JP4601809B2 - Biosensor and substrate measurement method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a biosensor and a substrate measuring method capable of simply and quickly carrying out rapid and highly accurate quantification of a measurement object in a sample.
[0002]
[Prior art]
Conventionally, as a method for easily quantifying a specific component in a sample without diluting or stirring the sample solution, a method using the following biosensor has been proposed (Japanese Patent Laid-Open No. 2-062952). ).
The biosensor used here forms an electrode system consisting of a measurement electrode, a counter electrode and a reference electrode on an insulating substrate by a method such as screen printing, and on this electrode system, a hydrophilic polymer, an oxidoreductase, and An enzyme reaction layer containing an electron mediator is formed. A buffering agent is added to the enzyme reaction layer as necessary.
When a sample solution containing a substrate is dropped onto the enzyme reaction layer of the biosensor produced in this way, the enzyme reaction layer dissolves and the enzyme and the substrate react, and the electron mediator is reduced accordingly. After the completion of the enzyme reaction, the reduced electron mediator is electrochemically oxidized, and the substrate concentration in the sample solution can be determined from the oxidation current value obtained at this time.
Such a biosensor can in principle measure various substances by selecting an enzyme that uses the substance to be measured as a substrate.
For example, if glucose oxidase is used as the oxidoreductase, a biosensor that measures glucose concentration in blood can be constructed. This sensor is widely used as a glucose sensor.
Further, if cholesterol oxidase is used, a biosensor for measuring cholesterol in serum can be constructed.
[0003]
Usually, the serum cholesterol level used as a diagnostic guide is the sum of the concentrations of cholesterol and cholesterol ester. Cholesterol esters cannot be a substrate for oxidation reaction by cholesterol oxidase.
Therefore, in order to measure serum cholesterol level as a diagnostic guideline, a process of changing cholesterol ester to cholesterol is required. Cholesterol esterase is known as an enzyme that catalyzes this process.
By using a biosensor containing the cholesterol esterase and cholesterol oxidase in the enzyme reaction layer, the total cholesterol concentration in the serum can be measured.
[0004]
However, for example, the measurement of cholesterol can be influenced by cholesterol present in the cell membrane. In addition, the cholesterol esterase in the reaction reagent requires a surfactant to increase the reactivity. Surfactants often destroy cell membranes, so intracellular substances can directly or indirectly affect enzymatic or electrode reactions. For this reason, in the cholesterol sensor, the enzyme reaction and the subsequent electrode reaction need to be performed in plasma or serum. In addition to the cholesterol sensor, the presence of blood cells in the blood may affect the response value. Therefore, it is ideal that the enzyme reaction and the electrode reaction are performed in a solution that does not contain blood cells.
Centrifugation is well known as a method for separating plasma or serum from whole blood. However, the method using centrifugation is time consuming and complicated.
[0005]
U.S. Pat. No. 3,607,092 discloses a membrane for testing blood. This membrane has a thin film layer that is permeable to solutions but impermeable to solids such as blood cells and macromolecules such as proteins. It is possible to remove blood cells with such a thin film. However, since solid components accumulate on the thin film as blood passes through, a thin film layer having a large area is required to obtain an amount of filtrate necessary for the reaction of the biosensor. Therefore, the thin film is not necessarily suitable.
U.S. Pat. No. 4,477,575 discloses an apparatus and method for separating serum by passing whole blood through a glass fiber filter. Such a method of separating serum from whole blood using a filter made of fiber or porous material can be used for the biosensor. However, this method does not hold blood cells with a filter, but simply delays the flow to separate blood cells and plasma. Therefore, in order to use this method for the above biosensor, it is necessary to obtain more plasma or serum filtered by the filter than necessary for the reaction of the biosensor while blood cells do not flow out of the filter.
[0006]
A biosensor having blood cell filtration ability by installing a filter satisfying such conditions between a part where the electrode system and reaction reagent system of the biosensor are arranged and a part supplying blood as a sample Can be configured. An example is shown in FIG. FIG. 4 shows an exploded perspective view excluding the reaction reagent layer. In FIG. 4, a silver paste is printed by screen printing on an insulating substrate 1 made of polyethylene terephthalate to form leads 2, 3 and a base for an electrode system. Further, an electrode system including the measuring electrode 6 and the counter electrode 7 is formed on the substrate 1 by printing a conductive carbon paste containing a resin binder, and the insulating layer 10 is formed by printing the insulating paste. Each is formed. The measurement electrode 6 is connected to the lead 2 and the counter electrode 7 is connected to the lead 3. The insulating layer 10 keeps the exposed areas of the measurement electrode 6 and the counter electrode 7 constant, and partially covers the leads.
[0007]
The positional relationship as shown by the alternate long and short dash line in FIG. 4 is the insulating substrate 1 on which the electrode system is formed in this way, the cover 11 having the air holes 12, the spacer 13, and the filter 16 having blood cell filtration ability. To make a biosensor. The filter 16 is cut between the cover 11 and the insulating substrate 1 so as to be fitted into the sample solution supply path formed by the slit 14 of the spacer 13. Reference numeral 16 a denotes a portion where the filter 16 contacts the insulating substrate 1. The filter 16 is disposed between the electrode system and the sample supply unit 15 without covering the electrode system including the measurement electrode 6 and the counter electrode 7 in the sample solution supply path.
[0008]
In the biosensor having such a configuration, when blood is dropped on the sample supply unit 15, blood penetrates into the filter from the end of the filter 16 on the sample supply unit side. In the filter, the blood cell penetration rate is slower than that of the liquid component plasma, so that the plasma oozes out from the end of the filter on the electrode system side. The soaked plasma is composed of an enzyme or the like, and the sample solution is supplied from the vicinity of the electrode system to the air hole 12 portion while dissolving the reaction reagent carried on the electrode covering position or on the cover rear surface immediately above the electrode system. Fill the whole road. When the entire sample liquid supply path is filled with the liquid, the flow of the liquid in the filter 16 also stops, and at that time, the blood cells do not reach the end of the filter on the electrode system side, but remain in that position. Through such a blood cell filtration process, a chemical reaction occurs between the reaction reagent layer dissolved in plasma and the measurement component in plasma, or cholesterol in the case of a cholesterol sensor. By measuring this, the components in plasma are measured.
[0009]
However, as described above, in this method, the blood cells are not held by the filter, but are simply separated from the blood cells by delaying the flow. It is necessary to obtain more plasma or serum filtered by the method than necessary for the reaction of the biosensor. For this purpose, the amount of liquid that can be absorbed by the filter must be considerably larger than the volume of the sample liquid supply path.
Due to the restriction of the ratio of the volume of the sample liquid supply path and the amount of liquid absorbed by the filter, a part of the reaction reagent dissolved by the liquid such as plasma that reaches the sample liquid supply path diffuses into the filter. As a result, the concentration of the reaction reagent in the sample solution supply path is lowered, and the response may be lowered. In particular, when blood containing a high concentration of lipids is used, the viscosity of plasma is relatively high, so that the penetration of plasma into the reaction reagent layer and the dissolution of the reaction reagent layer also proceed relatively slowly. There was also a phenomenon that the reagent was diffused sequentially into the filter.
[0010]
[Problems to be solved by the invention]
In view of the above problems, the present invention, in a biosensor including a filter having the ability to filter solid components such as blood cells, prevents the reaction reagent dissolved by the sample liquid filtered by the filter from diffusing into the filter, An object of the present invention is to provide a biosensor exhibiting stable responsiveness.
Another object of the present invention is to provide a method for measuring a substrate using such a biosensor.
[0011]
[Means for Solving the Problems]
  The biosensor of the present invention includes an insulating substrate, a first electrode system having at least a measurement electrode and a counter electrode provided on the substrate, and a sample supply unit on the substrate between the substrate and the substrate. A cover member that forms a sample solution supply path for supplying the sample solution to the first electrode system, at least an electron mediator and an oxidation-reductionenzymeA reaction reagent system disposed on or near the first electrode system, a filter installed between the first electrode system and the sample supply unit in the sample liquid supply path,andA second electrode comprising a pair of electrodes in which at least one electrode for electrolyzing the sample solution upstream of the first electrode system in the sample solution supply path is disposed between the first electrode system and the filter. With electrode systemThe other electrode of the second electrode system is provided on the sample supply unit side.It is characterized by that. It is preferable to have a liquid junction detection electrode on the downstream side of the first electrode system in the sample liquid supply path..
[0012]
In the substrate measuring method of the present invention, the step of adding a sample solution to the sample supply unit using the biosensor, the sample solution completely covers the first electrode system via the filter. At this point, a voltage sufficient to generate gas from the electrode disposed between the first electrode system and the filter is applied to the second electrode system, so that the sample liquid on the first electrode system side A step of generating bubbles at a contact portion with the filter, and a step of obtaining a predetermined response by the first electrode system.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
  The biosensor of the present invention includes an insulating substrate, a first electrode system having at least a measurement electrode and a counter electrode provided on the substrate, and a sample supply unit on the substrate between the substrate and the substrate. A cover member that forms a sample solution supply path for supplying the sample solution to the first electrode system, at least an electron mediator and an oxidation-reductionenzymeAnd a reaction reagent system disposed on or near the first electrode system, and a filter disposed between the first electrode system and the sample supply unit in the sample liquid supply path And at least one electrode for electrolyzing the sample solution upstream of the first electrode system in the sample solution supply path comprises a pair of electrodes disposed between the first electrode system and the filter. The second electrode systemAnd the other electrode of the second electrode system is provided on the sample supply unit side.It is characterized by that.
  In the measurement method using this biosensor, when the sample solution is added to the sample supply unit and the sample solution completely covers the first electrode system via the filter, A voltage sufficient to generate a gas from an electrode disposed between one electrode system and the filter is applied to the second electrode system. As a result, bubbles are generated at the contact portion of the sample solution on the first electrode system side with the filter, and the contact between the sample solution in which the reaction reagent is dissolved by the bubbles and the filter is interrupted, and Prevent diffusion. Thereafter, a predetermined response is obtained by the first electrode system.
[0014]
As described above, the present invention generates a gas in the sample solution, thereby cutting the substantial contact between the sample solution that has reached the first electrode system side and the filter, and the reaction reagent dissolved in the sample solution can be obtained. It is possible to prevent diffusion in the filter and obtain a stable response. The electrode for generating the gas, that is, the electrode provided between the first electrode system and the filter is equal to or larger than the width of the sample liquid supply path in order to cut off the contact between the sample liquid and the filter more completely. It is desirable to have a width of
The other electrode constituting the second electrode system is preferably arranged on the sample supply unit side upstream of the one electrode. The lower part of the filter may be in contact with the filter. When a voltage of 2.5 to 3 V is applied to the second electrode system, oxygen gas is generated from the anode and hydrogen gas is generated from the cathode. When a voltage is applied to the second electrode system, an electrode provided between the first electrode system and the filter is a cathode, and the other electrode is an anode. It is also possible to make the electrode provided between the first electrode system and the filter an anode. However, in that case, oxygen gas generated from the electrode may affect the enzyme reaction system, which is not preferable.
[0015]
In the measurement, the sample solution is added to the sample supply unit, filtered through a filter, and whether or not the sample solution has completely covered the first electrode system is determined by visual observation or using a liquid junction detection electrode. Detect electrically. When visually observed, the cover needs to be made of a transparent material. Electrical detection is performed by a change in electrical conductivity between the liquid junction detection electrode and any electrode of the second electrode system. That is, when the sample liquid completely covers the first electrode system, the liquid junction detection electrode and one of the electrodes of the second electrode system are in liquid junction, and the electrical conductivity between the two electrodes rapidly increases. Therefore, it can be confirmed easily.
The biosensor of the present invention is arranged in the order of the filter, the counter electrode, and the measurement electrode in the order that the positional relationship between the measurement electrode and the counter electrode is closer to the sample supply unit among the electrodes constituting the filter and the first electrode system. It is desirable. This is because, when bubbles are generated at the end of the filter on the first electrode system side using the second electrode system, if the filter and the measurement electrode are adjacent to each other, the generated bubbles contact the measurement electrode. This may hinder accurate measurement.
[0016]
Various oxidoreductases constituting the reaction reagent system can be used.
For example, glucose oxidase, lactate oxidase, cholesterol oxidase and the like can be mentioned.
When measuring serum cholesterol levels, an enzyme having cholesterol oxidase and cholesterol ester hydrolyzing ability is used. Examples of the enzyme having the ability to hydrolyze cholesterol ester include cholesterol esterase and lipoprotein lipase. In particular, cholesterol esterase is advantageous because it can rapidly convert cholesterol ester to cholesterol by using an appropriate surfactant.
When using an enzyme having cholesterol ester hydrolyzing ability, it is preferable to include a surfactant having an effect of improving the activity of the enzyme in the reaction reagent because the time required for the enzyme reaction can be shortened. For example, surfactants that improve the activity of cholesterol esterase include n-octyl-β-D-thioglucoside, polyethylene glycol monododecyl ether, sodium cholate, dodecyl-β-maltoside, sucrose monolaurate, deoxychol Acid sodium, sodium taurodeoxycholate, N, N-bis (3-D-gluconamidopropyl) coleamide, N, N-bis (3-D-gluconamidopropyl) deoxycholamide, polyoxyethylene-p- t-Octylphenyl ether (“Triton X-100”) and the like can be arbitrarily used.
[0017]
When the electrode system of the biosensor is formed using an electrochemically stable metal such as platinum, the obtained oxidation current value does not include an error. However, since such a metal is expensive, in a disposable sensor, a silver electrode is formed using a silver paste or the like, and then this is covered with a carbon paste to form an electrode system. However, when a surfactant is contained in the sample solution, the sample solution infiltrates between the carbon particles by the action of the surfactant. As a result, the activity of the carbon electrode may decrease. In addition, the sample solution comes into contact with the silver electrode. For this reason, when a voltage is applied to the measurement electrode in this state, the silver electrode causes an oxidation reaction to generate a current, which may give a positive error to the measured current value.
In order to suppress such a phenomenon, there is a method of coating the surface of the electrode system with a hydrophilic polymer. This hydrophilic polymer becomes a viscous layer even when the sample solution is introduced, and suppresses the sample solution from contacting the electrode.
[0018]
  Such hydrophilic polymers include carboxymethyl cellulose, polyvinyl pyrrolidone, polyvinyl alcohol, ethyl cellulose, hydroxypropyl cellulose, gelatin, polyacrylic acid and its salts, starch and its derivatives, maleic anhydride polymer and its salts, polyacrylamide , Methacrylate resin, poly-2-hydroxyethyl methacrylateTExamples include chlorate.
  In addition to the method using the hydrophilic polymer as described above, the influence of the surfactant as described above can be suppressed by forming the part that contacts the sample liquid of the electrode system only with the carbon paste, and ensuring conductivity. The silver paste used in the above can also be obtained by using a printed electrode only in the portion covered with the insulating layer. In this case, the hydrophilic polymer layer is not necessary. However, these hydrophilic polymers also have the effect of preventing the protein in the sample solution or the mixture of the sample solution and the reaction reagent from adsorbing to the electrode surface and reducing the activity of the electrode reaction. Even when a printed electrode is used, it is preferably used.
[0019]
When the biosensor electrode system is formed of silver and carbon, an electron mediator is contained in the reagent supported on the carrier.
As such an electron mediator, a water-soluble compound such as potassium ferricyanide, p-benzoquinone, phenazine methosulphate, and a ferrocene derivative (oxidized form) that can mediate electron transfer between an enzyme and an electrode can be arbitrarily used.
As a method of measuring the oxidation current, there are a first electrode system, a two-electrode system with only a measuring electrode and a counter electrode, and a three-electrode method with a reference electrode, and the three-electrode method allows more accurate measurement is there.
[0020]
【Example】
Hereinafter, the present invention will be described in detail with reference to specific examples. Note that the drawings are schematic and the relative sizes of the elements are not necessarily accurate.
FIG. 1 is an exploded perspective view excluding a reagent layer of a biosensor according to an embodiment of the present invention, and FIG. 2 is a longitudinal sectional view of the main part.
A silver paste is printed by screen printing on an insulating substrate 1 made of polyethylene terephthalate to form leads 2, 3, 4, 5 and a base for an electrode system. Further, by printing a conductive carbon paste containing a resin binder on the substrate 1, a first electrode system composed of the measurement electrode 6 and the counter electrode 7 and a second electrode system composed of the cathode 8 and the anode 9 are formed. Insulating layers 10 are formed by printing and printing an insulating paste. The measurement electrode 6 is connected to the lead 2, the counter electrode 7 is connected to the lead 3, the cathode 8 is connected to the lead 4, and the anode 9 is connected to the lead 5. The insulating layer 10 keeps the exposed areas of the measurement electrode 6 and the counter electrode 7 constant, and partially covers the leads. In this way, the insulating substrate 1 on which the electrode system is formed, the cover 11 having the air holes 12, the spacer 13, and the filter 16 having blood cell filtering ability are in a positional relationship as shown by a one-dot chain line in FIG. To make a biosensor.
[0021]
A sample solution supply path is formed between the insulating substrate 1 and the cover 11 by the slit 14 of the spacer 13. The filter 16 is cut so as to fit into the sample solution supply path. Reference numeral 16 a denotes a portion where the filter 16 contacts the insulating substrate 1. The filter 16 is disposed between the first electrode system including the measurement electrode 6 and the counter electrode 7 and the sample supply unit 15 in the sample solution supply path. The cathode 8 of the second electrode system is located between the first electrode system and the filter 16, and the anode 9 is disposed on the sample supply unit 15 side. The secondary side end of the filter 16 may or may not be in contact with the cathode 8.
When the sample solution is dropped on the sample supply unit 15 and brought into contact with the end of the filter 16 on the sample supply unit side, the sample solution is sucked into the filter 16, and the solid solution such as blood cells is removed by the filter 16. It moves through the supply path and is introduced into the sensor. In the present embodiment, the length from the end of the slit 14 on the sample supply part side to the outer periphery of the air hole 12 is 12.5 mm, the width of the slit 14 is 2.0 mm, and the depth of the slit 14 is 0.1 mm. is there. In order to define the overall dimensions of the biosensor, the above dimensions are specified, but the dimensions are not necessarily limited thereto.
[0022]
As shown in FIG. 2, a hydrophilic polymer layer 17 and an electron mediator layer 18 are formed on the first electrode system so as to cover it. These layers may or may not be in contact with the cathode 8.
On the back surface of the cover 11 in the sample solution supply path, an enzyme / surfactant layer 19 composed of an enzyme and a surfactant is formed in a region extending from the first electrode system to the vicinity of the air hole 12. If the enzyme / surfactant layer 19 and the end of the filter 16 on the secondary side are in contact with each other, the sample solution is likely to flow in, but the contact is not essential.
Next, FIG. 3 is an exploded perspective view of a biosensor according to another embodiment of the present invention. The configuration is the same as that shown in FIG. 1 except that a liquid junction detection electrode 21 and a silver lead 20 connected thereto are provided on the downstream side of the first electrode system.
[0023]
Example 1
In order to produce a cholesterol sensor which is an example of a biosensor, first, a sodium salt of carboxymethyl cellulose (hereinafter referred to as CMC), which is a hydrophilic polymer, is formed on the electrode system on the insulating substrate 1 of FIG. A 5 wt% aqueous solution was dropped and dried in a hot air dryer at 50 ° C. for 10 minutes to form a CMC layer 17. Subsequently, 4 μl of an aqueous solution of potassium ferricyanide as an electron mediator (corresponding to 70 mM potassium ferricyanide) is dropped on the CMC layer so as to cover the CMC layer 17 and dried in a hot air dryer at 50 ° C. for 10 minutes. A potassium ferricyanide layer 18 was formed.
[0024]
On the other hand, 2 μl of a 2 wt% ethanol solution of Triton X-100, which is a surfactant, is dropped into a recess formed in the slit 14 portion of the cover member where the cover 11 and the spacer 13 are bonded together, and dried at room temperature for 3 minutes. As a result, a surface active layer was formed. Subsequently, Triton X was added to an aqueous solution in which nocardia-derived cholesterol oxidase (EC 1.1.3.6, hereinafter abbreviated as ChOD) and Pseudomonas-derived cholesterol esterase (EC 3.1.1.13, hereinafter abbreviated as ChE) were dissolved. -100 was added. 1.5 μl of this mixed aqueous solution was dropped on the surfactant layer, frozen in liquid nitrogen, and then dried overnight in a large lyophilizer to obtain 1 unit (U) / sensor cholesterol oxidase. An enzyme / surfactant layer 19 containing 2.5 U / sensor cholesterol esterase and 2 wt% surfactant was formed.
Subsequently, a glass filter 16 (GC50 manufactured by ADVANTEC, thickness 0.19 mm) cut into a 2 mm × 8 mm rectangle was installed at the position shown in FIG. 1 in the sample liquid supply path.
Thereafter, the cover member was bonded to the substrate 1 to produce a biosensor as shown in FIGS.
[0025]
  As a sample liquid, 20 μl of a whole blood sample is dropped on the sample liquid supply unit 15, and it is visually confirmed that the filtered liquid has reached the outer periphery of the air hole 12 in the sample liquid supply path. A voltage of 2.75 V was applied between the anodes 9 for 1 second. As a result, generation of bubbles was recognized from both the cathode 8 and the anode 9. Three minutes later, a + 0.5V pulse voltage was applied to the measuring electrode in the anode direction with reference to the counter electrode 7, and 5 seconds later.Measuring poleThe value of the current flowing between the electrode and the counter electrode was measured. As a result, it was possible to obtain a response depending on the cholesterol concentration in serum.
  In this example, the enzyme / surfactant is formed by freeze-drying, but it can also be formed by air-drying. In this case, however, the solubility of the reaction reagent layer is greatly deteriorated. Therefore, it takes a long time until the reaction is completed after the filtered liquid reaches the outer periphery of the air hole 12 in the sample liquid supply path. Cost.
[0026]
Example 2
  Using the electrode system of FIG. 3, a reaction reagent layer was formed in the same manner as in Example 1 to configure a sensor. Before the sample supply, the resistance value between the liquid junction detection electrode 21 and the anode 9 formed on the primary side of the filter, that is, the end on the sample supply unit side, is continuously measured. Before the sample is added, since there is no conductor between the liquid junction detection electrode 21 and the anode 9, the resistance value is almost infinite. Then, when the whole blood sample was added to the sample supply section in the same manner as in Example 1, the resistance value suddenly decreased at the moment when the filtered sample solution (plasma) reached the liquid junction detection electrode 21. After confirming this, the measurement of the resistance value was stopped, and a voltage of 2.75 V was applied between the cathode 8 and the anode 9 in the cathode direction for 1 second as in Example 1. As a result, generation of bubbles was recognized from both the cathode 8 and the anode 9. Three minutes later, a pulse voltage of +0.5 V was applied to the measurement electrode in the anode direction with reference to the counter electrode, and 5 seconds later.Measuring poleThe value of the current flowing between the electrode and the counter electrode was measured. As a result, it was possible to obtain a response depending on the cholesterol concentration in serum.
[0027]
The process from the measurement of the resistance value between the liquid junction detection electrode 21 and the anode 9 to the application of a voltage between the anode 9 and the cathode 8 is automatically performed by automating the circuit switching of the measuring instrument. It is also possible.
Further, the resistance value may be measured between the liquid junction detection electrode 21 and the cathode 8.
In the above embodiment, the width of the slit 14 for forming the sample liquid supply path between the portion where the filter is fitted and the portion where the first electrode system is present and the sample filtered by the filter flows in. Are the same, but either one may be narrow.
In addition, the arrangement and loading method of the reagents constituting the reaction reagent system are shown in the above examples as long as the reagents constituting the reaction reagent system rapidly dissolve and satisfy the requirement that the enzyme reaction proceeds smoothly. It is not limited by the conditions.
[0028]
【The invention's effect】
As described above, according to the present invention, a filter can be used for a sample solution containing a solid component such as blood cells, and this can be filtered and used for measurement, and the reaction reagent can diffuse into the filter. It is also possible to prevent a decrease in responsiveness due to. Therefore, highly accurate measurement is possible, and variations in response values are reduced.
[Brief description of the drawings]
FIG. 1 is an exploded perspective view of a biosensor according to an embodiment of the present invention, with a reagent layer removed.
FIG. 2 is a longitudinal sectional view of a main part of the sensor.
FIG. 3 is an exploded perspective view of a biosensor according to another embodiment of the present invention excluding a reagent layer.
FIG. 4 is an exploded perspective view of the biosensor in the comparative example with the reagent layer removed.
[Explanation of symbols]
1 Insulating substrate
2, 3, 4, 5, 20 leads
6 Measuring pole
7 Counter electrode
8 Cathode
9 Anode
10 Insulating layer
11 Cover
12 Air holes
13 Spacer
14 Slit
15 Sample supply section
16 filters
16a The part where the filter contacts the insulating substrate
17 Hydrophilic polymer layer
18 Layer containing electron mediator
19 Layer containing surfactant and enzyme
21 Liquid junction detection electrode

Claims (7)

Insulating substrate,
A first electrode system having at least a measurement electrode and a counter electrode provided on the substrate;
A cover member that forms a sample solution supply path for supplying a sample solution to the first electrode system from a sample supply unit on the substrate in combination with the substrate;
At least a first electrode system on or arranged reaction reagent system near the includes the electron mediator and oxidoreductase,
For electrolyzing the sample liquid in the upstream side of the first electrode system in the installed filters, and the sample solution supply pathway between the first electrode system and the sample supply unit in said sample solution supply pathway, at least A first electrode comprising a second electrode system comprising a pair of electrodes disposed between the first electrode system and the filter;
A biosensor in which the other electrode of the second electrode system is provided on the sample supply unit side .   The biosensor according to claim 1, wherein a liquid junction detection electrode is provided downstream of the first electrode system in the sample solution supply path. The measuring electrode, biosensor of claim 1, wherein arranged downstream from the counter electrode. Insulating substrate,
A first electrode system having at least a measurement electrode and a counter electrode provided on the substrate;
A cover member that forms a sample solution supply path for supplying a sample solution to the first electrode system from a sample supply unit on the substrate in combination with the substrate;
At least a first electrode system on or arranged reaction reagent system near the includes the electron mediator and oxidoreductase,
For electrolyzing the sample liquid in the upstream side of the first electrode system in the installed filters, and the sample solution supply pathway between the first electrode system and the sample supply unit in said sample solution supply pathway, at least A first electrode comprising a second electrode system comprising a pair of electrodes disposed between the first electrode system and the filter ;
A method for measuring a substrate using a biosensor in which the other electrode of the second electrode system is provided on the sample supply unit side ,
Adding a sample solution to the sample supply unit;
A voltage sufficient to generate a gas from the electrode disposed between the first electrode system and the filter when the sample solution completely covers the first electrode system through the filter. Is applied to the second electrode system to generate bubbles in the contact portion of the sample liquid on the first electrode system side with the filter, and to obtain a predetermined response by the first electrode system. Substrate measurement method. The biosensor has a liquid junction detection electrode on the downstream side of the first electrode system in the sample liquid supply path, and electricity between the liquid junction detection electrode and one of the electrodes of the second electrode system. 5. The method for measuring a substrate according to claim 4, wherein it is detected that the sample solution has reached the first electrode system due to a change in conductivity. The method for measuring a substrate according to claim 4 , wherein the voltage applied to the second electrode system is a voltage for electrolyzing water in the sample, and the gas is hydrogen gas. The method for measuring a substrate according to any one of claims 4 to 6 , wherein the oxidoreductase is cholesterol oxidase, the reaction reagent system includes an enzyme having cholesterol ester hydrolyzing ability, and the substrate is cholesterol.

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