JP5170803B1 - Multi-biosensor chip assembly kit, multi-biosensor chip manufacturing method, and multi-biosensor chip - Google Patents

Abstract

Provided is a multi-type biosensor chip having a plurality of working electrodes and capable of measuring one sample simultaneously or in parallel.
A first member comprising a first substrate having a first electrode and a second electrode embedded therein and having a plurality of flow path openings extending radially from the center of the opening and the center of the opening, to be measured A biosensor assembly kit including a second member having a through-hole for introducing a subject. A biomaterial for the biosensor is fixed to the exposed surface of the second electrode in the channel opening, the surface on the tip side of the second electrode of each channel opening, or the exposed surface and the surface on the tip side. 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, each flow channel opening of the first member and each flow channel opening of the second member A space is formed between the surfaces facing each other.
[Selection] Figure 1b

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 WO2006 / 135061

  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.

  Accordingly, an object of the present invention is to provide a chip for a biosensor such as an enzyme sensor which does not require a complicated correction function, and provide a multi-type biosensor chip in a form suitable for a small amount and a 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. Therefore, a biosensor chip manufactured only by injection molding is not known.

  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.

  An object of the present invention is a biosensor chip comprising a structure divided into two parts that includes a metal electrode with good electrical conductivity and can be produced by injection molding, under conditions that do not adversely affect the activity of an enzyme, etc. An object of the present invention is to provide means for providing a biosensor chip in which the dimensional variation is reduced by joining these two divided structures. 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, it is possible to produce a two-part structure including a metal electrode with good conductivity by injection molding, and these injections are performed under conditions that do not adversely affect the activity of enzymes and the like. Multi-type biosensor that has a dimensional variation reduced by joining a molded product (two-part structure) and has at least two working electrodes and can measure one sample simultaneously or in parallel The present invention has been completed by finding means for obtaining chips.

  In the present invention, an injection molded product in which a metal electrode is integrally molded using a resin composition containing a polypropylene resin described in Patent Document 7 and a hydrogenated derivative of a block copolymer represented by the general formula XY ( It has been found that a biosensor chip can be obtained by joining a bipartite structure) with a new method not described in Patent Document 4 without adversely affecting the activity of an enzyme or the like.

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 electrode embedded in the first substrate and exposed at least partially at the opening center portion; A second electrode that is embedded in the first substrate, is at least partially exposed at the flow path opening, and is disposed independently for each flow path opening in a non-contact state with the first electrode. A first wiring connected to the first electrode and connectable to the outside of the first member; and a second wiring connected to each of the second electrode and connectable to the outside of the first member, the opening The first member having the first joint surface to be joined to the second joint surface of the second member, and the portion corresponding to the opening center portion of the first member, comprising the center and the surface surrounding the channel opening Has a through-hole for introducing an analyte to be measured by the biosensor that extends in the thickness direction. And each of the corresponding portions on the front end side of the second electrode of each flow path opening is made of a second substrate having a ventilation through hole extending in the thickness direction, and the first joint surface of the first member A biosensor chip assembly kit including a second member having a second joint surface to be joined,
At least a part of the exposed surface in the at least one flow path opening of the second electrode, the surface on the tip side of the second electrode of each flow path opening, or the exposed surface and the surface on the tip side are bio Used to immobilize biological materials for sensors,
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 second member is positioned on the tip side of the second electrode of each flow path opening, and each flow path opening of the first member and each flow of the second member A space is formed between the road opening and the opposite surface,
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 first member and the second member that are stacked are assembled by attaching the joint surface to a joint under a temperature condition that does not deactivate the biomaterial for the biosensor without using an adhesive.
The biosensor chip assembly kit.
[2]
The first electrode is divided into a number corresponding to the number of second electrodes, and is connected to the first electrode independently and has a first wiring connectable to the outside of the first member,
The kit for assembling a biosensor chip according to [1].
[3]
The first member is embedded in the first substrate, a part of the first member is exposed on the front end side of the second electrode of the flow path opening, and is in a non-contact state with the first electrode and the second electrode. A third electrode disposed independently for each flow path opening, 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 stacked and bonded so that the first bonding surface and the second bonding surface face each other, each ventilation through hole of the second member overlaps with each third electrode or each The kit for assembling a biosensor chip according to [1] or [2], which is located on the tip side from the third electrode.
[4]
The biosensor chip assembly kit according to [1], wherein at least the first member is formed by insert molding the first electrode, the first wiring, the second electrode, and the second wiring by injection molding.
[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]
At least a part of at least one exposed surface of the second electrode of the first member of the first member of the kit for assembling the biosensor chip according to [1], a surface on the tip side of the second electrode of each channel opening, or the above Fix biomaterial for biosensor on the exposed surface and the surface on the tip side,
The through hole for introducing the subject of the second member is positioned at the opening center of the first member, and each through hole for ventilation of the second member is distal to the second electrode of each channel opening A biosensor comprising: laminating a first joint surface and a second joint surface so as to be positioned on a side; and joining the joint surfaces of the first member and the second member under a temperature condition that does not deactivate the biological material. Chip manufacturing method.
[7]
At least a part of at least one exposed surface of the second electrode of the first member of the first member of the biosensor chip assembling kit according to [3], a surface on the tip side of the second electrode of each channel opening, or the Fix biomaterial for biosensor on the exposed surface and the surface on the tip side,
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 through hole of the second member overlaps with each third electrode or each third Including laminating the first joint surface and the second joint surface so as to be positioned on the tip side from the electrode, and joining the joint surfaces of the first member and the second member under a temperature condition in which the biological material is not deactivated. The manufacturing method of a biosensor chip.
[8]
[7] or [8], 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 electrode embedded in the first substrate and exposed at least partially at the opening center portion; A second electrode that is embedded in the first substrate, is at least partially exposed at the flow path opening, and is disposed independently for each flow path opening in a non-contact state with the first electrode. A first wiring connected to the first electrode and connectable to the outside of the first member; and a second wiring connected to each of the second electrode and connectable to the outside of the first member, the opening The first member having the first joint surface to be joined to the second joint surface of the second member, and the portion corresponding to the opening center portion of the first member, comprising the center and the surface surrounding the channel opening Has a through-hole for introducing an analyte to be measured by the biosensor that extends in the thickness direction. In addition, each of the corresponding portions on the distal end side from the second electrode of each flow path opening has a ventilation through hole extending in the thickness direction and is joined to the first joint surface of the first member. A second member having a second joining surface of
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),
At least a part of the exposed surface in the at least one flow path opening of the second electrode, the surface on the tip side of the second electrode of each flow path opening, or the exposed surface and the surface on the tip side are bio Biological material for sensor 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 second member is located on the tip side from the second electrode of each channel opening, and for each channel opening of the first member and each channel of the second member The bonding surface is bonded without using an adhesive so that a space is formed between the opening and the surface facing the opening.
Biosensor chip.
[12]
The first electrode is divided into a number corresponding to the number of second electrodes, and is connected to the first electrode independently and has a first wiring connectable to the outside of the first member,
[11] The biosensor chip according to [11].
[13]
The first member is embedded in the first substrate, a part of the first member is exposed on the front end side of the second electrode of the flow path opening, and is in a non-contact state with the first electrode and the second electrode. A third electrode disposed independently for each flow path opening, 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 so that each ventilation through hole of the second member overlaps with each third electrode or is located on the tip side from each third electrode, [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, there is provided a chip for a biosensor such as an enzyme sensor that does not require a complicated correction function, which has at least three working electrodes and can measure one sample simultaneously or in parallel. It is possible to provide a kit for assembling a biosensor that makes it possible to provide a chip of a type in a form suitable for a small variety of products. Furthermore, a chip for a biosensor such as an enzyme sensor can be provided using this kit.

The first substrate 100 of the first member 10, which is an example of the first member of the biosensor assembly kit of the present invention, is viewed from the surface having the opening center and the channel opening radially extending from the opening center. FIG. The first substrate 100 of the first member 10, which is an example of the first member of the biosensor assembly kit of the present invention, is viewed from the surface having the opening center and the channel opening radially extending from the opening center. FIG. The perspective view which extracted only the opening part for flow paths which extends radially from the opening center part and opening center part from FIG. 1b is shown. FIG. 3 is a plan view showing an example of electrodes and wiring embedded in the first member of the biosensor assembly kit of the present invention. FIG. 3 is a plan view showing an example of electrodes and wiring embedded in the first member of the biosensor assembly kit of the present invention. The top view from the 2nd member 20 side when it laminates | stacks so that the 1st joining surface 150 of the 1st member 10 of the biosensor assembly kit of this invention and the 2nd joining surface 220 of the 2nd member 20 may oppose Indicates. The upper part is a perspective view of the second member 20, which is an example of the second member of the biosensor assembly kit of the present invention, and is joined to the first joint surface 150 of the first member 10 of the second member 20. FIG. 6 is a diagram depicting the second joint surface 220 side of The lower part of FIG. 2a is a perspective view of the first member 10 which is an example of the first member of the biosensor assembly kit of the present invention. The upper left part is a plan view of the second member 20 opposite to the second joint surface 220, the left middle part is an AA sectional view of the upper left part, and the lower left part is a plane of the second member 20 on the second joint surface 220 side. FIG. The upper right portion is a perspective view of the second member 20 on the second bonding surface 220 side, and the lower right portion is a cross-sectional perspective view seen from the opposite side of the second bonding surface 220 of the second member 20. FIG. 2 is a perspective view showing a state in which the second member 20 is joined to the first member 10, and the side opposite to the second joining surface 220 of the second member 20 is drawn. FIG. 4 is a front view (middle left), a side surface (middle right), a plane (upper right), and a back surface (lower right) showing a state in which the second member 20 is joined to the first member 10. The first substrate 101 of the first member 11, which is another example of the first member of the biosensor assembly kit of the present invention, has a surface having an opening center portion and a flow passage opening extending radially from the opening center portion. It is the top view seen from. The first substrate 101 of the first member 11, which is another example of the first member of the biosensor assembly kit of the present invention, has a surface having an opening center portion and a flow passage opening extending radially from the opening center portion. It is the figure seen from. FIG. 4 is a perspective view of a second member 21, which is another example of the second member of the biosensor assembly kit of the present invention, for joining with the first joint surface 151 of the first member 11 of the second member 21. FIG. 6 is a perspective view illustrating the second bonding surface 221 side. FIG. 3 is a perspective view showing a state where the first member 11 and the second member 21 of the biosensor assembly kit of the present invention are assembled. The photograph in the state where the 1st member obtained by injection molding in Example 1 and the 2nd member were united is shown. The photograph of the state which cut | disconnected the unnecessary part from the integrated product shown in FIG. 4a and made it the 1st member (right side) and the 2nd member (left side) is shown. The photograph of what stuck the 1st member and 2nd member which were shown in FIG. (a), (b), (c), and (d) are the results of simultaneous measurement of current values with 4 CH for each measurement solution with Phe concentrations of 0 mM, 0.1 mM, 0.5 mM, and 1.0 mM, respectively. It is. The result of FIG. 5 is shown by the relationship between the Phe density | concentration in a measurement liquid, and the average (4CH average) electric current value of a chip | tip. This is a result of simultaneous measurement of current values with four CHs for each measurement solution with Phe and Tyr concentrations of 0 mM, Phe concentration of 1.0 mM, and Tyr concentration of 1.0 mM. The result of FIG. 7 is shown by the relationship between the Phe density | concentration in a measuring solution, and the average (even or odd 2CH average) electric current value of a chip | tip.

[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 center of the opening;
A second electrode that is embedded in the first substrate, is at least partially exposed at the flow path opening, and is disposed independently for each flow path opening in a non-contact state with the first electrode. ,
A first wiring connected to the first electrode and connectable to the outside of the first member; and a second wiring connected to each of the second electrode and connectable to the outside of the first member, the opening center A first member having a first joint surface to be joined to the second joint surface of the second member, and a portion corresponding to the opening center portion of the first member Each having a through-hole for introducing a subject to be measured by the biosensor extending in the thickness direction and corresponding to the tip side from the second electrode of each flow path opening. And a second member having a through hole for ventilation extending in the thickness direction and having a second joint surface for joining with the first joint surface of the first member.

  FIG. 1a is a plan view of the first substrate 100 of the first member 10 as seen from the center of the opening and the surface having the channel opening extending radially from the opening center, and FIG. 1b is the first member. FIG. 10 is a perspective view of ten first substrates 100 as viewed from a surface having an opening center portion and a channel opening portion extending radially from the opening center portion. In FIG. 1b, the electrodes and wiring are depicted as perspective images. FIG. 1c is a perspective view in which only the opening center portion and the channel opening portion extending radially from the opening center portion are extracted from FIG. 1b.

  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 the number and shape of the first electrodes provided so as to expose the surface at the opening center portion 110, and the amount of the sample that can be used. When the planar dimension of the opening center part 110 is circular, it can be in a range of 1 to 10 mm, for example. 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. Similarly to the width, the depth of the channel opening 111 is suitably a size that allows the sample supplied to the center of the opening to easily flow into each channel due to capillary action. For example, the width is 0.1 It can be in the range of ~ 5mm. 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 and a second electrode are embedded in the first substrate.
The first electrode 120 is embedded in the first substrate 100, and at least a part of the first electrode 120 is exposed at the opening center portion 110. The first electrode 120 can be a single electrode, but can also be an electrode divided into a number equal to or less than the number of second electrodes. The divided first electrodes 120 are not in contact with each other. For example, a biosensor having first electrodes divided into a number equal to the number of second electrodes can independently perform electrochemical measurements at each second electrode. In the aspect shown in FIG. 1, since it has four flow-path openings and four second electrodes, four first electrodes 120A, 120B, 120C, and 120D are also arranged. The electrode area (exposed area) of each first electrode can be appropriately determined in consideration of the amount of sample that can be used, the accuracy required for measurement, the electrochemical measurement conditions, and the like. For example, it can be in the range of 1 to 50 mm ² . However, it is not intended to be limited to this range.

The second electrode 130 is embedded in the first substrate 100, and a part of the second electrode 130 is exposed at the channel opening 111, and is independent of each channel opening that is in non-contact with the first electrode 120. Arranged. In the embodiment shown in FIG. 1, since there are four flow path openings, four second electrodes 130A, 130B, 130C, and 130D are also disposed independently for each flow path opening. The At least part of the exposed surface of at least one flow path opening of the second electrode, the surface of each flow path opening on the tip side from the second electrode, that is, between the second electrode 130A and the third electrode 140A Between the flow path opening surfaces 114A, 130B and the third electrode 140B between the flow path opening surfaces 114B, 130C and the third electrode 140C. The flow path opening surface 114D in between, or the exposed surface and the tip-side surface are used to fix the biomaterial for the biosensor. In the embodiment shown in FIG. 1, for example, a biomaterial for a biosensor can be fixed to each of the four second electrodes 130A, 130B, 130C, and 130D. Alternatively, in the embodiment shown in FIG. 1, for example, a biological material for a biosensor may be fixed to each of the flow path opening surfaces 114A, 114B, 114C, and 114D between the four second electrodes and the third electrode. it can. 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 second electrode and the surface of the channel opening between each second electrode and the third electrode are required for the type and amount of the biological material to be fixed, the amount of the sample that can be used, and the measurement. It can be appropriately determined in consideration of the accuracy to be obtained, electrochemical measurement conditions, and the like. For example, it can be in the range of 1 to 50 mm ² . 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.

  A first wiring 121 that is connected to the first electrode 120 and is connectable to the outside of the first member 10 is provided. In the aspect shown in FIG. 1, since it has four 1st electrodes 120A-120D, it can have 1st wiring 121A-121D which can be connected to a 1st electrode independently and can be connected with the exterior of a 1st member. The second electrodes 130A to 130D also include second wirings 131A to 131D that are connected to the respective electrodes and connectable to the outside of the first member 10. The ends of the first wirings 121A to 121D and the second wirings 131A to 131D opposite to the electrodes can be aligned to form a terminal 160 for connection with a measuring instrument.

  The first member 10 is embedded in the first substrate 100, and at least part of the first member 10 is exposed at the front end side from the second electrode 130 of the flow path opening 111, and is not in contact with the first electrode 120 and the second electrode 130. The third electrode 140 can be included independently for each flow path 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. Similarly to the first wiring and the second wiring, the third wiring 141A, 141B, 141C, 141D can be arranged at the end opposite to the electrode to serve as a terminal 160 for connection with a measuring instrument.

  First electrode 120A, 120B, 120C, 120D, first wiring 121A, 121B, 121C, 121D connected to the first electrode, second electrode 130A, 130B, 130C, 130D, second wiring 131A connected to the second electrode, 131B, 131C, 131D, third electrodes 140A, 140B, 140C, 140D, and third wirings 141A, 141B, 141C, 141D connected to the third electrode are shown on the left in FIG. 1d. The lower side of the figure is the terminal 160 side for connection with the measuring instrument. In FIG. 1d, each electrode and the wiring are integrally formed. The first electrodes 120A, 120B, 120C, 120D, the second electrodes 130A, 130B, 130C, 130D, the third electrodes 140A, 140B, 140C, which are exposed at the opening center 110 and the channel opening 111 on the right side of FIG. 140D is shown. FIG. 1e shows a type of electrode and wiring in which the first electrode is not divided. The first electrode 120 and the first wiring 121 connected to the first electrode 120 are provided. The second electrode, the second wiring, the third electrode, and the third wiring are formed in the form shown in FIG. Although they are different, they are almost the same. Each electrode and wiring are integrally formed.

  Further, as will be described later, in the embodiment having the third electrode 140, as shown in FIG. 1f, the first member 10 and the second member 20 are arranged so that the first joint surface 150 and the second joint surface 220 face each other. When laminated and joined, the ventilation through holes 221A to 221D of the second member 20 are located at positions that overlap with the third electrodes 140A to 140D, respectively, or at the tip side from the third electrodes.

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 120 and the second electrode 130 are exposed only in the opening center part 110 and the flow path opening part 111, and there are no exposed parts of the electrodes in other parts. A wide surface for joining with the second joint surface of the second member is secured. 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 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 upper part of FIG. 2 a is a perspective view 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. The lower part of FIG. 2 a is a perspective view of the first member 10. 2b is a plan view of the second member 20 opposite to the second joint surface 220, the left middle portion is an AA sectional view of the upper left portion, and the lower left portion is a second joint surface 220 of the second member 20. FIG. FIG. 2b is a perspective view of the second member 20 on the second joint surface 220 side, and a lower right portion is a cross-sectional perspective view of the second member 20 as viewed from the opposite side of the second joint surface 220. FIG. FIG. 2C is a perspective view showing a state in which the second member 20 is joined to the first member 10, and the side opposite to the second joining surface 220 of the second member 20 is depicted. FIG. 2d is a front view (middle left), a side view (middle right), a plane (upper right), and a rear view (lower right) showing a state in which the second member 20 is joined to the first member 10.

  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. Ventilation through-holes 221A, which have through-holes 210 for introduction, and extend in the thickness direction in the corresponding portions on the distal end side of the second electrodes 130A-130D of the flow passage openings 111A-111D, respectively. Has 221D. 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. The second substrate 200 corresponds to the opening central portion 110 of the first member 10 and the flow passage openings 111A to 111D extending radially from the opening central portion 110, and these engaging protrusions 230 and 231A to 231D are respectively provided. Can have. Since the second member 20 has the protrusions 230 and 231A to 231D, 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 increased. However, even if the second member 20 does not have the protrusions 230 and 231A to 231D, a flow path can be formed and a desired bonding strength between the first member and the second member can be obtained. The protrusions 230 and 231A to 231D have a height lower than the depth of the opening center portion 110 and the flow passage openings 111A to 111D, and when the first member 10 and the second member 20 are joined, the opening center portion The protrusions 230 and 231A to 231D enter the vicinity of the opening upper end of the opening 110 and the flow path openings 111A to 111D, respectively, and the tip surfaces of the protrusions 230 and 231A to 231D are together with the opening center part 110 and the flow path openings 111A to 111D. A flow path is formed. 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 protrusions 231A to 231D.

  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.

  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 first member 10 is located in the opening center part 110, and the ventilation through holes 221A to 221D of the second member 20 are located on the front end side of the second electrodes 130A to 130D of the flow path opening parts, respectively. A space is formed between each of the ten channel openings 111A to 111D and the surface of the second member facing the channel openings. Since this space has ventilation through-holes 221A to 221D 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, and the second electrode 130A Reach ~ 130D.

  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 embodiment shown in FIG. 2, the first member 10 has a groove-shaped recess 180 in a part of the first joint surface 150, and the second member 20 is in the part of the second joint surface 220. A ridge-like convex portion 240 corresponding to the concave portion 180 of the surface 150 is provided.

  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, ethanol treatment, etc. in order to make it easier for the liquid that is the subject to flow through the space by capillary action. Furthermore, it is preferable that the inner surface of the through hole for introducing the analyte of the second member is similarly made hydrophilic by, for example, plasma treatment, ethanol treatment, or the like.

  FIG. 3 shows a first substrate 101 of the first member 11, which is another example of the first member of the biosensor assembly kit of the present invention shown in FIG. 3A, extending radially from the opening center and the opening center. The top view seen from the surface which has the existing flow-path opening part is shown. FIG. 3B shows a perspective view of the first substrate 101 of the first member 11 as seen from the surface having the opening central portion and the flow passage opening extending radially from the opening central portion. However, the first member 11 shown in FIG. 3B has stepped portions 190 that can engage with the protrusions 250 on the outer periphery of the second member 21 on the three sides of the outer periphery of the first joint surface 151. FIG. 3c is a perspective view of the second member 21, which is another example of the second member of the biosensor assembly kit of the present invention, and the first joint surface 151 of the first member 11 of the second member 21 The figure which drawn the 2nd joining surface 221 side for joining is shown. On the three sides of the outer periphery of the second member 21, there are protrusions 250 that can engage with the stepped portions 190 provided on the three sides of the outer periphery of the first member 11. FIG. 3d is a perspective view showing a state where the first member 11 and the second member 21 of the biosensor assembly kit of the present invention are assembled.

  Another example of the first member of the biosensor assembly kit of the present invention shown in FIG. 3 has an opening center 112 and a channel opening 113 extending radially from the opening center 112, and the channel opening. The section has eight of 113A to 113H. The eight channel openings 131A to 131H extend radially from the opening center 112. Similarly, the number of the first wiring, the second electrode, the second wiring, the third electrode, and the third wiring connected to the first electrode and the first electrode is eight. Further, the number of ventilation through holes provided in the second member 21 is also eight.

  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 instruments used for biochemical research, a part (reaction part) that is in direct contact with an object to be measured (subject) is required to have excellent biocompatibility, do not adsorb polypeptides or the like, and do not affect biological substances. 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 that have been laminated can be used to join the first joint surface 150 and the second joint surface 220 under a temperature condition that does not deactivate the biomaterial for the biosensor without using an adhesive. It is attached 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 is exposed only at the opening center portion 110 and the flow passage opening 111, the first electrode 120 and the second electrode 130 are exposed. Instead, the 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 a through-hole 210 and ventilation through-holes 221A to 221D, and a surface between the first member 10 and each flow passage opening. In other parts, the joint surface with the first joint surface 150 of the first member 10 is secured. 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 first member 10 includes the first electrode and the first wiring, the second electrode and the second wiring, and, if desired, the third electrode and the third wiring as described above, the resin composition, the electrode and the wiring. These materials can be used for injection molding. That is, the first member can be formed by insert molding at least the first electrode, the first wiring, the second electrode, and the second 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. The second member can also be produced by injection molding.

  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) At least a part of at least one exposed surface of the second electrode of the first member of the first member of the biosensor chip assembly kit of the present invention, the surface on the tip side of the second electrode of each channel opening, or the above Fixing a biological material for a biosensor to the exposed surface and the surface on the tip side;
(2) 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 second member is the second of each channel opening. A step of laminating the first bonding surface and the second bonding surface so as to be positioned on the tip side from the electrode, and bonding the bonding surfaces of the first member and the second member under a temperature condition in which the biological material is not deactivated.

  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, this enzyme solution is mixed one by one in order, or two or more enzyme solutions are mixed in an arbitrary volume at a time, and the upper surface of the second electrode (working electrode) or the flow channel section outside the second electrode. It is dropped by using a micropipette, a dispenser, or an ink jet spotter on the bottom surface, the top surface of the third electrode, etc., and dried 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 through hole of the second member is Then, the first bonding surface and the second bonding surface are laminated so as to be positioned on the tip side of the second 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 first member 10 is located in the opening center part 110, and the ventilation through holes 221A to 221D of the second member 20 are located on the front end side of the second electrodes 130A to 130D of the flow path opening parts, respectively. A space is formed between each of the ten channel openings 111A to 111D and the surface of the second member facing the channel openings. Since this space has ventilation through-holes 221A to 221D 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, and the second electrode 130A Reach ~ 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 bonding of the bonding surfaces, but is maintained at a temperature condition that does not inactivate the biosensor biomaterial fixed to the electrode, the channel opening surface, or both. Is preferred. 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.

<Example 1>
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. The masterbatch resin composition is put into a hopper of an injection molding machine, and after the electrodes are placed in the mold so that the electrodes are arranged on the first member, the first member and the second member are injected. Molded. 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. 4a shows a photograph of a state in which the first member and the second member obtained by injection molding are integrated. Further, FIG. 4b shows a state in which unnecessary parts are cut from the integrated product shown in FIG. 4a to form a first member (right side) and a second member (left side).

  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 on the second electrode of the first member were performed as follows. Phenylalanine dehydrogenase (hereinafter abbreviated as PheDH) 1-50 U is dissolved in 1 mL of Gly-KOH buffer (pH 9.5), and diaphorase 1-50 U is dissolved in 1 mL of phosphate buffer (pH 7.0). Dissolve in Next, 1 μL of each of the two enzyme solutions is mixed and added dropwise onto the second electrode of each channel using a micropipette, followed by natural drying, so that phenylalanine dehydrogenase is added to the second electrode of each channel. Immobilized to.

  The bonded surface of the second member was brought into contact with the bonded surface of the first member that was injection-molded to form a laminate, which was then stored in a dryer at 35 ° C. for 4 hours in a state of being tightened by a storage screw. FIG. 4c shows a photograph of the first member and the second member bonded together 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.

<Example 2>
Measurement of Phenylalanine (Phe) Concentration Using the same first member and second member as in Example 1, a chip in which PheDH was immobilized in each of the four channels (CH) was produced. However, PheDH was immobilized not on the second electrode but on the flow path surface between the second electrode and the third electrode. Specific enzyme immobilization conditions are as follows. 0.5 μl of mixed enzyme solution 25 U / ml of PheDH / Gly-KOH buffer (pH 9.5) + DI / PB (pH 7.0), second electrode (working electrode) and third electrode (sensing electrode) of each CH of the chip ) And air-dried for 1 hour to immobilize PheDH (Bacillus badius) and DI (diaphorase) in all 4CH. A 0 to 1 mM Phe / PBS solution was added from the injection port at the center of the chip, and CA measurement was performed under the following conditions to measure an increase in the oxidation current of the electron mediator (1-methoxy PMS) due to the Phe enzyme reaction.

<Voltage application conditions>
1st: 0 V, 5 sec (enzyme reaction)
2nd: 0.4 V, 5 sec (electrochemical measurement)
Inhalation (filling) voltage: 0.1 V
Inhalation (filling) current: 0.1μA
<Composition of measurement solution>
25 mM NAD + / Gly-KOH buffer (pH 9.5) (final 5 mM) 1 μL
0.5 M KCl / Gly-KOH buffer (pH 9.5) (final 0.1M) 1 μL
12.5 mM 1-methoxy PMS / Gly-KOH buffer (pH 9.5) (final 5 mM) 2 μL
5 mM Phe / PBS (pH7.4) (final 1 mM) 1 μL
Total volume 5μL

  The results are shown in FIGS. FIG. 5 shows the results of simultaneous measurement of current values using four CHs for each measurement solution with a Phe concentration of 0 mM, 0.1 mM, 0.5 mM, and 1.0 mM. FIG. 6 shows this result as a relationship between the Phe concentration in the measurement liquid and the average (4CH average) current value of the chip. It was possible to measure Phe with a variation coefficient (relative standard deviation) of about 5% with four CHs. From this result, it was possible to realize measurement of 0.1 to 1 mM Phe dissolved in PBS. (Phe in plasma is about 65 μM in normal persons, and phenylketonuria patients are 0.2 mM or more, and severely 0.6 mM or more.)

<Example 3>
Simultaneous measurement of phenylalanine (Phe) and tyrosine (Tyr) concentrations The same first member and second member as in Example 1 were used. However, the depth of the flow path was 0.2 mm (the depth of the flow path in Example 1 was 0.1 mm). Phe-selective Bacillus badius (Bb) PheDH for odd channels of each of the four channels (CH), Bacillus sphaericus (Bs) selective for Phe and Tyr for even-numbered channels PheDH was immobilized, and a chip on which PheDH was immobilized was produced. However, PheDH was immobilized not on the second electrode but on the flow path surface between the second electrode and the third electrode, as in Example 2. The voltage application conditions were the same as in Example 2.

<Composition of measurement solution>
25 mM NAD + 1 μL
0.5 M KCl 1 μL
12.5 mM 1-methoxy PMS 2 μL
+ 5 mM Phe or Tyr (or PBS) 1 μL
Total volume 5μL

  The results are shown in FIGS. FIG. 7 shows the results of simultaneous measurement of current values with four CHs for each measurement solution in which the Phe and Tyr concentrations were 0 mM, the Phe concentration was 1.0 mM, and the Tyr concentration was 1.0 mM. FIG. 8 shows the relationship between the Phe concentration in the measurement liquid and the average (average of even or odd 2CH) current values of the chip. Odd CH responded only to Phe. Even CH responded to both Phe and Tyr. That is, it was shown that only Phe can be measured in the odd-numbered channel in which PheDH derived from Bb is immobilized, and Phe and Tyr can be measured in the even-numbered channel in which PheDH derived from Bs is immobilized. From this result, it can be seen that two amino acids can be measured simultaneously.

  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.

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 electrode embedded in the first substrate and exposed at least partially at the opening center portion; A second electrode that is embedded in the first substrate, is at least partially exposed at the flow path opening, and is disposed independently for each flow path opening in a non-contact state with the first electrode. A first wiring connected to the first electrode and connectable to the outside of the first member; and a second wiring connected to each of the second electrode and connectable to the outside of the first member, the opening Corresponding to the first member having the first joint surface to be joined to the second joint surface of the second member, the first member having a surface surrounding the center portion and the flow path opening, and the opening center portion of the first member. The part has a through-hole that extends in the thickness direction to introduce the analyte to be measured by the biosensor. And the first joint surface of the first member comprising a second substrate having a through hole for ventilation extending in the thickness direction in each of the corresponding portions on the distal end side of the second electrode of each flow path opening. A biosensor chip assembly kit including a second member having a second joint surface to be joined with
At least a part of the exposed surface in the at least one flow path opening of the second electrode, the surface on the tip side of the second electrode of each flow path opening, or the exposed surface and the surface on the tip side are bio Used to immobilize biological materials for sensors,
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 second member is positioned on the tip side of the second electrode of each flow path opening, and each flow path opening of the first member and each flow of the second member A space is formed between the road opening and the opposite surface,
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 first member and the second member that are stacked are assembled by attaching the joint surface to a joint under a temperature condition that does not deactivate the biomaterial for the biosensor without using an adhesive.
The biosensor chip assembly kit. The first electrode is divided into a number corresponding to the number of second electrodes, and is connected to the first electrode independently and has a first wiring connectable to the outside of the first member,
The biosensor chip assembly kit according to claim 1. The first member is embedded in the first substrate, a part of the first member is exposed on the front end side of the second electrode of the flow path opening, and is in a non-contact state with the first electrode and the second electrode. A third electrode disposed independently for each flow path opening, 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 stacked and bonded so that the first bonding surface and the second bonding surface face each other, each ventilation through hole of the second member overlaps with each third electrode or each The biosensor chip assembling kit according to claim 1, wherein the kit is located on a tip side of the third electrode. The biosensor chip assembly kit according to claim 1, wherein at least the first member is formed by insert molding the first electrode, the first wiring, the second electrode, and the second wiring by injection molding. 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. The at least one exposed surface of at least one second electrode of the first member of the first member of the biosensor chip assembling kit according to claim 1, the surface on the tip side of the second electrode of each channel opening, or the Fix biomaterial for biosensor on the exposed surface and the surface on the tip side,
The through hole for introducing the subject of the second member is positioned at the opening center of the first member, and each through hole for ventilation of the second member is distal to the second electrode of each channel opening A biosensor comprising: laminating a first joint surface and a second joint surface so as to be positioned on a side; and joining the joint surfaces of the first member and the second member under a temperature condition that does not deactivate the biological material. Chip manufacturing method. The biosensor chip assembly kit according to claim 3, wherein at least a part of at least one exposed surface of the second electrode of the first member of the first member, a surface on the tip side of the second electrode of each channel opening, or the Fix biomaterial for biosensor on the exposed surface and the surface on the tip side,
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 through hole of the second member overlaps with each third electrode or each third Including laminating the first joint surface and the second joint surface so as to be positioned on the tip side from the electrode, and joining the joint surfaces of the first member and the second member under a temperature condition in which the biological material is not deactivated. The manufacturing method of a biosensor chip. The production method according to claim 7 or 8, 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 electrode embedded in the first substrate and exposed at least partially at the opening center portion; A second electrode that is embedded in the first substrate, is at least partially exposed at the flow path opening, and is disposed independently for each flow path opening in a non-contact state with the first electrode. A first wiring connected to the first electrode and connectable to the outside of the first member; and a second wiring connected to each of the second electrode and connectable to the outside of the first member, the opening The first member having the first joint surface to be joined to the second joint surface of the second member, and the portion corresponding to the opening center portion of the first member, comprising the center and the surface surrounding the channel opening Has a through-hole for introducing an analyte to be measured by the biosensor that extends in the thickness direction. In addition, each of the corresponding portions on the distal end side from the second electrode of each flow path opening has a ventilation through hole extending in the thickness direction and is joined to the first joint surface of the first member. A second member having a second joining surface of
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),
At least a part of the exposed surface in the at least one flow path opening of the second electrode, the surface on the tip side of the second electrode of each flow path opening, or the exposed surface and the surface on the tip side are bio Biological material for sensor 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 second member is located on the tip side from the second electrode of each channel opening, and for each channel opening of the first member and each channel of the second member The bonding surface is bonded without using an adhesive so that a space is formed between the opening and the surface facing the opening.
Biosensor chip. The first electrode is divided into a number corresponding to the number of second electrodes, and is connected to the first electrode independently and has a first wiring connectable to the outside of the first member,
The biosensor chip according to claim 11. The first member is embedded in the first substrate, a part of the first member is exposed on the front end side of the second electrode of the flow path opening, and is in a non-contact state with the first electrode and the second electrode. A third electrode disposed independently for each flow path opening, 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 so that each ventilation through hole of the second member overlaps with each third electrode or is located on the tip side from each third electrode. The biosensor chip according to 1. 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.