WO2022039185A1

WO2022039185A1 - Reagent composition and sensor

Info

Publication number: WO2022039185A1
Application number: PCT/JP2021/030143
Authority: WO
Inventors: 陽介鉞
Original assignee: キッコーマン株式会社
Priority date: 2020-08-18
Filing date: 2021-08-18
Publication date: 2022-02-24
Also published as: JPWO2022039185A1

Abstract

The present invention provides a biosensor.　Provided are: a biosensor including an oxidoreductase, a ruthenium compound, and a non-PMS electron transfer accelerator; and a method in which the biosensor is used.

Description

Reagent compositions and sensors

The present disclosure relates to a biosensor containing an electron transfer promoter, an oxidoreductase, and a metal complex compound, and a method using the same.

Patent Document 1 discloses a novel electron transfer promoter containing a phenylenediamine-based compound.

Patent Document 2 discloses a novel electron transfer promoter containing a phenylenediamine-based compound.

Patent Document 3 discloses a novel electron transfer promoter containing a phenylenediamine-based compound.

The electron transfer promoter disclosed in Patent Documents 1-3 has few reports, and its characteristics and uses have not been sufficiently clarified.

In this specification, a large number of documents including patent applications and manufacturer's manuals are cited. The disclosure of these documents is not considered to be relevant to the patentability of the invention, but is incorporated herein by reference in its entirety. More specifically, all reference documents are incorporated herein by reference as if each individual document was specifically and individually indicated to be incorporated by reference.

International Publication No. 2019/198359 Pamphlet Patent No. 6484741 Patent No. 6484742

An object of the present invention is to provide a new use of the electron transfer promoter disclosed in Patent Documents 1-3.

The present inventor has conducted various studies on electron transfer promoters, and found that electron transfer promoters are used together with metal complex compounds having a higher oxidation-reduction potential than the electron transfer promoters for oxidoreductases for biosensors. By doing so, or by using it together with a metal complex compound having a lower oxidation-reduction potential than the electron transfer promoter, it has been found that electron transfer from an oxidoreductase to an electrode can be promoted, and this is set as an embodiment. The invention to be included has been completed.

The present disclosure includes the following embodiments.
[1] A biosensor comprising (i) an electron transfer promoter (excluding PMS), (ii) an oxidoreductase, (iii) a metal complex compound, and (iv) an electrode.
[2] Electron transport chain promoter

[During the ceremony,
R ¹ is -NR ⁷ R ⁸ and
R ² is −NR ¹⁰ R ¹¹ and
R ⁷ and R ⁸ are each independently substituted with hydrogen, optionally one or more X or V, linear or branched C _1-7 alkyl, C _1-7 alkenyl, C _1- . ₇ alkynyl, C _3-9 cycloalkyl, phenyl, 1-naphthyl, 2-naphthyl, anthracenyl, phenanthrenyl, acetyl, carboxy, furanylformyl, pyrazolylformyl, 1-methyl-1H-pyrazole-5-ylformyl, 9,9 -Dimethylfluorene-2-yl or benzyl,
R ¹⁰ is hydrogen, optionally one or more X or V, linear or branched C _1-7 alkyl, C _1-7 alkenyl, C _1-7 alkynyl, C _3-9 . Cycloalkyl, phenyl, 1-naphthyl, 2-naphthyl, anthracenyl or phenanthrenyl,
R ³ , R ⁴ , R ⁵ and R ⁶ are each independently substituted with hydrogen, optionally one or more Y, linear or branched C _1-7 alkyl, C _1-7 alkoxy. , C _1-7 alkynyl, C _1-7 alkoxy, halo, nitro, cyano, carboxy, sulfo, hydroxy or amino.
Alternatively, R ³ and R ⁴ , or R ⁵ and R ⁶ may be optionally substituted with one or more oxos, Xs, together with the benzene ring containing them, the benzene ring, or

, Where * is bonded to the carbon atom to which R ³ is bonded, ** is bonded to the carbon atom to which R ⁴ is bonded, or * is bonded to the carbon atom to which R ⁵ is bonded, ** Bonds to the carbon atom to which R ⁶ bonds,
R ¹¹ is a straight or branched C _1-7 alkyl or optionally substituted with one or more Xs or Zs, phenyl, 1-naphthyl, 2-naphthyl, anthrasenyl and phenanthrenyl. Selected from the group consisting of
Where V is —O-acryloyl, acetylamino, or phenyl, optionally substituted with C _1-7 alkyl.
X may optionally be substituted with a substituent selected from the group consisting of one or more halos, aminos, cyanos, carboxys, carbonyls, alkoxys, alkylaminos, nitrosos, nitros and sulfos, linear or branched. , C _1-7 alkyl, C _1-7 alkenyl, C _1-7 alkynyl, C _1-7 alkoxy, halo, hydroxy, nitro, carboxy, cyano, sulfo, amino or alkylamino.
Y is selected from the group consisting of halo, amino, cyano, carboxy, carbonyl, hydroxy, alkoxy and sulfo.
Z is -SO ₂ -CH = CH ₂ , -SO ₂ -C ₂ H ₄ -O-SO ₃ H, or 4,6-dichlorotriazine-2-ylamino]
The biosensor according to the first embodiment.
[3] The oxidase is FAD-type glucose dehydrogenase (FAD-GDH), L- or D-lactic acid oxidase (LOD), glucose oxidase, glucose dehydrogenase, amadriase, peroxidase, galactose oxidase, birylbin oxidase, pyruvate oxidase, D. -Or L-Amino acid oxidase, amine oxidase, cholesterol oxidase, choline oxidase, xanthin oxidase, sarcosine oxidase, ascorbic acid oxidase, cytochrome oxidase, alcohol dehydrogenase, cholesterol dehydrogenase, aldehyde dehydrogenase, aldehyde oxidase, fructose dehydrogenase (FDH), sorbitol dehydrogenase, D- or L-lactic dehydrogenase, malic acid dehydrogenase, glycerol dehydrogenase, 17B hydroxysteroid dehydrogenase, estradiol 17B dehydrogenase, D- or L-amino acid dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, 3-hydroxysteroid dehydrogenase, diahorase, catalase , Glutathion reductase, titochrome b5 reductase, adrenoxin reductase, cytochrome b5 reductase, adrenodoxin reductase, nitrate reductase, phosphate dehydrogenase, birylbin oxidase, lacquerse, polyamine oxidase, formate dehydrogenase, pyranose oxidase, pyranose dehydrogenase, The biosensor according to embodiment 1 or 2, selected from the group consisting of.
[4] The electrode includes an electrode portion having a working electrode and a counter electrode, the electrode portion is arranged on an insulating substrate, and a reagent layer is further arranged on the electrode portion.
The biosensor according to any one of embodiments 1 to 3, wherein the oxidoreductase, the metal complex compound, and the electron transfer promoter are contained in the reagent layer.
[5] A method for producing a biosensor, which comprises forming a reagent layer containing an oxidoreductase, a metal complex compound, and an electron transfer promoter on an electrode.
[6] Measuring the concentration of a target compound in a sample, including the biosensor according to any one of embodiments 1 to 4, a means for applying a voltage to the electrode of the biosensor, and a means for measuring a current. System to do.
[7] A method for measuring the concentration of a target compound in a sample using the system according to the sixth embodiment.
[8] A step of preparing a biosensor comprising (i) an electron transfer promoter (excluding PMS), (ii) an oxidoreductase, (iii) a metal complex compound, and (iv) an electrode.
A method for measuring the concentration of a target compound in a sample, which comprises a step of applying a voltage to the electrode of the biosensor and a step of measuring a response current.
This specification includes the disclosure of Japanese Patent Application No. 2020-138212, which is the basis of the priority of the present application.

According to the present disclosure, as one embodiment, it is possible to provide a biosensor in which an electron transfer promoter, an oxidoreductase, an electrode and a metal complex compound are combined, and a measurement method using the same. Further, according to the present disclosure, as one embodiment, it is possible to provide a glucose sensor in which an electron transfer promoter, a GDH, an electrode and a metal complex compound are combined, and a glucose measuring method using the same.

Glucose dehydrogenase (GDH) and ruthenium compound (Ru) and BGLB are used, and the response currents when various concentrations of glucose are added are shown. The control does not contain BGLB. The response currents when glucose dehydrogenase, ruthenium compound and TMPD are used and various concentrations of glucose are added are shown. The control does not include TMPD. The response currents when glucose dehydrogenase, ruthenium compound and APPD are used and various concentrations of glucose are added are shown. Controls do not include APPD. Glucose dehydrogenase and potassium ferricyanide and BGLB, DPPA or APPD as an electron transfer promoter are used, and the response currents when various concentrations of glucose are added are shown. The control does not contain an electron transfer promoter. The response currents when lactic acid oxidase (LOD), ruthenium compound (Ru) and BGLB are used and various concentrations of lactic acid are added are shown. The control does not contain BGLB.

In certain embodiments, the present disclosure provides a biosensor containing an electron transfer promoter, an oxidoreductase, a metal complex compound, and an electrode. The biosensor can measure a compound that can be recognized as a substrate by the oxidoreductase, depending on the oxidoreductase used. For example, when FAD-GDH is used as an oxidoreductase, a glucose sensor having FAD-GDH is provided. PMS is removed from the electron transfer promoter. As used herein, unless otherwise specified, a biosensor means a sensor containing an electron transfer promoter (excluding PMS), an electrode, an oxidoreductase, and a metal complex compound. It can be used as a sensor for substrates recognized by oxidoreductase. In certain embodiments, the electrode comprises an electrode portion having an working electrode and a counter electrode. In another embodiment, the electrode comprises an electrode portion having an working electrode, a counter electrode and a reference electrode. The electrode may be, for example, a triode electrode or a printed electrode. In certain embodiments, the electrodes may be placed on an insulating substrate.

In the present specification, the mediator refers to a compound that participates in electron transfer. For example, in a system using an electrode, the mediator receives an electron from an oxidoreductase to become a reduced form, and passes the electron to the electrode to return to the oxidized form. .. From this point of view, a compound that functions as a mediator can also be called an electronic mediator. These terms are synonymous herein.

Electron Transfer Promoter In the present specification, a compound that promotes the transfer of electrons from an oxidoreductase to an electrode is referred to as an “electron transfer promoter”. The "electron transfer promoter" can be used for a biosensor equipped with an oxidoreductase, for example, a glucose sensor, and promotes the generation of a response current depending on the concentration of the target compound when it is added. In certain embodiments, the electron transfer enhancer can alter the function of the mediator. As used herein, the term "electron transfer promoter modifies the function of a mediator" refers to an electron transfer promoter for a mediator in which electrons are hardly or hardly transferred from an oxidoreductase to an electrode in the absence of the electron transfer promoter. In the presence of, electrons are transferred from the oxidoreductase to the electrode.

In certain embodiments, the electron transfer promoter can be a compound of the following general formula I or II or a salt, anhydride or solvate thereof:

[During the ceremony,
R ¹ is -NR ⁷ R ⁸ and
R ² is −NR ¹⁰ R ¹¹ and
R ⁷ and R ⁸ are each independently substituted with hydrogen, optionally one or more X or V, linear or branched C _1-7 alkyl, eg, C _1-3 alkyl, C _{1 -7} alkenyl, C _1-7 alkynyl, C _3-9 cycloalkyl, phenyl, 1-naphthyl, 2-naphthyl, anthrasenyl, phenanthrenyl, acetyl, carboxy, furanylformyl, pyrazolylformyl, 1-methyl-1H-pyrazole- 5-Ilformyl, 9,9-dimethylfluorene-2-yl, or benzyl,
R ¹⁰ is a straight or branched C _1-7 alkyl, eg, C _1-3 alkyl, C _1-7 alkenyl, C _1- which may be substituted with hydrogen, optionally one or more Xs or Vs. ₇ alkynyl, C _3-9 cycloalkyl, phenyl, 1-naphthyl, 2-naphthyl, anthrasenyl or phenanthrenyl,
R ³ , R ⁴ , R ⁵ and R ⁶ may be independently substituted with hydrogen, optionally one or more Ys, with a straight or branched C _1-7 alkyl such as C _1-3 . Alkyl, C _1-7 alkenyl, C _1-7 alkynyl, C _1-7 alkoxy, halo, nitro, cyano, carboxy, sulfo, hydroxy or amino.
Alternatively, R ³ and R ⁴ , or R ⁵ and R ⁶ may be optionally substituted with one or more oxos, Xs, together with the benzene ring containing them, the benzene ring, or

, Where * is bonded to the carbon atom to which R ³ is bonded, ** is bonded to the carbon atom to which R ⁴ is bonded, or * is bonded to the carbon atom to which R ⁵ is bonded, ** Bonds to the carbon atom to which R ⁶ bonds,
R ¹¹ is a straight or branched C _1-7 alkyl, eg, C _1-3 alkyl, or may optionally be substituted with one or more Xs or Zs, phenyl, 1-naphthyl,. Selected from the group consisting of 2-naphthyl, anthracenyl and phenanthrenyl,
Where V is —O-acryloyl, acetylamino, or phenyl, optionally substituted with C _1-7 alkyl.
X may optionally be substituted with a substituent selected from the group consisting of one or more halos, aminos, cyanos, carboxys, carbonyls, alkoxys, alkylaminos, nitrosos, nitros and sulfos, linear or branched. With C _1-7 alkyl, eg C _1-3 alkyl, C _1-7 alkenyl, C _1-7 alkynyl, C _1-7 alkoxy, halo, hydroxy, nitro, carboxy, cyano, sulfo, amino or alkylamino. can be,
Y is selected from the group consisting of halo, amino, cyano, carboxy, carbonyl, hydroxy, alkoxy and sulfo.
Z is -SO ₂ -CH = CH ₂ , -SO ₂ -C ₂ H ₄ -O-SO ₃ H, or 4,6-dichlorotriazine-2-ylamino].

In certain embodiments, the electron transfer promoter can be a compound having the structure of the following general formula Ia or IIa or a salt, anhydride or solvate thereof:

[During the ceremony,
R ^1a is −NR ^7a R ^8a .
R ^2a is −NR ^10a R ^11a .
R ^7a and R ^8a are each independently substituted with hydrogen, optionally one or more Xa, linear or branched C _1-6 alkyl, eg C _1-3 alkyl, C _1-6 . Alkenyl, C _1-6 alkynyl, C _3-9 cycloalkyl, phenyl, 1-naphthyl, 2-naphthyl, anthrasenyl, or phenanthrenyl.
R ^10a is a straight or branched C _1-6 alkyl, eg, C _1-3 alkyl, C _1-6 alkenyl, C _1-6 alkynyl, which may be substituted with hydrogen, optionally one or more Xa. , C _3-9 cycloalkyl, phenyl, 1-naphthyl, 2-naphthyl, anthrasenyl or phenanthrenyl,
R ^3a , R ^4a , R ^5a and R ^6a are linear or branched C _1-6 alkyl, C _1-6 alkoxy, respectively, which may be independently substituted with hydrogen, optionally one or more Ys. , C _1-6 alkynyl, C _1-6 alkoxy, halo, nitro, cyano, carboxy, sulfo, hydroxy or amino.
Alternatively, R ^3a and R ^4a , or R ^5a and R ^6a are benzene rings, or

, Where * is bonded to the carbon atom to which R ^3a is bonded, ** is bonded to the carbon atom to which R ^4a is bonded, or * is bonded to the carbon atom to which R ^5a is bonded, ** Bonds to the carbon atom to which R ^6a bonds,
R ^11a is a straight or branched C _1-7 alkyl, eg C _1-3 alkyl, or may optionally be substituted with one or more Xa, phenyl, 1-naphthyl, 2-naphthyl. , Anthracenyl and phenylanthrenyl, selected from the group
Xa may optionally be substituted with a substituent selected from the group consisting of one or more halos, aminos, cyanos, carboxys, carbonyls, hydroxys, alkoxys, alkylaminos, nitroso, nitros and sulfos, linear or fractional. Branch chains of C _1-6 alkyl such as C _1-3 alkyl, C _1-6 alkenyl, C _1-6 alkynyl, C _1-6 alkoxy, halo, hydroxy, nitro, carboxy, cyano, sulfo, amino or alkyl Amino and
Y is selected from the group consisting of halo, amino, cyano, carboxy, carbonyl, alkoxy and sulfo].

In certain embodiments, the electron transfer promoter can be a compound having the structure of the following general formula Ib or IIb or a salt, anhydride or solvate thereof:

[During the ceremony,
R ^7b , R ^8b and R ^10b are each independently substituted with hydrogen, optionally one or more Xb, linear or branched C _1-6 alkyl, eg C _1-3 alkyl, C. _1-6 alkenyl, C _1-6 alkynyl, C _3-9 cycloalkyl, phenyl, 1-naphthyl, 2-naphthyl, anthrasenyl or phenanthrenyl, with R ^3b , R ^4b , R ^5b and R ^6b independently. , Hydrogen, optionally straight or branched C _1-6 alkyl, C _1-6 alkenyl, C _1-6 alkynyl, C _1-6 alkoxy, halo, nitro. , Cyano, carboxy, sulfo or amino,
R ^11b is a straight or branched C _1-7 alkyl, eg C _1-3 alkyl, or may optionally be substituted with one or more Xb, phenyl, 1-naphthyl, 2-naphthyl. , Anthracenyl and phenylanthrenyl, selected from the group
Here, Xb may be optionally substituted with a substituent selected from the group consisting of one or more halos, aminos, cyanos, carboxys, carbonyls, alkoxys and sulfos, C _1- of linear or branched chains. ₆ alkyl, eg C _1-3 alkyl, C _1-6 alkenyl, C _1-6 alkynyl, C _1-6 alkoxy, halo, hydroxy, nitro, carboxy, cyano, sulfo or amino.
Y is selected from the group consisting of halo, amino, cyano, carboxy, carbonyl, alkoxy and sulfo].

As used herein, alkyl refers to a straight-chain or branched-chain hydrocarbon having, for example, 1 to 7 carbon atoms, for example, 1 to 6 carbon atoms, for example, 1 to 3 carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl, n-butyl, tert-butyl, isopentyl, n-pentyl and heptyl.

The number of atoms (such as carbon atoms) used in the present specification is expressed as, for example, "Cx-Cy alkyl", which means an alkyl group having x to y carbon atoms. The same notation is used for other substituents and ranges.

The alkenyl used in the present specification refers to a straight-chain or branched-chain aliphatic hydrocarbon having one or more carbon-carbon double bonds. Examples include, but are not limited to, vinyl, allyl, and the like.

The alkynyl used in the present specification refers to a straight-chain or branched-chain aliphatic hydrocarbon having one or more carbon-carbon triple bonds. Examples include, but are not limited to, ethynyl and the like.

Cycloalkyl as used herein refers to a substituted or unsubstituted non-aromatic cyclic hydrocarbon ring. Cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

The halo used in the present specification refers to a group of Group 17 elements such as -Cl, -Br, -I and the like. As used herein, halogen refers to fluorine, chlorine, bromine, or iodine.

The haloalkyl used herein refers to an alkyl group substituted with at least one halogen. Haloalkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, and t-, which are independently substituted with one or more halogens, such as fluoro, chloro, bromo, and iodine. Butyl and the like can be mentioned.

The halosulfonyl used in the present specification refers to a sulfonyl group substituted with at least one halogen, and examples thereof include chlorosulfonyl (Cl _- SO2-) and bromosulfonyl (Br _- SO2-). Not limited to.

Phenyl as used herein refers to a substituted or unsubstituted benzene ring system. As used herein, naphthyl refers to a substituted or unsubstituted naphthalene ring system, and examples thereof include 1-naphthyl and 2-naphthyl. As used herein, anthracene refers to an anthracene ring system that has been substituted or not substituted. As used herein, phenanthrenel refers to a substituted or unsubstituted phenanthrene ring system.

As used herein, acetyl means CH ₃ CO-. As used herein, trifluoroacetyl means CF ₃ CO-. As used herein, carboxy means -COOH. As used herein, furanyl refers to a monovalent group of furan, such as 2-furanyl or 3-furanyl. In the present specification, formyl is also referred to as aldehyde and refers to -COH. In the present specification, the furanylformyl means a formyl group (furanyl-CO-) to which a furanyl group is bonded. As used herein, pyrazolyl refers to a pyrazole ring system. As used herein, the pyrazolyl formyl refers to a formyl group (pyrazolyl-CO-) to which a pyrazolyl group is bonded. As used herein, fluorenyl refers to a substituted or unsubstituted fluorene ring system.

As used herein, nitro refers to _two -NO groups. As used herein, nitroso refers to the -N = O group. As used herein, cyano refers to a -CN group. As used herein, sulfo refers to -SO _3H . In the present specification, hydroxy means -OH. In the present specification, oxo means = O.

As used herein, amino refers to a -NR'R'' group, and R'and R'' may be the same or different. R'and R'' can be, but are not limited to, for example, H, alkyl, alkenyl, alkynyl, cycloalkyl, phenyl, naphthyl, anthracenyl, phenanthrenyl, and the like. As used herein, the aminoalkyl has an alkylene linker to which an amino group is linked. Examples of the aminoalkyl include, but are not limited to- (CH ₂ ) _n NH ₂ . As used herein, the term alkylamino means an amino to which an alkyl group is linked. Examples of the alkylamino include, but are not limited to, C _1-7 alkyl-NH-. As used herein, acetylamino means an amino to which an acetyl group is linked. Examples of acetylamino include, but are not limited to, acetyl-NH-.

As used herein, acryloyl means H2C = CH _- C (= O)-. In the present specification, -O-acryloyl means H2C = CH _- C (= O) -O-. As used herein, isothiocyanate means −N = C = S. In the present specification, succinimidyl means (CH ₂ CO) _2N- . As used herein, carbonyl means −C (= O) −. Alkoxy used in the present specification refers to an —O-alkyl group.

As used herein, "possibly substituted" and "substituted or not substituted" mean any substitution with one or more substituents, including multiple substitutions. Is done.

In certain embodiments, the electron transfer promoter is TMPD (N, N, N', N'-tetramethylphenylenediamine).

Can be. The redox potential E of TMPD is about 50 mV.

In certain embodiments, the electron transfer promoter is APPD (2,4-Diaminodiphenyllamine).

Can be. The redox potential E of APPD is about 20 mV.

In certain embodiments, the electron transfer promoter is BGLB (Bindschedler's Green Leuco Base).

Can be. The redox potential E of BGLB is about 17 mV.

In certain embodiments, the electron transfer promoter is DPPA (4,4'-Diaminodiphenyllamine).

Can be. The redox potential E of DPPA is about 20 mV.

The electron transfer promoter may have a redox state and an ionization state. In the above chemical formula, the compounds are described in neutral and reduced form. However, not only in this form, the electron transfer promoter can be an oxidized type (diimine type), a semi-oxidized type, or a reduced type (diamine type). Further, the electron transfer promoter may be a neutral type or a cationic type. For convenience, the term electron transfer enhancer includes neutral or cationic, oxidized, semi-oxidized, or reduced forms. For example, after adding a neutral and oxidized compound as an electron transfer promoter to the measurement system, it may change to an oxidized and cationic compound depending on the pH of the solution and electron transfer. Such compounds shall also be included in the electron transfer promoter. Further, when referring to an electron transfer promoter, it shall include its salt, acid addition salt, anhydride and solvate. Examples of the salt include, but are not limited to, salts of Group 1 elements and salts of Group 17 elements, such as Na salt, K salt, Cl salt, Br salt and the like. Examples of the acid addition salt include, but are not limited to, hydrochloride, sulfate, sulfite, and nitrate.

The electron transfer promoter may be artificially synthesized or a natural product may be obtained. It may also be commercially available. In the case of synthesis, organic synthesis can be performed using a conventional organic synthesis method, and the product can be confirmed by NMR, IR, mass spectrometry and the like. Unless otherwise specified, the practice of the present invention uses prior arts of chemistry, organic synthesis, biochemistry, molecular biology and electrochemical, which are within the ability of those skilled in the art. Such techniques are described in the literature. For example, Organic Chemistry (Jonathan Clayden (editing), Nick Greeves (Author), Stuart Warren (Author), Peter Wothers (Author)), and Oxford Univ Pr, 2000, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure (See Michael B. Smith (Author), Jerry March (Author)) Wiley-Interscience, 6th edition, 2007. Each of these general texts is incorporated herein by reference.

In the present specification, PMS refers to phenazinemethsulfate and its derivatives. Typical examples of PMS include 1-methoxy-5-methylphenazinenium methyl sulfate (1-methoxy PMS). The term "electron transfer promoter" herein does not include PMS unless otherwise noted. That is, when the term "electron transfer promoter" is used, PMS is excluded. In certain embodiments, from the "electron transfer promoters" of the present disclosure, any one of the specific compounds of the general formula I or II, eg, general formula Ia or IIa, eg, general formula Ib or IIb. For example, known compounds can be excluded. In certain embodiments, alternative elements (options) positively described in the general formula I or II, such as the general formula Ia or IIa, such as the general formula Ib or IIb, from the "electron transfer promoter" of the present disclosure. Compounds identified by) can be excluded.

The content of the electron transfer promoter per biosensor can be, for example, 10 pmol to 1000 nmol, 10 pmol to 100 nmol, 10 pmol to 60 nmol, 10 pmol to 10 nmol, 10 pmol to 1 nmol, 40 to 900 pmol, 50 to 500 pmol, for example, 100 to 300 pmol. However, it is not limited to this.

When the content of the electron transfer promoter per biosensor is 5 nmol, the content of FAD-GDH is 1 to 100 U, 1 to 10 U, 1 to 6 U, for example, 1 to 4 U in a certain embodiment. .. The content of FAD-GDH is, in a certain embodiment, 0.1 to 100 μg, 1 to 50 μg, for example, 1 to 20 μg. In other redox enzymes, the content per sensor is, in a certain embodiment, 0.1 to 1000 μg, 1 to 500 μg, 1 to 50 μg, for example, 1 to 20 μg. The content of the metal complex compound may, in certain embodiments, be an amount that reaches a saturated concentration, or may be 5-50 μg, 10-40 μg, eg 15-25 μg. The content of potassium ferricyanide can be 10 to 1000 mM, 100 to 800 mM, for example 200 to 500 mM in certain embodiments.

Metal Complex Compound Examples of the metal complex compound include, but are not limited to, a ruthenium compound, an osmium compound, and an iron compound (for example, potassium ferricyanide and a ferrocene compound). In certain embodiments, the content of the metal complex compound per glucose sensor can be the amount typically used for glucose sensors, eg 0.1 μg-100 mg, 1 μg-50 mg, 1 μg-10 mg, It can be 1 μg to 1 mg, 1 to 100 μg, 5 to 50 μg, 10 to 40 μg, for example 15 to 25 μg. The redox potential of the metal complex compound may be a redox potential in a predetermined range, for example, the redox potential is -200 mV or more, -190 mV or more, -180 mV or more, -170 mV or more, -160 mV or more, -150 mV or more. , -140 mV or more, -130 mV or more, -120 mV or more, -110 mV or more, -100 mV or more, -90 mV or more, -80 mV or more, -70 mV or more, -60 mV or more, -50 mV or more, -40 mV or more, -30 mV or more,- 20mV or more, -10mV or more, -0mV or more, for example, +400mV or less, +380mV or less, +360mV or less, +340mV or less, +320mV or less, +300mV or less, +280mV or less, +260mV or less, +240mV or less, +220mV Below, +200 mV or less, +180 mV or less, +160 mV or less, +140 mV or less, +120 mV or less, +100 mV or less, +80 mV or less, +60 mV or less, +40 mV or less, +0 mV or less, for example, any upper and lower limits of these Combinations of, for example -200 mV to +400 mV, -200 mV to +300 mV, -200 mV to +200 mV, -100 mV to + 400 mV, for example -0 mV to + 400 mV, can be used.

Ruthenium compound As the ruthenium compound, ruthenium compounds used in conventional glucose sensors and biosensors, and their equivalents to be developed in the future can be used. In certain embodiments, the ruthenium compound can be present in the reaction system as an oxidized ruthenium complex. The ruthenium complex is not particularly limited. In a certain embodiment, the ruthenium complex has the following general formula,
[Ru (NH ₃ ) 5X] n ⁺
(In the formula, X is NH ₃ , CN, pyridine, halogen ion, nicotinamide, or H ₂ O) complex. Examples of the halogen ion include Cl ⁻ , F ⁻ , Br ⁻ , and I ⁻ . In the formula, n ⁺ represents the valence of the oxidized ruthenium (III) complex, which is determined by the type of X. For example, if X is NH ₃ , the compound is a hexaammine ruthenium complex compound, and if the halogen is Cl ⁻ , the compound is hexaammine ruthenium chloride.

Ruthenium Complex and / or Osmium Complex In certain embodiments, the metal complex compound can be a ruthenium complex or an osmium complex. In certain embodiments, the metal complex compound is the general formula III below.
[M (A) _x (B) _y ] ^m (Z ^o ) _n (Equation III)
In the formula, M is osmium, ruthenium, or iron and has an oxidation state of 0, 1, 2, 3 or 4.
x and n are integers selected from 1 to 6 independently of each other, y is an integer selected from 1 to 5, z is an integer of -2 to +1 and m is an integer of -5 to -5. It is an integer of +4,
A is a monodentate or bidentate aromatic ligand containing one or two nitrogen atoms.
B is independently selected to be one or more suitable ligands excluding nitrogen-containing heterocyclic ligands.
X is the counterion
In some cases, A is a substituted or unsubstituted alkyl, alkenyl or aryl group, -CN, -NO ₂ , -F, -Cl, -Br, -I, -CO ₂ H, -SO ₃ H, -NHNH ₂ , -SH, -OH, -NH2, alkoxy, alkoxycarbonyl, alkylamino, alkylaminocarbonyl, dialkylaminocarbonyl, dialkylamino, alkanoylamino, arylcarboxamide, alkylhydrazino, hydroxylamino, alkoxyamino, alkylthio, independently selected It may be substituted with 1 to 8 groups,
The number of coordinating atoms is 6.

The ligand A may be a monodentate ligand substituted with one or more CO2R ^B groups, or a bidentate or tridentate ligand optionally substituted with one or more CO2R ^B groups. The ^RB of the one or more ^CO2RB groups can be H.

The ligand A is 2,2'-bipyridine-5,5'-bis-carboxylic acid, 2,2'-bipyridine-4,4'-bis-carboxylic acid, 2,2'-bipyridine, 1,10. It can be selected from the group consisting of -phenanthroline-3,9-bis-carboxylic acid, nicotinic acid, isonicotinic acid, 5-carboxy-nicotinic acid, and 6-pyridyl-nicotinic acid. In certain embodiments, M can be osmium or ruthenium.

In another embodiment, the metal complex compound is the general formula IV below.
[M (A) _X (B) _y ] ^m (Z ^o ) _n (Equation IV)
In the formula, M is ruthenium, osmium, or iron and has an oxidation state of 0, 1, 2, 3 or 4.
x and n are integers independently selected from 1 to 6, y is an integer selected from 0 to 5, z is an integer of -2 to +1 and m is an integer of -5 to +4. And
A is a bidentate, tridentate, tetradentate, pentadentate or hexadentate ligand with the formula ^RA RN (C ₂ H ₄ NR) _w ^RA having a linear or formula (RNC ₂ H ₄ ). It may be a cyclic compound having _v , (RNC ₂ H ₄ ) _p (RNC ₃ H ₆ ) _q or [(RNC ₂ H ₄ ) (RNC ₃ H ₆ )] _s , and w is an integer of 1 to 5. Yes, v is an integer of 3 to 6, p and q are integers of 1 to 3, the sum of p and q is 4, s is 2 or 3, and R and ^RA are hydrogen or Methyl and
B is independently selected to be the appropriate ligand,
X is the counterion
In some cases, A is a substituted or unsubstituted alkyl, alkoxy or aryl group, -F, -Cl, -Br, -I, -NO2, -CN, -CO ₂ H, -SO ₃ H, -NHNH ₂ ,- Independently selected from SH, -OH, -NH2, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy, alkylamino, dialkylamino, alkanoylamino, arylcarboxamide, alkylhydrazino, hydroxylamino, alkoxyamino, alkylthio It may be substituted with 1 to 7 groups.
The number of coordinating atoms is 6.

The ligand A can be a bidentate, tridentate or tetradentate ligand and has the formula ^RA RN (C ₂ H ₄ NR) _w ^RA with a linear or formula (RNC ₂ H ₄ ) _v . , (RNC ₂ H ₄ ) _p (RNC ₃ H ₆ ) _q or [(RNC ₂ H ₄ ) (RNC ₃ H ₆ )] _s may be a ring, w is an integer of 1 to 3. , V is 3 or 4, p and q are integers of 1 to 3, the sum of p and q is 4, and s can be 2 or 3.

Ligand A is 1,1,4,7,10,10-hexamethyltriethylenetetramine, 1,4,7-trimethyl-1,4,7-triazacyclononane or 1,2-dimethylethylenediamine or It can be selected from the group consisting of 1,1,2,2-tetramethylethylenediamine. In certain embodiments, M can be osmium or ruthenium.

Ligand B can be from an amine ligand such as NH ₃ or NMe ₃ , or CO, CN, halogen, acetylacetonato (acac), 3-bromo-acetylacetonato (Bracac), oxalate, 1 , 4,7-Triethylene crown ether, oxalate or 5-chloro-8-hydroxyquinoline can be selected.

If the ligand A or B is chosen to be bidentate, the coordination of the complex can be cis or trans. The oxidation state of the metal in the complex of formula III or formula IV can be selected to be 2+ or 3+.

The ligands A and B can be selected such that the total charge on the complex of formula III or formula IV is +2, +1, 0, -1, -2 or -3. Counterions are F-, ^Cl- ^, Br- ^, I- ^, NO _3- ^, NH ₄ ⁺ , NR ₄ ⁺ , PF _6- ^, CF ₃ SO _3- , SO ₄ ^2- , _ClO ^4- ^, K ⁺ , You can choose from Na ⁺ and Li ⁺ . These combinations may be used as counterions.

The complex of formula III or formula IV is [Ru ^III (NH ₃ ) ₅ (pyridine-3-COOH)] (PF ₆ ) ₂ (CF ₃ SO ₃ ), [Ru ^III (2,4-pentandionate) ₂ (Pyridin-3-COOH) (Pridin-3-COO)], [Ru ^III (3-bromo-2,4-pentandionate) ₂ (Pridin-3-COOH) (Pridin-3-COO)], [ Ru ^III (2,4-pentandionate) ₂ (2,2'-bipyridine-5,5'-(COOH) (COO)], [Ru ^III (2,4-pentandionate) ₂ (2,2) '-Bipyridine-4,4'-(COOH) (COO)], [Ru ^III (2,4-pentanionate) ₂ (2,2'-bipyridine)] Cl, [Ru ^III (2,4-pentane) Dionate) ₂ (Pyridin-4-COOH) (Pyridin-4-COO)], [Ru ^III (5-Chloro-8-hydroxyquinoline) ₂ (Pyridin-3-COOH) (Pridin-3-COO)], [Ru ^III (1,1,4,7,10,10-hexamethyltriethylenetetramine) (2,4-pentandionate)] (PF ₆ ) (CF ₃ SO ₃ ), [Ru ^III (1, 1) , 4,7,10,10-hexamethyltriethylenetetramine) (2,4-pentandionate)] Cl ₂ , [Os ^II (2,2'-bipyridine) ₂ (2,4-pentandionate)] Cl, [Ru ^II (2,2'-bipyridine) ₂ (2,4 _- pentandionate)] Cl, [Ru ^II (2,2'-bipyridine) ₂ (C2O ₄ )], K [Ru ^III (C ₂ O ₄ ) ₂ (Pridin-3-COOH) ₂ ], and [Ru ^III (1,4,7-trimethyl-1,4,7-triazacyclononan) (2,4-pentanionate). (Ppyridine)] (NO ₃ ) ₂ can be selected.

In certain embodiments, the metal complex is [Ru ^III (acac) ₂ (py-3-COOH) (py-3-COO)], [Ru ^III (3-Bracac) ₂ (py-3-COOH) (py). -3-COO], [Ru ^III (acac) ₂ (py-4-COOH) (py-4-COO)], or [Ru ^III (acac) ₂ (py-3-COOH) (py-3-COO)] COO)].

In certain embodiments, the metal complexes are [Os ^III (2,4-pentandionate) ₂ (Pyridine-3-COOH) (Pyridine-3-COO)], [Os ^III (NH ₃ ) ₅ (Pyridine-3). -COOH)] (PF ₆ ) ₂ (CF ₃ SO ₃ ), [Os ^III (3-bromo-2,4-pentandionate) ₂ (pyridine-3-COOH) (pyridine-3-COO)], [ Os ^III (2,4-pentangionate) ₂ (2,2'-bipyridine-5,5'-(COOH) (COO)], [Os ^III (2,4-pentangionate) ₂ (2,2) '-Bipyridine-4,4'-(COOH) (COO)], [Os ^III (2,4-pentanionate) ₂ (2,2'-bipyridine)] Cl, [Os ^III (2,4-pentane) Zionate) ₂ (Pyridine-4-COOH) (Pyridine-4-COO)], [Os ^III (5-Chloro-8-hydroxyquinoline) ₂ (Pyridine-3-COOH) (Pyridine-3-COO)], [Os ^III (1,1,4,7,10,10-hexamethyltriethylenetetramine) (2,4-pentandionate)] (PF ₆ ) (CF ₃ SO ₃ ), [Os ^III (1, 1) , 4,7,10,10-hexamethyltriethylenetetramine) (2.4-pentangionate)] Cl ₂ , [Os ^II (2,2'-bipyridine) ₂ (2,4-pentandionate)] Cl, [Os ^III (1,4,7-trimethyl-1,4,7-triazacyclononan) (2,4-pentandionate) (pyridine)] (NO ₃ ) ₂ , or [Os ^II (2) .2'-Bipyridine) ₂ (C ₂ O ₄ )], K [Os ^III (C ₂ O ₄ ) ₂ (Pyridine-3-COOH) ₂ ], but not limited to metal in certain embodiments. The complex may be as described in WO 2007/072018. By reference, the description of WO 2007/072018 regarding the metal complex is incorporated herein.

Oxidases Oxidases include various oxidases classified in EC Group 1, such as glucose oxidase, glucose dehydrogenase, amadriase (also referred to as fructosyl peptide oxidase or fructosyl amino acid oxidase), peroxidase, galactose oxidase, and the like. Bilylvin oxidase, pyruvate oxidase, D- or L-amino acid oxidase, amine oxidase, cholesterol oxidase, choline oxidase, xanthin oxidase, sarcosin oxidase, D- or L-lactic oxidase (LOD), ascorbate oxidase, cytochrome oxidase, alcohol dehydrogenase. , Cholesterol dehydrogenase, aldehyde dehydrogenase, aldehyde oxidase, fructose dehydrogenase (FDH), sorbitol dehydrogenase, D- or L-lactic acid dehydrogenase, malic acid dehydrogenase, glycerol dehydrogenase, 17B hydroxysteroid dehydrogenase, estradiol 17B dehydrogenase, D- or L-amino acid dehydrogenase. , Glyceraldehyde 3-phosphate dehydrogenase, 3-hydroxysteroid dehydrogenase, diaholase, catalase, glutathione reductase, cytochrome b5 reductase, adrenoxine reductase, cytochrome b5 reductase, adrenodoxin reductase, nitrate reductase, phosphate dehydrogenase, bilirubin oxidase. , Lacquerse, polyamine oxidase, formate dehydrogenase, pyranose oxidase, pyranose dehydrogenase, tauropine dehydrogenase and the like, but are not limited thereto. Examples of the coenzyme of the above-mentioned enzyme include nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide (FAD), and pyroquinolinquinone. The activity of the redox enzymes listed above can be measured using various substrates by the method described in, for example, Methods in Energy (Volumes 1 to 602). The origin of the oxidoreductases listed above (eg glucose dehydrogenase, LOD, amadriase, etc.) is not particularly limited, and those derived from prokaryotes, eukaryotes, microorganisms, fungi, plants, or animals are used. Can be.

In certain embodiments, the redox enzyme can be FAD-GDH. As used herein, FAD-GDH refers to flavin adenine dinucleotide-dependent glucose dehydrogenase or flavin adenine dinucleotide-bound glucose dehydrogenase. In certain embodiments, FAD-GDH may be commercially available. In another embodiment, a variant of commercially available FAD-GDH or an equivalent thereof may be used. As used herein, the term "Mucole-type FAD-GDH" means a wild-type FAD-GDH of the genus Mucor and / or a variant thereof, and includes either a wild-type or a variant thereof unless otherwise specified. As used herein, the term Botriotinia genus FAD-GDH means wild-type FAD-GDH of the genus Botriotinia and / or a variant thereof, and includes either wild-type or variants thereof unless otherwise specified. .. As used herein, Aspergillus genus FAD-GDH means wild-type FAD-GDH of the genus Aspergillus and / or a variant thereof, and includes both wild-type and variants thereof unless otherwise specified. .. As used herein, the term Penicillium-type FAD-GDH means wild-type FAD-GDH of the genus Penicillium and / or a variant thereof, and includes either wild-type or a variant thereof unless otherwise specified. .. As used herein, the term FAD-GDH of the genus Circinella means wild-type FAD-GDH of the genus Circinella and / or a variant thereof, and includes both wild-type and variants thereof unless otherwise specified. ..

The microorganisms of the genus Mucor are not limited, but Mucor plainii, Mucor javanicus, Mucor silcineroides f. Mucor circinelloides f. Circinelloides, Mucor guilliermondii, Mucor hiemalis f. Examples include Mucor himalis f. Silvaticus, Mucor subtilissimus, and Mucor dimorphosporus. Examples of the microorganism belonging to the genus Botriotinia include, but are not limited to, Botriotinia fuckeliana. Aspergillus genus microorganisms include, but are not limited to, Aspergillus oryzae, Aspergillus sojae, Aspergillus niger, and Aspergillus ter. .. Examples of the microorganism belonging to the genus Penicillium include, but are not limited to, Penicillium sclerotiorum, Penicillium janzinellum, and Penicillium paneum.

As used herein, a variant of the wild-type FAD-GDH of the genus Mucor has a sequence in which the amino acid residue of the amino acid sequence of the wild-type FAD-GDH is substituted, and has FAD-dependent glucose dehydrogenase activity. Say something. In certain embodiments, the variants have low substrate specificity for maltose and galactose and high substrate specificity for glucose. The number of the substituted amino acid residues is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 such as 1 to 10, 1 to 5, 1 to 3, for example 1. It can be up to two.

In the present specification, the variant of the wild-type FAD-GDH of Mucor flavinii has a sequence in which the amino acid residue of the amino acid sequence of the wild-type FAD-GDH is substituted, and has FAD-dependent glucose dehydrogenase activity. Say something. In certain embodiments, the variants are less reactive with maltose, xylose and galactose and more reactive with glucose. The number of the substituted amino acid residues is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 such as 1 to 10, 1 to 5, 1 to 3, for example 1. It can be up to two.

For example, when the enzyme used is a variant of wild-type FAD-GDH of the genus Botriotinia, it has a sequence in which the amino acid residue of the amino acid sequence of the wild-type FAD-GDH is replaced and has a FAD-dependent glucose dehydrogenase. Those with activity. In certain embodiments, the variants are less reactive with maltose and galactose and more reactive with glucose. The number of the substituted amino acid residues is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 such as 1 to 10, 1 to 5, 1 to 3, for example 1. It can be up to two.

For example, in the case where the enzyme used is a variant of the wild-type FAD-GDH of Botriotinia fuccheliana, the enzyme has a sequence in which the amino acid residue of the amino acid sequence of the wild-type FAD-GDH is substituted, and the sequence is substituted. Those having FAD-dependent glucose dehydrogenase activity. In certain embodiments, the variants are less reactive with maltose and galactose and more reactive with glucose. The number of the substituted amino acid residues is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 such as 1 to 10, 1 to 5, 1 to 3, for example 1. It can be up to two.

For example, when the enzyme used is a variant of wild-type FAD-GDH of the genus Aspergillus, it has a sequence in which the amino acid residue of the amino acid sequence of the wild-type FAD-GDH is substituted, and has a FAD-dependent glucose dehydrogenase. Those with activity. In certain embodiments, the variants are less reactive with maltose and galactose and more reactive with glucose. The number of the substituted amino acid residues is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 such as 1 to 10, 1 to 5, 1 to 3, for example 1. It can be up to two.

For example, when the enzyme used is a variant of wild-type FAD-GDH of the genus Penicillium, it has a sequence in which the amino acid residue of the amino acid sequence of wild-type FAD-GDH is replaced, and FAD-dependent glucose dehydrogenase. Those with activity. In certain embodiments, the variants are less reactive with maltose and galactose and more reactive with glucose. The number of the substituted amino acid residues is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 such as 1 to 10, 1 to 5, 1 to 3, for example 1. It can be up to two.

In certain embodiments, the redox enzyme can be amadriase. As used herein, amadriase refers to flavin adenine dinucleotide-dependent fructosyl peptide oxidase or flavin adenine dinucleotide-dependent fructosyl amino acid oxidase, or flavin adenine dinucleotide-bound fructosyl peptide oxidase or flavin adenine dinucleotide-bound fructosyl. Refers to amino acid oxidase. In certain embodiments, amadriase may be commercially available. In another embodiment, a variant of commercially available amadriase or an equivalent thereof may be used. In the present specification, the origin of amadriase is not particularly limited, and for example, the genus Coniochaeta, the genus Eupenicillium, the genus Pyrenochaeta, the genus Arthrinium, the genus Curvularia. Neocosmospora, Cryptococcus, Phaeosphaeria, Aspergillus, Emericella, Emericella, Urocladium, Pilium Achaetomiella, Achaetomium, Thielavia, Chaetomium, Gelasinospora, Microascus, Litoporas The genus Pleospora, the genus Coniochaetidium, the genus Pichia, the genus Debaryomyces, the genus Corynebacterium, the genus Aglobacterium, or the genus Arsuro. It means amadriase and includes both wild type and its variants unless otherwise specified.

In certain embodiments, the redox enzyme can be lactate oxidase. As used herein, the term lactate oxidase refers to flavin adenine mononucleotide-dependent lactate oxidase or flavin adenine mononucleotide-bound lactate oxidase. In certain embodiments, lactate oxidase may be commercially available. In another embodiment, a variant of commercially available lactic acid oxidase or an equivalent thereof may be used. In the present specification, the origin of lactic acid oxidase is not particularly limited, and means, for example, lactic acid oxidase derived from Aerococcus, Streptococcus, Pediococcus, or Enterococcus. However, unless otherwise specified, both wild type and its variants are included.

In certain embodiments, FAD-GDH has a tag sequence for enzyme purification, a peptide sequence, a signal sequence, a recognition sequence for cleavage, and / or a cleavage residue of those sequences added to the N-terminus or C-terminus of the amino acid sequence. Recombinant FAD-GDH that has been subjected to FAD-dependent glucose dehydrogenase activity may be included.

In certain embodiments, the content of FAD-GDH per glucose sensor can be, for example, 0.1-50U, 0.5-20U, 1-10U, for example 1-5U, but is not limited to this. .. In the present specification, the enzyme unit U of FAD-GDH is an enzyme amount that converts 1 μmol of glucose at 37 ° C. for 1 minute.

As used herein, the content of FAD-GDH per glucose sensor refers to the amount of FAD-GDH used in a glucose sensor having one electrode system having an working electrode and a counter electrode in a specific embodiment. .. In another embodiment, the content refers to the amount of FAD-GDH contained in the reagent layer arranged on one electrode system. In another embodiment, the content refers to the amount of FAD-GDH to be added to the reagent to be included in the reaction system when the sample is added.

In certain embodiments, the LOD content per lactic acid sensor can be, for example, 0.1 to 50 U, 0.5 to 20 U, 1 to 10 U, for example 1 to 5 U, but is not limited to this. In the present specification, the enzyme unit U of LOD is an enzyme amount that oxidizes 1 μmol of lactic acid at 37 ° C. for 1 minute.

In the present specification, the content of LOD per lactic acid sensor refers to the amount of LOD used in a lactic acid sensor having one electrode system having an working electrode and a counter electrode in a specific embodiment. In another embodiment, the content refers to the amount of LOD contained in the reagent layer arranged on one electrode system. In another embodiment, the content refers to the amount of LOD to be added to the reagent to be included in the reaction system when the sample is added.

In certain embodiments, the biosensor has the usual size used for a measurement sample. For example, the glucose sensor has the usual size used for a sample that may contain glucose, such as blood, in certain embodiments. The sample added to the sensor, such as a blood sample, can be, for example, 0.1-2 μL, 0.2-1 μL, for example 0.2-0.5 μL. The sensor can be designed according to the volume of the sample or reaction system.

Reagent Layer In certain embodiments, the biosensors of the present disclosure include electron transfer promoters (excluding PMS), electrodes, oxidoreductases, and metal complex compounds. In certain embodiments, the biosensor comprises an electrode and a reagent layer, the reagent layer comprising an electron transfer promoter, an oxidoreductase, and a metal complex compound. In certain embodiments, the electrode comprises an electrode portion having an working electrode and a counter electrode. In certain embodiments, the electrodes may be placed on an insulating substrate. In certain embodiments, a reagent layer may be placed on top of the electrode section.

In certain embodiments, the present disclosure provides a method for producing a biosensor, which comprises forming a reagent layer containing an oxidoreductase, a metal complex compound, and an electron transfer promoter on an electrode.

In certain embodiments, the present disclosure provides a system for measuring the concentration of a target compound in a sample, comprising a biosensor, means for applying a voltage to the electrodes of the biosensor, and means for measuring an electric current. .. The means for applying the voltage may include a contact portion capable of contacting the electrode and a power source (eg, a DC power source). The system of the present disclosure may include potentiostats and galvanostats. In certain embodiments, the biosensor can be a glucose sensor. Also, in certain embodiments, the target compound in the sample may be glucose. That is, in certain embodiments, the present disclosure provides a method for measuring glucose concentration using a glucose concentration measuring system. In certain embodiments, the biosensor can be a lactate sensor. Also, in certain embodiments, the target compound in the sample may be lactic acid. That is, in certain embodiments, the present disclosure provides a method for measuring a lactate concentration using a lactate concentration measuring system.

In certain embodiments, the present disclosure relates to contacting a sample containing a compound to be measured with an oxidoreductase, applying a voltage to an electrode, and a metal complex compound and an electron transfer promoter (excluding PMS). Provided are methods for measuring the concentration of a compound to be measured, including measuring the response current in the presence. The voltage to be applied is not particularly limited, but when a ruthenium compound is used as the metal complex compound, it may be, for example, 10 to 1000 mV, 10 to 800 mV, 50 to 500 mV, for example, 0 to 100 mV. When potassium ferricyanide is used as the metal complex compound, it may be, for example, 200 to 1000 mV, 300 to 800 mV, or 300 to 500 mV.

As a method for measuring the concentration of the compound to be measured, the sample may be brought into contact with the oxidoreductase, and then the voltage may be applied after holding the sample without applying the potential for a certain period of time, and the voltage is applied at the same time as the contact. You can also do it. The time to hold without applying the potential can be greater than 0 seconds and less than 1 minute, eg 1-30 seconds, eg 1-10 seconds. For example, the redox enzyme can be FAD-GDH and the compound to be measured can be glucose. Further, the redox enzyme can be LOD, and the compound to be measured can be lactic acid. Similarly, for various oxidoreductases classified into the EC Group 1 listed above, the substrate recognized by these oxidoreductases can be used as the measurement target compound, and a system for measuring the concentration of the measurement target compound. Are provided respectively.

In certain embodiments, the reagent layer may further comprise a buffer, a surfactant, an inorganic compound, and other components.

The buffering agent is not particularly limited, and examples thereof include an amine-based buffering agent and a buffering agent having a carboxyl group. Examples of the amine-based buffer include Tris, ACES, CHES, CAPSO, TAPS, CAPS, Bis-Tris, TAPSO, TES, Tricine and ADA. Examples of the buffer having a carboxyl group include acetic acid-Na acetate buffer, malic acid-Na acetate buffer, malonic acid-Na acetate buffer, and succinic acid-Na acetate buffer. The buffer may be used alone or in combination.

The surfactant is not particularly limited, and examples thereof include nonionic, anionic, cationic, and amphoteric surfactants. Amphoteric surfactants include, but are not limited to, carboxybetaine, sulfobetaine, and phosphobetaine. Examples of the sulfobetaine include CHASPS (3-[(3-colamidepropyl)) dimethylammonio] propanesulfonate) and CHASPS (3-[(3-colamidepropyl) dimethylammonio] -2-hydroxy-1. -Propane sulfonate), and alkyl hydroxysulfobetaine, but not limited to.

In certain embodiments, the inorganic compound may be a layered inorganic compound, and those conventionally used for glucose sensors or equivalents thereof which will be developed in the future can be used. Examples of the inorganic compound include swellable clay minerals having ion exchange ability, smectite, bentonite, synthetic fluorine mica, vermiculite, synthetic hectorite, synthetic saponite and other synthetic smectites; swellable synthetic mica containing synthetic fluorine mica; Na-type mica. Examples include, but are not limited to, synthetic mica including, and combinations thereof.

In certain embodiments, the reagent layer may have an enzyme layer. In certain embodiments, the enzyme layer may have FAD-GDH. In certain embodiments, the enzyme layer containing FAD-GDH may contain additives such as sodium polyacrylate, trehalose, glucomannan and the like.

The reagent layer may have a single layer structure or a multi-layer structure. Each layer may have one or more components. In certain embodiments, the reagent layer can be an inorganic gel layer and an enzyme layer containing FAD-GDH laminated on top of the inorganic gel layer. The reagent layer can be placed dry on the electrodes.

The sample to be measured may be a biological sample (for example, blood, body fluid, urine, etc.) or another liquid sample.

In certain embodiments, the glucose sensor has a working electrode containing FAD-GDH, a counter electrode, and optionally a reference electrode. As the working electrode, a carbon electrode, a gold electrode, a platinum electrode and the like can be used. FAD-GDH may or may not be immobilized on the electrodes. The counter electrode can be a conventional electrode such as a platinum electrode or Pt / C. The reference electrode can be a conventional electrode such as an Ag / AgCl electrode. When FAD-GDH is immobilized, the immobilization method includes a method using a cross-linking reagent, a method of encapsulating in a polymer matrix, a method of coating with a dialysis membrane, a photocrosslinkable polymer, a conductive polymer, a redox polymer and the like. Or, it may be fixed in a polymer or adsorbed and fixed on an electrode together with an electron mediator typified by ferrocene or a derivative thereof, or these may be used in combination. Typically, FAD-GDH is immobilized on a carbon electrode with glutaraldehyde and then treated with a reagent having an amine group to block glutaraldehyde. Other redox enzymes can be immobilized in the same manner.

A method for manufacturing a glucose sensor is available in publicly known documents, for example, Liu, et. al. , Anal. Chem. 2012, 84, 3403-3409 and Tsujimura, et. al. , J. Am. Chem. Soc. 2014, 136, 14432-14437 (all of which are incorporated herein by reference).

In one embodiment, the glucose sensor may include a printed electrode. In this case, electrodes can be formed on the insulating substrate. Specifically, the electrodes can be formed on the substrate by printing techniques such as photolithography or screen printing, gravure printing or flexographic printing. Examples of the material constituting the insulating substrate include silicon, glass, ceramic, polyvinyl chloride, polyethylene, polypropylene, polyester and the like. Materials that are highly resistant to various solvents or chemicals can be used.

The glucose concentration can be measured as follows. Put buffer in a constant temperature cell and keep it at a constant temperature. Metal complex compounds (eg, ruthenium compounds, etc.) and electron transfer promoters are used for electron transfer. FAD-GDH is used as an oxidoreductase. A carbon electrode is used as the working electrode, and a counter electrode (for example, a platinum electrode) and a reference electrode (for example, an Ag / AgCl electrode) are used. A constant voltage is applied to the carbon electrode, and after the current becomes steady, a sample containing glucose is added and the increase in current is measured. The glucose concentration in the sample can be calculated according to the calibration curve prepared with the standard concentration glucose solution. Similarly, potassium ferricyanide can be used instead of the ruthenium compound to calculate the glucose concentration in the sample. In the present specification, unless otherwise specified, the redox potential is the redox potential (vs. Ag / AgCl) when silver silver chloride is used as a reference electrode.

The glucose sensor is used in combination with a measuring device equipped with a means for applying a predetermined voltage at a fixed time, a means for measuring an electric signal transmitted from a biosensor, a means for converting the electric signal into a concentration of an object to be measured, and the like. obtain. The same applies to biosensors including lactic acid sensors.

The electrochemical measurement method of the present disclosure may be a current measurement method, a potentiometric method, or a coulometric method. In one embodiment, the electrochemical measurement method measures the current value when the electron transfer substance in the reduced state is in the oxidized state by applying a potential.

In certain embodiments, the method of this disclosure does not include medical practice. In certain embodiments, the methods of the present disclosure do not include diagnostic activity by a physician. In certain embodiments, the methods of the present disclosure may aid in the diagnosis of diabetes. In certain embodiments, the methods of the present disclosure can be utilized for blood glucose monitoring. These do not require the judgment of a doctor.

Hereinafter, the present disclosure will be further described with reference to Examples and Comparative Examples. However, this disclosure is not construed as being limited to the following examples.

[Example 1]
Glucose Dehydrogenase (manufactured by Kikkoman Biochemifa, derived from the genus Mucor, Product Code: FADGDH-AA, hereinafter referred to as GDH), hexaammine ruthenium chloride (manufactured by Merck, hereinafter referred to as Ru) and BGLB are mixed. Chronoamperometry was performed by measuring the printed electrodes. Specifically, 100 μL of a solution containing GDH at a final concentration of 0.1 mg / ml, Ru at 300 mM, and BGLB at 12 mM in PBS was applied onto the print electrode. The printed electrode uses a carbon working electrode (12.6 mm2) and a silver reference electrode printed on it, SCREEN-PRINTED ELECTRODES (Drop Sense, DRP-110), and a dedicated connector (Drop Sense, DRP-). It was connected to the ALS electrochemical analyzer 814D (manufactured by BAS) using CAC). Then, the applied voltage was set to +100 mV (vs. Ag / AgCl). FIG. 1 shows the results of adding glucose and measuring the current value 5 seconds after the start of chronoamperometry measurement. As shown in FIG. 1, a higher response current was observed when BGLB was added as compared with the case where BGLB was not mixed, and the current value increased in a glucose concentration-dependent manner.

Subsequently, the same measurement was performed using 1 mM TMPD or APPD instead of BGLB. Specifically, 100 μL of a solution containing 0.5% ethanol, a final concentration of 0.1 mg / ml GDH, 300 mM Ru, 1 mM TMPD or APPD in PBS was applied onto the printed electrode. However, since APPD was not completely dissolved, it was actually measured at a concentration of less than 1 mM. Then, the applied voltage was set to +100 mV (vs. Ag / AgCl). The results of adding glucose and measuring the current value 20 seconds after the start of chronoamperometry measurement are shown in FIGS. 2 and 3. As shown in FIGS. 2 and 3, a higher response current was observed when TMPD or APPD was added as compared with the case where TMPD or APPD was not mixed, and the current value increased in a glucose concentration-dependent manner.

[Example 2]
The measurement was carried out using potassium ferricyanide (hereinafter referred to as FeCN) instead of Ru used in Example 1. Specifically, 100 μL of a solution containing 2.5% ethanol, a final concentration of 0.1 mg / ml GDH, 300 mM Ru, 1 mM BGLB or DPPA in PBS was applied onto the printed electrode. Similarly, 100 μL of a solution containing 0.5% ethanol, a final concentration of 0.1 mg / ml GDH, 300 mM Ru, and less than 1 mM APPD in PBS was applied onto the print electrode. FIG. 4 shows the results of adding glucose and measuring the current value 5 seconds after the start of chronoamperometry measurement. As shown in FIG. 4, a higher response current was observed when APPD was added as compared with the case where APPD was not mixed, and the current value increased in a glucose concentration-dependent manner. In particular, when 10 mM to 20 mM glucose was added, which is near the blood glucose level of mildly diabetic patients, the response current was low with FeCN alone, and a high response current was observed when BGLB, DPPA or APPD was added, and the sensor sensitivity was improved.

[Example 3]
Chronoamperometry by print electrode measurement was performed using lactic acid oxidase (manufactured by Toyobo Co., Ltd., Product Code: LCO-301, hereinafter referred to as LOD) instead of FADGDH-AA used in Example 1. Specifically, 100 μL of a solution containing a final concentration of 0.1 mg / ml LOD, 300 mM Ru, and 1 mM BGLB in PBS was applied onto the printed electrode. FIG. 5 shows the results obtained by measuring the current value 5 seconds after the start of chronoamperometry measurement with the addition of lactic acid and subtracting the current value when no lactic acid was added. As shown in FIG. 5, a higher response current was observed when BGLB was added as compared with the case where BGLB was not mixed, and the current value increased in a lactic acid concentration-dependent manner.

The biosensors of the present disclosure can be used in the fields of biochemistry, medicine and medicine. For example, the glucose sensor of the present disclosure is useful for glucose measurement. For example, the lactate sensor of the present disclosure is useful for measuring lactate.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

A biosensor comprising (i) an electron transfer promoter (excluding PMS), (ii) an oxidoreductase, (iii) a metal complex compound, and (iv) an electrode.
Electron transfer promoter

[During the ceremony,
R 1 is -NR 7 R 8 and
R 2 is −NR 10 R 11 and
R 7 and R 8 are each independently substituted with hydrogen, optionally one or more X or V, linear or branched C 1-7 alkyl, C 1-7 alkenyl, C 1- . 7 alkynyl, C 3-9 cycloalkyl, phenyl, 1-naphthyl, 2-naphthyl, anthracenyl, phenanthrenyl, acetyl, carboxy, furanylformyl, pyrazolylformyl, 1-methyl-1H-pyrazole-5-ylformyl, 9,9 -Dimethylfluorene-2-yl or benzyl,
R 10 is hydrogen, optionally one or more X or V, linear or branched C 1-7 alkyl, C 1-7 alkenyl, C 1-7 alkynyl, C 3-9 . Cycloalkyl, phenyl, 1-naphthyl, 2-naphthyl, anthracenyl or phenanthrenyl,
R 3 , R 4 , R 5 and R 6 are each independently substituted with hydrogen, optionally one or more Y, linear or branched C 1-7 alkyl, C 1-7 alkoxy. , C 1-7 alkynyl, C 1-7 alkoxy, halo, nitro, cyano, carboxy, sulfo, hydroxy or amino.
Alternatively, R 3 and R 4 , or R 5 and R 6 may be optionally substituted with one or more oxos, Xs, together with the benzene ring containing them, the benzene ring, or

, Where * is bonded to the carbon atom to which R 3 is bonded, ** is bonded to the carbon atom to which R 4 is bonded, or * is bonded to the carbon atom to which R 5 is bonded, ** Bonds to the carbon atom to which R 6 bonds,
R 11 is a straight or branched C 1-7 alkyl or optionally substituted with one or more Xs or Zs, phenyl, 1-naphthyl, 2-naphthyl, anthracenyl and phenanthrenyl. Selected from the group consisting of
Where V is —O-acryloyl, acetylamino, or phenyl, optionally substituted with C 1-7 alkyl.
X may optionally be substituted with a substituent selected from the group consisting of one or more halos, aminos, cyanos, carboxys, carbonyls, alkoxys, alkylaminos, nitrosos, nitros and sulfos, linear or branched. , C 1-7 alkyl, C 1-7 alkenyl, C 1-7 alkynyl, C 1-7 alkoxy, halo, hydroxy, nitro, carboxy, cyano, sulfo, amino or alkylamino.
Y is selected from the group consisting of halo, amino, cyano, carboxy, carbonyl, hydroxy, alkoxy and sulfo.
Z is -SO 2 -CH = CH 2 , -SO 2 -C 2 H 4 -O-SO 3 H, or 4,6-dichlorotriazine-2-ylamino]
The biosensor according to claim 1.
The oxidase is FAD-type glucose dehydrogenase (FAD-GDH), L- or D-lactic acid oxidase (LOD), glucose oxidase, glucose dehydrogenase, amadriase, peroxidase, galactose oxidase, birylbin oxidase, pyruvate oxidase, D- or L. -Amino acid oxidase, amine oxidase, cholesterol oxidase, choline oxidase, xanthin oxidase, sarcosin oxidase, ascorbic acid oxidase, cytochrome oxidase, alcohol dehydrogenase, cholesterol dehydrogenase, aldehyde dehydrogenase, aldehyde oxidase, fructose dehydrogenase (FDH), sorbitol dehydrogenase, D- or L-lactic dehydrogenase, malic acid dehydrogenase, glycerol dehydrogenase, 17B hydroxysteroid dehydrogenase, estradiol 17B dehydrogenase, D- or L-amino acid dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, 3-hydroxysteroid dehydrogenase, diahorase, catalase, glutathione reductase , Citochrome b5 reductase, adrenoxine reductase, cytochrome b5 reductase, adrenodoxin reductase, nitrate reductase, phosphate dehydrogenase, birylbin oxidase, lacquerse, polyamine oxidase, formate dehydrogenase, pyranose oxidase, pyranose dehydrogenase, and tauropine dehydrogenase. The biosensor according to claim 1 or 2, which is selected from the above.
The electrode includes an electrode portion having a working electrode and a counter electrode, the electrode portion is arranged on an insulating substrate, and a reagent layer is further arranged on the electrode portion.
The biosensor according to any one of claims 1 to 3, wherein the oxidoreductase, the metal complex compound, and the electron transfer promoter are contained in the reagent layer.
A method for producing a biosensor, which comprises forming a reagent layer containing an oxidoreductase, a metal complex compound, and an electron transfer promoter on an electrode.
For measuring the concentration of a target compound in a sample, which comprises the biosensor according to any one of claims 1 to 4, a means for applying a voltage to the electrode of the biosensor, and a means for measuring a current. system.
A method for measuring the concentration of a target compound in a sample using the system according to claim 6.
A step of preparing a biosensor comprising (i) an electron transfer promoter (excluding PMS), (ii) an oxidoreductase, (iii) a metal complex compound, and (iv) an electrode.
A method for measuring the concentration of a target compound in a sample, which comprises a step of applying a voltage to the electrode of the biosensor and a step of measuring a response current.