JP3770757B2 - Biosensor - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a biosensor capable of simply and quickly quantifying a specific component in a sample.
[0002]
[Prior art]
Conventionally, various biosensors have been proposed as a method for easily quantifying a specific component in a sample without diluting or stirring the sample solution. As an example of a biosensor, the following sensor is known (Japanese Patent Laid-Open No. 2-062952).
In this biosensor, an electrode system consisting of a working electrode, a counter electrode and a reference electrode is formed on an insulating substrate by a method such as screen printing, and a hydrophilic polymer, an oxidoreductase, and an electron acceptor are in contact with the electrode system. An enzyme reaction layer containing a body is formed.
[0003]
When a sample solution containing a substrate is dropped onto the enzyme reaction layer of the biosensor thus produced, the enzyme reaction layer dissolves and the enzyme reacts with the substrate, and the electron acceptor is reduced accordingly. After completion of the enzymatic reaction, the reduced electron acceptor is electrochemically oxidized, and the substrate concentration in the sample solution can be determined from the oxidation current value obtained at this time.
The biosensor as described above can in principle measure various substances by selecting an enzyme that uses the substance to be measured as a substrate.
Enzymes are mainly composed of proteins and are usually held in the sensor in a dry state.
However, depending on conditions such as air temperature and humidity, moisture in the air enters and exits the enzyme from the enzyme surface. Therefore, when the enzyme and air are in contact with each other for a long time, a very small amount of water present in the enzyme is changed, the enzyme is denatured, and the activity of the enzyme is reduced.
If the activity of the enzyme contained in the sensor during storage decreases over time after the sensor is produced, the amount of enzyme that reacts with the substrate becomes insufficient. Therefore, there arises a problem that the obtained response current value is not proportional to the concentration of the substrate.
[0004]
In order to solve this problem, it is important to prepare an environment in which the activity of the enzyme is maintained for a long time in the vicinity of the enzyme.
In addition, it is also necessary to increase the responsiveness of the sensor by smoothly moving electrons and substrates during the enzyme reaction.
[0005]
[Problems to be solved by the invention]
In view of the above problems, an object of the present invention is to provide a biosensor with high storage stability by suppressing a decrease in the activity of an enzyme contained in a sensor during storage.
The present invention provides a disposable biosensor that is particularly small and inexpensive.
[0006]
[Means for Solving the Problems]
The biosensor of the present invention comprises an insulating substrate, an electrode system having at least a working electrode and a counter electrode provided on the substrate, and a reaction layer containing at least an enzyme and a saccharide formed on the electrode system, The enzyme is glucose dehydrogenase using pyrroloquinoline quinone as a coenzyme, the saccharide is trehalose, and the reaction layer contains 5 to 20 units of glucose dehydrogenase and 40 to 200 nanomoles of trehalose per sensor chip. And
[0007]
DETAILED DESCRIPTION OF THE INVENTION
The biosensor according to the present invention contains saccharides in the reaction layer as described above. When a saccharide is added to the enzyme solution and this solution is dropped and dried to form a reaction layer, the enzyme surface is covered with the saccharide. Therefore, the enzyme can be protected from environmental changes such as temperature and humidity, and the enzyme activity can be stabilized for a long period of time.
In addition, since saccharides are easily dissolved in water, adding a sample solution to the reaction layer is convenient because the reaction layer immediately dissolves and the enzyme reaction and electrode reaction can proceed smoothly.
There are various sugars that can be expected to have such an effect. In addition, various enzymes can be selected as enzymes applicable to the biosensor of the present invention.
When using at least one enzyme selected from the group consisting of glucose oxidase, glucose dehydrogenase and fructose dehydrogenase, the reaction layer contains at least one sugar selected from the group consisting of trehalose, sucrose, glycerol, mannitol and ribose. Is preferred.
[0008]
In particular, when glucose dehydrogenase using pyrroloquinoline quinone as a coenzyme is used as an enzyme, using trehalose as a saccharide has a significant effect on maintaining the activity of the enzyme, and a good sensor response even after storing the sensor for a long period of time. Sex is obtained.
When the enzyme is glucose dehydrogenase using pyrroloquinoline quinone as a coenzyme and the saccharide is trehalose, the amount of the enzyme and trehalose per disposable sensor chip in which the sample solution is 3 to 4 μl of blood is 1 to 40, respectively. Units and 4-400 nanomoles are suitable, more preferably 5-20 units and 40-200 nanomoles , respectively.
[0009]
Examples of the electron acceptor include ferricyanide ion, p-benzoquinone and derivatives thereof, phenazine methosulfate, methylene blue, ferrocene and derivatives thereof. A sensor response can also be obtained when dissolved oxygen in the sample solution is used as an electron acceptor.
[0010]
The reaction layer of the biosensor according to the present invention may contain a hydrophilic polymer in addition to the enzymes, saccharides and electron acceptor.
By adding a hydrophilic polymer in the reaction layer, peeling of the reaction layer from the surface of the electrode system can be prevented. Furthermore, it also has the effect of preventing the reaction layer surface from cracking, which is effective in enhancing the reliability of the biosensor.
Examples of such hydrophilic polymers include carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, ethyl hydroxyethyl cellulose, carboxymethyl ethyl cellulose, polyvinyl pyrrolidone, polyvinyl alcohol, polylysine and other polyamino acids, polystyrene sulfonic acid, gelatin and A derivative thereof, a polymer of acrylic acid or a salt thereof, a polymer of methacrylic acid or a salt thereof, starch and a derivative thereof, a polymer of maleic anhydride or a salt thereof, an agarose gel and a derivative thereof are preferably used.
[0011]
The arrangement of the reaction layer in the biosensor has various forms other than the formation on the electrode system formed on the electrically insulating substrate. For example, a sample other than the electrode system of the substrate or a cover member that is combined with the substrate and forms a sample solution supply path for supplying the sample solution to the electrode system between the substrate and the sample of the cover member It can arrange | position to the surface exposed to a liquid supply path.
As a method for measuring the oxidation current, there are a two-electrode method using only a working electrode and a counter electrode, and a three-electrode method including a reference electrode. The three-electrode method allows more accurate measurement.
[0012]
【Example】
Hereinafter, the present invention will be described in more detail with reference to specific examples.
FIG. 1 is a schematic plan view in which a reaction layer of a biosensor in an embodiment of the present invention is removed. A silver paste is printed by screen printing on an electrically insulating substrate 1 made of polyethylene terephthalate to form leads 2 and 3. Next, a conductive carbon paste containing a resin binder is printed on the substrate 1 to form the working electrode 4. The working electrode 4 is in contact with the lead 2. Furthermore, an insulating layer 6 is formed on the substrate 1 by printing an insulating paste. The insulating layer 6 covers the outer periphery of the working electrode 4, thereby keeping the area of the exposed portion of the working electrode 4 constant. Then, a conductive carbon paste containing a resin binder is printed on the substrate 1 so as to come into contact with the leads 3 to form a ring-shaped counter electrode 5.
FIG. 2 is a longitudinal sectional view of the biosensor of FIG. A hydrophilic polymer layer 7 and a reaction layer 8 containing at least an enzyme and a saccharide are formed on the electrode system created as shown in FIG.
[0013]
Example 1
A 0.5 wt% aqueous solution of a sodium salt of carboxymethyl cellulose, which is a hydrophilic polymer (hereinafter abbreviated as CMC), is dropped on the electrode of the substrate 1 in FIG. 1 and is placed in a hot air dryer at 50 ° C. for 10 minutes. It was made to dry and the CMC layer 7 was formed. Subsequently, a mixed solution in which 5000 units of glucose dehydrogenase (hereinafter abbreviated as GDH), pyrroloquinoline quinone (hereinafter abbreviated as PQQ), 20 micromoles of trehalose and 50 micromoles of potassium ferricyanide are dissolved in 1 ml of water. 4 μl was dropped on the CMC layer 7 and dried to form a reaction layer 8 to produce a glucose sensor.
As the sample solution, glucose aqueous solutions adjusted to various concentrations were prepared. Then, the prepared sample solution was dropped on the reaction layer 8. When a sample solution containing glucose is supplied to the reaction layer, glucose in the sample is oxidized by GDH. At the same time, potassium ferricyanide in the reaction layer is reduced to potassium ferrocyanide.
[0014]
One minute after dropping the sample solution, a voltage of +0.5 V was applied to the working electrode 4 with respect to the counter electrode 5 to oxidize potassium ferrocyanide. Then, the current value flowing between the counter electrode and the working electrode was measured after 5 seconds. Similarly, current values were measured for aqueous glucose solutions having various concentrations, and the glucose concentration was plotted on the horizontal axis and the current value was plotted on the vertical axis to create a response characteristic diagram of the sensor. The result is shown in FIG.
Moreover, after storing the biosensor produced in the same manner for 6 months, a response characteristic diagram was created in the same manner. The result is shown in FIG.
From FIG. 3, there was a certain correlation between the concentration and the current value, and excellent linearity was obtained. Moreover, there was no change immediately after sensor preparation and after storage for 6 months, showing good storage stability.
[0015]
<< Comparative Example 1 >>
A glucose sensor was produced in the same manner as in Example 1 except that trehalose was not added to the reaction layer 8. Then, in the same manner as in Example 1, response characteristic diagrams of the sensor were created immediately after the sensor was produced and after storage for 6 months. The result is shown in FIG.
From FIG. 3, the obtained response current value was lower than that of Example 1. Further, in the sensor after storage for 6 months, the correlation between the concentration and the current value was lowered, and the response current value was also lower than that immediately after fabrication.
[0016]
Next, the amount of trehalose in the reaction layer is kept constant (80 nmoles ), and sensors are prepared by changing the amount of GDH containing PQQ as a coenzyme. After storage for 6 months, sample solutions with various glucose concentrations are used in the reaction layer. The sensor response was examined by feeding. The result is shown in FIG.
In addition, sensors were prepared in which the amount of GDH using PQQ as a coenzyme was constant (20 units) and the amount of trehalose was 4, 40, 200, and 400 nmol . Then, it was examined sensor response after supplying a sample liquid glucose concentration 10 m M each first-time and 6 months of storage. The ratio of the response value after storage to the initial response value is shown in FIG.
[0017]
From these results, it can be seen that when the amount of the enzyme is 1 unit, the glucose concentration and the sensor response show linearity at a glucose concentration of 600 mg / dl or less. If the amount of enzyme exceeds 40 units, it is disadvantageous in terms of cost. From these, the amount of enzyme per sensor chip is suitably 1 to 40 units, more preferably 5 to 20 units. On the other hand, the amount of trehalose is slightly reduced in storage stability at 4 nmol and 400 nmol . Accordingly, the range of 4 nmol to 400 nmol is appropriate, and more preferably 40 nm to 200 nmol .
[0018]
Example 2
A glucose sensor was prepared in the same manner as in Example 1 except that glucose oxidase was used instead of glucose dehydrogenase. Then, in the same manner as in Example 1, a response characteristic diagram of the sensor was created immediately after the production of the sensor and after storage for 6 months.
As a result, there was a high correlation between the glucose concentration and the current value. In addition, even after storage for 6 months, there was no difference from that immediately after the production of the sensor, indicating good storage stability.
[0019]
<< Comparative Example 2 >>
A glucose sensor was produced in the same manner as in Example 2 except that trehalose was not added to the reaction layer 8. Then, in the same manner as in Example 1, response characteristic diagrams of the sensor immediately after the sensor fabrication and after storage for 6 months were fabricated.
As a result, the obtained response current value was lower than that of Example 2. Further, in the sensor after storage for 6 months, the correlation between the concentration and the current value was lowered, and the response current value was also lower than that immediately after fabrication.
[0020]
Example 3
A sensor was produced in the same manner as in Example 1 except that fructose dehydrogenase was used instead of glucose dehydrogenase.
Fructose solutions with various concentrations were prepared as sample solutions. Then, using this sample solution, in the same manner as in Example 1, a sensor response characteristic diagram was created immediately after the sensor was produced and after storage for 6 months.
As a result, there was a high correlation between the fructose concentration and the current value. In addition, even after storage for 6 months, there was no difference from that immediately after fabrication of the sensor, and good storage was exhibited.
[0021]
<< Comparative Example 3 >>
A sensor was produced in the same manner as in Example 3 except that trehalose was not added to the reaction layer 8. Then, in the same manner as in Example 3, response characteristic diagrams of the sensor immediately after the sensor fabrication and after storage for 6 months were fabricated.
As a result, the obtained response current value was lower than that of Example 3. Further, in the sensor after storage for 6 months, the correlation between the concentration and the current value was lowered, and the response current value was also lower than that immediately after fabrication.
[0022]
【The invention's effect】
As described above, according to the present invention, a biosensor with excellent long-term storage stability can be obtained.
[Brief description of the drawings]
FIG. 1 is a schematic plan view of a biosensor according to an embodiment of the present invention excluding a reaction layer.
FIG. 2 is a longitudinal sectional view of a main part of the biosensor.
FIG. 3 is a diagram showing response characteristics of biosensors of examples of the present invention and comparative examples.
FIG. 4 is a diagram showing response characteristics of sensors having reaction layers with different enzyme amounts.
FIG. 5 is a diagram comparing the ratio of response values after storage of sensors having reaction layers with different amounts of trehalose to initial response values.
[Explanation of symbols]
1 Insulating substrate 2, 3 Lead 4 Working electrode 5 Counter electrode 6 Insulating layer 7 CMC layer 8 Reaction layer

Claims (1)

Comprising an insulating substrate, an electrode system having at least a working electrode and a counter electrode provided on the substrate, and a reaction layer containing at least an enzyme and a saccharide formed on the electrode system;
The enzyme is glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme,
The saccharide is trehalose;
The biosensor according to claim 1, wherein the reaction layer contains 5 to 20 units of glucose dehydrogenase and 40 to 200 nanomoles of trehalose per sensor chip .

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