JP2011196846A - Detection container and method for detecting fine particulate sample using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To hold a fine particulate sample to a sample detection part, without fail, so as to obtain a proper detection result, even in a case where the substitution of a sample treatment solution in a detection container is required a plurality of times.SOLUTION: The detection container has a branched microflow channel which has a flow channel having a branch part for forming a sample holding part and the recessed sump-like sample holding part arranged to the branch part for forming the sample holding part, formed thereto, and the fine particulate sample is detected by a detection method by using the detection container.

Description

  The present invention relates to a detection container for detecting various particulate samples by qualitative, quantitative reaction, physical property measurement, or the like, and a detection method using the same. In particular, the present invention relates to the detection of minute biological samples (biological samples), trace chemical substances attached to fine particles, and the like.

  As a technique for analyzing or detecting the above-mentioned fine biological sample, a disk-shaped micro reaction container called a microwell array having a plurality of microchannels and microchambers serving as sample detection units is known ( Patent Document 1). In addition, in the immunoassay method, an antibody is immobilized on microbeads, and the antigen-antibody reaction is carried out with the microchannel and sample detection part having a predetermined shape so that the microbeads can be efficiently held in the sample detection part. The technique to perform is introduced (nonpatent literature 1).

JP 2008-185423 A

N. Matsunaga, S.M. Furutani, I.K. Kubo, “CENTRIFUGAL FLOW DEVICE FOR HIGH-THROUGHPUT DETECTION OF CANCER MARKER CEA”, ECS Transactions, 2008, 16 (11), p. 123-128

  When detecting a particulate sample, it may be necessary to replace (replace) a so-called sample processing solution such as a cleaning solution, a reaction solution, or a detection solution after introducing the particulate sample into the sample detection unit. In such a case, if the flow path in the micro flow path and the sample detection unit have an arrangement relationship as described in Patent Document 1, the particulate sample is discharged from the sample detection unit by replacing the sample processing solution a plurality of times. There is a problem that detection becomes impossible.

  Non-Patent Document 1 describes that in order to hold microbeads on which antibodies are immobilized in a sample detection unit, the height of the flow path beyond the sample detection unit is equal to or smaller than the diameter of the microbeads, that is, the clearance is extremely small. Technology is used. However, with such a structure, when the sample is a cell or the like, there is a problem that the cell dies due to being sandwiched by this clearance.

  Accordingly, an object of the present invention is to ensure that the particulate sample is reliably held in the sample detection unit (sample holding unit) even when the sample processing solution is replaced a plurality of times, thereby enabling good sample detection. The object is to provide a detection container and a detection method.

  In order to solve the above-mentioned problems, the present inventors have conducted extensive research, and as a result, a detection container having a branched microchannel in which a sample holding unit for sample detection is arranged at a branching portion of the channel, and the detection A method for detecting a particulate sample using a container was developed and the present invention was completed.

  That is, according to the present invention, the plate having the sample injection part is connected to the sample injection part and has a flow path having the sample holding part forming branch part and the recess arranged in the sample holding part forming branch part. There is provided a detection container in which a branched microchannel is formed, which has a reservoir-like sample holder and a sample discharge port at an end opposite to a connection end between the channel and the sample injection unit. The branched microchannel is preferable because it is formed inside a plate-like plate, thereby preventing leakage or evaporation of the sample liquid or the like. In this application, the term “branch microchannel” refers to the entire basic configuration including the channel, the sample holding unit, the branching unit, and the discharge port. When not included, it is simply referred to as “channel”.

  Further, the plate is a disk-shaped plate, the sample injection portion is disposed in an area on the center side of the disk-shaped plate, and the sample discharge port of the branch microchannel is on an outer peripheral portion of the disk-shaped plate. The flow path is divided into two flow paths at the sample holding portion forming branch portion, and the sample holding portion is recessed in the outer peripheral direction of the disk-shaped plate at the sample holding portion forming branch portion. It is suitable to arrange in a shape.

  Further, the two flow paths branched by the sample holding part forming branch part may be configured to join before reaching the sample discharge port.

  Further, the branch microchannel has a plurality of branch parts in a tournament type hierarchy, and the branch part for forming the sample holding part and the sample holding part are arranged in a layer part closest to the outer peripheral part of the disc-shaped plate. It is suitable that it is arranged.

  The branch microchannel may have a branch portion where the sample holding portion is not disposed.

  Alternatively, a configuration may be adopted in which the sample holders are arranged in branch portions other than the sample holder formation branch portion of the branch portions arranged in a tournament type hierarchy, and all of the branch portions are arranged. A sample holder may be arranged.

  Furthermore, it is preferable that the detection container is formed with a plurality of branched microchannels having the respective configurations.

  According to another aspect of the present invention, a sample injection part, a flow path having a sample holding part forming branch part, a recessed sample holding part arranged in the sample holding part forming branch part, and A step of injecting a sample liquid containing a particulate sample into the sample injection portion using a detection container formed with a branched microchannel having a sample discharge port; and A step of feeding in the direction, a step of holding the particulate sample in the sample holder, a step of filling the sample solution in the branch microchannel, and a sample processing solution having different components from the sample solution Injecting the sample from the sample injection section, replacing the inside of the branch microchannel with the sample processing liquid, causing the sample processing liquid to act on the particulate sample held in the sample holding section, Capturing a response due to action Method of detecting a particulate sample is provided.

  For detection of the particulate sample, it is preferable to use a disc-shaped plate detection container. At this time, the liquid feeding of the sample liquid and the sample processing liquid is preferably performed by rotating the disk-shaped plate and feeding it by centrifugal force.

  Further, the branch microchannel has a plurality of branch portions in a tournament type hierarchy, and the branch portion for forming the sample holding portion and the sample holding portion are arranged in a hierarchy portion closest to the outer peripheral portion of the disc-shaped plate. It is preferable to use a detection container in which is disposed.

  Still further, it is preferred that the particulate sample to be detected is a biological sample.

  According to the detection container and the detection method of the present invention, the particulate sample is reliably held in the sample holding part, which is the detection part, even when the sample processing liquid is replaced a plurality of times in the detection container. There is an effect that the particulate sample can be detected well. For example, it is possible to easily detect various cells, bacteria, fungi, viruses, genes, or the like as they are or on a microcarrier such as microbeads.

FIG. 3 is a schematic diagram schematically showing a flow of a sample holder forming branch connected to a flow path, a sample holder, a particulate sample held in the sample holder, and a sample liquid. (B) is a figure which shows the direction of the force which acts on the branch part for the said sample holding part formation. It is a schematic diagram which shows roughly the branch microchannel connected to a sample injection part. It is a mimetic diagram showing roughly one embodiment of a disc shaped plate type detection container concerning the present invention. It is a schematic diagram which shows schematically another embodiment of the disc-shaped plate type | mold detection container which concerns on this invention which has a some branch part in the tournament-type hierarchy. It is a mimetic diagram showing roughly an example of a branch microchannel which has a branching part where a sample holding part is not arranged. It is a schematic diagram which shows roughly an example of a branch microchannel by which a sample holding | maintenance part is arrange | positioned at all the branch parts. It is a figure which shows a mode that a microbead is hold | maintained in a sample holding | maintenance part by a microscope picture, when liquid exchange (substitution) is performed in multiple times. It is a figure which shows a mode that a cell is hold | maintained in the state which survived in the case where liquid exchange (substitution) of multiple times was implemented with a fluorescence micrograph. It is a schematic diagram which shows roughly the arrangement | positioning relationship between the microchannel of a prior art, and a sample holding part (microchamber). FIG. 6 is a schematic diagram schematically showing the arrangement relationship between a microchannel and a sample holding unit (microchamber) and a state in which a particulate sample is held in the sample holding unit in another conventional technique. (A) is the top view seen from the top, (b) is sectional drawing of the arrow direction of a XX line.

  In the following, embodiments of the present invention will be described with reference to the drawings, with respect to important points for solving the problems and features that are different from the prior art.

  FIG. 1 is a diagram showing main components representing the features of the present invention. First, as shown by the arrows in FIG. 1, the sample liquid is fed from the flow channel 1 on the sample injection unit side toward the flow channel 2 after branching. At this time, the particulate sample 4 is transferred to the sample holding unit 3. Captured and retained. After the particulate sample 4 is held, a sample 6 such as a cleaning solution or a reaction solution passes through the channel 1 while a force 6 such as gravity or centrifugal force acts in the direction of the wide arrow shown in FIG. When the liquid is fed, the sample liquid in the sample holder is gradually replaced with the sample processing liquid. However, since the particulate sample 4 has the force 6 acting in the direction of the wide arrow, it can be maintained in the sample holding unit 3 without being discharged to the channel 2 side by the sample processing liquid. it can.

  As a result, the particulate sample 4 remains in the sample holder 3 and can react with the sample processing solution. It is possible to qualitatively identify or quantify the particulate sample 4 by using a sample processing solution in which a response unique to the particulate sample 4 is seen and capturing the response. Become.

  FIG. 2 shows an overall view of one (one set) of branched microchannels connected to the sample injection section 7. A sample solution containing the particulate sample 4 is injected into the sample injection unit 7, and the sample is discharged through the channel 1, the sample holding unit forming branch unit 5, and the channel 2 by a force such as compressive force, gravity, or centrifugal force. It leads to exit 9. FIG. 2 (b) shows a branched micro flow channel in a form in which the two flow channels branched by the sample holding portion forming branch portion 5 join before reaching the sample discharge port 9. In the present invention, the branched micro flow channel in any form of FIGS. 2A and 2B may be used.

  On the other hand, FIG. 9 shows an arrangement relationship between the flow path 31 and the microchamber (sample holding part) 33 in the microwell array (microreaction vessel) according to the prior art. 34 is discharged from the microchamber at the time of replacement with the sample processing solution. That is, in such a structure, since the flow of the liquid is a flow that scoops up the particulate sample 34, it cannot be applied when it is necessary to detect a target substance by performing a plurality of liquid replacements.

  An embodiment of the detection container according to the present invention will be described with reference to FIG. FIG. 3 is a schematic plan view of the detection container 10 as viewed from above. A plurality of branch microchannels (24) in which one branch portion 15 for forming a sample holding portion is formed on a disk-shaped plate. This is an example. Note that the branching micro flow path including the flow paths 11 and 18 and the sample holding portion forming branching portion 15 is formed inside the disc-shaped plate, and is arranged on the outer periphery of the sample injection portion 17 and the detection container. The sample outlet 19 is open to the atmosphere. In addition, although the flow paths 11 and 18 are drawn by one line, and the sample injection part 17 and the sample holding | maintenance part 13 are painted black, it is the expedient for drawing many branch micro flow paths, Actually, as shown in FIG. 2B, there is a space in which the sample liquid or the like flows and is held. The same applies to FIGS. 4, 5 and 6.

  Taking the detection container 10 as an example, the detection container and the method for detecting the particulate sample 4 of the present invention will be described in detail. First, as the material of the detection container 10, any material such as a polymer material such as a thermoplastic resin or a thermosetting resin, ceramics, glass, silicon, or metal can be used. Glass, silicon, polymer material, or a combination thereof is preferable in terms of hermeticity and fine workability. As the polymer material, for example, siloxanes such as polydimethylsiloxane (PDMS) can be used. The detection container 10 is preferably configured so that at least one surface is transparent in order to visually or optically observe the state of the branched microchannel including the sample holding portion forming branch portion 15 and the like. . In other words, it is conceivable to use a transparent material for all, or select a method for producing a disc-shaped transparent material and an opaque material.

  In the present invention, it is preferable that the sample solution containing the particulate sample 4 and the sample processing solution are injected into the sample injection portion 17 and then fed by centrifugal force by rotating the disc-shaped detection container 10. . This is because the particulate sample can be captured and held efficiently and uniformly by the sample holder 3 (13), and the working time can be shortened. Therefore, the detection container 10 is preferably a thin disk shape, but is not limited to a disk shape (disk shape), and is a polygon such as a top view, a triangle, a quadrangle (for example, a square), a regular hexagon, a regular octagon, and the like. It may be a thin plate.

  Although the detection container 10 of FIG. 3 has 24 independent branch microchannels, the number of branch microchannels may be 1 to 23 or more than 24 in the detection container. Multiple independent branches in that one sample vessel can analyze many samples simultaneously or prepare and analyze a series of sample solutions or sample treatment solutions with different conditions It is preferable to have a microchannel. Further, in order to form as many channels as possible, it is preferable that the plurality of independent branch microchannels be formed in the detection container in a symmetrical or equivalent positional (or arrangement) relationship. For example, it is preferable that the sample injection unit and the sample injection unit are arranged symmetrically with respect to the center point of the disk-shaped detection container as in the detection container 10. In the case of rotational symmetry, the number of times is determined by the number of branch microchannels. For example, when four branched microchannels are arranged, it is preferable to arrange them four times in rotational symmetry.

  Further, the detection container 10 has a configuration in which the flow channel 12 branched by the sample holding portion forming branching portion 15 joins before reaching the sample discharge port 19 to form the flow channel 18, but the flow channel 12 joins. Instead, it may be a form that reaches the sample discharge port 19. In FIG. 3, the sample discharge port 19 is arranged on the outer peripheral side surface of the detection container. It may be allowed to be discharged. The same applies to the detection container of the form shown in FIG. 4 described later and the detection container according to the scope of the present invention.

  An example of a method for detecting the particulate sample 4 using the detection container 10 is as follows, for example. First, a sample solution containing the particulate sample 4 is injected into the sample injection unit 17, and the detection container 10 is set on a rotation stage. Alternatively, the sample solution may be injected after the detection container 10 is placed on the stage. Examples of the fine sample 4 include a fine sample in which a chemical substance is supported on a microbead, a cell, or a virus, and the fine sample 4 is suspended or dispersed in the sample solution. In addition, the cells or the like may be used as they are or may be supported on a very small carrier such as microbeads.

  Subsequently, the detection container 10 is rotated for several hundred to several thousand rotations for about several seconds to one minute, and the sample liquid containing the particulate sample 4 is fed by centrifugal force. The rotation is, for example, a procedure for rotating a CD for reproduction, or a procedure for spin coating in a semiconductor manufacturing process. For example, a spin coater or a similar device can be used as the rotation device. By the rotation operation, the sample solution containing the particulate sample 4 is fed, and the particulate sample 4 and the sample solution are held in the sample holder 13. Note that the sample liquid that does not enter the sample holder 13 passes through the flow path 12 and the like and is discharged from the sample discharge port 19.

  When it is necessary to wash the particulate sample 4 held in the sample holder 13, the cleaning liquid is sent from the sample injection part 17 in the same manner, the sample liquid in the sample holder 13 is replaced with the cleaning liquid, and the particulate form is used. Sample 4 is washed. What is necessary is just to determine suitably the liquid feeding frequency (replacement frequency) of a washing | cleaning liquid so that the cleaning degree required for each detection may be achieved.

  When the desired cleaning is completed, a sample processing solution such as a reaction solution for detecting the particulate sample 4 is injected into the sample injection unit 17 and sent in the same manner as described above. At this time, the sample processing solution may be sent several times as necessary. As a result, the cleaning liquid in the sample holder 13 is replaced with the sample processing liquid, and the sample processing liquid acts on the particulate sample 4, and a response peculiar to the combination of the sample processing liquid and the particulate sample 4 is obtained.

  In addition, although the case where the cleaning operation with the cleaning liquid is performed has been described, naturally the cleaning operation may be unnecessary. Since the degree of substitution with the cleaning liquid and the sample processing liquid varies depending on each detection reaction and the like, the number of times of feeding the cleaning liquid and the sample processing liquid may be determined so as to satisfy the required conditions.

  By capturing a response peculiar to each detection, the target particulate sample 4 can be detected and qualitative or quantitative can be obtained. Examples of the response include fluorescence emission specific to the particulate sample 4, and examples of the capture include observation with a fluorescence microscope, camera photography, computer recording, and analysis. By the above method, accurate detection of the particulate sample 4 can be performed efficiently and simply.

  The maximum feature of the above-described detection method illustrating the detection container 10 will be described with reference to FIG. That is, the feature is that even if the cleaning liquid and the sample processing liquid are supplied a plurality of times and liquid replacement in the sample holding unit 3 is performed a plurality of times, the particulate sample 4 is not discharged to the flow channel 2 side, and the sample is held. This is because the state held in the portion 3 can be maintained. This is because the centrifugal force causes the force to act in the direction of the force 6 indicated by the wide arrow in FIG. 1B, so that even if the cleaning liquid or the sample processing liquid is sent to the sample holder 3, the particulate sample 4 It is thought that this is because there is no need to be drowned in the flow. That is, in the branch part 5 for forming the sample holding part, the flow path 1 before branching, the flow path 2 after branching, and the sample holding part 3 are arranged as shown in FIG. Only the retained liquid is replaced, and the effect that the particulate sample 4 is not discharged therefrom is obtained. FIG. 1 illustrates a case where the two flow paths 2 are divided into both sides at an angle of 180 ° immediately after the flow path 1 is branched into two flow paths 2. The angle on the sample holding unit 3 side (outer peripheral side) is preferably 180 ° or an obtuse angle from the viewpoint of the holding property of the particulate sample 4 described above.

  The detection method according to the present invention has been described above by taking the detection container 10 of FIG. 3 as an example. However, the detection container is not limited to the form of the detection container 10, and the detection container of FIG. A form such as 20 is also suitable. Furthermore, a detection container having a branched microchannel as shown in FIGS. 5 and 6 can also be suitably used, and the appearance of the detection container is not limited to a disk shape, and may be the polygonal shape described above. .

  As a detection method using these other types of detection containers, the method exemplified for the detection container 10 can be similarly applied. In the description of the detection method using the detection container 10 described above, the method using the centrifugal force due to rotation has been described. However, depending on the form of the detection container, for example, gravity may be used instead of the centrifugal force. . Alternatively, a method of applying a compressive force using a micropipette or the like when injecting a sample solution or a sample processing solution may be used. In view of simplicity of operation, homogeneity of processing operations, and capability of operation in a short time, it is preferable to use the centrifugal force of the rotation.

  Next, the detection container 20 of FIG. 4 which is another embodiment of the detection container according to the present invention will be described. FIG. 4 is a schematic diagram of a plan view of the detection container 20 as viewed from above, as in FIG. The detection container 20 of FIG. 4 is formed so that the flow path has a plurality of branch parts in a tournament type hierarchy from the sample injection part 27, and a sample holding part is formed in the hierarchy part closest to the outer peripheral part of the detection container 20. This is a configuration in which the branching portion 25 and the sample holding portion 23 are arranged. One (one set) of branched microchannels branches in three stages and has eight sample holding part forming branch parts 25. Twelve (12 sets) of these independent branching micro flow paths are formed point-symmetrically (12-fold rotational symmetry) with respect to the center point of the detection container, so that a total of 96 sample holding part forming branch parts 25 are provided. Is formed.

  The form as shown in FIG. 4 has an effect that the working time for injecting the sample liquid or the like into the sample injection part can be greatly shortened. The material of the detection container 20 is the same as that of the detection container 10 described above. Further, regarding the number of branches, the detection container 20 has three stages, but is not limited to this, and may have two stages or four or more stages. The practical size of the disc-shaped detection container is several cm to several tens of cm, preferably about 5 cm to 30 cm. Therefore, the number of stages of the above branching is also practically limited, and seven stages or less are considered common sense. . However, since it is influenced by the size of the detection container, it is not limited to seven steps or less.

  3 and 4 are examples having a plurality of branch microchannels, but one (one set) of branch microchannels may be provided in the detection container. However, in terms of efficiency, it is preferable that a plurality of lines are formed as shown in FIGS. In addition, the upper limit at which the branch microchannel can be formed is influenced by the size of the detection container, for example, the diameter in the case of a disk-shaped plate type, and the number of branch layers, and thus cannot be determined unconditionally. . Practically, the number of the flow paths 18 (28) connected to the discharge ports 19 (29) is about 200 or less.

  As the form of the branching micro flow path, the form shown in FIGS. 5 and 6 may be used in addition to the one described above. The branched micro flow channel in FIG. 5 is an example having a branch portion where the sample holding unit 3 is not arranged, and one flow channel branches into three or more at the branch portion. In FIG. 6, the flow path is formed so as to have a plurality of branch portions in a tournament type hierarchy, and all the branch portions are the branch portions 5 for forming the sample holding portion, that is, the sample holding portion 3 has all the branches. It is a thing of the form arrange | positioned at the part. When using the detection container in which the branched microchannel in the form of FIG. 6 is formed, the force 6 shown in FIG. 1B is controlled when the sample liquid containing the particulate sample 4 is sent. Thus, the particulate sample 4 can be distributed to the sample holders 3 of the branched portions in a hierarchical manner.

  Regarding the form, size, and number (number of sets) of the branched microchannels and detection containers described above, those that are suitable for the properties of the particulate sample 4, the detection method, and the like are appropriately used.

  Here, the size of each channel and the sample holding unit 3 forming the branch microchannel will be described with reference to FIG. First, the flow path 1 has a width of 50 to 1000 μm and a height of 20 to 100 μm. The width of the sample holder 3 is 50 to 1000 μm, and is preferably about the same as the width of the flow path 1 in terms of the holding ability of the particulate sample 4. Further, the depth H in the outer circumferential direction of the concave reservoir (indentation) of the sample holder 3 is 50 to 1000 μm, and the width of the sample holder 3 is balanced in terms of the balance between the liquid replacement and the holding ability of the particulate sample 4. It is preferable that it is 70 to 100%. The width of the flow path 2 is preferably 30 to 300 μm, and preferably 20 to 50% of the width of the flow path 1 from the viewpoint of easy liquid replacement. The height of the sample holder 3 and the channel 2 is 20 to 100 μm, and the height of the channel 1, the sample holder 3 and the channel 2 may be different from each other. Preferably they are the same height.

  The shape of the recess (dent) of the sample holder 3 is not particularly limited, but it is a rounded shape as shown in FIGS. 1 and 2 in terms of the balance between liquid replacement and retention of the particulate sample 4. It is preferable that For example, the lower part of the concave reservoir can be exemplified as a semicircular shape.

  Furthermore, although the volume of the sample injection unit 7 varies depending on the number of branch microchannels and the size of the detection container, it is preferable that the liquid amount for one liquid feeding can be held. The volume is about 3 to 5 μL.

  Next, a method for manufacturing the detection container according to the present invention will be described. For example, it can be manufactured by laminating a sheet or plate of the above-described material having a pattern of each flow path and sample holding part and a flat sheet or plate having a sample injection part. Further, the sample injection section may be formed on a sheet or plate having a pattern of each flow path and sample holding section. Alternatively, a detection container is manufactured by bonding together patterns (for example, substantially plane-symmetric shapes) in which each flow path, sample holding portion, and sample injection portion pattern is divided into two sheets or plates. Also good. In terms of being suitable for optical observation and analysis, the thickness of the detection container is preferably 1 mm to 3 mm.

  A sheet having a pattern such as each channel and a sample holding part can be produced by pressing with a mold or by pouring a fluid material such as a thermoplastic resin into the mold. Alternatively, a convex pattern may be formed by thick film lithography, and a fluid material such as a thermoplastic resin may be poured onto the pattern, and machining or photolithography can be used directly on the sheet or plate. It may be produced by conventional etching.

  According to the detection container of the present invention, for example, by holding a sample that needs to be processed a plurality of times in the sample holding unit 3, it can be detected accurately, efficiently and simply qualitatively or quantitatively. This detectable particulate sample 4 is a trace chemical substance adhering to a biological material or fine particles as described above, and the biological material is supported as it is or on a microcarrier such as a microbead. It is possible to detect in the state. Examples of the biological material include various cells, bacteria, fungi, viruses or genes.

  When microbeads are used in the present invention, the microbeads may be in the form of a sphere, a polyhedron, an egg, a rugby ball, or a sphere having a large number of dimples on the surface, such as a golf ball. The shape is not limited. In the case where the microbead is a sphere, the diameter is 1 μm or more and 50 μm or less, and 5 μm or more is preferable in terms of effective holding by the sample holding unit 3. For the same reason, it is preferably 30 μm or less. Even when the microbead has a shape other than a sphere, the size is preferably approximately the same as the above size.

  Further, the material of the microbead is not particularly limited as long as cells and the like are suitably supported, and glass, resin such as polystyrene or polycarbonate, metal such as gold or silver, and metal compound such as stainless steel may be used. it can. Resin-made microbeads are preferable because they are inexpensive and lightweight.

  Examples of trace chemical substances adhering to fine particles include fine paint fragments collected at the site of accidents and crimes, fine substances adhering to body fluids, etc. Is possible.

<Embodiment>
Hereinafter, the present invention will be further described in detail by exemplifying specific embodiments with reference to the drawings.

Production of Detection Container First, a branch microchannel mold was produced using a silicon wafer having a diameter of 4 inches. Photoresist (SU-850) was dropped on a silicon wafer, and was spread to a uniform thickness (40 μm) by rotating at 3000 rpm for 30 seconds with a spin coater. This was heated (prebaked) on a hot plate at 65 ° C. for 5 minutes and at 95 ° C. for 15 minutes. Then, it set to the mask aligner with the photomask of the flow path pattern, and irradiated with UV (350 nm) for 90 seconds. Furthermore, it was heated (post-baked) at 65 ° C. for 1 minute and at 95 ° C. for 1 minute on a hot plate. Thereafter, the substrate was washed in a SU-8 developer to remove unpolymerized resist, and then dried with dry nitrogen gas to complete a mold.

  A prepolymer of polydimethylsiloxane (PDMS) (Silgard 184 manufactured by Dow Corning) was poured into the mold and thermally cured at 70 ° C. After the cured PDMS sheet is taken out from the mold and molded so that the sample injection part is opened to the atmosphere, smooth glass of the same shape as the sheet is laminated on the sheet surface on which the recesses are formed, as shown in FIG. A detection container 10 having a branched microchannel as shown was produced. Further, the detection container 20 shown in FIG. 4 was produced in the same manner as described above except that the pattern of the branched microchannel was changed.

1. Example 1: Retention of microbeads by liquid replacement In this example, an experiment was conducted using the detection container 10 having the configuration shown in FIG. A channel 11 having a width of 500 μm, a sample holding part 13 having a width of 500 μm, a recess H depth of 400 μm, a channel 12 having a width of 100 μm, and each channel and the sample holding part 13 having a height of 40 μm was used. The experiment was performed using one branching microchannel.

  First, a microbead suspension in which microbeads made of polystyrene having a diameter of 10 μm (particulate sample 4) are suspended in ultrapure water (sample solution) is prepared, and the microbead suspension is used as a sample in the detection container 10. 1 μL was injected into the injection part 17. Subsequently, the detection container 10 is rotated at 1000 rpm for 10 seconds and further at 3000 rpm for 30 seconds by a spin coater (Mikasa Co., Ltd. Opticoat MS-A100), and the microbead suspension is fed and the sample holder 13 is also supplied. To hold microbeads. At this time, 10 microbeads were held. The water (sample liquid) that could not enter the sample holder 13 was discharged from the sample discharge port 19.

  Subsequently, in order to observe the state of the liquid replacement, 1 μL of the fluorescent dye solution was injected into the sample injection unit 17, and the detection container 10 was rotated under the same conditions and sent. As the fluorescent dye solution, a 0.1 M carboxyfluorescein aqueous solution was used. By this operation, a part of the water in the sample holder 13 was replaced with the fluorescent dye solution, and a yellow fluorescent color was exhibited.

Thereafter, 1 μL of PBS-EDTA solution (NaCl 137 mM, KCl 3 mM, Na ₂ HPO ₄ 7.6 mM, KH ₂ PO ₄ 1.4 mM, EDTA 1 mM) is injected into the sample injection unit 17, and the detection container 10 is subjected to the same conditions. The solution was fed by rotating. The liquid feeding operation of the PBS-EDTA solution was repeated, and the disappearance of the yellow fluorescent color, that is, the state of liquid replacement at the sample holding unit 13 was observed. At the same time, a change in the number of microbeads held in the sample holder 13 was observed.

  As a result, it was confirmed that the yellow color almost disappeared after 3 times of feeding the PBS-EDTA solution (3 times of washing), and that the liquid replacement was performed well. In addition, even after the fluorescent dye solution was fed and after the PBS-EDTA solution was fed 5 times (after washing 5 times), 10 microbeads were held in the sample holding unit 13. In other words, it was confirmed that the liquid replacement can be satisfactorily performed while the particulate sample 4 is held in the sample holder.

  In addition, the mode of the retention of the microbeads by liquid replacement in Example 1 is shown in a photograph in FIG. In addition, in the photograph of FIG. 7, since 10 microbeads overlap, the presence of 10 is not recognized, but it was confirmed by microscopic observation that 10 were held.

2. Example 2: Cell retention and viability by liquid replacement In this example, an experiment was conducted using the detection container 20 having the configuration shown in FIG. A channel 21 having a width of 500 μm, a sample holding part 23 having a width of 500 μm, a recess H depth of 400 μm, a channel 22 having a width of 100 μm, and each channel and the sample holding part 23 having a height of 40 μm was used. The experiment was conducted using one branch microchannel (one set).

  Human leukocyte T cells (Jurkat cell) were used as the cells. Human leukocyte T cells are floating eukaryotic cells and have a diameter of about 10 μm.

  First, 1 μL of the human leukocyte T cell suspension suspended in the medium was injected into the sample injection portion 27 of the detection container 20. Subsequently, the detection vessel 20 is rotated by a spin coater for 10 seconds at 1000 rpm, and further for 30 seconds at 3000 rpm to feed the human leukocyte T cell suspension, and the sample holder 23 holds the human leukocyte T cells. It was. At this time, the number of cells held in one sample holder 23 was confirmed with a microscope (20 cells). In addition, the medium that could not enter each sample holder 23 was discharged from the sample discharge port 29.

As the medium of this example, RPMI1640 medium manufactured by Sumitomo Dainippon Pharma Co., Ltd. was used, and the concentration of human leukocyte T cells was adjusted to about 1 × 10 ⁵ to 10 ⁶ cells / mL.

  As described above, human leukocyte T cells were held in the sample holder 23, and then 1 μL of PBS-EDTA solution was injected into the sample injector 27, and the detection container 20 was rotated and fed. The liquid feeding operation (washing) of the PBS-EDTA solution was repeated three times, and the medium in the sample holder 23 was replaced with the PBS-EDTA solution.

  Subsequently, 1 μL of the cell staining solution was injected into the sample injection unit 27, and the detection container 20 was rotated and fed. In this example, the conditions of the number of rotations of washing with PBS-EDTA solution and feeding of the cell staining solution were as follows: (1) 1000 rpm for 10 seconds, 4500 rpm for 30 seconds, and (2) 1000 rpm for 10 seconds. Two types of experiments were performed at 2500 rpm for 30 seconds.

  The cell staining solution used in this example was a combination of a fluorescent dye Calcein-AM for staining live cells and a fluorescent dye PI (Propium Iodide) for dead cell staining. Specifically, 2 μL of Calcein-AM and 3 μL of PI were added to 1 mL of PBS-EDTA solution to prepare and use a cell staining solution of Calcein-AM; 2 μM, PI; 4 μM.

  The above-mentioned cell staining solution is fed, the human leukocyte T cells retained in the sample retaining part 23 whose number of cells has been confirmed are stained and observed with a fluorescence microscope (Axiovert 135 manufactured by Carl Zeiss) and the number of living cells It was confirmed.

  As a result, approximately 100% of the white blood cell T cells are held in the sample holder 23 under the condition (1) with a rotational speed of 4500 rpm, and approximately 90% under the condition (2) with 2500 rpm. It was confirmed that about 95% were living cells.

  In addition, the state of human leukocyte T cell retention and viability by liquid replacement in Example 2 is shown by a photograph in FIG. FIG. 8A is a bright-field image, and FIG. 8B is a live cell fluorescence image. This is a photographic image obtained as a result of the rotation under the condition (1).

3. Comparative Example 1: Cell retention and viability using the microchannel shown in FIG. 10 As shown in FIG. 10, the height of the channel 42 after the sample holder 43 (microchamber) is very small, and the fine particles Using a detection container in which the sample 44 is trapped at the boundary between the sample holder 43 and the flow path 42, the retention and viability of human leukocyte T cells were tested. Liquid feeding, washing, and staining of human leukocyte T cells to the sample holder 43 were performed in substantially the same manner as in Example 2.

  As a result, the retention of cells was almost the same as in Example 2, but the proportion of living cells was 1% or less, and most of them were dead. This is probably because the cell died due to a very narrow clearance in the flow path 42.

  According to the present invention, various cells, bacteria, fungi, viruses, genes, and the like can be easily detected as they are or in a state where they are supported on a microcarrier such as microbeads. Therefore, the present invention can be applied to food inspection, clinical inspection, water quality inspection, or drug development that requires a complicated detection reaction.

1, 11, 21 Channels 2, 2a, 2b, 2c, 2d, 12, 22 before branching at the sample holding portion forming branching channels 3, 3a, after branching at the sample holding portion forming branching portions 3b, 3c, 3d, 13, 23 Sample holder 4, 34, 44 Particulate sample 5, 5a, 5b, 5c, 5d, 15, 25 Sample holder forming branch 6 Force 7 acting on the particulate sample, etc. 17, 27 Sample injection part 8, 18, 28 Flow path 9, 19, 29 after merging Sample outlet 10, 20 Detection container 30 Detection container 40 Prior art micro flow path 31, 41, 42 Prior art micro flow path 31, 41, 42 Channels 33, 43 Conventional sample holder (microchamber)
37, 47 Prior art sample injection section 49 Prior art sample discharge section

Claims (11)

To the plate with the sample injection part,
A flow path connected to the sample injection portion and having a branch portion for forming a sample holding portion;
A recessed sample holding part disposed in the branch part for forming the sample holding part; and
A sample discharge port at the end opposite to the connection end of the flow path with the sample injection portion;
A branch microchannel was formed,
Detection container. The plate is a disc-shaped plate;
The sample injection portion is arranged in a region on the center side of the disc-shaped plate,
The sample outlet of the branch microchannel is disposed on the outer periphery of the disc-shaped plate,
One flow path is branched into two flow paths at the sample holding part forming branch part,
The sample holder is disposed in a concave shape in the outer peripheral direction of the disk-shaped plate in the sample holder forming branch.
The detection container according to claim 1. The two flow paths branched by the sample holding part forming branch part merge before reaching the sample discharge port,
The detection container according to claim 2. The branch microchannel has a plurality of branch portions in a tournament type hierarchy, and the sample holding portion forming branch portion and the sample holding portion are arranged in a layer portion closest to the outer peripheral portion of the disc-shaped plate. The
The detection container according to claim 2 or 3. The branch microchannel has a branch part where the sample holding part is not disposed.
The detection container as described in any one of Claims 1-4. The sample holding part is also disposed in the branch part other than the sample holding part forming branch part,
The detection container according to claim 4. A plurality of the branched microchannels;
The detection container as described in any one of Claims 1-6. A sample injection section, a flow path having a branch section for forming a sample holding section, a concave sample holding section arranged in the branch section for forming the sample holding section, and a branched micro flow path having a sample discharge port Using the formed detection container,
Injecting a sample liquid containing a particulate sample into the sample injection portion;
Feeding the sample liquid toward the sample discharge port;
Holding the particulate sample in the sample holder;
Filling the branch microchannel with the sample solution;
A step of injecting a sample processing solution having a component different from that of the sample solution from the sample injection section and sending the solution, and replacing the inside of the branched microchannel with the sample processing solution
Allowing the sample treatment liquid to act on the particulate sample held in the sample holder;
Capturing a response due to the action.
A method for detecting a particulate sample. The detection container is a disc-shaped plate, and the sample solution and the sample processing solution are fed by a centrifugal force by rotating the disc-shaped plate.
A method for detecting the particulate sample according to claim 8. The branch microchannel has a plurality of branch portions in a tournament type hierarchy, and the sample holding portion forming branch portion and the sample holding portion are arranged in a layer portion closest to the outer peripheral portion of the disc-shaped plate. ing,
A method for detecting the particulate sample according to claim 9. The particulate sample is a biological sample;
A method for detecting a particulate sample according to any one of claims 8 to 10.