JP5794375B2 - Microchip and manufacturing method of microchip - Google Patents

Description

  The present invention relates to a microchip used for separation, synthesis, extraction, analysis and the like of a trace amount of reagent and a method for producing the microchip.

In recent years, separation, synthesis, and extraction of trace amounts of reagents using a microreactor consisting of microchips, in which microscale analysis channels are formed on a small substrate made of, for example, silicon, silicone, glass, etc. Analysis has been carried out (for example, see Patent Document 1 and Patent Document 2 for the microchip and its manufacture).
In a microchip, a chip suitable for various applications can be configured by providing regions having various functions such as a reaction region in which a reagent is arranged in a flow path also called a microchannel. Typical applications of microchips include chemical analysis such as genetic analysis, clinical diagnosis, and drug screening, biochemistry, pharmacy, medicine, veterinary analysis, compound synthesis, and environmental measurement.

  The above-described microchip typically has a structure in which a pair of substrates are bonded to face each other, and a fine channel (for example, a width of 10 to several hundreds μm, a depth of 10 to 10 μm) is provided on the surface of at least one substrate. About several hundred μm). Conventionally, a glass substrate is mainly used for a microchip because it can be easily manufactured and optically detected. Recently, development of a microchip using a resin substrate, which is light in weight but is less likely to be damaged than a glass substrate and inexpensive.

In the medical field, measurements using intermolecular interactions such as immune reactions in clinical examinations (surface plasmon resonance (SPR) measurement technology, crystal oscillator microbalance (QCM) measurement technology, from colloidal gold particles to ultrafine particles) In a microchip used in a measurement technique using a functionalized surface), for example, an antibody is immobilized in advance in a channel. And the measurement regarding the antibody antigen reaction which generate | occur | produces by distribute | circulating the reagent containing an antigen in a flow path is performed using the said microchip.
FIG. 9A is a schematic diagram of the microchip 10. FIG.9 (b) is AA sectional drawing of Fig.9 (a). As shown in FIG. 9A, the microchip 10 has a structure in which a pair of substrates (a first microchip substrate 11 and a second microchip substrate 12) are bonded to face each other. In the microchip 10, a fine flow path 14 having an inflow port 13a and a discharge port 13b, for example, having a width of 10 to several 100 μm and a depth of about 10 to several 100 μm is formed. Specifically, as shown in FIG. 9B, the flow path 14 is configured by the fine groove formed in the first microchip substrate 11 and the surface of the second microchip substrate 12. A metal thin film 15 is installed in the flow path. The metal thin film 15 is provided on the surface of the second microchip substrate 12 in the flow path (that is, the bonding surface of the first and second microchip substrates 11 and 12). The metal thin film 15 has a structure in which a gold (Au) thin film is laminated on a chromium (Cr) thin film.

For example, the antibody is immobilized in the channel 14 of the microchip as follows.
As shown in FIG. 10A, the reagent solution injection tube 101 is set in the inlet 13 a of the microchip 10. Similarly, a reagent solution discharge tube 102 is set in the discharge port 13 b of the microchip 10. Joints 103 are provided at the tips of the reagent solution injection tube 101 and the reagent solution discharge tube 102, and each joint 103 is connected to the inlet 13a and the outlet 13b.

  Phosphate buffered saline (hereinafter referred to as PBS) is injected into the channel 14 of the microchip 10 from the reagent solution injection tube 101, and the channel 14 is washed. The PBS that has passed through the flow path 14 is discharged to the outside through the reagent solution discharge pipe 102 connected to the discharge port 13 b of the flow path 14.

Next, as shown in FIG. 10 (b), a SAM-forming liquid (for example, an alkanethiol-containing solution) is injected into the channel 14 of the microchip 10 from the reagent solution injection tube 101. The alkanethiol in the alkanethiol-containing solution reacts with the Au thin film, and a self-assembled film (Self-Assembled Monolayer: SAM film 16) is formed on the Au thin film. The alkanethiol-containing solution that has not contributed to the SAM film formation is discharged to the outside through the reagent solution discharge tube 102.
Next, as shown in FIG. 10C, PBS is injected from the reagent solution injection tube 101 into the channel 14 of the microchip 10, and the alkanethiol-containing solution remaining in the channel 14 is removed. The PBS that has passed through the flow path 14 is discharged to the outside through the reagent solution discharge pipe 102.

Next, as shown in FIG. 11 (d), the antibody-containing solution is injected from the reagent solution injection tube 101 into the channel 14 of the microchip 10. The antibody in the antibody-containing solution reacts with the alkanethiol SAM film 16 and chemically binds thereto, and is immobilized on the SAM film 16. That is, antibody Ig is immobilized on the metal thin film 15.
In addition, since an antibody not fixed on the surface of the antibody Ig fixed to the SAM film 16 remains, or an antibody remains in a region other than the SAM film 16 in the flow path 14, FIG. As shown, PBS is injected into the flow path 14 of the microchip 10 from the reagent solution injection tube 101, and such residual antibodies are purged with PBS. The PBS containing the residual antibody is discharged to the outside through the reagent solution discharge tube 102 connected to the discharge port 13b of the flow path 14.

  Many antibodies are inactivated when they come into contact with air. Therefore, in order to prevent contact with air, the flow path 14 after the purge of the residual antibody with PBS is filled with PBS as shown in FIG. 11 (f), and the inlet 13a and outlet 13b of the microchip 10 are Sealed with a sealing material 104 such as a paraffin film.

JP 2006-187730 A Japanese Patent No. 3714338

  As shown in FIG. 9B, the shape of the flow path in the vicinity of the inlet 13a and the outlet 13b of the microchip 10 is often a right-angled shape. When the antibody-containing solution is circulated through such a channel 14, the antibody-containing solution flowing through the channel 14 becomes turbulent. Due to the influence of this turbulent flow, as shown in FIG. 12, the contact between the antibody in the antibody-containing solution and the alkanethiol SAM film 16 is disturbed, and the reaction between the antibody and the SAM film 16 is inhibited. It becomes difficult to fix the antibody.

  As shown in FIG. 13, when the reagent solution injection tube 101 and the reagent solution discharge tube 102 are detached from the flow path 14 of the microchip 10, the reagent solution injection tube 101 and the reagent solution are connected from the inlet 13a and the discharge port 13b. If the joint 103 provided at the tip of the discharge pipe 102 is removed, bubbles are likely to be generated at the inlet 13a and the outlet 13b.

Similarly, prior to distributing the reagent containing the antigen to the antibody immobilized in the flow path 14, a sealing material 104 such as a paraffin film sealing the inlet 13a and the outlet 13b of the microchip 10 is provided. Also during removal, as shown in FIG. 14, bubbles are likely to be generated at the inlet 13a and the outlet 13b.
Further, when the reagent solution injection tube 101 and the reagent solution discharge tube 102 are attached to the microchip flow channel 14 in order to distribute the reagent containing the antigen in the flow channel 14, the reagent solution injection tube is supplied from the inlet 13 a and the discharge port 13 b. 101, even when a joint provided at the tip of the reagent solution discharge pipe 102 is connected, bubbles are likely to be generated at the inlet 13a and the outlet 13b.

When bubbles are generated in the flow channel 14 and the bubbles move in the flow channel 14 when the reagent solution or the like is discharged from the flow channel 14, the bubble and the antibody come into contact with each other. In that case, since the antibody comes in contact with air, some antibodies are inactivated.
That is, when the antibody-containing solution is circulated in the flow channel 14 and the antibody is fixed in the flow channel 14 using the conventional microchip 10, the flow channel is affected by the influence of the turbulent flow generated in the flow channel 14. There arises a problem that it is difficult to immobilize the antibody in 14.
Further, when the reagent solution injection tube 101 and the discharge tube 102 are attached / detached to circulate the reagent in the flow channel 14, or for temporarily storing the microchip 10, the inlet 13 a and the discharge port 13 b of the flow channel 14. When the sealing material 104 is removed, bubbles are likely to be generated in the flow path 14, and if the antibody is fixed in the flow path 14, the antibody is deactivated.

  The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a microchip having a reagent arrangement area for immobilizing an anaerobic antibody, with the reagent arrangement area almost in contact with air, and An object of the present invention is to provide a microchip capable of stably supplying an anaerobic reagent such as an anaerobic antibody without variation and a method for producing the microchip.

In order to solve the above-described problems, in the present invention, in a microchip having a flow path formed as a space having a reagent arrangement region therein and having an flow inlet and a discharge port communicating with the flow path, the flow inlet and the discharge port are self-adjusted. Airtightly closed by a silicone gel having a restorative function.
A silicone gel provided at the inflow port and the discharge port includes a fluid discharge unit having a tip formed in a needle shape and having an opening serving as a fluid discharge port, and a fluid recovery unit having the same shape as the fluid discharge unit. Are passed through the space having the reagent arrangement area, and a reagent such as anaerobic antibody is supplied to the flow path that is the reagent arrangement area from the opening of the fluid discharge means. The supplied reagent was recovered from the opening of the fluid reforming means.
Thereby, a reagent such as an anaerobic antibody can be supplied to the reagent placement region and the reagent can be recovered from the region with almost no contact with air.
Since silicone gel is difficult to mold with a mold, as will be described later, for example, a recess (step) is formed around the inlet and outlet, and the silicone gel is poured into this recess (step). The inlet and outlet may be closed. In this case, when the silicone gel is poured into the outlet and the outlet, the inlet and outlet are blocked by a thin plate member, for example, so that the silicone gel does not flow into the flow path. Is desirable.
It is desirable that the thin plate member has a thickness that allows the fluid discharge means and the fluid recovery means to be easily penetrated. For example, when forming the inflow port and the discharge port, the thin plate member is integrated with the members constituting the microchip. The inflow port and the discharge port may be covered with a thin plate member.
Further, the reagent supply device for supplying the reagent to the microchip includes the fluid discharge means, the fluid recovery means, and the fluid discharge means and the fluid recovery means that are arranged through the silicone gel having the self-repairing function. Means for entering into and leaving from a space having a region.
By configuring the reagent supply device as described above, the reagent, such as anaerobic antibodies, can be supplied to and recovered from the reagent placement region of the microchip stably with little or no contact with air. Can do.
That is, this invention solves the said subject as follows.
(1) In the microchip, a flow path is formed as a space having a reagent arrangement area inside, an opening serving as an inlet and an outlet that communicates with the channel is provided, and the inlet and the outlet are formed by thin plate members. The step is formed in the peripheral portions of the inlet and outlet, and a silicone gel having a self-repairing function is poured into the step, and the inlet and outlet are airtightly closed.
(2) A groove, an inlet and an outlet communicating with the groove are provided, the inlet and the outlet are closed by a thin plate member, and a stepped portion is formed around the inlet and the outlet. A step in which a silicone gel having a self-repairing function is poured into the stepped portion of the first microchip substrate, a surface on which the groove portion of the first microchip substrate is formed, and a second microchip substrate A microchip is manufactured by joining one surface of the chip substrate and obtaining a microchip containing a flow path.

In the present invention, the following effects can be obtained.
(1) In a microchip having a flow path formed as a space having a reagent arrangement area inside, and having an inlet and an outlet connected to the channel, the inlet and the outlet are made of a silicone gel having a self-repairing function. Since it is airtightly closed, the reagent is obtained by penetrating the silicone gel provided at the inlet and outlet through the fluid discharge means and the fluid recovery means having an opening that is formed in a needle shape and serves as a fluid discharge port. By entering the space having the arrangement region, an anaerobic reagent such as an anaerobic antibody can be supplied to the reagent arrangement region and the reagent can be recovered from the region with almost no contact with air.
Moreover, since it is not necessary to attach a reagent solution injection tube and a reagent solution discharge tube as in the conventional example, bubbles can be prevented from being generated in the flow path.
In addition, silicone gel has strong adhesiveness and it is difficult to perform injection molding using a mold. However, by pouring silicone gel into the stepped part, a microchip substrate itself can be used instead of a mold to achieve adhesion. A gel can be formed.
Further, since the inlet and outlet are closed by the thin plate portion, the silicone gel can be prevented from flowing into the flow path (the space serving as the flow path) when the silicone gel is poured into the stepped portion.
(2) The silicone gel that closes the inlet and outlet has a self-healing function and deforms when a force is applied, and returns to the shape before the force is applied when the force is released. Even when the fluid discharge means and the fluid recovery means penetrate the silicone gel and enter the reagent arrangement region, the contact property between the silicone gel and the fluid discharge means and the fluid recovery means is good. Therefore, it is possible to prevent the outside air from entering the reagent arrangement area which is a closed space.
(3) When an anaerobic antibody-containing solution is contained in the reagent sequentially injected into the flow path, the liquid level of the reagent is such that the anaerobic antibody fixed in the reagent arrangement region is completely immersed. By injecting into the air, the anaerobic antibody can be prevented from coming into contact with air.
(4) As a reagent supply device to a microchip in which openings serving as an inlet and an outlet are closed with a silicone gel having a self-repairing function, the tip is made of a hollow cylindrical member, and the tip is needle-like A fluid discharge means formed on the side surface of the cylindrical portion of the hollow cylindrical member and a fluid recovery means having the same shape as the fluid discharge means. A reagent supply device configured such that the fluid discharge means and the fluid recovery means penetrate the silicone gel, enter the space having the reagent placement region of the microchip, and leave the space. By using it, an anaerobic reagent such as an anaerobic antibody is supplied to the reagent placement region of the microchip stably or almost without any contact with air, and the reagent is recovered from the region. Door can be.
(5) In the above (4), by arranging the opening of the fluid discharge means and the opening of the fluid recovery means so as to face each other, the reagent supplied from the fluid discharge means can be used as a flow path in the microchip. It is possible to suppress the reagent flowing in the fluid recovery means from the opening of the fluid recovery means and flowing into the flow path from becoming turbulent.
Further, the position of the lower end of the opening of the fluid recovery means is set so that the liquid level of the reagent supplied to the flow path is sufficiently high to immerse the anaerobic antibody fixed in the reagent arrangement region. By doing so, the anaerobic antibody can be prevented from coming into contact with air.

It is a figure which shows the structure of the microchip of the Example of this invention. It is a figure (1) explaining the manufacturing method of the microchip of the Example of this invention. It is a figure (2) explaining the manufacturing method of the microchip of the Example of this invention. It is a figure which shows the structural example of the reagent supply apparatus which supplies an anaerobic reagent in the flow path of the microchip of the Example of this invention. It is an enlarged view of the fluid discharge | release means and fluid collection | recovery means shown in FIG. It is a figure (1) explaining the fixation procedure of the antibody in a microchip flow path in this invention. FIG. 3 is a diagram (2) illustrating a procedure for immobilizing an antibody in a microchip channel in the present invention. FIG. 3 is a diagram (3) illustrating the procedure for immobilizing an antibody in a microchip channel in the present invention. It is the schematic diagram and sectional drawing which show the structure of a microchip. It is a figure (1) explaining the fixation procedure of the antibody in a flow path in the conventional microchip. It is a figure (2) explaining the fixation procedure of the antibody in a flow path in the conventional microchip. It is a figure explaining a mode that the contact of the antibody in an antibody containing solution and a SAM film | membrane is disturbed by the influence of a turbulent flow. It is a figure explaining a mode that a bubble generate | occur | produces when removing a reagent solution injection tube and a reagent solution discharge tube. It is a figure explaining a mode that a bubble generate | occur | produces when removing the sealing material which has sealed the inflow port and the discharge port.

FIG. 1 shows a configuration example of a microchip having a reagent arrangement region of the present invention.
FIG. 1A is an external view of a microchip according to the present invention, and FIG. 1B is a cross-sectional view taken along the line AA in FIG.
As shown in FIG. 1, the microchip 10 of the present invention has a structure in which a pair of substrates (a first microchip substrate 11 and a second microchip substrate 12) are bonded to face each other. The first microchip substrate 11 is a silicone resin substrate made of, for example, PDMS (polydimethylsiloxane), and the second microchip substrate 12 is a glass substrate.
In the microchip 10, a fine flow path 14 having an inflow port 13a and an exhaust port 13b and having a width of about 10 to several 100 μm and a depth of about 10 to several 100 μm is formed. Specifically, the flow path 14 is configured by the fine groove formed in the first microchip substrate 11 and the surface of the second microchip substrate 12. A metal thin film 15 is installed in the flow path 14. The metal thin film 15 is provided on the surface of the second microchip substrate 12 in the channel 14 (that is, the bonding surface of the first and second microchip substrates 11 and 12). The metal thin film 15 has a structure in which a gold (Au) thin film is laminated on a chromium (Cr) thin film.

In the microchip of the present invention, the inlet 13a and the outlet 13b of the flow path 14 are further closed by a thin plate portion 11a having a thickness of 100 μm or less, and a self-recoverable sealing material is further formed on the upper surface of the first microchip substrate 11. 17 has a structure provided. As the self-healing sealing material 17, a material that deforms when a force is applied and returns to a shape before the force is applied when the force is released is used. For example, a silicone gel that is an adhesive gel is employed.
This time, silicone adhesive X-40-3331-2 manufactured by Shin-Etsu Silicone Co., Ltd. was used as the silicone gel. In the following description, the inflow port 13a and the discharge port 13b blocked by the self-repairing sealing material 17 will be referred to as a microchip 10 here.

Hereinafter, the manufacture example of the microchip of this invention is demonstrated using FIG. 2, FIG.
The first microchip substrate 11 is a silicone resin substrate made of, for example, a silicone resin X-32 manufactured by Shin-Etsu Silicone, and the second microchip substrate 12 is a glass substrate.
As shown in FIG. 2A, first, the silicone resin (X-32) is molded by the first mold 71 and the second mold 72 to form the first microchip substrate 11.
Next, as shown in FIG. 2B, after the silicone resin 73 is solidified, the second mold 72 is removed.

Next, as shown in FIG. 2C, an adhesive gel (for example, X-40-3331-2 manufactured by Shin-Etsu Silicone Co., Ltd.) 17 is poured into the step portion 11 b formed on the upper surface of the silicone resin 73. Thereafter, the adhesive gel 17 and the silicone resin 73 (for example, X-32 made by Shin-Etsu Silicone) are integrated by thermoforming.
The adhesive gel has strong adhesiveness, and when a mold is used, the mold and the adhesive gel are bonded to each other and the mold cannot be removed. That is, it is difficult to perform injection molding using a mold.
Therefore, this time, the adhesive gel was formed by using the silicone resin substrate 73 itself instead of the mold.

Next, as shown in FIG. 3D, after the adhesive gel 17 and the silicone resin 73 are integrated by thermoforming, the first mold 71 is removed, and an adhesive gel (silicone gel) is formed on the upper part. Thus, the first microchip substrate 11 provided with the self-healing sealing material 17 made of is obtained.
Next, as shown in FIG. 3 (e), the first microchip substrate 11 provided with the self-healing sealing material 17 on the upper side and the second microchip substrate 12 which is a glass substrate are bonded together. 3 (f) (FIG. 1 (b)), the microchip 10 of the present invention is obtained.
The bonding between the first microchip substrate 11 and the second microchip substrate 12 is performed, for example, as shown in Patent Document 2, on the bonding surfaces of both microchip substrates with ultraviolet light having a wavelength of 220 nm or less (for example, xenon). This is performed by irradiating ultraviolet rays having a central wavelength of 172 nm emitted from an excimer lamp and bringing the bonding surfaces irradiated with the ultraviolet rays into close contact with each other.

That is, the self-healing sealing material 17 is arranged and integrated on the step portion 11b provided on the upper portion of the first microchip substrate 11 in which the fine groove portion is formed, and the integrated first microchip substrate. 11 and the second microchip substrate 12 are joined to form a microchip 10 having a flow path 14 therein, thereby obtaining the microchip of the present invention.
In the microchip of the present invention shown in FIG. 1B, the reason why the inlet 13a and the outlet 13b of the flow path 14 are closed by the thin plate portion 11a having a thickness of 100 μm or less is as shown in FIG. In this case, when the adhesive gel (X-40-3331-2) is poured into the step portion 11b provided on the upper surface of the silicone resin surface, if there is no thin plate portion 11a, the adhesiveness to the flow path 14 (the space serving as the flow path) is obtained. This is because the gel (self-healing sealing material 17) flows in.

As will be described later, when the needle-like fluid discharge means and the needle-like fluid recovery means of the reagent supply device are allowed to enter the reagent placement region through the self-healing sealing material 17, the thin plate portion 11a has a thickness of 100 μm. Since it is thin as follows, the needle-like fluid discharge means and the needle-like fluid recovery means can easily penetrate the thin plate portion.
In the microchip of the present invention shown in FIG. 1, the reagent arrangement area where an anaerobic reagent such as an anaerobic antibody can be arranged is a space in the flow path, and more specifically, installed in the flow path 14. This is the region of the metal thin film 15.

As described above, the microchip 10 of the present invention has a structure in which the inlet 13a and the outlet 13b of the flow path 14 are sealed with the self-healing sealing material 17 made of, for example, silicone gel. That is, the microchip 10 of the present invention has a structure in which the reagent arrangement area (space in the flow path) is closed by the self-healing sealing material 17. Therefore, it is possible to prevent the inflow of air from the outside into the closed space.
In addition, even when the needle-like fluid discharge means and the needle-like fluid recovery means of the reagent supply device described later penetrate the microplate thin plate portion 11a and the self-healing sealing material 17 and enter the reagent placement region, The self-healing sealing material 17 is deformed when a force is applied, and returns to the shape before the force is applied when the force is released. Therefore, the self-healing sealing material 17 and the injection needle-like fluid are released. The adhesiveness at the contact portion with the means and the needle-like fluid recovery means is good, and outside air hardly enters the reagent arrangement region which is a closed space from this contact portion.

  Further, even if the needle-like fluid discharge means and the needle-like fluid recovery means of the reagent supply device described later penetrate the microchip thin plate portion 11a and the self-healing sealing material 17 and then detach again, the self-healing seal The stopper 17 is deformed when a force is applied, and returns to the shape before the force is applied when the force is released. Therefore, the injection needle-like fluid is released through the self-recoverable sealing material 17. The holes generated in the self-healing sealing material 17 are quickly closed by the means and the needle-like fluid recovery means. Therefore, it is possible to prevent the inflow of air from the outside to the reagent arrangement area which is a closed space even after the injection needle-like fluid discharge means and the injection needle-like fluid recovery means have left the self-repairing sealing material 17. .

  Therefore, when the anaerobic reagent such as anaerobic antibody is fixed to the reagent placement region using the needle-like fluid discharge means and the needle-like fluid recovery means of the reagent supply device described later, the needle-like fluid discharge means, By purging the air remaining in the reagent placement area, which is a closed space, using a needle-like fluid recovery means, the reagent placement area removes anaerobic reagents such as anaerobic antibodies with almost no contact with air. It becomes possible to arrange in.

Next, an embodiment of a reagent supply device for supplying an anaerobic reagent such as an anaerobic antibody to the reagent placement region of the microchip 10 of the present invention with almost no contact with air will be described.
4 and 5, examples of a configuration block diagram of a reagent supply device that supplies an anaerobic reagent (here, an anaerobic antibody is taken as an example) into the flow path 14 of the microchip 10 of FIG. Show. The reagent supply apparatus shown in FIG. 4 includes a reagent injection mechanism 40, a specimen holding mechanism 20, a reagent recovery mechanism 50, and a control unit 60. FIG. 5 is an enlarged view for facilitating understanding of the needle-like fluid discharge means 21 and the needle-like fluid recovery means 22 shown later.

I. Reagent Injection Mechanism As shown in FIG. 4, the reagent injection mechanism 40 includes an injection needle fluid discharge means 21, a fluid discharge means drive mechanism 23, a joint 28 b, a reagent solution injection pipe 26, a joint 28 a, a temperature control unit 31, and a control. A valve 42 and a reagent storage unit 41 are included.
The injection needle-like fluid discharge means 21 penetrates the thin plate portion 11a of the microchip provided in the inlet 13a of the flow path 14 of the microchip 10 and the self-recoverable sealing material 17, and supplies the reagent to the flow path of the microchip 10. 14 to be discharged. As shown in FIG. 5, the injection needle-like fluid discharge means 21 is made of a hollow cylindrical member made of stainless steel. The distal end portion 21b of the injection needle-shaped fluid discharge means 21 is closed, and the distal end portion has a needle shape (for example, a bevel shape (an oblique shape) like an injection needle). The opening 21a that communicates with the internal cavity of the hollow cylindrical member and discharges the reagent supplied from the internal cavity into the flow path 14 is provided at a position as close as possible to the tip 21b on the side of the cylindrical part of the hollow cylindrical member. Yes. Hereinafter, the needle-like fluid discharge means 21 is simply referred to as fluid discharge means 21.

  That is, since the fluid discharge means 21 has a beveled tip portion 21b like an injection needle, it can easily penetrate the thin plate portion 11a of the microchip and the self-recoverable sealing material 17. Further, since the distal end portion 21b is closed and the opening portion 21a is provided on the side surface of the cylindrical portion 21c of the hollow cylindrical member, when the fluid discharge means 21 penetrates the self-recoverable sealing material 17, it Chips of the restorable sealing material 17 are hardly generated, and the opening 21a is not clogged with the chips of the self-healing sealing material 17.

The fluid discharge means 21 is driven in the vertical direction by a fluid discharge means drive mechanism 23. That is, in the fluid discharge means driving mechanism 23, the opening 21 a of the fluid discharge means 21 penetrates the thin plate portion 11 a of the microchip 10 and the self-recoverable sealing material 17 and enters the flow path 14 of the microchip 10. The fluid discharge means 21 is driven so that the fluid discharge means 21 is completely detached from the microchip 10 via the thin plate portion 11a of the microchip 10 and the self-healing sealing material 17. To drive.
The fluid discharge means driving mechanism 23 is connected to, for example, a joint 28b that connects the fluid discharge means 21 and the reagent solution injection pipe 26 to which the reagent is transported.

  As described above, one of the reagent solution injection pipes 26 is connected to the fluid discharge means 21 via the joint 28b, and the other is a temperature control unit for controlling the temperature of the reagent delivered from the reagent storage unit 41. 31 is connected to a pipe whose temperature is controlled. Since the fluid discharge means 21 and the joint 28b are driven in the vertical direction by the fluid discharge means drive mechanism 23, the reagent solution injection pipe 26 is also constituted by a flexible pipe so as to be able to cope with these operations.

The reagent storage unit 41 stores a reagent to be supplied into the flow path 14 of the microchip 10. In the example illustrated in FIG. 4, the reagent storage unit 41 includes a PBS storage unit 41 a, an alkanethiol-containing solution storage unit 41 b, an antibody-containing solution storage unit 41 c, an antigen-containing solution storage unit 41 d, and a temperature control unit 33.
Here, since the antibody is generally stable when stored in a low temperature state, the temperature of the antibody-containing solution storage unit 41 c is controlled by the temperature control unit 33. The storage temperature of the antibody is, for example, 4 ° C.

  Each storage unit of the reagent storage unit 41 is connected to a control valve 42 including a three-way valve. In the example shown in FIG. 4, the reagent storage unit 41 includes four storage units, so that the piping system including the reagent storage unit 41 and the control valve 42 is four systems. As the control valve 42, a three-way solenoid valve or the like is employed. In FIG. 4, the three-way valve that is the control valve 42 has three ports a, b, and c, and here, the a-c flow path and the bc flow path are switched. Each control valve 42 is connected in a manifold shape so that each bc flow path becomes one flow path. On the other hand, the a port of each control valve 42 is connected to the reagent storage unit 41. Further, a sealing plug is connected to the b port of the control valve 42 connected to the PBS storage unit 41a, and the c port of the control valve 42 connected to the antigen-containing solution storage unit 41d It is connected to a pipe whose temperature is controlled by the temperature controller 31.

That is, as shown in FIG. 4, the four piping systems of the reagent storage unit 41 are finally integrated into one piping system and connected to the piping whose temperature is controlled by the temperature control unit 31. By controlling the switching of the flow path of each control valve 42, it is possible to switch the reagent released from the fluid discharge means 21 to the flow path 14 of the microchip 10 via the temperature control unit 31.
The temperature control unit 31 is for controlling the temperature of a reagent such as an antibody. Specifically, the temperature control unit 31 controls the temperature of a pipe connected to the pipe from the reagent storage unit 41 integrated in the one pipe system described above. Control. One of the pipes whose temperature is controlled by the temperature control unit 31 is connected to the reagent storage unit 41 as described above, and the other is connected to the reagent solution injection pipe 26 by the joint 28a.

II. Test Specimen Holding Mechanism As shown in FIG. 4, the test specimen holding mechanism 20 includes a temperature control stage 34 including a temperature control unit 32. The temperature adjustment stage 34 has a function of adjusting the temperature of the microchip 10 while the microchip 10 is placed thereon. Specifically, the temperature control unit 32 controls the temperature of the temperature adjustment stage 34 to adjust the temperature of the microchip 10 placed on the temperature adjustment stage 34.

III. Reagent Recovery Mechanism As shown in FIG. 4, the reagent recovery mechanism 50 includes an injection needle fluid recovery means 22, a fluid recovery means drive mechanism 24, a joint 28 c, a reagent solution discharge pipe 27, a pump 51, and a waste liquid tank 52. .
The needle-like fluid recovery means 22 penetrates through the self-healing sealing material 17 provided in the discharge port 13b of the microchip channel 14 and removes at least a part of the reagent remaining in the microchip channel 14. It is collected in the waste liquid tank 52. As shown in FIG. 5, the needle-like fluid recovery means 22 is made of a hollow cylindrical member made of stainless steel, like the fluid discharge means 21. The distal end portion 22b of the fluid recovery means 22 is closed, and the distal end portion has a needle shape (for example, a bevel shape (an oblique shape) like an injection needle). The opening 22a that communicates with the internal cavity of the hollow cylindrical member and discharges the reagent supplied from the internal cavity into the fluid is provided at a position as close as possible to the tip 22b on the side surface of the cylindrical part 22c of the hollow cylindrical member. Yes. Hereinafter, the needle-like fluid recovery means 22 is simply referred to as fluid recovery means 22.

  That is, the fluid recovery means 22 can be easily penetrated through the thin plate portion 11a of the microchip and the self-healing sealing material 17 because the tip 22b has a bevel shape like an injection needle. Furthermore, since the tip 22b is closed and the opening 22a is provided on the side of the cylindrical portion 22c of the hollow cylindrical member, the self-healing when the fluid recovery means 22 penetrates the self-healing sealing material 17 The chip of the sealing material 17 is hardly generated, and the opening 22a is not clogged with the chip of the self-healing sealing material 17.

The fluid recovery means 22 is driven in the vertical direction by a fluid recovery means drive mechanism 24. That is, in the fluid recovery means driving mechanism 24, the opening 22 a of the fluid recovery means 22 penetrates through the thin plate portion 11 a of the microchip 10 and the self-recoverable sealing material 17 and enters the flow path 14 of the microchip 10. The fluid recovery means 22 is driven so that the fluid recovery means 22 is completely detached from the microchip 10 via the thin plate portion 11a of the microchip 10 and the self-healing sealing material 17. To drive.
The fluid recovery means drive mechanism 24 is connected to, for example, a joint 28 c that connects the fluid recovery means 22 and a reagent solution discharge pipe 27 that transports at least a part of the reagent in the flow path 14 to the waste liquid tank 52.

As described above, one of the reagent solution discharge pipes 27 is connected to the fluid recovery means 22 via the joint 28 c and the other is connected to the pump 51. Since the fluid recovery means 22 and the joint 28c are driven in the vertical direction by the fluid recovery means drive mechanism 24, the reagent solution discharge pipe 27 is also composed of a flexible pipe so as to be able to cope with these operations.
As described above, the pump 51 supplies the reagent stored in the reagent storage unit 41 into the flow path 14 of the microchip via the fluid discharge means 21, and at least a part of the reagent in the flow path 14 is disposed in the waste liquid tank. The reagent (waste liquid) sent from the pump 51 is stored in the waste liquid tank 52.

IV. The control unit 60 includes a fluid discharge means driving mechanism 23, a temperature control unit 31, a control valve 42, a temperature control unit 33, a temperature control unit 32 of the specimen holding mechanism 20, and a reagent recovery mechanism belonging to the reagent injection mechanism 40. The operation of the fluid recovery means drive mechanism 24 and the pump 51 belonging to 50 is controlled.

V. Procedure for Immobilizing Antibody in Microchip Channel The antibody is immobilized in the microchip channel 14 shown in FIG. 1 as follows, for example. This fixing procedure will be described with reference to FIGS. 4, 5, 6, 7, and 8.

(1) Setting of injection needle-like fluid discharge means and injection needle-like fluid recovery means on microchip The microchip 10 as a test body is placed on the temperature control stage 34. The microchip 10 may be placed on the temperature control stage 34 by an operator, or a well-known transport mechanism (not shown) may be used. Note that when the transport mechanism is used, the control unit 60 may control the transport mechanism.

Prior to placing the microchip 10 on the temperature adjustment stage 34, the temperature control unit 32 of the temperature adjustment stage 34 determines that the temperature of the temperature adjustment stage 34 is a predetermined temperature based on a command from the control unit 60. For example, the temperature is controlled to be 25 to 37 ° C.
Similarly, based on the command of the control unit 60, the temperature control unit 31 shown in FIG. 4 performs control so that the temperature of the piping of the temperature control unit 31 becomes a predetermined temperature, for example, 25 to 37 ° C. in advance.

  Based on the command of the control unit 60, the fluid discharge means driving mechanism 23 drives the fluid discharge means 21 (injection needle fluid discharge means) downward to a predetermined position. As shown in FIG. 6A, by this driving, the opening 21a of the fluid discharge means 21 passes through the self-healing sealing material 17 provided in the inflow port 13a of the microchip 10, and the microchip 10 It enters the flow path 14. The predetermined position is a position where the tip of the fluid discharge means 21 does not contact the second microchip substrate 12 of the microchip 10.

  Similarly, based on a command from the control unit 60, the fluid recovery means drive mechanism 24 drives the fluid recovery means 22 (injection needle fluid recovery means) downward to a predetermined position. As shown in FIG. 6A, by this driving, the opening 22a of the fluid recovery means 22 penetrates the thin plate portion 11a provided in the discharge port 13b of the microchip 10 and the self-recoverable sealing material 17 and It enters into the channel 14 of the microchip. Note that the above-described predetermined position is a position where the tip of the fluid recovery means 22 does not contact the second microchip substrate 12 of the microchip 10.

The controller 60 sets the fluid discharge means 21 and the fluid recovery means 22 so that the position of the lower end of the opening 22a of the fluid recovery means 22 is above the position of the upper end of the opening 21a of the fluid discharge means 21. To do.
Here, the opening 21a of the fluid discharge means 21 and the opening 22a of the fluid recovery means 22 are set to face each other.

(2) Cleaning in the flow path with PBS In FIG. 4, it is assumed that the flow path of each control valve 42 is set to a bc flow path.
The control part 60 switches the flow path of the control valve 42 which belongs to the piping system (henceforth a PBS piping system) connected to the PBS storage part 41a among several control valves 42 to ac flow path.

  Next, the control unit 60 starts driving the pump 51. As a result, the air in the flow path 14 of the microchip 10 is first sucked from the opening 22a of the fluid recovery means 22, and then the PBS stored in the PBS storage unit 41a is a- of the control valve 42 belonging to the PBS piping system. PBS flows from the opening 21a of the fluid discharge means 21 into the flow path 14 of the microchip 10 via the c flow path, the bc flow path of the other control valve 42, the temperature control section 31, and the reagent solution injection pipe 26. Inflow. When the liquid level of PBS that has flowed into the flow path 14 reaches the opening 22a of the fluid recovery means 22, the PBD is sucked from the opening 22a of the fluid recovery means 22 and passes through the reagent solution discharge pipe 27 to be a waste liquid tank 52. Is sent out.

By the above procedure, as shown in FIG. 6B, the PBS injected into the microchip channel 14 from the opening 21a of the fluid discharge unit 21 passes through the channel 14 while washing the fluid, and the fluid recovery unit 22 is discharged from the opening 22 a to the outside of the flow path and sent to the waste liquid tank 52. That is, a PBS flow for washing the flow path is generated in the flow path.
Since the position of the lower end of the opening 22a of the fluid recovery means 22 is set to be higher than the position of the upper end of the opening 21a of the fluid discharge means 21, it flows in the flow path 14 as shown in FIG. The liquid level of the PBS is the position of the lower end of the opening 22a of the fluid recovery means 22.

(3) SAM film formation After cleaning is performed for a certain period of time, the control unit 60 switches the flow path of the control valve 42 belonging to the PBS piping system to the bc flow path among the plurality of control valves 42, and The flow path of the control valve 42 belonging to the piping system (hereinafter referred to as SAMs piping system) connected to the alkanethiol-containing solution storage unit 41b that stores the alkanethiol-containing solution that is the forming solution is switched to the ac flow path.
It is assumed that the cleaning time in the PBS (the above-described fixed time) and the flow path switching timing of the control valve 42 belonging to the PBS piping system and the SAMs piping system are stored in the control unit 60 in advance.

  By such channel switching, the PBS remaining in the channel 14 of the microchip is sucked from the opening 22a of the fluid recovery means 22, and the alkanethiol stored in the alkanethiol-containing solution storage unit 41b. The ac-flow path of the control valve 42 belonging to the SAMs piping system, the piping system (hereinafter referred to as AB piping system) connected to the antibody-containing storage unit 41c, and the piping system (hereinafter referred to as AB-containing solution storage unit 41d) Into the flow path 14 of the microchip 10 from the opening 21a of the fluid discharge means 21 via the bc flow path of the control valve 42 belonging to the AC piping system), the temperature control section 31, and the reagent solution injection pipe 26. Inflow. Since a sealing plug is connected to the b port of the control valve 42 connected to the PBS storage section 41a, the alkanethiol-containing solution flows through the bc flow of the control valve 42 belonging to the PBS piping system. It does not flow on the roadside.

  By the above procedure, as shown in FIG. 6C, the alkanethiol-containing solution injected into the microchip channel 14 from the opening 21a of the fluid discharge means 21 is initially flowed before the alkanethiol-containing solution flows. It passes through the channel 14 while being mixed with the PBS remaining in the channel 14, discharged from the opening 22 a of the fluid recovery means 22 to the outside of the channel 14, and sent to the waste liquid tank 52. Eventually, the concentration of PBS gradually decreases, and finally, a flow consisting almost of an alkanethiol-containing solution is generated in the flow path 14. The alkanethiol in the alkanethiol-containing solution reacts with the Au thin film described above, and a self-assembled film (Self-Assembled Monolayer: SAM film) is formed on the Au thin film.

  Since the position of the lower end of the opening 22a of the fluid recovery means 22 is set to be higher than the position of the upper end of the opening 21a of the fluid discharge means 21, as shown in FIG. The liquid level of the flowing alkanethiol-containing solution is the position of the lower end of the opening 22 a of the fluid recovery means 22.

(4) Cleaning in the flow path with PBS After the SAM film 16 is formed on the Au film after a certain period of time, the control unit 60 selects the control valve 42 belonging to the SAMs piping system among the plurality of control valves 42. While switching the flow path to the bc flow path, the flow path of the control valve 42 belonging to the PBS piping system is switched to the ac flow path.
It should be noted that the time for the alkanethiol-containing solution to flow through the microchip flow path 14 until the SAM film 16 is formed on the Au film (a certain period of time described above), the control valves 42 belonging to the SAMs piping system and the PBS piping system. It is assumed that the flow path switching timing is stored in the control unit 60 in advance.

  By such channel switching, the alkanethiol-containing solution remaining in the channel 14 of the microchip is sucked from the opening 22a of the fluid recovery means 22, and the PBS is connected to the control valve 42 belonging to the PBS piping system. Flow into the microchip channel 14 from the opening 21a of the fluid discharge means 21 via the ac channel, the bc channel of the other control valve 42, the temperature control unit 31, and the reagent solution injection pipe 26. To do.

  Through the above procedure, as shown in FIG. 7 (d), the PBS injected into the microchip channel 14 from the opening 21a of the fluid discharge means 21 is initially an alkanethiol-containing solution remaining in the channel 14. The fluid passes through the flow path 14 while being mixed with the fluid, and is discharged from the opening 22 a of the fluid recovery means to the outside of the flow path 14 and sent to the waste liquid tank 52. Eventually, the concentration of the alkanethiol-containing solution gradually decreases, and finally, a flow consisting almost of PBS is generated in the flow path 14. That is, the alkanethiol-containing solution that has not contributed to the formation of the SAM film 16 is discharged to the outside through the reagent solution discharge pipe 27 together with the PBS.

  Since the position of the lower end of the opening 22a of the fluid recovery means 22 is set to be higher than the position of the upper end of the opening 21a of the fluid discharge means 21, it flows in the flow path 14 as shown in FIG. The liquid level of the PBS is the position of the lower end of the opening 22a of the fluid recovery means 22.

(5) Antibody fixation After washing is performed for a certain period of time, the control unit 60 switches the flow path of the control valve 42 belonging to the PBS piping system to the bc flow path among the plurality of control valves 42 and contains the antibody. The flow path of the control valve 42 belonging to the piping system (AB piping system) connected to the antibody-containing solution storage unit 41c that stores the solution is switched to the ac channel.
It is assumed that the cleaning time in the PBS (the above-described fixed time) and the flow path switching timing of the control valve 42 belonging to the PBS piping system and the AB piping system are stored in the control unit 60 in advance.

By such channel switching, PBS remaining in the channel 14 of the microchip is sucked from the opening 22a of the fluid recovery means 22, and the antibody-containing solution is a in the control valve 42 belonging to the AB piping system. -C flow path, fluid discharge means 21 via the bc flow path of the control valve 42 belonging to the piping system (AB piping system) connected to the antigen-containing solution storage section 41c, the temperature control section 31, and the reagent solution injection pipe 26 Flows into the microchip channel 14 from the opening 21a.
In addition, since a sealing plug is connected to the b port of the control valve 42 connected to the PBS storage unit 41a, the antibody-containing solution is transferred to the bc flow path side of the control valve 42 belonging to the SAMs piping system. And the control valve 42 belonging to the PBS piping system does not flow to the bc flow path side.

In the antibody-containing solution storage unit 41c, for example, the antibody-containing solution stored in a low temperature state of 4 ° C. is connected to the pipe of the temperature control unit 31 that is temperature-controlled by the temperature control unit 31 shown in FIG. By passing, it is heated to, for example, 25 to 37 ° C.
As described above, the temperature of the microchip 10 placed on the temperature control stage 34 is maintained at, for example, 25 to 37 ° C. by the temperature control stage 34 controlled in temperature by the temperature control unit 32. The temperature of the antibody-containing solution that has flowed into the flow path 14 does not drop.

  By the above procedure, as shown in FIG. 7E, the antibody-containing solution injected into the microchip channel 14 from the opening 21a of the fluid discharge means 21 is initially flow channel 14 before the antibody-containing solution flows. The fluid passes through the flow path 14 while being mixed with the PBS remaining therein, and is discharged from the opening 22 a of the fluid recovery means 22 to the outside of the flow path 14 and sent to the waste liquid tank 52. Eventually, the concentration of PBS gradually decreases, and finally, a flow consisting essentially of an antibody-containing solution is generated in the flow path 14. The antibody in the antibody-containing solution reacts with the alkanethiol SAM film 16 and chemically binds thereto, and is immobilized on the SAM film 16. That is, antibody Ig is immobilized on the metal thin film 15.

Since the position of the lower end of the opening 22a of the fluid recovery means 22 is set to be higher than the position of the upper end of the opening 21a of the fluid discharge means 21, it flows in the flow path 14 as shown in FIG. The liquid level of the antibody-containing solution is at the lower end of the opening 22a of the fluid recovery means 22.
Here, the position of the lower end of the opening 22a of the fluid recovery means 22 is set so that the level of the antibody Ig fixed to the SAM film 16 is completely immersed in the antibody-containing solution. The antibody Ig immobilized on the SAM membrane 16 does not come into contact with air.

Further, as described above, since the opening 21a of the fluid discharge means 21 is provided on the side surface of the hollow cylindrical member, the reagent (here, the antibody-containing solution) supplied from the fluid discharge means 21 is hollow. It is discharged from the opening in a direction orthogonal to the axial direction of the cylindrical member. That is, the reagent is released in the lateral direction in FIGS.
As described above, the opening 21a of the fluid discharge means 21 and the opening 22a of the fluid recovery means 22 are set so as to face each other, and the antibody-containing solution released from the opening 21a in the lateral direction. By setting the position of the opening 21a of the fluid discharge means 21 so as not to directly collide with the corner of the flow path, the antibody-containing solution flowing in the flow path 14 is suppressed from becoming turbulent.

That is, the setting of the fluid discharge means 21 and the fluid recovery means 22 in the procedure (1) is such that the position of the lower end of the opening 22a of the fluid recovery means 22 and the position of the opening 21a of the fluid discharge means 21 are as described above. Done to be set.
(6) Cleaning in the flow path with PBS After a certain period of time has passed and the antibody Ig is fixed on the SAM film 16, the control unit 60 selects the control valve 42 belonging to the AB piping system from among the plurality of control valves 42. While switching the flow path to the bc flow path, the flow path of the control valve 42 belonging to the PBS piping system is switched to the ac flow path.
It should be noted that the time required for the antibody-containing solution to flow through the microchip channel 14 until the antibody Ig is immobilized on the SAM membrane 16 (a certain period of time described above), the flow of the control valve 42 belonging to the AB piping system and the PBS piping system. It is assumed that the path switching timing is stored in the control unit 60 in advance.

By such channel switching, the alkanethiol-containing solution remaining in the channel 14 of the microchip is sucked from the opening 22a of the fluid recovery means 22, and the PBS is connected to the control valve 42 belonging to the PBS piping system. Flow into the microchip channel 14 from the opening 21a of the fluid discharge means 21 via the ac channel, the bc channel of the other control valve 42, the temperature control unit 31, and the reagent solution injection pipe 26. To do.
By the above procedure, as shown in FIG. 7 (f), the non-fixed antibody Ig remaining on the surface of the antibody Ig fixed to the SAM film 16, or the antibody remaining in the region other than the SAM film 16 Ig is discharged to the outside through the reagent solution discharge pipe 27 together with PBS.

  Since the position of the lower end of the opening 22a of the fluid recovery means 22 is set to be higher than the position of the upper end of the opening 21a of the fluid discharge means 21, it flows in the flow path 14 as shown in FIG. The liquid level of the PBS is the position of the lower end of the opening 22a of the fluid recovery means 22. Since the level of the liquid is such that the antibody Ig fixed to the SAM film 16 is completely immersed in PBS, the antibody Ig fixed to the SAM film 16 does not come into contact with air.

The antibody Ig is fixed to the reagent arrangement region (SAM film forming region on the metal thin film 15) in the microchip flow path 14 by the above procedures (1) to (6).
As is clear from the above procedure, in the procedure (2), after the inside of the flow path 14 is washed with PBS, the metal thin film 15 is always PBS, a SAM film forming solution (alkanethiol-containing solution in the above example), Immerse in the antibody-containing solution. The SAM film formation in the procedure (3) is performed without air contacting the metal thin film 15. Then, the antibody fixation to the SAM film 16 in the procedure (5) is performed without contact of air with the antibody Ig under the condition that no air remains in the SAM film 16. Furthermore, the discharge of the antibody Ig not fixed to the SAM film 16 in the procedure (6) is also performed without contact of air with the antibody Ig fixed to the SAM film 16.
As described above, this is because the liquid level of the reagent flowing in the flow path 14 is such that the antibody Ig fixed to the SAM film 16 is completely immersed in the reagent by the opening of the fluid recovery means 22. This is because the position of the lower end of the portion 22a is set.

That is, the reagent supply apparatus using the microchip 10 of the present invention can supply an anaerobic reagent such as an anaerobic antibody to the reagent arrangement region of the microchip 10 without contacting air. That is, it becomes possible to fix the anaerobic antibody to the reagent arrangement region of the microchip 10 without deactivating it.
In addition, since the reagent is supplied to the flow path 14 of the microchip 10 mechanically by the control unit 60, the reagent injection mechanism 40, and the reagent recovery mechanism 50, the flow path 14 of the microchip 10 can be stably distributed. The reagent can be supplied to

  Further, the opening 21a of the fluid discharge means 21 and the opening 22a of the fluid recovery means 22 are set so as to face each other, and the antibody-containing solution released from the opening in the lateral direction is directly at the corner of the flow path 14. By setting the position of the opening 21a of the fluid discharge means 21 so as not to collide with the part, the antibody-containing solution flowing in the flow path 14 is suppressed from becoming turbulent. Therefore, the contact between the antibody Ig in the antibody-containing solution and the SAM film 16 of alkanethiol is hardly disturbed, the reaction between the antibody Ig and the SAM film 16 proceeds well, and the antibody Ig is stably immobilized on the SAM film 16. It becomes possible.

  Since the microchip 10 has a structure in which the inlet 13a and the outlet 13b of the flow path 14 of the present invention are sealed with a self-recoverable sealing material 17 made of, for example, silicone gel. Even when the fluid discharge means 21 and the fluid recovery means 22 of the reagent supply apparatus of the present invention penetrate the microchip thin plate portion 11a and the self-healing sealing material 17 and enter the reagent arrangement region, the self-healing seal Since the stopper 17 is deformed when a force is applied and returns to a shape before the force is applied when the force is released, the self-restoring sealing material 17, the fluid discharge means 21, and the fluid recovery means 22 are provided. Adhesiveness at the contact portion is good, and outside air hardly enters the reagent arrangement region which is a closed space from this contact portion.

VI. Procedure for generating antibody-antigen reaction in microchip flow channel Following the antibody fixation to the microchip 10 described above, when an antigen is supplied to the immobilized antibody Ig to generate an antibody-antigen reaction, for example, Perform the procedure.

(7) Antibody-antigen reaction In the above-described procedure (6), after washing is performed for a predetermined time, the control unit 60 sets the flow path of the control valve 42 belonging to the PBS piping system among the plurality of control valves 42 to b- While switching to the c flow path, the flow path of the control valve 42 belonging to the piping system (AC piping system) connected to the antigen-containing solution storage unit 41d that stores the antigen-containing solution is switched to the ac flow path.
It is assumed that the cleaning time in PBS (the above-described fixed time) and the flow path switching timing of the control valve 42 belonging to the PBS piping system and the AC piping system are stored in the control unit 60 in advance.

  By such channel switching, the PBS remaining in the channel of the microchip 10 is sucked from the opening 22a of the fluid recovery means 22, and the antigen-containing solution a of the control valve 42 belonging to the AC piping system. It flows into the microchip channel 14 from the opening 21a of the fluid discharge means 21 via the -c channel, the temperature control unit 31, and the reagent solution injection tube 26. Since a sealing plug is connected to the b port of the control valve 42 connected to the PBS storage section, the antigen-containing solution is transferred to the bc flow path side of the control valve 42 belonging to the AB piping system, It does not flow to the bc flow path side of the control valve 42 belonging to the SAMs piping system and to the bc flow path side of the control valve 42 belonging to the PBS piping system.

  The antigen-containing solution is heated to, for example, 25 to 37 ° C. by passing through the piping of the temperature control unit 31 that is temperature-controlled by the temperature control unit 31 shown in FIG.

By the above procedure, as shown in FIG. 8G, the antigen-containing solution injected from the opening 21a of the fluid discharge means 21 into the channel 14 of the microchip is initially channeled before the antigen-containing solution flows. The fluid passes through the flow path 14 while being mixed with the PBS remaining therein, and is discharged from the opening 22 a of the fluid recovery means 22 to the outside of the flow path 14 and sent to the waste liquid tank 52. Eventually, the concentration of PBS gradually decreases, and finally, a flow consisting almost of an antigen-containing solution is generated in the flow path 14. The antigen in the antigen-containing solution undergoes an antibody-antigen reaction with the antibody Ig immobilized on the SAM film 16 and chemically binds thereto. As described above, the antibody-antigen reaction is performed in an antigen-containing solution that does not contain air, depending on the settings of the fluid discharge means 21 and the fluid recovery means 22 in the procedure (1).
As described above, the temperature of the microchip 10 placed on the temperature control stage 34 is maintained at, for example, 25 to 37 ° C. by the temperature control stage 34 controlled in temperature by the temperature control unit 32. The antibody-antigen reaction performed in the flow path 14 is performed under a temperature condition of 25 to 37 ° C. This temperature condition conforms to human body temperature.

(8) Washing in the flow path with PBS After a certain time has passed and the antibody-antigen reaction is completed, the control unit 60 sets the flow path of the control valve 42 belonging to the AC piping system to b− While switching to the c channel, the channel of the control valve 42 belonging to the PBS piping system is switched to the ac channel.
It should be noted that the time for the antigen-containing solution to flow through the microchip flow path 14 until the antibody-antigen reaction is completed (a certain period of time as described above), and the flow path switching timing of the control valve 42 belonging to the AC piping system and the PBS piping system are as follows. Suppose that it is stored in the control unit 60 in advance.

By such channel switching, the antigen-containing solution remaining in the channel 14 of the microchip is sucked from the opening 22a of the fluid recovery means 22, and the PBS of the control valve 42 belonging to the PBS piping system is a. -C flow path, bc flow path of other control valve 42, temperature control section 31, and reagent solution injection pipe 26, and flows into the microchip flow path 14 from the opening 21a of the fluid discharge means 21. .
By the above procedure, as shown in FIG. 8 (h), the antigen that has not contributed to the antibody-antigen reaction remaining in the flow path is discharged to the outside through the reagent solution discharge pipe 27 together with PBS.

By the procedures (7) to (8) described above, an antigen is supplied to the antibody Ig fixed in the flow path 14 of the microchip, and an antibody-antigen reaction is generated.
As is clear from the above procedure, the generation of the antibody antigen reaction in the procedure (7) is performed without contact of air with the antibody Ig fixed to the SAM membrane 16. And the discharge | emission of the antigen which did not contribute to the antibody antigen reaction which remained in the flow path in a procedure (8) is also performed, without air contacting antibody Ig.
As described above, this is because the level of the reagent flowing in the flow path is such that the antibody Ig fixed to the SAM film 16 is completely immersed in the antibody-containing solution. This is because the lower end position is set.

That is, the reagent supply apparatus using the microchip 10 of the present invention can supply the antigen to the reagent arrangement region of the microchip 10 without bringing air into contact therewith. Therefore, it is possible to generate an antigen-antibody reaction without deactivating the anaerobic antibody Ig fixed in the reagent arrangement region of the microchip 10.
In addition, since the reagent is supplied to the microchip flow path 14 mechanically by the control unit 60, the reagent injection mechanism 40, and the reagent recovery mechanism 50, the microchip flow path 14 can be stably supplied to the microchip. Reagents can be supplied.

(9) After the needle-like fluid discharge means and the needle-like fluid recovery means are separated and cleaned from the microchip for a certain period of time, the control unit 60 controls among the plurality of control valves 42 that belong to the PBS piping system. The flow path 14 of the valve 42 is switched to the bc flow path. It is assumed that the cleaning time with PBS (the above-described fixed time) and the flow path switching timing of the control valve 42 belonging to the PBS piping system are stored in the control unit 60 in advance.

Next, the control unit 60 stops driving the pump 51. As a result, the flow consisting essentially of PBS in the flow path 14 of the microchip stops. As described above, the position of the lower end of the opening of the fluid recovery means 22 (injection needle fluid recovery means) is higher than the position of the upper end of the opening of the fluid discharge means 21 (injection needle fluid discharge means). Since it is set, as shown in FIG. 5, the liquid level of the PBS is the position of the lower end of the opening 22a of the fluid recovery means 22.
Here, the position of the lower end of the opening 22a of the fluid recovery means 22 is set so that the level of the antibody Ig fixed on the SAM film 16 is completely immersed in PBS. The antibody Ig that has completed the antibody-antigen reaction fixed to the membrane 16 does not come into contact with air.

  Based on the command of the control unit 60, the fluid discharge means driving mechanism 23 drives the fluid discharge means 21 upward to a predetermined position. As shown in FIG. 8 (i), the opening 21a of the fluid discharge means 21 leaves the microchip flow path 14 by this driving. The predetermined position is such that the fluid discharge means 21 is completely detached from the microchip 10 via the thin plate portion 11a provided at the injection port of the microchip 10 and the self-recoverable sealing material 17. Position.

Similarly, based on a command from the control unit 60, the fluid recovery means driving mechanism 24 drives the fluid recovery means 22 upward to a predetermined position. As shown in FIG. 8 (i), this driving causes the opening 22a of the fluid recovery means 22 to leave the microchip channel 14. Note that the above-mentioned predetermined position means that the fluid recovery means 22 is completely detached from the microchip 10 via the thin plate portion 11 a provided in the discharge port 13 b of the microchip 10 and the self-recoverable sealing material 17. It is the position.

The fluid discharge means 21 and the fluid recovery means 22 that have penetrated the thin plate portion 11a and the self-healing sealing material 17 of the microchip 10 are detached via the thin plate portion 11a and the self-healing sealing material 17. However, since the self-healing sealing material 17 is deformed when a force is applied and returns to the shape before the force is applied when the force is released, the thin plate portion 11a and the self-healing sealing material 17 are restored. The holes generated in the thin plate portion 11a are maintained by the fluid discharge means 21 and the fluid recovery means 22 that pass through and leave, but the holes generated in the self-healing sealing material 17 are quickly closed. Therefore, the inflow of air from the outside into the flow path is prevented even after the fluid discharge means 21 and the fluid recovery means 22 are separated from the thin plate portion 11a of the microchip and the self-healing sealing material 17.

The microchip 10 placed on the temperature control stage 34 is carried out to a measuring instrument for measuring the antibody-antigen reaction. It should be noted that the operator may carry out the microchip 10 to the measuring instrument or may use a known transport mechanism that is not shown. Note that when the transport mechanism is used, the control unit 60 may control the transport mechanism.
Subsequently, when the antibody is not fixed to the next microchip 10 or the like, the temperature control unit 32 of the temperature adjustment stage 34 stops the temperature control of the temperature adjustment stage 34 based on a command from the control unit 60. Similarly, based on a command from the control unit 60, the temperature control unit 31 illustrated in FIG. 14 stops the temperature control of the piping of the temperature control unit 31.

  In the above description, the case where the reagent supply to the microchip is automatically performed under the control of the control unit 60 has been described. However, a part or all of the operations of the above procedure may be performed manually by a person. Good.

DESCRIPTION OF SYMBOLS 10 Microchip 11 1st microchip board | substrate 11a Thin plate part 12 2nd microchip board | substrate 13a Outlet 13b Inlet 14 Channel 15 Metal thin film 16 SAM film 17 Self-healing sealing material 20 Test body holding mechanism 21 Injection needle Fluid discharge means 21a, 22a Openings 21b, 22b Tip portions 21c, 22c Cylindrical portion 22 Injection needle fluid recovery means 23 Fluid discharge means drive mechanism 24 Fluid recovery means drive mechanism 26 Reagent solution injection pipe 27 Reagent solution discharge pipe 28a Joint 28b Joint 28c Joint 31 Temperature control unit 32 Temperature control unit 33 Temperature control unit 34 Temperature control stage 40 Reagent injection mechanism 41 Reagent storage unit 41a PBS storage unit 41b Alkanethiol-containing solution storage unit 41c Antibody-containing solution storage unit 41d Antigen-containing solution storage Part 42 Control valve 50 Reagent recovery mechanism 5 Pump 52 waste liquid tank 60 controller 71 first die 72 second mold 73 silicone resin Ig antibody

Claims (2)

A flow path is formed as a space having a reagent arrangement area inside, and an opening serving as an inlet and an outlet communicating with the flow path is provided,
The inlet and outlet are closed by a thin plate member, and a step portion is formed around the inlet and outlet, and a silicone gel having a self-repairing function is poured into the step portion. A microchip characterized in that an inlet and an outlet are hermetically closed. A groove, an inlet and an outlet communicating with the groove are provided, the inlet and the outlet are closed by a thin plate member, and a stepped portion is formed around the inlet and the outlet. A step in which a silicone gel having a self-repairing function is poured into the stepped portion of the first microchip substrate;
Joining the surface of the first microchip substrate on which the groove is formed and one surface of the second microchip substrate to obtain a microchip containing the flow path. A method for manufacturing a microchip.

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