WO2002018924A1

WO2002018924A1 - Method for determination of substrate and biosensor

Info

Publication number: WO2002018924A1
Application number: PCT/JP2000/005789
Authority: WO
Inventors: Naoki Shinozuka; Toru Yokoyama; Kenji Nakamura
Original assignee: Sapporo Immuno Diagnostic Laboratory
Priority date: 2000-08-28
Filing date: 2000-08-28
Publication date: 2002-03-07
Also published as: AU2000267324A1

Abstract

A method for determining a substrate wherein a substrate in a sample is determined by providing a biosensor having a structure in which electrodes are formed on the same plane by the use of electrically conductive materials, and reaction reagents including at least a dehydrogenase, a coenzyme, an electronic mediator and a tetrazolium salt are fixed on absorbent carriers and arranged on or above the electrodes, and detecting electrochemically a formazan produced by the supply of a sample; and a biosensor used therein. The method and the biosensor can be used for determining a substrate in a sample specifically, rapidly and with ease, without the need of a complicated pretreatment of the sample.

Description

Specification

FIELD OF THE INVENTION Field of the Invention

The present invention relates to a method and a biosensor for specifically and simply quantifying a substrate in a sample without requiring complicated pretreatment of the sample. Specifically, a reaction reagent comprising at least a dehydrogenase, a coenzyme, an electron mediator, and a tetrazolium salt was immobilized on an absorbent carrier and placed on an electrode formed on the same plane using a conductive material. A method for quantifying a substrate and a biosensor are provided. Background of the Invention

In recent years, various biosensors have been devised and put into practical use that simply and rapidly quantify a substrate in a sample without diluting or stirring the sample solution.

A biosensor is a technology that enables quantification by combining a molecular recognition function that specifically reacts with a substrate contained in a sample, a transducer that can electrically convert information, and electronics technology. Are classified into enzyme sensors, immunosensors, DNA sensors, etc. Among them, enzyme sensors have been the most studied since ancient times and widely used.

Enzyme sensors are also classified into reaction principles using oxidases or dehydrogenases. Enzyme sensors using oxidases have been applied to many blood glucose measurement sensors. However, an enzyme sensor using an oxidase is greatly affected by dissolved oxygen, which poses a major problem when measuring low-concentration regions. For this reason, enzymatic sensors using dehydrogenase that are not affected by dissolved oxygen are being actively studied.

Since the reduced coenzyme produced from the dehydrogenase reaction is a non-conductive protein, direct electrochemical detection requires a high applied potential. Therefore, a method of electrochemically detecting the reduced coenzyme generated from the enzymatic reaction via electronic media has been used. (Japanese Unexamined Patent Publication No. 10-165 1999). In the electron media reaction, a reduced electron mediator is generated by a redox reaction with a reduced coenzyme, and a redox reaction is easily performed by applying an electric potential to the electrode to determine the substrate concentration in the sample. Amount is possible. From this, it is possible to produce enzyme sensors by using electronic media.

We have also developed a biosensor (special feature) that integrates oxidized nicotinamide adenine dinucleotide (NAD +) of various dehydrogenases and coenzymes and 1-methoxy PMS of electron mediator with an electrode as a reaction reagent. We devised Nipponhei 10—2 0 15 5 3, WO 00/04 4 378) and produced biosensors for various substrates. One of the features of these biosensors is that they are biosensors in which an absorbent carrier carrying all the reaction reagents is placed between the working electrode formed by printing a conductive material and the electrode facing the counter electrode. A linear and good response current depending on the concentration was confirmed. However, subsequent studies showed that these biosensors had large variations in the low concentration region of the substrate, and needed to be improved.

Therefore, in addition to an electrode formed using a conductive material, at least a dehydrogenase, a coenzyme, and an electron mediator, a biosensor using a reagent consisting of a tetrazolium salt has been developed (PC TZ JP99 / 0 1 3 9 2). Compared to biosensors using reduced coenzyme direct oxidation or various electronic media, this biosensor ultimately produces chemically stable formazan, resulting in less responsive response. A current is obtained. In addition, a large increase in response current and an improvement in detection sensitivity were observed, enabling highly sensitive quantification of the substrate in a low concentration range. The biosensor fabricated at that time had a structure in which both electrodes faced each other, and the reaction reagent was immobilized on both electrodes. However, subsequent studies revealed that this method was structurally difficult to produce in large quantities, and this time the production method was improved for mass production. Summary of the Invention

In order to solve the above problems, at least a dehydrogenase, a coenzyme, an electron mediator and a tetrazolium salt are formed on at least an electrode composed of a working electrode and a counter electrode formed on the same plane using a conductive material. It is intended to provide a method for quantifying a substrate and a biosensor on which an absorptive carrier in which a reaction reagent consisting of:

The biosensor of the present invention is on the same plane The electrode system is formed in a simple manner.Since it has a simple structure in which an absorptive carrier on which all the reaction reagents are immobilized is simply laminated on the electrodes, the biosensor can be easily manufactured, Production has become possible. Description of the invention

In the present invention, an electrode system comprising at least a working electrode and a counter electrode formed on the same plane using a conductive material, and absorbing a reaction reagent comprising at least a dehydrogenase, a coenzyme, an electron mediator, and a tetrazolium salt. To provide a method for quantifying a substrate in a sample having a structure immobilized on an absorbent carrier, and a biosensor capable of simple and rapid quantification by integrating an electrode and an absorptive carrier on which a reaction reagent is immobilized Is what you do.

In the present invention, the reducing coenzyme is generated by a specific enzymatic reaction of a substrate in a sample with a dehydrogenase and a coenzyme as reaction reagents, and further, a redox reaction with an electron mediator and a tetrazolium salt. Progress and eventually produce chemically stable formazan. Subsequently, when a potential is applied to the electrode, formazan is oxidized, and the concentration of the substrate in the sample can be determined from the obtained oxidation current.

The feature of the present invention is that by disposing the absorbent carrier on both electrodes formed on the same plane on the insulating substrate, an effect of the filter by the absorbent carrier can be obtained, and the measurement contained in the sample can be performed. Separation of the influential solid components etc. becomes possible, and only the separated sample reaches both electrodes. Further, the separation effect can be further improved by using several kinds of absorbent carriers and gradually reducing the pore size. In addition, since it has a simple structure in which an absorbent carrier is simply laminated on the electrode, the biosensor can be easily manufactured and mass production is possible. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a method for manufacturing an electrode according to one embodiment of the present invention;

FIG. 2 is a configuration diagram of a method (1) for manufacturing a sensor chip in Example 2, and FIG. 3 is a configuration diagram of a method (2) of manufacturing a sensor chip in Example 3. FIG. It is a block diagram of the manufacturing method (3). FIG. 5 is a configuration diagram of a method (4) for manufacturing a sensor chip in Example 5; FIG. 6 is a rough drawing showing a result of an addition test of glucose in saliva in Example 6;

FIG. 7 is a graph showing the results of a dilution test of glucose in saliva in Example 7.

The symbols in the above figures are explained as follows:

1 is an insulating support; 2 is a lead; 3 is an insulating layer; 4 is a working electrode; 5 is a counter electrode; 6 is a cover; 7 is a supply port; 8 is an absorption layer; 11 is a sponsor. Description of the preferred embodiment

In the present invention, the structures of the substrate to be measured, the reaction reagent, and the sensor chip will be described in order.

The measurable substrate of the present invention is not particularly limited as long as it can generate a reduced coenzyme using dehydrogenase as a catalyst, and any substrate can be quantified. For example, alanine, alcohol, aldehyde, isochenoic acid, peridine-5, -diphosphoglucose, galactose, formic acid, dariceraldehyde-13-monophosphate, glycerol, glycerol-13-phosphate, glucose, glucose-16 —Phosphoric acid, glutamic acid, cholesterol, sarcosine, sorbitol, carbonic acid, lactic acid, 3-hydroxybutyric acid, pyruvic acid, phenylalanine, flux 1, 6-phosphogluconic acid, formaldehyde, mannitol, malic acid, mouth isocyanate Etc. are available.

The dehydrogenase used in the present invention is not particularly limited as long as it is an enzyme that produces a reduced coenzyme, and the origin is not particularly limited. For example, alanine dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, isocenoic acid dehydrogenase, peridine-1,5-diphosphoglucose dehydrogenase, galactose dehydrogenase, formate dehydrogenase, darisel Aldehyde-13-phosphate dehydrogenase, glycerol dehydrogenase, glycerol-13-phosphate dehydrogenase, glucose dehydrogenase, glucose-16-phosphate dehydrogenase, glutamate dehydrogenase, kore Sterol dehydrogenase, sarcosine dehydrogenase, sorbitol dehydrogenase, carbonic acid dehydrogenase, lactate dehydrogenase, 3-hydroxybutyrate dehydrogenase, pyruvate dehydrogenase, phenylalanine dehydrogenase, fructose dehydration Enzyme, 6-phosphodalconate dehydrogenase, formaldehyde dehydrogenase, mannitol dehydrogenase, malate dehydrogenase, leucine dehydrogenase and the like can be used.

There is no particular limitation on the electron mediator as long as it is a substance that promptly undergoes an oxidative reduction reaction with a reduced coenzyme and a tetrazolium salt. For example, quinones, diaphorases, cytochromes, porogens, phenazines, 'phenoxazines, phenothiazines, ferricyanides, ferredoxins, phenocens and derivatives thereof can be used. Among them, phenazines show stable response here, and in particular, 1-methoxy PMS has good storage stability and excellent reactivity with reduced coenzyme and tetrazolium salt. —I found it to be a good evening.

The tetrazolium salt is not particularly limited as long as it forms formazan, and among them, 2- (4-1-dophenyl) -1,3- (412-trophenyl) 1,5-1 (2,4-disulfophenyl) 1-1 2H-tetrazolium, monosodium salt (WST-1) is a water-soluble and chemically stable formazane produced upon reduction, and the produced formazan shows good response in electrochemical detection methods From this, it has been found that the tetrazolium salt in the present invention is preferable.

By pre-fixing the above reaction reagents, a method can be constructed in which a series of reactions proceed to finally produce formazan by dissolving and mixing just by bringing the sample into contact.

The electrode used in the present invention is a conductive substance and is not particularly limited as long as it is electrochemically stable. The material is carbon, gold, silver, silver / silver chloride, nickel, platinum, platinum black, and platinum black. Palladium, etc., and their alloys can be used. As a result of examining various materials among them, they found that carbon materials are inexpensive and chemically stable, and are preferable as working electrodes of the electrodes in the present invention.

Here, the carbon material means all materials containing carbon. The carbon materials that can be used are not particularly limited, and are used in conventional carbon electrodes. Any material can be used, for example, pon fiber, carbon black, bon paste, glass, bonbon, graphite, and the like. Such a carbon material is formed as an electrode portion on an insulating support by a conventional method. Usually, it can be formed by printing a paste made from a carbon fiber material using a resin binder or the like and then heating and drying the paste.

The printing method is not particularly limited to screen printing, and gravure printing, offset printing, inkjet printing, and the like can be applied.

Since the counter electrode also serves as the reference electrode, silver / silver silver is selected here.The lead that is the connection between the electrode reaction part and the electrochemical detection circuit is the most conductive and inexpensive silver. Was selected and formed by printing.

In the present invention, a two-electrode system including only the working electrode and the counter electrode is used. However, if a three-electrode system including a reference electrode is used, more accurate quantification can be performed.

Examples of the insulating support include glass, glass epoxy, ceramics, and plastic. However, there is no particular limitation as long as it is a material that is not attacked when printing the electrode portion or adding a sample. For example, plastic films such as polyester, polyethylene, polyethylene terephthalate (PET), polystyrene, and polypropylene are inexpensive, and PET films are preferred here because of their good adhesion to conductive inks and good workability. I found it.

The cover, spacer, and the material that adheres to it can be shaped as long as the sample can be retained, the absorbent carrier can be retained, there is no hindrance to the series of reactions, and external contact with the reaction area can be protected. There are no particular restrictions on the material, etc.

The absorbent carrier used in the present invention is not particularly limited as long as it is a material that can absorb the reaction reagent solution and does not affect the reaction reagent. Examples of the material include glass fiber, silica fiber, and cellulose fiber. Fibers and carboxymethylcellulose, getylaminoethylcellulose, cellulose acetate, cellulose mixed ester, nylon woven fabric, nitrocellulose, polypropylene, polyether sulfone, polyester nonwoven fabric, polytetrafluoroethylene (PTFE), Polypropylene or the like can be used.

In the present invention, the three types of absorbent carriers of the absorption layer, the spreading layer, and the reaction layer are respectively It uses polypropylene, cellulose fiber, and nitrocellulose, and forms a reaction reagent layer by layering them. A single-layer structure is also possible, in which case the constituent materials can be reduced. Example

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto. Example 1 Method for producing electrode

FIG. 1 shows the electrode manufactured in this example. It should be noted that this electrode can be manufactured more easily by screen printing two types of electrodes on the same plane.

Screen printing of conductive silver ink (Nippon Azizon Co., Ltd.) on the insulating support 1 of PET film (Toray Co., Ltd.), followed by heating and drying (120 ° C, 15 minutes), Form 2 was formed. Next, the insulating layer 3 was formed (cured by ultraviolet irradiation) using an insulating ink (manufactured by Nippon Acheson Co., Ltd.) while leaving the connection portions to the working electrode and the counter electrode. The working electrode 4 is screen printed using conductive graphite ink (manufactured by Nippon Acheson Co., Ltd.), and the counter electrode 5 is printed using conductive silver chloride silver ink (manufactured by Nippon Acheson Co., Ltd.). After drying (120, 15 minutes), electrodes were laminated. Example 2 Fabrication method of sensor chip (1)

FIG. 2 shows the sensor chip manufactured in this example. This sensor chip is composed of the electrode prepared in Example 1 and three kinds of absorbent carriers.The sample is supplied into the sensor chip by bringing the sample into contact with the supply port 7 of the force par 6. It is a structure that can be used.

A polypropylene membrane (manufactured by Nippon Millipore Co., Ltd.) is used as the absorbent carrier 8 of the absorption layer, and a buffer component for adjusting the optimum pH of the enzyme reaction is absorbed in a solution and dried (at 40 ° C). , 15 minutes).

Cellulose fiber (Advantech Toyo Co., Ltd.) as absorbent carrier 9 in the spreading layer Then, 1-methoxy PMS (made by Dojindo Laboratories, Inc.), which is an electronic media overnight, is dissolved in ultrapure water, then absorbed and dried (40, 15 minutes) and fixed. Has become

A nitrocellulose membrane (manufactured by Nippon Millipore Co., Ltd.) was used as the absorbent carrier 10 in the reaction layer, and a tetrazolium salt WST-1 (manufactured by Dojin Chemical Lab.) And a dehydrogenase (Toyobo) Co., Ltd.) and oxidized nicotinamide adenine dinucleotide (NAD '+) (manufactured by Oriental Co., Ltd.) as a coenzyme were dissolved in a phosphate buffer solution (pH 7.4, 2 O mM). After absorption, drying (40. C, 10 minutes), the cells were immobilized.

Absorbent carriers 8, 9, and 10 on which the reaction reagents were immobilized were placed on the electrode prepared in Example 1 via a spacer 11 and covered with a cover 16, thereby producing a sensor chip. did. Example 3 Sensor chip fabrication method (2)

FIG. 3 shows the sensor chip manufactured in this example. The sensor chip is composed of the electrode prepared in Example '1 and one kind of absorbent carrier', and the sample is supplied into the sensor chip by bringing the sample into contact with the supply port 7 on the side of the sensor chip. It is a structure that can do it.

Buffer solution, 1-methoxy PMS, WST-1 and enzyme are absorbed into each part of the nitrocellulose membrane of absorbent carrier 10 and dried (40 ° C, 10 minutes) and fixed. It has become.

The electrode prepared in Example 1 was arranged so that the position where the WST-1 of the absorbent carrier 10 and the enzyme were immobilized was in contact with each other, and the cover 6 was covered with the cover 11 through the spacer 11 to make the sensor. A sub chip was prepared. Example 4 Manufacturing method of sensor chip (3)

FIG. 4 shows the sensor chip manufactured in this example. This sensor chip is composed of the electrode prepared in Example 1 and three types of absorbent carriers. By bringing the sample into contact with the supply port 7 on the side of the sensor chip, the sample is immediately supplied into the sensor chip. Do Can be.

The reaction reagent was immobilized using the same absorptive carrier as in Example 2.The three absorptive carriers were placed over the electrode from the supply port so that a part of the absorptive carriers partially overlapped. 6 to form a sensor chip. Example 5 Sensor chip fabrication method (4)

FIG. 5 shows the sensor chip manufactured in this example. This sensor chip is composed of the electrode prepared in Example 1 and three types of absorbent carriers. By bringing the sample into contact with the supply port 7 on the side of the sensor chip, the sample can be supplied into the sensor chip. It has a structure that can be used.

The same reagent as in Example 2 was used to immobilize the reaction reagent, the absorption layer was arranged from the supply port to the electrode, and the other developing layer and the reaction layer were arranged on the electrode. The results of measurement of glucose in saliva using the biosensor produced by the above-described method in which a sensor chip was produced by covering with a cover 6 via a spacer 11 are shown below. Example 6 Addition test of gafflecose in saliva

FIG. 6 shows the results of measurement using a biosensor produced using glucose dehydrogenase in Example 2 and saliva samples adjusted to various concentrations by adding glucose to saliva.

A saliva sample was added 70, and after 60 seconds, a potential of +50 OmV was applied with reference to the counter electrode, and the response current value was measured 7 seconds after the potential was applied. This result shows the concentration obtained by calculating the obtained response current value by the conversion formula.

From these results, no inhibition of sensor response by saliva components was observed, and a linear response current value dependent on glucose concentration was obtained. In addition, high recoverability was obtained with respect to the added concentration, so that glucose can be quantified with high sensitivity even using saliva samples. Example 7 Dilution test of glucose in saliva

FIG. 7 shows the results of measurement using the biosensor prepared in Example 6 and a sample obtained by diluting saliva having a high glucose concentration with physiological saline.

In the same manner as in the measurement of Example 6, a sample containing glucose was immersed in 70 L, and after 60 seconds, a potential of +50 OmV was applied with reference to the counter electrode. The response current value was measured. This result shows the concentration obtained by calculating the obtained response current value by the conversion formula.

From these results, it was confirmed that glucose in saliva was specifically detected because the sample showed good linearity passing through the origin. In the above embodiment, the electrode shape, the shape of the absorbent carrier, the arrangement method, and the like have been described, but the present invention is not limited thereto. In addition, although one or three absorbent carriers have been described, a biosensor can be manufactured using more absorbent carriers.

Claims

The scope of the claims

1. At least a reaction reagent consisting of a dehydrogenase, a coenzyme, an electron mediator, and a tetrazolium salt is immobilized in the sample supply section and the electrode reaction section, and the formazan generated by the reaction caused by the supply of the sample is subjected to electricity in the electrode reaction section. A method for quantifying a substrate, which is characterized by being chemically detected.

2. The method for quantifying a substrate according to claim 1, wherein the reaction reagent is immobilized on an absorptive carrier and arranged in the sample supply section and the electrode reaction section.

3. The substrate according to claim 1 or 2, wherein the absorbent carrier has a smaller pore size than impurities contained in the sample, or uses several types of the absorbent carriers having different pore sizes. Quantitation method.

4. The method according to claim 1, wherein the electrode reaction section has at least a working electrode and a counter electrode on the same plane of the insulating substrate.

5. The method of claim 1, wherein the sample is a biological sample such as blood, urine, saliva, or sweat, a food sample, and an environmental sample.

6. The substrate is alanine, alcohol, aldehyde, isochenoic acid, peridine-1,5, diphosphoglucose, galactic acid, formic acid, dariceraldehyde-3-phosphate, glycerol, glycerol-3-phosphate. , Glucose, darcos-6-phosphate, glutamic acid, cholesterol, sarcosine, sorbitol, carbonic acid, lactic acid, 3-hydroxybutyric acid, pyruvate, phenylalanine, fruc! The method for quantifying a substrate according to any one of claims 1 to 5, wherein the substance is mannitol, malic acid, glycine, or the like.

7. At least dehydrogenase, coenzyme, electronic media and tetrazolium salt By immobilizing and integrating a reaction reagent consisting of the following into the sample supply section and the electrode reaction section, and electrochemically detecting the generated formazan by a reaction that occurs only by supplying the sample. A biosensor using the quantification method according to item 1.

8. The biosensor according to claim 7, wherein the reaction reagent is immobilized on an absorbent carrier, and is disposed in the sample supply unit and the electrode reaction unit.

9. The biomaterial according to claim 7, wherein the absorbent carrier has a smaller pore size than impurities contained in the sample, or uses several kinds of the absorbent carriers having different pore sizes. Sensor.

10. The biosensor according to claim 7, wherein the electrode reaction unit has at least a working electrode and a counter electrode on the same plane of the insulating substrate.

11. The claim, wherein the sample is a biological sample such as blood, urine, saliva, sweat, a food sample and an environmental sample. The biosensor according to any one of Items 1 to 10.

1 2. The substrate is alanine, alcohol, aldehyde, isocenoic acid, peridine-1 5'-diphosphoglucose, galactose, formic acid, glyceraldehyde — 3-phosphate, glycerol, glycerol 3- Phosphoric acid, glucose, glucosyl monophosphate, glutamic acid, cholesterol, sarcosine, sorbitol, carbonic acid, lactic acid, 3-hydroxybutyric acid, pyruvate, phenylalanine, fructose, 6-phosphogluconate, formaldehyde, The biosensor according to any one of claims 7 to 11, wherein the biosensor is mannitol, malic acid, lactic acid, or the like.