JP4806699B2 - Microchip and antibody immobilization method using the microchip - Google Patents

Description

  The present invention relates to a microchip, and more particularly to a microchip capable of achieving homogenization in antibody immobilization and an antibody immobilization method using the microchip.

Bovine mastitis develops when microorganisms such as bacteria invade the breast of a milking cow. In milking cows infected with this causative mastitis causative agent, a decrease in milk yield and quality is observed, and when it becomes severe, not only milk is not produced at all, but the cow itself may die. It is particularly important to deal with mastitis in milking cows at the very early stages of onset, since it is very difficult to cure once they are affected. Therefore, establishment of an efficient early diagnosis method is strongly desired.
Conventionally, a culture test that is conducted as a search for microorganisms (causative bacteria) that causes bovine mastitis requires at least several days. In addition, since the optimal therapeutic drug and coping method differ depending on the type of causative bacteria, development of a method capable of detecting specific bacteria quickly and quantitatively from a plurality of types of bacteria has been awaited.

  Regarding the identification of causative bacteria, in addition to the conventional identification method through culture, identification method by staining, and method of measuring substances related to bacterial metabolism such as ATP, SPR (Surface using surface plasmon resonance method) Bacterial identification methods have been developed by immunoassay. In particular, the SPR immunoassay is being applied in various forms because it can be measured quickly with high sensitivity using a small amount of measurement sample.

  In SPR immunoassay, a chip (antibody-immobilized chip) in which an antibody is immobilized on the surface of a thin film is used for detection of a measurement object, as disclosed in, for example, Patent Document 1 and Patent Document 2. In this antibody immobilization chip, a gel layer of 100 nm or less is formed on a gold thin film, and a method of immobilizing an antibody on this gel layer, or an antibody solution mainly composed of whole type immunoglobulin (antibody), It is produced by a physical adsorption method in which it is dropped on a clean gold thin film surface and dried. In SPR immunoassay, the detection sensitivity of the refractive index is highest on the surface of the gold thin film, which is the reflection surface of the total reflected light. The immobilization chip is advantageous from the viewpoint of sensitivity.

In the antibody-immobilized chip prepared by such a simple physical adsorption method, the immunoglobulin directly contacting the gold thin film surface is adsorbed on the gold thin film in various directions, and the Fab portion, which is an antigen capture site, is adsorbed on the gold thin film surface. It is said that the activity can be maintained only about 20% because it cannot be contacted with the antigen by adsorption or the Fab part is denatured by contact with gold. However, even in this case, since immunoglobulin can be adsorbed in multiple layers, another antibody that maintains its activity is superimposed on the inactivated antibody. Therefore, the antibody immobilization chip produced by the physical adsorption method is multiplied by the measurement principle that the density of the antibody is highly sensitive to the surface nearest the surface of the gold thin film in SPR measurement. Sensitivity comparable to that obtained when an antibody immobilization layer such as a gel layer is provided on the surface of the gold thin film can be obtained.
JP 2005-98770 A JP 2004-125462 A

  However, when the microscopic state of the gold thin film surface in this antibody-immobilized chip is examined, the portion where the antibody is stacked like a mountain with respect to a certain nucleus (antibody) (thickness, hemisphere having a diameter of about several μm) And a portion where the gold thin film surface is exposed are mixed. In the stacked portion of the antibody, the antibody is first adsorbed to form a nucleus due to nano level roughness, kink, etc. on the gold thin film surface. It is formed by further adsorption of the antibodies around the antibody that is the nucleus. Furthermore, when the aqueous solution containing the antibody is dried, water is adsorbed on the hydrophilic protein surface, and the inter-protein adsorption of the antibody is promoted by the surface tension of the water. Therefore, on the gold thin film, antibody aggregation portions having a shape like a mountain on the micrometer scale are finally dispersed and arranged, and antibodies of other shapes are also arranged, resulting in low uniformity of antibody distribution. An antibody-immobilized chip thus formed is formed.

In the antibody immobilization chip in which the antibody is immobilized non-uniformly as described above, the following problems occur.
1: In order to carry out SPR immunoassay with high sensitivity, it is necessary to prepare a pile of antibodies in a form that is uniformly dispersed within a measurement region (in the order of several hundred μm ² ). For this purpose, it is necessary to drop an antibody solution containing a certain concentration on the surface of the gold thin film. As a result, the amount of antibody necessary for immobilization becomes large, resulting in an increase in cost.
2: Since it is difficult to control the formation of a mountain that is an antibody aggregation portion, it is difficult to control the production reproducibility of antibody-immobilized chips used for SPR immunoassay and individual differences between antibody-immobilized chips. Therefore, the reproducibility and quantitativeness of SPR immunoassay may be unreliable.

  The present invention has been made in view of the above circumstances, and the antibody can be immobilized at a desired position on the surface of the gold thin film with a small amount of antibody, and the uniformity of the antibody immobilized on the surface of the gold thin film An object of the present invention is to provide a microchip capable of improving the above.

The microchip according to claim 1 of the present invention is formed by laminating one surface of the first substrate and one surface of the second substrate so that the first substrate has an introduction portion into which a sample solution is introduced. , A reservoir for temporarily blocking the sample solution added to the introduction unit, a flow path in which one end is connected to the reservoir, the other end is opened on one surface of the first substrate, and the sample solution is transferred The second substrate is provided with a through-hole arranged to connect to the other end of the flow path and a detection chip having a gold thin film on the other surface of the second substrate. The through hole is opened in at least a part of the gold thin film.
A microchip according to a second aspect of the present invention is the microchip according to the first aspect, wherein a plurality of introduction pipes each including the introduction section, the storage section, and the flow path and the through holes connected to the introduction pipe are arranged. It is characterized by being.
A microchip according to a third aspect of the present invention is the microchip according to the first or second aspect, wherein the first substrate is further provided with a mixing section for mixing the sample solution between the introduction section and the storage section. It is characterized by being.
A microchip according to a fourth aspect of the present invention is the microchip according to the third aspect, wherein a plurality of the introduction parts are connected to the mixing part.
According to a fifth aspect of the present invention, in the microchip according to the fourth aspect, the mixing unit includes a mixing path refracted a plurality of times and a plurality of bypasses connecting the mixing paths.
The microchip according to a sixth aspect of the present invention is the microchip according to the fourth aspect, wherein the mixing portion comprises a mixing path bent a plurality of times and a plurality of convex portions formed on an inner wall surface of the mixing path. And
A microchip according to a seventh aspect of the present invention is characterized in that, in any one of the first to sixth aspects, the storage section is a filter.

  An antibody immobilization method according to claim 8 of the present invention is an antibody immobilization method for immobilizing an antibody on at least a part of a gold thin film using the microchip according to any one of claims 1 to 7. , After introducing an antibody into the introduction part of the microchip, converting the antibody into F (ab), moving the F (ab) -converted antibody into the flow path, and moving the F into the flow path And (ab) a step of immobilizing the antibody by moving it to the gold thin film surface through a through-hole.

  According to the microchip of the present invention, since the gold thin film is incorporated into the microchip, the antibody can be simply immobilized at a desired position with a smaller amount of antibody than in the past.

  Hereinafter, the present invention will be described in detail with reference to the drawings. However, the present invention is not limited thereto, and various modifications can be made without departing from the gist of the present invention.

"Microchip"
<First Embodiment>
FIG. 1 is a diagram schematically showing a microchip 1A according to the first embodiment of the present invention. FIG. 1A is a plan view, and FIG. 1B is an LL cross-sectional view in FIG.
The microchip 1A (1) according to the first embodiment of the present invention is formed by laminating one surface 10a of the first substrate 10 and one surface 20a of the second substrate 20 facing each other,
The first substrate 10 has an introduction part 11 into which the sample solution is introduced, a storage part 12 that temporarily blocks the sample solution added to the introduction part 11, and one end 13a connected to the storage part 12, and the other end 13b. Is opened in one surface 10a of the first substrate 10, and a flow path 13 through which the sample solution is transferred is formed. Further, the second substrate 20 has a through-hole 21 disposed so as to be connected to the other end 13 b of the flow path 13, and a detection chip 30 having a gold thin film 32 on the other surface 20 b of the second substrate 20. Is arranged. Further, the through hole 21 is opened in at least a part of the gold thin film 32. Hereinafter, each will be described in detail.

  For the first substrate 10, polymers such as PDMS (Polydimethylsiloxane), polyacrylic acid, polyethylene terephthalate, polystyrene, polyolefin, polyvinyl chloride, polyester, polyurethane, polyacrylonitrile, silane cross-linked polyolefin, and the like can be applied. In addition, in the case of a polymer having the characteristics of a thermoplastic elastomer among the above-mentioned polymers, patterning by molding using a mold is possible in addition to lithography by UV. The size of the first substrate 10 is not less than 35 mm and not more than 100 mm in the longitudinal direction of the flow path 13. Moreover, thickness is 1 mm or more and 8 mm or less, for example. One surface 10 a where the first substrate 10 is in contact with the second substrate 20 is connected to the introduction portion 11, the storage portion 12 that temporarily blocks the sample solution added to the introduction portion 11, and one end 13 a to the storage portion 12. An introduction pipe 14 composed of the flow path 13 is formed.

The introduction part 11 is a part to which various solutions such as antibodies are added. The size of the micropipette tip is sufficient as long as the tip of the micropipette tip can be inserted. It is 2 mm × 2 mm or less, preferably about 1 mm × 1 mm.
In addition, one end 11 a of the introduction part 11 opens to the side surface of the first substrate 10 to form an opening part 11 c, and the other end 11 b communicates with the storage part 12. In the microchip 1 of the present invention, a cap (not shown in the figure) that fits into the opening 11c can be used.

  The storage unit 12 suppresses the plurality of sample solutions added to the introduction unit 11 from being freely transferred to the flow path 13. The reservoir 12 may have a meniscus structure as shown in FIG. As a size of the storage part 12, a part communicating with the introduction part 11 can be equal to or more than a cross section of the introduction part 11, for example, 0.5 mm × 0.5 mm or more and 2 mm × 2 mm. It is as follows. The cross section of the portion communicating with the flow path 13 is the same as that of the flow path 13. By setting the storage part 12 as such a structure, it can suppress that the solution transferred from the introduction part 11 to the storage part 12 moves to the flow path 13 freely.

  The flow path 13 has one end 13a connected to the reservoir 12 and communicated with a through hole 21 provided in the second substrate 20 around the other end 13b. As the size of the flow path 13, the cross section in the short direction of the flow path 13 is about 0.3 mm × 0.3 mm. Further, the length from the storage portion 12 to the through hole 21 is not particularly limited, and can be appropriately adjusted according to the length to the through hole 21 opened at a desired position on the gold thin film 32. .

  As with the first substrate 10, for example, polymers such as PDMS, polyacrylic acid, polyethylene terephthalate, polystyrene, polyolefin, polyvinyl chloride, polyester, polyurethane, polyacrylonitrile, and silane-crosslinked polyolefin can be applied to the second substrate 20. In addition, in the case of a polymer having the characteristics of a thermoplastic elastomer among the above-mentioned polymers, patterning by molding using a mold is possible in addition to lithography by UV. Its size is the same as that of the first substrate. The second substrate 20 is provided with a through hole 21, and one end 21 a of the through hole 21 communicates with the other end 13 b of the flow path 13. Further, the other end 21 b of the through hole 21 is open to the surface 32 a of the gold thin film 32 of the detection chip 30 disposed on the other surface 20 b of the second substrate 20.

  The through hole 21 is formed in the thickness direction of the second substrate 20, and the gold thin film 32 surface 32 a of the detection chip 30 disposed on the flow path 13 disposed on the first substrate 10 and the other surface 20 b of the second substrate 20. It communicates with. The size of the through hole 21 is such that the cross section in the overlapping direction of the first substrate 10 and the second substrate 20 is 0.1 mm × 0.3 mm or more and 0.8 mm × 0.3 mm or less. If the other end 21b is on the surface of the gold thin film 32, the through-hole 21 can be provided at a desired position.

  The detection chip 30 has a gold thin film 32 and can be freely detached from the second substrate 20. After the antibody is immobilized on the surface of the gold thin film 32, it is detached from the second substrate 20, and can be used for various measurements, for example, measurements using SPR, ELISA, and the like. At this time, the gold thin film 32 is preferably disposed and supported on the third substrate 31. When removing from the second substrate 20, stress is applied to the gold thin film 32, and damage due to bending or the like can be suppressed.

Although it does not specifically limit as the 3rd board | substrate 31, For example, glass, a plastics, a silicon wafer etc. can be used.
When the detection chip 30 is used for measurement using, for example, SRP, a third substrate 30 having transparency is used. At that time, the third substrate 31 is preferably made of glass, plastic or the like.

  The gold thin film 32 has a thiol group of an antibody immobilized on its surface 32a by a covalent bond, and is used for various measurements such as SPR and ELISA.

  When the detection chip 30 is used for SPR, it is necessary to exude the evanescent wave, and therefore the thickness of the gold thin film 32 is preferably 20 nm or more and 100 nm or less. Furthermore, the surface 32a of the gold thin film 32 is preferably flat, and the surface roughness is, for example, 5 nm or less.

  According to the microchip 1A of the present embodiment, the antibody can be uniformly immobilized at a desired position on the surface 32a of the gold thin film 32 with a small amount of antibody. In addition, since the detection chip 30 is incorporated in the microchip 1, the antibody can be simply immobilized on the surface 32a of the gold thin film 32 to obtain an antibody-immobilized chip.

Second Embodiment
FIG. 2 is a diagram schematically showing a microchip 1B according to the second embodiment of the present invention. 2A is a plan view, and FIG. 2B is an LL cross-sectional view in FIG. 2A.
The microchip 1B of this embodiment is different from the microchip 1A of the first embodiment in that a plurality of introduction pipes 14 and through-holes 21 each including an introduction part 11, a storage part 12, and a flow path 13 are arranged. .

  According to the microchip 1B of the present embodiment, by arranging a plurality of introduction tubes 14 and through-holes 21, it is possible to easily immobilize different types of antibodies simultaneously at desired positions on the surface 32a of the gold thin film 32. An antibody array can be easily prepared. The detection chip thus obtained is particularly effective in identifying the causative bacteria of the infection because a plurality of antibodies are arranged on the surface thereof.

<Third Embodiment>
FIG. 3 is a diagram schematically showing a microchip 1C (1) according to the second embodiment of the present invention. 3A is a plan view, and FIG. 3B is an LL cross-sectional view in FIG. 3A.
The difference between the microchip 1C of the present embodiment and the microchip 1A of the first embodiment is that a mixing unit 15 is arranged between the introduction unit 11 and the storage unit 12, and the introduction unit 11 and the mixing unit 15 Is a point communicating with the second flow path 16.

The mixing unit 15 is a part that mixes and reacts various solutions added to the introduction unit 11. The size of the mixing unit 15 is preferably 0.5 mm × 0.5 mm or more and 5 mm × 5 mm or less, more preferably about 1 mm × 1 mm, in the short-side direction of the channel 13 (first channel 13). Moreover, as for the length in the longitudinal direction of the 1st flow path 13, 10 mm or more and 20 mm or less are desirable, for example. The shape of the mixing portion 15 is shown as a square in FIG. 3, but is not particularly limited to this shape. For example, the mixing portion 15 has a bowl shape that has a curved surface on the side communicating with the first flow path 13. The solution added to the mixing unit 15 can be mixed efficiently.
Providing the mixing unit 15 separately requires a step of transferring the solution from the introduction unit 11 to the mixing unit 15, so that it is easy to control the reaction time in the mixing unit 15.

  As the second flow path 16, the cross section in the short direction of the second flow path 16 is preferably 0.1 mm × 0.1 mm to 0.8 mm × 0.8 mm, more preferably 0.3 mm × 0.3 mm. Degree. Moreover, the length from the introduction part 11 of the second flow path 16 to the mixing part 15 is preferably, for example, 5 mm or more and 30 mm or less, and more preferably about 10 mm.

  According to the microchip 1C of the present embodiment, since the step of transferring the solution from the introduction unit 11 to the mixing unit 15 is required by separately providing the mixing unit 15, the reaction time in the mixing unit 15 is controlled. Becomes easy. In the present embodiment, similarly to the microchip 1B of the second embodiment, the introduction pipe 14 including the introduction part 11, the second flow path 16, the mixing part 15, the storage part 12, and the first flow path 13 and the A plurality of through holes 21 communicating with the introduction pipe 14 may be provided. The same effect as the microchip 1B of the second embodiment described above can be obtained.

<Fourth embodiment>
FIG. 4 is a view schematically showing a microchip 1D (1) according to the third embodiment of the present invention. 4A is a plan view, and FIG. 4B is an LL cross-sectional view in FIG. 4A.
The microchip 1D of the present embodiment is different from the microchip 1C of the third embodiment in that a plurality of introducing portions 11A and 11B communicate with one mixing portion 15.

By providing a plurality of introduction parts 11A and 11B in this way, different types of solutions, for example, solutions containing antibodies, and solutions for treating the antibodies (antibody treatment solutions) to the introduction parts 11A and 11B, for example. It can be added and mixed in the mixing unit 15 to be reacted. Accordingly, the reaction time can be easily controlled, and the solution added to each of the introducing portions 11A and 11B can be simultaneously transferred to the mixing portion, so that the solution of the introducing portions 11A and 11B is transferred to the mixing portion 15. This process can be simplified as compared with the microchip 1C of the third embodiment.
Although FIG. 4 shows an example in which two introduction parts are provided, three or more introduction parts may be provided. Similarly to the microchip 1 </ b> B of the second embodiment, the introduction pipe 14 including the introduction parts 11 </ b> A and 11 </ b> B, the second flow path 16, the mixing part 15, the storage part 12, and the first flow path 13, A plurality of through holes 21 communicating with each other can also be provided. The same effect as the microchip 1B of the second embodiment described above can be obtained.

<Fifth Embodiment>
FIG. 5 is a diagram schematically showing a microchip 1E according to the fifth embodiment of the present invention. 5A is a plan view, FIG. 5B is an LL cross-sectional view in FIG. 5A, and FIG. 5C is an enlarged view of α in FIG. 5A.
The microchip 1E of this embodiment is different from the microchip 1C of the third embodiment in that the mixing unit 15 includes a mixing path 55 refracted a plurality of times and a plurality of bypasses 56 connecting the mixing paths 55. It is.

  The mixing path 55 is preferably bent at 45 ° with respect to the first flow path 13. The cross section in the short direction of the first flow path 13 of the mixing path 55 is, for example, 100 μm × 100 μm or more and 400 μm × 400 μm or less.

The bypass 56 communicates between the bent mixing paths 55 and is composed of a plurality of flow paths having different lengths. Further, the respective bypasses 56 are linear and are arranged substantially in parallel with each other. The cross section in the short direction of the first flow path 13 of the bypass 56 is smaller than the mixing path 55 and is preferably about 1/5 to 1/6 of the mixing path 55. For example, when the width of the mixing path 55 is about 27 μm, the width of each bypass 56 is preferably about 5 μm. When the width of the bypass 56 is too thick, or when the width of the bypass 56 is narrow, mixing between the solutions added to the introduction portions 11A and 11B is difficult to occur.
In addition, it is preferable that the depth (height in the thickness direction of the 1st board | substrate 10) of the mixing path 55 and the bypass 56 is the same.

  According to the present embodiment, when the solution travels along the wall surface due to the surface tension with the wall surface of the mixing unit 15 (mixing path 55), a large number of bypasses 56 having different lengths are arranged, so that each introduction unit Mixing of the sample solution added to 11A and 11B is promoted, and the sample solution can be mixed with a small amount of liquid. In the present embodiment, similarly to the microchip 1B of the second embodiment, the introduction pipe 14 including the introduction part 11, the second flow path 16, the mixing part 15, the storage part 12, and the first flow path 13 and the A plurality of through holes 21 communicating with the introduction pipe 14 may be provided. The same effect as the microchip 1B of the second embodiment described above can be obtained.

<Sixth Embodiment>
FIG. 6 is a view schematically showing a microchip 1F according to the sixth embodiment of the present invention. 6A is a plan view, and FIG. 6B is an LL cross-sectional view in FIG. 6A.
The microchip 1F of the present embodiment is different from the microchip 1C of the third embodiment in that the mixed layer 15 includes a mixed path 65 bent a plurality of times and a plurality of convex portions formed on the inner wall surface 65a of the mixed path 65. 66.

  The mixing path 65 is a flow path having a plurality of bent portions C. For example, the cross section in the short direction is 200 μm × 500 μm or more and 500 μm × 500 μm or less. On the inner wall 65a of the mixing path 65, a plurality of convex portions 66 are arranged outside the bent portion C.

  The shape of the convex portion 66 is not particularly limited as long as sample droplets added to the introduction portions 11A and 11B are mixed in the mixing path 65. However, if the surface is a curved surface, the droplet is not limited. A plurality of types of liquids contained therein can be efficiently mixed, which is preferable. Further, the size of the convex portion 66 is not particularly limited as long as the fluidity of the droplet flowing through the mixing path 65 is not significantly reduced, and a plurality of types of liquids contained in the droplet can be mixed efficiently. For example, the height from the inner wall 65a of the mixing path 65 is, for example, not less than 100 μm and not more than 250 μm.

  According to the present embodiment, a droplet made of the sample solution added to the introduction portions 11A and 11B flows through the mixing path 65 while hitting the projection 66 formed on the inner wall 65a of the mixing path 65. Mixing of the two liquids is promoted. Therefore, mixing between solutions can be promoted even with a small amount of liquid. In the present embodiment, similarly to the microchip 1B of the second embodiment, the introduction pipe 14 including the introduction part 11, the second flow path 16, the mixing part 15, the storage part 12, and the first flow path 13 and the A plurality of through holes 21 communicating with the introduction pipe 14 may be provided. The same effect as the microchip 1B of the second embodiment described above can be obtained.

<Seventh embodiment>
FIG. 7 is a view schematically showing a microchip 1G according to the seventh embodiment of the present invention. 7A is a plan view, and FIG. 7B is an LL cross-sectional view in FIG. 7A.
The microchip 1G of the present embodiment is different from the microchip 1A of the first embodiment in that a filter 72 is used as the storage unit 12.

The filter 72 is not particularly limited as long as it prevents the sample solution added to the introduction part 11 from freely flowing into the flow path 13, but for example, a fractionation filter according to the molecular weight, etc. Can be used.
Note that the filter 72 can be used as the reservoir 12 not only in the microchip 1A of the first embodiment but also in the microchips of the second to sixth embodiments described above.

"Antibody immobilization method of antibody immobilization chip"
<Production Method when Using Microchip 1A of First Embodiment>
Next, an antibody immobilization method using the microchip 1A according to the first embodiment of the present invention will be described.
First, a predetermined amount of F (ab ′) 2 antibody solution is added to the introduction part 11. The antibody solution added to the introduction unit 11 is transferred to the storage unit 12 and temporarily held in the storage unit 12.

The antibody is not particularly limited and can be appropriately changed depending on the measurement object. For example, IgG such as cow, sheep, horse, pig, goat, human, mouse, rat, rabbit, etc. is preferably used. . Depending on the measurement target, IgE, IgM, IgA, or IgD may be used.
The F (ab ′) 2 antibody may be prepared by a conventionally known method or may be a purchased one.
The concentration of the antibody is appropriately adjusted with, for example, PBST, but is preferably 0.1 mg / ml which is generally used.
The amount of antibody added is about 5 μl.
As described above, when the amount of antibody conventionally used in the range of 5 to 7 μg is about 0.5 μg when the microchip of the present invention is used, an antibody-immobilized chip is prepared. In this case, the cost required for the antibody can be greatly reduced.

  Subsequently, the antibody treatment solution is added to the introduction part 11. The antibody treatment solution is a buffer solution containing DTT or the like that reduces the disulfide bond of the antibody. For example, DTT, EDTA, and HEPES are mixed in water. The antibody treatment solution added to the introduction unit 11 is transferred to the storage unit 12 and mixed with the antibody solution in the storage unit 12.

Next, the disulfide bond of the antibody is reduced to make F (ab ′) 2 into a Fab. In this reaction, it is preferable to mix with a shaker or the like at room temperature. The reaction time is appropriately adjusted, but it is usually sufficient to carry out the reaction for 60 minutes. After that, for example, by centrifuging at × 2000 G, the antibody that has been made into Fab is transferred from the reservoir 12 to the flow path 13, and the antibody reaches the surface 32 a of the gold thin film 32 through the through hole 21.
When centrifuging, it is preferable to install the microchip 1 in a centrifuge tube 81, for example, as shown in FIG. FIG. 8 is a diagram schematically showing an example in which the microchip 1 is installed in the centrifuge 80. FIG. 8A is a top view, and FIG. 8B is a view as seen from the direction of the arrow in FIG. In FIG. 8B, only the centrifuge tube 81 and the microchip 1 arranged in the centrifuge tube 81 are shown. As shown in FIG. 8, the microchip 1 is placed in a centrifuge 80 and centrifuged to minimize the amount of the antibody-containing solution adhering to the flow path 13 and the through-holes 21. It can be transferred to the surface 32a.

  The thiol group of the antibody that has reached the surface 32a of the gold thin film 32 is immobilized on the surface 32a of the gold thin film 32 by covalently bonding with gold. Therefore, the immobilization of the antibody is completed on the surface 32a of the gold thin film 32 before the aggregation of the antibodies occurs. At this time, since the thiol group is in the vicinity of the end opposite to the antigen capturing portion of the antibody, the Fab antigen capturing portion immobilized on the surface 32a of the gold thin film 32 is a portion not immobilized on the gold thin film 32, that is, It comes to exist in the direction which is easy to capture an antigen.

According to the antibody solidification method using the microchip 1A of the present invention, the antibody can be immobilized on the gold thin film surface at a desired position with a small amount of antibody. In particular, since the antibody is expensive, the cost can be reduced. In addition, the antibody is not physically adsorbed to the gold thin film by drying, but the antibody thiol group is immobilized to the gold thin film by covalent bond. There is no influence, and aggregation of antibodies hardly occurs. Therefore, the uniformity of the antibody solidified on the gold thin film surface can be improved.
Further, since the antibody can be immobilized at a desired position on the gold thin film surface by centrifugation, the antibody can be simply immobilized without requiring a spotter or the like as in the prior art.

<Antibody immobilization method when using the microchip 1B of the second embodiment>
Next, an antibody immobilization method using the microchip 1B of the second embodiment will be described. When using the microchip 1B of this embodiment, it is the same as when using the microchip 1A of the first embodiment, except that different antibody solutions are added to each introduction part 11.
By using the microchip 1B of the present embodiment, a plurality of types of antibodies can be simultaneously immobilized on the surface 32a of the gold thin film 32, and an antibody array can be easily produced.

<Antibody immobilization method when using the microchip 1C of the third embodiment>
Next, an antibody immobilization method using the microchip 1C of the third embodiment will be described.
First, an antibody solution is added to the introduction part 11, and the antibody solution is transferred to the mixing part 15 through the second flow path 16 by centrifugation. Regarding the centrifugation, for example, the antibody solution can be transferred from the introduction unit 11 to the mixing unit 15 and the storage unit 12 through the second flow path 16 by centrifuging at 500 G for 1 to 3 minutes.

  Subsequently, the antibody treatment solution is added to the introduction unit 11, and the antibody treatment solution is transferred to the mixing unit 15 and the storage unit 12 while being centrifuged. For centrifugation, for example, it is sufficient to carry out at 500 G for 10 minutes.

  Thereafter, the antibody solution and the antibody treatment solution are reacted at room temperature for 60 minutes in the mixing unit 15 and the storage unit 12 to reduce the disulfide bond of the antibody, and F (ab ′) 2 is converted to Fab. In that case, it is preferable to mix using a shaker similarly to 1st Embodiment. Thereafter, for example, by centrifuging at × 2000 G, the Fab antibody is transferred from the mixing unit 15 and the storage unit 12 to the first flow path 13, and the Fab antibody reaches the gold thin film 32 surface 32 a through the through hole 21. The antibody that has reached the surface 32a of the gold thin film 32 is immobilized on the gold thin film 32 by covalent bonding in the same manner as in the first embodiment, and a detection chip 30 (antibody immobilization chip) on which the antibody is immobilized is produced. The

  By using the microchip 1C of the present embodiment, the addition of the antibody and the antibody treatment solution and the treatment of the antibody are performed separately, so that the antibody can be reliably made into a Fab, and the gold thin film 32 can be more efficiently produced. An antibody can be immobilized on the surface 32a.

<Antibody immobilization method when using microchip 1D of the fourth embodiment>
Next, an antibody immobilization method using the microchip 1D of the fourth embodiment will be described. When the microchip of this embodiment is used, an antibody solution is added to the introduction part 11A, and an antibody treatment solution is added to the introduction part 11B, both are transferred to the mixing part 15 and the storage part 12 by centrifugation, and the mixing part 15 and the storage part are stored. The antibody is made into Fab by reacting in part 12. The reaction and subsequent steps are the same as when the microchip 1C of the third embodiment is used.

  By using the microchip 1D of the present embodiment, the solutions can be individually added and both can be mixed in the mixing unit by centrifugation. Therefore, since the addition of the antibody and the antibody treatment solution and the treatment of the antibody are performed separately, the antibody can be surely made into a Fab, and the antibody can be more efficiently immobilized on the gold thin film surface. Furthermore, since each solution can be transferred to the mixing part 15 and the storage part 12 by one centrifugation, the frequency | count of centrifugation reduces and the operation of an antibody fixed chip | tip can be simplified.

<Antibody immobilization method when microchip 1E of the fifth embodiment is used>
Next, an antibody immobilization method using the microchip 1E of the fifth embodiment will be described. First, an antibody solution and an antibody treatment solution are respectively added to the introduction parts 11A and 11B, and the antibody solution and the antibody treatment solution are transferred to the mixing part 15 (first mixing path 51) through the second flow path 16 by centrifugation. . Regarding the centrifugation, for example, the antibody solution and the antibody treatment solution can be transferred from each of the introduction portions 11A and 11B to the mixing portion 15 via the second flow path 16 by centrifuging at 500 G for 1 to 3 minutes. The antibody solution and the antibody treatment solution that have reached the mixing unit 15 are mixed when traveling by surface tension along the wall surfaces of the mixing path 55 and the bypass 56, and are transferred to the storage unit 12. At this time, the reaction is allowed to stand at room temperature for 60 minutes to reduce the disulfide bond of the antibody, and F (ab ′) 2 is converted to Fab. Thereafter, for example, by centrifuging at × 2000 G, the Fab antibody is transferred from the mixing unit 15 and the storage unit 12 to the first flow path 13, and the antibody reaches the surface 32 a of the gold thin film 32 through the through hole 21. The antibody reaching the surface 32a of the gold thin film 32 is immobilized on the gold thin film 32 by covalent bonding as in the first embodiment, and the detection chip 30 on which the antibody is immobilized is produced.

  By using the microchip 1E of the present embodiment, the antibody can be immobilized on the surface 32a of the gold thin film 32 with a small amount of liquid, so that the amount of antibody to be used is reduced and the cost required for producing the antibody-immobilized chip is reduced. Can do.

<Antibody immobilization method when using microchip 1F of sixth embodiment>
Next, an antibody immobilization method using the microchip 1F of the sixth embodiment will be described. First, an antibody solution and an antibody treatment solution are added to the introduction parts 11a and 11b, respectively, and the antibody solution and the antibody treatment solution are transferred to the mixing part 15 (mixing path 65) via the second flow path 16 by centrifugation. Regarding the centrifugation, for example, the antibody solution and the antibody treatment solution can be transferred from the introduction unit 11 to the mixing unit 15 (mixing channel 65) through the second flow channel 16 by centrifuging at 500 G for 1 to 3 minutes. . The antibody solution and the antibody treatment solution that have reached the mixing portion 15 form droplets, and flow through the mixing path 65 while hitting the convex portions 66 formed on the inner wall 65a of the bending portion C of the mixing path 65, whereby the droplets The mixing of the two liquids is promoted and transferred to the reservoir 12. At this time, the reaction is allowed to stand at room temperature for 60 minutes to reduce the disulfide bond of the antibody, and F (ab ′) 2 is converted to Fab. Thereafter, for example, by centrifuging at × 2000 G, the Fab antibody is transferred from the mixing unit 15 and the storage unit 12 to the first flow path 13, and the antibody reaches the surface 32 a of the gold thin film 32 through the through hole 21. The antibody reaching the surface 32a of the gold thin film 32 is immobilized on the gold thin film 32 by covalent bonding as in the first embodiment, and the detection chip 30 on which the antibody is immobilized is produced.

  By using the microchip 1E of the present embodiment, the antibody can be immobilized on the surface 32a of the gold thin film 32 with a small amount of liquid, so that the amount of antibody to be used is reduced and the cost required for producing the antibody-immobilized chip is reduced. Can do.

<Antibody immobilization method when using the microchip 1G of the seventh embodiment>
Next, an antibody immobilization method using the microchip 1G of the seventh embodiment will be described. Since the filter 72 is arranged as the reservoir 12 in the microchip 1G of the present embodiment, the centrifugation when transferring the antibody solution and the antibody treatment solution to the flow path 13 in the first embodiment is set to × 2000G. Thus, it can be similarly manufactured.
By using the microchip 1G in which the filter 72 is arranged as the reservoir 12, the unreacted antibody F (ab ′) whose disulfide bond is not reduced reaches the gold thin film surface, and aggregation between the antibodies occurs. Can be suppressed more reliably. Therefore, the antibody immobilized on the surface 32a of the gold thin film 32 can be more evenly immobilized.

<Production of microchip>
A microchip 1C of the third embodiment shown in FIG. 9 was produced. However, a filter 72 was used as the storage unit 12.
The 1st board | substrate 10 with which the introducing | transducing part 11, the mixing part 15, the 1st flow path 13, and the 2nd flow path 16 was distribute | arranged was produced by injection molding. Similarly, PDMS was used to produce the second substrate 20 provided with the through holes 21 by injection molding.
Thereafter, the surfaces of the first substrate 10 and the second substrate 20 are subjected to plasma treatment, the filter 72 is disposed at a predetermined position, and the one surface 10a of the first substrate 10 and the one surface 20a of the second substrate 20 are bonded to each other. Microchip 1 was obtained. Thereafter, a gold thin film 32 was disposed on the glass substrate 31 to produce a detection chip 30, and a plasma surface treatment was performed. Next, the detection chip 30 was pressed and attached to the other surface 20b of the second substrate 20 so that the surface 32a of the gold thin film 32 overlapped with the through hole 21, and the microchip shown in FIG. 9 was obtained.

<Immobilization of antibody>
10 μl of 0.1 mg / ml F (ab ′) 2 (manufactured by Sigma: I9885 anti-Human IgG, Goat) was added to the introduction part 11 by pipetting, centrifuged at 1000 g for 1 minute, and the antibody solution was mixed. Moved to 15. Then, 30 μl of Ar-purged antibody treatment solution (6.25 mM DTT, 5 mM EDTA, 10 mM HEPES) just before use is pipetted into the introduction part 11 and centrifuged at room temperature at × 500 g for 10 minutes to mix the antibody reaction solution It was made to react with an antibody, moving to part 15. Thereafter, the centrifuge tube provided with the microchip 1 was placed on a shaker and reacted at room temperature for 60 minutes. Next, the mixture was centrifuged for 2 minutes at × 5000 G at room temperature, the antibody solution was passed through the filter 72, the antibody was moved to the surface 32 a of the gold thin film 32 through the first flow path 13 and the through hole 21, and was immobilized.
In addition, the molecular weight of the separately prepared antibody was confirmed by electrophoresis (SDS-PAGE). The result is shown in FIG. Lane (1) is a marker, lane (2) is the F (ab ′) 2 antibody used above, and lane (3) is an antibody prepared with the microchip 1C of the present invention. As shown in FIG. 10, the antibody prepared with the microchip 1C of the present invention has a molecular weight about half that of the F (ab ′) 2 antibody, and it was confirmed that the antibody was made into a Fab.

<SPR measurement>
The prepared antibody-immobilized chip (detection chip 30) was removed from the microchip and blocked with a protein-free buffer (Protein-Free T20 (TBS) Blocking Buffer) at room temperature for 2 hours. Thereafter, antigen-antibody reaction was measured by SPR using this antibody-immobilized chip. As the antigen, Human IgG (Sigma: 14506) and rabbit IgG (Sigma: R2004) were used. In the SPR measurement, the surface plasmon resonance angle was measured using HandySPR (manufactured by NTT-AT). The antibody-immobilized chip was attached to the SPR device via matching oil, and 50 μL of 0.1 M phosphate buffer solution (pH 8) containing 0.1 mg / ml Human IgG was dropped, and the SPR angle was observed. The result is shown in FIG. FIG. 11 (a) shows the results of using rabbit IgG as the antigen, and FIG. 11 (b) shows the results of using Human IgG as the antigen.
From FIG. 11, it was observed that the reaction to human IgG, an antigen, was observed, and it was confirmed that the antibody-immobilized chip can be produced with a small amount of antibody according to the microchip of the present invention.

  The present invention can be applied to an antibody-immobilized chip used for SPR or ELISA.

It is the figure which showed typically the microchip which concerns on 1st Embodiment of this invention. It is the figure which showed typically the microchip which concerns on 2nd Embodiment of this invention. It is the figure which showed typically the microchip which concerns on 3rd Embodiment of this invention. It is the figure which showed typically the microchip which concerns on 4th Embodiment of this invention. It is the figure which showed typically the microchip which concerns on 5th Embodiment of this invention. It is the figure which showed typically the microchip which concerns on 6th Embodiment of this invention. It is the figure which showed typically the microchip which concerns on 7th Embodiment of this invention. It is the figure which showed typically an example at the time of installing a microchip in a centrifuge. It is sectional drawing which showed the microchip of the Example typically. It is an image when the antibody is electrophoresed. It is the figure which showed the measurement result of SPR.

Explanation of symbols

  1 (1A, 1B, 1C, 1D, 1E, 1F) Microchip, 10 First substrate, 11 Introducing section, 12 Reserving section, 13 Channel (first channel), 14 Introducing pipe, 15 Mixing section, 16 Second Flow path, 20 Second substrate, 21 Through hole, 30 Detection chip, 31 Third substrate, 32 Gold thin film, 55 First mixing path, 56 Second mixing path, 65 Third mixing path, 66 Projection, 72 Filter, 80 centrifuge, 81 centrifuge tube C bent part.

Claims (8)

One surface of the first substrate and one surface of the second substrate are laminated facing each other,
The first substrate has an introduction part into which the sample solution is introduced, a storage part for temporarily blocking the sample solution added to the introduction part, one end connected to the storage part, and the other end of the first substrate An opening is formed on one surface of the substrate, and a flow path for transferring the sample solution is formed,
The second substrate is provided with a through-hole arranged to connect to the other end of the flow path, and a detection chip having a gold thin film on the other surface of the second substrate,
The microchip according to claim 1, wherein the through hole is opened in at least a part of the gold thin film.   2. The microchip according to claim 1, wherein a plurality of introduction pipes including the introduction section, the storage section, and the flow path, and a plurality of the through holes connected to the introduction pipe are arranged.   3. The microchip according to claim 1, wherein a mixing unit that mixes a sample solution is further disposed on the first substrate between the introduction unit and the storage unit.   The microchip according to claim 3, wherein a plurality of the introduction parts are connected to the mixing part.   The microchip according to claim 4, wherein the mixing unit includes a mixing path refracted a plurality of times and a plurality of bypasses connecting the mixing paths.   The microchip according to claim 4, wherein the mixing portion includes a mixing path bent a plurality of times and a plurality of convex portions formed on an inner wall surface of the mixing path.   The microchip according to claim 1, wherein the storage unit is a filter. An antibody immobilization method for immobilizing an antibody on at least a part of a gold thin film using the microchip according to claim 1,
A step of introducing an antibody into the introduction part of the microchip and then converting the antibody into F (ab);
Moving the F (ab) antibody to the channel;
A method of immobilizing an antibody, comprising the step of moving the F (ab) antibody transferred to the flow path to the gold thin film surface through a through-hole and immobilizing the antibody.

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