JP6612651B2 - Manufacturing method of biosensor chip - Google Patents

Description

  The present invention relates to a method for manufacturing a biosensor chip. More specifically, the present invention relates to a method for manufacturing a biosensor chip in which a working electrode is selectively modified.

  In recent years, biosensors for detecting or quantifying substances contained in biological samples by immune reactions or enzymatic reactions have been developed. Usually, these sensors use biological materials that interact with the substance to be measured, such as DNA. Oligonucleotides, antibodies, receptors, enzymes, etc. are fixed to the sensor. As a method for immobilizing these biomaterials, a self-assembled monolayer (SAM) is produced by using thiol solution or the like on the surface of the electrode or part of it using gold or platinum, and crosslinked. A method of immobilizing a biomaterial through an agent or the like has been proposed. Various materials have been proposed as SAM materials, and processing methods suitable for each SAM are employed.

  On the other hand, this type of biosensor chip is not reused after measurement and is disposable. For this reason, it is desired to reduce the cost of the biosensor chip, and miniaturization is progressing. For example, there are biosensor chips that have a flat or thin-film working electrode, counter electrode, and reference electrode patterned on the same substrate, and in these biosensor chips, the distance between the working electrode and the counter electrode or reference electrode is reduced. Moreover, since they are formed on the same plane, it is difficult to perform the above-described modification only on the surface of the working electrode by treatment with a solution. Patent Document 1 discloses a small sensor chip used in an electrochemical immunoassay for measuring cortisol, in which a pair of comb-shaped electrodes is used as a working electrode, a cortisol antibody is fixed to at least one of the electrodes, Although a sensor chip in which a reference electrode is formed is disclosed, a method for selectively modifying only at least one working electrode with an antibody is not disclosed.

  On the other hand, Patent Document 2 discloses a method in which a solution is dropped onto a working electrode for modification, and a region having a relatively low affinity for the solution is provided around the working electrode so that the solution selectively stays at the working electrode portion. Is disclosed.

JP 2011-002430 A JP 2007-278981 A

  However, the method of Patent Document 1 complicates the substrate manufacturing process, and the method of Patent Document 2 requires treatment with droplets. Therefore, when manufacturing a large number of sensor chips at one time, extra equipment is required. There is a problem that it is necessary. In addition, with a planar biosensor chip, it is difficult to measure a certain volume of sample.

  Furthermore, in the method of forming the working electrode and the other electrode as separate members and combining them, there is a problem that a plurality of members are required, the assembly process is increased, and it is difficult to reduce the size and cost.

  Therefore, an object of the present invention is to provide a manufacturing method capable of easily mass-producing a biosensor chip in which only a working electrode is modified without affecting a counter electrode or a reference electrode.

  As a result of intensive studies, the inventors have formed electrodes other than the working electrode on the lid, which is a separate member, a biosensor chip that forms only the working electrode on the base substrate, and a plurality of base substrates in a matrix. A first collective substrate arranged and a second collective substrate in which a plurality of lids are arranged in a matrix are prepared, and these are bonded together to produce individual pieces, thereby reducing the size of a small biosensor chip. The present invention has been completed by finding that it can be mass-produced at low cost.

That is, the present invention
[1] (A) A step of preparing a first aggregate substrate in which a plurality of base substrates having recesses for forming a reaction region are continuously formed in a matrix through the planned cutting region;
(B) forming a working electrode, an extraction electrode connected to the working electrode, and a counter electrode connection extraction electrode on each base substrate of the first collective substrate;
(C) modifying the working electrode;
(D) When a plurality of lids are continuously formed in a matrix through the scheduled cutting region and are stacked on the first collective substrate, a reaction region is formed by covering the concave portion, and the base Preparing a second aggregate substrate that exposes at least a part of each of the extraction electrode connected to the working electrode on the substrate and the extraction electrode for counter electrode connection;
(E) forming a counter electrode and an extraction electrode connected to the counter electrode at a position facing the working electrode when the lid portion of the second aggregate substrate is overlaid on the base substrate;
(F) The first collective substrate and the second collective substrate, wherein the working electrode and the counter electrode face each other, and the counter electrode connecting lead electrode of the base substrate is connected to the counter electrode of the lid portion Bonding the corresponding scheduled cutting region of the first collective substrate and the planned cutting region of the second collective substrate so as to be connected to an electrode, and obtaining a biosensor chip collective substrate;
(G) cutting the biosensor chip assembly substrate in the planned cutting region and dividing it into individual pieces,
[2] At least one of a sample supply port or a vent is provided in the first collective substrate, and the sample supply port or the vent communicates with the concave portion of the adjacent base substrate. The method for producing a biosensor chip according to [1] above,
[3] The method for producing a biosensor chip according to [1] or [2] above, wherein the step of modifying the working electrode is a step of selectively modifying the working electrode; 4] Any of the above-mentioned [1] to [3], further comprising a step of performing a hydrophilic treatment on the concave portion for forming the reaction region and the region corresponding to the concave portion for forming the reaction region of the lid portion. The present invention relates to a method for producing the biosensor chip according to claim 1.

  According to the method for manufacturing a biosensor chip of the present invention, a small-sized biosensor chip in which only the working electrode is selectively modified can be mass-produced at low cost, so that the biosensor chip can be disposable for each measurement (disposable). Can be provided. For this reason, it can be easily measured with a small amount of sample such as saliva, and when used as a stress sensor with a large individual difference, it is easy to accumulate individual trends and feed back to subsequent evaluation of measurement results Have

It is a flowchart explaining the manufacturing method of the biosensor chip of this invention. It is plane explanatory drawing of S1a process of the manufacturing method of FIG. It is plane explanatory drawing of S2a process of the manufacturing method of FIG. It is a plane explanatory view of S1b process of the manufacturing method of FIG. It is a plane explanatory view of S2b process of the manufacturing method of FIG. It is plane explanatory drawing of S4 process of the manufacturing method of FIG. It is a plane explanatory view of an example of a biosensor chip manufactured with the manufacturing method of FIG. It is explanatory drawing of the AA cross section of the biosensor chip | tip of FIG. It is a disassembled perspective view of the cover part and base substrate of the biosensor chip of FIG. It is a plane explanatory view of other examples of the biosensor chip manufactured with the manufacturing method of FIG. FIG. 7 is an explanatory plan view of a base substrate of the biosensor chip of FIG. 6. It is a plane explanatory view of the surface facing the working electrode of the lid of the biosensor chip of FIG. It is explanatory drawing of the BB cross section of the biosensor chip | tip of FIG. It is a disassembled perspective view of the cover part and base substrate of the biosensor chip of FIG. It is a plane explanatory view of another example of the biosensor chip manufactured by the manufacturing method of FIG. It is a plane explanatory view of the base substrate of the biosensor chip of FIG. It is a plane explanatory view of the surface facing the working electrode of the lid of the biosensor chip of FIG. It is explanatory drawing of CC cross section of the biosensor chip | tip of FIG. It is a disassembled perspective view of the cover part and base substrate of the biosensor chip of FIG. It is a plane explanatory view of still another example of the biosensor chip manufactured by the manufacturing method of FIG. FIG. 17 is an explanatory plan view of a base substrate of the biosensor chip of FIG. 16. FIG. 17 is an explanatory plan view of a surface facing the working electrode of the lid portion of the biosensor chip of FIG. 16. FIG. 10 is an explanatory plan view of still another example of the biosensor chip manufactured by the manufacturing method of FIG. 1. FIG. 20 is an explanatory plan view of a base substrate of the biosensor chip of FIG. 19. FIG. 20 is an explanatory plan view of a surface facing the working electrode of the lid portion of the biosensor chip of FIG. 19. It is explanatory drawing which shows one embodiment of the 1st aggregate substrate of the base substrate of the biosensor chip of FIG.

  The manufacturing method of the biosensor chip 100 of the present invention will be described with reference to FIGS. 1 to 5 attached hereto, but the present invention is not limited to these embodiments. FIG. 1 is a flowchart showing a method for manufacturing a biosensor chip of the present invention, and FIGS. 2A to 2E are plan explanatory views of the respective steps.

  In the biosensor chip manufacturing method of the present invention, first, as shown in step S1a in the flowchart of FIG. 1, a first collective substrate 200 having a plurality of base substrates 101 is prepared. In the first collective substrate 200, a plurality of base substrates 101 each having a recess 106a for forming the reaction region 106 are continuously formed in a matrix through the planned cutting region 400 (see FIG. 2A).

  The material of the first collective substrate serving as the base substrate 101 is not particularly limited as long as it is an insulating material that does not affect the measurement of the target substance. For example, ceramic, glass, silicon, polychlorinated Examples thereof include thermoplastic resins such as vinyl, polypropylene, polystyrene, polycarbonate, acrylic resin, polybutylene terephthalate and polyethylene terephthalate (PET), thermosetting resins such as epoxy resins, and synthetic resin materials such as UV curable resins.

  The shape of the base substrate 101 can be at least thicker than the recess 106a for forming the reaction region, and can be a rectangle, a circle, an ellipse, or the like, but a rectangle is preferable from the viewpoint of manufacturing in a lattice shape. . Moreover, the pitch of the matrix of the base substrate 101 in the first collective substrate can be appropriately set so as to have a size suitable for the biosensor chip.

  The recess 106 a for forming the reaction region 106 is provided to form the reaction region 106 that is a space of a certain capacity by joining the base substrate 101 and the lid 102. The formation of the recess 106a for forming the reaction region 106 is preferably performed simultaneously with the production of the substrate when the first aggregate substrate 200 is produced by molding. Moreover, after preparing the plate-shaped 1st aggregate substrate 200, a recessed part can also be mechanically formed by press or cutting. Furthermore, when the vent hole 108 is formed as a recess on the base substrate 101 as shown in FIG. 2A, or when the sample supply port 107 is formed as a recess on the base substrate 101 as shown in FIG. This step can be performed simultaneously with the formation of the recess 106a for forming the reaction region 106.

  The depth of the recess 106a for forming the reaction region 106 is preferably set so that the height of the reaction region 106 to be formed facilitates the introduction of the sample by capillary action. In addition, as shown in FIG. 2A, the vent hole 108 preferably has a structure communicating with the recess 106a for forming the reaction region 106 of the adjacent base substrate 101. This is suitable for selective modification of the working electrode 103 described later.

  Next, as shown in step S2a of FIG. 1, the working electrode 103 is formed on each base substrate 101 of the first collective substrate 200. The working electrode 103 is formed on the bottom surface of the recess 106 a for forming the reaction region 106. In addition to the working electrode 103, an extraction electrode 109 connected to the working electrode 103 and a counter electrode connecting extraction electrode 110 are also formed on each base substrate 101. When the reference electrode 105 is provided, it is preferable that the reference electrode connection extraction electrode 111 is also formed on each base substrate 101 in this step (see FIG. 2B). The shape of each electrode is not particularly limited, but is preferably a flat plate or a thin film from the viewpoint of miniaturization of the sensor chip, and includes a sputtering method, a vacuum deposition method, a plating method, various printing methods, and the like. Thus, film formation and pattern formation can be performed. These methods are the same as those used in general semiconductor device manufacturing processes.

  A known electrode material can be used as the working electrode 103, and examples thereof include materials made of copper, aluminum, gold, silver, silver chloride, platinum, chromium, nickel, iron, and carbon. Depending on the type, an appropriate material can be selected. For example, when the working electrode 103 is modified via a thiol group as described later, the working electrode 103 can be selected from the group consisting of gold, platinum, silver, and copper.

  The extraction electrode 109 connected to the working electrode 103 formed on the base substrate 101 extends from the working electrode 103 to the end of the base substrate, for example, as shown in FIG. can do. Similarly, for example, as shown in FIG. 3 and the like, the extraction electrode 110 for connecting the counter electrode is formed so as to be connected to the extraction electrode 110a connected to the counter electrode 104 of the lid 102, and is connected to the working electrode 103. Similarly, the reference electrode connection extraction electrode 111 is connected to the extraction electrode 111a connected to the reference electrode 105 of the lid portion 102 in the same manner as the extraction electrode for the counter electrode connection. The same material as that of the extraction electrode 109 connected to the working electrode 103 is extended to the end of the base substrate.

  In step S3 in FIG. 1, the working electrode 103 is modified. The modification of the working electrode 103 means immobilization of a material that interacts with a measurement target such as DNA, oligonucleotide, antibody, receptor, enzyme, and the like. Various methods known in the field of biosensors can be used as a method for immobilizing a material that interacts with these measurement objects. For example, a method using a self-assembled monolayer (SAM) can be given. It is done. In SAM, a functional group of an organic molecule, such as a thiol group, is chemically adsorbed on the substrate surface by being immersed in a solution containing the organic molecule or placed in a vapor containing the organic molecule, and spontaneously formed on the substrate surface. It is a high density and highly oriented monomolecular film. Specifically, SAMs composed of alkyl thiols and disulfide compounds, SAMs with thiols as fixed groups and oriented polyethylene glycol (PEG), SAMs with benzene rings, and alkyl thiols with different carbon chain lengths are mixed. SAM and the like. These SAM constituent molecules preferably have a functional group for imparting characteristics to the SAM surface separately from the functional group that is chemically adsorbed to the substrate, and a material that interacts with the measurement target using this functional group. Can be immobilized. In order to selectively modify the working electrode 103, the solution is injected only into the recess 106a for forming the reaction region 106, or the region where the extraction electrodes 109, 110, 111 are formed is masked. What is necessary is just to immerse in. When a solution is injected only into the recess 106a for forming the reaction region 106, the vent 108 is structured to communicate with the recess 106a for forming the reaction region 106 of another adjacent base substrate 101. The solution can be injected at once into the recesses 106a forming the plurality of reaction regions 106 of the first collective substrate 100, and the solution can be easily injected into the recesses 106a forming the reaction regions 106 throughout. Of course, although not shown, a recess is provided in the planned cutting region 400, and the recess 106 a for forming the reaction regions 106 of all the base substrates 101 of the first collective substrate 100 is formed by the vent 108 and / or the sample supply port 107. It is also possible to communicate (for example, see FIG. 22).

  On the other hand, as shown in step S1b of FIG. 1, a second aggregate substrate 300 having a plurality of lids 102 is prepared. On the second collective substrate 300, a plurality of lids 102 are continuously formed in a matrix form with the same vertical and horizontal pitches as the matrix of the base substrate 101 through the planned cutting region 400, and are superimposed on the first collective substrate 200. Then, the reaction region 106 is formed by covering the recess 106a, and at least a part of the extraction electrode 109 connected to the working electrode 103 and the extraction electrode 110 for counter electrode connection (and the reference electrode 105) are connected to the base substrate 101. In the case where it is provided, it is provided with an opening 301 that exposes at least a part of the reference electrode connecting extraction electrode 111 (see FIG. 2C). By setting the matrix shape of the lid portion 102 in the second collective substrate 300 to the same vertical and horizontal pitch as the matrix shape of the base substrate 101 in the first collective substrate 200, the first and second collective substrates (200, 300) are connected to each other. It is possible to obtain a biosensor chip assembly substrate 500 (FIG. 2E) in which a plurality of biosensor chips 100 are continuously formed in a matrix through the planned cutting region 400 by overlapping and joining. As described above, according to the present embodiment, it is not necessary to join the lid portions 102 to the first collective substrate 200 of the base substrate 101 one by one.

  The material of the second collective substrate that becomes the lid 102 can be selected from the same materials as the first collective substrate that becomes the base substrate 101 described above. From the viewpoint of manufacturing, it is preferable to use the same material as that of the first collective substrate to be the base substrate 101.

  Here, each of the extraction electrodes 109, 110, and 111 on the base substrate 101 needs to be at least partially exposed for connection to a measuring instrument or the like. An opening 301 for exposing at least a part of each extraction electrode 109, 110, and 111 is provided (FIG. 2C). The second aggregate substrate 300 of the lid portion 102 having the opening 301 for exposing the extraction electrode may be formed by mold formation as described for the first aggregate substrate 200, or the second aggregate After preparing the substrate, the opening 301 exposing the extraction electrode may be formed by machining or the like. The opening 301 that exposes at least a part of each of the extraction electrodes 109, 110, and 111 is provided as an opening 301 that exposes the extraction electrode in a region sufficient to be exposed to the extent that a measuring instrument can be connected. However, at least one end thereof is preferably designed on the planned cutting region 400 of the second collective substrate 300. In this case, since the end of the opening 301 that exposes the extraction electrode is placed on the planned cutting region 400, the edge of the opening 301 that exposes the extraction electrode when the biosensor chip 100 is separated by cutting. 2C to 2E, for example, as shown in FIGS. 2C to 2E, when the opening 301 that exposes the extraction electrode reaches the region to be cut 400 in three directions, the end of the lid 102 is connected to the base substrate 101. The upper extraction electrodes 109, 110 and 111 are formed so as to be exposed so that the end sides are exposed, and access to the extraction electrodes is facilitated. Furthermore, as shown in FIG. 2C, when the sample supply port 107 is formed in the lid portion 102, or when the vent hole 108 is formed in the lid portion 102 as shown in FIG. 11 or the like, the reaction region 106 is also formed. In the case where the concave portion 106b (FIGS. 11 to 15) is formed in the lid portion 102, the opening portion 301 that exposes at least a part of each of the extraction electrodes 109, 110, and 110 on the base substrate 101 in this step is formed. Can be done at the same time. Note that the opening 301 for exposing the extraction electrode has a large shape that exposes the extraction electrodes 109, 110, and 111 on the base substrate 101, instead of a via hole shape in which a conductor is formed. Alternatively, the lead electrodes 109, 110 and 111 may be drawn out.

  Next, as shown in step S2b of FIG. The counter electrode 104 and the extraction electrode 110a connected to the counter electrode are formed at a position facing the working electrode 103 when the lid 102 is overlaid on the base substrate 101 (FIG. 2D). Here, the reference electrode 105 and the extraction electrode 111 a connected to the reference electrode 105 are also formed at a position where they are connected to the extraction electrode 111 formed earlier. Thus, the reaction region 106 is formed between the working electrode 103, the counter electrode 104, and the reference electrode 105 by joining the base substrate 101 and the lid portion 102, and measurement with high accuracy can be performed. The reference electrode 105 is not necessarily formed, but is preferably formed from the viewpoint of measurement accuracy. The shape of each electrode is not particularly limited, but is preferably a flat plate or a thin film from the viewpoint of miniaturization of the sensor chip, and includes a sputtering method, a vacuum deposition method, a plating method, various printing methods, and the like. Thus, film formation and pattern formation can be performed.

  A known electrode material can be used for the counter electrode 104 and the reference electrode 105, and examples thereof include materials made of copper, aluminum, gold, silver, silver chloride, platinum, chromium, nickel, iron, and carbon.

  The extraction electrode 110a connected to the counter electrode 104 only needs to extend continuously from the counter electrode 104 toward the end of the lid portion to the extent that it can be connected to the extraction electrode 110 for connecting the counter electrode of the base substrate 101 (FIG. 2D, FIG. 8), and can be formed integrally with the counter electrode 104. Similarly, the extraction electrode 111a connected to the reference electrode 105 only needs to extend continuously from the reference electrode 105 in the direction toward the end of the lid so that it can be connected to the extraction electrode 111 for connecting the reference electrode of the base substrate 101. (See FIG. 2D, FIG. 8, etc.) and can be formed integrally with the reference electrode 105.

  Next, as shown in step S4 of FIG. 1, the first collective substrate 200 and the second collective substrate 300 are bonded together. In bonding the first collective substrate 200 and the second collective substrate 300, the working electrode 103 and the counter electrode 104 face each other, and the counter electrode connecting extraction electrode 110 of the base substrate 101 and the counter electrode 104 of the lid 102 are connected. In addition, the reference electrode 105 is connected to the reference electrode 105 of the lid 102 and the reference electrode 105 of the lid 102 so that the extraction electrode 110a is connected to the connection electrode 110b (and the reference electrode 105 is provided). This is performed in such a manner that the planned cutting area 400 of the first collective substrate 200 and the planned cut area 400 of the second collective substrate 300 correspond to each other (so that the extraction electrode 111a is connected by the connecting portion 111b) (FIG. 2E). Thereby, the biosensor chip aggregate substrate 500 is obtained (FIG. 2E). For bonding the first collective substrate 200 and the second collective substrate 300, a bonding method or an adhesive suitable for the material of the base substrate 101 or the lid 102 may be employed. As the adhesive, for example, an epoxy adhesive, an acrylic adhesive, a polyurethane adhesive, a thermosetting adhesive, a UV curable adhesive, or the like can be used. For example, when a known bonding means such as an adhesive is applied to either or both of the base substrate 101 and the lid 102, and the end of the extraction electrode 110 a connected to the counter electrode 104 and the reference electrode 105 are used, the reference electrode 105, a known electrode connection means such as a conductive paste is applied to the end of the extraction electrode 111a connected to 105, or to the connection side end of the extraction electrode 110 for counter electrode connection or the extraction electrode 111 for reference electrode connection on the base substrate 101. It can be performed by pasting and pressing as necessary.

  Here, by bonding the first collective substrate 200 and the second collective substrate 300, a reaction region 106 that is a space of a certain capacity is formed between each base substrate 101 and each lid portion 102. The capacity of the reaction region 106 is not particularly limited as long as it can detect the measurement target and can fill the reaction region 106 with a sample by capillary action. By setting the reaction area to a constant volume, it becomes possible to fix the electrolyte solution in the reaction area in advance and keep the electrolyte concentration constant, and it is possible to perform a stable quantitative analysis of the sample.

  As one embodiment of the method for producing a biosensor chip of the present invention, it is preferable that the inner surface of the reaction region 106 is further subjected to a hydrophilic treatment in order to introduce the sample more smoothly into the reaction region 106. Examples of a method for hydrophilic treatment of the inner surface of the reaction region 106 include a method of introducing a hydrophilic functional group by plasma treatment or UV treatment, and a coating of siloxane with a silanol group. This hydrophilic treatment can be performed on the concave portion 106a for forming the reaction region of the base substrate 101 in the first collective substrate 200 after the S1a step shown in FIG. 1 and before the electrode formation. After the S1b step shown, the region corresponding to the concave portion for forming the reaction region of the lid portion 102 (if there is a concave portion 106b for forming the reaction region 106 in the lid portion 102, the concave portion 106b (FIG. 11 To FIG. 15)).

  As shown in step S5 of FIG. 1, the obtained biosensor chip assembly substrate 500 is cut at the planned cutting region 400 and separated into individual pieces (see FIG. 2E). This step can be performed by using a known dicing saw or the like, for example, by running the dicing saw through the planned cutting area 400 indicated by an arrow in FIG. 2E to remove the planned cutting area 400. 3 is a plan view of an individualized biosensor chip, FIG. 4 is an explanatory view of the AA cross section of FIG. 3, and FIG. 5 is a view of a lid portion and a base substrate of the biosensor chip of FIG. The exploded perspective view of each is shown. In the biosensor chip of FIG. 3, it can be seen that the vents 108 are opened on the side surfaces by the separation. Here, in the case where at least one of the sample supply port 107 and the vent port 108 is provided in the second collective substrate 300 (see, for example, FIGS. 4 and 14), before this step, The surface opposite to the surface in contact with the first aggregate substrate 200 of the second aggregate substrate 300 is covered with a sheet material, and the biosensor chip aggregate substrate 500 is cut from the first aggregate substrate 101 side so as not to cut the sheet material in this step. It is preferable to do. Thus, by covering the openings of the sample supply port 107 and / or the vent port 108 with the sheet material, not only can the individualization be facilitated, but also contamination of the reaction region 106 can be prevented.

  As the sheet material, a dicing tape or the like provided with an adhesive layer capable of eliminating the adhesive force by UV curing or heating on a polyvinyl chloride base material or a polyolefin base material is preferably used.

  In addition to the above embodiments, various biosensor chips can be manufactured by the manufacturing method of the present invention described above. An explanatory view of this type of biosensor chip 100 is shown in FIGS.

  In the example shown in FIGS. 6 to 10, the sample supply port 107 is provided in the base substrate 101. With this structure, the sample enters the inside simply by bringing the sample into contact with the sample supply port 107 of the biosensor chip, and the sample can be easily supplied to the reaction region 106. At this time, the air in the reaction region 106 is exhausted from the vent 108. Further, the shape of the counter electrode connection extraction electrode 110 and the reference electrode connection extraction electrode 111, the extraction electrode 110a connected to the counter electrode of the lid portion 102, and the extraction electrode 111a connected to the reference electrode are not formed up to the end of the lid portion. However, this is merely a variation of the shape. Since other configurations are the same as those in FIGS. 3 to 5 described above, the same portions are denoted by the same reference numerals and description thereof is omitted. The sample supply port 107 communicates with the recess 106a for forming the reaction region 106 of another adjacent base substrate 101 through the recess provided in the planned cutting region 400 in the first collective substrate, for example. Then, the concave portion 106 a for forming the reaction region 106 of each adjacent base substrate 101 communicates with both the sample supply port 107 and the vent port 108, which is suitable for selective modification of the working electrode 103. . Also in this case, the sample supply port 107 and the vent port 108 are opened on the side surfaces by cutting the planned cutting region. 6 is a plan view of the biosensor chip 100, FIG. 7 is a plan view of the base substrate 101 of the biosensor chip 100 of FIG. 6, and FIG. 8 is an operation of the lid portion 102 of the biosensor chip 100 of FIG. 9 is a plan view of the surface facing the pole, FIG. 9 is an explanatory view of a BB cross section of FIG. 6, and FIG. 10 is an exploded perspective view of the lid portion 102 and the base substrate 101 of the biosensor chip 100 of FIG. Respectively.

  In the example shown in FIGS. 11 to 15 which is another embodiment, the lid portion 102 is provided with a recess 106b for forming the reaction region 106 and a vent hole 108 communicating from the reaction region 106 to the outside. Region 106 is cylindrical. Further, the arrangement of the counter electrode 104 and the reference electrode 105 is opposite to each other, but this is merely one variation. Since other configurations are the same as those in FIGS. 6 to 10 described above, the same portions are denoted by the same reference numerals and description thereof is omitted. In this case, since the vent hole 108 is provided in the lid portion 102, the sample supply port 107 in the first collective substrate has a reaction region 106 of another base substrate 101 adjacent to it through a recess provided in the planned cutting region. It can be set as the structure connected to the recessed part 106 for forming. 11 is a plan view of the biosensor chip 100, FIG. 12 is a plan view of the base substrate 101 of the biosensor chip 100 of FIG. 11, and FIG. 13 is a lid portion 102 of the biosensor chip 100 of FIG. 14 is a plan view of a surface facing the working electrode of FIG. 11, FIG. 14 is an explanatory view of a CC cross section of FIG. 11, and FIG. 15 is an exploded perspective view of the lid portion 102 and the base substrate 101 of the biosensor chip 100 of FIG. Respectively.

  In the example shown in FIGS. 16 to 18 which is another embodiment, the working electrode 103 and the counter electrode 104 are comb electrodes, and the reference electrode 105 is not used. Since other configurations are the same as those in FIGS. 6 to 10 described above, the same portions are denoted by the same reference numerals and description thereof is omitted. In this case, the sample supply port 107 and the vent hole 108 communicate with the recess 106 for forming the reaction region 106 of another adjacent base substrate 101 through the recess provided in the cutting region 400 in the first collective substrate. It can be set as the structure to do. 16 is a cross-sectional view of the biosensor chip 100, FIG. 17 is a plan view of the base substrate 101 of the biosensor chip 100 in FIG. 16, and FIG. 18 is a plan view of the lid portion 102 of the biosensor chip 100 in FIG. The top view of the surface facing a working electrode is shown, respectively.

  In the example shown in FIGS. 19 to 21, which is another embodiment, the base substrate 101 is provided with a sample supply port 107 and a vent hole 108 communicating from the reaction region 106 to the outside. Since other configurations are the same as those in FIGS. 11 to 15 described above, the same portions are denoted by the same reference numerals and description thereof is omitted. In this case, the sample supply port 107 and the vent port 108 are provided at positions facing the concave portion 106a forming the reaction region 106, and the sample supply port 107 and the vent port 108 can be manufactured by a continuous cutting. Further, as shown in the first collective substrate 200 of FIG. 22, in this case, the sample supply port 107 and the vent port 108 are connected to the reaction region 106 of another base substrate 101 adjacent to each other via the recess 401 provided in the planned cutting region 400. It can be set as the structure connected to the recessed part 106a for forming. Accordingly, when the solution is injected only into the recess 106 a for forming the reaction region 106 for modification of the working electrode 103, the solution is efficiently filled into the recess 106 a for forming the plurality of reaction regions 106. The amount of solution can be adjusted easily. In FIG. 22, the concave portion 401 is provided in a part of the planned cutting region, but the concave portion 401 may be provided in all the planned cutting regions 400 surrounding the base substrate 101. 19 is a plan view of the biosensor chip 100, FIG. 20 is a plan view of the base substrate 101 of the biosensor chip 100 of FIG. 19, and FIG. 21 is a lid portion 102 of the biosensor chip 100 of FIG. FIG. 22 is an explanatory view showing one embodiment of the first collective substrate 200 of the base substrate 101 of the biosensor chip 100 of FIG. 19.

  The sample inlet 107 may have an opening having a certain size to absorb necessary droplets or a structure capable of absorbing droplets squeezed from absorbent cotton by capillary action.

  On the other hand, the vent 108 is more preferably subjected to hydrophobic treatment. By such processing, when a sample is applied to the sample supply port 107, the sample is smoothly guided into the reaction region 106 by capillary action, and leakage of the sample from the vent 108 to the outside can be prevented. The reaction region 106 is easily filled with the sample.

  Examples of the method of hydrophobically processing the vent 108 include a method of coating the surface of the opening of the vent 108 with a hydrophobic material such as siloxane having an alkyl group.

  Examples of the sample include body fluid, blood, saliva and the like, and saliva is preferable.

  The measurement target by the biosensor chip 100 is not particularly limited, and examples thereof include glucose and cortisol. For example, if cortisol can be easily measured as a stress index, it is preferable to provide a simple measurement method for stress check accompanying the recent mandatory stress check.

  The sample is supplied from the sample supply port 107 to the reaction region 106 by a capillary phenomenon, or by applying pressure, for example, absorbent cotton containing the sample to the sample supply port 107 to handle the reaction region. 106 can be pushed. A vent 108 is provided at the end of the reaction region 106, and the air that has filled the reaction region 106 is discharged from the vent 108 in accordance with the injection of the sample, and the reaction region 106 is filled with the sample.

DESCRIPTION OF SYMBOLS 100 Biosensor chip 101 Base substrate 102 Cover part 103 Modified working electrode 104 Counter electrode 105 Reference electrode 106 Reaction area 106a Recess 106b for forming the reaction area 106b Recess for forming the reaction area 106 107 Sample supply port 108 Ventilation Port 109 Extraction electrode connected to working electrode 110 Extraction electrode for counter electrode connection 110a Extraction electrode connected to counter electrode 110b Connection portion between extraction electrode 110a connected to counter electrode and extraction electrode 110 for connection with counter electrode 111 Reference electrode connection extraction Electrode 111a Extraction electrode 111b connected to reference electrode 111b Connection between extraction electrode 111a connected to reference electrode and extraction electrode 111 for reference electrode connection 200 First collective substrate 300 Second collective substrate 301 For exposing extraction electrode Opening 400 Cut scheduled area 401 Cut scheduled area 4 0 provided in the recess 500 biosensor chip assembly substrate

Claims (4)

(A) preparing a first collective substrate in which a plurality of base substrates having recesses for forming a reaction region are continuously formed in a matrix through the planned cutting region;
(B) forming a working electrode, an extraction electrode connected to the working electrode, and a counter electrode connection extraction electrode on each base substrate of the first collective substrate;
(C) modifying the working electrode;
(D) When a plurality of lids are continuously formed in a matrix through the scheduled cutting region and are stacked on the first collective substrate, a reaction region is formed by covering the concave portion, and the base Preparing a second aggregate substrate that exposes at least a part of each of the extraction electrode connected to the working electrode on the substrate and the extraction electrode for counter electrode connection;
(E) forming a counter electrode and an extraction electrode connected to the counter electrode at a position facing the working electrode when the lid portion of the second aggregate substrate is overlaid on the base substrate;
(F) The first collective substrate and the second collective substrate, wherein the working electrode and the counter electrode face each other, and the counter electrode connecting lead electrode of the base substrate is connected to the counter electrode of the lid portion Bonding the corresponding scheduled cutting region of the first collective substrate and the planned cutting region of the second collective substrate so as to be connected to an electrode, and obtaining a biosensor chip collective substrate;
(G) A method of manufacturing a biosensor chip, including a step of cutting the biosensor chip assembly substrate into the cut-scheduled region and separating the substrate. 2. The sample supply port or the vent port is provided in the first collective substrate, and the sample supply port or the vent port communicates with the concave portion of the adjacent base substrate. A method for producing the biosensor chip according to claim. The method of manufacturing a biosensor chip according to claim 1, wherein the step of modifying the working electrode is a step of selectively modifying the working electrode. 4. The method according to claim 1, further comprising a step of performing a hydrophilic treatment on a concave portion for forming the reaction region and a region corresponding to the concave portion for forming the reaction region of the lid portion. Manufacturing method of biosensor chip.

Images