JP5164193B1 - Multi-biosensor chip assembly kit, multi-biosensor chip manufacturing method, and multi-biosensor chip - Google Patents

Abstract

[PROBLEMS] To provide a biosensor chip assembly kit for providing a multi-type biosensor chip capable of obtaining a relatively high measurement accuracy, a method of manufacturing a biosensor chip using the kit, and a kit manufactured using the kit. Provided biosensor chip.
A first member comprising a first substrate having a first electrode embedded therein and a plurality of flow path openings extending radially from the center of the opening and the center of the opening, a through-hole for introducing a measurement object A biosensor assembly kit comprising: a second electrode embedded with a second member. A biological material for a biosensor is fixed on the surface of the flow path. When the first member and the second member are laminated and joined, the subject introduction through hole is positioned at the opening center of the first member, and each ventilation through hole is the first electrode of each flow path opening. A space is formed between the flow path openings of the first member and the surfaces facing the flow path openings of the second member, which are located on the more distal side. The laminated first member and second member are assembled by being attached to each other under a temperature condition that does not deactivate the biological material.
[Selection] Figure 7

Description

  The present invention relates to a multi-biosensor chip assembly kit, a multi-biosensor chip manufacturing method, and a multi-biosensor chip. The multi-biosensor chip of the present invention is a “multi” -type biosensor chip having a plurality of working electrodes that function as a biosensor.

  Biosensors include enzyme sensors, microbial sensors, and immunosensors. These can selectively identify and quantify specific organic substances (measuring objects, analytes) even in a solution in which a large number of organic substances are mixed, utilizing the reaction specificity of enzyme groups and antigen antibodies. The biosensor chip is a part that directly stores the measurement target in the biosensor. The biosensor chip body that forms a reaction part (space) that specifically acts on the measurement target and a phenomenon that changes depending on the reaction in the reaction part. Means (electrode and wiring) for electrically detecting and transmitting to the outside are provided.

  Currently, a widely used enzyme electrochemical measurement is a glucose sensor that measures a blood glucose level. The chip including the electrode for the sensor and the reagent such as an enzyme is produced by a film and a printing technique. Specifically, it is produced by printing and bonding a reagent such as an electrode, an adhesive, and an enzyme on a film (for example, Patent Documents 1 and 2).

  Although this type of biosensor chip has the advantage of being inexpensive and can be manufactured in large quantities, the dimensional variation due to differences in film production lots can reach 10%. Directly linked to the variation in the amount of target. In addition, the printed electrodes are based on carbon and have poor electrical conductivity, leading to a reduction in measurement accuracy due to adverse effects on sensitivity. Currently, it is necessary to provide a complicated correction function to alleviate these problems (for example, Patent Document 3). Therefore, in the end, productivity and operability are sacrificed. In addition, the above production method is not suitable for a small variety and a variety.

  Biosensor chips that can measure a plurality of physical properties with a single biosensor chip have also been proposed (for example, Patent Documents 4 to 6). These biosensor chips have two or more working electrodes in order to perform a plurality of physical property measurements.

JP 2008-209219 A JP 2004-147845 A Special table 2008-511841 gazette JP 2007-327965 A JP 2007-256092 A JP 2001-215208 A WO2011 / 125520

  The biosensor chip having two or more working electrodes for performing a plurality of physical property measurements described in Patent Documents 4 to 6 has a more complicated structure than a biosensor chip having a single working electrode, and is inexpensive. It was difficult to mass-produce, and it was not in a form suitable for a small variety of products. For a single sample, for example, blood, there is a need for a multi-type biosensor chip that can be measured at the same time, concurrently or in parallel, for different components in the blood. However, such a multi-type biosensor chip is not known in a form that can be mass-produced at low cost and that is suitable for a small variety of products.

  Conventional biosensor chips are basically produced using a film and a printing technique. In order to eliminate dimensional variations due to differences in film production lots, the provision of substrates by injection molding is promising. Although it is possible to perform injection molding with a single structure, in this case, it is difficult to accurately fix an enzyme or the like inside, and such a sensor cannot be realized.

  Moreover, in the case of a structure in which the injection molded product is divided into two, it is necessary to join them. However, it has not been possible to achieve a hollow structure necessary for the sensor because it cannot be joined by a method that does not adversely affect the activity of an enzyme or the like.

  In view of such a situation, the present inventors have previously described a biosensor chip comprising a two-part structure including a metal electrode with good electrical conductivity and capable of being produced by injection molding, Thus, a means for providing a biosensor chip with reduced dimensional variation by joining these two-divided structures under conditions that do not adversely affect the activity of the above has been provided (Patent Document 7). However, this biosensor chip was not a biosensor chip (hereinafter referred to as a multi-type biosensor chip) having two or more working electrodes capable of simultaneously performing a plurality of physical property measurements.

  The present inventors worked on the development of a multi-type biosensor chip based on the biosensor chip described in Patent Document 7, and applied for a patent separately. However, the multi-type biosensor chip developed based on the biosensor chip described in Patent Document 7 has found a new problem that has not been realized in the biosensor chip described in Patent Document 7.

  As described above, the biosensor chip is mainly intended for measurement of biological samples such as blood, and it is desired that any biological sample can be measured with as little sample as possible. Even in the case of a multi-type biosensor chip, it is desired that the amount of the biological sample be measured with an amount of the biological sample that is the same as that of a single biosensor chip. On the other hand, the multi-type biosensor chip has two or more working electrodes, and the amount of biological sample that can be distributed per working electrode is reduced by the number of working electrodes.

  The newly discovered problem in the newly developed multi-type biosensor chip is that the amount of change in current or voltage obtained by one electrochemical measurement becomes much smaller, and the measurement accuracy decreases. It is that. This is because the amount of the biological sample that can be used for one measurement does not change greatly even if the number of working electrodes increases. In the multi-type biosensor chip, compared to the biosensor chip described in Patent Document 7. Thus, the electrode area such as the working electrode is reduced, and the volume of the flow path per working electrode leading to the electrode such as the working electrode is reduced, leading to a decrease in the measurement accuracy. I guessed.

  Therefore, an object of the present invention is a multi-type biosensor chip in a form suitable for a small amount and a variety of products, such as an enzyme sensor that does not require a complicated correction function and can obtain a relatively high measurement accuracy. It is to provide a chip for a biosensor.

  More specifically, an object of the present invention is to provide a biosensor chip assembly kit that can be used to form the biosensor chip, a method of manufacturing a biosensor chip using the kit, and further using the kit. It is to provide a biosensor chip to be manufactured. Moreover, the present invention is to provide a multi-type biosensor chip that satisfies these conditions and has at least two working electrodes and can measure one sample simultaneously or in parallel.

  As a result of the study by the present inventors, the working electrode is embedded in one of the two divided structures that can be manufactured by injection molding, and the counter electrode is embedded in a position facing the working electrode of the other structure. With this structure, it is possible to increase the area of the counter electrode and bring it closer to the working electrode, and as a result, a multi-type biosensor chip that can obtain the same measurement accuracy as a single-type biosensor chip And the present invention was completed. Moreover, as described in Patent Document 7, a multi-type biosensor chip in which dimensional variations are reduced by joining these injection-molded products (two-part structure) under conditions that do not adversely affect the activity of enzymes and the like. Can provide.

The present invention is as follows.
[1]
A first substrate having an opening center portion and at least three flow passage openings extending radially from the opening center portion; a first substrate embedded in the first substrate and exposed at least partially at the flow passage opening portion; 1st electrode, 2nd joint surface of 2nd member which consists of the surface surrounding the said opening center part and flow-path opening part including the 1st wiring which can connect with the said 1st electrode and the exterior of the 1st member In order to introduce a subject to be measured by the biosensor that extends in the thickness direction into the first member having the first joint surface and the portion corresponding to the opening center of the first member. A second substrate having a through hole, having a second bonding surface for bonding to the first bonding surface of the first member, and embedded in the second substrate, at least a part of the opening for the flow path Exposed at the part facing the part and arranged independently for each part facing the channel opening. A biosensor chip assembly kit including a second member connected to each of the second electrode and the second electrode and including a second member connectable to the outside of the second member,
The exposed area of the second electrode is in the range of 1 to 10 times the exposed area of the first electrode to the channel opening,
(a) at least part of the surface of the first member from which the first electrode is exposed, (b) at least part of the surface of the flow path opening other than the surface of the first member from which the first electrode is exposed, c) at least part of the exposed surface of the second electrode of the second member, (d) at least part of the surface of the second member facing the flow channel opening other than the exposed surface of the second electrode; Or (e) two or more of the above (a) to (d) are used for fixing a biological material for a biosensor,
The first member or the second member has a ventilation through hole extending in the thickness direction on each of the front end side of the first electrode of each flow passage opening,
When the first member and the second member are laminated and bonded so that the first bonding surface and the second bonding surface face each other, the through hole for introducing the subject of the second member is the opening center of the first member Each through hole for ventilation of the first member or the second member is located on the front end side of the first electrode of each channel opening, and each channel opening of the first member and the second A space is formed between each second electrode of the member,
The first substrate and the second substrate are a hydrogenated derivative of a polypropylene resin and a block copolymer represented by the general formula XY (where X is a polymer block that is incompatible with the polypropylene resin, Y is an elastomeric property of a conjugated diene) A resin block containing a polymer block),
The laminated first member and the second member are assembled by being attached to a joint under a temperature condition that does not deactivate the biomaterial for the biosensor without using an adhesive on the joint surface.
The biosensor chip assembly kit.
[2]
When the first member and the second member are laminated and joined, the first electrode is provided at a position facing the second electrode, and the first electrode and the second electrode facing the first electrode The biosensor chip assembling kit according to [1], wherein the distance between the surfaces is 5 mm or less.
[3]
The first member is embedded in the first substrate, a part of the first member is exposed at a tip side of the first electrode of the channel opening, and is in a non-contact state with the first electrode. A third electrode disposed independently for each part, and a third wiring connected to each of the third electrodes and connectable to the outside of the first member;
When the first member and the second member are laminated and joined so that the first joint surface and the second joint surface face each other, each ventilation through hole of the first member or the second member overlaps with each third electrode. The kit for assembling a biosensor chip according to [1] or [2], which is located on the tip side from the position where the third electrode is formed.
[4]
At least the first member is formed by insert molding the first electrode and the first wiring by injection molding, and the second member is formed by insert molding the second electrode and the second wiring by injection molding. The kit for assembling a biosensor chip according to [1].
[5]
The biosensor chip assembly kit according to [4], wherein at least the first member is formed by insert molding the third electrode and the third wiring by injection molding.
[6]
In the biosensor chip assembly kit according to [1], (a) at least a part of a surface of the first member from which the first electrode is exposed, and (b) a surface other than the surface of the first member from which the first electrode is exposed. At least part of the surface of the opening for the flow path, (c) at least part of the exposed surface of the second electrode of the second member, and (d) the flow other than the exposed surface of the second electrode of the second member. A biological material for a biosensor is fixed to at least a part of the surface facing the road opening, or (e) two or more of the above (a) to (d),
The through hole for introducing the subject of the second member is located at the opening center of the first member, and each ventilation through hole of the first member or the second member is the first of the opening for each flow path. The first bonding surface and the second bonding surface are laminated so as to be positioned on the tip side from the one electrode, and the bonding surfaces of the first member and the second member are bonded under a temperature condition in which the biological material is not deactivated. A method for producing a biosensor chip.
[7]
[3] The biosensor chip assembly kit according to [3], wherein (a) at least part of a surface of the first member from which the first electrode is exposed, (b) other than a surface of the first member from which the first electrode is exposed. At least part of the surface of the opening for the flow path, (c) at least part of the exposed surface of the second electrode of the second member, and (d) the flow other than the exposed surface of the second electrode of the second member. A biological material for a biosensor is fixed to at least a part of the surface facing the road opening, or (e) two or more of the above (a) to (d),
A position where the through hole for introducing the subject of the second member is located at the center of the opening of the first member, and the respective through holes for ventilation of the first member or the second member overlap with the respective third electrodes Alternatively, the first bonding surface and the second bonding surface are stacked so as to be positioned on the tip side from each third electrode, and the bonding surfaces of the first member and the second member are bonded under a temperature condition that does not deactivate the biological material. A method for producing a biosensor chip, comprising:
[8]
The production method according to [6] or [7], wherein the biological material is at least one material selected from the group consisting of enzymes, antigens, antibodies, peptides, proteins, and nucleic acids.
[9]
The production method according to any one of [6] to [8], wherein a temperature condition in which the biological material is not deactivated is in a range of 30 to 40 ° C.
[10]
A biosensor chip produced by the method according to any one of [6] to [9].
[11]
A first substrate having an opening center portion and at least three flow passage openings extending radially from the opening center portion; a first substrate embedded in the first substrate and exposed at least partially at the flow passage opening portion; A second joint surface of the second member, comprising a first wire connected to the first electrode and connected to the outside of the first member, and comprising a surface surrounding the opening center portion and the flow passage opening portion; A first member having a first joint surface to be joined, and a penetration for introducing a subject to be measured by the biosensor that extends in the thickness direction into a portion corresponding to the opening center of the first member A second substrate having a hole, having a second bonding surface to be bonded to the first bonding surface of the first member, and embedded in the second substrate, at least a part of which is in the flow path opening. The second exposed at the facing portion and independently arranged for each portion facing the flow path opening. Connected to each pole and the second electrode includes a second member including a second wiring connectable to the outside of the second member,
The exposed area of the second electrode is in the range of 1 to 10 times the exposed area of the first electrode to the channel opening,
The first substrate and the second substrate are a hydrogenated derivative of a polypropylene resin and a block copolymer represented by the general formula XY (where X is a polymer block that is incompatible with the polypropylene resin, Y is an elastomeric property of a conjugated diene) A resin block containing a polymer block),
(a) at least part of the surface of the first member from which the first electrode is exposed, (b) at least part of the surface of the flow path opening other than the surface of the first member from which the first electrode is exposed, c) at least part of the exposed surface of the second electrode of the second member, (d) at least part of the surface of the second member facing the flow channel opening other than the exposed surface of the second electrode; Or (e) two or more of the above (a) to (d), the biological material for the biosensor is fixed,
The first member and the second member are stacked so that the first bonding surface and the second bonding surface face each other, and a through hole for introducing the subject of the second member is formed in the opening center portion of the first member. Each through-hole for ventilation of the first member or the second member is located on the tip side from the first electrode of each flow passage opening, and each flow passage opening of the first member and the second member The bonding surface is bonded without using an adhesive so that a space is formed between each channel opening and the surface facing the channel opening.
Biosensor chip.
[12]
The first electrode is provided at a position facing the second electrode, and a distance between surfaces of the first electrode and the second electrode facing the first electrode is 5 mm or less, according to [11]. Biosensor chip.
[13]
The first member is embedded in the first substrate, a part of the first member is exposed at a tip side of the first electrode of the channel opening, and is in a non-contact state with the first electrode. A third electrode disposed independently for each part, and a third wiring connected to each of the third electrodes and connectable to the outside of the first member;
The first member and the second member are laminated such that each ventilation through-hole of the first member or the second member is located at a position overlapping the third electrode or at the tip side from the third electrode, The biosensor chip according to [11] or [12].
[14]
The biosensor chip according to any one of [11] to [13], wherein the biological material is at least one material selected from the group consisting of enzymes, antigens, antibodies, peptides, proteins, and nucleic acids.
[15]
[11] A biosensor comprising the biosensor chip according to any one of [14].

  According to the present invention, a multi-type biosensor chip capable of obtaining relatively high measurement accuracy can be obtained. Moreover, as described in Patent Document 7, a multi-type biosensor chip in which dimensional variations are reduced by joining these injection-molded products (two-part structure) under conditions that do not adversely affect the activity of enzymes and the like. Can provide. The biosensor chip of the present invention is a chip for a biosensor such as an enzyme sensor that does not require a complicated correction function, and has at least three working electrodes and can measure one sample simultaneously or in parallel. This multi-type chip can provide a kit for assembling a biosensor, which can be provided in a form suitable for a small quantity and a wide variety. Furthermore, a chip for a biosensor such as an enzyme sensor can be provided using this kit.

2 is an example of a first member of the biosensor assembly kit of the present invention. FIG. 3 is a plan view of the first substrate 100 of the first member 10 as viewed from the center of the opening and the surface having the flow path opening extending radially from the opening center. It is the figure which expanded the opening center part 110 and the one flow-path opening part 111 of the 1st member shown in FIG. The side view (left side) and back view (right side) of the first substrate 100 of the first member 10 are shown. The upper part is an example of the second member of the biosensor assembly kit of the present invention. FIG. 4 is a plan view of the second member 20, depicting the second bonding surface 220 side to be bonded to the first bonding surface 150 of the first member 10 of the second member 20. FIG. 5 is an enlarged view of an opening center portion 210 and one flow passage opening 211 of the second member shown in FIG. 4. A side view (right side) and a rear view (left side) of the second substrate 200 of the second member 20 are shown. A state in which the first member 10 and the second member 20 are joined and assembled as a biosensor chip is shown. In the embodiment in which the third electrode is provided in the first member 10, an embodiment in which a ventilation through hole is provided is shown. An embodiment in which the second member 20 is provided with a ventilation through hole is shown. The top view of the stamping molded object for shaping | molding containing an electrode and wiring is shown. FIG. 11 shows a state in which a first substrate and a second substrate are formed on the stamping molded body shown in FIG. 10 and the first member and the second member are integrated. FIG. 10 shows a rear view (right) and a side view (left) in a state where the first substrate and the second substrate are formed on the stamping molded body shown in FIG. 10 and the first member and the second member are integrated. . The 1st board | substrate and the 2nd board | substrate are shape | molded in the stamping molded object for shaping | molding shown in FIG. 11 and 12, and the photograph of the state in which the 1st member and the 2nd member were united is shown. The measurement result in PBS only (no Phe addition) in the examples is shown. The measurement result of Phe (final concentration 1 mM) in an Example is shown. The summary of the measurement result in an Example is shown.

[Biosensor assembly kit]
The biosensor assembly kit of the present invention comprises:
A first substrate having an opening center portion and at least three flow passage openings extending radially from the opening center portion;
A first electrode embedded in the first substrate and at least a part of which is exposed at the flow path opening;
A first wiring connected to the first electrode and connectable to the outside of the first member;
Corresponding to the first member having the first joint surface to be joined with the second joint surface of the second member, which consists of the surface surrounding the opening center portion and the flow passage opening portion, and the opening center portion of the first member Consisting of a second substrate having a through-hole for introducing a subject to be measured by the biosensor, extending in the thickness direction,
The second member has a second bonding surface to be bonded to the first bonding surface of the first member, and is embedded in the second substrate, and at least a part thereof is exposed at a portion facing the channel opening, A second electrode disposed independently for each portion facing the road opening and a second member connected to each of the second electrodes and including a second wiring connectable to the outside of the second member .
Furthermore, the first member or the second member has a ventilation through-hole extending in the thickness direction at each of the distal end side of the first electrode of each flow path opening.

  FIG. 1 is a plan view of the first substrate 100 of the first member 10 as viewed from the center of the opening and the surface having the flow passage opening extending radially from the center of the opening. FIG. 2 is an enlarged view of one flow path opening 111. FIG. 3 shows a side view (left) and a rear view (right) of the first substrate 100 of the first member 10.

  The first substrate 100 has an opening center part 110 and a channel opening part 111 extending radially from the opening center part 110. In the embodiment shown in FIG. 1, there are four channel openings 111A, 111B, 111C, and 111D. The four channel openings 111A, 111B, 111C, 111D extend radially from the opening center 110. The number of flow path openings extending radially is at least three, and can be four or more. The number of channel openings in the example shown in FIG. 1 is four. The upper limit of the number of openings for the flow path is not particularly limited, but the planar dimensions of the entire first member, the diameter of the opening center, the width of the opening for the flow path, the dimensions of the electrodes and wiring, and the measurement results are obtained simultaneously. The number is selected appropriately in consideration of the number of physical properties (that is, the number of second electrodes, which corresponds to the number of flow path openings). From a practical viewpoint, the upper limit of the number of flow path openings can be, for example, 10. However, it is not intended to prevent the passages having a larger number of openings.

  Each of the radially extending flow path openings is adjacent to each other with the same angle or an arbitrary angle. If the number of channel openings is three at the same angle, the angle between adjacent channel openings is 120 °, and if the number of channel openings is four, the adjacent flow The angle between the road openings is 90 °. Since the angles between the adjacent channel openings are equal in this way, the sample supplied to the center of the opening can flow evenly in each channel, and the measurement accuracy can be improved. In addition, the adjacent flow channel openings are adjacent to each other at an arbitrary angle, so that the sample supplied to the central portion of the opening can flow to the flow channel openings at an arbitrary distribution.

  The planar shape of the opening center 110 is circular or a polygon equal to the number of the openings for the flow path, or a polygon that is a multiple thereof, from the viewpoint that the supplied sample can flow uniformly into each flow path. It is appropriate to be. However, it is not intended to be limited thereto. The planar dimension of the opening center portion 110 can be appropriately determined depending on, for example, the amount of a sample that can be used. When the planar dimension of the opening center part 110 is a square, the length of one side can be, for example, in the range of 1 to 10 mm, and in the case of a circular shape, the diameter is in the range of, for example, 1 to 10 mm. be able to. However, it is not intended to be limited to this range. The flow path opening 111 extending radially from the opening center 110 is appropriate to have a width that allows the sample supplied to the opening center to easily flow into each flow path by capillary action. It can be in the range of 0.1-5 mm. However, it is not intended to be limited to this range. The depth of the flow channel opening 111 is suitably a size that allows the sample supplied to the central portion of the opening to easily flow into each flow channel due to capillary action, similar to the width. For example, the depth is It can be in the range of 0.1-5 mm. However, it is not intended to be limited to this range. The amount of the sample supplied to the center of the opening is not particularly limited, but can be in the range of 0.1 to 5 mL, for example. However, it is not intended to be limited to this range. The length of the flow path opening 111 (depth from the opening center 110) is appropriately determined in consideration of the exposed dimension of the second electrode provided so that the surface of the flow path opening 111 is exposed. . As will be described later, when an additional third electrode that functions as a sensing electrode for sensing the inflow of the sample is provided, the third electrode provided so that the surface is exposed in the flow path opening 111. It is determined as appropriate in consideration of the exposure dimension and the like. The length of the flow path opening 111 (depth from the opening center 110) can be set in the range of 2 to 15 mm, for example. However, it is not intended to be limited to this range.

At least a first electrode is embedded in the first substrate.
The first electrode 130 is embedded in the first substrate 100, and a part of the first electrode 130 is exposed at the channel opening 111, and is disposed independently for each channel opening. In the embodiment shown in FIG. 1, since there are four flow path openings, four first electrodes 130A, 130B, 130C, and 130D are also disposed independently for each flow path opening. The

  The place where the biomaterial for the biosensor is to be fixed is (a) at least a part of the surface where the first electrode of the first member is exposed, and (b) other than the surface where the first electrode of the first member is exposed. At least part of the surface of the opening for the channel, (c) at least part of the exposed surface of the second electrode of the second member, (d) for the channel other than the exposed surface of the second electrode of the second member It is at least a part of the surface facing the opening, or two or more of (e) (a) to (d). In the case of (a), the biosensor biological material is fixed to at least a part of the exposed surfaces of the first electrodes 130A, 130B, 130C, and 130D. In the case of (b), the surface on the tip side of the first electrode of each flow path opening, that is, the flow path opening surface 114A between the first electrode 130A and a third electrode 140A described later, Flow path opening surface 114B between 1 electrode 130B and third electrode 140B, flow path opening surface 114C between first electrode 130C and third electrode 140C, and between first electrode 130D and third electrode 140D The channel opening surface 114D is used to immobilize the biomaterial for the biosensor. In the embodiment shown in FIG. 1, each of the four first electrodes 130A, 130B, 130C, and 130D, and each of the four channel opening surfaces 114A, 114B, 114C, and 114D between the first electrode and the third electrode. Biological material for biosensor can be fixed to both. Further, in this case, a biomaterial for biosensor may be fixed to at least a part of the surface of the third electrode. The third electrode will be described later.

The biological materials to be fixed may be different from each other, or a part or all of them may be common. The electrode area (exposed area) of each first electrode can be appropriately determined in consideration of the type and amount of biological material to be fixed, the amount of sample that can be used, the accuracy required for measurement, the electrochemical measurement conditions, and the like. For example, it can range from 1 to 50 mm ^2, preferably in the range of 1 to 30 mm ^2, more preferably from 1 to 10 mm ^2. Not only the first electrode but also a smaller electrode dimension enables measurement with a smaller amount of sample, which is preferable from this viewpoint. However, it is not intended to be limited to this range. The biosensor assembly kit of the present invention has a second electrode serving as a working electrode in each of at least three channel openings, and can be used as a multi-biosensor chip after assembly.

  Further, the first member 10 has through holes 115A to 115D for ventilation extending in the thickness direction on the tip side from the first electrodes 130A to 130D of the openings 111A to 111D for the flow paths (see FIGS. 1 and 2). (Not shown). The through hole for ventilation will be described in detail later.

The first member 10 has a first joint surface 150 to be joined to the second joint surface of the second member, which is a surface surrounding the opening center portion 110 and the flow path opening portion 111. In the first bonding surface 150, the first electrode 130 is exposed only at the opening center part 110 and the flow path opening part 111, and there is no exposed part of the electrode in the other parts, and the second part of the second member 2 The surface to be joined with the joining surface is secured widely. The first joint surface 150 is preferably smooth in order to achieve a good joint state with the second joint surface, and the area is suitably, for example, 10 mm ² or more, more preferably 50 mm ^2. That's it. The upper limit of the area of the second bonding surface is not particularly limited, but is preferably 200 mm ² or less, and more preferably 150 mm ² or less, from the viewpoint of realizing a compact chip.

  The thickness of the first member 10 is not particularly limited, but is appropriately determined in consideration of the fact that the electrode and the wiring can be maintained in a sealed state except for the exposed portion to the outside and can be molded well. The thickness of the first member 10 can be, for example, in the range of 1 to 10 mm.

  The first electrodes 130A to 130D include first wirings 131A to 131D that are connected to each of the first electrodes 130A to 130D and can be connected to the outside of the first member 10. The ends of the first wirings 131A to 131D opposite to the electrodes can be aligned to serve as the first terminal 160 for connection with the measuring instrument (see FIG. 3).

  The first member 10 is embedded in the first substrate 100, and at least a part of the first member 10 is exposed at the front end side of the first electrode 130 of the channel opening 111, and is in a non-contact state with the first electrode 130. The third electrode 140 may be included independently for each opening. Further, each of the third electrodes 140 may have a third wiring 141 that can be connected to the outside of the first member 10. In the embodiment shown in FIG. 1, four third electrodes 140A, 140B, 140C, and 140D are provided, and four third wirings 141A, 141B, 141C, and 141D are provided. As with the first wiring, the third wiring 141A, 141B, 141C, 141D can be arranged as the first terminal 160 for connection with the measuring device by aligning the end opposite to the electrode (FIG. 3). reference).

  FIG. 4 is a plan view of the second substrate 200 of the second member 20, and is a view depicting the second bonding surface 220 side to be bonded to the first bonding surface 150 of the first member 10 of the second member 20. It is. FIG. 5 is an enlarged view of the portion of the through-hole 210 and the second electrode 215, which are portions facing the opening center portion 110 of the first member 10 and the one flow passage opening portion 111 in FIG. FIG. 6 shows a side view (right side) and a rear view (left side) of the second substrate 200 of the second member 20.

  The second member 20 is composed of the second substrate 200, and the second substrate 200 extends in the thickness direction to a portion corresponding to the opening center portion 110 of the first member 10 and the subject to be measured by the biosensor. It has a through hole 210 for introduction. Further, the second member 20 has ventilation through holes 221A to 221D extending in the thickness direction in the corresponding portions on the tip side of the first electrodes 130A to 130D of the flow path openings 111A to 111D, respectively. (See FIG. 9). Further, the second substrate 200 of the second member 20 has a second bonding surface 220 for bonding to the first bonding surface 150 of the first member 10.

A second electrode 215 is embedded in the second substrate 200, and at least a part of the second electrode 215 is exposed on the surface of the second bonding surface 220. The second electrode 215 corresponds to the flow passage openings 111A to 111D extending radially from the opening center part 110 of the first member 10, and is formed in a protruding shape as 215A to 215D so that these can be engaged with each other. You can also. When the second electrodes 215A to 215D are formed in a protruding shape, there is an advantage that the sealing property of the flow path and the bonding strength of the first member and the second member are enhanced. The second electrodes 215A to 215D are not in contact with each other. A biosensor having second electrodes divided into a number equal to the number of first electrodes can perform electrochemical measurements independently at each second electrode. In the embodiment shown in FIGS. 1 to 6, since there are four channel openings and four first electrodes, four second electrodes 215A, 215B, 215C, and 215D are also arranged. The electrode area (exposed area) of each second electrode can be appropriately determined in consideration of the amount of sample that can be used, the accuracy required for measurement, electrochemical measurement conditions, and the like. However, considering the measurement accuracy, it is appropriate that the exposed area of the second electrode is in the range of 1 to 10 times the exposed area of the first electrode to the channel opening, preferably 1.5 to 10 times. More preferably, the range is 2 to 10 times, and the area can be, for example, 1 to 50 mm ² . However, it is not intended to be limited to this range.

  However, the flow path can be formed even if the second electrode 215 is not a protrusion, and a desired bonding strength between the first member and the second member can be obtained. The projecting portion of the second electrode 215 has a height lower than the depth of the opening center portion 110 and the flow passage openings 111A to 111D, and the center of the opening when the first member 10 and the second member 20 are joined. Each of the second electrodes 215A to 215D enters the vicinity of the opening upper end of the portion 110 and the flow passage openings 111A to 111D, and the surfaces of the second electrodes 215A to 215D form a flow path together with the flow path openings 111A to 111D be able to. The cross-sectional shape and dimensions perpendicular to the longitudinal direction of the flow path can be appropriately determined according to the cross-sectional shapes and dimensions of the flow path openings 111A to 111D and the cross-sectional shapes and dimensions of the second electrodes 215A to 215D.

  A second wiring 121 that is connected to the second electrode 215 and connectable to the outside of the second member 20 is provided. In the aspect shown in FIG. 4, since the four second electrodes 215A to 215D are provided, the second wirings 216A to 216D that are independently connected to the second electrode and can be connected to the outside of the second member can be provided. The ends of the second wirings 216A to 216D opposite to the electrodes can be aligned to serve as the second terminal 230 for connection with the measuring instrument (see FIG. 6). FIG. 5 shows an enlarged view of a portion including the second electrode 215, the second wiring 216, and the through hole 210. As shown in FIG. 7, the second terminal 230 of the second member 20 goes to the outside of the chip when the first member 10 and the second member 20 are laminated and joined together with the first terminal 160 of the first member 10. The terminal is formed.

  The thickness of the second member 20 is not particularly limited, but is appropriately determined in consideration of easy assembly to the first member 10 and good molding. The thickness of the second member 20 can be, for example, in the range of 1 to 10 mm.

  As shown in FIG. 7, when the first member 10 and the second member 20 are stacked and bonded so that the first bonding surface 150 and the second bonding surface 220 face each other, the subject of the second member 20 is introduced. The through hole 210 for the first member 10 is located in the opening center part 110, and the ventilation holes 112A to 112D (see FIG. 8) of the first member 10 or the ventilation through holes 221A to 221D of the second member 20 (Refer to FIG. 9) are positioned on the front end side of the first electrodes 130A to 130D of the flow path openings, and for the flow paths of the first member 10 and the flow paths of the second member 111A to 111D, respectively. A space is formed between the opening and the surface of the second electrodes 215A to 215D facing each other.

  Since this space has the through holes 221A to 221D for ventilation on the front end side of the space for each flow path opening, the sample solution introduced from the through hole 210 is sucked by capillary action, and the first electrode 130A To 130D and the second electrodes 215A to 215D. In other words, the first member 10 and the second member 20 are stacked and bonded so that the first bonding surface 150 and the second bonding surface 220 face each other, and each of the flow paths formed on the tip side of each flow path is formed. In addition, it is only necessary to have at least one ventilation through hole extending in the thickness direction of the first member or the second member. The through hole for ventilation may be provided on either side of the first member and the second member. However, in consideration of the shape and structure of the flow channel opening and the arrangement, shape, and structure of the electrode, the first member It is more preferable to provide in. However, it can be provided on the second member or on both the first member and the second member. FIG. 8 shows an embodiment in which the first electrode 10 is provided with the third electrode and the ventilation through hole 112 is provided. FIG. 9 shows a mode in which the second member 20 is provided with a ventilation through hole 221. When the second member has each ventilation through hole, in the aspect having the third electrode 140, the first member 10 and the second member 20 so that the first bonding surface 150 and the second bonding surface 220 face each other. It is appropriate that the ventilation through holes 221A to 221D of the second member 20 are positioned so as to overlap with the third electrodes 140A to 140D or on the tip side from the third electrodes, respectively. is there.

  The first member 10 can have a convex portion or a concave portion in a part of the first joint surface 150, and the second member 20 can be a convex portion of the first joint surface 150 or a part of the second joint surface 220. A concave portion or a convex portion corresponding to the concave portion can be provided. The first member 10 and the second member 20 are engaged by the engagement of the convex portion of the first joint surface 150 and the concave portion of the second joint surface 220 or the concave portion of the first joint surface 150 and the convex portion of the second joint surface 220. And positioning with ease.

  In the biosensor assembly kit of the present invention, an inner surface forming a space formed between each channel opening of the first member and a surface facing each channel opening of the second member. The surface composed of the resin composition is preferably hydrophilized by, for example, plasma treatment or ethanol treatment in order to make it easier for the liquid as the subject to flow through the space due to capillary action. Furthermore, the inner surface of the through hole for introducing the analyte of the second member is also preferably made hydrophilic by, for example, plasma treatment or ethanol treatment.

  The first substrate 100 and the second substrate 200 are made of a polypropylene resin and a hydrogenated derivative of a block copolymer represented by the general formula XY (where X is a polymer block incompatible with the polypropylene resin, Y is a conjugated diene) It is an elastomeric polymer block).

  Here, as the polypropylene resin, a homopolymer or a random copolymer containing an α-olefin such as ethylene, butene-1, and hexene-1 can be used. Examples of the polymer block X include polymerized polymers such as vinyl aromatic monomers (for example, styrene), ethylene, or methacrylate (for example, methyl methacrylate). In addition, the hydrogenated derivatives of the block copolymer represented by the general formula XY include those in the range of (X—Y) n where n = 1 to 5, XYX, YXY. Etc. are included.

  As the polymer block X of the hydrogenated derivative, there are polystyrene type and polyolefin type, and those of polystyrene type are styrene, α-methylstyrene, o-methylstyrene, m-methylstyrene, p-methylstyrene, 2, A polymer block composed of one or more vinyl aromatic compounds selected from 4-dimethylstyrene, vinylnaphthalene, and vinylanthracene as monomer units can be raised. In addition, polyolefin-based materials include copolymers of ethylene and α-olefins having 3 to 10 carbon atoms. Further, a non-conjugated diene may be conjugated polymerized. Examples of the olefin include propylene, 1-butene, 3-methyl-1-butene, 1-pentene, 4-methyl-1-pentene, 1-hexene, 1-pentene, 1-octene and 1-decene. Examples of the non-conjugated diene include 1,4-hexadiene, 5-methyl-1,5-hexadiene, 1,4-octadiene, cyclohexadiene, cyclooctadiene, cyclopentadiene, 5-ethylidene-2-norbonel, 5 -Butylidene-2-norbornel, 2-isopropenyl-5-nerbornene and the like. Specific examples of the copolymer include an ethylene-propylene copolymer, an ethylene-1-butene copolymer, an ethylene-1-octene copolymer, an ethylene-propylene-1,4-hexadiene copolymer, and an ethylene-propylene. Examples include -5-ethylidene-2-norbornene copolymer.

  As the polymer block Y before hydrogenation, polybutadiene having at least one group selected from the group consisting of 2-butene-1,4-diyl group and vinylethylene group as a monomer unit, And polyisoprene composed of at least one group selected from the group consisting of methyl-2-butene-1,4-diyl group, isopropenylethylene group and 1-methyl-1-vinylethylene group as a monomer unit. It is done. Further, the polymer block Y before hydrogenation is an isoprene / butadiene copolymer composed of monomer units mainly composed of isoprene units and butadiene units, in which the isoprene units are 2-methyl-2-butene-1,4-diyl groups, What is at least one group selected from the group consisting of propenylethylene group and 1-methyl-1-vinylethylene group, and whose butadiene unit is 2-butene-1,4-diyl group and / or vinylethylene group Can be mentioned. The arrangement of the butadiene unit and the isoprene unit may be any form of random, block, and tapered block.

  The polymer block Y before hydrogenation is a vinyl aromatic compound / butadiene copolymer composed of a vinyl aromatic compound unit and a monomer unit mainly composed of a butadiene unit, wherein the vinyl aromatic compound unit is styrene, α -A monomer unit selected from methyl styrene, o-methyl styrene, m-methyl styrene, p-methyl styrene, 2,4-dimethyl styrene, vinyl naphthalene, vinyl anthracene, and butadiene unit is 2 -A copolymer which is a butene 1,4-diyl group and / or a vinylethylene group. The arrangement of the vinyl aromatic compound unit and the butadiene unit may be any form of random, block, and tapered block. The state of hydrogenation in the polymer block Y as described above may be partial hydrogenation or complete hydrogenation.

  The raw material is obtained when the polymer block X of the hydrogenated derivative is polystyrene and the polymer block Y before the hydrogenation is 1,2-bond, 3,4-bond and / or 1,4-bond polyisoprene. Cheap. Since the styrene component is incompatible with polypropylene resin and the like, it takes time to mix with polypropylene when the ratio is high, so when using a hydrogenated derivative with a large amount of styrene component, make a masterbatch and mix well beforehand Is good.

  The raw material is also readily available when the polymer block X of the hydrogenated derivative is polystyrene and the polymer block Y before hydrogenation is 1,2-bond and / or 1,4-bond polybutadiene.

  In an instrument used for biochemical research, a portion (reaction portion) that directly contacts a measurement target (subject) is required to have excellent biocompatibility, do not adsorb polypeptides or the like, and do not affect biological materials. Furthermore, in the biosensor chip made of synthetic resin, it is necessary that the additive added for the modification of the synthetic resin does not elute in various operation processes. A resin composition comprising a polymer alloy formed of the above polypropylene-based resin and a hydrogenated derivative of a block copolymer represented by the general formula XY includes a spherical microdomain of several tens of nanometers of the hydrogenated derivative, It consists of a crystalline lamella and an amorphous matrix of polypropylene forming a matrix, and the size of the crystal part of polypropylene is 1/100 or less compared to the case of polypropylene alone.

  The first member 10 and the second member 20 stacked as shown in FIG. 7 do not deactivate the biomaterial for the biosensor without using an adhesive on the first joint surface 150 and the second joint surface 220. It is attached to the joint under temperature conditions and assembled. The first substrate 100 and the second substrate 200 constituting the first member 10 and the second member 20 are both resins containing the polypropylene resin and a hydrogenated derivative of a block copolymer represented by the general formula XY It consists of a composition. In addition, the first joint surface 150 has the first electrode 130 exposed only at the opening center portion 110 and the flow passage opening 111, and in the case where it is present, the first bonding surface 150 only has the through holes 112A to 112D for ventilation. In the other portions, there is no exposed portion of the electrode, and a joint surface with the second joint surface 220 of the second member 20 is secured. Similarly, the second joint surface 220 of the second member 20 also has the through-hole 210 and, if present, the ventilation through-holes 221A to 221D and faces the respective channel openings of the first member 10. It is configured so that a space is formed between the surfaces of the second electrodes 215A to 215D, and a bonding surface with the first bonding surface 150 of the first member 10 is secured in the other portions. ing. As described above, the first bonding surface 150 and the second bonding surface 220 have almost no electrodes and wiring exposed, and a wide surface for bonding is secured. For this reason, the first member 10 and the second member 20 are assembled by being attached to and assembled at a temperature condition where the biomaterial for the biosensor is not deactivated, for example, 30 to 50 ° C., preferably 40 to 50 ° C. It can be assembled and assembled as a biosensor chip. The assembled state as a biosensor chip is shown in FIG.

  The first member 10 is formed by injection molding using the resin composition and the electrode and wiring material in the state described above for the first electrode and the first wiring, and further, if desired, the third electrode and the third wiring. Can be molded. That is, the first member can be formed by insert molding at least the first electrode and the first wiring by injection molding. Furthermore, the first member may be formed by insert molding the third electrode and the third wiring by injection molding. When injection molding is performed, the electrodes, wiring, and substrate are integrally formed by insert molding. Similarly, the second member can be produced by molding the second electrode and the second wiring by injection molding using the resin composition and the electrode and wiring material in the state described above.

  FIG. 10 shows a plan view of a stamped molded body for molding including electrodes and wiring. The lower half of FIG. 10 is a set including the first electrodes 130A to 130D, the first wirings 131A to 131D, the third electrodes 140A to 140D, and the third wirings 141A to 141D, and the upper half is the second electrodes 215A to 215D and the first electrodes A set including two wirings 216A to 216D.

  FIG. 11 shows a state where the first substrate and the second substrate are formed on the stamping molded body for molding shown in FIG. 10, and the first member and the second member are integrated, and the first member is the first member. The top view of the surface where the electrode and the 3rd electrode were exposed and the surface where the 2nd electrode was exposed in the 2nd member is shown. FIG. 12 shows a rear view (right) and a side view (left) in a state in which the same first member and second member are integrated. The integrated member is cut from the first member and the second member so that each wiring forms a connection terminal 160 in the state shown in FIGS. 3 and 6, and the first member and the second member are cut. Get.

  The electrodes and wirings are not particularly limited as long as they are conductive materials, and various metal electrodes and wirings can be used. For example, electrodes or wirings made of copper or copper alloy, which are non-ferrous metals having good conductivity, are used. And surface treatment (for example, plating of Ag, Au, etc.). Preferably, a copper thin plate is nickel-plated and then gold-plated and punched into a predetermined shape. The first member can be manufactured by fixing the stamped electrode and wiring to the mold cavity and injecting the resin composition. In addition, it is preferable that the thickness of the electrode is, for example, in the range of 0.05 to 0.08 mm so that the resin does not cover the exposed portion of the electrode in consideration of shrinkage of the resin constituting the substrate.

[Biosensor chip]
The present invention includes a biosensor chip manufacturing method and a biosensor chip obtained by this manufacturing method.
The manufacturing method of a biosensor chip includes the following steps.
(1) In the biosensor chip assembly kit of the present invention, (a) at least part of the surface of the first member from which the first electrode is exposed, (b) other than the surface of the first member from which the first electrode is exposed. At least part of the surface of the opening for the flow path, (c) at least part of the exposed surface of the second electrode of the second member, and (d) the flow other than the exposed surface of the second electrode of the second member. A step of fixing a biomaterial for a biosensor to at least a part of a surface facing the road opening, or (e) two or more of the above (a) to (d),
(2) The through hole for introducing the subject of the second member is located in the center of the opening of the first member, and each ventilation through hole of the first member or the second member is an opening for each flow path The first joint surface and the second joint surface are stacked so that the first joint surface and the second joint surface are positioned on the tip side from the first electrode of the part, and the joint surfaces of the first member and the second member are joined under a temperature condition that does not deactivate the biological material. Process.

  The biological material for the biosensor is not particularly limited, and examples thereof include solutions and solids of enzymes, antigens, antibodies, peptides, proteins, nucleic acids, and the like. The amount of the biomaterial for biosensor is not particularly limited due to the difference in the size of the biosensor chip specimen inhalation part or the difference in the reactivity of the biomaterial, but a liquid of several picoliters to several tens of microliters. Or immobilizing a solid of several femtograms to several micrograms.

  For example, when the biomaterial for the biosensor is an enzyme, one or two or more enzymes corresponding to the substance to be measured are dissolved in a buffer solution having an appropriate pH at an appropriate concentration to prepare an enzyme solution. In order to stabilize enzyme protein or to act as a binder when performing cross-linking immobilization, proteins other than enzymes can be added to this solution. Next, the enzyme solutions are mixed one by one in order, or two or more enzyme solutions are mixed in an arbitrary volume at a time, and any one of the above (a) to (e) A micropipette, dispenser, or inkjet spotter on the surface of the electrode, the top surface of the first electrode (working electrode), the bottom surface of the flow channel outside the first electrode, the surface of the third electrode, the surface of the second electrode, etc. Use and drop to dry naturally. If necessary, a thickener can be added to the enzyme solution to make it easy to dissolve when the measurement solution flows in. After application of the enzyme solution, 0.1 to several percent glutaraldehyde is also possible. By further applying the solution, it is possible to firmly cross-link and immobilize the enzyme protein so that it does not dissolve easily.

In the lamination and joining of the first member and the second member, the through hole for introducing the subject of the second member is located at the center of the opening of the first member, and each ventilation of the first member or the second member The first joint surface and the second joint surface are laminated so that the through-hole for use is positioned on the tip side from the first electrode of each flow path opening.
When the first member 10 and the second member 20 are laminated and bonded so that the first bonding surface 150 and the second bonding surface 220 face each other, a through-hole 210 for introducing the subject of the second member 20 is provided. The ventilation through holes 112A to 112D of the first member 10 or the ventilation through holes 221A to 221D of the second member 20 are located at the opening center part 110 of the first member 10, respectively, and the first electrodes of the flow path opening parts, respectively. 130A to 130D located on the front end side, spaces between the respective channel openings 111A to 111D of the first member 10 and the surfaces of the second electrodes 215A to 215D facing the respective channel openings of the second member Is formed. In this space, since there are through holes 112A to 112D or 221A to 221D for ventilation on the distal end side of the space of each channel opening, the analyte solution introduced from the through hole 210 is aspirated by capillary action, It reaches the first electrodes 130A to 130D.

  The first member and the second member are laminated, and it is preferable to pressurize so as to promote the bonding. For example, the pressurization is performed by sandwiching the first member and the second member laminated between two plates, Pressure can be applied from both sides. Furthermore, a plurality of laminated first members and second members can be sandwiched between the two plates to simultaneously create a plurality of biosensors. The pressurization promotes the joining of the joining surfaces, but is preferably maintained at a temperature condition that does not deactivate the biomaterial for the biosensor fixed to the electrode. The said temperature conditions are the range of 30-40 degreeC, for example, Preferably it is about 35 degreeC. Although the time for joining changes with conditions, it is the range of 10 minutes-10 hours, for example.

  The present invention relates to a biosensor including the biosensor chip of the present invention. The biosensor of the present invention can include, in addition to the biosensor chip, a device for performing electrochemical measurement using the biosensor chip, for example, a constant current measuring device or a constant voltage measuring device. A recording device or the like may be included to record the signal from the device for performing the measurement.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not intended to be limited to these examples.

<First embodiment>
As a resin composition for molding the first member and the second member, a homopolymer MA04A manufactured by Nippon Polypro was used as a polypropylene resin, and a Kyura Hibler 7311 was used as a hydrogenated derivative. The mixing ratio of the two was 50:50 in mass ratio, and a master batch was prepared in order to mix them in advance. Place the masterbatch resin composition into the hopper of the injection molding machine, and install the electrodes in the mold so that the first and third electrodes and the wiring for them are arranged on the first member. An electrode or the like was placed in the mold so that the second member and the wiring for them were arranged on the second member, and then injected to mold the first member and the second member. The mold used here was mirror-polished on the molding surface of the cavity, in particular the surface on which the joining surfaces of the first member and the second member were molded. FIG. 13 shows a photograph of a state in which the first member and the second member obtained by injection molding are integrated. Furthermore, unnecessary parts were cut from the integrated product shown in FIG. 13 to obtain a first member and a second member.

  The MA04A and Hibler 7311 keep the amount of modifier added to a very low level, and the polymer alloy formed by both has a spherical microdomain of several tens of nanometers of Hibler 7311, a crystalline lamella of polypropylene, and It is composed of an amorphous matrix of polypropylene forming a matrix, and the size of the crystal part of polypropylene is 1/100 or less compared to the case of polypropylene alone. Since the polymer alloy is excellent in precision transferability, when the first member and the second member are molded with the mold, the joint surfaces thereof are approximately the same as the degree of mirror polishing (planar smoothness) of the mold. A smooth joint surface can be formed.

  Application and immobilization of the enzyme-containing solution between the first electrode (working electrode) and the third electrode (detection electrode) of the first member were performed as follows. Phenylalanine dehydrogenase (PheDH) (15.4 U / mg) /0.1 MGly-KOH buffer (pH 9.5) (3.2 mg / ml) and diaphorase (DI) (22.4 U / mg) /0.1 M phosphate buffer (pH 7.0) Add 1.0 μL of the mixed enzyme solution (2.2 mg / ml) to the surface of the channel between the first electrode (working electrode) and the third electrode (detection electrode) of each channel using a micropipette. Then, by naturally drying for 1 hour, phenylalanine dehydrogenase (PheDH) and diaphorase (DI) were immobilized in each channel.

  The bonded surface of the second member with the second electrode (counter electrode) embedded in contact with the bonded surface of the injection-molded first member is made into a laminate, and is held in a bonding auxiliary jig and tightened with a screw in the dryer. Stored at 35 ° C. for 4 hours. The first member and the second member were bonded to form a chip shape. Thereby, the enzyme chip | tip for phenylalanine measurement was producible.

  The chip taken out from the dryer could not be peeled unless a considerable force was applied even if it was peeled off in the direction of separating the joint surface with a finger. The obtained chip could withstand repeated use.

Using the above chip, the phenylalanine concentration in the sample liquid was measured under the following conditions.
<Measurement solution>
PBS (pH 7.4) 1mL
NAD + (25mM) 1mL
KCl (0.5M) 1mL
1-methoxyPMS (12.5mM) 1mL
Phe (5mM) or PBS 1mL
Total volume 5μL
<Starting conditions for automatic measurement>
Inhalation (filling) voltage: 0.1 V
Inhalation (filling) current: 0.1μA
Phe measurement: measured with a working electrode and a counter electrode
<Voltage application conditions>
1st: 0 V, 5 sec
2nd: 0.4 V, 5 sec

  The results are shown in FIGS. When PheDH and DI derived from Bacillus badius (Bb) were immobilized on 4CH and measurement of 1 mM Phe was attempted, an increase in oxidation current was observed, and the variation of 4CH was 4% or less. Almost the same current value was obtained between each channel on the same chip.

  The present invention is useful in fields related to biosensors. In particular, it is particularly useful in the field where it is necessary to easily measure a plurality of physical properties and substance concentrations for a single sample.

10 First part
100 1st board
110 Center of opening
111, 111A to 111D Channel opening
112, 112A-112D Venting through hole
114, 114A-114D Flow path opening surface
130, 130A ~ 130D 1st electrode
131, 131A to 131D 1st wiring
140, 140A ~ 140D 3rd electrode
141, 141A-141D 3rd wiring
150 First joint surface
160 Terminal 1
20 Second part
200 2nd board
210 Through hole
215, 215A ~ 215D 2 electrodes
216, 216A to 216D 2nd wiring
220 Second joint surface
221, 221A to 221D Ventilation through hole
230 Terminal 2

Claims (15)

A first substrate having an opening center portion and at least three flow passage openings extending radially from the opening center portion; a first substrate embedded in the first substrate and exposed at least partially at the flow passage opening portion; 1st electrode, 2nd joint surface of 2nd member which consists of the surface surrounding the said opening center part and flow-path opening part including the 1st wiring which can connect with the said 1st electrode and the exterior of the 1st member In order to introduce a subject to be measured by the biosensor that extends in the thickness direction into the first member having the first joint surface and the portion corresponding to the opening center of the first member. A second substrate having a through hole, having a second bonding surface for bonding to the first bonding surface of the first member, and embedded in the second substrate, at least a part of the opening for the flow path Exposed at the part facing the part and arranged independently for each part facing the channel opening. A biosensor chip assembly kit including a second member connected to each of the second electrode and the second electrode and including a second member connectable to the outside of the second member,
The exposed area of the second electrode is in the range of 1 to 10 times the exposed area of the first electrode to the channel opening,
(a) at least part of the surface of the first member from which the first electrode is exposed, (b) at least part of the surface of the flow path opening other than the surface of the first member from which the first electrode is exposed, c) at least part of the exposed surface of the second electrode of the second member, (d) at least part of the surface of the second member facing the flow channel opening other than the exposed surface of the second electrode; Or (e) two or more of the above (a) to (d) are used for fixing a biological material for a biosensor,
The first member or the second member has a ventilation through hole extending in the thickness direction on each of the front end side of the first electrode of each flow passage opening,
When the first member and the second member are laminated and bonded so that the first bonding surface and the second bonding surface face each other, the through hole for introducing the subject of the second member is the opening center of the first member Each through hole for ventilation of the first member or the second member is located on the front end side of the first electrode of each channel opening, and each channel opening of the first member and the second A space is formed between each second electrode of the member,
The first substrate and the second substrate are a hydrogenated derivative of a polypropylene resin and a block copolymer represented by the general formula XY (where X is a polymer block that is incompatible with the polypropylene resin, Y is an elastomeric property of a conjugated diene) A resin block containing a polymer block),
The laminated first member and the second member are assembled by being attached to a joint under a temperature condition that does not deactivate the biomaterial for the biosensor without using an adhesive on the joint surface.
The biosensor chip assembly kit. When the first member and the second member are laminated and joined, the first electrode is provided at a position facing the second electrode, and the first electrode and the second electrode facing the first electrode The biosensor chip assembling kit according to claim 1, wherein the distance between the surfaces is 5 mm or less. The first member is embedded in the first substrate, a part of the first member is exposed at a tip side of the first electrode of the channel opening, and is in a non-contact state with the first electrode. A third electrode disposed independently for each part, and a third wiring connected to each of the third electrodes and connectable to the outside of the first member;
When the first member and the second member are laminated and joined so that the first joint surface and the second joint surface face each other, each ventilation through hole of the first member or the second member overlaps with each third electrode. The kit for assembling a biosensor chip according to claim 1, wherein the kit is located on a distal end side with respect to a position where the third electrode is disposed. At least the first member is formed by insert molding the first electrode and the first wiring by injection molding, and the second member is formed by insert molding the second electrode and the second wiring by injection molding. The biosensor chip assembly kit according to claim 1. The biosensor chip assembly kit according to claim 4, wherein at least the first member is formed by insert molding the third electrode and the third wiring by injection molding. 2. The biosensor chip assembly kit according to claim 1, wherein (a) at least part of a surface of the first member from which the first electrode is exposed, (b) other than a surface of the first member from which the first electrode is exposed. At least part of the surface of the opening for the flow path, (c) at least part of the exposed surface of the second electrode of the second member, and (d) the flow other than the exposed surface of the second electrode of the second member. A biological material for a biosensor is fixed to at least a part of the surface facing the road opening, or (e) two or more of the above (a) to (d),
The through hole for introducing the subject of the second member is located at the opening center of the first member, and each ventilation through hole of the first member or the second member is the first of the opening for each flow path. The first bonding surface and the second bonding surface are laminated so as to be positioned on the tip side from the one electrode, and the bonding surfaces of the first member and the second member are bonded under a temperature condition in which the biological material is not deactivated. A method for producing a biosensor chip. The biosensor chip assembly kit according to claim 3, wherein (a) at least a part of a surface of the first member from which the first electrode is exposed, and (b) a surface other than the surface of the first member from which the first electrode is exposed. At least part of the surface of the opening for the flow path, (c) at least part of the exposed surface of the second electrode of the second member, and (d) the flow other than the exposed surface of the second electrode of the second member. A biological material for a biosensor is fixed to at least a part of the surface facing the road opening, or (e) two or more of the above (a) to (d),
A position where the through hole for introducing the subject of the second member is located at the center of the opening of the first member, and the respective through holes for ventilation of the first member or the second member overlap with the respective third electrodes Alternatively, the first bonding surface and the second bonding surface are stacked so as to be positioned on the tip side from each third electrode, and the bonding surfaces of the first member and the second member are bonded under a temperature condition that does not deactivate the biological material. A method for producing a biosensor chip, comprising: The production method according to claim 6 or 7, wherein the biological material is at least one material selected from the group consisting of enzymes, antigens, antibodies, peptides, proteins, and nucleic acids. The manufacturing method according to any one of claims 6 to 8, wherein a temperature condition in which the biological material is not deactivated is in a range of 30 to 40 ° C. The biosensor chip obtained by manufacturing with the method in any one of Claims 6-9. A first substrate having an opening center portion and at least three flow passage openings extending radially from the opening center portion; a first substrate embedded in the first substrate and exposed at least partially at the flow passage opening portion; A second joint surface of the second member, comprising a first wire connected to the first electrode and connected to the outside of the first member, and comprising a surface surrounding the opening center portion and the flow passage opening portion; A first member having a first joint surface to be joined, and a portion that corresponds to the opening center portion of the first member for introducing a subject to be measured by a biosensor that extends in the thickness direction. The flow path opening includes a second substrate having a through hole, has a second bonding surface to be bonded to the first bonding surface of the first member, and is embedded in the second substrate. Is exposed at the part facing the first channel, and is disposed independently for each part facing the channel opening. Connected to each of the two electrodes and the second electrode, including a second member including a second wiring connectable to the outside of the second member,
The exposed area of the second electrode is in the range of 1 to 10 times the exposed area of the first electrode to the channel opening,
The first substrate and the second substrate are a hydrogenated derivative of a polypropylene resin and a block copolymer represented by the general formula XY (where X is a polymer block that is incompatible with the polypropylene resin, Y is an elastomeric property of a conjugated diene) A resin block containing a polymer block),
(a) at least part of the surface of the first member from which the first electrode is exposed, (b) at least part of the surface of the flow path opening other than the surface of the first member from which the first electrode is exposed, c) at least part of the exposed surface of the second electrode of the second member, (d) at least part of the surface of the second member facing the flow channel opening other than the exposed surface of the second electrode; Or (e) two or more of the above (a) to (d), the biological material for the biosensor is fixed,
The first member and the second member are stacked so that the first bonding surface and the second bonding surface face each other, and a through hole for introducing the subject of the second member is formed in the opening center portion of the first member. Each through-hole for ventilation of the first member or the second member is located on the tip side from the first electrode of each flow passage opening, and each flow passage opening of the first member and the second member The bonding surface is bonded without using an adhesive so that a space is formed between each channel opening and the surface facing the channel opening.
Biosensor chip. The first electrode is provided at a position facing the second electrode, and a distance between surfaces of the first electrode and the second electrode facing the first electrode is 5 mm or less. Biosensor chip. The first member is embedded in the first substrate, a part of the first member is exposed at a tip side of the first electrode of the channel opening, and is in a non-contact state with the first electrode. A third electrode disposed independently for each part, and a third wiring connected to each of the third electrodes and connectable to the outside of the first member;
The first member and the second member are laminated such that each ventilation through-hole of the first member or the second member is located at a position overlapping with each third electrode or at the tip side from each third electrode, Item 13. The biosensor chip according to Item 11 or 12. The biosensor chip according to any one of claims 11 to 13, wherein the biological material is at least one material selected from the group consisting of enzymes, antigens, antibodies, peptides, proteins, and nucleic acids. The biosensor containing the biosensor chip in any one of Claims 11-14.