JPH09297121A - Cholesterol sensor - Google Patents

Abstract

(57) Abstract: An object of the present invention is to provide a sensor capable of highly accurately measuring cholesterol concentration in serum in a short time. An insulating substrate, an electrode system including a measurement electrode and a counter electrode provided on the substrate, and a sample liquid supply path which is arranged on the substrate from the end of the substrate to the electrode system. And a reaction layer including a reaction reagent system provided in a portion exposed to the sample liquid supply path, wherein at least cholesterol dehydrogenase, nicotinamide adenine dinucleotide, and electrons are included in the reaction reagent system. Cholesterol sensor containing mediator.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cholesterol sensor capable of easily and rapidly quantifying cholesterol in a sample.

[0002]

2. Description of the Related Art Conventionally, various biosensors have been proposed as a system for simply quantifying a specific component in a sample without diluting or stirring the sample solution. First, a glucose sensor will be described as an example of a biosensor. As a method for quantifying glucose using an enzyme electrode,
A method in which glucose oxidase is combined with an oxygen electrode or a hydrogen peroxide electrode is generally known. Glucose oxidase uses oxygen as an electron mediator to selectively oxidize a substrate β-D-glucose into D-glucono δ-lactone. With this reaction, oxygen is reduced to hydrogen peroxide. Glucose is quantified by measuring the oxygen consumption at this time with an oxygen electrode or the hydrogen peroxide production amount with a hydrogen peroxide electrode using a platinum electrode or the like.

However, with the above method, measurement is impossible under oxygen-free conditions. Therefore, a type of glucose sensor has been developed which does not use oxygen as an electron mediator, but uses a metal complex such as potassium ferricyanide, a ferrocene derivative, a quinone derivative or an organic compound as an electron mediator. In this type of sensor, the reduced form of the electron mediator produced as a result of the enzymatic reaction is oxidized at the electrode, and the glucose concentration is obtained from the oxidation current. This method is widely applied not only to glucose but also to quantification of other substrates.

As an example of this type of biosensor, the following glucose sensor is known (Japanese Patent Laid-Open No. 2-0).
No. 62952). That is, an electrode system consisting of a measuring electrode, a counter electrode and a reference electrode is formed on an insulating substrate by a method such as screen printing, and on this electrode system, a hydrophilic polymer and a hydrophilic polymer as a reaction reagent system in contact with the electrode system. A reaction layer containing an oxidoreductase, an electron mediator, and a buffer added as necessary is formed. When the sample solution containing the substrate is dropped onto the reaction layer, the reaction layer dissolves, the pH is adjusted to the highest enzyme activity by the buffering action of the buffer, the enzyme reacts with the substrate, and the electron mediator is further reduced. It
After the completion of the enzymatic reaction, this reduced electron mediator is electrochemically oxidized, and the concentration of the substrate in the sample solution is determined from the oxidation current value obtained at this time.

In principle, such a biosensor can measure various substances by using an enzyme whose substrate is a substance to be measured. By using cholesterol oxidase as the redox enzyme, a biosensor for measuring cholesterol in serum can be constructed. However, the serum cholesterol value used as a diagnostic guideline is the sum of the concentrations of cholesterol and cholesterol ester. Since cholesterol ester cannot serve as a substrate for the oxidation reaction by cholesterol oxidase, a process of converting cholesterol ester into cholesterol is necessary to measure serum cholesterol level as a diagnostic guide. Cholesterol esterase is known as an enzyme that catalyzes this process. Therefore, the method currently used is carried out based on the following general reaction formula.

[0006]

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[0007]

However, when a compound other than oxygen is used as an electron mediator in the reaction of oxidizing cholesterol by cholesterol oxidase as described above, the secondary reaction rate between oxygen and the enzyme is Since the secondary reaction rate between the mediator and the enzyme is large, when oxygen is dissolved in the sample solution, the oxidation current value of the electron mediator obtained from the electrode depends on the oxidation reaction of the substrate in the sample solution. The value tended to be lower than the value expected to be obtained when coupled with the reduction reaction of. Therefore, there is a problem that the response may not be accurate especially when the substrate concentration in the sample solution is low. In addition, there is also a problem that the time required for the reaction becomes long, and increasing the amount of cholesterol oxidase carried in order to avoid this causes an increase in manufacturing cost, and an increase in the amount of reagent carried by the sensor is There was a problem of making the configuration of the physical configuration difficult.

[0008] The present invention solves such problems, and an object of the present invention is to provide a cholesterol sensor capable of high-speed and highly accurate quantification.

[0009]

To achieve this object, the cholesterol sensor of the present invention comprises an electrode system having a measuring electrode and a counter electrode, and a reaction reagent system, and the reaction reagent system contains at least cholesterol dehydrogenase and nicotinamide. It has a structure containing an adenine dinucleotide and an electron mediator. With this configuration, it is possible to obtain a biosensor that can quickly measure the cholesterol concentration without being affected by oxygen.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The cholesterol sensor of the present invention may be configured such that the above-mentioned reaction reagent is used as a solution and the electrode system is immersed in this solution. By arranging them in the vicinity, a cholesterol sensor that can be manufactured at low cost and that is easy to use can be obtained. That is,
An insulative substrate, an electrode system including a measurement electrode and a counter electrode provided on the substrate, and a sample liquid supply path which is disposed on the substrate and between the substrate and an end of the substrate to the electrode system. The sensor main body is constituted by the cover member having the groove, and the reaction layer containing the reaction reagent system is formed in the portion exposed to the sample liquid supply path.

With respect to the cholesterol sensor having such a structure, some preferable modes for arranging the reaction layer will be described below. The first configuration example has a reaction layer on the electrode system of the substrate. When forming this reaction layer, a layer of a hydrophilic polymer such as CMC is formed on the electrode system to prevent the enzyme or electron mediator, which is a component of the reaction layer, from directly contacting the surface of the electrode system. Is preferred. As a result, adsorption of proteins on the surface of the electrode system and changes in the characteristics of the electrode system due to the chemical action of substances having an oxidizing ability such as potassium ferricyanide are less likely to occur.

In the second configuration example, the reaction layer is divided and formed on the electrode system and on the cover member side. In particular, as a reaction reagent, an electron mediator such as potassium 1,2-naphthoquinone-4-sulfonate, which is relatively unstable under high pH, and a tris (hydroxymethyl) aminomethane-hydrochloric acid (hereinafter abbreviated as Tris hydrochloride) buffer agent. When such a buffering agent is included, it is preferable to arrange the both separately. For example, a layer of an electron mediator such as 1,2-naphthoquinone-4-potassium sulfonate is formed on a portion of the cover member exposed to the sample liquid supply path, and a buffer such as Tris hydrochloride is formed on the electrode system of the substrate. A reaction layer containing the agent is formed.

The third structural example is 1,2-naphthoquinone-
An electron mediator such as potassium 4-sulfonate and a buffering agent such as Tris hydrochloride are separately formed on the same substrate. For example, a reaction layer containing the above buffer is formed on the electrode system, and a layer containing an electron mediator such as 1,2-naphthoquinone-4-potassium sulfonate on the opening side of the sample liquid supply path than the reaction layer. To provide. It is preferable to form a layer of a hydrophilic polymer on the base of the latter layer, or to mix a hydrophilic polymer to these layers.

In the fourth configuration example, a hydrophilic polymer layer is provided on the electrode system on the substrate, and the reaction layer is formed on the portion of the cover member exposed to the sample liquid supply path. In addition, also in the second and third configuration examples, it is preferable that the electrode system is covered with a layer of a hydrophilic polymer. In addition, in the second and third configuration examples, when a layer containing an electron mediator or the like is formed on the cover member side, a hydrophilic polymer layer is formed underneath or a hydrophilic high layer is formed on these layers. It is preferred to mix the molecules. Further, it is preferable to provide a lecithin layer on or near the above reaction layer to facilitate the introduction of the sample solution into the electrode system.

In the present invention, preferred electron mediators are ferricyanide, 1,2-naphthoquinone-4-sulfonic acid, 2,6-dichlorophenolindophenol, dimethylbenzoquinone and 1-methoxy-.
It is at least one selected from the group consisting of 5-methylphenazinium sulfate, methylene blue, galocyanine, thionine, phenazine methosulfate, and meldra blue. Further, it is preferable that the reaction reagent system contains diaphorase. Diaphorase catalyzes the reaction between an electron mediator and a reduced form of nicotinamide adenine dinucleotide, and serves to shorten the measurement time.
Diaphorase is especially necessary when using an electron mediator, such as potassium 1,2-naphthoquinone-4-sulfonate, which performs a very fast redox reaction with a reduced form of nicotinamide adenine dinucleotide without the catalytic action of diaphorase. Not. It is preferable to include cholesterol esterase and a surfactant in the reaction reagent system.

[0016]

Embodiments of the present invention will be described below. Example 1 FIG. 1 shows a cholesterol sensor of this example. Reference numeral 1 is a glass cell, in which a stirrer 2 is placed and fixed on a stirrer machine 8. A measuring electrode 4, a counter electrode 5, and a reference electrode 6 are set in the glass cell 1 by an electrode fixing device 3. The measurement electrode 4 is a glassy carbon electrode, and the counter electrode 5 is a platinum wire. The reference electrode 6 is composed of a silver / silver chloride electrode,
It is connected to a glass cell via a salt bridge in which an agarose gel is infiltrated with a KCl solution. These electrodes are connected to the recording device 10 via the constant potential setting device 9, respectively. A measuring device system is composed of the above-mentioned elements.

The glass cell 1 is filled with the reaction solution 7, and the reaction solution 7 is stirred by the stirrer 2. The reaction solution 7 is a buffered aqueous solution of Tris hydrochloride containing cholesterol dehydrogenase (hereinafter abbreviated as ChDH), nicotinamide adenine dinucleotide (hereinafter abbreviated as NAD), potassium ferricyanide as an electron mediator, and diaphorase. The desirable concentration of each reaction reagent is as follows. ChDH = 10 units / ml, NAD = 25 mM /
1, diaphorase = 20 units / ml, potassium ferricyanide = 100 mM / l, Tris hydrochloride = 0.3
M / l. It is desirable that the concentration is the one mentioned here, but if necessary, an appropriate response can be obtained even if a partially different concentration is used depending on the use conditions. The pH is preferably in the range of 8.0 to 9.0. If the pH is lower than 8.0, the reaction time will increase remarkably, and if the pH is 9.0 or higher, the current value per unit concentration of cholesterol will decrease, and conversely the residual current value that is considered to be due to a non-enzymatic chemical reaction. Will increase.

The measuring electrode 4 has 5 parts with respect to the reference electrode 6.
A potential of 00 mV is applied. The sample solution containing cholesterol is fed from the through hole 11 of the electrode fixing device 3 to the reaction solution 7
When added dropwise, ChDH oxidizes cholesterol to cholestenone, which is coupled with this reaction to give NA.
D is reduced, and its reduced form, NADH, is produced. The generated NADH exchanges electrons with the ferricyanide ion and returns to NAD which is an oxidant. The ferricyanide ion receives an electron from NADH and is reduced to a ferrocyanide ion. The concentration of the ferrocyanide ion thus produced is proportional to the cholesterol concentration in the sample solution. This redox reaction is catalyzed by diaphorase. The reaction proceeds spontaneously without diaphorase, but the presence of diaphorase allows the reaction to proceed very quickly.

As described above, since a potential of 500 mV is applied to the measuring electrode 4 with respect to the reference electrode 6, the ferrocyanide ion transfers electrons to the measuring electrode 4 and returns to the ferricyanide ion. Since the measurement electrode 4 is a glassy carbon electrode and the potential in the vicinity of the measurement electrode 4 is 500 mV with respect to the silver / silver chloride electrode, it is considered that the amount of NADH electrochemically oxidized in the measurement electrode 4 is extremely small. . By observing the movement of electrons received by the measuring electrode 4 as a current, the cholesterol concentration in the sample liquid can be quantified. When the sample solution contains cholesterol ester like serum, by adding cholesterol esterase and a surfactant for promoting the catalytic activity of cholesterol esterase to the reaction solution 7, in addition to the above components, The total concentration of cholesterol ester and cholesterol can be measured.

2 and 3 show measurement examples of this embodiment. FIG. 2 is a graph showing the change in current value over time by the recording device 10, and FIG. 3 is a graph showing the relationship between the current value and cholesterol concentration in FIG. As shown in these figures, the current value increases with the addition of cholesterol,
The steady-state value was reached within a minute, and the current value and cholesterol concentration showed a very good linear relationship.

<Embodiment 2> FIG. 4 is a vertical sectional view of the cholesterol sensor of this embodiment from which the cover and the spacer are removed, and FIG. 5 is an exploded perspective view of the cholesterol sensor from which the reaction layer is removed. Reference numeral 12 denotes an insulating substrate made of polyethylene terephthalate. On this substrate 12,
Lead 1 by printing silver paste by screen printing
3 and 14 are formed. On the substrate 12, a conductive carbon paste containing a resin binder is further printed to form an electrode system including the measuring electrode 15 and the counter electrode 16, and an insulating paste is printed to form an insulating layer 17. . The insulating layer 17 keeps the areas of the exposed portions of the measurement electrode 15 and the counter electrode 16 constant and partially covers the leads.

After the electrode portion was prepared in this way, a 0.5 wt% aqueous solution of the sodium salt of hydrophilic polymer carboxymethyl cellulose (hereinafter abbreviated as CMC) was dropped on the electrode system and dried with warm air at 50 ° C. CMC layer 18 was formed by drying in an oven for 10 minutes. Next, 10 units / ml of Nocardia-derived ChDH and NA which is a coenzyme
D is 50 mM / l, Pseudomonas-derived enzyme diaphorase is 10 units / ml, potassium ferricyanide as an electron mediator is 50 mM / l, Pseudomonas-derived cholesterol esterase (hereinafter abbreviated as ChE) is 1 k unit / ml, and a surfactant. PH of a mixed aqueous solution containing 0.5 wt% of n-octyl-β-D-thioglucoside and 0.3 M / l of Tris hydrochloride as a buffer was adjusted to pH 8.5. 5 μl each of this aqueous solution was dropped onto the CMC layer 18 and dried in a room temperature dry air device for 30 minutes to obtain ChDH-ChE-potassium ferricyanide-surfactant-NAD-diaphorase-.
The buffer layer 19 was formed.

In this case, CMC and ChDH, ChE, potassium ferricyanide, n-octyl-β-D-thioglucoside, NAD, and the buffer are in a partially mixed state in a thin film with a thickness of several microns. ing. That is, when the aqueous solution is dropped onto the CMC layer, the CMC layer formed first is once dissolved, and the layer is formed in a form mixed with an enzyme or the like in the subsequent drying process. However, since it is not accompanied by stirring or the like, it is not in a completely mixed state, and the electrode system surface is in a state of being covered only with CMC. Therefore, since the enzyme and electron mediator do not contact the electrode system surface, adsorption of proteins on the electrode system surface,
It becomes difficult for the characteristics of the electrode system to change due to the chemical action of a substance having an oxidizing ability such as potassium ferricyanide.
As a result, a sensor having a highly accurate sensor response can be obtained. This ChDH-ChE-potassium ferricyanide-surfactant-NAD-diaphorase-buffer layer 1
Onto No. 9, 5 μl of a 0.5 wt% toluene solution of phosphatidylcholine was dropped and dried to form a lecithin layer 20. By providing this lecithin layer, the sample solution can be smoothly introduced. This lecithin layer is not essential for the enzymatic reaction.

As described above, the CMC layer 18, ChDH
-ChE-potassium ferricyanide-surfactant-NA
D-diaphorase-buffer layer 19 and lecithin layer 2
After forming the reaction layer consisting of 0, the cover 25 and the spacer 24 are adhered in a positional relationship as shown by the alternate long and short dash line in FIG. 4 to complete the cholesterol sensor. In the sensor thus assembled, the sample liquid supply passage 28 is formed in the slit 26 portion of the spacer 24. This cholesterol sensor is a simple operation of bringing the sample solution into contact with the opening of the sample solution supply path 28 at the tip of the sensor, and the sample solution is easily introduced into the reaction layer portion. The supply amount of the sample liquid depends on the volume of the sample liquid supply passage 28, and therefore does not need to be quantified in advance. Furthermore, evaporation of the sample liquid during measurement can be minimized, and highly accurate measurement can be performed. In FIG. 4, 27 is an air hole provided in the cover 25. Here, when a transparent polymer material is used for the cover 25 and the spacer 24, the state of the reaction layer and the introduction state of the sample solution can be easily observed from the outside.

To the cholesterol sensor thus prepared, 3 μl of a cholesterol standard solution was supplied as a sample solution from the opening of the sample solution supply passage, and after 3 minutes, a pulse voltage of +0.5 V was applied to the measurement electrode in the anode direction with the counter electrode as a reference. The voltage was applied and the current value was measured after 5 seconds. When the sample solution reaches the reaction layer, the lecithin layer 20 is dissolved and the ChDH-C continues.
hE-potassium ferricyanide-surfactant-NAD-
The diaphorase-buffer layer 19 was dissolved. The cholesterol ester in the sample solution is ChDH-ChE-potassium ferricyanide-surfactant-NAD-diaphorase-
It is redispersed by n-octyl-β-D-thioglucoside in the buffer layer 19, becomes cholesterol by the catalytic action of ChE, is oxidized by ChDH, and NAD is reduced in conjugation with this oxidation reaction, and NA is reduced.
DH is generated, and NADH is again oxidized to NAD by the catalytic action of diaphorase. Ferricyanide ion is reduced to ferrocyanide ion in conjugation with the oxidation reaction of NADH. Next, by applying the above-mentioned pulse voltage, an oxidation current of the generated ferrocyanide ion and part of NADH is obtained, and this current value corresponds to the concentration of cholesterol as a substrate. Note that the observed current value may partially include the oxidation current of NADH, but the addition of diaphorase to the reaction reagent system shortens the time required for measurement. The ratio of the highest degree of electrochemical oxidation to the measured value is
It seems to be extremely low.

Example 3 Similar to Example 2, the CMC layer 18 was formed on the electrode system on the substrate. Next, by combining the spacer 24 and the cover 25, 0.5 wt% of CMC is also contained in the recess formed in the slit 26.
The aqueous solution was dropped and dried to form the CMC layer 23.
To cover the CMC layer 23, 5 μl of a 50 mM solution of potassium 1,2-naphthoquinone-4-sulfonate (hereinafter abbreviated as NQS), which is an electron mediator, was dropped, and the NQS layer 21 was dried at room temperature in air. Formed.
When the upper reaction layer is formed on the cover member side in which the spacer 24 and the cover 25 are combined, the reaction layer is easily peeled off without the CMC layer 23. Therefore, the CMC layer 23 is preferably provided. Instead of providing the CMC layer 23 and the NQS layer 21, a solution containing NQS dissolved in a CMC aqueous solution may be dropped to provide a layer containing both CMC and NQS. NQS is relatively unstable at high pH, so T
Although it cannot be dissolved in a solution containing a buffer such as ris hydrochloride and left for a long time, it can be incorporated into the sensor as an electron mediator by separating from the buffer.

Next, 10 units / ml of Nocardia-derived ChDH, 50 mM of coenzyme NAD, 1 kunits / ml of Pseudomonas-derived ChE, and a surfactant of n-octyl-β-D-thioglucoside were added. 0.
5 wt% and buffer Tris hydrochloride 0.3M
The pH of the mixed aqueous solution containing was adjusted to 8.5. 5 μl each of this aqueous solution was dropped onto the CMC layer 18 on the electrode system, and dried in normal temperature dry air for 30 minutes to obtain ChD.
The H-ChE-surfactant-NAD-buffer layer 22 was formed. When using an electron mediator, such as NQS, which carries out a very fast redox reaction with NADH even without the catalytic action of diaphorase, diaphorase can be omitted as in this example. When it is necessary to further shorten the measurement time, diaphorase can be added to the constitution of this example. This ChDH-ChE-surfactant-NA
In the same manner as in Example 2, 5 μl of a 0.5 wt% toluene solution of phosphatidylcholine was dropped on the D-buffer layer 22 and dried to form the lecithin layer 20. The lower reaction layer of the cholesterol sensor is formed as described above.

A cholesterol sensor is completed by combining the above-mentioned combination of the spacer having the upper reaction layer and the cover with the insulating substrate having the lower reaction layer. The structure of the cholesterol sensor thus obtained is shown in FIG. The response to the cholesterol standard solution obtained by the cholesterol sensor thus produced was linear with respect to the cholesterol concentration.

<Embodiment 4> FIG. 7 is a longitudinal sectional view of a cholesterol sensor of this embodiment. First, in the same manner as in Example 2, the CMC layer 18 was formed on the electrode system on the substrate. Further, between the CMC layer 18 and the tip of the substrate corresponding to the opening of the sample liquid supply path 28, the CMC layer 1 is provided so as not to come into contact with the CMC layer 18 at a position where the electrode portion is not covered.
A CMC layer 29 was formed in the same manner as in No. 8. The CMC layer 29 and the CMC layer 18 do not have to be the same. Next, 10 units / m of ChDH derived from Nocardia
1, co-enzyme NAD 50 mM, Pseudomonas-derived enzyme diaphorase 10 units / ml, electron mediator potassium ferricyanide 50 mM,
A mixed aqueous solution containing 1 k unit / ml of Pseudomonas-derived ChE, 0.5 wt% of n-octyl-β-D-thioglucoside which is a surfactant, and 0.3 M of Tris hydrochloride which is a buffer, has a pH of 8.5. Adjusted to. 5 μl each of this mixed aqueous solution was dropped onto the CMC layer 18 so as not to come into contact with the CMC layer 29, and dried in a room temperature dry air dryer for 30 minutes, whereby ChDH-ChE-potassium ferricyanide-surfactant-NAD- The diaphorase-buffer layer 19 was formed.

Further, so as to cover the CMC layer 29,
5μ of 50 mM solution of NQS which is an electron mediator
1 drops and dried in air at room temperature to form an NQS layer 21. Instead of providing the CMC layer 29 and the NQS layer 21,
A solution of NQS dissolved in a CMC aqueous solution is added dropwise to CM.
It is also possible to provide a layer containing C and NQS. Since NQS is relatively unstable at high pH, it cannot be dissolved in a solution containing a buffer such as Tris hydrochloride and left for a long time, but by separating from NQS in this way,
It can be incorporated into the sensor as an electronic mediator. When the NQS solution is dropped on the CMC layer 29 to form the NQS layer 21, the NQS solution is added to the ChDH layer.
-ChE-potassium ferricyanide-surfactant-NA
It is necessary to avoid contact with the D-diaphorase-buffer layer 19. This ChDH-ChE-potassium ferricyanide-surfactant-NAD-diaphorase-buffer layer 19 and N
On the QS layer 21, 0.5 wt of phosphatidylcholine
5% toluene solution was added dropwise and dried to form lecithin layer 2
Formed 0. By providing this lecithin layer, the sample solution can be smoothly introduced, but the lecithin layer is not essential for the enzymatic reaction. The lecithin layer 20 is a recess on the cover member side in which the spacer 24 and the cover 25 are combined,
That is, it may be formed on the surface exposed to the sample liquid supply path.

CMC layer 18, CMC layer 29, ChDH-ChE-potassium ferricyanide-surfactant-NAD-diaphorase-buffer layer 19, NQ as described above.
After forming the reaction layer including the S layer 21 and the lecithin layer 20, the cover 25 and the spacer 24 are adhered in a positional relationship as shown by the one-dot chain line in FIG.
The cholesterol sensor is completed.

Example 5 In the same manner as in Example 2, a 0.5 wt% aqueous solution of CMC was dropped on the electrode system on the substrate and dried to form a CMC layer 18. Next, in the same manner as in Example 3, a 0.5 wt% aqueous solution of CMC was dropped into the recess on the cover member side in which the spacer 24 and the cover 25 were combined, and dried to form the CMC layer 23. Continuing,
ChDH 10 unit / ml, NAD 50 mM, diaphorase 10 unit / ml, electron mediator potassium ferricyanide 50 mM, Pseudomonas-derived ChE 1 k unit / ml, surfactant n-octyl-β-D- 0.5w thioglucoside
The pH of a mixed aqueous solution containing t% and Tris hydrochloride 0.3M as a buffer was adjusted to 8.5. 5 μl of this mixed aqueous solution was dropped so as to cover the CMC layer 23, and dried in a room temperature dry air device for 30 minutes to obtain ChDH-
ChE-potassium ferricyanide-surfactant-NAD
-A diaphorase-buffer layer 19 was formed.

A cholesterol sensor is completed by adhering a cover member consisting of the spacer 24 and the cover 25 having the reaction layer thus formed to the above-mentioned substrate.
As described above, when the reaction layer is provided separately from the electrode portion and only the CMC layer is formed on the electrode surface, the current value per unit substrate concentration is higher than when the reaction layer is provided on the electrode surface.

The loading amount of each reagent shown in the above embodiment is an example of the embodiment, and the present invention is not limited to these. Further, like the lecithin layer 20 shown in Example 2, in the cholesterol sensor of the present invention, a layer containing lipid is provided on the surface of the reaction layer to facilitate introduction of the sample solution into the reaction reagent system. be able to. As the lipid used for this purpose, in addition to the phosphatidylcholine used in the above examples, amphipathic lipids such as phospholipids such as phosphatidylserine and phosphatidylethanolamine can be used. Further, as the hydrophilic polymer for forming the reaction layer, in addition to carboxymethylcellulose and polyvinylpyrrolidone used in the above examples, polyvinyl alcohol, water-soluble cellulose derivative, particularly ethyl cellulose, hydroxypropyl cellulose; gelatin, polyacryl An acid and its salt, starch and its derivative, maleic anhydride and its salt, polyacrylamide, a methacrylate resin, poly 2-hydroxyethyl methacrylate, etc. can be used.

In the above examples, Pseudomonas-derived ChE was used, and n was used as the surfactant.
-Octyl-β-D-thioglucoside is used,
When using ChE derived from Pseudomonas, Lubr
Good response is also obtained using ol PX, sodium cholate, dodecyl-β-maltoside and DK-ester.
Further, as ChE, one derived from mammalian pancreas can also be used, and in this case, such as sodium cholate,
By using a surfactant having a bile acid skeleton, very good responsiveness is exhibited. Further, in the above-mentioned Examples 2 to 5, the bipolar electrode system having only the measurement electrode and the counter electrode was described, but more accurate measurement is possible by using the three-electrode system in which the reference electrode is added.

[0036]

As described above, according to the present invention, it is possible to obtain a cholesterol sensor capable of measuring the cholesterol concentration in a sample solution in a shorter time without being affected by oxygen.

[Brief description of drawings]

FIG. 1 is a diagram showing a configuration of a cholesterol sensor according to an embodiment of the present invention.

FIG. 2 is a diagram showing an example of measurement results by the cholesterol sensor.

FIG. 3 is a diagram showing a relationship between a cholesterol concentration and a current value in a measurement example of the cholesterol sensor.

FIG. 4 is a cross-sectional view showing a configuration of a main part of a cholesterol sensor according to another embodiment of the present invention.

FIG. 5 is an exploded perspective view of the cholesterol sensor excluding a reaction layer.

FIG. 6 is a cross-sectional view showing a configuration of a main part of a cholesterol sensor according to still another embodiment of the present invention.

FIG. 7 is a cross-sectional view showing a configuration of a main part of a cholesterol sensor according to still another embodiment of the present invention.

FIG. 8 is a cross-sectional view showing a configuration of a main part of a cholesterol sensor according to still another embodiment of the present invention.

[Explanation of symbols]

1 Glass Cell 2 Stirrer 3 Electrode Fixture 4 Measurement Electrode 5 Counter Electrode 6 Reference Electrode 7 Reaction Solution 8 Stirrer Machine 9 Constant Potential Setting Device 10 Recording Device 11 Through Hole 12 Insulating Substrate 13, 14 Lead 15 Measurement Electrode 16 Counter Electrode 17 Insulating layer 18 CMC layer 19 ChDH-ChE-potassium ferricyanide-surfactant-NAD-diaphorase-buffer layer 20 Lecithin layer 21 NQS layer 22 ChDH-ChE-surfactant-NAD-buffer layer 23 CMC layer 24 Spacer 25 cover 26 slit 27 air hole 28 sample liquid supply path

Front page continuation (72) Inventor Shiro Nankai 1006 Kadoma, Kadoma, Osaka Prefecture Matsushita Electric Industrial Co., Ltd. (72) Inko Junko Iwata, 1006 Kadoma, Kadoma City, Osaka Matsushita Electric Industrial

Claims (8)

[Claims] 1. A cholesterol sensor comprising an electrode system having a measurement electrode and a counter electrode, and a reaction reagent system, wherein the reaction reagent system contains at least cholesterol dehydrogenase, nicotinamide adenine dinucleotide, and an electron mediator. . 2. An insulating substrate, an electrode system including a measurement electrode and a counter electrode provided on the substrate, and a sample liquid supply from the end of the substrate to the electrode system disposed on the substrate. A cover member having a groove forming a passage, and a reaction layer including a reaction reagent system provided in a portion exposed to the sample liquid supply passage, wherein at least cholesterol dehydrogenase and nicotinamide adenine dinucleotide are contained in the reaction reagent system. , And a cholesterol sensor comprising an electron mediator. 3. The electron mediator is ferricyanide, 1,2-naphthoquinone-4-sulfonic acid, 2,6-
Dichlorophenol indophenol, dimethylbenzoquinone, 1-methoxy-5-methylphenazinium sulfate, methylene blue, galocyanine, thionine,
2. At least one selected from the group consisting of phenazine methosulfate and Meldola blue.
Alternatively, the cholesterol sensor described in 2. 4. The cholesterol sensor according to claim 1, wherein the reaction reagent system contains diaphorase. 5. The cholesterol sensor according to claim 1, wherein the reaction reagent system contains cholesterol esterase and a surfactant. 6. The cholesterol sensor according to claim 2, wherein the reaction layer is provided separately on the electrode system and on the cover member side. 7. The cholesterol sensor according to claim 2, which has a layer containing a hydrophilic polymer in at least a part of the reaction layer. 8. The cholesterol sensor according to claim 2, wherein at least a part of the reaction layer has a layer in which a hydrophilic polymer and an electron mediator are mixed.