JP2010279908A - Three-dimensional sheath flow forming structure and method for collecting fine particles - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a three-dimensional sheath flow forming structure which can be produced easily by an extremely simple method and in which a sample solution can be collected in the central part of a flow path only by pouring a sheath liquid thereinto. <P>SOLUTION: The sheath flow forming structure for collecting a sample solution flow to the central direction of the flow path includes such a first structure that sheath liquid introduction flow paths, each of which has a plane common with a first plane of the flow path including the sample solution flow and which are arranged at different steps (depths), are joined from both sides of the flow path including the sample solution flow to collect the sample solution flow to the central direction of the flow path by a sheath liquid flow, in at least one region of the first collection region and the second collection region. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention has a three-dimensional sheath flow structure, particularly a three-dimensional sheath flow forming structure that is a multistage structure that does not include a hollow structure and can be economically manufactured by processing by a single transfer process, and the structure. The present invention relates to a microsystem (such as a microfluidic device or a microchip), and a particle focusing method using these.

With the recent development of microfabrication technology, a micron-order liquid flow path is formed on a chip such as glass or resin, and the sample is allowed to flow through the liquid flow path to perform sample analysis, reaction, or separation. System development is underway. Such a microsystem has an advantage that the sample can be analyzed even if the amount of the sample is small, and the sample can be operated while being confined in the chip.

By the way, in general, when cells are fluorescently labeled, the fluorescence is extremely weak and it is very difficult to detect this fluorescence without blurring or blurring. Therefore, in order to perform detection reliably, it is essential to use a high-power lens and increase the S / N ratio. However, a high-power lens has a shallow depth of focus, and a fluorescent material that is far from the focal plane appears blurry, resulting in a decrease in luminance.

Further, in such a micro system, there is a possibility that the detected object is detected twice if the detected object passes through the detection area at a low speed and is not detected if it passes through the detection area at a high speed. Thus, the detection object passing through the detection area at different speeds prevents accurate detection.

Furthermore, polydimethylsiloxane (PDMS), which is most commonly used as a material for forming a channel in such a microsystem, has a strong adsorptive property, and if only a sample stream is flowed into the channel, most of the detected substance flows. It can be considered to adsorb to the road wall. Further, if the collected material is stuck to the wall surface in the detection area, the fluorescence is always detected in the detection area, so that sorting is not possible.

In the sheath flow, the sample solution and the fine particles are focused in the vicinity of the top of a parabolic flow velocity distribution known as a Poiseuille flow through the microchannel, so that the time for the fine particles to pass through a specific region can be made uniform. is there. Therefore, in such a microsystem, particularly when flowing fine particles such as cells and analyzing individually, by using a sheath flow configuration in which the sample solution and the fine particles are focused on the center of the microchannel by the sheath flow, the depth of focus can be reduced. The width can be reduced, the shearing force on the channel wall surface is not received, the sample flow rate can be kept constant, the sample adsorption by PDMS can be prevented, and the shearing force on the channel wall surface can be avoided. Can be resolved.

As described above, the sheath flow is important for quantitative analysis of optical information generated from, for example, individual fine particles and high-precision separation of individual fine particles.

A number of methods for forming a sheath flow and fine particle focusing in a microsystem have been reported so far. For example, a method of processing a microchannel three-dimensionally and enclosing a sample solution and fine particles from four directions by a sheath liquid flow (for example, Patent Document 1, Non-Patent Documents 1 and 2) or two-dimensional processing For example, there is a method of focusing fine particles on the center of a microchannel using a flow velocity distribution (Non-patent Document 3). Further, the method of focusing the sample solution and the fine particles by a multi-stage structure that can be processed by a single transfer process (Non-patent Document 4) is that the sample solution and the fine particles are placed in the center of the lower part of the flow path from the three directions of the upper and side of the flow path. Provides a single-sided structure that converges to

JP 2007-514522 A

Micro Total Analysis systems 2000, 209-212 Lab on a chip, 2009, 9, 972-981, DOI: 10.1039 / b808336c Lab on a chip, 2007,7, 1260-1262, DOI: 10.1039 / b711155j Sensors and Actuators A: 135 (2007) 99-105, DOI: 10.1016 / j.sna.2006.06.074

However, the conventional method using a micro flow path as described in Patent Document 1 or Non-Patent Documents 1 and 2 processes a step structure on both substrates when processing, or a hollow structure on one substrate. There is a drawback that it takes a lot of time and effort. In particular, when a double-sided substrate is processed, a step (“alignment” step) is required in which the positions of the structures on both sides are aligned with high accuracy. On the other hand, the method using a micro flow path subjected to two-dimensional processing as described in Non-Patent Document 3 is easy to process and does not need to be aligned. The body has the difficulty of requiring a high flow rate of 0.837 mL / min (= 1.86 m / sec) in order to focus fine particles. In addition, the method described in Non-Patent Document 4 cannot narrow the sample solution and the fine particles from the lower part of the flow path toward the center of the flow path.

  As a result of research to solve the above problems, the present inventor has conducted a sheath flow for focusing the sample solution and the fine particles on the center of the flow path only by a single-stage multi-step (step) structure that does not include a hollow structure by a single transfer process. The present invention has been completed by finding that it can be formed.

That is, the present invention relates to the following aspects.
[Aspect 1]
A sheath flow forming structure for focusing a sample solution stream toward the center of the flow path, having at least a first focusing area or a second focusing area:
(1) At least one of the first focusing area and the second focusing area has a surface common to the first surface of the flow path containing the sample solution (sample) flow, and a step (depth). A first structure in which a sheath liquid (carrier) introduction flow path having a different flow rate is joined from both sides of the flow path including the sample solution flow, and the sample solution flow is focused toward the center of the flow path by the sheath liquid flow;
(2) In the first focusing region or the second focusing region not having the first structure, the sheath liquid is merged from three surfaces other than the first surface of the flow path including the sample solution flow, A second structure for converging the sample solution flow from the three directions of the second surface facing the first surface and the side surface with respect to the first surface to the center direction of the flow path by the sheath liquid flow; The sheath flow forming structure.
[Aspect 2]
A microsystem having the sheath flow forming structure according to aspect 1.
[Aspect 3]
A method for manufacturing a sheath flow forming structure according to aspect 1 or a microsystem according to aspect 2, which includes joining a substrate provided with a step structure and a planar substrate.
[Aspect 4]
A method for focusing particles using the sheath flow forming structure of Aspect 1 or the microsystem of Aspect 2.

The sample solution can be focused on the central portion of the flow path simply by pouring the sample solution and the sheath liquid into the sheath flow forming structure of the present invention. Furthermore, the sheath flow forming structure of the present invention can be easily manufactured by an extremely simple method of joining a substrate provided with a step structure and a flat substrate, and the microchip having the sheath flow forming structure The number of steps for manufacturing a microsystem such as the above is drastically reduced, and reproducibility and productivity in the manufacturing process are extremely high.

The specific example of the 1st structure contained in the sheath flow formation structure of this invention is shown. The specific example of the sheath flow formation structure of this invention is shown. One specific example of the sheath flow formation structure of this invention is typically shown. Here, the first focusing region, the second focusing region, and the first focusing region correspond to a, b, and c in the drawing, respectively, and the carrier flow is a sheath liquid flow. An example of the sheath flow formation structure of this invention containing each flow path is shown typically. Here, “focus” means a focusing area. The simulation of the function of the 1st structure in the sheath flow formation structure of the present invention using fluid simulation software (Coventor Ware, Marubeni Information Systems Co., Ltd.) is shown. An example in which a sheath flow is actually formed using a fluorescent dye as a sample in the sheath flow forming structure of the present invention will be described. An example in which a sheath flow is actually formed using fluorescent beads as a sample in the sheath flow forming structure of the present invention will be described. It is the graph which compared the linear velocity of the fluorescent bead by the presence or absence of a sheath flow. The result of the optimization of the ratio of the depth ratio of the flow path including the sheath liquid introduction flow path and the sample solution flow in the first structure of the sheath flow forming structure is shown. Here, Y axis: position (boundary) in the channel height direction (boundary) (μm), X axis: height of carrier channel (μm), and%: relative concentration of the sample defining the boundary. In each graph in the figure, the arrow indicates the height of the carrier flow path where the amount of transition upward in the flow path when the sample relative concentration is defined as 0.00% is reduced. The sample relative concentrations are 0.50, 0.25, 0.10, 0.05, 0.01, and 0.00% in order from the top in each graph.

The main feature of the sheath flow forming structure for focusing the sample solution flow according to the present invention toward the center of the flow path is that the flow path including the sample solution flow in at least one of the first focusing area and the second focusing area. The seed liquid introduction flow path having a surface common to the first surface and having different steps ("depth" or "height") is joined from both sides of the flow path including the sample solution flow. And having a first structure that focuses the sample solution flow toward the center of the flow path by the sheath liquid flow.

In the first structure described above, in the downstream region after the sample solution flow and the sheath liquid flow merge, the sample solution flow is reduced as long as the sample solution flow can be focused toward the center of the flow path by the sheath liquid flow. There is no particular limitation on the angle at which the sheath liquid introduction flow path having the same surface as the first face of the flow path including the difference and the step difference joins the flow path including the sample solution flow. In order to more efficiently focus the sample solution flow toward the center of the flow path by the sheath liquid flow, the sheath liquid introduction flow path is at an angle of approximately 45 degrees to approximately orthogonal to the flow path including the sample solution flow. It is preferable to join together. A specific example is shown in FIG.

Depending on the conditions of the microsystem having a sheath flow forming structure (for example, a microchip), its purpose of use and state, etc., the first surface of the first structure has a flow path including a sample solution flow. It corresponds to either the upper surface, the lower surface, or the side surface.

In the first structure, in order to focus the sample solution flow toward the center of the flow path by the sheath liquid flow, preferably to a position slightly below the center (50 μm) in the flow path height direction. The depth of the sheath liquid introduction channel is preferably shallower than that of the channel containing the sample solution flow. That is, it usually includes the sample solution flow, although it depends on the conditions of the microsystem type (for example, microchip), its purpose and state of use, and the relative proportions of the sheath liquid flow and the sample solution flow. The step (depth) of the flow path is 5 times or more, preferably about 5 to 20 times the step of the sheath liquid introduction flow path. The relative ratio of the sheath liquid flow (total amount from both sides) and the flow rate of the sample solution flow can be set as appropriate, but is usually in the range of 0.5: 1 to 2: 1. Preferably it is about 1: 1.

In the sheath flow forming structure of the present invention, in the first focusing region or the second focusing region that does not have the first structure, the three surfaces other than the first surface of the flow path including the sample solution flow are used as a shim. A second structure for converging the sample solution from the three directions of the second surface facing the first surface and the side surface with respect to the first surface by the sheath liquid flow, toward the center of the flow path; Take.

The first structure can be provided in either the first focusing region or the second focusing region. For example, FIG. 2 shows an example in which the first structure is provided in the second focusing area (second stage narrowing) and the second structure is provided in the first focusing area (first stage narrowing).

  Alternatively, both the first focusing area and the second focusing area can be the first structure. In such a case, the first surface where the flow path including the sample solution flow and the sheath liquid introduction flow path are common to each other in each focusing area (for example, the upper surface in the first focusing area, And the lower surface in the second focusing region.

Furthermore, in the sheath flow forming structure of the present invention, in addition to the first focusing area and the second focusing area, a third focusing area is provided, and a step in the flow path (in which the sheath flow formed in the second focusing area flows ( It is also possible to reduce the depth) so that the sample solution flow flows more centrally in the sheath flow. An example of such a structure is schematically shown in FIG.

The sheath liquid that merges with the flow path including the sample solution flow in the first focusing area and the second focusing area may be different paths, but usually the first focusing area after branching upstream. In addition, it is preferable to have a structure that joins the flow path including the sample solution flow in the second focusing region.

Such a sheath flow forming structure can be used for various purposes and applications by being incorporated into various types of microsystems for performing sample analysis, reaction, or separation. Examples of such microsystems include, for example, FACS (Fluorescence Activated Cell Sorter) used for cell purification / separation, which is an important technique in proteomic analysis, and temperature sensitive gels (eg, Meviol (registered trademark)). Can be mentioned as a μ-TAS cell sorter. As the sample, for example, various particles and fine particles such as cells and various organelles can be used.

  In general, in the sheath flow forming structure or microsystem of the present invention, the width, depth, etc. of each flow path of the sample solution flow, the sheath liquid flow, and the sheath flow flow are determined depending on the type of the sample solution and the particles contained therein, the micro Although those skilled in the art can appropriately determine the system according to the purpose and application of the system, the channel width is usually 10 to 100 μm and the channel depth is 5 to 100 μm.

  FIG. 4 schematically shows an example of the sheath flow forming structure of the present invention including each flow path (the depth is three types of 100 μm, 50 μm, and 5 μm).

The microsystem includes optional elements such as a sample solution injection path, a sample solution discharge path, a sheath liquid injection path, a waste liquid port, a recovery flow path, and a sample detection unit, depending on the purpose and application. It is also possible to consist of

For example, in the detection of the sample separated in the microsystem of the present invention, for example, light from a light source is incident on an analysis channel, and fluorescence in the analysis channel is detected by a detector, and an electrode is installed in the channel. Any means known to those skilled in the art can be used, such as one that inserts and measures the amount of electrochemical change, or one that introduces and measures the solution from the flow path to the mass spectrometer.

  The sheath flow forming structure or microsystem of the present invention can be produced by methods known to those skilled in the art. In particular, when the first structure is provided only in one of the first focusing area and the second focusing area and the second structure is provided in the other area, the hollow structure is included. It is possible to manufacture a single-sided substrate provided with a non-multistage (step) structure by an extremely simple method of bonding to the other planar substrate.

For example, a template is produced by microfabrication using photolithography on various glass, quartz, and Si substrates, and this is transferred to an appropriate resin such as polydimethylsiloxane (PDMS). By bonding a substrate having a structure to a flat substrate by an arbitrary method such as plasma bonding, the sheath flow forming structure or the microsystem of the present invention can be easily manufactured.

For example, photolithographic molds can be prepared by, for example, applying photoresist, patterning separation channels (exposure, development), Si etching, resist removal, photoresist coating, injection hole patterning, Si etching, resist removal, surface oxidation, anodic bonding. This process can be performed.

Polydimethylsiloxane (PDMS) is a kind of silicone rubber, and a microstructure can be manufactured by molding (molding), and a sub-micron structure can be transferred. Because of its self-adsorbing property, it can be sealed by simply sticking it to the substrate when large internal pressure is not required. In addition, it is colorless and transparent, has little absorption in the visible light region, hardly shows autofluorescence, and is a biocompatible material that does not adversely affect cells and tissues. Therefore, it is suitable for fluorescence detection used in the bio field. Furthermore, unlike glass and plastic chips, it can be made relatively easily without the need for wet etching or high temperature bonding.

  Hereinafter, the present invention will be described in detail with reference to reference examples and examples. However, the technical scope of the present invention is not limited to these examples.

Simulation is performed using fluid simulation software (Coventor Ware, Marubeni Information Systems Co., Ltd.), and the first solution in the sheath flow forming structure of the present invention causes the sample solution flow to flow in the center direction of the flow path. It was confirmed that it converged to (Fig. 5). In the figure, the range in which the sample solution flows is shown as the concentration of the sample in the flow path cross-sectional view when the sheath liquid and the sample solution are injected at the same flow rate. In addition, the direction of the flow in the cross section of the flow path at that time is indicated by →. This simulation showed that most of the sample solution was shifted toward the upper part of the flow channel downstream by injecting the sheath liquid from the lower part.

In the sheath flow forming structure of the present invention shown in FIG. 4, a sheath flow was actually formed using an aqueous solution of a fluorescent dye (rhodamine 6G) as a sample solution and water as a sheath solution. It can be seen that rhodamine 6G is focused near the center of the channel (FIG. 6).
Carrier solution flow rate: Sample solution flow rate = 20 μL / min: 1 μL / min.

An experiment similar to that of Example 2 was performed using an aqueous solution of fluorescent beads having a diameter of 10 μm as the sample solution. The beads are focused near the center of the flow path (FIG. 7). Carrier solution flow rate: Sample solution flow rate = 5 μL / min: 1 μL / min.

The same experiment as in Example 2 was performed using an aqueous solution of fluorescent beads having a diameter of 2 μm as a sample solution, and the linear velocity of each fluorescent bead with or without narrowing by sheath flow was measured in the same microsystem. It can be seen that the variation in the linear velocity of the fluorescent beads passing through the flow path is narrowed by narrowing down by the sheath flow (FIG. 8). Carrier solution flow rate: Sample solution flow rate = 3.5 μL / min: 0.5 μL / min or 0 μL / min: 4 μL / min.
Note that “no sheath flow” was performed in the same system by stopping the flow of the sheath liquid, and instead increasing the flow rate of the sample solution so that the final flow rate after merging was the same.

  Further, in the first structure of the sheath flow forming structure of the present invention, the ratio of the depth of the flow path including the sheath liquid (carrier) introduction flow path and the sample solution (sample) flow is optimized as in the first embodiment. The simulation was performed using the same software. That is, the shredding was performed under the following conditions. The sample concentration was displayed as a color map, and the boundary with the carrier was calculated from the concentration.

Sample channel width: 50 μm Sample channel height: 100 μm Carrier channel width: 50 μm
Carrier channel height: 2.5, 5.0, 10, 15, 20, 25, 30, and 50μm
Convergence shape: 90 degrees (no significant difference in the final distribution even at 45 degrees)
Flow rate: Carrier: Sample: Carrier (3 types below)
(1) 1.0 μl / min: 1.0 μl / min: 1.0 μl / min = 1: 1: 1
(2) 0.5μl / min: 1.0μl / min: 0.5μl / min = 0.5: 1: 0.5
(3) 0.25μl / min: 1.0μl / min: 0.25μl / min = 0.25: 1: 0.25

  From the results shown in FIG. 9, it was found that the greater the inflow amount of the carrier, the easier the transition of the central portion of the sample flow upwards the flow path even if the height of the carrier flow path to be merged is large. Further, at the carrier flow path height at which the center portion of the sample flow cannot be shifted upward, both ends of the sample flow are shifted upward. In this simulation, the carrier flow rate to sample flow rate ratio of 0.5: 1 is 5 μm deep, 1: 1 is 15 μm, and 2: 1 is 30 μm. Was shown to increase. When actually forming a sheath flow, it is desirable to make the sample flow transition to a position slightly below the center (50 μm) in the flow path height direction, so a simulation with a flow ratio of about 0.5: 1: 0.5 is suitable. Yes. From this, it can be said that it is desirable that the flow path of the sample solution has a height of 5 times or more than the carrier flow path to be joined.

The sheath flow forming structure of the present invention can be used for various purposes in fields such as microsystem biochemistry, pharmacy, and medicine.

Claims (13)

A sheath flow forming structure for focusing a sample solution stream toward the center of the flow path, having at least a first focusing area or a second focusing area:
(1) At least one region of the first focusing region or the second focusing region has a surface common to the first surface of the flow path including the sample solution flow and has a different step (depth). -A first structure in which the flow of the sample solution is merged from both sides of the flow path including the sample solution flow, and the sample solution flow is converged toward the center of the flow path by the sheath liquid flow;
(2) In the first focusing region or the second focusing region not having the first structure, the sheath liquid is merged from three surfaces other than the first surface of the flow path including the sample solution flow, A second structure for focusing the sample solution flow from the three directions of the second surface facing the first surface and the side surface of the first surface by the sheath liquid flow toward the center of the flow path. The sheath flow forming structure. In the first structure, the sheath liquid introduction flow path having a surface common to the first face of the flow path including the sample solution flow and having different steps is provided between the flow path including the sample solution flow and the flow path direction. The sheath flow forming structure according to claim 1, wherein the sheath flow is joined from a substantially orthogonal direction. The sheath flow forming structure according to claim 1 or 2, wherein the first surface corresponds to an upper surface or a lower surface of a flow path containing a sample solution flow. The sheath according to any one of claims 1 to 3, wherein, in the first structure, the step of the flow path including the sample solution flow is at least five times the step of the sheath liquid introduction flow path. Flow forming structure. The sheath flow forming structure according to any one of claims 1 to 4, wherein the first structure is provided in the second focusing region, and the second structure is provided in the first focusing region. 6. The sheath flow forming structure according to claim 1, wherein after the sheath liquid branches upstream, the sheath liquid joins the flow path including the sample solution flow in the first focusing region and the second focusing region. Further, the third converging has a third structure in which the step (depth) of the flow path through which the sheath flow formed in the second converging region flows is reduced, and the sample solution flow flows in the central part of the sheath flow. The sheath flow forming structure according to claim 1, further comprising a region. A microsystem having the sheath flow forming structure according to any one of claims 1 to 7. The method for manufacturing a sheath flow forming structure according to any one of claims 1 to 7 or a method for manufacturing a microsystem according to claim 8, comprising joining a substrate provided with a step structure and a planar substrate. 9. The manufacturing method according to claim 8, wherein the substrate provided with the step structure is made of a resin transferred from a mold manufactured using photolithography, and the planar substrate is a glass substrate. The production method according to claim 9, wherein the resin is polydimethylsiloxane. A particle focusing method using the sheath flow forming structure according to any one of claims 1 to 7 or the microsystem according to claim 8. The particle focusing method according to claim 12, wherein the particle is a cell or various organelles.