JP4866208B2 - Micro reactor - Google Patents

Description

  The present invention relates to a microreactor for performing a large number of tests on a very small amount of sample using μTAS (Micro Total Analysis Systems) technology.

  For example, regarding an analyzer for cells for evaluating the effects of cells on drugs and toxicants, when measuring the reactions of many types of drugs on cells, there are generally 24 to 384 cells called microplates. These are formed on a single plate. In recent years, it has been reported that more than 1000 containers are formed on a plate of about several centimeters using microfabrication technology, and cell analysis is performed, but each plate has an open opening in each container. It is formed as a recessed portion.

  When introducing a cell such as a microplate having such an open container into an analysis container, cells are injected into the container manually or by a robot using a dispensing instrument such as a micropipette.

  However, in the case of such a cell introduction method, the time required for cell introduction increases in proportion to the number of containers. Further, as the number of containers on one plate increases, the volume per container decreases, so the time until the cell culture solution evaporates and the culture solution concentration changes is shortened. Therefore, since the time required for cell introduction must be shortened, the cell introduction time becomes a problem as the number of containers on one plate increases.

  In order to solve the problem of such an open-type container, in order to be able to dispense cells into a plurality of analysis containers while suppressing evaporation of the cell culture solution, a minute amount is used by using μTAS technology. Many microreactors for efficiently analyzing samples have been reported. Such a microreaction apparatus includes a plurality of cell analysis containers formed in a substrate, a sample introduction unit that distributes the cell sample introduced from the sample introduction port to all cell analysis containers while flowing in the substrate, A sample discharge channel connected to the sample discharge port from all cell analysis containers is provided.

  In order to further improve the efficiency of analysis in such a microreaction apparatus, it is desirable to increase the number of cell analysis containers to be accumulated. However, if the number of cell analysis containers is increased, the analysis cells are evenly distributed in each cell analysis container. It becomes difficult to distribute.

  For the purpose of evenly distributing the sample to a plurality of analysis containers formed in the substrate, the present inventors have proposed a passive sample introduction method (see Patent Document 1 and Non-Patent Document 1). . Passive sample introduction means that the sample flowing through the flow path is distributed by branching the flow path stepwise without applying external force or energy to the sample flowing through the flow path. ing.

FIGS. 6A and 6B are conceptual diagrams of the proposed method. The cell introduction portion 4 in the substrate repeats equally from one sample introduction port (in this case, the cell introduction port) 2 and is connected to a plurality of cell analysis containers 6 which are analysis wells in the substrate. The cells in the fine particle dispersed fluid sample introduced from the sample introduction port 2 are introduced into the plurality of cell analysis containers 6 while being evenly distributed through the flow path of the cell introduction part 4. At that time, the introduced cells are distributed almost evenly at each branch point of the cell introduction part 4, and the cells are introduced almost uniformly into the plurality of analysis wells 6 by one introduction operation.
In this method, unlike the microplate, each analysis container is covered, and thus evaporation of a liquid such as a cell culture solution can be suppressed even when the volume of the analysis container is reduced.

  By using the proposed method of FIG. 6, it is possible to simultaneously introduce samples into a large number of analysis wells 6 in one operation, but the fluid sample is a solid component and a transport liquid like a cell dispersion solution. And the diffusion coefficient of the solid component is not sufficiently large, the amount (for example, the number of cells) introduced into each analysis well 6 varies. As shown in FIGS. 7A and 7B, there is a difference in the concentration of the sample in the cross-sectional direction of the flow channel after the first branch, and the sample is distributed at an equal concentration in the subsequent branches. The cause is not being distributed. That is, the sample introduced from the sample introduction port 2 has a symmetric concentration distribution 14 in which the concentration is increased at the central portion in the flow path cross-sectional direction at the first branch portion 8. This is because a large amount of sample flows at the center of the channel due to friction with the channel wall. Therefore, in the branching portion 8, the sample is divided at the center in the cross-sectional direction of the flow path so that the sample is divided into equal parts, but the concentration distributions 16-1 and 16- in the cross-sectional direction of the sample after branching are divided. 2 is an asymmetric concentration distribution. Since the concentration distributions 16-1 and 16-2 are maintained, the next branching unit 10 is divided at the center in the cross-sectional direction of the flow path. As shown as 2, there is a difference in sample concentration. Furthermore, when there is a branch portion after the next stage, the non-uniformity of the sample concentration after branching is emphasized.

Therefore, in the microreactor using such a sample distribution method, the present inventors once branched the flow channel after each branch for the purpose of suppressing non-uniformity of the sample concentration after branching at the branching portion. Proposed a method to correct the deviation of the sample concentration in the direction of the cross section of the flow channel after branching by crossing again after three-dimensional intersection, and finally improve the deviation of the amount of sample introduced into each analysis well (See Non-Patent Document 2).
JP 2006-087336 A M. Kanai, et.al. “A Multi Cellular Diagnostic Device for High-throughput Analysis”, Proceeding of μTAS2004, pp.126-128, 2004 K. Kawai, et.al, “Improved Passive Cell Distributing Method for Micro Cellular Diagnostic Well Array”, Japanese Journal of Applied Physics, Vol. 45, No. 6B, 2006, pp. 5607-5613

  By using the method proposed in Non-Patent Document 2, the deviation of the sample concentration in the cross-sectional direction of the flow channel after branching is corrected, and the deviation of the amount of sample finally introduced into each analysis well is improved. Can do.

  The present invention is also directed to a microreactor using such a sample distribution method, and the concentration of the sample after branching at the branching portion is determined by a method different from the method proposed in Non-Patent Document 2. The purpose is to suppress uniformity.

  Such a problem is not limited to the case where the target is a cell, but is a common problem when handling a microparticle-dispersed fluid sample in which microparticles are dispersed in a liquid. Intended for fluid samples.

  The present invention provides a plurality of analysis wells formed in a substrate and a sample distributed from all sample analysis wells while flowing a sample-dispersed fluid sample introduced from a sample inlet and dispersed in a transport fluid. A microreaction apparatus having an introduction part and a sample discharge channel that leads from all the analysis wells to the sample discharge port, wherein the sample introduction part has at least two stages of branch parts and performs all analysis from the sample introduction port. Concentration distribution that adjusts the concentration distribution of fine particles in the direction of the cross-section of the flow path in the flow path between the branch sections in the sample introduction section. An adjustment unit is provided.

  The concentration distribution adjusting unit includes a main channel through which the fine particle-dispersed fluid sample flows, and at least one side wall of the main channel, and a main channel is separated by a partition on the upstream side and joined on the downstream side. Is provided with a gap that does not allow the particulates in the sample to pass through, so that only the transport fluid flows from the main channel into the sub-channel, and downstream, the transport fluid in the sub-channel flows to the side of the main channel flow. It has come to join.

  In one form of the concentration distribution adjusting unit, the sub-channel is provided on both side walls of the main channel, and the fine particle concentration in the channel cross-sectional direction of the fluid sample before entering the main channel is high. At least one of the size of the sub-channel and the size of the gap between the partition walls is adjusted so that the flow rate from the sub-flow channel to the main flow channel is greater than the flow rate from the other sub-flow channel to the main flow channel Has been.

In another form of the concentration distribution adjusting unit, the sub-channel is provided only on one side wall side of the main channel on the side where the fine particle concentration in the channel cross-sectional direction of the fluid sample before entering the main channel is high. ing.
The sample discharge channel preferably has a channel structure that repeats merging stepwise and evenly toward the sample discharge port.

  One preferred application of this microreactor is a cell analyzer. In that case, the sample becomes a cell dispersion solution in which living cells are dispersed in the cell culture solution, and the analysis well becomes a cell analysis container for analyzing the sample cells.

  In the present invention, a concentration distribution adjusting unit that adjusts the particle concentration distribution in the channel cross-sectional direction in the channel is provided in the channel between the branch portions in the sample introduction unit, and the main flow in which the particle dispersion fluid sample flows through the concentration distribution adjusting unit. Since the flow path and the secondary flow path that merges with the main flow path and the downstream flow path are configured, the flow of the solid sample that flows through the main flow path is positioned on the center side of the flow path by the transport liquid that flows through the secondary flow path. Thus, the deviation of the sample concentration in the cross-sectional direction of the flow channel after each branch is corrected, and the deviation of the amount of the sample finally introduced into each analysis container can be improved.

  In addition, since the concentration distribution adjusting unit of the present invention has the sub-flow path arranged on the side of the main flow path along the flow, the flow path structure is constituted by the same planar flow path structure, so that photolithography In the case of manufacturing using a method and an etching method, it is possible to manufacture the channel with only one photomask, and it can be manufactured at a lower cost than a structure including a three-dimensional intersection. Further, it can be produced by an injection molding method using a resin material or a hot embossing method, and can be produced at a lower cost.

Embodiments of the present invention will be described with reference to the drawings.
Embodiments of the present invention will be described below with reference to the drawings.
FIG. 1 shows a portion on the sample introduction side of one embodiment. The overall schematic structure is the same as that shown in FIGS. 6 (A) and 6 (B), and a plurality of analysis wells 6 formed in the substrate and the sample introduction port 2 are introduced into the transport fluid. A sample introduction part 4a for distributing a fine particle-dispersed fluid sample in which fine particles are dispersed in the substrate to all the analysis wells 6 while flowing in the substrate, and a sample discharge channel 9 connected to the sample discharge ports 7 from all the analysis wells 6. Yes.

  The sample introduction part is shown as 4a in FIG. The sample introduction part 4a has at least two stages of branch parts 8, 10, and 12 and has a flow channel structure that branches from the sample introduction port 2 to all the analysis wells 6 in a stepwise and even manner. In this embodiment, since eight analysis wells 6 are provided as an example, three stages of branch portions 8, 10, and 12 are provided in order to distribute samples to the eight analysis wells 6. .

  The sample introduction part 4a includes concentration distribution adjusting parts 24 in the flow paths 20a and 20b between the branch parts 8 and 10 along the flow and the flow paths 22a to 20d between the branch parts 10 and 12, respectively. The concentration distribution adjusting unit 24 has a function of adjusting so that the fine particle concentration distribution in the channel cross-sectional direction in the channel in which it is arranged is symmetric.

  An example of the density distribution adjusting unit 24 is shown in FIG. The concentration distribution adjusting unit 24 includes a main channel 32a through which the fine particle-dispersed fluid sample flows, and a sub channel that is separated from the main channel 32a by the partition walls 30a and 30b on the side walls of the main channel 32a on the upstream side and merges on the downstream side. 32b and 32c. The partition walls 30a and 30b are provided with a gap 34 having a size that prevents the fine particles in the sample from passing therethrough. Only the transport fluid flows from the main channel 32a into the sub channels 32b and 32c, and the sub channels 32b and 32c are downstream. These transport fluids join the side of the flow of the main flow path 32a. The particles exaggerated in size as 36 in FIG. 2 represent fine particles such as cells dispersed in the transport fluid of the fine particle dispersed fluid sample, and the gaps 34 between the partition walls 30a and 30b are fine particles. It is smaller than 36.

  In this embodiment, the sub-channels 32b and 32c are provided on both side walls of the main channel 32a, and the side of the fluid sample before entering the main channel 32a has a higher particle concentration in the channel cross-sectional direction. The width of the sub-flow channel 32b is larger than the flow rate of the flow from the other sub-flow channel 32b to the main flow channel 32a than that from the other sub-flow channel 32c. It is set larger than the width of. In this case, since the branch portions 8 and 10 are T-shaped branch portions that branch one flow path into two flow paths, the rear is viewed from the flow path on the fluid introduction side (flow path before branching) in the branch section. Since the fine particle concentration is relatively high in the portion on the side, the width of the sub-channel 32b on that side is larger than the width of the sub-channel 32c. All the gaps 34 provided in the partition walls 30a and 30b have the same size and are arranged uniformly.

  In the concentration distribution adjusting unit 24, the solid sample in the fluid sample passes only through the central flow path, that is, the main flow path 32a, and the transport fluid passes through the gap 34 provided in the partition wall, and the sub flow paths 32b and 32c on both sides. , And joins the main flow path 32a again on the downstream side. At this time, by setting the widths of the sub-channels 32b and 32c on both sides so that the width of the sub-channel 32b is larger than the width of the sub-channel 32c, the flow rate at the junction point to the main channel 32a. The balance can be adjusted. After the three flow paths 32a, 32b, and 32c merge, the solid particulate flow is sandwiched between the transport liquid flows from the sub-flow paths 32b and 32c on both sides. At this time, the flow of particles that is biased to one side in the cross-sectional direction of the flow path can be corrected to the center of the flow path by the flow rate balance of the transport fluid.

  Since the concentration distribution adjusting unit 24 has such a main channel and sub-channel structure, a fluid sample passes through the concentration distribution adjusting unit 24, whereby a high concentration region is led to the center of the channel, and as a result. Is corrected as indicated by reference numeral 17.

  The method for producing the microreaction apparatus of the present invention is not particularly limited, and can be realized using a microfabrication technique. FIG. 3 shows an example of a flow channel structure actually fabricated as an observation image by a scanning electron microscope (SEM). An image surrounded by a frame in the figure is an image showing the partition walls 30a and 30b and the gap 34 in an enlarged manner.

  In this embodiment, the analysis well 6, the flow path of the sample introduction unit 4, the concentration distribution adjusting unit 24, and the sample are formed as a pattern having a bottom as shown in FIG. 3 by a photolithography process and a dry etching process on the surface of the silicon substrate. A discharge channel 9 was formed. In the concentration distribution adjusting unit 24, partition walls 30a and 30b are formed as micro pillars in which rod-like bodies having a cross section of 10 μm on a side and upright at a height of 50 μm in a direction perpendicular to the bottom surface are arranged in two rows at equal intervals. did. Each rod-like body arrangement was arranged so that one side of the cross-sectional triangle was arranged on a straight line at an interval of 5 μm. Therefore, the gap 34 between the partition walls 30a and 30b is 5 μm. The distance between the partition walls 30a and 30b, which is the arrangement of the respective micro pillars, that is, the width of the main flow path 32a was 50 μm. The microreactor was formed by anodically bonding a borosilicate glass plate having the sample introduction port 2 and the sample discharge port 7 to the surface of the silicon substrate thus formed.

Since the analysis well 6, the sample introduction unit 4, the concentration distribution adjusting unit 24, and the sample discharge channel 9 can be formed only on one side of the substrate, the step of forming the channel shape in the resist layer using the photolithography method is performed. Fabrication can be performed with only one mask.
Further, as an inexpensive manufacturing method, an injection molding using a resin material or a hot embossing method can be used.

  FIG. 4 shows the result of calculating the fluid flow by computer simulation in the device structure of the embodiment of FIG. By this simulation, it can be seen that the fluid flows from the sub-channels 32b and 32c on both sides of the main channel 32a on the downstream side of the main channel 32a.

  When this embodiment is used as a cell analyzer and a cell dispersion solution in which living cells are dispersed as a sample is injected from the sample introduction port 2 into the cell culture solution, the cells in the introduced cell dispersion solution flow in the sample introduction section 4a. Through the path, the concentration distribution is evenly distributed while being corrected by the concentration distribution adjusting unit 24 and introduced into the analysis well 6 which is a cell analysis container. In this way, cells are evenly introduced into the plurality of analysis wells 6 by a single introduction operation.

  In FIG. 5, the microreaction apparatus of this example was used, and an introduction experiment was actually performed 10 times using a solution in which polystyrene fluorescent beads having a diameter of 20 μm were dispersed as a sample, and introduced into each analysis well 6. The result of comparing the amount of fluorescent beads is shown. The number of analysis wells 6 is 8, and they are numbered sequentially from the end. (A) is a comparative example showing a case where the density distribution adjustment unit 24 is not provided, and (B) is a case where the density distribution adjustment unit 24 of the embodiment is provided. From the results of FIG. 5, the coefficient of variation of the average number of fluorescent beads introduced in the eight analysis wells 6 was 94.9% when the concentration distribution adjustment unit was not provided (A), whereas In the case where the density distribution adjusting unit 24 according to the example is provided, (B) is improved to 29.3%. It is apparent that the variation in the number of fluorescent beads introduced in the analysis well 6 is reduced by providing the concentration distribution adjusting unit 24.

  In the present embodiment, the secondary flow channels 32b and 32c are provided on both side walls of the main flow channel 32a in the concentration distribution adjusting unit 24, but the sub flow channel is a flow channel cross section in the fluid sample before entering the main flow channel 32a. In this case, only the secondary flow path 32b may be provided only on the side where the fine particle concentration in the direction is high. The fine particle concentration in the cross-sectional direction of the fluid sample before entering the main channel 32a is high on the sub-channel 32b side. It is possible to correct the concentration of fine particles to be higher in the center of the flow path by joining the transport fluid from the sub flow path 32b to the main flow path 32a.

  The microreaction apparatus of the present invention can be used as a reaction apparatus for conducting a large number of tests on a small amount of sample at the same time, for example, as a cell analysis apparatus for evaluating the influence of cells on drugs and poisons.

1 is a plan view schematically showing a main part of a microreaction apparatus of one embodiment, and shows a sample introduction side into an analysis well. FIG. It is the top view which expanded and showed one density distribution adjustment part vicinity in the Example. It is a SEM image which shows the one branch part vicinity of the Example. It is a simulation figure which shows the flow of the fluid in the Example. It is a figure which shows the result of the sample introduction | transduction experiment in the Example, (A) is a comparative example which did not provide a concentration distribution adjustment part, (B) is a case of the Example which provided the concentration distribution adjustment part. It is a figure which shows schematically the micro reaction apparatus which employ | adopted the passive sample introduction method in proposal, (A) is a perspective view shown as a perspective view, (B) is a principal part perspective view which shows the flow of a sample. It is a figure which shows the change of concentration distribution in the same passive sample introduction method, (A) is a top view which shows a sample introduction part, (B) is a principal part top view which shows the change of concentration distribution in a branch part.

Explanation of symbols

2 Sample introduction port 4a Sample introduction unit 6 Analysis well 7 Sample discharge port 9 Sample discharge channel 8, 10, 12 Branch part 20a, 20b, 22a-20d Channel between branch unit 24 Concentration distribution adjustment unit 30a, 30b Partition wall 32a Main flow path 32b, 32c Sub flow path 34 Clearance

Claims (5)

A plurality of analysis wells formed in the substrate, and a sample introduction unit that distributes all of the analysis wells while flowing a particle dispersion fluid sample introduced from the sample inlet and dispersed in the transport fluid in the substrate. In a microreaction apparatus having a sample discharge channel connected to a sample discharge port from all the analysis wells,
The sample introduction part has a flow path structure that has at least two stages of branch parts and branches from the sample introduction port to all the analysis wells stepwise and evenly.
The flow path between the branch parts in the sample introduction part is provided with a concentration distribution adjusting part for adjusting the fine particle concentration distribution in the flow path cross-sectional direction in the flow path,
The concentration distribution adjusting unit includes a main flow channel through which the fine particle dispersed fluid sample flows, and a sub flow channel separated from the main flow channel by a partition on the upstream side and joined on the downstream side on at least one side wall of the main flow channel. Is provided with a gap that does not allow the passage of fine particles in the sample, so that only the transport fluid flows from the main channel into the sub-channel, and the transport fluid in the sub-channel flows downstream of the main channel flow downstream. A microreactor characterized in that it merges with the above.   The sub-channel is provided on both side walls of the main channel, and joins the main channel from the sub-channel on the side where the fine particle concentration in the cross-sectional direction of the fluid sample before entering the main channel is high 2. The at least one of the size of the sub flow channel and the size of the gap between the partition walls is adjusted so that the flow rate to be generated is larger than the flow rate of the flow from the other sub flow channel to the main flow channel. Micro reactor.   2. The micro of claim 1, wherein the sub-channel is provided only on one side wall side of the main channel on the side where the fine particle concentration in the channel cross-sectional direction of the fluid sample before entering the main channel is high. Reactor.   The microreaction apparatus according to any one of claims 1 to 3, wherein the sample discharge channel has a channel structure that repeats merging stepwise and evenly toward the sample discharge port.   The sample is a cell dispersion solution in which living cells are dispersed in a cell culture solution, the analysis well is a cell analysis container for analyzing the sample cell, and the microreaction apparatus constitutes a cell analysis apparatus. The microreaction apparatus according to any one of 1 to 4.