JPH09166571A - Biosensor and manufacture thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To make a biosensor easily measurable to a small quantity of sample by forming such structure that an electrode formed on one face of an insulating base plate and a connecting terminal formed on the other face are made conductive in the front and rear of the plate through one through hole or more provided in the plate, and separating the electrode and the connecting terminal from each other. SOLUTION: An electrode 22 provided with a reaction layer 3 containing organismic substance such as enzyme becomes a working electrode. The electrode 22 has electrode shape equal to the hole shape of an opening part of a through hole 7, and is made conductive in the front and rear of an insulating base plate 1a through the through hole 7 and conductive to a connecting terminal 231 for the working electrode through a lead 21 on the outer surface side of the plate 1a. A counter electrode provided on the same face side as the reaction layer 3 is composed of two pairs of electrodes 222a, 222b, and formed in such a way as to hold the working electrode 221 from both sides. Two pairs of electrodes 222a, 222b for the counter electrode are respectively formed on one ends of leads 21a, 21b. The other ends of these leads are connected to form a connecting terminal for the counter electrode, and an exposure unnecessary part is covered with an insulating layer 5.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to, for example, glucose in blood and specific components in a liquid sample quickly and easily,
The present invention also relates to a biosensor that can be accurately quantified, and more specifically to an easy-to-use and inexpensive biosensor electrode structure that can be easily measured with a small amount of sample, and a manufacturing method thereof.

[0002]

2. Description of the Related Art Conventionally, there have been known biosensors for measuring the abundance of various substances to be measured by utilizing the high molecular recognition ability of bio-related substances such as enzymes in the quantification of specific components in a sample solution. For example, a glucose sensor for quantifying glucose has been put into practical use as an enzyme sensor using an enzyme as a biologically relevant substance. The enzyme sensor
An enzyme having a high substrate specificity for a substance to be measured is immobilized on a substrate such as a polymer membrane, the substance to be measured in a sample is brought into contact with the enzyme, and a substance produced by an enzymatic reaction is electrochemically detected, It can be quantified.

Biosensors are used in various fields, and, for example, in the case of the above-mentioned glucose sensor, it has been commercialized for controlling the blood glucose level of diabetic patients and process control of food processing.
In addition, a biosensor that uses microorganisms (microorganism sensor)
Has been put to practical use, and is used for river water quality analysis and safety management of factory wastewater. In this way, biosensors can be used for medical treatment in the determination of specific components in sample liquids.
Widely used in food analysis, fermentation control, environmental measurement, etc. The measurement at the beginning of practical use had a problem that the device was large and time-consuming and costly, but it is often used in facilities such as hospitals and factories, and its excellent molecular recognition ability makes it unacceptable. There wasn't. However, especially when an individual measures certain components in blood or urine to check their health, illness, or effectiveness of treatment,
Due to the running cost and the difficult operation method, a heavy burden is placed on the user, and an inexpensive sensor that can perform simple on-site measurement has been desired.

For example, a biosensor that can be used as a glucose sensor or the like proposed in Japanese Examined Patent Publication No. 6-76984 is one that enables measurement in a short time by simply dropping a small amount of sample liquid from above. In the same publication, a platinum electrode embedded in a resin-made cylindrical base material is exposed to the electrode portion from the upper and bottom surfaces thereof, a liquid-retaining layer of rayon paper, a filtration layer of a porous membrane, and an enzyme in an annular frame. There has been proposed a biosensor having a structure in which a measuring chip having a reaction layer of a non-woven fabric impregnated with it is installed. However, the structure is complicated, there are many manufacturing processes and components, and it is inevitable that the cost will increase no matter how the device is devised. In addition, Japanese Examined Patent Publication 6-58338
The publication proposes a biosensor that can be used as a glucose sensor or the like, which is of a disposable type.
As shown in FIGS. 6 to 8, this biosensor has a structure in which one end of a lead printed and formed on a resin sheet 91a is used as a connection terminal 923, and the other end of the lead serving as an electrode is used. After printing the electrode material on the top,
An insulating layer 94 is printed so as to expose the connection terminal 923 and the electrodes 922 and 922a to cover the remaining leads, and the reaction layer 9 in which an enzyme is fixed on the electrodes 922 and 922a.
5 is formed as a lower substrate, and a sheet 91b serving as a cover is laminated on the lower substrate through a spacer sheet 97 having a space 96 around the reaction layer 95, and the sample solution is introduced from the inlet port 98 at the tip to a capillary tube. Due to the phenomenon, the space enters the space, and the gas in the space is pushed out from the discharge port 99 by the introduction of the sample solution, so that a small amount of the sample solution can be smoothly exchanged with the gas in the space. It was done.

[0005]

However, although the biosensor as proposed in Japanese Patent Publication No. 6-58338 is disposable and facilitates individual use, it is a skilled and exclusive subject. Unlike the case where an analyst uses it, when it is used by an ordinary individual, it is not always easy to use. It has multiple connection terminals
It is provided adjacent to the same surface of the substrate sheet,
This is because if the sample liquid or the like to be measured by mistake is adhered to another place of the sensor and it wets the connection terminal portion, the adjacent connection terminals may be electrically connected to each other. Further, in addition to the two sheets, a spacer sheet cut into a predetermined shape to form a space is required as a component, and there is room for improvement in the structure, and it is possible to avoid an increase in cost. There wasn't.

[0006]

Therefore, in the biosensor of the present invention, a reaction facing at least an insulating substrate, an electrode system provided on the substrate, and a space formed between two insulating substrates. In a biosensor having a layer, an electrode formed on one surface of an insulating substrate and its connection terminal formed on the other surface are provided on the insulating substrate. 1
As a structure that conducts on the front and back through the above through holes,
The electrode and its connecting terminal were separated on both sides of the insulating substrate. Further, in the second embodiment of the present invention, as the conduction means of the through hole, the through hole has a structure in which one or both of the electrode forming substance or the lead forming substance forming the electrode system is provided inside the through hole to conduct the front and back. went.

Further, in the third mode, in the second mode,
The electrode forming substance inside the through hole is exposed from at least the entire area inside the hole diameter of the through hole on the electrode forming surface side, and the electrode having the opening shape is formed by using the through hole. The shaped electrode was used as a working electrode having a reaction layer formed on the electrode, and the working electrode and its connection terminal were connected on the front and back sides. In the fifth mode, in the fourth mode, the working electrode and the counter electrode are connected to the connection terminal on the front and back sides, and the working electrode and the counter electrode are formed on one surface of one insulating substrate, and the counter electrode functions as the working electrode. It consists of two pairs of electrodes sandwiched from both sides, and the connection terminals for the counter electrode are formed on the same surface side as these electrodes, and the connection terminals for the working electrode and the counter electrode are separated on the front and back of one insulating substrate. It was

The sixth embodiment of the biosensor of the present invention has a structure in which the electrodes connected to the connection terminals on the front and back sides through the through holes have a counter electrode structure, and the seventh embodiment is the sixth embodiment. Conduction between the front surface and the back surface of the through hole was performed by the lead forming substance contained therein. In the eighth embodiment, the working electrode according to the fourth embodiment and the counter electrode according to the sixth or seventh embodiment are provided, and two pairs of the counter electrodes are formed so as to sandwich the working electrode from both sides. Electrodes, which are a working electrode and a counter electrode, and these connection terminals are separated on the front and back sides of a single insulating substrate. Further, in the ninth mode, in the above various modes, a space where the two insulating substrates to be used are bonded and laminated by a spacer formed by printing or coating on at least one of the insulating substrates to face the reaction layer. Secured a section.

Further, the method for manufacturing a biosensor according to the present invention includes at least an insulating substrate, an electrode system provided on the substrate, and a reaction layer facing a space formed between two insulating substrates. And a connection terminal formed on the other surface of the insulating substrate, wherein the electrode is formed on one surface of the insulating substrate and at least one through hole is formed on the insulating substrate. In order to manufacture a biosensor that is electrically connected on the front and back sides via, an insulating substrate having one or more through holes is prepared, and a back film is attached to one surface of the insulating substrate to cover the through holes. After printing a predetermined conductor pattern with a conductive paste for electrode formation so as to fill the through hole from the other surface, the back film is peeled off, and the electrode forming substance inside the through hole is at least the electrode forming surface side. From the entire area inside the hole diameter of the through hole of Out, together with an electrode in which the opening shape and a connection terminal electrode and the other surface of the one surface of the insulating substrate, and a manufacturing method for conducting the front and back. In contrast to the first manufacturing method of the first embodiment, in the second embodiment, a reaction layer is formed on an electrode having an opening shape of a through hole to serve as a working electrode, and the working electrode is made of an insulating substrate. It was formed as an electrode that is electrically connected to the connection terminals on the other surface on the front and back sides.

A third mode of the present manufacturing method is a biosensor having at least an insulating substrate, an electrode system provided on the substrate, and a reaction layer, which is formed on one surface of the insulating substrate. 1 or more in order to manufacture a biosensor in which the formed electrode and the connection terminal formed on the other surface are electrically connected to each other through one or more through holes provided in the insulating substrate. After preparing an insulating substrate having a through hole, the back film is attached to one surface of the insulating substrate to cover the through hole, and the conductive material for lead formation is formed so as to enter the inside of the through hole from the other surface. After printing a predetermined lead pattern with a conductive paste, peel off the back film and then lead again from the exposed side of the insulating substrate so that it will be connected to the conductive paste already inside the through hole. With conductive paste for formation By printing a lead pattern of the through holes made conductive by the read-forming material on the front and back, and a manufacturing method of forming a lead to conduct at front and back.

The fourth mode of the present manufacturing method is the third mode of the above manufacturing method, in which the counter electrode is provided on the lead which is electrically connected to the connection terminal on the other surface of the insulating substrate.
Further, according to a fifth mode of the present manufacturing method, a working electrode is formed on one surface of one insulating substrate by the second mode of the manufacturing method, and two pairs of counter electrodes sandwich the working electrode from both sides. Similarly, the connection terminal for the counter electrode was formed on the same surface side as the counter electrode, and the connection terminals for the working electrode and the counter electrode were separately formed on the front and back surfaces of one insulating substrate. Then, in the sixth mode of the present manufacturing method, a working electrode is formed on one surface of one insulating substrate by the second mode of the manufacturing method, and two pairs of counter electrodes sandwich the working electrode from both sides. As described above, according to the fourth mode of the manufacturing method, the electrodes of the working electrode and the counter electrode, and the connection terminals thereof are separately formed on the front and back of one insulating substrate. Further, a seventh mode of the present manufacturing method is the various modes of the above manufacturing method, in which a spacer is formed on at least one of the insulating substrates by printing or coating, and two insulating substrates are bonded and laminated by the spacer. Then, the space portion facing the reaction layer was secured.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the biosensor of the present invention and a method for manufacturing the same will be described below with reference to the drawings. 1 and 2 are views showing an embodiment of the biosensor of the present invention. FIG. 1 (a) is a plan view seen from above, and FIG. 1 (b) is a line AA of FIG. 1 (a). 1A is a sectional view taken along line BB in FIG. 1A.
1 (d) is a plan view seen from below, FIG. 1 (e) is a plan view seen from above a lower substrate having a working electrode, and FIG. 2 is a bottom view utilizing conduction through holes in a working electrode. It is a perspective view which shows the conductor pattern about the board in three dimensions.

The biosensor 10 shown in FIGS. 1 and 2.
Is a structure in which two insulating substrates 1a and 1b are adhered and laminated so as to leave a space 6 facing the reaction layer 3 via a spacer 4, and the insulating property of the lower side in a state before lamination. Board 1a
In addition, all the electrode systems, the reaction layer and the spacers are formed by printing or coating, and the upper insulating substrate 1b as a cover sheet is adhered and laminated by the spacers. The electrode system formed on the lower insulating substrate 1a is composed of leads, electrodes, connection terminals, and an insulating layer. It should be noted that the insulating layer is shown only in FIG. 1 (e) for the sake of explanation because the drawing becomes complicated. The electrode 22 provided with the reaction layer 3 containing a biological substance such as an enzyme serves as a working electrode 221, and the electrode has an electrode shape equal to the hole shape of the opening of the through hole 7 and is insulated via the through hole. The conductive substrate is electrically connected to the front and back, and is electrically connected to the working electrode connection terminal 231 via the lead 21 on the outer surface side of the substrate. On the other hand, the counter electrode provided on the same side as the reaction layer has two pairs of linear electrodes 222a.
And 222b, and is formed so as to sandwich the working electrode 221 from both sides (away from the covering reaction layer 3). These two pairs of electrodes for the counter electrodes are leads 21a and 21b, respectively.
Is formed on one end of each of the leads and the other ends of these leads are connected to each other to form (also serve as) a connection terminal 232 for the counter electrode, and the unnecessary portion is covered with the insulating layer 5. As a result, the working electrode and the counter electrode are provided on the inner surface side of one insulating substrate, and only the connecting terminals of the working electrode are provided on the outer surface side of the substrate. The biosensor has a structure provided on both sides of.

Further, the spacer 4 is formed on the lower insulating substrate as a linear pattern on both sides so as to leave the space 6, the inlet 61 and the outlet 62 for bringing the reaction layer into contact with the sample solution. It is used. As a result, the introduction port 61 for introducing the sample liquid into the side end surface portion on the side where the connection terminal is not provided.
Has a structure in which both sides are sandwiched between the spacers and opened, and a discharge port 62 for discharging the gas in the space is opened in the side end surface portion of the upper insulating substrate on the connection terminal side.

As a result of the above, even if the sample solution that has reached the discharge port overflows and the connection terminals are inadvertently wetted, there is only one connection terminal and both connection terminals are formed separately on both sides of the substrate. Therefore, it is possible to avoid electrical continuity between both connection terminals.

As described above, the biosensor of the present invention is usually composed of two insulating substrates, and at least one electrode has its electrode forming surface and connection terminal forming surface formed on both sides of the substrate by a through hole. There is a basic feature in the structure provided separately. Then, by this basic structure, the electrode forming surface side on the side where the sample liquid contacts, and the connection terminal side for conduction with the measuring device are separately formed on the front and back of one substrate,
Even if the connection terminals are inadvertently wetted by the sample liquid or the like, the connection terminals can be prevented from conducting. In the conventional biosensor, two insulating substrates are used to form a working electrode and a counter electrode on the inner surface side of one substrate, and all electrode systems for both working electrode and counter electrode connection terminals. When the terminal portion gets wet, the connecting terminals are likely to be electrically connected to each other. However, if the connection terminals of at least one of the electrodes are formed on the other surface as in the present invention, both connection terminals are separated on both surfaces, and even if they get wet, conduction between both connection terminals can be prevented. As a result, even if the undesired portion gets wet with liquid, the conduction at the connection terminal portion can be prevented, and the advantage that it is easy to use even for an unfamiliar person can be obtained. Further, if all electrodes and all connection terminals formed on one insulating substrate are separately formed on both sides, the electrode side surface that is also in contact with the sample solution,
Since the side surface of the connection terminal is different, liquid wetting can be prevented. Furthermore, the above is not limited to the case where all the electrode systems are formed on one insulating substrate. Opposite 2
Even when the electrode system is separately provided on both of the insulating substrates, the connection terminal on each substrate is on the back surface side with respect to the electrode side in contact with the sample solution, and the same thing can be said about the relationship between the electrode and the connection terminal. I can say.

In the biosensor of the present invention, the through-holes described above may be skillfully utilized to form the through-holes into electrode shapes. Conventionally, even if an electrode is formed of, for example, carbon paste or the like, a method of covering an unnecessary portion of the electrode with an insulating layer and controlling the electrode shape by forming the insulating layer may be adopted in order to keep the area constant. . However, if the through hole is used, the shape of the electrode can be controlled by the size of the through hole, and the insulating layer can serve the purpose of insulating the lead portion. Therefore, this structure is suitable for an electrode that is covered with a reaction layer and requires area accuracy.

Materials such as the insulating substrate, the electrode system, the reaction layer, and the spacers and their formation may be appropriately selected from materials and methods known in the art according to their applications. For example, a resin sheet made of polyethylene terephthalate (hereinafter PET) or the like is used for the insulating substrate. In the electrode system, the leads and the connection terminals may be conductive paste containing a metal such as silver or gold, the electrodes may be carbon paste, and the insulating layer may be an insulating paste, which may be formed by screen printing. If the enzyme sensor is used as a glucose sensor, the reaction layer may be a layer on which glucose oxidase is immobilized, and the reaction layer is formed by screen-printing an enzyme-containing ink or applying a coating liquid with a dispenser. In addition to this, there may be a layer for controlling the permeation of the sample liquid sample, for example.

Next, regarding the front and back conduction means by the through holes, the means is not particularly limited as long as electrical connection can be obtained, but the above-mentioned constituent materials are used as an electrode forming substance using a carbon paste or a silver paste. It is convenient to use a lead forming material such as gold or gold paste so that it exists inside and conducts electricity on the front and back sides, and at the same time conduction on the through hole can be achieved at the same time when the electrodes and leads are formed. In particular, if the electrode forming material is filled inside and the through hole is seen from the electrode surface side and exposed in the entire area inside the hole diameter, it will be an electrode having an area corresponding to the hole diameter of the through hole. You can This simplest case is a case where the electrode forming substance inside the through hole is exposed from at least the opening of the through hole on the electrode forming surface side from the entire area inside the hole diameter in a plane continuous to the substrate surface. . Therefore, it is not always necessary that the electrode forming substance completely fills the inside of the through hole. Further, the leads may be electrically connected to the front and back sides through the through holes. In this case, if the lead forming material is used as the penetrating means, it is convenient to conduct the leads to the front and back sides at the same time. Further, it is not always necessary to completely fill the inside of the through hole with the conductive material as the conducting means of the through hole even for the conduction of the lead portion. Further, the number of through holes forming one front and back conduction path or the electrode is not limited to one, and may be a plurality of through holes, for example, a group of through holes formed in a certain portion.

To form a continuous electrode exposed portion on the substrate surface, a back film may be temporarily attached, an electrode forming substance may be embedded from the back surface side, and then the back film may be peeled off. As the back film, considering the heat resistance when the electrodes and leads are screen-printed and dried and baked (thermosetting, etc.), the same quality as the insulating substrate used, for example, a PET film heat-resistant acrylic adhesive Those coated with (for example, ultraviolet curing type) can be used. The back film can be peeled off after firing if it can withstand firing,
If the printed matter does not collapse, peeling after drying may be used. In addition, especially when printing from the front and back sides at the lead part to connect the front and back sides, in the relationship between the through hole pore diameter and the paste viscosity, the undried paste after printing does not flow out from the through holes and wet the back surface, the back film is It can be omitted.

Next, in the present invention, PET is used as a spacer.
A sheet or the like may be used, and the insulating substrates may be bonded and fixed to each other with an adhesive through the sheet, but at least one of the insulating substrates is required in that a sheet having a predetermined shape is required to form a space. It is advantageous to use the one formed by printing or coating (in a pattern that forms a space portion) in terms of reducing the number of parts and cost. The application is preferably a partial application. However, even the entire surface coating can be used in combination with printing or partial coating. That is, one of the two insulating substrates may be a cover sheet without providing an electrode system or the like, and a heat-sensitive adhesive or the like may be applied to the entire surface of the cover sheet, while the other substrate may be sufficient to form a space. It can be used in a form in which the convex portions are formed by printing, and these convex portions are adhered and laminated.

The spacer forming means for printing or partial application may be appropriately selected depending on the material used. Screen printing is preferable because it can be formed thick, but a material that is not suitable for printing is partially applied by an applicator such as hot melt or a plate shape for application. Manufacturing is usually carried out in multiple faces, but if continuous stripes that are connected to adjacent sensors or intermittent stripes that are discontinuous between adjacent sensors are used, it is relatively easy to apply and form materials that are not suitable for printing. . As the spacer material by printing or partial application, for example, a hot-melt adhesive such as a heat-activatable thermosetting adhesive, or a hot-melt adhesive such as a hot-melt adhesive such as a thermoactive thermosetting adhesive. Can be used, and as these resin components, for example, acrylic resins, silicone elastomers, etc. are used. These can also be applied to the above-mentioned entire surface coating. In addition, additives such as fillers are added as appropriate to improve printability and coating suitability. Then, two insulating substrates are laminated and heat and, if necessary, an appropriate pressure are applied, the formed spacers are activated by heat, and both insulating substrates are bonded and fixed.
In addition, with a single printing or partial application of the adhesive or the adhesive, a desired space can be provided between the insulating substrates (a space capable of accommodating the thickness of the electrode system in the space and allowing the sample solution to reach and contact the reaction layer). If it is not possible to form a film having a thickness of about 50 to 300 μm), printing or partial application may be repeated a plurality of times. In this case, the layers having the thickness function may be sandwiched between the layers having the thickness function, instead of the same material. The layer that is responsible for the thickness function does not need the function of developing an adhesive force by heat, and may be a screen printing ink or the like capable of forming a thick layer.
An insulating paste or the like that can be thickly applied may be used. When the spacer is formed after the reaction layer is formed, it may be covered with a protective layer so that the reaction layer can be protected even if a measurement-interfering substance comes out of the spacer. For example, in the case of a reaction layer containing glucose oxidase, an ink or coating liquid containing phospholipid is printed or applied.

Next, one embodiment of the biosensor manufacturing method of the present invention will be described with reference to FIG. This figure is an example of manufacturing a sensor in which the working electrode and the connection terminal thereof shown in FIGS. 1 and 2 are separately formed on the front and back sides. First, FIG. 3 (a) [hereinafter,
[FIG. 3 is omitted], as shown in FIG.
Drilling. Next, the back film 8 is attached to the inner surface when the sensor is assembled (the other side in the drawing).
(B) Next, an electrode forming material is printed from the outer side (front side of the drawing) so as to fill the through holes 7, and the electrode 22 for the working electrode is then printed.
And the back film is peeled off after printing (c). Further, the lead 21 which also serves as the connection terminal 231 for the working electrode is formed (d). Then, the insulating substrate is turned upside down and the lead 21 which also serves as the connection terminal 232 for the counter electrode is provided on the inner surface.
Form a and 21b (e). In addition, in (e), it can be seen that the electrode 22 for the working electrode has the shape of the through hole 7 and is exposed from the portion where the through hole was formed. Next, electrodes 222a and 222b for counter electrodes are formed (f), and then the insulating layer 5 is formed to form an electrode system as a final counter electrode (exposed) shape (g). Next, the reaction layer 3 is formed on the electrode 22 for the working electrode formed in the through hole portion (h), and the spacer 4 is formed (i). Then, an insulating substrate 1c having a cutout portion 11 is prepared as a cover sheet (j), which are superposed on each other and heat pressure is applied to the both substrates by a spacer.
Fix and obtain biosensor (k). Notch 1
The shape of 1 may be intentionally made asymmetric, and it may be used as one of measures for preventing erroneous connection (working electrode and counter electrode) when the sensor is set in the measuring device. It is also possible to visually recognize the structurally different shape on the measuring device side.

Next, as another embodiment of the biosensor of the present invention, FIG. 4 shows a sensor having a structure in which the counter electrode and the connection terminal are separately formed on the front and back sides. 4A is an inner side surface of the insulating substrate, FIG. 4B is an outer side surface thereof, and FIG. 4C is a cross-sectional view taken along line AA and line BB in FIG. 4A. Indicates. The counter electrode is electrically connected on the front and back sides by a through hole in the middle of the lead, and the lead is provided on both sides. The counter electrode 222a is the lead 21e,
It conducts to the connection terminal 232a through the through hole 7b and the lead 21c. The counter electrode 222b includes the lead 21f, the through hole 7c,
It conducts to the connection terminal 232b through the lead 21d. In this way, the working electrode and the counter electrode can be provided on the inner surface side of the insulating substrate, and their connecting terminals can all be provided on the outer surface side. Even if the sample liquid overflows from the outlet, the connection terminals are on the opposite back surface side, so that the conduction between the connection terminals can be effectively prevented. Further, in FIG. 4, front and back conduction by the through hole is performed in the lead portion. In addition,
Since the drawing of the insulating layer is complicated, B- in FIG.
It is shown only in the sectional view taken along line B.

FIG. 5 is an explanatory view of a manufacturing process showing one embodiment of a method of manufacturing a sensor having a structure in which the counter electrode is also formed so as to be separated from the front and back sides of the connection terminal as in FIG. However, in FIG. 5, the counter electrode shape is such that the circumference of the circular working electrode and the reaction layer are surrounded by a certain distance in a ring shape with a certain distance. In the process explanatory view of FIG.
First, as shown in FIG. 5A (hereinafter, “FIG. 5” is omitted), after the through holes 7a, 7b and 7c are formed in one insulating substrate,
The back film 8 is attached to the inner surface (the other side in the drawing on the drawing) (b), the electrode 22 for the working electrode is formed so as to fill the through hole 7a (c), and the connection terminal 231 for the working electrode is further formed.
Lead 21c that also serves as the counter terminal 232a and lead 21c that also serves as the counter terminal 232a, and counter terminal 232b that also serves as the counter electrode
The lead 21d which also serves as the lead is formed, and the back film is peeled off (d). Next, the insulating substrate is turned upside down, and leads 21e and 21f extending to the working electrode are formed on the inner surface by connecting to the through holes 7b and 7c, respectively (e). Next, after the electrodes 222c and 222d for the counter electrode are formed (f), the insulating layer 5 is formed to form an electrode system (g).
Next, after forming the reaction layer 3 on the electrode 22 for the working electrode (h), the spacer 4 is formed (i). Then, an insulating substrate 1d to be a cover sheet is prepared (j), and the substrates are stacked and heat pressure is applied to bond and fix both substrates with a spacer to obtain a biosensor (k).

[0026]

EXAMPLES Next, the present invention will be explained with reference to examples. This example is for manufacturing a biosensor having the structure shown in FIGS. 1 and 2, and a structure in which a working electrode and its connecting terminals are separately provided on both sides of an insulating substrate can be obtained. First, as an insulating substrate, a 250 μm-thick PET sheet having one through hole with a diameter of 1 mm for use as a working electrode is prepared. Next, an ultraviolet-curing acrylic adhesive was applied to one side of the inner surface of the biosensor during assembly with the electrodes inside 25
A 0 μm thick PET sheet is attached as a back film. Then, after printing the carbon paste by screen printing so that the inside of the through hole is made slightly larger than the through hole at the time of assembly to completely fill the inside, after drying and peeling the back film (the adhesive is attached to the back film Peeling) and firing to form a working electrode. Next, a silver paste (on the outer surface side) is screen-printed and then fired to form leads and connection terminals for the working electrode. Similarly, silver paste is also screen-printed on the inner surface side and then fired to form a counter electrode lead and connection terminal, and then carbon paste is screen-printed and fired to form a counter electrode. Further, an insulating paste is screen-printed on the inner surface side and then baked to form an insulating layer, and an electrode system serving as a working electrode and a counter electrode is formed on one insulating substrate. Then, a mixed ink of glucose oxidase and ferrocenecarboxylic acid is screen-printed on the electrode portion exposed from the through hole and then dried to form a reaction layer. Further, an ink composed of a heat-activatable thermosetting adhesive is screen-printed and dried to form spacers. Next, another insulating substrate that serves as a cover sheet having a cutout portion in the connection terminal portion is overlaid, and a biosensor is obtained by bonding and stacking and fixing with a spacer by heat and pressure.

[0027]

Industrial Applicability According to the present invention, a disposable type biosensor can be easily measured with a small amount of sample, easy to use and inexpensive. Electrodes and their connecting terminals can be separately formed on the front and back of the board by front and back conduction with through holes,
This prevents the connection terminal from getting wet accidentally, and makes it a sensor that is easy to use. No special material is required for the conduction in the through hole as long as it is used as a material for forming electrodes and leads. Further, if the through-hole is filled with an electrode forming substance and exposed from the opening, the through-hole shape provides an electrode having a high area accuracy, which can be applied to a working electrode that does not require area control by an insulating layer. If the connecting terminal of the working electrode is formed on the back surface side of the insulating substrate and the counter electrode and the connecting terminal thereof are formed on the working electrode side, the connecting terminals of both electrodes can be formed separately on both surfaces, even if the electrode surface side Even if the connection terminal gets wet with the sample liquid overflowing from the discharge port, it is possible to prevent conduction between both connection terminals. Also, with respect to the counter electrode, it is possible to prevent the connection terminal of the counter electrode from getting wet by conducting the front and back sides of the lead portion through the through holes and forming the connection terminal of the counter electrode on the back surface side. Furthermore, if the spacers are printed or partially applied spacers, a spacer sheet is not required and the number of constituent parts can be reduced, so that an inexpensive sensor can be obtained. Further, according to the manufacturing method of the present invention, the biosensor can be efficiently manufactured,
In particular, an electrode having a continuous smooth surface can be formed on the surface of the insulating substrate by the electrode-forming substance placed inside the through hole, and an electrode suitable for a working electrode that requires electrode area accuracy can be obtained.

[Brief description of the drawings]

FIG. 1 is an explanatory view showing a structure of an embodiment of a biosensor of the present invention (front and back connection of a working electrode with its connection terminal through a through hole). (A) is a plan view seen from above, (b) is a cross-sectional view taken along line AA of (a), (c) is a cross-sectional view taken along line BB of (a), and (d) is downward. And (e) is a plan view of the lower substrate having a working electrode as viewed from above.

FIG. 2 is a perspective view three-dimensionally showing a conductor pattern of a lower substrate using conduction through holes for working electrodes.

FIG. 3 is a process explanatory view according to an embodiment of a biosensor manufacturing method of the present invention. (A) Perforation of through-holes, (b) Adhesion of back film, (c) Filling of through-holes with electrode material and peeling of back film, (d) Lead formation of working electrode, (e) Lead formation of counter electrode, ( f) counter electrode formation, (g) insulating layer formation,
(H) Reaction layer formation, (i) Spacer formation, (j) Upper insulating substrate prepared, (k) Stacking and bonding of upper and lower substrates.

FIG. 4 is an explanatory view showing the structure of another embodiment of the biosensor of the present invention (the working electrode and the counter electrode and their connecting terminals are all connected to each other by through holes on the front and back sides).

FIG. 5 is a process explanatory diagram for manufacturing a front-back connected biosensor similar to FIG. 4 as another embodiment of the manufacturing method of the present invention.
(A) Perforation of through-holes, (b) Adhesion of back film, (c)
(D) Filling the through hole for the working electrode with the electrode material, (d) filling the through hole for the counter electrode with the lead material, forming the outer surface side lead for the counter electrode, forming the working electrode lead and peeling the back film, (e) the inner surface side lead of the counter electrode Formation, (f) counter electrode formation, (g) insulation layer formation, (h) reaction layer formation, (i) spacer formation, (j)
Prepare the upper insulating substrate, (k) stack and bond the upper and lower substrates.

FIG. 6 is an exploded perspective view showing an example of the structure of a conventional biosensor.

FIG. 7 is an external view of the conventional biosensor of FIG.

8 is a line AA of the conventional biosensor of FIG. 6 (FIG. 7).
Sectional view at.

[Explanation of symbols]

1, 1a to 1d Insulating substrate 11 Notch portion 21 Lead 21a to 21f Counter electrode lead 22 Electrode 221 Working electrode 222a to 222d Counter electrode 23 Connection terminal 231 Working electrode connection terminal 232, 232a, 232b Counter electrode connection terminal 3 Reaction Layer 4 Spacer 5 Insulating layer 6 Space 61 Inlet 62 Outlet 7 Through hole 7a Through hole that also serves as an electrode (for working electrode) 7b, 7c Through hole that serves as a lead (for counter electrode) ) 8 back film 10 biosensor 91a sheet (substrate) 91b sheet (cover sheet) 921 lead 922, 922a electrode 923 connection terminal 94 insulating layer 95 reaction layer 96 space 97 spacer sheet 98 inlet 99 outlet

Claims (16)

[Claims] 1. A biosensor having at least an insulating substrate, an electrode system provided on the substrate, and a reaction layer facing a space formed between the two insulating substrates. An electrode formed on one surface and a connection terminal formed on the other surface are electrically connected on the front and back sides through one or more through holes provided on the insulating substrate. Biosensor. 2. The biosensor according to claim 1, wherein the through-hole has an electrode forming substance and / or a lead forming substance forming an electrode system therein, so that the through hole is electrically connected to the front and back sides. 3. The electrode forming substance inside the through hole is exposed from at least the entire area inside the hole diameter of the through hole on the electrode forming surface side to form an electrode having the opening shape. Biosensor. 4. The biosensor according to claim 3, wherein the electrode connected to the connection terminal on the front and back sides is a working electrode having a reaction layer formed on the electrode. 5. A working electrode and a counter electrode are formed on one surface of a single insulating substrate, and the counter electrode is composed of two pairs of electrodes so that the working electrode is sandwiched from both sides. The biosensor according to claim 4, wherein a connection terminal is formed, and the connection terminal is divided into front and back surfaces of one insulating substrate for the working electrode and the counter electrode. 6. The biosensor according to claim 1, wherein the electrodes connected to the connection terminal on the front and back sides are counter electrodes. 7. The biosensor according to claim 6, wherein conduction between the front surface and the back surface of the through hole is formed by a lead forming substance contained therein. 8. A working electrode and a counter electrode are formed on one surface of one insulating substrate, and the counter electrode according to claim 6 or 7 sandwiches the working electrode from both sides with respect to the working electrode according to claim 4. A biosensor comprising two pairs of electrodes formed, wherein a working electrode and a counter electrode, and these connection terminals are separated on the front and back sides of a single insulating substrate. 9. The insulating substrate according to any one of claims 1 to 8 and another insulating substrate are formed by printing or coating on at least one of the insulating substrates with the electrode formation surface inside. A biosensor characterized in that the spacers are adhered and laminated so that a space facing the reaction layer is left. 10. A biosensor having at least an insulating substrate, an electrode system provided on the substrate, and a reaction layer facing a space formed between the two insulating substrates. A biotechnology in which an electrode formed on one surface of an insulating substrate and a connection terminal formed on the other surface are electrically connected on the front and back sides through one or more through holes provided in the insulating substrate. A method of manufacturing a sensor, comprising preparing an insulating substrate having one or more through holes, bonding a back film to one surface of the insulating substrate to cover the through holes, and then filling the through holes from the other surface. After printing a predetermined conductor pattern with a conductive paste for electrode formation as described above, the back film is peeled off, and the electrode forming substance inside the through hole is at least the entire area inside the hole diameter of the through hole on the electrode forming surface side. Exposed from above and used as the electrode having the opening shape. With method of the biosensor, characterized in that the connection terminal electrode and the other surface of the one surface of the insulating substrate, thereby turning on the front and back. 11. A working electrode is formed by forming a reaction layer on an electrode having an opening shape of a through hole, and the working electrode is formed as an electrode electrically connected to a connection terminal on the other surface of the insulating substrate on the front and back sides. The method for manufacturing a biosensor according to claim 10, wherein: 12. A biosensor having at least an insulating substrate, an electrode system provided on the substrate, and a reaction layer. An electrode formed on one surface of the insulating substrate and a biosensor formed on the other surface. A method for manufacturing a biosensor in which the connected connection terminal is electrically connected to the front and back sides through one or more through holes provided in the insulating substrate, the insulating substrate having one or more through holes is provided. Prepare a back film on one surface to cover the through hole, and then print a predetermined lead pattern with a conductive paste for lead formation so that it will enter the inside of the through hole from the other surface. After that, the back film is peeled off, and then a predetermined amount of conductive paste for lead formation is used to connect again from the exposed side of the insulating substrate with the conductive paste already inside the through hole. Print the lead pattern , Through holes made conductive by the read-forming material on the front and back, a manufacturing method of a biosensor and forming a lead to conduct at front and back. 13. The method for manufacturing a biosensor according to claim 12, wherein the counter electrode is provided on a lead which is electrically connected to the connection terminal on the other surface of the insulating substrate. 14. A working electrode is formed on one surface of one insulating substrate by the manufacturing method according to claim 11, and two pairs of counter electrodes are formed so as to sandwich the working electrode from both sides. The method of manufacturing a biosensor, wherein the connection terminal is formed on the same surface side as the counter electrode, and the connection terminal is separately formed for the working electrode and the counter electrode on the front and back sides of one insulating substrate. 15. The manufacturing method according to claim 13, wherein a working electrode is formed on one surface of one insulating substrate by the manufacturing method according to claim 11, and two pairs of counter electrodes are sandwiched between the working electrodes from both sides. A method for manufacturing a biosensor, which comprises forming electrodes for a working electrode and a counter electrode, and these connecting terminals separately on the front and back of a single insulating substrate. 16. A spacer is formed on at least one of the insulative substrates by printing or coating, and the two insulative substrates are bonded and laminated by the spacer so as to leave a space facing the reaction layer. The method for manufacturing the biosensor according to claim 10, wherein