JP2016109537A - Liquid feeding method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a liquid feeding method capable of easily controlling inflow and outflow of a liquid in a minute reaction field.SOLUTION: A liquid feeding method is configured so that: a micro fluid device is used, in which the micro fluid device comprises a main body part 4 comprising a first inner wall face w1 forming a first space part S1, a second inner wall face w2 forming a second space part S2, and an inside face Y having one or more minute penetration holes T having a first opening Ta and a second opening Tb, and spatially connecting the first space part S1 and the second space part S2; a liquid Q is introduced to the first space part S1, then part of the liquid Q is flown from the first opening Ta into the minute penetration holes T; then, the liquid Q is discharged from the first space part S1 for forming a membrane M formed of the liquid Q on the first inner wall face w1; then, the part of the liquid Q is flown from the first openings Ta of the minute penetration hole T to the first space part S1, by tension of the membrane M and the part of the liquid Q which remains temporarily in the minute penetration holes T, following to a drying step of the membrane M.SELECTED DRAWING: Figure 4

Description

  The present invention relates to a liquid feeding method in a microfluidic device having fine through holes.

  In recent years, micro-TAS, microfluidic devices, micromixers, etc. for performing immunological measurement, chemical reaction, cell culture, etc. using antigen-antibody reaction in microscale to nanoscale micro reaction fields have attracted attention. . In order to perform home medical care, point-of-care (POC), clinical examination, cell culture, etc. quickly with a small number of samples, downsizing of these devices is important.

  As a conventional microfluidic device, there has been disclosed a flow channel chip having a space in which cells can be cultured in the middle of a flow channel and having a blocking portion so that cells do not flow out downstream (Patent Document 1). A fine flow path is formed in the upper part of the damming portion, so that cells do not easily pass through and only the culture solution is likely to flow out. Since the culture space and the channel are formed of a transparent plastic substrate, a desired drug can be administered to the cultured cells and the reaction can be optically observed.

JP 2012-185008 A

  A syringe pump is generally used as a liquid feeding method for allowing a liquid containing a target substance to flow into a reaction field provided in a microfluidic device or for flowing a liquid from the reaction field. However, in recent microfluidic devices, not only the flow path but also the reaction field connected to the flow path has been miniaturized (miniaturization). In the case of liquid feeding, an expensive pump capable of precise pressure control is required. For this reason, there exists a problem that the whole cost for using a microfluidic device rises. Furthermore, it is common for air as well as liquid to flow into the flow path that constitutes the microfluidic device. However, liquid feeding in the flow path of such a gas-liquid mixing system is precisely controlled only by a pump. There is a problem that it is difficult to do.

  This invention is made | formed in view of the said situation, and makes it a subject to provide the liquid feeding method which can control easily the inflow and outflow of the liquid in a micro reaction field.

(1) A method for delivering a liquid in a microfluidic device, wherein a first inner wall surface constituting a first space portion, a second inner wall surface constituting a second space portion, and an opening in the first inner wall surface And having at least one fine through hole that spatially connects the first space portion and the second space portion. Using a microfluidic device including a main body portion including an inner surface, and introducing the liquid into the first space portion, and the liquid from the first opening into the one or more fine through holes. After the flow of a part of the film, the liquid is discharged from the first space portion to form a film made of the liquid on the first inner wall surface, and subsequently, the film accompanying the drying process of the film And the tension of the liquid partially remaining in the one or more fine through holes. Te, feeding method, characterized in that said part of the liquid, to flow out into the first space portion from said first opening of said one or more micro through holes.

  According to the liquid feeding method of (1) above, the capillary force (capillary phenomenon) inherently possessed by the fine through-hole is used to allow the liquid to flow into the fine through-hole that can be used as a minute reaction field. ing. Further, in order to allow the liquid to flow out from the inside of the fine through hole, the difference in tension between the film and the liquid in the fine through hole and the fact that the tension difference changes as the film is dried are utilized. Therefore, since it is not necessary to apply high pressure generated by the pump to the fine through hole, a desired liquid can be gently and surely flowed into and out of the fine through hole.

(2) A method for feeding a liquid in a microfluidic device, the main body including a first inner wall surface constituting a first space and a second inner wall constituting a second space, and the first space A first opening that faces the portion and a second opening that opens to face the second space, and spatially connects the first space and the second space. Using a microfluidic device comprising a sub-body part including an inner surface constituting one or more fine through-holes, and introducing a liquid into the first space part, Forming a film made of the liquid on the first inner wall surface by discharging a part of the liquid into the fine through-holes and then discharging the liquid from the first space; Temporarily remained in the film and the one or more fine through-holes during the drying process of the film. Serial by the tension of the part of the liquid, feeding method, characterized in that said part of the liquid, to flow out into the first space portion from said first opening of said one or more micro through holes.

According to the liquid feeding method of the above (2), as in the case of the above (1), in order to allow the liquid to flow into the fine through holes that can be used as a minute reaction field, Capillary force (capillary phenomenon) is used. Further, in order to allow the liquid to flow out from the inside of the fine through hole, the difference in tension between the film and the liquid in the fine through hole and the fact that the tension difference changes as the film is dried are utilized. Therefore, since it is not necessary to apply high pressure generated by the pump to the fine through hole, a desired liquid can be gently and surely flowed into and out of the fine through hole.
Further, in the microfluidic device used in the liquid feeding method of (2) above, the fine through hole is provided in the sub-main body portion. For this reason, if the form of the sub body part is a form that can be removed from the main body part, after carrying out the liquid feeding method, by removing the sub body part from the body part and attaching a new sub body part, The liquid feeding method can be carried out again by reusing the main part of the microfluidic device.

(3) A method for delivering a liquid in a microfluidic device, the main body including a first inner wall surface constituting a first space portion and a second inner wall surface constituting a second space portion, and the first space A first sub-inner wall surface that constitutes a portion and a second sub-inner wall surface that constitutes the second space, and further includes a first opening that opens in the first sub-inner wall surface and a second sub-inner wall surface. A micro main body having a second opening that opens, and a sub-body portion including an inner surface that constitutes one or more fine through holes that spatially connect the first space and the second space. Using a fluid device, a liquid is introduced into a first space portion constituted by the first inner wall surface and the first sub inner wall surface, and the one or more fine penetrations are caused by capillary action from the first opening portion. After flowing a part of the liquid into the hole, the liquid is discharged from the first space portion. In this way, a film made of the liquid is formed on the first inner wall surface, and then the part of the film temporarily remaining in the film and the one or more fine through-holes during the drying process of the film. The liquid feeding method, wherein the part of the liquid is caused to flow out from the first opening of the one or more fine through holes to the first space by tension with the liquid.

  According to the liquid feeding method of (3) above, as in the case of (1) above, in order to allow the liquid to flow into the fine through holes that can be used as a minute reaction field, Capillary force (capillary phenomenon) is used. Further, in order to allow the liquid to flow out from the inside of the fine through hole, the difference in tension between the film and the liquid in the fine through hole and the fact that the tension difference changes as the film is dried are utilized. Therefore, since it is not necessary to apply high pressure generated by the pump to the fine through hole, a desired liquid can be gently and surely flowed into and out of the fine through hole. Further, in the microfluidic device used in the liquid feeding method of (3) above, a fine through hole is provided in the sub-main body as in the case of (2) above. For this reason, if the form of the sub-main part is removable from the main part, after carrying out the liquid feeding method, the sub-main part is detached from the main part and a new sub-main part is attached. The liquid feeding method can be performed again by reusing the main body of the fluidic device.

(4) A method for delivering a liquid in a microfluidic device, the first inner wall surface constituting the first space portion, the second inner wall surface constituting the second space portion, and the nth space portion. (N represents an integer greater than or equal to 3) n inner wall surfaces including the first inner wall surface, the first opening portion opening in the first inner wall surface, and the second inner wall surface. A first inner surface having a second opening and constituting one or more fine through-holes spatially connecting the first space and the second space; and the first inner wall surface One or more fine through holes that spatially connect the first space portion and the n-th space portion, having a first opening portion that opens to the second wall portion and a second opening portion that opens to the n-th inner wall surface. Using a microfluidic device comprising a body portion comprising (n-1) inner side surfaces, and (n-1) inner side surfaces comprising A liquid is introduced into the first space portion, and the liquid is introduced into each fine through-hole from the first opening provided in each fine through-hole constituted by the (n-1) inner side surfaces. A film made of the liquid is formed on the n-th inner wall surface by discharging the liquid from the first space portion, and then the film is dried along with the film drying process. And the part of the liquid temporarily remaining in each fine through hole, the part of the liquid flows out from the first opening of the fine through hole to the first space part. A liquid feeding method characterized by causing the liquid to flow.

  According to the liquid feeding method of (4) above, in the same manner as in (1) above, in order to allow liquid to flow into each of a plurality of fine through holes that can be used as a minute reaction field, Capillary force inherent in the pores (capillary phenomenon) is used. Further, in order to allow the liquid to flow out from the inside of the fine through hole, the difference in tension between the film and the liquid in the fine through hole and the fact that the tension difference changes as the film is dried are utilized. Therefore, since it is not necessary to apply high pressure generated by the pump to each fine through-hole, a desired liquid can be flowed into and out of each fine through-hole gently and reliably.

  According to the liquid feeding method of the present invention, when a desired liquid is introduced into the first space part included in the microfluidic device, the capillary force causes each of the one or more fine through-holes opened in the first space part. A part of the liquid can be allowed to flow gently and reliably. Next, when the liquid is discharged from the first space portion, the part of the liquid temporarily remains in each fine through hole at the time of the discharge, and then automatically the one liquid in each fine through hole. The liquid in the part can flow out to the first space part. In this way, since it is possible to control the inflow and outflow of the liquid in each fine through-hole opened in the first space by simply controlling the inflow and outflow of the liquid in the first space, use an inexpensive pump or By using the syringe manually, the liquid feeding method according to the present invention can be easily carried out.

In addition, by using the fine through-holes provided in the microfluidic device used in the present invention as a minute reaction field, the inter-substance distance (intermolecular distance) between the reactants (reactive molecules) is dramatically shortened. be able to. For this reason, for example, it is possible to carry out ELISA (enzyme-linked immunosorbent assay), which required several hours in the conventional method, in about ten minutes to several tens of minutes.
Therefore, the liquid feeding method of the present invention is suitable for uses such as home medical care, point-of-care, clinical examination, and food inspection.

It is typical sectional drawing of the microfluidic device 10 which can be used in the liquid feeding method of this invention. FIG. 2 is a diagram illustrating a main part of FIG. 1 and a schematic diagram illustrating a state of liquid feeding in a microfluidic device 10. FIG. 2 is a diagram illustrating a main part of FIG. 1 and a schematic diagram illustrating a state of liquid feeding in a microfluidic device 10. FIG. 2 is a diagram illustrating a main part of FIG. 1 and a schematic diagram illustrating a state of liquid feeding in a microfluidic device 10. It is typical sectional drawing showing the 1st space part, the 2nd space part, and several fine through-holes with which the microfluidic device was equipped. It is the schematic diagram which showed an example of the cross-sectional shape of each micro through-hole when the some micro through-hole in the microfluidic device of FIG. 5A is cut | disconnected by A-A 'direction. It is the schematic diagram which showed an example of the cross-sectional shape of each micro through-hole when the some micro through-hole in the microfluidic device of FIG. 5A is cut | disconnected by A-A 'direction. It is the schematic diagram which showed an example of the cross-sectional shape of each micro through-hole when the some micro through-hole in the microfluidic device of FIG. 5A is cut | disconnected by A-A 'direction. It is the schematic diagram which showed an example of the cross-sectional shape of each micro through-hole when the some micro through-hole in the microfluidic device of FIG. 5A is cut | disconnected by A-A 'direction. It is typical sectional drawing of the microfluidic device 20 which can be used in the liquid feeding method of this invention. It is typical sectional drawing of the microfluidic device 30 which can be used in the liquid feeding method of this invention. It is typical sectional drawing of the microfluidic device 40 which can be used in the liquid feeding method of this invention. It is typical sectional drawing of the 1st board | substrate and 2nd board | substrate which comprise a microfluidic device. It is typical sectional drawing of the 1st board | substrate and 2nd board | substrate which comprise a microfluidic device. It is typical sectional drawing of the principal part containing the fine through-hole T of a microfluidic device. It is typical sectional drawing of the principal part containing the fine through-hole T of a microfluidic device. It is typical sectional drawing of the principal part containing the fine through-hole T of a microfluidic device. It is typical sectional drawing of the principal part of a microfluidic device which showed a mode that an example of the biochemical test | inspection using the liquid feeding method of this invention was performed. It is typical sectional drawing of the principal part of a microfluidic device which showed a mode that an example of the biochemical test | inspection using the liquid feeding method of this invention was performed. It is typical sectional drawing of the principal part of a microfluidic device which showed a mode that an example of the biochemical test | inspection using the liquid feeding method of this invention was performed. It is typical sectional drawing of the principal part of a microfluidic device which showed a mode that an example of the biochemical test | inspection using the liquid feeding method of this invention was performed. It is typical sectional drawing of the principal part of a microfluidic device which showed a mode that an example of the biochemical test | inspection using the liquid feeding method of this invention was performed. It is typical sectional drawing of the principal part of a microfluidic device which showed a mode that an example of the biochemical test | inspection using the liquid feeding method of this invention was performed. It is typical sectional drawing of the principal part of a microfluidic device which showed a mode that an example of the biochemical test | inspection using the liquid feeding method of this invention was performed. It is typical sectional drawing of the principal part of a microfluidic device which showed a mode that an example of the biochemical test | inspection using the liquid feeding method of this invention was performed. It is typical sectional drawing of the principal part of a microfluidic device which showed a mode that an example of the biochemical test | inspection using the liquid feeding method of this invention was performed.

《Liquid feeding method》
A microfluidic device 10 shown in FIG. 1 is an example of a microfluidic device that can be used in the first embodiment of the liquid feeding method according to the present invention. FIG. 1 shows a schematic cross-sectional view of a microfluidic device 10.

  The microfluidic device 10 includes a first inner wall surface w1 constituting the first space portion S1, a second inner wall surface w2 constituting the second space portion S2, and a first opening portion Ta opened to the first inner wall surface w1. And an inner side surface Y that forms one or more fine through holes T that spatially connect the first space S1 and the second space S2 with a second opening Tb that opens to the second inner wall surface w2. And at least a main body portion 4 including: In the microfluidic device 10, the first space portion S <b> 1 and the second space portion S <b> 2 included in the main body portion 4 are spatially connected (communicated) with each other by a plurality of fine through holes T.

  In the liquid feeding method of the first embodiment, an arbitrary liquid is introduced into the first space S1, and a part of the liquid is introduced into each fine through hole T from the first opening Ta by a capillary phenomenon (capillary force). The liquid is discharged from the first space S1, and then the liquid partially temporarily left in the fine through holes T when the liquid is discharged. In this method, the liquid is automatically discharged from the first opening Ta of the hole T to the first space S1.

  The description of the liquid feeding method of the first embodiment is divided into three steps of an inflow step, a discharge step, and an outflow step.

In the inflow step, an arbitrary liquid is introduced into the first space portion S1, and from the first opening portion Ta of each fine through hole T that opens to the first space portion S1, into each fine through hole T by capillary action. A step of allowing a part of the liquid to flow in;
The discharging step is a step of discharging the liquid from the first space portion S1 while leaving the part of the liquid flowing into each fine through hole T.
The outflow step is a step of automatically flowing out the liquid remaining in each fine through hole T from the first opening Ta of each fine through hole T to the first space S1 after the discharge. .
Hereinafter, each step will be described in order.

(Inflow step)
As shown in FIG. 2, when the liquid Q is introduced into the first space S1 from the opening opened on the surface of the main body 4 constituting the microfluidic device 10, each fine through hole T opened in the first space S1. Part of the liquid Q flows into each fine through hole T from the first opening Ta by capillary action (capillary force). At this time, the air in each fine through hole T is naturally pushed out to the second space S2.

  The hole diameter of the fine through hole T (the diameter or the short diameter of the cross section perpendicular to the longitudinal direction) is not particularly limited as long as the capillary phenomenon occurs, and depends on the surface tension and viscosity of the liquid Q used, Usually, it is preferably in the range of 200 nm to 10 μm.

  The tip of the liquid Q that flows in from the first opening Ta of the fine through hole T may or may not reach the second opening Tb that opens in the second space S2. When the second opening Tb is reached, part of the liquid Q constituting the tip may flow out of the second opening Tb, but basically flows out of the second opening Tb. Instead, the progress of the tip of the liquid Q stops at the second opening Tb. This is because the capillary force keeps the liquid Q in the fine through-hole T and the surface tension acting on the tip of the liquid Q in the second opening Tb keeps the liquid Q in the fine through-hole T. .

  When the liquid Q is sufficiently introduced into the first space S1, the inside of each fine through hole T is also filled with a part of the liquid Q (see FIG. 3). At this time, the liquid Q may be continuously introduced into the first space S1, or the liquid Q in the first space S1 is stopped and the first space S1 is filled with the liquid Q. May be kept.

(Discharge step)
Next, as shown in FIG. 4, the liquid Q is discharged from the first space S <b> 1 while leaving the part of the liquid Q flowing into each fine through hole T. The discharge method is not particularly limited, and may be natural discharge using gravity, or forced discharge using a syringe pump, a peristaltic pump, or the like. The discharge speed is not particularly limited, and the first space part S1 may be discharged with a negative pressure. However, it is usually preferable to discharge more gently. That is, by discharging the liquid Q, the first space S1 may or may not be a negative pressure or a positive pressure.

(Outflow step)
When the liquid Q is discharged from the first space portion S1, the liquid Q is discharged and air flows into the first space portion S1, and in order of contact with the air, from the first opening portion Ta of each fine through hole T, The liquid Q remaining in each fine through hole T automatically flows out to the first space S1.

  As one of the factors that cause this automatic outflow, immediately after the liquid Q is discharged from the first space portion S1, the first space portion S1 of the first space portion S1 in which the first opening portion Ta of each fine through-hole T opens. A thin film M made of the liquid Q is formed on the inner wall surface w1. The film M is gradually dried by the air flowing into the first space S1, and the thickness of the film M is gradually reduced, and finally the film M disappears. Along with this drying process, tension continues to work between the liquid Q in each fine through hole T and the film M, and the liquid Q in each fine through hole T is drawn in the direction of the film M, so that the first opening The mechanism of outflow from Ta works. By this mechanism, in order from the fine through hole T having the first opening Ta that is fast in contact with air (in FIG. 4, among the multiple fine through holes T arranged from the top to the bottom of the paper, In order from the through-hole T), the liquid Q remaining inside each fine through-hole T automatically flows out into the first space S1 with a certain time difference.

  Through each of the above steps, a part of the liquid Q introduced into the first space portion S1 is caused to flow into each fine through hole T, and the state is maintained as desired. Subsequently, the liquid is discharged from the first space portion S1. By discharging Q, a part of the liquid Q that is temporarily left in each fine through hole T when the liquid Q is discharged can flow out to the first space S1.

  In the above description, the case where the liquid Q is introduced into the first space portion S1 has been described. However, if the liquid Q is introduced into the second space portion S2 in the same manner as this case, each opening to the second space portion S2 is performed. A part of the liquid Q can be caused to flow into each fine through hole T from the second opening Tb of the fine through hole T. Next, by discharging the liquid Q in the second space portion S2, the liquid Q in each fine through hole T can flow out from the second opening portion Tb to the second space portion S2. The structures and configurations of the first space portion S1 and the second space portion S2 may be the same or different from each other.

  By implementing the liquid feeding method according to the present invention using the microfluidic device 10, each fine through hole T can be used as a reaction field relating to physics, chemistry, and biology (biotechnology). Specifically, for example, the microfluidic device 10 can be used as a biochemical test device using an immunochemical reaction.

<Microfluidic device>
The plurality of fine through-holes T constituting the microfluidic device 10 respectively include a first opening Ta that opens to the first inner wall surface w1 that forms the first space S1, and a second that forms the second space S2. And a second opening Tb that opens to the inner wall surface w2. Each fine through hole T is in the main body 4 constituting the microfluidic device 10 and forms a first flow path S1 and a second flow path in the main body 4 The second space portion S2 to be connected is spatially connected (communicated). That is, the first end of each fine through hole T constitutes a first opening Ta, and the second end of each fine through hole T constitutes a second opening Tb.

  The first space S <b> 1 inherent in the main body 4 is formed by a first inner wall surface w <b> 1 included in the main body 4. The first space S <b> 1 has at least one opening at an arbitrary location on the surface of the main body 4. When any liquid Q is injected from any of the openings, the liquid Q is introduced into the first space S1. Thereafter, the liquid Q in the first space S1 is discharged from any opening at a desired timing. A pump or valve may be attached to the microfluidic device 10 in order to control the introduction and discharge of the liquid Q in the first space S1.

The shape of the first space portion S1 is not particularly limited as long as it has the first inner wall surface w1 in which the first opening portion Ta is opened. For example, the shape of the first space portion S1 is the same shape as the flow path that configures a known fluid device. Alternatively, any solid shape such as a cube, a rectangular parallelepiped, a sphere, and a spheroid may be used. When the shape of the first space S1 is a shape having a longitudinal direction, the shape of the cross section orthogonal to the longitudinal direction may be an arbitrary shape such as a rectangle, a circle, or an ellipse. The cross-sectional area of the cross section is not particularly limited, for ^example, preferably 1mm ² ~400mm 2 about. Within this range, the liquid Q can flow in and out gently and reliably into each fine through-hole T opened in the first space S1.

  The shape and size of the second space S2 may be the same as or different from the first space S1. The path of the second flow path formed by the second space part S2 and the path of the first flow path formed by the first space part S1 are different from each other, but some of both paths overlap or intersect. It does not matter. Control of the flow of the liquid Q in each flow path may be performed by a known method, and can be controlled by, for example, a valve or a pump (not shown) provided on the flow path. Since the description regarding other 2nd space part S2 is the same as that of said 1st space part S1, it abbreviate | omits.

  The hole diameter of the fine through hole T, that is, the diameter or the short diameter (minimum diameter) of the cross section perpendicular to the longitudinal direction of the fine through hole T facilitates the inflow of the liquid Q and the automatic outflow of the liquid Q by the capillary force. Therefore, it is preferable that the first opening Ta is uniform from the second opening Tb. As described above, the pore diameter is preferably in the range of about 200 nm to 10 μm. Within this range, the inflow of the liquid Q by the capillary force and the automatic outflow of the liquid Q can be performed gently and reliably.

  The shapes of the first opening Ta and the second opening Tb of the fine through-hole T (the contours of the edges formed by the both ends of the fine through-hole T on the first inner wall surface w1 and the second inner wall surface w2, respectively) are as follows: The shape is not particularly limited as long as it does not prevent the inflow of the liquid Q by the capillary force and the automatic outflow of the liquid Q. Since the inflow and outflow occur more easily, the shape of the first opening Ta and the second opening Tb is a cross section perpendicular to the longitudinal direction of the fine through hole T having the openings Ta and Tb at both ends. The shape is preferably the same.

  The shapes of the first opening Ta and the second opening Tb of the fine through hole T may be the same or different from each other. Further, when a plurality of fine through holes T are provided in the device as in the microfluidic device 10, the shape of the cross section perpendicular to the longitudinal direction of each fine through hole T and the opening of each fine through hole T are provided. The shapes of the parts Ta and Tb may be the same or different from each other.

  A specific example of the cross-sectional shape of each micro through-hole T when the micro through-hole T included in the microfluidic device is cut in the A-A ′ direction of FIG. 5A is shown below. FIG. 5B is an example in which a plurality of fine through holes T having an elliptical cross section are linearly arranged, and FIG. 5C is an example in which a plurality of fine through holes T having a rectangular cross section are linearly arranged. 5D is an example in which a plurality of fine through-holes T having an elliptical cross section are arranged in three straight lines, and FIG. 5E is an elliptical cross section that is flatter than that of FIG. 5B and has a different major axis direction. This is an example in which a plurality of fine through holes T are linearly arranged. The plurality of fine through holes T provided in the microfluidic device may have the same cross-sectional shape as each other, or may have different cross-sectional shapes.

  The length in the longitudinal direction of the fine through-hole T is not particularly limited as long as the liquid Q flows in and the automatic liquid Q flows out due to the capillary force. For example, the length is about 0.5 mm to 15 mm. preferable. Within this range, the inflow and outflow of the liquid Q in the fine through hole T can be caused gently and reliably. The length is preferably a length capable of optically detecting the inflow or outflow of the liquid Q in the fine through hole T.

  When there are a plurality of fine through holes T provided in the microfluidic device 10, the distance between the fine through holes T (separation distance) and the distance between the openings Ta and Tb of each fine through hole T (separation distance) are It does not restrict | limit in particular, For example, about 1 micrometer-100 micrometers are preferable. The direction of the central axis along the longitudinal direction of each fine through hole T (the direction indicated by the central axis) may be parallel to each other or non-parallel.

  In the main body 4 of the microfluidic device 10, the first space S <b> 1 and the second space S <b> 2 with which the fine through-hole T communicates form two parallel flow paths. In each flow path, the diameter of the flow path in the direction in which the central axis along the longitudinal direction of the fine through hole T passes through each flow path is not particularly limited, but the viewpoint that the liquid easily flows into the fine through hole T from each flow path. Therefore, for example, about 0.5 mm to 20 mm is preferable.

  The “angle formed” between the central axis along the longitudinal direction of each flow path and the central axis along the longitudinal direction of each fine through hole T is not particularly limited, and may be 90 degrees or an obtuse angle. Or an acute angle. The reason why the angle formed is not particularly limited is that the force dominant to the inflow and outflow of the liquid Q in each fine through hole T is the capillary force and the first space portion around each first opening Ta. This is the tension generated when the film M made of the liquid Q formed on the first inner wall surface w1 constituting S1 moves (drys), and the contribution of the angle made is small.

  In the microfluidic device 10, the first inner wall surface w1 and the second inner wall surface w2 constituting the first space portion S1 and the second space portion S2, and the inner side surface Y constituting the plurality of fine through holes T are the main body portion. 4 included in the base material (substrate).

Next, as a modification of the microfluidic device 10, a microfluidic device 20 (see FIG. 6) and a microfluidic device 30 (see FIG. 7) will be described.
The microfluidic devices 20 and 30 can be used to send liquid by the inflow step, the discharge step, and the outflow step, as in the microfluidic device 10 described above.

  The microfluidic device 20 shown in FIG. 6 includes a main body portion 4 including a first inner wall surface w1 constituting the first space portion S1 and a second inner wall surface w2 constituting the second space portion S2, and the first space portion S1. The first opening portion Ta that faces and the second opening portion Tb that opens facing the second space portion S2 and spatially connects the first space portion S1 and the second space portion S2. And a sub main body portion 5 (chip) including an inner surface Y constituting one or more fine through holes T (in communication).

  In the microfluidic device 20, the sub body 5 is installed inside the body 4. The first surface 5u of the sub main body 5 is integrated with the first inner wall surface w1 of the main body 4 to constitute the first space S1. The first opening Ta of each fine through-hole T included in the sub-main body 5 is open to the first surface 5u so as to face the first space S1. Similarly, the second surface 5v of the sub main body 5 is integrated with the second inner wall surface w2 of the main body 4 to constitute a second space S2. The second opening Tb of each fine through-hole T included in the sub-main body 5 is open to the second surface 5v so as to face the second space S2.

  The microfluidic device 30 shown in FIG. 7 includes a main body portion 4 including a first inner wall surface w1 constituting the first space portion S1 and a second inner wall surface w2 constituting the second space portion S2, and the first space portion S1. The first auxiliary inner wall surface ww1 and the second auxiliary inner wall surface ww2 forming the second space S2 are included, and the first opening Ta and the second auxiliary inner wall surface ww2 that open to the first auxiliary inner wall surface ww1 are included. And includes an inner surface Y that constitutes one or more fine through holes T that spatially connect (communicate) the first space S1 and the second space S2. A sub main body 5 (chip).

  In the microfluidic device 30, the sub main body 5 is installed inside the main body 4. The first sub inner wall surface ww1 of the sub main body 5 is connected to the first inner wall surface w1 of the main body 4 to form one first space S1 as a whole. The first opening portion Ta of each fine through hole T included in the sub body portion 5 opens to the first sub inner wall surface ww1 so as to face the first space portion S1. Similarly, the second sub inner wall surface ww2 of the sub main body portion 5 is connected to the first inner wall surface w2 of the main body portion 4 to form one second space portion S2 as a whole. The second opening Tb of each fine through-hole T included in the sub main body 5 opens to the second sub inner wall surface ww2 so as to face the second space S2.

  The sub main body 5 (chip) constituting the microfluidic devices 20 and 30 may be installed so that it can be detached from the main body 4 and attached to the main body 4 or removed from the main body 4. It may be installed in a state where it is bonded or bonded so that it is not possible.

  The shape of the base constituting the sub main body 5 is not particularly limited as long as it can be installed on the main body 4. Examples of the shape of the substrate include a box shape (chip shape) such as a rectangular parallelepiped and a cube. The material of the substrate is not particularly limited, and the same material as the material of the substrate constituting the main body 4 can be applied.

  A method for forming the fine through hole T, the first space portion S1, and the second space portion S2 in the base body constituting the main body portion 4 or the sub main body portion 5 is not particularly limited, and a known fine processing technique can be applied. A manufacturing method of the microfluidic device 10 will be described in detail later as a representative example.

The microfluidic devices 10, 20, and 30 described above are devices including two space portions S1 and S2.
Hereinafter, a microfluidic device 40 having a plurality of spaces will be exemplified with reference to FIG.

  The microfluidic device 40 includes a first inner wall surface w1 constituting the first space portion S1, a second inner wall surface w2 constituting the second space portion S2, and an nth inner portion constituting the nth space portion Sn. A total of n inner wall surfaces including the wall surface wn are included. including. The “n” represents an integer (ordinal number) of 3 or more.

  Furthermore, the microfluidic device 40 has a first opening Ta that opens to the first inner wall surface w1 and a second opening Tb that opens to the second inner wall surface w2, and includes the first space S1 and the second space. A first inner surface Y1 constituting one or more fine through-holes T that spatially connect the portion S2, and a first opening Ta and an nth inner wall surface wn that open to the first inner wall surface w1. The (n−1) th (n−1) th one or more fine through holes T having a second opening Tb that opens and spatially connect (communicate) the first space S1 and the nth space Sn. A total of (n−1) inner surfaces including the inner surface Y (n−1) are included. One inner surface constitutes one or more fine through holes. including. The “n” represents an integer (ordinal number) of 3 or more.

  Here, “a first inner wall surface w1 constituting the first space portion S1, a second inner wall surface w2 constituting the second space portion S2, and an nth inner wall surface wn constituting the nth space portion Sn. "..." in the notation of "is the third inner wall surface w3 constituting the third space portion S3, the fourth inner wall surface w4 constituting the fourth space portion S4, and the fifth inner portion constituting the fifth space portion S5. It represents that any n number of space portions are repeated in the order of the wall surfaces w5,. The “n” represents an integer (ordinal number) of 3 or more, and FIG. 8 illustrates the case of n = 5.

Similarly, “... Has a first opening Ta that opens to the first inner wall surface w1 and a second opening Tb that opens to the nth inner wall surface, and the first space portion S1 and the nth space portion Sn. "..." in the notation of the (n-1) th inner surface Y (n-1) constituting one or more fine through-holes T that spatially connect (communicate) with each other,
It has a first opening Ta that opens to the first inner wall surface w1 and a second opening Tb that opens to the third inner wall surface, and spatially connects the first space portion S1 and the third space portion S3 ( A second inner surface Y2 constituting one or more fine through-holes T (in communication);
The first opening portion Ta has a first opening portion Ta that opens on the first inner wall surface w1 and a second opening portion Tb that opens on the fourth inner wall surface, and spatially connects the first space portion S1 and the fourth space portion S4 ( A third inner surface Y3 constituting one or more fine through-holes T (in communication);
In this order, any (n-1) inner surfaces are repeated. The “n” represents an integer (ordinal number) of 3 or more, and is the same integer as “n” in the nth space portion Sn. Therefore, FIG. 8 illustrates the case where n = 5.

  The microfluidic device 40 communicates the first micro space group G1 composed of one or more micro through holes T communicating the first space portion S1 and the second space portion S2, and the first space portion S1 and the third space portion S3. A second micro space group G2 composed of one or more micro through holes T, (n-1) of one or more micro through holes T communicating the first space portion S1 and the n th space portion Sn. It has a minute space group G (n-1). Here, the notation “...” Represents that any (n−1) minute space groups are repeated in the same order as described above. The “n” represents an integer (ordinal number) of 3 or more, and is the same integer as “n” in the nth space portion Sn. Therefore, FIG. 8 illustrates the case where n = 5.

  The first liquid feeding method in the microfluidic device 40 can be performed by the inflow step, the discharge step, and the outflow step, similarly to the microfluidic device 10 and the like described above.

  In the first liquid feeding method, the inflow step introduces the liquid Q into the first space S1, and opens to the first space S1, and the first through holes T of the fine through holes T constituting the first minute space group G1. From the first opening Ta of each fine through hole T that opens into the first opening Ta,... And the first space S1, and constitutes the (n−1) th minute space group G (n−1). In this step, a part of the liquid Q is caused to flow into each fine through hole T by capillary action. The discharging step is a step of discharging the liquid Q from the first space portion S1 while leaving the part of the liquid Q flowing into each fine through hole T. The outflow step is a step of automatically flowing out the liquid Q in each fine through hole T from the first opening Ta of each fine through hole T to the first space S1 after the discharge. Note that n represents an integer (ordinal number) of 3 or more.

  In the first liquid feeding method described above, the first opening Ta of each micro through hole T constituting the first micro space group to the (n-1) micro space group of the microfluidic device 40 is commonly used. By introducing the liquid Q into the open first space S1, the same liquid Q flows into the fine through holes T of the first to (n-1) th minute space groups, and then Leaked. As a second liquid feeding method different from the first liquid feeding method, it is also possible to cause different liquids to flow into and out of each of the first micro space group to the (n-1) micro space group. Is possible.

  In the second liquid feeding method using the microfluidic device 40, each of the first microspace group to the (n-1) th microspace group independently performs an inflow step, an exhaust step, and an outflow step. In the inflow step, the second opening Tb of each fine through hole T constituting each group is individually opened for each group, and each of the second space part S2 to the nth space part Sn is desired. Introduce liquid.

  When the predetermined liquid A is allowed to flow into and out of each fine through-hole T constituting the first minute space group G1, the liquid A may be introduced and discharged in the second space portion S2. Independently of the inflow and outflow of the liquid A in the first microspace group G1, the nth space portion is used when the liquid B flows in and out of the (n-1) th microspace group G (n-1). The liquid B may be introduced into Sn and then discharged.

A part of the liquid A introduced into the second space S2 flows into the fine through holes T from the second openings Tb of the fine through holes T constituting the first minute space group G1. Next, when the liquid A is discharged from the second space portion S2, a part of the liquid A temporarily remaining in each minute through hole T constituting the first minute space group G1 is second from the second opening portion Tb. It flows out to space part S2.
The inflow and outflow of the liquid B introduced into the nth space portion Sn into the (n-1) th minute space group G (n-1) are the same as in the case of the liquid A introduced into the second space portion S2. It is.

  Therefore, the second liquid feeding method includes the main body part 4, the first space part S1, the second space part S2,... And the nth space part Sn (n is an integer of 3 or more). And the first group G1 composed of one or more fine through-holes T that are inherent in the main body part 4 and communicate with the first space part S1 and the second space part S2. And a microfluidic device 40 including the (n-1) th group G (n-1) composed of one or more fine through holes T communicating with the nth space Sn. Thus, the liquid feeding method in which the liquid feeding in the first micro space group G1,..., The liquid feeding in the (n-1) th micro space group G (n-1) is performed independently.

  Here, the notation “...” Represents that any (n−1) minute space groups are repeated in the same order as described above. The “n” represents an integer (ordinal number) of 3 or more, and is the same integer as “n” in the nth space portion Sn. Therefore, FIG. 8 illustrates the case where n = 5.

  The liquid feeding in the first micro space group G1 introduces the first liquid into the second space portion S2, and at the same time the first through holes T constituting the first micro space group G1 opening in the second space portion S2. From the second opening Tb, an inflow step for allowing a part of the liquid to flow into each fine through hole T by capillary action, and the one that has flowed into each fine through hole T constituting the first micro space group G1. A discharge step of discharging the liquid from the second space portion S2 while leaving the liquid of the portion, and after the discharge, from the second opening Tb of each fine through hole T constituting the first minute space group G1, An outflow step for automatically outflowing the liquid in each fine through hole T to the second space S2.

  In the (n−1) th minute space group G (n−1), liquid feeding introduces the (n−1) th liquid into the nth space portion Sn and opens the (n−1) th space portion Sn. -1) an inflow step for allowing a part of the liquid to flow into each micro through hole T by capillary action from the second opening Tb of each micro through hole T constituting the micro space group G (n-1); The liquid is discharged from the nth space portion Sn while leaving the part of the liquid flowing into each fine through hole T constituting the (n-1) th minute space group G (n-1). After the discharge step, and after the discharge, the liquid in each fine through hole T is supplied from the second opening Tb of each fine through hole T constituting the (n-1) th minute space group G (n-1). an outflow step for automatically flowing out into the n space portion Sn.

In the second liquid feeding method using the microfluidic device 40, the same liquid may be fed to each group independently, or different liquids may be fed. For example, an ELISA that detects different antigens in each group can be performed. Since ELISA in each group can be performed independently, a plurality of ELISAs can be performed simultaneously.
On the other hand, in the first liquid feeding method described above, the same liquid can be fed all at once to all the groups having the fine through holes T opened in the first space S1.

  In the use of the microfluidic device 40, a liquid feeding method combining the second liquid feeding method and the first liquid feeding method may be performed. By this liquid feeding method, it is possible to realize efficient liquid feeding utilizing the independence of each group. The following usage method is mentioned as an example.

First, by the first liquid feeding method, a solution containing protein A is fed to all groups at once, and protein A is adsorbed to the inner surface of each fine through hole T constituting all groups. After that, a solution containing skim milk is further fed to block the inner wall surfaces of the fine through holes T constituting all the groups.
Next, by the second liquid feeding method, a solution containing individual primary antibodies having a desired antigen specificity is fed independently to each group, and the inner wall surface of the fine through hole T included in each group Place an individual primary antibody in
Subsequently, the first liquid feeding method is used to send the cleaning liquid to all groups at once, and then the sample liquid to be inspected is sent to all groups at once. Independent ELISA can be performed in each group by sending a solution containing a secondary antibody to which a labeling substance is bound to the group in a lump.

<< Method for Manufacturing Microfluidic Device >>
The above-described microfluidic device can be manufactured by applying a known microfabrication technique.
The material in particular of the main-body part 4 of a microfluidic device is not restrict | limited, For example, glass, a plastic (resin), a semiconductor, a metal, ceramics etc. are mentioned. The shape of the main body 4 is not particularly limited, and it is easy to connect and install the main body 4 to other devices (for example, pumps, reagent bottles, waste liquid reservoirs, etc.). A box shape is preferable. The main body 4 having such a shape is referred to as a “substrate”. Hereinafter, although the manufacturing method of the microfluidic device when the main-body part 4 is comprised with the board | substrate is demonstrated, even if the main-body part 4 is shapes other than a board | substrate, it can manufacture similarly.

  As a method for forming the fine through hole T inherent in the substrate (main body portion 4), for example, two formation methods, a first formation method and a second formation method, can be exemplified.

(First forming method)
As shown in FIGS. 9 and 10, the first forming method is to form a groove Ya (concave portion) on the surface of the first substrate 4A, join the second substrate 4B to the surface, and place a ceiling in the groove Ya. In this method, fine through holes T are formed.

  9 and 10 are schematic cross-sectional views in which main parts including the fine through hole T of the main body 4 are enlarged. The main body 4 is formed by joining the first substrate 4A and the second substrate 4B. The fine through hole T is formed at the interface between the two substrates. In the case of FIG. 9, one fine through hole T having a rectangular cross section is formed. In the case of FIG. 10, a plurality of fine through holes T having a rectangular cross section are formed.

  As shown in FIG. 10, when the height H1 of the groove Ya is larger than the distance L2 between the adjacent fine through holes T (when H1> L2), the surface area of each fine through hole T (the fine through holes are The sum of the areas of the inner side surfaces to be formed is larger than the surface area of one large fine through hole T shown in FIG. 9 (the area of the inner side surface constituting the fine through holes). Therefore, when it is desired to increase the surface area of the fine through hole T in the main body 4, that is, the contact area between the liquid Q and the inner surface constituting the fine through hole T, the fine through hole T as shown in FIG. A plurality of layers may be formed. In order to further increase the surface area (contact area), the aspect ratio (height H1 / base L1) of each fine through hole T may be made larger than 1. The surface area can be increased as the aspect ratio is increased.

  As a method of forming the groove Ya in the first substrate 4A, various known methods can be cited depending on the material of the first substrate 4A.

  In the case of using a resin substrate as the first substrate 4A, for example, a forming method in which known methods such as molding, nanoimprinting, injection molding, and the like for transferring and forming a fine pattern produced by a soft lithography technique are appropriately combined.

  When a glass substrate is used as the first substrate 4A, for example, a method of appropriately combining known methods such as photolithography, laser processing, machining, dry etching, and wet etching can be used. In the case of forming the nanoscale groove Ya, a combination of substrate modification by short pulse laser processing and wet etching is preferable.

  Examples of a method for bonding the first substrate 4A and the second substrate 4B include known methods such as a method of bonding with an adhesive, an anodic bonding method, a self-welding method, and a low-temperature pressure bonding method using surface modification. .

  The materials of the first substrate 4A and the second substrate 4B may be the same or different from each other. Specifically, both substrates may be glass substrates, a combination of a glass substrate and a silicon substrate, a combination of a glass substrate and a plastic substrate, or both substrates. It may be a resin substrate.

  The kind of plastic substrate is not particularly limited. For example, PET (polyethylene terephthalate), PS (polystyrene), PMMA (polymethyl methacrylate), PDMS (polydimethylsiloxane), AS (acrylonitrile styrene) resin, PC (polycarbonate), PLA (Polylactic acid) and the like. The kind of glass which comprises a glass substrate is not specifically limited, For example, quartz glass, borosilicate glass, soda-lime glass, etc. are mentioned.

(Second forming method)
The second forming method is a method of directly forming the fine through hole T in the first substrate 4C as shown in FIGS. By connecting the focal point (condensing part) of short pulse laser light inside the substrate and scanning the part where the fine through hole T is formed in the substrate, the etching resistance of the scanned part (modified part) is weakened. To reform. Thereafter, the modified through hole T can be formed in the substrate by removing the modified portion from the substrate by wet etching.

  11, 12, and 13 are schematic cross-sectional views of a main part including a plurality of fine through holes T formed in the main body 4. The main body 4 is constituted by a single substrate 4C. The fine through hole T is formed inside the substrate 4C.

  In the case of FIG. 11, the fine through holes T having a plurality of elliptical cross sections are arranged in a line in a direction (plane direction of the substrate) orthogonal to the substrate thickness direction K. Since the major axis of the ellipse is along the substrate thickness direction K, the integration density of the fine through holes 4 in the plane direction of the substrate can be increased as compared with the case where the cross section is a perfect circle. By increasing the integration density, the number of measurement samples per unit volume increases, so that measurement accuracy can be improved.

  The case of FIG. 12 is a case where the fine through holes T having a plurality of elliptical cross sections are arranged in two rows in a direction orthogonal to the substrate thickness direction K (plane direction of the substrate). In the drawing, the fine through holes T arranged in the upper stage and the fine through holes T arranged in the lower stage are arranged so as not to overlap each other when viewed in the thickness direction K of the substrate. By arranging in this way, the integration density of the plurality of fine through holes T can be increased without impairing the ease of observing each fine through hole T in the substrate thickness direction K.

  In the case of FIG. 13, the fine through holes T having a plurality of elliptical cross sections are arranged in a line in the substrate thickness direction K. When arranged in this way, since the major axis of the fine through hole T is along the substrate plane direction (the minor axis of the minute through hole T is along the substrate thickness direction K), light ( For example, when the fine through-hole T is irradiated with excitation light, a backlight for observing the inside, or the like, the irradiation light is hardly refracted and the irradiation light is easily transmitted. Therefore, it is easy to irradiate light inside the fine through hole T, and light emission inside the fine through hole T can be easily observed from the thickness direction K of the substrate.

(Specific example of the second forming method)
In this specific example, an apparatus that generates a titanium sapphire laser beam, which is a femtosecond laser, is used, but an apparatus that generates another type of laser beam may be used.
First, laser light is incident on the first surface of a quartz glass substrate placed on a precision stage, the laser light is focused on a predetermined position inside the substrate, and perpendicular to the propagation direction (optical axis) of the laser light. The focal point of the laser beam is scanned in the direction of. At this time, if the laser polarization is perpendicular to the scanning direction, it is possible to easily form the fine through hole T having a minor diameter of nano order (for example, about 10 nm to 500 nm). Moreover, it is preferable that the irradiation intensity of a laser beam is set to be equal to or higher than the processing lower limit threshold and lower than the processing upper limit threshold. The optimum irradiation intensity is preferably examined in advance using the same type of quartz glass substrate.
As an example, the following irradiation conditions are mentioned, for example.

Wavelength (center wavelength) = 800 nm, spectrum width = 10 nm (± 5 nm), pulse time width = ˜250 fs, numerical aperture (NA) of objective lens = 0.5, polarization = linear polarization, optical axis and scanning direction Angle of about 90 degrees / peak intensity (laser fluence per pulse / pulse time width) = 9 TW / cm ²
・ Scanning speed (μm / sec) = 1,000 μm / sec, repetition frequency (kHz) = 200 kHz
・ Scan at a constant speed while shifting so that the focus of each pulse overlaps.

  By irradiating the quartz substrate with laser light as described above, a modified portion with reduced etching resistance is formed in the region scanned by the light collecting portion including the focal point of the laser light and its periphery. For example, it is possible to form a modified portion that extends substantially parallel to the substrate surface and has an elliptical (substantially rectangular) cross-sectional shape perpendicular to the extending direction. As an example, a modified portion having a long diameter (vertical length) (length in the substrate thickness direction K) of about 5 μm and a short diameter (horizontal length) (length in the substrate plane direction) of about 30 nm. Can be formed. By such laser processing, it is possible to form a plurality of modified portions arranged in parallel to each other.

  Next, the quartz substrate with both ends of each modified portion exposed on the surface is immersed in hydrofluoric acid or an aqueous potassium hydroxide solution for etching. In this etching, the etching solution permeates into each modified portion from both ends of each modified portion, and each modified portion is removed from the quartz substrate. As a result, a plurality of fine through holes T penetrating the quartz substrate can be formed. As an example, both end portions are open on the surface of the quartz substrate, the cross-sectional shape in a direction orthogonal to the longitudinal direction is an ellipse (substantially rectangular), and a long diameter (vertical length) (length in the substrate thickness direction K) ) Is about 5.5 μm, and a fine through hole having a short diameter (horizontal length) (length in the substrate plane direction) of about 300 nm can be formed.

  When the modified portion is formed on the quartz substrate, if both ends of the modified portion are not exposed on the surface of the substrate, the modified portion can be formed by a method such as photolithography, grinding, polishing, etc. before etching. What is necessary is just to pre-process so that the both ends of may be exposed to the substrate surface.

  The laser processing method described above can also be used for processing resin materials such as polystyrene, acrylic (PMMA), and PDMS. Since femtosecond laser processing of a resin material can form fine holes only by laser focused irradiation, the removal of the modified portion by etching is generally unnecessary.

<Use of microfluidic device>
The use of the microfluidic device is not particularly limited as long as the above-described liquid feeding method can be applied, and examples thereof include use as a biochemical test device using an antibody.

  Hereinafter, with reference to FIGS. 14A to 14I, the flow of the liquid Q1 to the liquid Q3 in an example in which the microfluidic device 10 is used as a biochemical test device is illustrated.

  First, when the first liquid Q1 containing the primary antibody Ab1 is introduced into the first space part S1, the liquid Q1 gradually fills the first space part S1, and the first through the fine through holes T by capillary force. The liquid Q1 flows into the inside from the opening Ta (FIG. 14A). The primary antibody Ab1 contained in the liquid Q1 flowing into each fine through hole T is adsorbed on the inner side surface Y of each fine through hole T.

  After the liquid Q1 is filled in all the fine through holes T, when the liquid Q1 is discharged from the first space S1, air flows into the first space S1. Among the first openings Ta of the fine through holes T that open to the first space S1, the first openings Ta that are in the order of contact with air are sequentially left in the fine through holes T in order. The liquid Q1 flows out to the first space S1 (FIG. 14B). At this time, the primary antibody Ab1 adsorbed on the inner side surface Y of each fine through hole T maintains the state adsorbed on the inner side surface Y. Even after all the liquid Q1 inside each fine through-hole T has been discharged, the primary antibody Ab1 remains adsorbed on the inner surface Y of each fine through-hole T (FIG. 14C).

  Subsequently, when the second liquid Q2 containing the antigen Ag is introduced into the first space portion S1, the liquid Q2 gradually fills the first space portion S1, and the first through the fine through-holes T by the capillary force. The liquid Q2 flows into the inside from the opening Ta (FIG. 14D). The antigen Ag contained in the liquid Q2 flowing into each fine through hole T is bound by an antigen-antibody reaction against the primary antibody Ab1 adsorbed on the inner surface Y of each fine through hole T. The primary antibody Ab1 is preliminarily provided with a binding property to the antigen Ag.

  After the liquid Q2 is filled in all the fine through holes T, when the liquid Q2 is discharged from the first space S1, air flows into the first space S1. Among the first openings Ta of the fine through holes T that open to the first space S1, the first openings Ta that are in the order of contact with air are sequentially left in the fine through holes T in order. The liquid Q2 flows out to the first space S1 (FIG. 14E). At this time, the primary antibody Ab1 adsorbed on the inner surface Y of each fine through-hole T maintains a state in which the antigen Ag is bound. Even after all the liquid Q2 inside each fine through-hole T has been discharged, the primary antibody Ab1 maintains a state in which the antigen Ag is bound (FIG. 14F).

  Here, when the liquid Q2 contains a foreign substance different from the antigen Ag, that is, a substance that does not bind to the primary antibody Ab1, when the liquid Q2 flows out of the fine through hole T, the fine through hole is formed together with the liquid Q2. Contaminants are excluded from T. That is, B / F separation (separation of a substance (antigen) bound to the primary antibody and a substance (contaminant) not bound) is efficiently performed.

  Next, when the third liquid Q3 containing the secondary antibody Ab2 to which the binding property to the antigen Ag is previously imparted is introduced into the second space S2, the liquid Q3 gradually fills the second space S2, The liquid Q3 flows into the inside from the second opening Tb of each fine through hole T by the capillary force (FIG. 14G). The secondary antibody Ab2 contained in the liquid Q3 flowing into each fine through-hole T recognizes and binds to the antigen Ag bound to the primary antibody Ab1 on the inner surface Y of each fine through-hole T, and the primary antibody-antigen-2 A ternary complex Comp. Form.

  After the liquid Q3 is filled in all the fine through holes T, when the liquid Q3 is discharged from the second space S2, air flows into the second space S2. Among the second openings Tb of the fine through holes T that open to the second space S2, the second openings Tb that are in the order of contact with air are sequentially left in the fine through holes T in order. The liquid Q3 flows out to the second space S2 (FIG. 14H). At this time, the ternary complex Comp. Maintains a state of being bound to the inner surface Y via the primary antibody Ab1. Even after all the liquid Q3 inside each fine through hole T is discharged, the three-component complex Comp. Maintains the state of binding to the inner surface Y via the primary antibody Ab1 (FIG. 14I).

  Since the luminescent labeling substance is bound (conjugate) in advance to the secondary antibody Ab2, for example, when the fine through-hole T is irradiated with excitation light, the three antibodies formed on the inner surface Y of the fine through-hole T Complex Comp. The luminescent labeling substance contained in fluoresces. The emission amount of this fluorescence is determined by the tripartite complex Comp. Depends on the abundance of Therefore, the content of the antigen Ag contained in the liquid Q2 can be quantified by measuring the fluorescence emission amount. However, there is a premise that the amount of the primary antibody Ab1 adsorbed on the inner surface Y of the fine through hole T and the amount of the secondary antibody Ab2 introduced into the fine through hole T are sufficiently larger than the amount of the antigen Ag.

  Before performing the fluorescence measurement by the excitation light irradiation, the cleaning liquid is placed in the fine through-hole T for the purpose of washing the inner surface Y of the fine through-hole T or the secondary antibody Ab2 adsorbed non-specifically to the primary antibody Ab1. After flowing in, it may be discharged.

In general, it is preferable that the inside of the fine through hole T is cleaned at any one or more of the following stages.
After the liquid Q1 containing the primary antibody Ab1 flows into and out of the fine through-hole T After the liquid Q2 containing the antigen Ag flows into and out of the fine through-hole T The liquid Q3 containing the secondary antibody Ab2 is fine through-hole After flowing into and out of T

  As the luminescent labeling substance to which the secondary antibody Ab2 binds, the luminescent labeling substance excited by receiving external light irradiation may be a fluorescent substance that emits fluorescence, or chemiluminescent to other substrates. It may be a catalytic substance that converts to a luminescent substance.

  Examples of the fluorescent substance include green fluorescent protein (GFP) and quantum dots. As the catalytic substance, for example, known enzymes used in ELISA can be applied, and specific examples include enzymes such as peroxidase, luciferase, and aequorin. Examples of substrates for this enzyme include 3- (p-hydroxyphenol) propionic acid and its analogs, luciferin and luciferin analogs, coelenterazine and coelenterazine analogs, and the like.

  One kind of luminescent labeling substance may be used alone, or two or more kinds of luminescent substances may be used in combination. Two or more kinds of fluorescent substances may be used in combination, two or more kinds of enzymes and substrates may be used in combination, or one or more kinds of fluorescent substances may be used in combination with one or more kinds of enzymes and substrates. Good. The method for binding various luminescent labeling substances to the secondary antibody Ab2 is not particularly limited, and known methods can be applied.

  The labeling substance for tracing the substance present in the fine through-hole T may be a luminescent labeling substance as described above or a non-luminescent labeling substance. Examples of non-luminescent labeling substances include radioactive labeling substances as used in known radioimmunoassay methods.

  The type of antigen Ag is not particularly limited and is appropriately selected according to the purpose of biochemical examination. Specific examples of the antigen Ag include proteins, peptides, nucleic acids, lipids, sugar chains and the like derived from pathogens such as viruses, bacteria, and the like that cause colds, hepatitis, acquired immune deficiency, and the like.

  The substance that captures the antigen Ag in the fine through-hole T is not particularly limited as long as it is a substance that can capture the antigen Ag in the state of being disposed in the fine through-hole T. The antibody (primary antibody Ab1 and secondary antibody Ab2 Other substances may be used instead of Examples of such substances include peptides (peptide aptamers), DNA aptamers, RNA aptamers, sugar chains, and the like. Among these substances, a substance having high specificity for antigen Ag (which can specifically recognize antigen Ag) is preferable.

  The embodiment described above qualitatively analyzes whether or not the antigen Ag is contained in the liquid Q2 to be examined, or quantitatively analyzes the content of the antigen Ag contained in the liquid Q2. For this purpose, a format generally called a sandwich immunoassay is adopted. For this reason, it is necessary to prepare in advance the primary antibody Ab1 and the secondary antibody Ab2 that specifically or non-specifically bind to the antigen Ag. Such antibodies are obtained by known methods.

  In the biochemical test using the microfluidic device according to the present invention, other known immunoassay formats such as indirect antibody immunoassay format, bridging immunoassay format, competitive format, etc. may be adopted instead of the above sandwich immunoassay format. Good. Moreover, you may implement the biochemical test | inspection using the interaction between other molecules which does not utilize at least any one of an antibody and an antigen. Since these basic methods of biochemical testing using these immunoassay formats and other intermolecular interactions are known, only a brief description will be given here.

  When the indirect antibody immunoassay format is employed, the primary antibody Ab1 and the secondary antibody Ab2 are sequentially introduced into the fine through-hole T after the antigen Ag is adsorbed on the inner surface Y of the fine through-hole T. According to this format, when there is a possibility that the primary antibody Ab1 is contained in the sample liquid to be examined, it is possible to analyze whether or not the primary antibody Ab1 is contained or contained.

  When the bridging immunoassay format is adopted, after the antigen Ag is adsorbed on the inner surface Y of the fine through-hole T, the primary antibody Ab1 and the antigen Ag having a labeling substance are sequentially introduced into the fine through-hole T. According to this format, the presence or content of the primary antibody Ab1 in the liquid to be examined can be analyzed.

  Known methods can be applied to the method of immobilizing anchor substances such as antibodies, antigens, and other proteins, peptides, nucleic acids, lipids, sugar chains, etc. in the fine through-holes T. For example, when a solution containing an arbitrary antigen Ag is caused to flow into the fine through hole T having a clean inner surface Y, the antigen Ag can be adsorbed to the inner surface Y. Then, it is preferable to perform the blocking process for preventing nonspecific adsorption | suction to the inner surface Y as needed. In addition, when there is a portion in the flow path where the antigen Ag is not desired to be adsorbed, unnecessary adsorption can be prevented by performing a surface treatment such as a coating for improving the water repellency of the portion in advance.

  In the use of the antibody, the antigen and the anchor substance, one type may be used alone, or two or more types may be used in combination.

  The configurations and combinations thereof in the embodiments described above are examples, and the addition, omission, replacement, and other modifications of the configurations can be made without departing from the spirit of the present invention. Further, the present invention is not limited by each embodiment, and is limited only by the scope of the claims.

  The microfluidic device according to the present invention can be widely used in the fields of medical inspection, food inspection, and the like.

10, 20, 30, 40 ... microfluidic device, S1 ... first space, S2 ... second space, S3 ... third space, S4 ... fourth space, S5 ... fifth space, w1 ... first 1 inner wall surface, w2 ... second inner wall surface, w3 ... third inner wall surface, w4 ... fourth inner wall surface, w5 ... fifth inner wall surface, Ta ... first opening, Tb ... second opening, T ... Fine through-hole, Y ... inside surface, 4 ... main body part, 5 ... sub-main body part, M ... film made of liquid, G1 ... first minute space group, G2 ... second minute space group, G3 ... third minute space group G4 ... fourth micro space group, Q ... liquid, Q1 ... first liquid, Q2 ... second liquid, Q3 ... third liquid, Ab1 ... primary antibody, Ab2 ... secondary antibody, Ag ... antigen, Comp . ... Tripartite complex

Claims (4)

A method for delivering a liquid in a microfluidic device, comprising:
A first inner wall surface constituting the first space portion, a second inner wall surface constituting the second space portion,
One having a first opening opening in the first inner wall surface and a second opening opening in the second inner wall surface, and spatially connecting the first space portion and the second space portion. Using a microfluidic device comprising a main body portion including the inner side surface constituting the fine through hole,
After introducing a liquid into the first space, and flowing a part of the liquid into the one or more fine through holes from the first opening,
A film made of the liquid is formed on the first inner wall surface by discharging the liquid from the first space, and then the film and the one or more fine through-holes accompanying the drying process of the film The partial liquid is caused to flow out from the first opening of the one or more fine through-holes to the first space by a tension with the partial liquid temporarily remaining in the first through-hole. A characteristic liquid delivery method. A method for delivering a liquid in a microfluidic device, comprising:
A main body portion including a first inner wall surface constituting the first space portion and a second inner wall surface constituting the second space portion;
A first opening that opens to face the first space and a second opening that opens to face the second space, the first space and the second space being a space; A microfluidic device comprising: a sub-body portion including an inner surface that constitutes one or more fine through-holes to be connected to each other;
After introducing a liquid into the first space, and flowing a part of the liquid into the one or more fine through holes from the first opening,
A film made of the liquid is formed on the first inner wall surface by discharging the liquid from the first space, and then the film and the one or more fine through-holes accompanying the drying process of the film The partial liquid is caused to flow out from the first opening of the one or more fine through-holes to the first space by a tension with the partial liquid temporarily remaining in the first through-hole. A characteristic liquid delivery method. A method for delivering a liquid in a microfluidic device, comprising:
A main body portion including a first inner wall surface constituting the first space portion and a second inner wall surface constituting the second space portion;
A first sub-inner wall surface constituting the first space portion; a second sub-inner wall surface constituting the second space portion; and a first opening and a second opening opening in the first sub-inner wall surface. A sub-main body portion including a second opening portion that opens in the sub-inner wall surface and including an inner surface that forms one or more fine through holes that spatially connect the first space portion and the second space portion; Using a microfluidic device comprising
A liquid is introduced into a first space formed by the first inner wall surface and the first sub inner wall surface, and a part of the liquid flows into the one or more fine through holes from the first opening. After letting
A film made of the liquid is formed on the first inner wall surface by discharging the liquid from the first space, and then the film and the one or more fine through-holes accompanying the drying process of the film The partial liquid is caused to flow out from the first opening of the one or more fine through-holes to the first space by a tension with the partial liquid temporarily remaining in the first through-hole. A characteristic liquid delivery method. A method for delivering a liquid in a microfluidic device, comprising:
A first inner wall surface constituting the first space portion, a second inner wall surface constituting the second space portion,... Nth space constituting the nth space portion (n represents an integer of 3 or more). N inner wall surfaces including a wall surface;
One having a first opening opening in the first inner wall surface and a second opening opening in the second inner wall surface, and spatially connecting the first space portion and the second space portion. A first inner surface constituting the fine through hole, and
One that has a first opening that opens in the first inner wall surface and a second opening that opens in the nth inner wall surface, and spatially connects the first space portion and the nth space portion. (N-1) inner side surfaces including the (n-1) th inner side surface constituting the fine through hole, and
Using a microfluidic device comprising a body comprising
A liquid is introduced into the first space portion, and one liquid is introduced into each fine through-hole from a first opening included in each fine through-hole constituted by the (n-1) inner surfaces. After flowing the part
By discharging the liquid from the first space portion, a film made of the liquid is formed on the nth inner wall surfaces, and subsequently, in the film and each fine through-hole in the drying process of the film The partial liquid is caused to flow out from the first opening of each fine through-hole to the first space due to the tension with the partial liquid temporarily remaining in the liquid. Method.

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