JP2017009550A - Biosensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a biosensor capable of measurement of higher accuracy than conventional.SOLUTION: A biosensor for quantifying a substrate contained in sample liquid includes: an insulation substrate; a first electrode provided on one surface of the insulation substrate for quantifying the substrate; a second electrode provided on one surface of the insulation substrate for quantifying objects other than the substrate by using an AC method; a reagent layer containing enzyme reacting to the substrate and an electrode active material formed on at least a part of a surface opposite the insulation substrate of the first electrode; a spacer which has a notch part for forming a cavity leading the sample liquid to the reagent layer and is arranged on the first electrode and the second electrode so that the reagent layer is positioned inside the notch part; and a cover provided on the opposite surface to the insulation substrate of the spacer in the way that it covers at least the notch part. On the surface of at least the second electrode, no electrode active material is present.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a biosensor using an electrochemical method.

  Various biosensors using electrochemical methods are known, such as glucose sensors such as blood glucose level sensors.

  For example, when a sample (blood, etc.) is introduced into a cavity (a space formed by a groove formed in a spacer) of a biosensor, a component (substrate) contained in the sample is converted into a mediator (electrode active material) via an enzyme. Reduce. Here, when a predetermined voltage is applied to the electrode, the reduced mediator is reversely oxidized by an electrochemical reaction. By measuring the oxidation current generated at this time, the amount of the component of interest can be detected.

  Such a biosensor generally includes two or more electrodes including at least a working electrode and a counter electrode. After a spacer for forming a cavity is bonded on the electrode, an enzyme, It has a structure in which a reagent layer containing a mediator or the like is formed and a cover is attached.

  Here, in the case of glucose measurement using a blood sample, the blood contains blood cells such as red blood cells. It is known that the magnitude of the oxidation current derived from glucose is affected by the magnitude of the hematocrit value that indicates the proportion of the volume of blood cells in the blood sample.

  In recent years, there has been a demand for quantitative analysis of a substrate in a blood sample in a short time using a small amount of blood sample, so that a small amount of blood sample is supplied to the biosensor (sensor chip). A smaller volume cavity is formed. For this reason, since the volume of the cavity of a biosensor is small and the blood sample used for a measurement is small, the magnitude | size of the oxidation current which is a measuring object is small.

  Therefore, the oxidation current measured by the biosensor includes an error depending on the hematocrit value of the blood sample, but the ratio of the error depending on the hematocrit value included in the oxidation current is small because the measured oxidation current is small. Since becomes relatively large, an error depending on the hematocrit value cannot be ignored.

  Therefore, these hematocrit values are measured by the alternating current method, the plasma or blood cell volume is measured, the hematocrit value is converted, and correction based on the converted hematocrit value is performed to improve the accuracy of quantitative analysis of the substrate in the blood sample. Attempts have been made (for example, Japanese Patent Application Laid-Open No. 62-25262 (Patent Document 1), Japanese Patent Application No. 63-133302 (Patent Document 2)).

Japanese Patent Laid-Open No. 62-25262 Japanese Patent Application No. 63-133302

  However, as a result of studies by the present inventors, when the hematocrit value is measured by the alternating current method as in Patent Documents 1 and 2, the electrode active material is measured when a mediator (electrode active material) is present in the sample liquid. Thus, it has been found that it is difficult to accurately measure blood cells (hematocrit value) and plasma.

  The present invention has been made in view of the above problems, and an object of the present invention is to provide a biosensor capable of measuring with higher accuracy than before.

[1] A biosensor for quantifying a substrate contained in a sample solution,
An insulating substrate;
A first electrode for quantifying a substrate, including a working electrode and a counter electrode provided on one surface of the insulating substrate;
A second electrode for quantifying an object other than the substrate using an alternating current method, including a working electrode and a counter electrode provided on one surface of the insulating substrate;
A reagent layer formed on at least a part of the surface of the first electrode opposite to the insulating substrate, the enzyme reacting with the substrate, and an electrode active material;
It has a notch for forming a cavity for guiding the sample solution to the reagent layer, and is disposed on the first electrode and the second electrode so that the reagent layer is located inside the notch. Spacers,
A cover provided on a surface opposite to the insulating substrate of the spacer so as to cover at least the notch,
A biosensor characterized in that no electrode active material is present on at least the surface of the second electrode.

[2] The cavity has an opening for introducing the sample solution,
The biosensor according to [1], wherein the second electrode is installed at a position closer to the opening of the cavity than the first electrode.

  [3] The biosensor according to [1] or [2], wherein the cavity has a constricted shape between the reagent layer and a portion of the second electrode exposed in the cavity.

  [4] The biosensor according to [3], wherein the frequency of the AC voltage used in the AC method is at least one frequency within a range of 1 kHz to 30 MHz.

  According to the present invention, it is possible to provide a biosensor capable of measuring with higher accuracy than before.

1 is an exploded perspective view showing a configuration of a biosensor of Embodiment 1. FIG. 1 is a perspective view illustrating a configuration of a biosensor according to Embodiment 1. FIG. 2 is a partially enlarged view showing the configuration of the biosensor of Embodiment 1. FIG. It is the elements on larger scale which show the structure of the modification of the biosensor of Embodiment 1. FIG. 6 is a flowchart for explaining an example of a measurement process of the biosensor of Embodiment 1. FIG. 3 is a schematic diagram for explaining a measurement method of Example 1. FIG. 3 is a graph showing measurement results of Example 1. 6 is a graph showing measurement results of Comparative Example 1. 6 is a graph showing measurement results of Example 2. 10 is a graph showing measurement results of Comparative Example 2. 10 is a graph showing measurement results of Example 3. 10 is a graph showing measurement results of Example 4. 10 is another graph showing the measurement results of Example 4. It is a graph which shows cv value in the measurement result of Example 4.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. In the drawings, the same reference numerals represent the same or corresponding parts. In addition, dimensional relationships such as length, width, thickness, and depth are changed as appropriate for clarity and simplification of the drawings, and do not represent actual dimensional relationships. Each embodiment is an exemplification, and needless to say, partial replacement or combination of configurations shown in different embodiments is possible.

[Embodiment 1]
1 to 3, the biosensor of this embodiment is a biosensor for quantifying a substrate contained in a sample liquid, and quantifies the insulating substrate 1 and the substrate electrochemically. A first electrode (working electrode 21 and counter electrode 22), a second electrode (working electrode 23 and counter electrode 24) for quantifying an object other than a substrate by an AC method, a reagent layer 3, a spacer 4, And a cover 5.

  Examples of measurement of an object other than a substrate quantified by the second electrode include measurement of blood cell volume (hematocrit), detection of insertion of a biosensor (sensor chip) into a measuring machine, detection of introduction of a sample into a biosensor, Measurement of ambient environment temperature (air temperature), etc.

  The first electrode includes a working electrode 21 and a counter electrode 22 provided on one surface of the insulating substrate 1. The second electrode includes a working electrode 23 and a counter electrode 24 provided on one surface of the insulating substrate 1. The reagent layer 3 is formed on a part of the surface of the first electrode opposite to the insulating substrate 1 and includes at least an enzyme that reacts with a substrate and an electrode active material (mediator).

  The spacer 4 has a notch 42 for forming a cavity 41 for guiding the sample solution to the reagent layer 3, and is arranged on the electrode so that the reagent layer 3 is located inside the cavity 41.

  The cover 5 is provided on the surface of the spacer 4 opposite to the insulating substrate 1 so as to cover at least the notch 42. The cover 5 has an air hole 5 a communicating with the cavity 41.

  The biosensor of this embodiment is characterized in that no electrode active material is present on the surface of the second electrode (working electrode 23 and counter electrode 24) used for measuring an object other than the substrate. In addition, it is preferable that an electrode active material does not exist in the part 23a, 24a exposed in the cavity 41 among the surfaces of an electrode.

  Thereby, when quantifying the object other than the substrate by the alternating current method with the second electrode, it is possible to prevent the occurrence of error due to the measurement of the blood electrode active material, and to increase the measurement accuracy of the object other than the substrate. Can do.

  In addition, the measurement of an object other than the substrate is often performed for the purpose of improving the measurement accuracy of the quantitative value of the substrate such as correcting the quantitative value of the substrate. By improving the accuracy, the measurement accuracy of the substrate can also be improved.

  The cavity 41 has an opening 41c (see FIG. 2) for introducing a sample solution, and the second electrode (working electrode 23 and counter electrode 24) is more cavity than the first electrode (working electrode 21 and counter electrode 22). It is installed at a position close to the opening 41 c of 41. As described above, the first electrode (substrate measurement electrode) is installed on the back side of the cavity 41, and the reagent layer including the mediator is formed on the first electrode. Therefore, the second electrode comes into contact with the sample solution before the first electrode. That is, it is possible to measure the hematocrit value of the sample solution before the electrode active material contained in the reagent layer 3 is dissolved in the sample solution. Therefore, the electrode active material can be prevented from being present in the vicinity of the hematocrit measurement electrode more reliably, and the measurement accuracy of the target object other than the substrate can be increased. As a result, the measurement accuracy of the substrate can also be improved. it can.

  As a modification of the present embodiment, as shown in FIG. 4, the cavity 41 is between the reagent layer 3 and the portions 23 a and 24 a exposed in the cavity 41 of the second electrode (working electrode 23 and counter electrode 24). The shape which has the constriction 41a may be sufficient. By providing such a constriction 41a, the electrode active material contained in the reagent layer 3 can be prevented from moving to the second electrode side, and the measurement accuracy of an object other than the substrate can be improved more reliably. As a result, the measurement accuracy of the substrate can also be improved.

  In the present embodiment, examples of the substrate (analyte) include glucose (blood glucose), lactic acid, cholesterol, alcohol, sarcosine, fructosylamine, pyruvic acid, lactic acid, and hydroxybutyric acid.

  The material of the insulating substrate 1 is not particularly limited, and examples thereof include a plastic material such as a PET (polyethylene terephthalate) film, a photosensitive material, paper, glass, ceramic, or a biodegradable material. These materials are also used as materials for the spacer 4 and the cover 5.

  On the insulating substrate 1, at least a first electrode (working electrode 21 and counter electrode 22) for electrochemically quantifying the substrate and a second electrode (action) for quantifying an object other than the substrate by the alternating current method. A pole 23 and a counter electrode 24) are provided. On the insulating substrate 1, in addition to the first electrode and the second electrode, a reference electrode serving as a potential reference when measuring the electrode potential, a detection electrode for detecting that the sample is supplied to the cavity 41, and the like. It may be provided.

  Materials for these electrodes (working electrode, counter electrode, reference electrode, detection electrode, etc.) include platinum, gold, palladium and other precious metals, carbon, copper, aluminum, nickel, titanium, ITO (Indium Tin Oxide) ), ZnO (zinc oxide) and the like. The electrode is formed by forming a conductive layer made of the above material on one surface of the insulating substrate 1 using, for example, screen printing or sputtering, and further patterning using laser processing, photolithography, etc. Can be produced. The electrode and the surface of the insulating substrate 1 may be subjected to plasma treatment.

  In the present embodiment, the reagent layer 3 is formed on part of the portions 21 a and 22 a exposed in the cavity 41 on the surface of the first electrode (working electrode 21 and counter electrode 22). The reagent layer 3 can be formed, for example, by dropping a reagent solution and drying the reagent solution. For example, the reagent solution 3 may be formed by dropping an enzyme solution, an electrode active material (mediator) solution, and the like in order, or after dropping a mixed solution containing the enzyme, the electrode active material, and the like. May be dried to form the reagent layer 3.

  Examples of the enzyme include glucose oxidase, glucose dehydrogenase, alcohol oxidase, alcohol dehydrogenase, lactate oxidase, lactate dehydrogenase, cholesterol esterase, cholesterol oxidase, sarcosine oxidase, fructosylamine oxidase, pyruvate oxidase, hydroxybutyrate dehydrogenase, creatininase , Creatinase and DNA polymerase. To produce various biosensors by selecting these enzymes according to the substrate (glucose, alcohol, lactic acid, cholesterol, sarcosine, fructosylamine, pyruvic acid, hydroxybutyric acid, etc.) that is the main measurement target substance Can do.

  For example, glucose oxidase or glucose dehydrogenase can be used to produce a glucose sensor that detects glucose in a blood sample, and alcohol oxidase or alcohol dehydrogenase can be used to produce an alcohol sensor that detects ethanol in a blood sample. For example, a lactic acid sensor for detecting lactic acid in a blood sample can be produced, and a total cholesterol sensor can be produced by using a mixture of cholesterol esterase and cholesterol oxidase.

  The electrode active material (mediator) is a compound (electron carrier) that mediates electron transfer between the working electrode 21 and the counter electrode 22, and is preferably a substance that itself performs a redox reaction. As the electrode active material, for example, potassium ferricyanide, ferrocene, ferrocene derivatives, benzoquinone, quinone derivatives, osmium complexes, ruthenium complexes, and the like can be used.

  The reagent layer 3 may contain a hydrophilic polymer. In this case, the reagent layer 3 can be easily fixed to the surface of the electrode layer. The hydrophilic polymer also functions as a filtering agent for filtering impurities (such as blood cells in blood) in the sample solution. However, in the biosensor of this embodiment, since the blood glucose level can be corrected according to the blood cell volume, it is not necessary to filter blood cells in the blood, and it is necessary to add a hydrophilic polymer to the reagent layer. Has the advantage of not.

  The hydrophilic polymer is not particularly limited, for example, carbonyl group, acyl group, carboxyl group, aldehyde group, sulfo group, sulfonyl group, sulfoxide group, tosyl group, nitro group, nitroso group, ester group, keto group, Examples thereof include hydrophilic polymers having a ketene group. Examples of the hydrophilic polymer having a carboxyl group include carboxymethyl cellulose and carboxymethyl ethyl cellulose, and carboxymethyl cellulose (CMC) is preferable.

  Further, the reagent layer 3 may contain a hydrophilizing agent for promoting the introduction of the specimen. Examples of the hydrophilizing agent include surfactants such as Triton X100, Tween 20, sodium bis (2-ethylhexyl) sulfosuccinate, and phospholipids such as lecithin. The hydrophilizing agent may be mixed with the above reagent and dropped, or may be further dropped from above the reagent layer. Moreover, you may form a hydrophilizing agent in the below-mentioned cover.

  The material of the cover 5 is preferably an insulating material. For example, a plastic such as a PET film, a photosensitive material, paper, glass, ceramic, or a biodegradable material can be used.

  The cover 5 preferably has an air hole 5 a communicating with the cavity 41 formed by the spacer 4. This is because not only the diffusion of the sample liquid but also the sample is sucked toward the air hole 5a by capillary action, so that the sample can be easily introduced into the cavity 41.

(How to use biosensor)
Hereinafter, as an example of a method for using the biosensor of the present embodiment, an example of a measurement process for quantifying glucose in a blood sample will be described with reference to FIG.

  First, the biosensor (biochip) of this embodiment is mounted on a dedicated measuring instrument. When the measuring device detects that the biosensor 100 is mounted, the blood sample detection electrode (not shown) for detecting that the blood sample is supplied to the cavity 41 of the biosensor 100 is used for blood sample detection. Is applied (step S1).

  Next, blood is brought into contact with the distal end portion (opening 41c) of the cavity 41, and the blood is introduced into the cavity 41 using capillary action. When a blood sample is supplied to the cavity 41 and the blood sample detection electrode is in liquid contact with the blood sample, the resistance value of the electrode changes and the current flowing through the electrode increases. Is detected (step S2).

  When it is detected that the blood sample is supplied to the cavity 41, an AC voltage for Hct measurement is applied between the working electrode 23 for measuring the hematocrit value (Hct) and the counter electrode 24 (step S3), and the response current Is measured at a predetermined timing (step S4). Thus, the alternating current method is used for measuring the hematocrit value in steps S3 and S4.

The frequency of the alternating voltage used in the alternating current method is preferably at least one frequency within the range of 1 kHz to 30 MHz, more preferably at least one frequency within the range of 1 kHz to 20 MHz, and more preferably 1 kHz. It is at least one frequency within the range of 10 MHz or less. By measuring the response current, impedance (ratio of phasor-displayed voltage and current in the AC circuit), etc., using an AC voltage with a frequency within this range, the concentration of the substance (other than the substrate) in the sample solution can be measured. It can measure with high reproducibility and high accuracy And the following formula:
Hematocrit (%) = 100− [volume ratio of plasma in blood] (%)
Thus, the hematocrit value can be obtained by converting the volume ratio of plasma in blood into hematocrit (volume ratio of blood cells in blood).

  In addition, it is preferable that the frequency of alternating current used for a measurement is only one. If there is only one AC generator, the number of measurements increases when there are multiple AC frequencies used for measurement. However, by using only one AC frequency for measurement, the number of measurements is one. Therefore, the measurement time can be shortened.

  Next, a blood glucose level measurement voltage (for example, 0.3 V) is applied between the working electrode 21 for measuring glucose concentration (blood glucose level) and the counter electrode 22 (step S5), and a predetermined time after the voltage is applied. The current value of the oxidation current after elapse of (for example, 3 to 5 seconds) is measured (step S6).

  That is, when a blood sample is supplied to the reagent layer 3 by diffusion or capillary action, a reducing substance is generated by the reaction of glucose in the blood sample with an enzyme and an electrode active material (mediator). Here, a voltage is applied between the working electrode 21 and the counter electrode 22 by a measuring device electrically connected to the working electrode 21 and the counter electrode 22 for measuring the blood glucose level, and this reducing substance (reduced form of the electrode active material). ) To measure the oxidation current obtained. In this way, glucose contained in the blood sample is quantified.

  Then, glucose contained in the blood sample is quantified based on the hematocrit value derived based on the measured current value of the response current, the measured current value of the oxidation current, and a predetermined conversion formula. . That is, the quantitative value of glucose corrected using the hematocrit value is obtained.

  As described above, the measurement process using the biosensor according to the present embodiment quantifies the measurement target substance based on the oxidation current and the hematocrit value of the reducing substance, thereby removing the influence of the hematocrit value of the blood sample. Glucose can be accurately quantified.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these.

Example 1
Basically, the biosensor having the configuration shown in FIGS. 1 to 3 described in the first embodiment was manufactured as the test sensor 1. However, in this example, since only the hematocrit value was measured, the reagent layer 3 was not formed.

  In the test sensor 1, gold is used as an electrode material, a metal film made of gold is formed by a sputtering method, and this is patterned to form a glucose measurement electrode (electrode film) and a hematocrit measurement electrode. (Electrode film) was prepared. The material of the insulating substrate 1 and the spacer is polyethylene terephthalate.

  Next, as a sample liquid (model sample of a blood cell-containing liquid) to be measured for the hematocrit value, beads having a concentration of any of 0, 20, 40, and 70% by volume (made of polystyrene, volume average particle diameter of 8 μm, approximately true A suspension (dispersion medium: 100 mM phosphate buffer) containing a spherical shape was prepared. The average particle size of the beads is close to that of blood cells.

As shown in FIG. 6, the measurement was performed in a state where a resistor (330 kΩ) was connected in series to the hematocrit measurement electrode of the biosensor (sensor chip) 100. The sample liquid (suspension) is supplied into the cavities of the biosensors of the test sensor 1 and the comparison sensor 1, and an AC voltage of 7.08 Vpp (between the working electrode 23 and the counter electrode 24 of the hematocrit measurement electrode) Input voltage V _in ) was applied, and the voltage applied to the resistor was measured with an oscilloscope as an output value (output voltage V _out ). This output voltage was measured by setting an average of 10 wavelengths for visual measurement. The measurement frequency was 1 kHz to 50 MHz, and the measurement temperature was room temperature (about 25 ° C.). Then, the current value was obtained from the output voltage _Vout and the resistance (330 kΩ) according to Ohm's law.

(Comparative Example 1)
For comparison, a current value was measured by an alternating current method in the same manner as in Example 1 using a comparative sample solution containing an electrode active material instead of the sample solution. In addition, the comparison sample solution is an electrode active material (potassium ferricyanide 280 mM and potassium ferrocyanide) so that the concentration of the electrode active material can be obtained when a normal blood glucose concentration of 100 mg / dL of glucose is reacted in a healthy person. 66 mM) is the same as the sample solution except that it is added.

  The measurement results of Example 1 (without electrode active material) and Comparative Example 1 (with electrode active material) are shown in FIGS. 7 and 8, respectively.

  As shown in FIG. 7 and FIG. 8, when the electrode active material is present (FIG. 8), this electrode active material is measured, so compared to the case where the electrode active material is not present (FIG. 7), Although the decrease in the current value with respect to the increase in the bead concentration does not change much, the total amount of current increases. From this, it can be seen that when an electrode active material is present, the responsiveness (measurement accuracy) to the amount of beads decreases.

  Further, from the results shown in FIG. 7 and FIG. 8, when the frequency of the AC voltage is in the range of 1 kHz to 20 MHz, the current value tends to decrease as the particle (bead) concentration in the sample liquid increases. It can be seen. In addition, this is considered to be because the response current according to the amount of the liquid part other than the beads in the sample liquid is measured when an AC voltage in this frequency range is applied. Rather than measuring the response current according to the amount of the liquid, the response current according to the amount of the liquid part is not affected by the shape or distribution of the solid, and is not affected by the shape or distribution of the solid. ) It is considered that the substance concentration can be measured with high reproducibility and high accuracy.

(Example 2)
The current value was measured by the alternating current method in the same manner as in Example 1 except that ovine blood containing blood cells having predetermined blood cell concentrations (0, 20, 40, and 70% by volume) was used instead of the sample solution. It was. As for ovine blood, ovine blood was once centrifuged at 1000 × g for 10 min, divided into blood cell components and liquid plasma components, and reconstituted so that the volume ratio would be 0, 20, 40 and 70%. A thing was used.

(Comparative Example 2)
As a comparison, the current value was measured by the alternating current method in the same manner as in Example 2 using a lamb fluid containing an electrode active material instead of the lamb blood. The electrode active material was added in the same manner as in Comparative Example 1.

  The measurement results of Example 2 (without electrode active material) and Comparative Example 2 (with electrode active material) are shown in FIGS. 9 and 10, respectively.

  As shown in FIGS. 9 and 10, when the electrode active material is present (FIG. 10), the decrease in the current value with respect to the increase in blood cell concentration is less than when the electrode active material is not present (FIG. 9). Although it does not change, it can be seen that the response to the amount of beads decreases because the total amount of current increases.

  Further, from the results shown in FIG. 9 and FIG. 10, when the frequency of the AC voltage is in the range of 1 kHz to 10 MHz, the current value tends to decrease as the blood cell concentration in the ovine blood increases.

(Example 3)
The current value was measured by the AC method in the same manner as in Example 2 except that human blood was used instead of the sheep blood. However, the center of the input voltage was 1.25 V, and the input voltage was 1.6 Vpp. In this experiment, the input voltage was changed from that in Example 2. In this experiment, the voltage was applied via a function generator and measured with an oscilloscope. This is because the above conditions are taken into consideration. The measurement results are shown in FIG.

  From the results shown in FIG. 11, it can be seen that even in the case of human blood, responsiveness similar to that in the case of ovine blood (FIG. 9: Example 2) is shown. In addition, when the frequency of the AC voltage is in the range of 1 kHz to 30 MHz, the current value tends to decrease as the blood cell concentration in human blood increases.

Example 4
About the case where the alternating voltage of the frequency around 100 kHz was used, it measured by the alternating current method similarly to Example 3. FIG. The frequency of the AC voltage was 0.1, 1, 3, 5, 10, 20, 50, 100, 200, and 500. Moreover, in order to evaluate reproducibility, 10 measurements were performed about all the measurements. However, the conversion from the output voltage to the current value was not performed, and the output voltage was shown as a result.

  FIG. 12 shows a graph created from the average value of 10 measurements with the frequency on the horizontal axis and the output voltage on the vertical axis. In FIG. 13, the horizontal axis represents the blood cell concentration, and the amount of change in impedance (value obtained by subtracting the output value at each blood cell concentration of 20, 40, and 70% from the output value at the blood cell concentration of 0%). A graph with axes is shown. Furthermore, in FIG. 14, in each measurement, the graph which shows the CV value about 10 measurements is shown.

  From the results shown in FIG. 12, it can be seen that the output voltage increases as the frequency of the AC voltage increases, but the output voltage reaches a plateau when the frequency of the AC voltage is about 50 kHz (or 100 kHz). Therefore, even if an AC voltage having a frequency higher than 100 kHz is used, it is considered that the measurement accuracy cannot be increased very efficiently.

  From the results shown in FIG. 13, it can be seen that if the frequency of the AC voltage is 1 kHz or more, there is responsiveness to the blood cell concentration. It can also be seen that when the frequency of the AC voltage is 10 kHz or more and 500 kHz or less, the proportionality to the blood cell concentration is good (the linearity of the graph is high).

  From the results shown in FIG. 14, it can be seen that the CV value is within 2% when the frequency of the AC voltage is 3 kHz or more.

  DESCRIPTION OF SYMBOLS 1 Insulating substrate, 100 Biosensor, 21 (1st electrode) working electrode, 22 (1st electrode) counter electrode, 23 (2nd electrode) working electrode, 24 (2nd electrode) counter electrode, 3 Reagent layer 4, spacer, 41 cavity, 41c opening, 42 notch, 5 cover, 5a air hole, 6 sample solution.

Claims (4)

A biosensor for quantifying a substrate contained in a sample solution,
An insulating substrate;
A first electrode for quantifying a substrate, including a working electrode and a counter electrode provided on one surface of the insulating substrate;
A second electrode for quantifying an object other than the substrate using an alternating current method, including a working electrode and a counter electrode provided on one surface of the insulating substrate;
A reagent layer formed on at least a part of the surface of the first electrode opposite to the insulating substrate, the enzyme reacting with the substrate, and an electrode active material;
It has a notch for forming a cavity for guiding the sample solution to the reagent layer, and is disposed on the first electrode and the second electrode so that the reagent layer is located inside the notch. Spacers,
A cover provided on a surface opposite to the insulating substrate of the spacer so as to cover at least the notch,
A biosensor characterized in that no electrode active material is present on at least the surface of the second electrode. The cavity has an opening for introducing the sample liquid,
The biosensor according to claim 1, wherein the second electrode is installed at a position closer to the opening of the cavity than the first electrode.   The biosensor according to claim 1, wherein the cavity has a constricted shape between the reagent layer and a portion of the second electrode exposed in the cavity.   The biosensor according to claim 3, wherein the frequency of the AC voltage used in the AC method is at least one frequency within a range of 1 kHz to 30 MHz.

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