JP2000131264A - Enzyme sensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel enzyme sensor, in particular a disposal type enzyme sensor, capable of easily determining quantitatively components in a body fluid sample such as blood and urine and component concentrations in food. SOLUTION: This sensor is an amperometric type enzyme sensor of which a working electrode 2 is a single coat applied layer made of a conductive paste containing at least an oxidoreductase, noble metal fine particulates, a conductive powder material and a binder, in an enzyme sensor having an electrode system comprising at least the working electrode 2 and a counter electrode 3 provided on an insulating substrate 1.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel enzyme sensor which can easily determine the concentration of components in body fluid samples such as blood and urine and the concentration of components in foods, and particularly to a disposable enzyme sensor.

[0002]

2. Description of the Related Art In recent years, disposable enzyme sensors which do not require calibration of a calibration curve or washing of electrodes and which can be miniaturized have been put to practical use. An enzyme sensor generally has a working electrode that detects an enzyme reaction (also called an enzyme electrode or a measurement electrode) and a counter electrode that is used in combination with the working electrode to form an electric circuit. Then, a change in a substance due to an enzyme reaction at the working electrode is taken out as a change in an electric signal by the electrodes, and the concentration of a substrate on which the enzyme specifically acts is measured from the change. An enzyme sensor is known which has a structure in which an enzyme reaction layer containing an enzyme and an electron acceptor is laminated on an electrode system comprising a working electrode and a counter electrode provided on an insulating substrate (for example, JP
2-157645 and JP-A-3-54447).

A working electrode provided on an insulating substrate is prepared by mixing a conductive powder such as carbon black, an enzyme, and an electron transfer material (also called an electron acceptor or mediator) together with a polymer binder and a solvent. A type of enzyme sensor produced by a printing method from the obtained mixture is also known (for example, see JP-A-6-281615, JP-A-7-270373 and JP-A-8-94573). Mediators act as mediators that transmit the amount of an enzymatic reaction to the working electrode.

[0004]

When an amperometric enzyme sensor is used to measure a biochemical substance in a biological sample serving as a substrate for an enzyme, hydrogen peroxide (H) generated from the reaction between the enzyme and the substrate is measured. ₂ O ₂ ) is often used as the detection substance.
In the measurement, H ₂ O ₂ generated by the progress of the enzyme reaction is oxidized by a voltage applied to the working electrode (referred to as applied voltage),
The generated current signal is detected by the electric device. Although it depends on the electrode material, the applied voltage is to promote the oxidation reaction of H ₂ O ₂ , and in the case of platinum, +0.4 V (vsAg / AgC
l) The above is necessary. Furthermore, in order to obtain a stable current, platinum requires +0.6 V or more, and carbon requires +0.9 V or more. However, such an applied voltage oxidizes not only H ₂ O ₂ but also reducing substances (for example, ascorbic acid and uric acid in blood) that coexist in the sample, and measures the substrate, which is the target substance. It becomes an error factor.

[0005] To solve this problem, two main methods are used. One is the introduction of mediators. The mediator functions as a substance that transmits the oxidation-reduction reaction of the enzyme to the electrode surface. A redox compound that functions as an electron transporter of a redox enzyme is usually used for an amperometric enzyme sensor. Specifically, ferrocene and its derivatives, benzoquinone, methylene blue, 2,6-dichloroindophenol, metal cyanide complexes and the like are used. By introducing such a mediator, in the case of a platinum working electrode, an enzymatic reaction can be detected at an applied voltage of +0.3 to +0.5 V (vsAg / AgCl). In particular, when preparing an enzyme sensor having a working electrode obtained by mixing a conductive paste material containing carbon as a conductive powder and an enzyme and applying the mixture by a screen printing method, adding a mediator to the working electrode material , +0.5
Even with an applied voltage of V, a sufficient electric signal can be obtained. Other co-reducing substances are less active under these conditions and are not oxidized or have a negligible effect from the oxidation current. There are several methods for using the mediator, such as mixing with the electrode material, modifying the electrode surface and fixing it, or fixing it in the enzyme macromolecule.

However, when a mediator is introduced, the following problems also appear. For example, there are problems such as a decrease in performance over time due to instability of the mediator, low stability of the mediator immobilization method, and contamination of the environment.

As another method, an applied voltage at which a stable oxidation current is obtained is set, the surface of the working electrode is devised, and a reducing substance is removed from the sample to be measured. There are means such as limiting diffusion. That is, the effect of coexisting substances is reduced by forming a multilayer structure. For example, a reducing membrane with a smaller hole diameter is interposed between the immobilized enzyme and its working electrode to block a reducing substance having a molecular weight greater than H ₂ O ₂ or a charged permeable membrane is used for the enzyme electrode. A method is used in which a substance having the same charge is rejected from the electrode by attaching it to the surface (see, for example, JP-A-3-28752). But,
In such a method, there is a limit in improving the performance and quality of the enzyme sensor, and the manufacturing process becomes complicated.

[0008] HP Bennetto et al. (See US Pat. No. 4,970,145) provided an enzyme sensor by immobilizing an enzyme on the surface of a porous conductive layer made of carbon black having adsorbed platinum group metal black. Y. Ikariyama et al. (See US Pat. No. 5,269,903) formed platinum black by electrodeposition on the surface of a conductive electrode substrate, introduced an enzyme into the porous layer, and immobilized the enzyme on the surface. Thus, a method for producing an enzyme sensor was examined. However, in these methods, the production process is complicated because the production of the electrode and the enzyme immobilization are performed in separate steps. In addition, a problem of inactivation of the enzyme due to immobilization of the enzyme such as a crosslinking treatment is also expected.

Therefore, for a disposable enzyme sensor which can immobilize the enzyme only by simple mixing and can be manufactured by one-time printing, it is not necessary to mix a mediator in its working electrode, and it coexists in the measurement sample. It is desired to provide a new enzyme sensor that can be operated at a low applied voltage in which the oxidation reaction of the reducing substance does not easily occur.

[0010]

The present inventors have conducted various studies for the purpose of constructing a new enzyme sensor that meets the above demand. As a result, a conductive paste containing, for example, carbon black or graphite as a conductive powder material, noble metal fine particles, for example, platinum (platinum) black, palladium black, or gold colloid, an oxidoreductase, and a binder is added without adding a mediator. Prepare a working electrode by applying a conventional printing method on an insulative substrate and drying to form a working electrode. It has been found that, by providing the enzyme sensor on a substrate, an enzyme sensor having stable operability and high performance even at a low applied voltage can be manufactured at low cost.

Therefore, according to the present invention, in an enzyme sensor having an electrode system comprising at least a working electrode and a counter electrode provided on an insulating substrate, the working electrode comprises at least oxidoreductase, noble metal fine particles, conductive powder material and binder. An amperometric enzyme sensor is provided, which is a single coating layer made of a conductive paste containing:

The oxidoreductase, noble metal fine particles, conductive powder material, and binder used in the enzyme sensor of the present invention are in the form of a single paste-like mixture, and the sensor is used as a single coating layer of the mixture. Working electrode can be formed.

In the method for producing an enzyme sensor according to the present invention, since the conductive paste can form an electrode by one printing, it is possible to save at least two steps of conventional electrode formation and enzyme immobilization to one step. be able to.
Therefore, the manufacturing process is simplified.

The conductive paste used for preparing the enzyme sensor according to the present invention preferably contains carbon black as a conductive powder material, a polymer binder, and a solvent such as water. It also contains noble metal fine particles in an amount of 1 to 50% by weight, preferably 2 to 20% by weight based on the solid content of the conductive paste.

The noble metal fine particles to be blended in the conductive paste are fine particles of noble metal such as platinum, ruthenium, palladium, iridium, rhodium, gold or silver. Since the specific surface area of these noble metal fine particles is several tens to several thousand times the surface area of the same metal in a lump, the addition thereof increases the effective area of the electrode (for example, see US Pat. No. 5,269,903). When these noble metal fine particles having a large specific surface area are blended with conductive carbon and a binder, it is presumed that they are adsorbed on the surface of the conductive carbon to form a porous and sponge-like layer having good conductivity. The noble metal fine particles also act as a catalyst for mediating electron transfer between the enzyme and the electrode in the working electrode of the enzyme sensor of the present invention.

Further, in the enzyme sensor of the present invention,
A base electrode made of a conductive metal, particularly silver, can be provided between the working electrode and the insulating substrate, and the base electrode is connected to the lead wire portion.

The conductive powder material contained in the conductive paste used in the present invention functions as a conductor,
Carbon, graphite and the like can be mentioned, but carbon black is preferred from the viewpoint of cost, hydrophilicity of the surface and printability. The content of the conductive powder material is determined by taking into account the conductivity and the printing characteristics (viscosity, fluidity, etc.) adhesion to the substrate, etc., and taking into account the balance with other mixed components, and is 30 to 70% (weight ), More preferably 40-60%.

Examples of the polymer binder contained in the conductive paste used in the present invention include natural polymers such as starch, cellulose, alginic acid, gums, proteins, acrylic, butyral, and vinyl acetate. Synthetic polymers such as a polymer system, a polyamide system, a polyester system, and a polyurethane system are exemplified. When a water-soluble binder is used, it is preferable to use it by mixing with a water-insoluble binder. The content of the binder is determined by the balance between the fluidity and printability of the paste, and is usually about 5 to 50% by weight of the total solid content of the conductive paste. More preferably, it is 10 to 30%. The solvent incorporated in the paste is mainly water. An appropriate amount of a phosphate buffer and / or a volatile organic solvent such as ethyl acetate, acetone, and ethanol can also be added.

The enzyme mixed with the conductive paste is not particularly limited as long as it is an oxidoreductase. For example, glucose oxidase, cholesterol oxidase,
Lactate oxidase, alcohol oxidase, xanthine oxidase, pyruvate oxidase, aldehyde oxidase and the like are used.

As the insulating substrate, ceramic, glass, glass epoxy, plastic, etc., have high insulating properties.
Any material having good heat conductivity may be used, but a plastic film such as polyvinyl chloride, polyester, polyethylene, or polypropylene that is inexpensive and easy to handle is preferable.

The working electrode provided on the insulating substrate can have a plane shape commonly used in an enzyme sensor. For example, it is circular, rectangular or rectangular. The shape of the counter electrode can be any suitable shape that matches the shape of the working electrode, for example a ring, rectangle or concave.

As a printing method for producing the electrode portion of the enzyme sensor, screen printing is suitable, but gravure printing, gravure offset printing, nozzle coating,
Dispenser printing, ink jet printing, etc. can also be applied.

When H ₂ O ₂ is used as the detection substance in the enzyme sensor of the present invention, the voltage for oxidizing H ₂ O ₂ is
It can be applied in the range of + 0.3V to + 0.7V for the Ag / AgCl counter electrode. Further, an applied voltage of +0.3 V to +0.5 V is preferable in order to effectively eliminate interference caused by a reducing substance coexisting in the sample.

Next, an example of the structure of the enzyme sensor of the present invention will be described with reference to the accompanying drawings.

FIG. 1 of the accompanying drawings shows a schematic plan view of an embodiment of the enzyme sensor according to the present invention. In FIG. 1, a working electrode 2 having a circular planar shape is provided at the center of a rectangular insulating substrate 1. The working electrode 2 is formed by coating a conductive paste containing carbon black, an enzyme, platinum black and a binder on a circular silver base electrode (not shown) provided directly on the substrate 1 by a printing method. , Dried.

The counter electrode 3 is provided in an annular shape so as to surround the working electrode 2. This counter electrode is a silver electrode (Ag / AgCl electrode) plated with silver chloride. The underlying electrode of the working electrode 2 is connected to a lead 4 of a conductive metal (preferably silver),
The counter electrode 3 is also connected to a similar lead wire portion 4 '. These lead wires 4, 4 'are produced by applying a silver paste by a printing method.

[0027]

Next, an enzyme sensor of the present invention will be specifically described with reference to Example 1 and Comparative Example 1.

Example 1 A glucose enzyme sensor for measuring glucose in a biochemical sample was prepared as follows. A carbon paste comprising a kneaded product of acetylene black and an aqueous solution of carboxymethyl cellulose was prepared. Further, 8.0 g of this carbon paste was platinum black [Kanto Chemical Co., Ltd.]
1.0 g and glucose oxidase (GOD) [Toyobo Co., Ltd.
By mixing with 1.0 g (125 IU / mg) of
A conductive paste was prepared in the form of a homogeneous mixture.

The above-mentioned conductive paste was screen-printed on a silver base electrode provided on an insulating substrate made of polyethylene, and dried at 45 ° C. for 1 hour, as shown in FIG. A working electrode having a layer structure was produced. Thus, a circular working electrode having a diameter of about 1.5 mm was formed on the substrate. The working electrode thus produced is referred to as a CP-Ptb-GOD electrode.

Further, on the substrate, as shown in FIG.
The silver base electrode provided in a ring shape was plated with AgCl to prepare a silver / silver chloride electrode (Ag / AgCl). Thus, an enzyme sensor was produced.

Comparative Example 1 For comparison, three types of electrodes were prepared as follows. (i) Platinum disk electrode (Pt): A platinum electrode in which a platinum wire having a diameter of 1.0 mm is sealed in glass and its end face is polished to expose a circular platinum surface having a diameter of 1.0 mm. (ii) Carbon paste electrode (CP): A carbon paste electrode having a diameter of 1.0 mm formed by printing only a carbon paste on a silver base electrode provided on an insulating substrate in the same manner as in Example 1. (iii) Carbon paste-platinum black electrode (CP-Pt
b): A 1.0 mm diameter circular electrode produced by adding the same amount of BSA (bovine serum albumin) instead of GOD added to the conductive paste of Example 1. Since BSA is an inactive protein, it is often used for evaluating the performance of an electrode substrate.
The CP-Ptb electrode manufactured in this way is the same as the CP-Pt electrode manufactured in Example 1.
It has the same structure as the tb-GOD electrode, and has no enzymatic activity.

Next, the performance evaluation of the electrode substrate used in the present invention and the enzyme sensor produced in Example 1 will be specifically described for Test Examples 1 and 2.

Test Example 1 In order to evaluate the performance of a carbon paste containing platinum black as an electrode substrate, the following test was conducted.

In the working electrode of an amperometric enzyme sensor using H ₂ O ₂ as a detection substance, platinum is generally suitable as an electrode material. This is because the response current corresponding to the change in the applied voltage applied to the working electrode clearly changes at the platinum electrode. The correlation between the change in the applied voltage and the change in the response current can be measured by a cyclic voltammetry scanning method [for example, see the section “Indicating electrodes for voltammetry” in the book “Electrochemical Measurement Method” (1984)). ].

Since the CP-Ptb electrode manufactured in Comparative Example 1 was the same as the electrode substrate of Example 1, a comparative test with the Pt electrode was performed. For reference, a CP electrode was also measured.

(I) Using a Pt electrode as a working electrode and an Ag / AgCl electrode as a reference electrode, the sample was placed in a 1.0 N aqueous sulfuric acid solution, and the applied voltage was measured by an electrochemical measurement apparatus (CS-1090 of Toa Denpa Co., Ltd.). The correlation waveform between the response current and the response current was measured by the scanning method. Scan speed is 10
0 mV / s. This response waveform is shown in FIG.

In FIG. 2A, a curve Pt indicates a change curve of the response current corresponding to the scanning voltage. By this potential scanning (cycle of voltage rise and fall), specific adsorption and desorption of hydrogen and formation and desorption of an oxide film on the surface of the platinum electrode are recognized. It is determined that these measurement conditions can be used as conditions for confirming the electrode characteristics of the platinum electrode.

(Ii) Next, the CP electrode of Comparative Example 1 was measured under the same conditions in place of the Pt electrode. The response waveform is shown by CP in FIG. From this waveform, it was found that a response current corresponding to the voltage applied to the CP electrode could not be confirmed. Therefore, it is determined that the CP electrode is unsuitable as an electrode substrate for the working electrode of an amperometric enzyme sensor using H ₂ O ₂ as a detection substance.

(Iii) Further, the same measurement as in (i) was performed using the CP-Ptb electrode of Comparative Example 1. The response waveform is shown in Figure 2 of the attached drawing.
(B) CP-Ptb. This response waveform is large, approximating the response waveform of Pt ((a) of FIG. 2), and the response current is Pt
It is recognized that it is about 10 times larger. Therefore, CP-Ptb
The electrodes are remarkably good in terms of response current sensitivity, and are also considered to have excellent performance. Then, it is determined that it can be sufficiently used as a working electrode base material of an amperometric enzyme sensor using H ₂ O ₂ as a detection substance.

Therefore, the CP-Ptb-GOD electrode of Example 1
Since tb and the electrode material are the same, it is recognized that the material used for CP-Ptb-GOD also has excellent performance as a working electrode substrate in an amperometric enzyme sensor.

Test Example 2 The following test was performed to examine the measurement performance of the enzyme sensor for measuring glucose having a single layer structure prepared in Example 1.

(I) The response characteristics of the glucose enzyme sensor were examined as follows. That is, the CP-Ptb-GOD enzyme sensor prepared in Example 1 described above was replaced with a 50 mM phosphate buffer (p
H7.0). Ag / AgCl for working electrode of enzyme sensor
A voltage of +0.5 V was applied to the reference electrode. An aqueous solution of glucose as a substrate was added dropwise to the above buffer solution so that the final glucose concentration was 5.0 mM, and the mixture was mixed uniformly to perform measurement. The solution was not agitated during the measurement. For comparison, the above enzyme sensor was thoroughly washed with a 50 mM phosphate buffer, placed again in a 50 mM phosphate buffer, and an aqueous ascorbic acid solution as a reducing substance as an interfering substance was used.
The measurement was carried out in the same manner by dropping ascorbic acid final concentration to 0.5 mM.

The response current (uA) flowing between the working electrode and the counter electrode (reference electrode) and the response time (seconds) (that is, the time elapsed from the time of dropping the substrate glucose or ascorbic acid) were measured, and the results were attached to the attached drawing. Is shown in FIG.

It can be seen from the comparison between the left and right curves in FIG. 3 that the response current in the case of adding the above concentration of glucose is larger than that in the case of adding the above concentration of ascorbic acid. It was also confirmed that the enzyme activity of the enzyme sensor did not change before and after the above measurement. And it turns out that the enzyme sensor of Example 1 of this invention showed high sensitivity with respect to glucose, and the response with respect to an interfering substance was low. On the other hand, usually, the glucose concentration in human serum is 4.
The ascorbic acid concentration is 0.
Since the concentration is 017 to 0.08 mM, the glucose concentration in human serum can be measured with low interference and high sensitivity using the glucose enzyme sensor of Example 1.

(Ii) The dynamic range for glucose of the glucose enzyme sensor prepared in Example 1 was examined as follows. That is, it was produced in Example 1 above.
The CP-Ptb-GOD enzyme sensor was used in a 50 mM phosphate buffer (pH 7.
0). A voltage of +0.5 V was applied to the working electrode of the enzyme sensor with respect to the Ag / AgCl reference electrode. An aqueous solution of glucose as a substrate was added dropwise to the above buffer solution so that the final glucose concentration was constant, and the mixture was mixed uniformly to measure a current response. The solution was not agitated during the measurement. Next, the enzyme sensor was washed thoroughly with 50 mM phosphate buffer,
Similar measurements were made. However, the amount of the aqueous glucose solution dropped at that time was adjusted so that the final glucose concentration was changed. The measurement was repeated in this manner, and the measurement was performed in a final glucose concentration range of 0 to 50.0 mM. The measurement results are shown in FIG. 4 of the accompanying drawings.

As a result, a wide range of linearity was obtained up to a final glucose concentration of 20 mM in the calculation by the end point method and up to 50 mM in the calculation by the initial velocity method. The endpoint method and the initial velocity method are described in “Revised Clinical Chemistry” 122-125.
Page, published in Kodansha 1984.

[Brief description of the drawings]

FIG. 1 is a schematic plan view of an enzyme sensor according to an embodiment of the present invention.

FIG. 2 (a) is a curve diagram of a response waveform of a platinum electrode in a 1.0N aqueous sulfuric acid solution by a cyclic voltammetric method. FIG. 2B shows a carbon paste electrode (CP electrode) similarly measured by the cyclic voltammetric method and an electrode (CP-P) made of carbon and platinum black.
FIG. 4 is a curve diagram of a response waveform with a tb electrode).

FIG. 3 shows the glucose measured in a phosphate buffer solution (50 mM, pH 7.0) of the glucose enzyme sensor (a sensor having a CP-Ptb-GOD electrode as a working electrode) manufactured in Example 1 of the present invention. And response curves for ascorbic acid and ascorbic acid.

FIG. 4 is a curve diagram for examining the linearity of a current response curve of the glucose enzyme sensor produced in Example 1 of the present invention. The left vertical axis is the result of measurement by the end point method, and the right vertical axis is the result of measurement by the initial velocity method.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Insulating substrate 2 Working electrode 3 Counter electrode 4, 4 'Metal lead part

Claims (7)

[Claims] 1. An enzyme sensor having an electrode system comprising at least a working electrode and a counter electrode provided on an insulating substrate, wherein the working electrode includes at least an oxidoreductase, noble metal fine particles, a conductive powder material, and a binder. An amperometric enzyme sensor characterized by a single coating layer made from a paste. 2. The enzyme according to claim 1, wherein the oxidoreductase, the noble metal fine particles, the conductive powder material and the binder are in the form of a single paste-like mixture, and the working electrode is a single coating layer of the mixture. sensor. 3. The noble metal fine particles are fine particles of colloidal, porous mass or powder of noble metal such as platinum, gold, ruthenium, palladium, iridium, rhodium, silver or the like, and the specific surface area of the noble metal is noble metal mass. The enzyme sensor according to claim 1, which is larger than a substance. 4. The amount of the noble metal fine particles in the conductive paste is 1 to 50% by weight based on the solid content of the conductive paste.
The enzyme sensor according to claim 1, which is: 5. The enzyme sensor according to claim 1, wherein the noble metal fine particles in the conductive paste are of a single type. 6. The enzyme sensor according to claim 1, wherein the conductive paste contains a plurality of types of noble metal fine particles. 7. When hydrogen peroxide at the working electrode is used as the detection substance, the DC voltage applied between the working electrode and the counter electrode is +0.3.
2. The enzyme sensor according to claim 1, wherein the range is from V to + 0.7V.