JP2001305095A - Biosensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a biosensor of uniform performance with a reagent layer uniformly arranged on electrodes regardless of the composition of a reagent solution. SOLUTION: This biosensor for determining substrate contained in a sample solution is provided with an insulator substrate; a plurality of electrodes formed by providing first slits in an electroconductive layer formed on a part or the whole surface of the insulator substrate; second slits for regulating a regent applied position and area; a spacer disposed on the electrodes and having a cutout part for forming a specimen supply path for supplying the sample solution to the electrodes; a reagent layer containing oxygen, provided on the electrodes in the specimen supply path; and a cover with an air hole leading to the specimen supply path.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a biosensor for quantifying a substrate contained in a sample solution.

[0002]

2. Description of the Related Art A biosensor is a sensor that utilizes a biological material, such as a microorganism, an enzyme, an antibody, or the like, and uses the biological material as a molecular identification element. That is, the reaction that occurs when the immobilized biological material recognizes the target substrate,
It utilizes oxygen consumption, enzymatic reaction, luminescence, etc. due to the respiration of microorganisms.

[0003] Among biosensors, enzyme sensors have been put into practical use. For example, enzyme sensors for glucose, lactose, urea, and amino acids are used in medical measurement and the food industry. The enzyme sensor reduces the electron acceptor by the electrons generated by the reaction between the substrate and the enzyme contained in the sample solution that is the specimen, and the measuring device electrochemically measures the reduction amount of the electron acceptor. Perform quantitative analysis of the sample.

Hereinafter, a conventional biosensor will be described with reference to the drawings. FIG. 3 is a diagram showing a perspective view of a conventional biosensor in the order of preparation steps, and FIG. 4 is a diagram showing a sample supply path of the conventional biosensor. Reference numeral 101 denotes an insulating substrate made of polyethylene terephthalate or the like. 10
Reference numeral 2 denotes an electrically conductive layer formed on the entire surface of the substrate 101 and made of carbon, a metal substance, or the like. 103a, 10
3b, 103c and 103d are slits formed in the electrically conductive layer 102. 105, 106, and 107 form slits 103a, 103b, and 103 in the electrically conductive layer 102.
The electrodes formed by dividing by c and 103d are a measurement electrode, a counter electrode, and a detection electrode. 10
Reference numeral 8 denotes a measurement electrode 105, a counter electrode 106, and a detection electrode 107.
Is a spacer that covers. Reference numeral 109 denotes a rectangular notch provided in the center of the front edge of the spacer 108 and forming a sample supply path. 110 is an inlet of the sample supply path. 111
Represents the measurement electrode 105, the counter electrode 106, and the detection electrode 10
7 is a reagent layer formed by applying a reagent containing an enzyme dropwise. A cover 112 covers the spacer 108. Reference numeral 113 denotes an air hole provided at the center of the cover 112.

As shown in FIG. 3A, an electrically conductive layer 102 is formed on the entire surface of a substrate 101 by a screen printing method or the like. Next, as shown in FIG. 3B, slits 103a, 103a are formed in the electrically conductive layer 102 using a laser.
3b, 103c, and 103d are formed, and the measurement electrodes 105,
The electrically conductive layer 10 is provided on the counter electrode 106 and the detection electrode 107.
Divide 2 Next, as shown in FIG.
In the case of a blood sugar level sensor, a reagent composed of glucose oxidase as an enzyme and potassium ferricyanide as an electron acceptor is applied to the counter electrode 106 and the detection electrode 107 by dropping to form a reagent layer 111. Next, a spacer 108 having a cutout 109 for forming a sample supply path is provided on the measurement electrode 105, the counter electrode 106, and the detection electrode 107. The sample supply path is in a state as shown in FIG. Cover 1 on top
12 is installed. Here, the notch 10 of the spacer 108
One end of 9 communicates with an air hole 113 provided in the cover 112.

According to this configuration, when a sample liquid, such as blood, is supplied to the inlet 110 of the sample supply path, the air hole 1
A certain amount of the sample is sucked into the sample supply path by the capillary action by 13, and reaches the counter electrode 106, the measurement electrode 105, and the detection electrode 107. Reagent layer 1 formed on electrode
Numeral 11 is dissolved by blood, for example, an oxidation-reduction reaction occurs between the reagent and the sample, and an electrical change occurs between the measurement electrode 105 and the counter electrode 106. At the same time, if the sample is correctly filled in the sample supply path, an electrical change occurs between the measurement electrode 105 and the detection electrode 107. When this is sensed and a voltage is applied to the measurement electrode 105 and the counter electrode 106, for example, in the case of a blood sugar level sensor, a current proportional to the glucose concentration is generated, and the blood sugar level can be measured from the value.

[0007]

However, in a conventional biosensor, even if a reagent is applied by drops to form a reagent layer on the electrode, the difference in the surface condition of the electrode and the spread of the reagent due to the composition of the reagent solution. Therefore, the reagent is not uniformly applied on the electrode, and the amount of the reagent on the electrode varies. That is, even if the same amount of the reagent is applied by dropping, the spread of the reagent varies, so that the position and area of the reagent layer vary. Therefore, there was a problem that the performance of the biosensor deteriorated.

The present invention has been made in view of the above problems, and has as its object to provide a biosensor in which a reagent layer is uniformly disposed on an electrode regardless of the composition of a reagent solution and the performance is uniform.

[0009]

To achieve the above object, a biosensor according to claim 1 is a biosensor for quantifying a substrate contained in a sample solution, comprising: an insulator substrate; A plurality of electrodes formed by providing a first slit in an electrically conductive layer formed on the entire surface or a part of the insulator substrate, and a sample arranged on the electrodes and supplying a sample liquid to the electrodes A spacer having a notch forming a supply path, disposed on the electrode in the sample supply path,
A reagent layer formed by dropping a reagent, and a cover having an air hole that is arranged on the spacer and communicates with the specimen supply path, around a position where the reagent on the electrode is dropped, A second slit surrounding the position where the reagent is applied is provided.

[0010] The biosensor according to claim 2 is
2. The biosensor according to claim 1, wherein the electrically conductive layer is formed on the insulating substrate by a sputtering method.

Further, the biosensor according to claim 3 is
In the biosensor according to claim 1 or 2, the first slit and the second slit are:
It is characterized by being formed by a laser.

Further, the biosensor according to claim 4 is
The biosensor according to any one of claims 1 to 3, wherein the second slit has an arc shape.

[0013]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Embodiment 1 The biosensor according to the first embodiment will be described with reference to the drawings. FIG.
FIG. 3 is a diagram illustrating a perspective view of the biosensor according to the first embodiment in the order of creation steps, and FIG. 2 is a diagram illustrating a sample supply path of the biosensor according to the first embodiment. Reference numeral 1 denotes an insulating substrate made of polyethylene terephthalate or the like. Reference numeral 2 denotes an electric conductive layer formed on the entire surface of the substrate 1 and made of an electric conductive substance such as a noble metal such as gold or palladium or carbon. Reference numerals 3a, 3b, 3c, and 3d are first slits provided in the electrically conductive layer 2. 5,
6, 7 connect the electrically conductive layer 2 to the first slits 3a, 3b,
These electrodes are formed by dividing by 3c and 3d, and are a measurement electrode, a counter electrode, and a detection electrode that is an electrode for confirming whether or not the sample is surely sucked into the sample supply path. Reference numerals 4a and 4b denote second slits that regulate the position and area of the electrode on which the reagent is applied.
Reference numeral 8 denotes a spacer that covers the measurement electrode 5, the counter electrode 6, and the detection electrode 7. Reference numeral 9 denotes a rectangular notch formed in the center of the front edge of the spacer 8 and forming a sample supply path. Reference numeral 10 denotes an inlet of the sample supply path. Reference numeral 11 denotes a reagent layer formed by applying a reagent containing an enzyme to the measurement electrode 5, the counter electrode 6, and the detection electrode 7 by dropping. 12 is a cover for covering the spacer 8. Reference numeral 13 denotes an air hole provided at the center of the cover 12.

As shown in FIG. 1A, a sputtering method for forming a thin film over the entire surface of the substrate 1
An electrically conductive layer 2 of a thin film of a noble metal such as gold or palladium is formed. The electrically conductive layer 2 may be formed not on the entire surface of the substrate 1 but only on a portion necessary for forming an electrode.

Next, as shown in FIG. 1B, the first slits 3a, 3b, 3
c, 3d are formed, and the electrically conductive layer 2 is divided into the measurement electrode 5, the counter electrode 6, and the detection electrode 7. Using a laser, arc-shaped second slits 4a and 4b are formed in the electrically conductive layer 2 so as to surround the position where the reagent is applied, around the position where the reagent is dropped.

The first slits 3a, 3b, 3c,
On the substrate 1 by a screen printing method or a sputtering method using a printing plate or a masking plate on which a pattern necessary for forming the electric conductive layer 2 having 3d and the second slits 4a and 4b is previously arranged. And the first slits 3a, 3b, 3c, 3d and the second slit 4
a and 4b may be formed.

The first slits 3a, 3b, 3c,
3d and the second slits 4a, 4b
As a method for providing the conductive layer 2, a part of the electrically conductive layer 2 may be shaved with a jig having a sharp tip or the like.

Next, as shown in FIG. 1 (c), the measuring electrode 5, the counter electrode 6 and the detecting electrode 7 are composed of glucose oxidase as an enzyme and potassium ferricyanide as an electron acceptor in the case of a blood sugar level sensor. The reagent is applied dropwise. The part to which the reagent is applied is the second slit 4
a, 4b, the second slit 4a,
4b can be used as a mark of the place where the reagent is applied. Further, since the applied reagent is a liquid, it spreads outward in a circle around the location where it is applied by dropping, but the second slits 4a and 4b function as breakwaters, and the position of the reagent layer 11 is reduced. And the area of the second slit 4
It does not spread beyond a and 4b. Therefore, the reagent layer 11 is formed at a predetermined position with a predetermined area.

Next, a notch 9 for forming a sample supply path on the measurement electrode 5, the counter electrode 6, and the detection electrode 7 is provided.
Is installed. The sample supply path is in a state as shown in FIG.

Next, the cover 12 is set on the spacer 8. Here, one end of the notch 9 of the spacer 8 communicates with an air hole 13 provided in the cover 12. In addition, after forming the spacer 8 on the electrodes of the measurement electrode 5, the counter electrode 6, and the detection electrode 7, the reagent is dropped on a portion of the measurement electrode 5, the counter electrode 6, and the detection electrode 7 which is exposed from the cutout 9. Thus, the reagent layer 11 may be formed.

According to this configuration, when blood is supplied to the inlet 10 of the sample supply channel as a sample liquid as a sample, the air hole 1
A certain amount of the sample is sucked into the sample supply path by capillary action by 3 and reaches the counter electrode 6, the measurement electrode 5, and the detection electrode 7. The reagent layer 11 formed on the electrode is dissolved by the blood sample, and an oxidation-reduction reaction occurs between the reagent and a specific component in the sample. Here, if the sample is correctly filled in the sample supply path, an electrical change occurs between the counter electrode 6 and the detection electrode 7. Thus, it is confirmed that the sample has been sucked up to the detection electrode 7. Since an electrical change also occurs between the measurement electrode 5 and the detection electrode 7, it may be confirmed that the sample has been sucked up to the detection electrode 7. After the sample is aspirated to the detection electrode 7, the reaction between the sample and the reagent is promoted for a certain period of time.
And a constant voltage is applied to the counter electrode 6 or both the counter electrode 6 and the detection electrode 7. Since it is a blood glucose level sensor, a current proportional to the glucose concentration is generated, and the blood glucose level can be measured from the value.

Although the blood glucose level sensor has been described in the first embodiment, the blood glucose level sensor can be used as a biosensor other than the blood glucose level sensor by changing the components and the sample of the reagent layer 11. In the first embodiment, the biosensor having three electrodes has been described, but the number of electrodes may be other than that.

In the first embodiment, the second slits 4a and 4b have an arc shape. However, unless the position and area of the reagent layer can be regulated and the accuracy of the electrodes is not reduced, this is not the case. It is not limited to the shape. For example, a straight line or a key shape may be used.

As described above, the biosensor according to the first embodiment is a biosensor for quantifying a substrate contained in a sample liquid, and includes an insulator substrate and an entire surface or one surface of the insulator substrate. A plurality of electrodes formed by providing a first slit in an electrically conductive layer formed by a sputtering method on a portion, and arcs for regulating a reagent application position and an area provided in the electrically conductive layer. Second of shape
And a spacer disposed on the electrode, having a notch for forming a sample supply path for supplying a sample liquid to the measurement electrode, and an enzyme provided on the electrode in the sample supply path. A reagent layer containing the reagent layer, and a cover disposed on the spacer and having an air hole communicating with the sample supply path.
When the reagent is applied on the electrode to form the reagent layer, the reagent spreads evenly, and a reagent layer with no variation in position and area is formed. There is an effect that accurate measurement without variation can be performed.

[0025]

As described above, according to the first aspect of the present invention,
According to the biosensor according to the above, a biosensor for quantifying the substrate contained in the sample solution, the insulator substrate, the electrically conductive layer formed on the entire surface or a part of the insulator substrate A plurality of electrodes formed by providing a first slit, and a spacer having a notch formed on the electrode and forming a sample supply path for supplying a sample liquid to the electrode,
A reagent layer disposed on the electrode in the sample supply path, formed by dropping a reagent, and a cover having an air hole disposed on the spacer and communicating with the sample supply path; A second slit is provided around the position where the reagent is applied around the position where the reagent is applied, so that the reagent is applied by drops onto the electrode for forming a reagent layer. In this case, the reagent spreads uniformly and a reagent layer having a predetermined area is formed at a predetermined position, so that a uniform reagent layer having no variation in position and area is formed, and accurate measurement without variation can be performed. Having.

According to the biosensor of the second aspect of the present invention, in the biosensor of the first aspect, the electrically conductive layer is formed on the insulating substrate by a sputtering method. As a result, a highly accurate thin film can be formed, a highly accurate electrode can be formed, and the accuracy of measurement can be increased.

According to the biosensor of the third aspect of the present invention, in the biosensor of the first or second aspect, the first slit and the second slit are formed by a laser. Therefore, there is an effect that the area of each electrode can be defined with high accuracy. In addition, there is an effect that the distance between the electrodes can be made very short and the size of the sample supply path can be reduced. Further, there is an effect that the position and area of the reagent layer can be defined with high precision.

According to a fourth aspect of the present invention, in the biosensor according to the first aspect, the second slit may have an arc shape. Therefore, since the spread of the reagent is regulated by the slit having the same shape as that of the reagent, the area and position of the reagent layer can be regulated more accurately.

[Brief description of the drawings]

FIG. 1 is a diagram showing a perspective view of a biosensor according to a first embodiment in the order of creation steps.

FIG. 2 is a diagram showing a sample supply path of the biosensor according to the first embodiment.

FIG. 3 is a diagram showing a conventional biosensor perspective view in the order of creation steps.

FIG. 4 is a diagram showing a sample supply path of a conventional biosensor.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Substrate 2 Electric conductive layer 3a 1st slit 3b 1st slit 3c 1st slit 3d 1st slit 4a 2nd slit 4b 2nd slit 5 Measurement electrode 6 Counter electrode 7 Detecting electrode 8 Spacer 9 Notch Part 10 Entrance of sample supply path 11 Reagent layer 12 Cover 13 Air hole 101 Substrate 102 Electrically conductive layer 103a Slit 103b Slit 103c Slit 103d Slit 105 Measurement electrode 106 Counter electrode 107 Detection electrode 108 Spacer 109 Cutout 110 Entrance of sample supply path 111 Reagent layer 112 Cover 113 Air hole

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 27/30 353R (72) Inventor Hiroyuki Tokunaga 8-1, Koshinmachi, Takamatsu City, Kagawa Prefecture Matsushita Hisashi Denko Kogyo Co., Ltd. Company F term (reference) 2G045 BB21 CB21 DA20 DA36 FB05 HA14 4B029 AA07 BB16 FA12 4B063 QA01 QA19 QQ03 QQ68 QR03 QR50 QS39 QX05

Claims (4)

[Claims] 1. A biosensor for quantifying a substrate contained in a sample liquid, comprising: an insulating substrate; and an electrically conductive layer formed on the entire surface or a part of the insulating substrate. A plurality of electrodes provided with slits, a spacer disposed on the electrode and having a cutout portion forming a sample supply path for supplying a sample liquid to the electrode, and a spacer disposed on the electrode in the sample supply path A reagent layer formed by dropping a reagent, and a cover disposed on the spacer and having an air hole communicating with the sample supply path, around a position where the reagent is dropped on the electrode. And a second slit surrounding the position where the reagent is applied. 2. The biosensor according to claim 1, wherein the electrically conductive layer is formed on the insulating substrate by a sputtering method. 3. The biosensor according to claim 1, wherein the first slit and the second slit are formed by a laser. 4. The biosensor according to claim 1, wherein the second slit has an arc shape.