JP3186862B2 - Catechol compound sensor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a sensor capable of detecting a low concentration of a catechol compound with high sensitivity.

[0002]

2. Description of the Related Art Adrenaline and noradrenaline secreted from the adrenal medulla are synthesized in vivo from tyrosine via dopa and dopamine. Each of the above catecholamine compounds ranging from dopa to adrenaline, blood, urine,
Although present in very small amounts in biological samples such as cerebrospinal fluid, accurately measuring these amounts is, for example,
It is important for examination of adrenal medulla function, examination of central amine level such as neurosis, or emotional stress examination. As a method for detecting or measuring these catecholamines, a method in which catecholamines are oxidized to corresponding quinones by an oxidase and the amount of oxygen consumed in the oxidation reaction is measured by an oxygen electrode can be considered. However, since the concentration of catecholamines in a biological sample is generally extremely low, the amount of oxygen reduction is close to the detection limit of an oxygen electrode, and there is a disadvantage that it is difficult to distinguish the noise from noise.

On the other hand, enzyme immunoassay (EI)
In the field of A) and ELISA, a method for measuring a trace amount of a catechol compound with high sensitivity is disclosed in JP-A-56-501.
384. In this method, a catechol compound is oxidized to a corresponding quinone compound by an oxidase, and the corresponding quinone compound obtained is reduced with a reducing agent NA
The original catechol compound is reduced again by DH, and these oxidation and reduction reactions are continuously circulated. Here, in the oxidation reaction, oxygen is irreversibly reduced to water at the same time as the oxidation of the catechol compound to the quinone compound. Also, in the above-mentioned reduction reaction, the reducing agent / NA is simultaneously added with the reduction of the quinone compound to the original catechol compound.
DH is irreversibly oxidized to NAD.
H consumption (NAD generation) is performed independently of the oxygen consumption described above. This circulation reaction is illustrated as follows.

[0004]

(Equation 1)

Therefore, the amount of the oxidase and the reducing agent, NADH, are compared with the amount of the catechol compound originally present in the sample.
Is present in an excess amount, the above-mentioned circulating reaction proceeds at a reaction rate corresponding to the amount of the catechol compound, and oxygen and NADH are cumulatively consumed, and NAD is also cumulatively produced at the same time. The NADH or NAD produced in this manner is measured by, for example, a spectrophotometric method or a fluorimetric method.

[0006]

However, Japanese Patent Publication No. 56-501384 described above specifically describes the measurement of oxygen which should be consumed by the circulating reaction and the means for measuring it. However, there is no suggestion that the oxidase can be immobilized and used. The present inventors have conducted intensive studies to apply the above-mentioned circulating reaction, and surprisingly, when a layer containing an oxidase and a reducing agent is supported on an oxygen permeable membrane of an oxygen electrode and used, It has been found that the amount of oxygen that changes according to the concentration of the catechol compound can be detected with high sensitivity. In addition, the present inventor has disclosed the above-mentioned Japanese Patent Publication No. 56-501384.
It has also been found that there is a reducing agent that is superior to NADH specifically described in the publication. The present invention is based on such findings.

[0007]

Accordingly, the present invention provides a layer containing a reducing agent and an oxidase together or separately (hereinafter referred to as a layer containing a reducing agent / oxidase) on an oxygen-permeable membrane of an oxygen electrode. A catechol compound sensor characterized by carrying a catechol compound sensor.

In the present specification, the catechol compound is
It refers to catechol (1,2-dioxybenzene) and derivatives thereof. The catechol derivative is particularly limited as long as it is a compound that can be converted to the corresponding quinone derivative by an oxidase and converted to the original catechol derivative by a reducing agent. Not done. As catechol derivatives, particularly aminoalkyl-substituted catechol present in vivo, for example,
Adrenaline (or epinephrine), noradrenaline (or norepinephrine), dopa or dopamine can be mentioned.

The sensor of the present invention has a structure in which a reducing agent / oxidase-containing layer is provided further outside the outermost layer of the oxygen permeable membrane of a conventionally known oxygen electrode. FIG. 1 schematically shows one embodiment of the sensor of the present invention. The oxygen electrode portion includes an oxygen permeable membrane 1 which is the outermost layer, a cathode 2 in contact with the oxygen permeable membrane 1, a lead wire 3 from the cathode 2, an electrolytic solution 4, an anode 5, and a housing 6. Then, a reducing agent-containing layer 7 and an oxidase-containing layer 8 are provided on the oxygen permeable membrane 1, and these three layers are fixed with a grip ring 9. The oxygen electrode used in the present invention is not particularly limited as long as it has an oxygen permeable membrane that comes in contact with the liquid sample as the outermost layer. Further, the reducing agent / oxidizing enzyme-containing layer is not limited to the embodiment shown in FIG. 1 and may include a reducing agent and an oxidizing enzyme together in one layer.
An oxidase-containing layer 8 may be provided on the oxygen permeable membrane 1, and a reducing agent-containing layer 7 may be further provided thereon.

The oxidase used in the method of the present invention is not particularly limited as long as it can convert a catechol compound into a corresponding quinone compound while consuming oxygen, and does not substantially deactivate even when immobilized. For example, tyrosinase or laccase can be mentioned, and tyrosinase is particularly preferable.

The reducing agent used in the method of the present invention is not particularly limited as long as it is a compound capable of converting the corresponding ketone compound into the original catechol compound. For example, NADH, potassium ferrocyanide, ascorbic acid or The salt can be mentioned. The present inventor has found that when ascorbic acid or a salt thereof is used as a reducing agent, a change in the amount of oxygen in the above-mentioned circulating reaction can be detected with particularly high sensitivity. Ascorbic acid
It is desirable to have the L-form, but it may be the D-form or the racemic form. The ascorbate is desirably an inorganic salt, for example, an alkali metal (for example, sodium or potassium) salt.

The oxidase-containing layer can be prepared by immobilizing an oxidase on a suitable support (for example, a polymer film such as a cellulose film or a nonwoven fabric) by a known method.
For example, in order to fix tyrosinase on an acetylcellulose membrane, it is desirable to use a so-called carrier binding method in which glutaraldehyde or carbodiimide resin is used for chemical fixation. When immobilized on a support having a relatively rough structure such as a nonwoven fabric, the above carrier binding method may be used.However, tyrosinase is retained in silk fibroin and formed into a film on the surface of the constituent fibers of the nonwoven fabric. It is better to use the law.

The reducing agent-containing layer can be prepared by supporting a reducing agent on a suitable support (for example, filter paper or nonwoven fabric) by a known method. For example, in order to fix ascorbate to a nonwoven fabric, dry ascorbate is bonded to the fibers with an adhesive, or the bonding fibers are mixed in the constituent fibers of the nonwoven fabric, and the fibers are bonded by the bonding fibers. Good. Alternatively, the ascorbate solution may be impregnated into the nonwoven fabric and dried to be applied. Ascorbate can be filled in a nonwoven fabric covered with a polymer membrane sheet or the like that does not transmit ascorbate powder.

In order to include an oxidase and a reducing agent in one layer, for example, the oxidase is immobilized on the surface of the constituent fibers of the nonwoven fabric by an entrapment method or the like, and the dry state is filled in the gaps between the constituent fibers of the nonwoven fabric. The reducing agent is filled and the nonwoven fabric is covered with a polymer film or the like so that the reducing agent does not leak.

When the sensor of the present invention is used to measure the amount of catecholamine in a blood sample, for example, a blood coagulation inhibitor is added immediately after blood collection, and cell components are removed by centrifugation to obtain plasma. Next, when the sensor of the present invention is immersed in the plasma, the plasma permeates the reducing agent / oxidase-containing layer provided on the oxygen permeable membrane, and an oxidation reaction occurs between catecholamine and the oxidase in the plasma. The corresponding quinone compound is formed, and then the reaction in which the quinone compound is reduced with a reducing agent to reduce it to the original catecholamine circulates,
Oxygen is amplified and consumed by repeating the oxidation reaction. Since the amplified oxygen consumption is detected by the oxygen electrode, the amount of catecholamine can be detected with extremely high sensitivity.

The sensor of the present invention is a liquid biological sample (for example,
It can be used not only for detecting or measuring a catechol compound in blood, serum, plasma, urine or cerebrospinal fluid) but also generally for detecting or measuring a catechol compound in any liquid sample.

[0017]

According to the sensor of the present invention, the amount of oxygen consumed according to the concentration of catechol and its related compounds contained in a trace amount in a sample can be detected with high sensitivity.

[Brief description of the drawings]

FIG. 1 is an explanatory view schematically showing one embodiment of a sensor according to the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 ... Oxygen permeable membrane 2 ... Cathode 3 ... Lead wire 4 ... Electrolyte 5 ... Anode 6 ... Housing 7 ... Reducing agent containing layer 8 ... Oxidase enzyme containing layer 9 ... Grip ring

Continuation of the front page (56) References JP-A-62-58157 (JP, A) JP-A-63-144245 (JP, A) JP-A-4-216453 (JP, A) JP-A-2-61549 (JP) JP-A-63-172950 (JP, A) JP-A-2-298855 (JP, A) JP-T-56-501384 (JP, A) JP-T-62-501171 (JP, A) (58) Fields surveyed (Int.Cl. ⁷ , DB name) G01N 27/327 G01N 27/404 G01N 27/416 JICST file (JOIS) WPI (DIALOG)

Claims (1)

(57) [Claims] 1. A catechol compound sensor characterized in that a layer containing a reducing agent and an oxidase together or separately is carried on an oxygen permeable membrane of an oxygen electrode.