the cross-sectional dimensions thereof are aligned. The channels other than the connection channel, the first resistance channel, and the second resistance channel in the microchannel network were rectangular grooves (square grooves) having a width of 100 μm and a depth of 50 μm. The cross-sectional area ratio of the connection channel, the first resistance channel, and the second resistance channel to the main channel is 25 times. Further, the microchannel network was designed so that the dilution rate D in the n-th mixing portion was ½.
A neucoccin aqueous solution having a concentration of 17 wt% was fed from the first liquid inlet to the prototyped microchip, and ultrapure water for dilution was fed from the second liquid inlet. In all cases, the liquid was fed at 75 kPa. After 12 hours, the solution discharged from the liquid outlet was collected, the absorbance was measured, and the concentration was calculated from the calibration dose. From the obtained results, the concentration gradient generation mechanism was evaluated. As a result of FIG. 9, the New Coxin concentration showed a monotonic decrease almost in agreement with the set dilution rate. In addition, it was suggested that by setting the residence time in the mixing part to be sufficiently long, even if the medicine is flowed at a high pressure (high flow rate), two-component diffusion mixing occurs sufficiently.

The microchannel network for preparing multi-stage concentration solutions disclosed by the present invention can be applied to a microchip for testing drug cytotoxicity in a wide concentration range.

It is a figure which shows typically the microchip of this invention and the microchannel network of this microchip. It is the figure which expanded the microchannel network of FIG. 1, and was shown typically. It is the figure which expanded and showed the vicinity of the 1st step | paragraph and the 2nd step | paragraph liquid feeding line of the microchannel network of FIG. It is a figure which shows the example which provided the narrow part only in a part of longitudinal direction of the connection flow path of the microchannel network. It is a graph which shows the density | concentration change of the mixing part of each stage after a liquid feeding stop. It is a graph which shows the relationship between the density | concentration of the mixing part of each stage 10 minutes after a liquid-feed stop, and the cross-sectional area ratio with respect to the mixing part of a connection flow path. It is a graph which shows the relationship between the cross-sectional area Ac of a connection flow path, and JN _{, C} / _{JN, D.} It is a graph which shows the relationship between the flow volume of the solution which flows through a connection channel, and a connection channel cross-sectional area. It is a graph which shows the result of having evaluated the concentration gradient production | generation mechanism of the prototyped microchip. (A)-(g) is a figure explaining the production | generation of a master chip used for manufacture of a microchip, and the manufacture (assembly) procedure of a master chip. (A), (b) is a figure which shows the example which formed the protrusion for connection flow path formation, and the protrusion for 2nd resistance flow path formation with the same resin layer in the master chip.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1 ... Microchip, 2 ... Chip board | substrate, 20 ... Microchannel network, 21 ... Liquid inlet, 211 ... Liquid inlet (1st liquid inlet), 212 ... Liquid inlet (1st liquid inlet), 23 , 231 to 238... Liquid feeding line, 24... Main flow path (mixing section), 25 .. connection flow path, 251... Narrow section, 26. 2 resistance flow path, 4 ... master chip, 41 ... base substrate, 42 ... resin layer, 43 ... resin layer (flow path forming resin layer), 44 ... photoresist layer (first photoresist layer), 46 ... photoresist Layer (second photoresist layer), 42a ... projection for forming connection channel, 42b ... projection for forming second resistance channel.

Claims (10)

A plurality of liquid inlets into which liquids having different concentrations are introduced and four or more liquid outlets are formed, and a microchannel network is formed for guiding the liquid introduced from the liquid inlet to the liquid outlet. The microchannel network has a plurality of branch points and a plurality of junction points, and the liquid introduced from the plurality of liquid inlet ports is repeated for each liquid discharge port by repeating the branching and the junction twice or more. A microchip configured to prepare solutions having different multi-stage concentrations.
The micro-channel network is configured by connecting main channels for mixing liquids having different concentrations through a connection channel having a narrow portion having a smaller channel cross-sectional area than the main channel,
The connection channel is a liquid between the main channel extending from the branch point to the upstream side in the liquid feeding direction and the main channel extending from the junction point to the downstream side in the liquid feeding direction in the micro channel network. A microchip comprising a flow path.   2. The microchip according to claim 1, wherein the narrowed portion of the connection channel is a groove formed with a depth smaller than that of the main channel. The microchip according to claim 1 or 2,
The microchannel network includes the main channel, a first resistance channel connected to the upstream end of the main channel in the liquid feeding direction, and a second resistance channel connected to the downstream end of the main channel in the liquid feeding direction. A plurality of liquid supply lines extending from the liquid inlet, the first resistance channel and the second resistance channel are formed with a channel cross-sectional area smaller than the main channel,
Each liquid feed line has an upstream end in the liquid feed direction communicated with the liquid introduction port, and a downstream end in the liquid feed direction communicated with the liquid discharge port,
The connection flow path is connected to the main flow path at the upstream end of the liquid flow direction at the branch point of the liquid flow direction downstream end of the main flow path of the liquid flow line. A microchip characterized in that it communicates with the main flow path at the junction point at the upstream end of the liquid flow line in the liquid flow direction.   4. The microchip according to claim 3, wherein the first resistance channel and the second resistance channel and the narrow portion of the connection channel are aligned with each other, and are deeper than the main channel. A microchip characterized by being a shallow groove.   5. The microchip according to claim 1, wherein a micro container is connected downstream of the liquid discharge port.   6. The microchip according to claim 5, wherein the micro container is a cell culture container.   The microchip according to any one of claims 1 to 6, wherein the microchannel network is designed so that a solution having a predetermined concentration can be adjusted by applying the same pressure to each liquid inlet. A microchip characterized by that. A master chip for producing the microchip according to any one of claims 1 to 7 by replica molding,
A main flow path forming ridge for forming the main flow path and a connection flow path forming ridge for forming the connection flow path by resin layers having different thicknesses formed on the base substrate. Formed,
The connection channel forming ridge is formed of a resin layer having a thickness smaller than that of the main channel forming ridge, and the projecting dimension from the base substrate is smaller than that of the main channel forming ridge. A master chip characterized by   The master chip according to claim 8, wherein the resin layer is a photoresist layer. A master chip according to claim 8 or 9 for producing the microchip according to claim 4,
Further, a first resistance channel forming ridge for forming the first resistance channel and a second resistance channel forming ridge for forming the second resistance channel form a main channel. The protrusions of the first and second resistance flow path forming protrusions from the base substrate are formed by the resin layer having a smaller thickness on the base substrate than the protrusions for use. Master chip characterized by being aligned with the ridge for use.

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