JPH07110313A - Biosensor and manufacture thereof - Google Patents

Abstract

PURPOSE:To obtain a quick and highly accurate sensor by arranging a layer mainly composed of an enzyme modified with a lipid of an amphipathic medium and a reaction layer comprising a layer mainly composed of an electron acceptor in an electrode system comprising a measuring electrode and a counter electrode provided on an insulating substrate. CONSTITUTION:Leads 2 and 3 are formed on an insulating substrate 1 by a screen printing with a silver paste. A measuring electrode 4 is printed by a conductive carbon paste, an insulation layer 6 by an insulating paste and a counter electrode 5 with a conductive carbon paste separately. Then, an aqueous solution of carboxymethyl cellulose(CMC) is dropped on an electrode system comprising the measuring electrode 4 and a counter electrode and dried to form a CMC layer. As electron acceptor. potassium ferricyanide is dissolved into a phosphoric acid buffer solution containing about 0.5wt.% of CMC. dropped and dried to form a first layer 7. Then, a benzene solution of glucose oxidase modified with amphipathic lipid is dropped on the layer 7 and dried to form a second layer 8. Thus. a highly accurate sensor can be obtained.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biosensor which enables rapid and highly accurate quantification of a specific component in a sample liquid and has high storage reliability, and a method for producing the same.

[0002]

2. Description of the Related Art Hitherto, as a method capable of performing a rapid and highly accurate quantification of a specific component in a sample solution,
There is a biosensor disclosed in JP-A-3-20276.

The biosensor will be described below.
FIG. 3 is a sectional view showing an example of the biosensor. After forming an electrode system consisting of the measurement electrode 4 and the counter electrode 5 on the insulating substrate 1 by a method such as screen printing, and further forming an insulating layer 6, a hydrophilic polymer and a redox enzyme are formed on the electrode system. And a reaction layer 25 composed of an electron acceptor.

When the sample solution containing the substrate is dropped onto the reaction layer 25, the reaction layer is dissolved, the enzyme reacts with the substrate, and the electron acceptor is further reduced. After the completion of the enzymatic reaction, this reduced electron acceptor is electrochemically oxidized, and the concentration of the substrate in the sample solution is determined from the oxidation current value obtained at this time.

[0005]

In the biosensor having such a conventional structure, since the enzyme and the electron acceptor are present in the same reaction layer, the reaction is promptly started when the sample solution is supplied. There was an advantage. However, since the enzyme is a biomaterial, the activity of the enzyme decreases when mixed with a chemical compound such as an electron acceptor, and the response characteristics of the sensor decrease. Therefore, a structure in which the enzyme and the electron acceptor are separated into separate layers is conceivable, but it is difficult to achieve a completely separated state because both the enzyme and the electron acceptor are water-soluble.

The present invention solves such a problem while taking advantage of the biosensor having the conventional structure, and provides a biosensor which is quick, highly accurate, and has high storage reliability.

[0007]

In order to solve the above problems, the present invention provides an electrode system comprising at least a measuring electrode and a counter electrode on an insulating substrate, and the electrode system is modified with an amphipathic lipid. In addition, a reaction layer composed of two layers, that is, a layer mainly containing an enzyme and a layer mainly containing an electron acceptor is provided.

[0008]

[Function] The enzyme becomes soluble in an organic solvent and insoluble in water by being modified with an amphipathic lipid. Therefore, a layer containing a solution containing an enzyme modified with an amphipathic lipid dissolved in an organic solvent was dropped on an electrode and dried, and an aqueous solution containing an electron acceptor was dropped and dried. When the layers are provided in any order, it is possible to form a uniform layer without affecting the enzyme and the electron acceptor contained in each layer. Therefore, the decrease in enzyme activity due to the inclusion is prevented, and thus the biosensor having the constitution of the present invention has high storage reliability.

[0009]

EXAMPLES The present invention will be described below with reference to examples.

Example 1 FIG. 1 is a sectional view of a glucose sensor manufactured as an example of the biosensor of the present invention. 2 is an exploded perspective view of FIG.

The method of manufacturing the glucose sensor will be described below. Silver paste was printed on the insulating substrate 1 made of polyethylene terephthalate by screen printing to form leads 2 and 3. Next, using a conductive carbon paste containing a resin binder, the measuring electrode 4 of the electrode system was printed, and subsequently the insulating layer 6 made of an insulating paste was printed.

The insulating layer 6 keeps the exposed area (about 1 mm ² ) of the measuring electrode 4 constant and partially covers the leads 2 and 3. Finally, the counter electrode 5 was printed using the same carbon paste as the measuring electrode.

Next, a 0.5 wt% aqueous solution of carboxymethyl cellulose (hereinafter abbreviated as CMC) as a hydrophilic polymer was dropped on the electrode system and dried to form a CMC layer. Then, a phosphate buffer solution (0.2M: K2HPO4 -0.2M :) containing 33 mg of potassium ferricyanide as an electron acceptor on the CMC layer and containing 0.5 wt% of CMC.
KH2PO4) 4 μl of a solution dissolved in 1 ml,
The first layer 7 was formed by drying in a warm air dryer at 50 ° C. for 10 minutes. Next, 4 μl of a solution prepared by dissolving 750 U of glucose oxidase (EC 1.1.3.4: hereinafter abbreviated as GOD) 750 U modified with an amphipathic lipid in 1 ml of benzene was dropped on the first layer and dried at room temperature. The second layer 8 was formed. Here, a method for modifying an enzyme with an amphipathic lipid will be briefly described. Acetate buffer containing 0.1 M KCl of GOD (0.02M: CH3COOH-0.02M:
The solution dissolved in CH3COOK) is mixed with an aqueous solution of amphipathic lipid and left standing at 4 ° C for 24 hours, and then separated by freeze-drying to obtain a lipid-modifying enzyme.

The reaction layer composed of the first layer and the second layer has a substantially circular shape with a diameter of about 3.6 mm and substantially coincides with the outer peripheral portion of the counter electrode.

In the above reaction layer forming step, when a mixed solution of a phosphate and an electron acceptor is dropped, the CMC layer composed of the hydrophilic polymer is once dissolved and then mixed in the subsequent drying process to form the first mixture. Form the layer 7. Therefore, the dissolution reaction with the sample solution is also rapidly performed. Moreover, since the organic solvent solution of the enzyme modified with the amphipathic lipid is dropped after forming the hydrophilic layer, it is not necessary to separate and purify the remaining lipid when the enzyme is modified with the lipid.

Although the enzyme modified with an amphipathic lipid was used in the above-mentioned examples, the enzyme separated by a method of dissolving the enzyme including the unused lipid after the modification in an organic solvent and extracting the enzyme was used. Also, a reaction layer could be prepared in the same process, and a highly accurate biosensor could be obtained.

After forming the reaction layer as described above, the cover 10 and the spacer 9 were adhered in a positional relationship as shown by the one-dot chain line in FIG. If a transparent material is used for the cover, the state of the reaction layer and the introduction state of the sample solution can be confirmed very easily from the outside.

Further, when the cover is attached, the sample liquid is easily introduced into the reaction layer portion by a simple operation of bringing the sample liquid into contact with the sample supply hole 11 at the tip of the sensor due to the capillary phenomenon in the space formed by the cover and the spacer.

Since the amount of the sample solution supplied depends on the space volume generated by the cover and the spacer, it is not necessary to quantify it in advance. Furthermore, evaporation of the sample liquid during measurement can be minimized, and highly accurate measurement can be performed.

3 μl of an aqueous glucose solution was supplied as a sample solution from the sample supply hole 12 to the glucose sensor manufactured as described above. The sample liquid quickly reached the air holes 12 by the capillary phenomenon, and the reaction layer on the electrode system was dissolved.

After a lapse of a certain time from the supply of the sample solution, the counter electrode 5 of the electrode system is used as a reference and the measurement electrode 4 is moved to the anode direction +0.
When a pulse voltage of 5 V was applied and the current value after 5 seconds was measured, a response current value proportional to the glucose concentration in the sample solution was obtained.

When the reaction layer is dissolved in the sample solution, glucose in the sample solution is oxidized by GOD. The electrons transferred by the oxidation reaction by GOD reduce potassium ferricyanide to potassium ferrocyanide. Next, by applying the above-mentioned pulse voltage, an oxidation current of the produced potassium ferrocyanide is obtained, and this current value corresponds to the concentration of glucose as a substrate.

(Embodiment 2) Since the first embodiment is the same as the first embodiment except for the composition of the first layer 7 and the second layer 8, it is omitted here and only the first layer 7 and the second layer 8 will be described. .

A 0.5 wt% CMC aqueous solution was dropped on the electrode system prepared in the same manner as in the above (Example 1) and dried to prepare CM.
The C layer was formed. Next, 4 μl of a solution of GOD750U modified with an amphipathic lipid in 1 ml of benzene was dropped on the CMC layer and dried at room temperature to form the first layer 7. The amphipathic lipid-modified enzyme used at this time was obtained by the method described in Example 1. However, since there is a step of developing an aqueous solution containing an electron acceptor, It is necessary to remove excess lipid not used for modification by repeatedly washing with water and freeze-drying. Subsequently, a phosphate buffer solution containing 0.2 mg of potassium ferricyanide as an electron acceptor and 0.5 wt% of CMC (0.2 M: K 2 HPO 4 -0.2 M: KH 2 PO 4; p
H = 7.4) 4 μl of a solution dissolved in 1 ml was dropped,
The second layer 8 was formed by drying in a warm air dryer at 50 ° C. for 10 minutes. The outer peripheral portion of the reaction layer composed of the first layer and the second layer has a diameter of about 3.6 mm, which is substantially the same as the diameter of the counter electrode.

In contrast to Example 1, in Example 2, since the surface of the electrode system and the layer containing the electron acceptor are separated, the characteristics of the electrode system due to the chemical action of substances having an oxidizing ability such as potassium ferricyanide and H2PO4-. It is considered that the change is unlikely to occur, and as a result, a biosensor having a highly accurate sensor response can be obtained.

To the glucose sensor manufactured as described above, 3 μl of an aqueous glucose solution was supplied as a sample solution from the sample supply hole 11. The sample liquid quickly reached the air holes 12 by the capillary phenomenon, and the reaction layer on the electrode system was dissolved.

After a fixed time from the supply of the sample solution, the counter electrode 5 of the electrode system was used as a reference and the measurement electrode 4 was moved to the anode direction +0.
When a pulse voltage of 5 V was applied and the current value after 5 seconds was measured, a response current value proportional to the glucose concentration in the sample solution was obtained.

In the above embodiment, C is used as the hydrophilic polymer.
Although MC was used, the present invention is not limited thereto, and other cellulose-based, vinyl alcohol-based, vinylpyrrolidone-based, gelatin-based, acrylate-based, starch-based, maleic anhydride-based, acrylamide-based, etc. are used respectively. However, the same effect was obtained.

On the other hand, as the electron acceptor, p-benzoquinone, a ferrocene compound, etc. can be used in addition to the potassium ferricyanide in the above-mentioned examples.

Further, in the above embodiment, the two-electrode system consisting of the measurement electrode and the counter electrode was described, but the three-electrode system including the reference electrode enables more accurate measurement.

[0031]

As described above, according to the present invention, a highly reliable biosensor can be obtained.

[Brief description of drawings]

FIG. 1 is a sectional view of a glucose sensor which is a biosensor of the present invention.

FIG. 2 is an exploded perspective view of the glucose sensor manufactured as an example of the biosensor of the present invention, except for the reaction layer, which is viewed obliquely from above in FIG.

FIG. 3 is a cross-sectional view of a glucose sensor manufactured as an example of a conventional biosensor.

[Explanation of symbols]

 1 Insulating Substrate 2, 3 Lead 4 Measuring Electrode 5 Counter Electrode 6 Insulating Layer 7 First Layer 8 Second Layer 9 Spacer 10 Cover 11 Sample Supply Hole 12 Air Hole 25 Reaction Layer

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Tomohiro Yamamoto 1006 Kadoma, Kadoma City, Osaka Prefecture Matsushita Electric Industrial Co., Ltd. 72) Inventor Shiro Nankai 1006 Kadoma, Kadoma City, Osaka Prefecture Matsushita Electric Industrial Co., Ltd.

Claims (3)

[Claims] 1. An enzyme comprising an electrode system mainly composed of a measuring electrode and a counter electrode formed on an insulating substrate and a reaction layer formed on the electrode system, the reaction layer being modified with an amphipathic lipid. A biosensor, wherein at least two layers of a layer containing ??? as a main component and a layer containing an electron acceptor as a main component are formed. 2. A step of providing an electrode system mainly composed of at least a working electrode and a counter electrode on an insulating substrate, a step of spreading an aqueous solution of a hydrophilic polymer on the insulating substrate and drying the same.
A step of providing an aqueous solution containing an electron acceptor on the hydrophilic polymer layer and drying it to provide a first layer containing a hydrophilic polymer and an electron acceptor, and an organic solvent solution containing an enzyme on the first layer. A method for manufacturing a biosensor, which comprises sequentially developing and drying to provide a second layer containing an enzyme. 3. A step of providing an electrode system mainly composed of at least a working electrode and a counter electrode on an insulating substrate, a step of spreading an aqueous solution of a hydrophilic polymer on the insulating substrate and drying the same.
A step of developing an organic solvent solution containing an enzyme on the hydrophilic polymer layer and drying the solution to form a first layer containing the enzyme; developing an aqueous solution containing an electron acceptor on the first layer and drying the solution to obtain an electron acceptor A method for manufacturing a biosensor, which comprises sequentially manufacturing a step of providing a second layer including a body.