JP2976652B2 - Biosensor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biosensor for determining the presence or absence of pregnancy by utilizing an antigen-antibody reaction with human chorionic gonadotropin.

[0002]

2. Description of the Related Art Conventionally, test papers utilizing an antigen-antibody reaction with human chorionic gonadotropin (hCG) have been used to determine the presence or absence of pregnancy by a subject himself. The test paper was moved from the tip of the test paper to the first antibody adsorption point and the second antibody adsorption point.
Are provided in this order. At the first antibody adsorption point, a monoclonal antibody which reacts with human chorionic gonadotropin as an antigen-antibody is adsorbed. At the second antibody adsorption point, an antigen-antibody reaction with human chorionic gonadotropin at the first adsorption point is performed. A labeled monoclonal antibody obtained by labeling a monoclonal antibody that causes an antigen-antibody reaction with an antigen-antibody reaction product generated through the above step with a coloring substance is adsorbed.

[0003] The presence or absence of pregnancy is determined as follows. When urine is applied to the tip of the test paper or when the tip of the test paper is immersed in urine in the urine collection container, the urine moves in the test paper toward the first and second suction points by capillary action. . Therefore, if human chorionic gonadotropin is contained in urine, the human chorionic gonadotropin first causes an antigen-antibody reaction with the monoclonal antibody at the first adsorption point to generate an antigen-antibody reaction product. When the antigen-antibody reaction product thus generated further moves through the test paper together with excess urine and reaches the second adsorption point, the antigen-antibody reaction product and the labeled monoclonal An antigen-antibody reaction occurs with the antibody to develop a second adsorption point. The presence or absence of pregnancy is determined based on the state of color development at the second suction point.

[0004]

However, the following problems have been pointed out in the conventional method using the test paper. For the purpose of utilizing the presence or absence of coloration of the test paper due to the antigen-antibody reaction, in order to allow the subject to visually recognize the color change of the test paper, the antigen-antibody reaction product generated at the first adsorption point is required. It is necessary to adsorb a sufficient amount of the monoclonal antibody capable of moving to the second adsorption point and a sufficient amount of the labeled monoclonal antibody to exhibit color on the test paper. The amount of this monoclonal antibody reaches about 1.5 to 3 μg in total.

[0005] In addition, there are the following problems when urine is applied to a test paper. Depending on how urine is applied, urine may be applied first from the second suction point side, or urine may be applied simultaneously to the first and second suction points. In such a situation, urine is directly held at the second suction point. For this reason, although the human chorionic gonadotropin in urine must move sequentially between the first and second adsorption points as described above, the antigen-antibody reaction generated at the first adsorption point is generated. The movement of the object to the second suction point is hindered. As a result, the antigen-antibody reaction product reaches the second adsorption point, causes an antigen-antibody reaction with the labeled monoclonal antibody, and the time until the second adsorption point develops color becomes uneven. Therefore,
The time required to determine the presence or absence of pregnancy varies, and in some cases may take tens of minutes to several hours.

In addition, depending on how urine is applied, the antibody adsorbed on the test paper may flow out of the adsorption point, and the antigen-antibody reaction may not proceed sufficiently. For this reason, the color development at the second suction point may not be sufficient, and there is a possibility that the presence or absence of pregnancy cannot be accurately determined.
That is, the accuracy of determination depends on how urine is applied.

In the case where urine is collected in a urine collecting container and the tip of the test paper is immersed in the urine in the container, the test paper is immersed in the urine for a predetermined period until the color of the test paper can be visually recognized. It needs to be kept in a state. For example, care must be taken to prevent the test paper from standing upright in the urine collection container.

Further, the test paper is attached in advance to a urine collection container provided inside the toilet or a urine collection position in the urine to which urine naturally flows during urination, and the pregnancy can be determined without holding the urine collection container or the like by hand. It is required that the inspection work be performed simply by performing the inspection. However, when the above-described test paper is used, even if it is attached to a urine collection container or the like, it is necessary to bend down and look into the toilet bowl in order to visually determine the color development of the test paper itself. That is, the conventional pregnancy judgment test paper is inconvenient because it requires paying attention to holding the test paper and looking into the toilet.

The present invention has been made to solve the above-mentioned conventional problems, and provides a biosensor which can accurately determine the presence or absence of pregnancy in a short time with a small amount of a monoclonal antibody and is easy to use. The purpose is to do.

[0010]

In order to achieve the above object, the present invention employs an electrolytic solution chamber filled with an electrolytic solution, a pair of electrodes immersed in the electrolytic solution, and a An identification layer carrying a biological substance having an identification function for the measurement substance, and the electrolyte solution and the identification layer are provided so as to seal the electrolyte solution chamber with the identification layer therebetween, and a predetermined substance to be permeated into the electrolyte solution chamber is provided. With a permeable membrane that allows permeation, a biosensor that measures the substance to be measured based on the amount of electrical change between the pair of electrodes caused by the amount of permeation of the permeation target substance into the electrolytic solution, The discriminating layer is a monoclonal anti-α-hC that generates a primary antigen-antibody reaction product by performing an antigen-antibody reaction with human chorionic gonadotropin (hCG).
A first discriminating membrane carrying a G antibody, and a monoclonal anti-β-hC that reacts antigen-antibody with the primary antigen-antibody reaction product.
A second discriminating membrane carrying a G antibody; and a second discriminating membrane disposed on the permeable membrane side, wherein the permeable membrane comprises a monoclonal anti-β-hCG antibody in the second discriminating membrane. The gist of the present invention is that a secondary antigen-antibody reaction product generated by the action of a permeation target substance is a permeable membrane that allows the permeation.

[0011]

When the biosensor of the present invention is immersed in urine containing human chorionic gonadotropin, or when the urine is applied to the biosensor of the present invention, the monoclonal anti-α-hCG carried on the first discriminating membrane is obtained. The antibody undergoes an antigen-antibody reaction with human chorionic gonadotropin to produce a primary antigen-antibody reaction product. This primary antigen-antibody reaction product is
An antigen-antibody reaction occurs with the monoclonal anti-β-hCG antibody carried on the second discriminating membrane. By the antigen-antibody reaction between the primary antigen-antibody reaction product and the monoclonal anti-β-hCG antibody, a secondary antigen-antibody reaction product is generated.

This secondary antigen-antibody reaction product comprises a monoclonal anti-α-hCG antibody and a monoclonal anti-β-hC
It is a complex product with G antibody and has a relatively large molecular weight (1
(About 0,000), which is known to have properties equivalent to hormones in living bodies. Then, such a secondary antigen-antibody reaction product permeates through the permeable membrane as a permeation target substance and diffuses into the electrolytic solution. For this reason, the amount of dissolved oxygen in the electrolytic solution changes due to the amount of the secondary antigen-antibody reaction product permeating into the electrolytic solution. This change in the amount of dissolved oxygen appears as an electrical change between a pair of electrodes immersed in the electrolyte, and the amount of human chorionic gonadotropin in urine is measured based on the electrical change. You can determine if you are pregnant.

As described above, according to the present invention, the amount of electrical change is determined from the change in the amount of dissolved oxygen in the electrolytic solution due to the antigen-antibody reaction of human chorionic gonadotropin and the monoclonal anti-α, anti-β-hCG antibodies. Finally, the presence or absence of pregnancy is determined based on the amount of electrical change. Accordingly, an amount of the antibody that causes an electrical change is required, and the amount may be extremely small.

Further, the first discriminating membrane carrying the monoclonal anti-α-hCG antibody and the second discriminating membrane carrying the monoclonal anti-β-hCG antibody are combined so that the second discriminating layer is on the permeable membrane side. Arranged and laminated. Therefore, urine inevitably comes into direct contact with the first discriminating membrane, and the antigen-antibody reaction between human chorionic gonadotropin and the monoclonal anti-α-hCG antibody proceeds first, and then, based on this antigen-antibody reaction, Since the antigen-antibody reaction between the generated primary antigen-antibody reaction product and the monoclonal anti-β-hCG antibody progresses, the measurement of the amount of human chorionic gonadotropin in urine, and thus the determination of the presence or absence of pregnancy, are accurately performed. be able to.

Further, since the determination result of the biosensor of the present invention is obtained by the amount of electrical change, a display device or the like for displaying the determination result can be provided at a position in the toilet away from the urine collection container. For this reason, by simply setting the biosensor in a urine collection container or a urine collection position and urinating, it is possible to determine the presence or absence of pregnancy using a display device remote from the toilet, thereby improving usability.

[0016]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In order to further clarify the structure and operation of the present invention described above, preferred embodiments of the present invention will be described below.

FIG. 1 shows that the diameter is about 5 mm and the total length is about 25 m.
It is a schematic sectional drawing which shows the sensitive part of the cylindrical biosensor of m. In the drawings used in the following description, the vertical and horizontal scales in the drawings are different for convenience of description. The sensitive part 1 of the biosensor uses the electrode type sensor 10,
An identification layer 30 carrying an antibody described below is laminated on the permeable membrane 11 below the sensor 10. The electrode type sensor 10 includes a cylindrical case 13 whose lower end opening is sealed with a permeable membrane 11, a lid 15 which seals the upper end opening of this case 13, and a KO of a predetermined concentration filled in the case 13.
An electrolyte 17 such as H or KCl, a cathode electrode 19 in close contact with the permeable membrane 11, and an anode electrode 21 supported by the lid 15 are provided.

The permeable membrane 11 is a porous membrane formed of a porous material such as Teflon having fine pores capable of transmitting hormones in a living body to a thickness of 10 μm to 30 μm. In the present embodiment, a permeable membrane having a thickness of about 30 μm was used. The cathode electrode 19 is formed using a noble metal such as Pt or Au as a material. On the other hand, the anode electrode 21 is formed of Ag / AgCl, Pb, saturated sweet potato, or the like. The discrimination layer 30 is formed by laminating a first discrimination film 33 and a second discrimination film 35 to a thickness of about 20 μm. Then, as shown in the figure, the first identification film 33 is held on the case 13 by using a fixing ring 41 at the peripheral portion.

The first discrimination film 33 is made of a polybutyral film,
A monoclonal anti-α-hCG antibody isolated and purified from spleen cells (antibody-producing cells) of mice, rabbits, sheep, etc., is supported on a polymer membrane such as acetylcellulose, collagen or the like or filter paper. This antibody reacts antigenically with human chorionic gonadotropin (hCG).

The second discriminating membrane 35 is, like the first discriminating membrane, a monoclonal anti-β-hCG antibody isolated and purified from antibody-producing cells such as a mouse, on a polymer membrane such as a polybutyral membrane or on filter paper. Is carried. This monoclonal anti-β-hCG antibody causes an antigen-antibody reaction to a primary antigen-antibody reaction product generated after the above-mentioned human chorionic gonadotropin has caused an antigen-antibody reaction with the monoclonal anti-α-hCG antibody. is there. In addition, these monoclonal antibodies are usually distinguished as mouse monoclonal antibodies, rabbit monoclonal antibodies, and the like depending on the antibody-producing cells to be purified. In the following description, mouse monoclonal antibodies are simply described as monoclonal antibodies.

The first discrimination film 33 and the second discrimination film 3
As a step of supporting each antibody (biological substance) on the above, a well-known method can be applied. For example,
Inclusion method for encapsulating biological materials in a polymer matrix,
A covalent bonding method in which a biological substance is immobilized using a substance covalently bonded to a biological substance, an adsorption method in which a biological substance is adsorbed on an insoluble membrane, and the like can be adopted. Further, the first identification film 33 and the second
The total amount of the antibody carried on the discriminating film 35 is about 1 μg.

The above-mentioned cathode electrode 19 and anode electrode 2
Numeral 1 is connected to an electric measurement unit (not shown), and a weak voltage for current detection is applied between both electrodes. Then, the electric measurement unit measures the amount of human chorionic gonadotropin based on the current value (saturation current value) flowing between the two electrodes, and the presence or absence of pregnancy is determined based on this. Specifically, the display unit (not shown) in the electrical measurement unit displays the positive / negative status of pregnancy, and displays the number of months of pregnancy estimated from the measured amount of human chorionic gonadotropin, the probability of positiveness, and the like. In addition, positive or negative pregnancy can be determined based on the differential value of the measured current value, not limited to the saturated current value.

Next, a measuring method using the biosensor will be described. When the sensitive part 1 of the biosensor is immersed in urine, the monoclonal anti-α-
The hCG antibody causes an antigen-antibody reaction with human chorionic gonadotropin in urine to generate a primary antigen-antibody reaction product. The primary antigen-antibody reaction product is supplied to the second discrimination membrane 3
5 to cause an antigen-antibody reaction with the monoclonal anti-β-hCG antibody carried on the second
The following antigen-antibody reaction product is produced.

In this manner, the 2 generated by the second identification film 35
The next antigen-antibody reaction product, as a permeation target substance to the permeable membrane 11, permeates through the permeable membrane 11 and diffuses into the electrolytic solution 17, thereby changing the concentration of dissolved oxygen.

The change in the concentration of dissolved oxygen in the electrolytic solution 17 is as follows:
It appears as a change in the current value between the cathode electrode 19 and the anode electrode 21, and the presence or absence of pregnancy can be accurately determined based on this change in the amount of electricity. Further, when urine is applied to the sensitive part 1 instead of immersing the sensitive part 1 of the biosensor in urine, urine inevitably comes into direct contact with the first identification film 33 and the primary antigen-antibody Since the reaction product is generated and diffused into the second discriminating film 35, the presence or absence of pregnancy can be accurately determined without depending on how urine is applied to the sensitive part 1.

In the above biosensor, human chorionic gonadotropin contained in urine is converted to monoclonal anti-α-hC
Using the antigen-antibody reaction with the G antibody and the monoclonal anti-β-hCG antibody, the change in the permeation amount of the secondary antigen-antibody reaction product generated in the reaction, that is, the change in the dissolved oxygen amount in the electrolyte solution Is converted into a change in current value. Then, based on the current value, human chorionic gonadotropin is measured in urine, and the measurement value of this human chorionic gonadotropin is
Ultimately, the pregnancy status is determined. Therefore, the amount of each of the above monoclonal antibodies which can provide a determinable amount of electricity may be sufficient, and a total amount of about 1 μg is sufficient, which may be extremely small as compared with the amount of antibodies in the conventional test paper. Monoclonal antibodies are isolated and processed through various processes.
It is generally expensive because it is purified. Therefore, in the biosensor according to the present embodiment, the amount of the monoclonal antibody is small and the cost can be reduced.

The biosensor of the above embodiment has the following effects. Since the first discriminating film 33 and the second discriminating film 35 are in close contact with each other, the second discriminating film 35 of the primary antigen-antibody reaction product generated in the first discriminating film 33 is formed.
Is spread within a short time, and the determination of the presence or absence of pregnancy can be made within a short time. Moreover, unlike the conventional test paper, it is not necessary to prevent the test paper from falling down while standing in the urine collection container, so that the usability is improved. The sensing part 1 which is a part in contact with urine is provided directly at a position where urine in the toilet is applied, or provided in a container where urine is collected, and a display device for displaying the determination result is separated from the toilet, for example, at a position separated from the urinal. By placing the device at the eye level of the user, it is not necessary to look into the toilet, so that the usability is also improved.

<Experimental Example> Next, main members used in the biosensor were prepared from the following materials, and an evaluation test was performed using a sample solution. That is, the first identification film 33 is
The second identification film 35 was formed from a polybutyral film carrying a monoclonal anti-β-hCG antibody, and the second discriminating film 35 was formed from a polybutyral film carrying a monoclonal anti-α-hCG antibody. The first identification film 33 and the second identification film 3
The film thickness of 5 and the amount of each biological substance carried thereon are as described above.
The case 13 is made of an electrolytic solution 17 made of 1N KOH.
Is filled with

As sample solutions to be measured, seven types of solutions having different amounts of human chorionic gonadotropin (hCG) per liter of distilled water, that is, 10 IU (international unit; 1 IU)
U = 2.5 × 10 ⁻⁷ g), 50 IU, 100 IU, 20
Each solution of 0 IU, 300 IU, 400 IU, and 500 IU was prepared. Then, the saturation current value when the sensor was immersed in each sample solution was measured. The result is shown in FIG.

As shown in FIG. 2, the current was not measured in the sample solution of 10 IU, and the current was measured in the sample solution of 50 IU or more. Also, the saturation current value and h in the sample solution
The hCG concentration is proportional to the CG concentration.
This proportional relationship is remarkable over a range of ４００400 IU / l. Usually, the concentration of hCG in urine of pregnant women is
Since it does not fall below 50 IU, according to the present biosensor, pregnancy can be accurately and reliably determined through accurate measurement of the amount of human chorionic gonadotropin in urine. Even if the biosensor of this example was replaced and the above measurement was repeated, the measurement result of FIG. 2 was reproduced. In addition, for the sample solution of each concentration, when the elapsed time from the start of the measurement until the saturation current value was obtained was measured,
The time was about 3 minutes for the 10 IU / l sample solution, about 2 minutes for 100 IU / l, and about 1 minute for 400 IU / l. From this, the determination of the presence or absence of pregnancy through measurement of the amount of human chorionic gonadotropin can be completed within about 2 minutes.

The monoclonal anti-α-h used at this time
The total amount of the CG antibody and the monoclonal anti-β-hCG antibody is only about 1 μg, which is extremely small as compared with 1.5 to 3 μg required for the test paper for judging pregnancy described in the related art. That is, it is possible to accurately determine pregnancy with a small amount of antibody.

Next, as a modification, a biosensor using a sensor that does not use a biological substance will be described. As shown in FIG. 3, the biosensor 50 includes two sensors 60 and 70 having the same configuration on a silicon substrate 51.
Then, the identification layer 81 is formed on the transmission film 61 of one sensor 60.
Are stacked to form the detection electrode unit 80, and the other sensor 7
A comparative film 91 is laminated on the transmission film 71 of the
0 is configured.

The discrimination layer 81 of the detection electrode section 80 includes a first discrimination film 82 having a thickness of 20 μm in which a monoclonal anti-α-hCG antibody is supported on a polybutyral film, and a polybutyral film having a monoclonal anti-β-hCG antibody. And a second discriminating film 83 having a thickness of 20 μm. On the other hand, the comparative film 91 of the comparative electrode section 90 is a polybutyral film formed of polybutyral alone, and is formed to have the same thickness (40 μm) as the identification layer 81.

As described above, the sensors 60 and 70 have the same configuration and are produced simultaneously in the same manufacturing process. That is, the sensors 60 and 70 have V-shaped grooves 62 and 72 formed on the silicon substrate 51 by etching using a photoresist, and a thickness 1000 formed on the upper surfaces of the V-shaped grooves 62 and 72 by a thermal oxidation method. Of silicon oxide films 63 and 73 of Å, protective films 64 and 74 of 500 Å thick made of Si ₃ N ₄ formed on the silicon oxide films 63 and 73 by sputtering, and V-shaped grooves 62 and 72. Protective film 6 at bottom
Anode electrodes 65 and 75 formed by sputtering on the U.S. 4, 74, cathode Au electrodes 66 and 76 formed by sputtering on the protective film 64 above the V-shaped grooves 62 and 72; 1N KOH electrolytes 67 and 77 filled in the V-shaped grooves 62 and 72, and a permeable membrane 61 provided to seal the electrolytes 67 and 77.
71.

The detection electrode section 80 and the comparison electrode section 9
The Ag electrodes 65 and 75 on the anode side and the Au electrodes 66 and 76 on the cathode side in the respective sensors 60 and 70 of 0 are connected to the electric measurement unit 100, respectively. The electric measurement unit 100 applies the same weak voltage for current detection between the anode side electrode and the cathode side electrode in each sensor, and determines the difference between the current values obtained from each sensor as the concentration of the substance to be measured, In this case, it is measured as hCG concentration.

Next, a measuring method using the biosensor 50 will be described. This biosensor 50 is connected to the detection electrode unit 80.
And when the reference electrode unit 90 is immersed in urine, the detection electrode unit 8
In the sensor 60 on the 0 side, the same reaction as in the electrode type sensor 10 in the sensitive part 1 of the biosensor described occurs. That is, an antigen-antibody reaction between the monoclonal anti-α-hCG antibody and human chorionic gonadotropin in urine occurs on the first discriminating film 82 of the discriminating layer 81, and a primary antigen-antibody reaction product, which is a product of the reaction, Diffusion into the second discriminating membrane 83 causes an antigen-antibody reaction with the monoclonal anti-β-hCG antibody at the second discriminating membrane 83 to generate a secondary antigen-antibody reaction product.

On the other hand, in the sensor 70 on the side of the comparison electrode section 90, the comparison film (polybutyral) having the same thickness as the identification layer 81 is used.
Since only 91 is formed on the upper surface of the permeable membrane 71, generation of a secondary antigen-antibody reaction product based on the antigen-antibody reaction with human chorionic gonadotropin in urine is not observed.
Therefore, only the dissolved oxygen in the urine permeates through the permeable membrane 71 and
The concentration of dissolved oxygen in the OH electrolyte 77 is changed.

Thus, the change in the oxygen concentration of the electrolytic solution in each of the sensors 60 and 70 appears as a change in the current value between the cathode side electrode and the anode side electrode. The difference is obtained. This difference in the current value is obtained by subtracting the current value measured only by the dissolved oxygen in the urine measured by the sensor 70 from the current value measured by the sensor 60, so that the amount of human chorionic gonadotropin in the urine is determined. To reflect exactly. Therefore, it is possible to accurately and extremely accurately determine the presence or absence of pregnancy while eliminating the influence of dissolved oxygen in urine. Further, even when urine is applied to the biosensor 50, generation of primary antigen-antibody reaction products,
Since the production of the secondary antigen-antibody reaction product occurs in this order, the presence or absence of pregnancy can be accurately determined without being influenced by the way of urine. Further, as in the case of the biosensor of the described embodiment, it is needless to say that a small amount of each of the above monoclonal antibodies may be used. Further, similarly to the biosensor of the described embodiment, not only the determination of the presence or absence of pregnancy can be made in a short time through the rapid diffusion of the primary antigen-antibody reaction product, but also the usability is improved. In addition, since both sensors 60 and 70 for accurately determining the presence or absence of pregnancy can be manufactured at the same time in the same process, an inexpensive sensor can be provided.

When an evaluation test of this biosensor 50 was performed in the same manner as the biosensor of the above embodiment, the current (current value of both biosensors) was measured in a sample solution having human chorionic gonadotropin (hCG) of 50 IU / l or more. Was measured, and a remarkable proportional relationship was found between the obtained current value and the hCG concentration in the sample solution when the hCG concentration was between 50 and 650 IU / l. 400 IU / l
The relationship between the elapsed time from the start of the measurement and the obtained current value was measured for the sample solution of (1). FIG. 4 shows the results. From FIG. 4, the determination of the presence or absence of pregnancy through measurement of the amount of human chorionic gonadotropin can be completed in about one minute.

The present invention is not limited to the above-described embodiment, but can be implemented in various modes without departing from the gist of the invention, and the following modifications are possible. For example, in the biosensor 50, the comparison electrode unit 90 provided with the sensor 70 not using a biological substance and the detection electrode unit 80 are used in combination. You may comprise. In addition, the first identification film 33 and the second identification film 35
In addition, when an electron acceptor that mediates the electrical change of the product in the electrolytic solution is supported together with the antibody, it is possible to more accurately and accurately determine the presence or absence of pregnancy. The electron acceptor is called a mediator,
It is appropriately selected from substances such as ferrocene, ubiquinone, and ferricium.

[0041]

As described above, the biosensor of the present invention comprises human chorionic gonadotropin and monoclonal anti-α,
Utilizing an antigen-antibody reaction followed by an anti-β-hCG antibody, the change in the amount of dissolved oxygen in the electrolyte due to the amount of permeate into the electrolyte resulting from the permeation of the product generated by the antigen-antibody reaction. The amount is obtained, and the presence or absence of pregnancy is finally determined based on the amount of electrical change. Therefore, according to the biosensor of the present invention, the presence or absence of pregnancy can be accurately and accurately determined with a small amount of a monoclonal antibody that causes an electrical change.

Further, since the determination result of the biosensor of the present invention is obtained by the amount of electrical change, a site such as an identification layer immersed in urine is provided in the urine collection container or directly in the toilet, and the determination result is displayed. Can be provided at a position remote from the urine collection container or the like. Furthermore, since the first discriminating membrane carrying the monoclonal anti-α-hCG antibody and the second discriminating membrane carrying the monoclonal anti-β-hCG antibody were brought into close contact with each other, the primary antigen generated on the first discriminating membrane was produced. The diffusion of the antibody reaction product to the second discriminating membrane can be performed within a short period of time. As a result, in combination with the determination of the presence or absence of pregnancy based on the amount of electrical change as described above, not only can the determination be made within a short time, but also the test can be performed on a urine collection container like a conventional test paper. Since there is no need to keep the paper standing upright or look into the test paper, the usability is improved.

[Brief description of the drawings]

FIG. 1 is a cross-sectional view illustrating a biosensor according to an embodiment of the present invention.

FIG. 2 is a graph showing the relationship between the saturation current value and the hCG concentration obtained when seven types of solutions having different addition amounts of human chorionic gonadotropin (hCG) are measured.

FIG. 3 is a cross-sectional view illustrating a biosensor according to another embodiment.

FIG. 4. Human chorionic gonadotropin (hCG) is 400I
7 is a graph showing the relationship between the elapsed time from the start of measurement and the obtained current value for a U / l sample solution.

[Explanation of symbols]

 Reference Signs List 10 electrode type sensor 11 permeable membrane 17 electrolyte 19 cathode electrode 21 anode electrode 30 identification layer 33 first identification film 35 second identification film 50 biosensor 61 transmission film 65 Ag electrode 66 Au electrode 67 electrolyte solution 71 transmission film 75 Ag electrode 76 Au electrode 77 electrolyte 80 detection electrode unit 81 identification layer 82 first identification film 83 second identification film 90 comparison electrode unit 91 comparison film 100 electric measurement unit

Claims (1)

(57) [Claims] 1. An electrolyte chamber filled with an electrolyte, a pair of electrodes immersed in the electrolyte, an identification layer carrying a biological substance having an identification function for a substance to be measured, A permeable membrane that is provided so as to seal the electrolytic solution chamber with a layer interposed therebetween, and that allows the permeation of a predetermined substance to be permeated into the electrolytic solution chamber, In a biosensor for measuring the substance to be measured based on an amount of electric change between the pair of electrodes caused by a permeation amount, the discriminating layer reacts with human chorionic gonadotropin (hCG) by an antigen-antibody reaction to perform primary reaction. A first discriminating membrane that carries a monoclonal anti-α-hCG antibody that produces an antigen-antibody reaction product, and a second discrimination membrane that carries a monoclonal anti-β-hCG antibody that reacts with the primary antigen-antibody reaction product. The identification membrane and The layers, wherein the transparent film side becomes to place the second identification layer, wherein the permeable membrane, monoclonal anti-beta-hCG in the second identification film
A biosensor characterized in that the biosensor is a permeable membrane that allows permeation of a secondary antigen-antibody reaction product produced by the involvement of an antibody as a permeation target substance.