Patent Application
20190271658
The present disclosure relates to a method of electrochemically measuring an analyte via an electron mediator, and an oxidant for an electron mediator for use in a biosensor using the electron mediator.
A method of electrochemically measuring an analyte in a sample by use of an electron mediator is broadly known. The electron mediator is a redox substance that mediates electron transfer, and shoulders responsibility for transfer of an electron produced by oxidation-reduction reaction of an analyte in a biosensor.
For example, Patent Literature 1 discloses a biosensor for measuring total hemoglobin using potassium ferricyanide as an electron mediator. Specifically, Patent Literature 1 discloses that potassium ferricyanide oxidizes hemoglobin iron (II) while potassium ferricyanide is reduced to potassium ferrocyanide. At the same time, an electrode system comprising a working electrode and a counter electrode is used to obtain an electrochemical signal from the potassium ferrocyanide, and a total hemoglobin amount is determined based on the signal.
Patent Literature 2 discloses a biosensor for measuring glucose concentration in blood (blood sugar level). Specifically, Patent Literature 2 discloses that potassium ferricyanide is used as an electron mediator, and when glucose and potassium ferricyanide are reacted in the presence of glucose dehydrogenase, the glucose is reduced by the catalytic action of glucose dehydrogenase while the potassium ferricyanide is reduced to potassium ferrocyanide. At the same time, an electrode system comprising a working electrode and a counter electrode is used to obtain an electrochemical signal from the potassium ferrocyanide, and the glucose amount is determined based on the signal.
However, development of an electrochemical technique with increased measurement accuracy is still needed.
Patent Literature 1: Japanese Patent No. 5812957
Patent Literature 2: Japanese Patent No. 5325574
The present inventors found out a technical problem that an electron mediator is reduced. depending on storage conditions of the electron mediator, independently of oxidation reaction of an analyte in electrochemical measurement process or in production process of an electrochemical sensor using electrochemical measurement technique. For example, in the case of using potassium ferricyanide as an electron mediator, it was found that when a solution of potassium ferricyanide was added dropwise to an electrode system and dried thereon, a part of the potassium ferricyanide was reduced to potassium ferrocyanide by influence of moisture in the air and light. Further, it was found that when a solution containing a reagent such as a high hygroscopic buffer component and potassium ferricyanide was added dropwise to an electrode system and dried thereon, a part of the potassium ferricyanide was reduced to potassium ferrocyanide depending on the storage conditions. Such unintended reduction of an electron mediator affects the measurement value of an analyte, resulting in decreased measurement accuracy.
Thus the present inventors intensively studied, and as a result, surprisingly found that use of an oxidant comprising a trivalent iodobenzene compound could solve the above-described problem.
The present disclosure provides the following aspects:
[1] an oxidant for an electron mediator, comprising a trivalent iodobenzene compound,
[2] the oxidant according to [1], wherein the trivalent iodobenzene compound is represented by any of formulae I to III:
wherein:
R1 to R5 are independently a hydrogen atom, halogen, an amino group, a nitro group, a hydroxyl group, a carboxyl group, an acetyl group, or an optionally substituted hydrocarbon group,
X and Y are independently a hydroxyl group, an aklylcarbonyloxy group, an alkylsulfonyloxy group, an alkylbenzenesulfonyloxy group, or an optionally substituted hydrocarbon group,
Z is halogen, a hydroxyl group, an alkylcarbonyloxy group, an alkylperoxy group, or an optionally substituted hydrocarbon group,
W1 and W2 are independently a methyl group, or W1 and W2 together form oxo (═O),
[3] the oxidant according to [2], wherein in formula I, R1 to R5 are independently a hydrogen atom, halogen, a nitro group, or a linear or branched, saturated or unsaturated, optionally substituted C1-4 hydrocarbon group,
X and Y are independently a hydroxyl group, an acetoxy group, a trifluoroacetoxy group, a tosyloxy group, a methanesulfonyloxy group, a trifluoromethanesulfonyloxy group, or a linear saturated halogenated C1-10 hydrocarbon group;
in formula II, R1 to R5 are independently a hydrogen atom, halogen, a nitro group, a carboxyl group, or a linear or branched, saturated or unsaturated, optionally substituted C1-4 hydrocarbon group; and
in formula III, R1 to R4 are independently a hydrogen atom, halogen, a nitro group, or a linear or branched, saturated or unsaturated, optionally substituted C1-4 hydrocarbon group,
Z is halogen, a hydroxyl group, an acetoxy group, a trifluoroacetoxy group, or a linear or branched C1-6 alkylperoxy group,
W1 and W2 are independently a methyl group, or W1 and W2 together form oxo (═O),
[4] the oxidant according to [1], wherein the trivalent iodobenzene compound is selected from the group consisting of iodosobenzene, 2,4,6-trimethyl(diacetoxyiodo)benzene, [bis(trifluoroacetoxy)iodo]pentafluorobenzene, [hydroxy(tosyloxy)iodo]benzene, 2-iodosobenzoic acid, 1-fluoro-3,3-dimethyl-1,2-benziodoxol, and [hydroxy(methanesulfonyloxy)iodo]benzene,
[5] the oxidant according to any one of [1]-[4], wherein the electron mediator is selected from the group consisting of a metal complex, a quinone compound, a phenazine compound, a viologen compound, a phenothiazine compound, and a phenol compound,
[6] the oxidant according to [5], wherein the electron mediator is at least one compound selected from the group consisting of potassium ferricyanide, hexaammineruthenium, ferrocene, poly(1-vinylimidazole)-bis-(bipyridine)chloroosmium, hydroquinone, 2-methyl-1,4-benzoquinone, 1,2-naphthoquinone-4-sulfonic acid or a salt thereof, 9,10-phenanthrenequinone-2-sulfonic acid or a salt thereof, 9,10-phenanthrenequinone-2,7-disulfonic acid or a salt thereof, 1,10-phenanthroline-5,6-dione, anthraquinone-2-sulfonic acid or a salt thereof, 1-methoxy-5-methylphenazinium methyl sulfate, hydroxyphenazine, methyl viologen, benzyl viologen, methylene blue, methylene green, 2-aminophenol, 2-amino-4-methylphenol, and 2,4-diaminophenol,
[7] a reagent for measurement of an analyte, containing an electron mediator, and the oxidant according to any one of [1]-[4],
[8] the reagent for measurement of an analyte according to [7], further containing an enzyme that catalyzes oxidation reaction,
[9] the reagent for measurement of an analyte according to [7] or [8], wherein the electron mediator is selected from the group consisting of a metal complex, a quinone compound, a phenazine compound, a viologen compound, a phenothiazine compound, and a phenol compound,
[10] the reagent for measurement of an analyte according to [9], wherein the electron mediator is at least one compound selected from the group consisting of potassium ferricyanide, hexaammineruthenium, ferrocene, poly(1-vinylimidazole)-bis-(bipyridine)chloroosmium, hydroquinone, 2-methyl-1,4-benzoquinone, 1,2-naphthoquinone-4-sulfonic acid or a salt thereof, 9,10-phenanthrenequinone-2-sulfonic acid or a salt thereof, 9,10-phenanthrenequinone-2,7-disulfonic acid or a salt thereof, 1,10-phenanthroline-5,6-dione, anthraquinone-2-sulfonic acid or a salt thereof, 1-methoxy-5-methylphenazinium methyl sulfate, hydroxyphenazine, methyl viologen, benzyl viologen, methylene blue, methylene green, 2-aminophenol, 2-amino-4-methylphenol, and 2,4-diaminophenol,
[11] a sensor comprising a working electrode, a counter electrode, and a reagent layer, wherein the reagent layer contains an electron mediator and the oxidant according to any one of [1]-[4],
[12] the sensor according to [11], wherein the reagent layer further contains an enzyme that catalyzes oxidation reaction,
[13] the sensor according to [11] or [12], wherein the electron mediator is selected from the group consisting of a metal complex, a quinone compound, a phenazine compound, a viologen compound, a phenothiazine compound, and a phenol compound,
[14] the sensor according to [13], wherein the electron mediator is at least one compound selected from the group consisting of potassium ferricyanide, hexaammineruthenium, ferrocene, poly(1-vinylimidazole)-bis-(bipyridine)chloroosmium, hydroquinone, 2-methyl-1,4-benzoquinone, 1,2-naphthoquinone-4-sulfonic acid or a salt thereof, 9,10-phenanthrenequinone-2-sulfonic acid or a salt thereof, 9,10-phenanthrenequinone-2,7-disulfonic acid or a salt thereof, 1,10-phenanthroline-5,6-dione, anthraquinone-2-sulfonic acid or a salt thereof, 1-methoxy-5-methylphenazinium methyl sulfate, hydroxyphenazine, methyl viologen, benzyl viologen, methylene blue, methylene green, 2-aminophenol, 2-amino-4-methylphenol, and 2,4-diaminophenol,
[15] a method of electrochemically measuring an analyte, the method comprising:
(A) bringing a sample containing the analyte into contact with a reagent containing an electron mediator and the oxidant according to any one of [1]-[4], thereby the analyte is oxidized while the electron mediator is reduced, and
(B) using an electrode system comprising at least a working electrode and a counter electrode to obtain an electrochemical signal from the reduced electron mediator,
[16] the method according to [15], wherein in step (A), the analyte is oxidized by the electron mediator while the electron mediator is reduced,
[17] the method according to [15], wherein in step (A), the reagent further contains an enzyme that catalyzes oxidation reaction, and the analyte is oxidized by the enzyme while the electron mediator is reduced,
[18] the method according to any one of [15]-[17], wherein the electron mediator is selected from the group consisting of a metal complex, a quinone compound, a phenazine compound, a viologen compound, a phenothiazine compound, and a phenol compound,
[19] the method according to [18], wherein the electron mediator is at least one compound selected from the group consisting of potassium ferricyanide, hexaammineruthenium, ferrocene, poly(1-vinylimidazole)-bis-(bipyridine)chloroosmium, hydroquinone, 2-methyl-1,4-benzoquinone, 1,2-naphthoquinone-4-sulfonic acid or a salt thereof, 9,10-phenanthrenequinone-2-sulfonic acid or a salt thereof, 9,10-phenanthrenequinone-2,7-disulfonic acid or a salt thereof, 1,10-phenanthroline-5,6-dione, anthraquinone-2-sulfonic acid or a salt thereof, 1-methoxy-5-methylphenazinium methyl sulfate, hydroxyphenazine, methyl viologen, benzyl viologen, methylene blue, methylene green, 2-aminophenol, 2-amino-4-methylphenol, and 2,4-diaminophenol,
[20] a use of the oxidant according to any one of [1]-[4] for decreasing degradation of an electron mediator,
[21] the use according to [20], wherein the electron mediator is selected from the group consisting of a metal complex, a quinone compound, a phenazine compound, a viologen compound, a phenothiazine compound, and a phenol compound,
[22] the use according to [21], wherein the electron mediator is at least one compound selected from the group consisting of potassium ferricyanide, hexaammineruthenium, ferrocene, poly(1-vinylimidazole)-bis-(bipyridine)chloroosmium, hydroquinone, 2-methyl-1,4-benzoquinone, 1,2-naphthoquinone-4-sulfonic acid or a salt thereof, 9,10-phenanthrenequinone-2-sulfonic acid or a salt thereof, 9,10-phenanthrenequinone-2,7-disulfonic acid or a salt thereof, 1,10-phenanthroline-5,6-dione, anthraquinone-2-sulfonic acid or a salt thereof, 1-methoxy-5-methylphenazinium methyl sulfate, hydroxyphenazine, methyl viologen, benzyl viologen, methylene blue, methylene green, 2-aminophenol, 2-amino-4-methylphenol, and 2,4-diaminophenol,
[23] a method of preventing degradation of an electron mediator, the method comprising using the oxidant according to any one of [1]-[4],
[24] the method according to [23], wherein the electron mediator is selected from the group consisting of a metal complex, a quinone compound, a phenazine compound, a viologen compound, a phenothiazine compound, and a phenol compound, and
[25] the method according to [24], wherein the electron mediator is at least one compound selected from the group consisting of potassium ferricyanide, hexaammineruthenium, ferrocene, poly(1-vinylimidazole)-bis-(bipyridine)chloroosmium, hydroquinone, 2-methyl-1,4-benzoquinone, 1,2-naphthoquinone-4-sulfonic acid or a salt thereof, 9,10-phenanthrenequinone-2-sulfonic acid or a salt thereof, 9,10-phenanthrenequinone-2,7-disulfonic acid or a salt thereof, 1,10-phenanthroline-5,6-dione, anthraquinone-2-sulfonic acid or a salt thereof, 1-methoxy-5-methylphenazinium methyl sulfate, hydroxyphenazine, methyl viologen, benzyl viologen, methylene blue, methylene green, 2-aminophenol, 2-amino-4-methylphenol, and 2,4-diaminophenol.
The accuracy of electrochemical measurement of an analyte is increased by using the oxidant disclosed herein.
The oxidant for an electron mediator as disclosed herein (hereinafter, also referred to as “the oxidant disclosed herein”) is an oxidant for oxidizing an electron mediator. The oxidant disclosed herein is particularly used for oxidizing an electron mediator that has been reduced independently of oxidation reaction of an analyte in electrochemical measurement of the analyte using an electron mediator or in a process of production of an electrochemical sensor using the electrochemical measurement technique. As used herein, the term “analyte” means a substance to be measured.
An electron mediator was found to be reduced depending on storage conditions including moisture in the air, light, components of a buffer for dissolving the electron mediator (in particular, high hygroscopic buffer components including many phosphates such as disodium hydrogenphosphate, and sodium dihydrogen phosphate, citric acid, etc.) and the like. The degradation of an electron mediator as described above can be decreased or prevented by making the oxidant disclosed herein coexist with the electron mediator.
Specifically, the oxidant disclosed herein comprises a trivalent iodobenzene compound. The oxidant disclosed herein may be a trivalent iodobenzene compound itself, or may comprise a trivalent iodobenzene compound and, as necessary, an additional component that does not adversely affect the oxidizing action of the trivalent iodobenzene compound, such as water, a solvent, a buffer, protein, a polymeric material, sugar, a salt, or a surfactant. The oxidant disclosed herein may comprise two or more kinds of trivalent iodobenzene compounds.
Examples of the trivalent iodobenzene compound used in the present disclosure include, but not limited to, compounds represented by formulae I to III:
wherein:
R1 to R5 are independently a hydrogen atom, halogen, an amino group, a nitro group, a hydroxyl group, a carboxyl group, an acetyl group, or an optionally substituted hydrocarbon group,
X and Y are independently a hydroxyl group, an aklylcarbonyloxy group, an alkylsulfonyloxy group, an alkylbenzenesulfonyloxy group, or an optionally substituted hydrocarbon group,
Z is halogen, a hydroxyl group, an alkylcarbonyloxy group, an alkylperoxy group, or an optionally substituted hydrocarbon group,
W1 and W2 are independently a methyl group, or W1 and W2 together form oxo (═O).
As used herein, examples of the halogen include bromine, chlorine, iodine, and fluorine. Preferably bromine or fluorine is used.
As used herein, the hydrocarbon group includes a saturated or unsaturated, linear, branched or cyclic hydrocarbon group. Examples of the hydrocarbon group include linear, branched or cyclic hydrocarbon groups containing 1 to 10 carbon atoms. Examples of a substituent that the hydrocarbon group optionally has include at least one substituent selected from the group consisting of halogen, an amino group, a hydroxyl group, a carboxyl group, and an acetyl group. It is preferable that the hydrocarbon group is substituted with halogen or unsubstituted. The number of a substituent(s) that the hydrocarbon group optionally has is not particularly limited. A part of the hydrogen atoms that the hydrocarbon group contains may be substituted with a substituent(s), or all of the hydrogen atoms that the hydrocarbon group contains may be substituted with substituents.
As used herein, alkyl may be substituted with halogen, and a part or all of the hydrogen atoms that the alkyl contains may be substituted with halogen. Examples of alkyl include linear or branched alkyl having one to six carbons, preferably one to four carbons.
As used herein, preferable examples of the trivalent iodobenzene compound include compounds represented by formula I, formula II and formula III, wherein R1 to R5 are independently a hydrogen atom, halogen, an amino group, a nitro group, a hydroxyl group, a carboxyl group, an acetyl group, or a linear or branched, saturated or unsaturated optionally substituted C1-4 hydrocarbon group;
X and Y are independently a hydroxyl group, an acetoxy group, a trifluoroacetoxy group, a tosyloxy group, a methanesulfonyloxy group, a trifluoromethanesulfonyloxy group, or a linear or branched, saturated or unsaturated, optionally substituted C1-10 hydrocarbon group;
Z is halogen, a hydroxyl group, an acetoxy group, a trifluoroacetoxy group, a linear or branched C1-6 alkylperoxy group, or a linear or branched, saturated or unsaturated, optionally substituted C1-4 hydrocarbon group;
W1 and W2 are independently a methyl group, or W1 and W2 together form oxo (═O).
In formula I, R1 to R5 are, more preferably, independently a hydrogen atom, halogen, a nitro group, or a linear or branched, saturated or unsaturated, optionally substituted C1-4 hydrocarbon group, and further preferably, R1 to R5 are independently a hydrogen atom, halogen, a nitro group, or a saturated or unsaturated, unsubstituted C1-2 hydrocarbon group; X and Y are, more preferably, independently a hydroxyl group, an acetoxy group, a trifluoroacetoxy group, a tosyloxy group, a methanesulfonyloxy group, a trifluoromethanesulfonyloxy group, or a linear saturated halogenated C1-10 hydrocarbon group, and further preferably, X and Y are the same and are an acetoxy group, a trifluoroacetoxy group, a tosyloxy group, a methanesulfonyloxy group, or a trifluoromethanesulfonyloxy group, or one of X and Y is a hydroxyl group or a linear saturated halogenated C1-10 hydrocarbon group and the other of X and Y is an acetoxy group, a trifluoroacetoxy group, a tosyloxy group, a methanesulfonyloxy group, or a trifluoromethanesulfonyloxy group. Still further preferably, in formula I, all of R1 to R5 are the same; R1, R3 and R5 are the same and are any one of the above-mentioned substituents other than a hydrogen atom, and R2 and R4 are the same and are a hydrogen atom; or R3 is any one of the above-mentioned substituents other than a hydrogen atom, and R1, R2, R4 and R5 are the same and are a hydrogen atom. When all of R1 to R5 are the same, they are preferably hydrogen or halogen. Further preferably, in formula I, X and y are the same and are an acetoxy group or a trifluoroacetoxy group; or X is a hydroxyl group and Y is a tosyloxy group or a methanesulfonyloxy group.
In formula I, when R3 is a vinyl group, the compound represented by formula I may take a polymer form as polystyrene. Such a trivalent iodobenzene compound in a polymer form is also included in the oxidant disclosed herein.
In formula II, more preferably, R1 to R5 are independently a hydrogen atom, halogen, a nitro group, a carboxyl group, or a linear or branched, saturated or unsaturated, optionally substituted C1-4 hydrocarbon group. Further preferably, in formula II, R1 to R5 are independently a hydrogen atom, halogen, a nitro group, a carboxyl group, or a saturated or unsaturated unsubstituted C1-2 hydrocarbon group.
In formula II, when iodosobenzene is substituted with a carboxyl group, the carboxyl group is preferably at an ortho position. In formula II, when iodosobenzene is ortho-substituted with a carboxyl group, the iodosobenzene compound may be cyclized to take a tautomeric form represented by formula III.
The iodosobenzene compound represented by formula II wherein R1 and R5 are not a carboxyl group is usually regarded as being in a form of a polymer consisting of a I-O-I-O chain. Such a iodosobenzene compound in a polymer form is also included in the oxidant disclosed herein.
In formula III, more preferably, R1 to R4 are independently a hydrogen atom, halogen, a nitro group, or a linear or branched, saturated or unsaturated, optionally substituted C1-4 hydrocarbon group, and further preferably, R1 to R4 are independently a hydrogen atom, halogen, a nitro group, or a saturated or unsaturated, unsubstituted C1_2 hydrocarbon group; more preferably, Z is halogen, a hydroxyl group, an acetoxy group, a trifluoroacetoxy group, or a linear or branched C1-6 alkylperoxy group, and further preferably, Z is fluorine, a hydroxyl group, an acetoxy group, or a linear or branched C1-4 alkylperoxy group; W1 and W2 are independently a methyl group, or W1 and W2 together form oxo (═O). Further preferably, in formula III, R2 is a hydrogen atom, halogen or a nitro group, and R1, R3 and R4 are the same and are a hydrogen atom.
Specific examples of the trivalent iodobenzene compound used as the oxidant herein include, but not limited to, iodosobenzene, 2,4,6-trimethyl(diacetoxyiodo)benzene, [bis(trifluoroacetoxy)iodo]pentafluorobenzene, [hydroxy(tosyloxy) iodo]benzene, 2-iodosobenzoic acid [also referred to as 1-hydroxy-1,2-benziodoxol-3(1H)-one], 1-acetoxy-5-bromo-1,2-benziodoxol-3(1H)-one, 1-acetoxy-5-nitro-1,2-benziodoxol-3(1H)-one, [bis(trifluoroacetoxy)iodo]benzene, 4-nitro(diacetoxyiodo)benzene, 1-(tert-butylperoxy)-1,2-benziodoxol-3(1H)-one, (diacetoxyiodo)benzene, [hydroxy(methanesulfonyloxy)iodo]benzene, poly[4-(diacetoxyiodo)styrene], 1-fluoro-3,3-dimethyl-1,2-benziodoxol, (perfluoropropyl)phenyliodonium trifluoromethanesulfonate, (perfluorohexyl)phenyliodonium trifluoromethanesulfonate, and (perfluoro-n-octyl)phenyliodonium trifluoromethanesulfonate.
The electron mediator to be oxidized by the oxidant disclosed herein is not particularly limited, and may be any electron mediator that is able to be used in electrochemical measurement. Examples of the electron mediator include, but not limited to, a metal complex (for example, an osmium complex, a ruthenium complex, a ferric complex, etc.), a quinone compound (for example, benzoquinone, naphthoquinone, phenanthrenequinone, phenanthrolinequinone, anthraquinone, and their derivatives), a phenazine compound, a viologen compound, a phenothiazine compound, and a phenol compound. For example, one or more compounds selected from the group consisting of potassium ferricyanide, hexaammineruthenium, ferrocene, poly(1-vinylimidazole)-bis-(bipyridine)chloroosmium, hydroquinone, 2-methyl-1,4-benzoquinone, 1,2-naphthoquinone-4-sulfonic acid or a salt thereof, 9,10-phenanthrenequinone-2-sulfonic acid or a salt thereof, 9,10-phenanthrenequinone-2,7-disulfonic acid or a salt thereof, 1,10-phenanthroline-5,6-dione, anthraquinone-2-sulfonic acid or a salt thereof, 1-methoxy-5-methylphenazinium methyl sulfate, hydroxyphenazine, methyl viologen, benzyl viologen, methylene blue, methylene green, 2-aminophenol, 2-amino-4-methylphenol, and 2,4-diaminophenol are used as the electron mediator. Examples of the above-mentioned salt include, but not limited to, a sodium salt, a potassium salt, a calcium salt, a magnesium salt, and a lithium salt.
The oxidant disclosed herein can be used in electrochemical measurement of an analyte by being made to coexist with an electron mediator. The coexistence of the oxidant disclosed herein with the electron mediator in electrochemical measurement of an analyte can lead to oxidation of the electron mediator that has been reduced depending on storage conditions including moisture in the air, light, components in a solution and the like, and thereby a decrease in the measurement accuracy caused by the reduction of electron mediator that is independent of the analyte is prevented and an accurate measurement value of the analyte can be obtained. Thus, use of the oxidant disclosed herein in electrochemical measurement of an analyte results in an increase in the measurement accuracy, and thereby a more accurate measurement value of the analyte can be obtained.
A method of electrochemically measuring an analyte which is characterized by using the oxidant disclosed herein (hereinafter, also referred to as “the measurement method disclosed herein”) comprises the following steps:
(A) bringing a sample containing the analyte into contact with a reagent containing an electron mediator and the oxidant disclosed herein, thereby the analyte is oxidized while the electron mediator is reduced, and
(B) using an electrode system comprising at least a working electrode and a counter electrode to obtain an electrochemical signal from the reduced electron mediator. As used herein, the term “measurement” includes “quantitative” measurement and “qualitative” measurement.
Examples of the analyte to be measured by the measurement method disclosed herein include, but not limited to, hemoglobin, glucose, ketone body (3-hydroxy butyric acid), lactic acid, glycated hemoglobin, and glycated albumin.
The sample to be subjected to the measurement method disclosed herein may be any sample containing the analyte, and is not particularly limited. Examples of the sample include a blood sample such as whole blood, plasma, a serum, a blood cell, diluted blood, etc. (for example, a diluted blood sample from human), a biological sample such as urine, spinal fluid, sweat, tears, saliva, skin, mucous membrane, hair, etc., various foods or extract thereof, and beverage such as alcohol and juice. The biological sample may not be derived from human. The blood sample may be subjected to hemolysis treatment. The hemolysis treatment may be carried out by a conventional method.
The electron mediator used in the measurement method disclosed herein is not particularly limited, and can be determined as appropriate depending on the kind of the analyte and measurement conditions. For example, the above-mentioned electron mediators can be used.
In step (A) of the measurement method disclosed herein, the analyte in the sample is oxidized by the electron mediator, or is made to coexist with an enzyme that catalyzes oxidation reaction and oxidized by the enzyme. In the latter case, the reagent containing an electron mediator and the oxidant disclosed herein further contains the enzyme that catalyzes oxidation reaction. The enzyme can be determined as appropriate depending on the kind of the analyte. Examples of the enzyme include oxidoreductase, such as oxidase and dehydrogenase. Specific examples thereof include, but not limited to, glucose dehydrogenase, glucose oxidase, 3-hydroxybutyrate dehydrogenase, lactate dehydrogenase, lactate oxidase, cholesterol oxidase, and fructosyl peptide oxidase. The enzyme may be dependent on a coenzyme. Examples of the coenzyme include, but not limited to, pyrroloquinoline quinone (PQQ), and flavin adenine dinucleotide (FAD).
For example, when the analyte is hemoglobin, the ferrous iron of hemoglobin is oxidized by an electron mediator sensitive to hemoglobin such as potassium ferricyanide, and at the same time, the electron mediator is reduced. For example, when the analyte is glucose and glucose dehydrogenase is used as the enzyme, the glucose dehydrogenase reacts with the glucose to generate gluconolactone and electrons, and an electron mediator is reduced by the electrons. For example, when the analyte is lactic acid and lactate oxidase is used as the enzyme, the lactate oxidase reacts with the lactic acid to generate pyruvic acid and electrons, and an electron mediator is reduced by the electrons. For example, when the analyte is 3-hydroxybutyric acid and 3-hydroxybutyrate dehydrogenase is used as the enzyme, the 3-hydroxybutyrate dehydrogenase reacts with the 3-hydroxybutyric acid to generate acetoacetic acid and electrons, and nicotinamide adenine dinucleotide is reduced by the electrons. Then, the reduced nicotinamide adenine dinucleotide reduces an electron mediator directly or via diaphorase.
The amount of the oxidant disclosed herein used in the measurement method disclosed herein is not particularly limited, and it is determined as appropriate depending on the sample to be measured, the kind or amount of the analyte or electron mediator, the kind of the oxidant, and other measurement conditions. Examples of the reaction final concentration of the oxidant disclosed herein include, but not limited to, ranges of about 10 μM to about 5 mM, about 30 μM to about 1 mM, and about 50 μM to about 500 μM.
The amounts of the electron mediator and the enzyme used in the measurement method disclosed herein are not particularly limited, and they may be determined as appropriate depending on the sample to be measured, the kind or amount of the analyte, oxidant or electron mediator, the kind of the enzyme, and other measurement conditions.
For the purpose of stabilization and the like of the reagent, the reagent may further contain an additional component that does not adversely affect the action of the electron mediator, oxidant and enzyme, such as a buffer, a surfactant, a polymeric material, organic acid, protein, sugar, or a salt, as necessary.
The electrode system used in the measurement method disclosed herein comprises at least a working electrode and a counter electrode. The electrode system may further comprise a reference electrode. The material of the electrode is not particularly limited, and may be any material usually used in electrochemical measurement, such as platinum, gold, silver, palladium, carbon, iridium, or iridium oxide. Preferably, gold, platinum, palladium or carbon is used as the electrode material. The electrode system may be loaded with a reagent containing an electron mediator and the oxidant disclosed herein, or a reagent containing an electron mediator, the oxidant disclosed herein and an enzyme that catalyzes oxidation reaction. The reagent may be preferably loaded into a reagent layer formed on the electrodes.
In the measurement method disclosed herein, the contact of the sample containing the analyte with the reagent containing an electron mediator and the oxidant disclosed herein is preferably carried out on electrodes. For example, the contact may be carried out by applying the sample to electrodes loaded with the reagent or by immersing the electrodes in the sample.
In the measurement method disclosed herein, the electrochemical signal reflects the amount of the analyte. Examples of the electrochemical signal include current, the quantity of electric charge, electric potential, and impedance. In the measurement method disclosed herein, the electrochemical signal can be acquired by applying a predetermined voltage across a working electrode and a counter electrode or, in the case of three-electrode system, applying a predetermined voltage based on a reference electrode to a working electrode to oxidize a reduced electron mediator. Specifically, oxidation reaction of a reduced electron mediator is induced by the applied voltage as described above, and then, the value of an electric current flowing along the working electrode or the quantity of the electric charge is detected.
The “predetermined voltage” means an electric potential at which a reduced electron mediator is oxidized. The predetermined voltage is determined as appropriate depending on various conditions such as the electrode material and pH. Examples of the predetermined voltage when the reduced electron mediator is potassium ferrocyanide (potassium hexacyanoferrate(II)) include, but not limited to, about 0.3 V to 0.6 V in the case of three-electrode system [a working electrode (for example, platinum or palladium), a counter electrode, and a reference electrode (for example, silver/silver chloride (saturated potassium chloride) electrode)], and about 0.2 V to 0.4 V in the case of two-electrode system (a working electrode and a counter electrode).
The present disclosure provides a reagent for analyte measurement containing an electron mediator and the oxidant disclosed herein (hereinafter, also referred to as “the reagent for measurement disclosed herein”). The reagent for measurement disclosed herein may further contain an enzyme that catalyzes oxidation reaction. The enzyme is an enzyme that catalyzes the oxidation reaction of an analyte. The reagent for measurement disclosed herein is used for electrochemical measurement of an analyte. The reagent for measurement disclosed herein is preferably used in the measurement method disclosed herein. The electron mediator, analyte and enzyme are as described above.
The reagent for measurement disclosed herein may further contain, as necessary, an additional component that does not adversely affect the action of the electron mediator, oxidant and enzyme, such as a buffer, a surfactant, a polymeric material, organic acid, protein, sugar, or a salt.
The present disclosure provides a sensor for measuring an analyte in a sample, which comprises an electrode system containing a working electrode and a counter electrode, and a reagent layer containing an electron mediator and the oxidant disclosed herein (hereinafter, also referred to as “the sensor disclosed herein”). The reagent layer may further contain an enzyme that catalyzes oxidation reaction. The enzyme is an enzyme that catalyzes the oxidation reaction of an analyte. In the sensor disclosed herein, the electrode system may further contain a reference electrode. The sensor disclosed herein is used for electrochemical measurement of an analyte. The sensor disclosed herein is preferably used in the measurement method disclosed herein. The electron mediator, analyte and enzyme as well as the working electrode, counter electrode and reference electrode are as described above. A method of producing the sensor disclosed herein may follow a known method in the art.
In the sensor disclosed herein, the reagent layer is formed near the electrodes or on the electrodes. The reagent layer is preferably formed on the electrodes. The reagent layer may be formed only on the working electrode, or may be formed on both the working electrode and the counter electrode. The electrodes may be formed on an insulating substrate. Examples of the insulating substrate include, but not limited to, resin such as polyethylene terephthalate, vinyl polymer, polyimide, polyester, and styrenics; glass, and ceramics. A method of loading the electron mediator and the oxidant disclosed herein, and, as necessary, the enzyme that catalyzes oxidation reaction into the reagent layer on the electrodes is not particularly limited, and a known method in the art can be used. For example, an ink-jet method may be used. The amounts of the electron mediator, oxidant and enzyme loaded into the reagent layer are not particularly limited, and can be determined as appropriate by a person skilled in the art.
For the purpose of stabilization and the like of reagents, the reagent layer may further contain an additional component that does not adversely affect the action of the electron mediator, oxidant and enzyme, such as a buffer, a surfactant, a polymeric material, organic acid, protein, sugar, or a salt, as necessary.
Hereinafter, the present disclosure is explained in detail based on Examples which the present invention is not limited to.
This Example demonstrated that the oxidant disclosed herein oxidized a reduced electron mediator.
As the oxidant, iodosobenzene (hereinafter, also referred to as “Compound 1”), 2,4,6-trimethyl(diacetoxyiodo)benzene (hereinafter, also referred to as “Compound 2”), [bis(trifluoroacetoxy)iodo]pentafluorobenzene (hereinafter, also referred to as “Compound 3”), [hydroxy(tosyloxy)iodo]benzene (hereinafter, also referred to as “Compound 4”), 2-iodosobenzoic acid (hereinafter, also referred to as “Compound 5”), 1-fluoro-3,3-dimethyl-1,2-benziodoxol (hereinafter, also referred to as “Compound 6”), and [hydroxy(methanesulfonyloxy)iodo]benzene (hereinafter, also referred to as “Compound 7”) (all manufactured by TOKYO KASEI KOGYO CO., LTD.; catalog numbers I0072, I0479, B1616, P1015, I0073, F0957, and P1298, respectively) were used. As the reduced electron mediator, potassium hexacyanoferrate(II) trihydrate (potassium ferrocyanide) (manufactured by Wako Pure Chemical Corporation) was used. As a control oxidant, hydroxypyridine N-oxide (manufactured by Dojindo Laboratories), WST-3 (manufactured by Dojindo Laboratories), and Dess-Martin periodinane (manufactured by TOKYO KASEI KOGYO CO., LTD.) were used.
Of the composition of a measurement solution shown in Table 1, all components other than the oxidant were previously mixed. Then, the oxidant was added to the mixture, and reacted for a predetermined time (30 seconds, 10 minutes, or 60 minutes). Then, the reaction mixture was applied to a sensor comprising a working electrode and a counter electrode that were palladium electrodes. A voltage of 0.4 V was applied to the working electrode and the counter electrode, and 30 seconds after, a current value was measured. As a control, a measurement solution containing no oxidant was prepared, and measured in the same manner as described above.
Results are shown in
As is evident from
As is evident from
Thus it was found that Compounds 1 to 7 are superior oxidants for oxidizing a reduced electron mediator.
An analyte was measured by using a sensor loaded with the oxidant disclosed herein, and the measurement accuracy was assessed.
As the oxidant, [bis(trifluoroacetoxy)iodo]pentafluorobenzene (Compound 3), and [hydroxy(tosyloxy)iodo]benzene (Compound 4) were used. As the electron mediator, potassium hexacyanoferrate(III) trihydrate (potassium ferricyanide) (manufactured by Wako Pure Chemical Corporation) was used. As the analyte, Certified Reference Material for Total Hemoglobin Measurement (purchased from Reference Material Institute for Clinical Chemistry Standards; 25-fold dilutions of JCCRM 912-2L, M ad H were used) was used.
A measurement solution having the composition shown in Table 2 was prepared, and applied to a sensor comprising a working electrode and a counter electrode that were palladium electrodes, loaded with any one of reagents (A) to (D) shown in Table 3. A voltage of 0.2 V was applied to the working electrode and the counter electrode, and 5 seconds after, a current value was measured. As a control, using a sensor loaded with a reagent having the same composition as described above except containing no oxidant, the above-described measurement solution was measured in the same manner as described above. Further, a measurement solution having the same composition as described above except containing no hemoglobin (in other words, containing 5.0% (w/v) of an anion surfactant, 300 mM of potassium glutamate, 30 mM of potassium phosphate buffer (pH 8)) was prepared and applied to the above-described sensor, and then, a blank current value was measured. In tables, “%” all means “% (w/v)”. As the anion surfactant, sodium N-dodecanoylsarcosinate was used. As excipient 1, AQUPAANA (partially neutralized polyacrylic acid; manufactured by SUMITOMO SEIKA CHEMICALS CO., LTD.) was used. As excipient 2, STABILIZE QM [(methyl vinyl ether/maleic acid) crosspolymer; manufactured by ISP Japan Ltd.] was used.
Results are shown in
In the figures, “Com.3” and “Com.4” mean Compound 3 and Compound 4 respectively, and “Hb” means hemoglobin.
As is evident from
Next, whether the effect of the oxidant disclosed herein depends on its concentration was examined. As seen from
As the oxidant, [hydroxy(tosyloxy)iodo]benzene (Compound 4) was used. As the electron mediator, sodium 1,2-naphthoquinone-4-sulfonate (manufactured by Wako Pure Chemical Corporation) was used. As the analyte, glucose was used.
A measurement solution was prepared by using phosphate buffered saline (PBS) to dissolve glucose at each concentration of 7.2 mg/dL (400 μM), 10.8 mg/dL (600 μM) or 14.4 mg/dL (800 μM). The measurement solution was applied to a sensor comprising a working electrode and a counter electrode that were palladium electrodes, loaded reagent (E) shown in Table 7. A voltage of 0.25 V was applied to the working electrode and the counter electrode, and 10 seconds after, a current value was measured. As a control, using a sensor loaded with a reagent having the same composition as described above except containing no oxidant, the above-described measurement solution was measured in the same manner as described above. Further, PBS was applied as a measurement solution to the above-described sensor to measure a blank current value. In the table, “%” all means “% (w/v)”.
Results are shown in
In the figure, “Com.4” means “Compound 4”.
As is evident from
As the oxidant, 1-fluoro-3,3-dimethyl-1,2-benziodoxol (Compound 6) was used. As the electron mediator, 1-hydroxyphenazine (manufactured by TOKYO KASEI KOGYO CO., LTD.) was used in the form of suspension to prepare a reagent. As the analyte, glucose was used.
A measurement solution was prepared by using phosphate buffered saline (PBS) to dissolve glucose at each concentration [0 μM, 25 μM (0.45 mg/dL) or 60 μM (1.08 mg/dL)]. The measurement solution was applied to a sensor comprising a working electrode and a counter electrode that were palladium electrodes, loaded with reagent (F) shown in Table 11. A voltage of 0.25 V was applied across the working electrode and the counter electrode, and 10 seconds after, a current value was measured. As a control, using a sensor loaded with a reagent having the same composition as described above except containing no oxidant, the above-described measurement solution was measured in the same manner as described above. Further, PBS was applied as a measurement solution to the above-described sensor to measure a blank current. In the table, “%” all means “% (w/v)”.
Results are shown in
In the figure, “Com.6” means “Compound 6”.
As is evident from
As the oxidant, 1-fluoro-3,3-dimethyl-1,2-benziodoxol (Compound 6) was used. As the electron mediator, sodium anthraquinone-β-sulfonate (manufactured by Wako Pure Chemical Corporation) was used. As a measurement solution, phosphate buffered saline (PBS) was used.
The measurement solution was applied to a sensor comprising a working electrode and a counter electrode that were palladium electrodes, loaded with reagent (G) shown in Table 15. A voltage of 0.25 V was applied across the working electrode and the counter electrode, and 10 seconds after, a blank current value was measured. As a control, using a sensor loaded with a reagent having the same composition as described above except containing no oxidant, the above-described measurement solution was measured in the same manner as described above. Results are shown in Table 16. In the table, “%” all means “% (w/v)”.
As is evident from Table 16, in the sensor loaded with Compound 6, the blank current value was decreased as compared with the sensor that was not loaded with the oxidant disclosed herein. This result shows that addition of the oxidant disclosed herein decreased the reduced electron mediator that was unintentionally generated when the sensor was produced.
As the oxidant, [hydroxy(tosyloxy)iodo]benzene (Compound 4) was used. As the electron mediator, sodium 9,10-phenanthrenequinone-2-sulfonate was used. The sodium 9,10-phenanthrenequinone-2-sulfonate was obtained by sulfonylation of commercially available 9,10-phenanthrenequinone with fuming sulfuric acid, sorting of isomers, and then conversion into the sodium salt. As a measurement solution, phosphate buffered saline (PBS) was used.
The measurement solution was applied to a sensor comprising a working electrode and a counter electrode that were palladium electrodes, loaded with reagent (H) shown in Table 17. A voltage of 0.25 V was applied across the working electrode and the counter electrode, and 10 seconds after, a blank current value was measured. As a control, using a sensor loaded with a reagent having the same composition as described above except containing no oxidant, the above-described measurement solution was measured in the same manner as described above. Results are shown in Table 18. In the table, “%” all means “% (w/v)”.
As is evident from Table 18, in the sensor loaded with Compound 4, the blank current value was decreased as compared with the sensor that was not loaded with the oxidant disclosed herein. This result shows that addition of the oxidant disclosed herein decreased the reduced electron mediator that was unintentionally generated when the sensor was produced.
The oxidant for an electron mediator as disclosed herein can be used in electrochemical measurement of various analytes, and increase the measurement accuracy.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2016-208024 | Oct 2016 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2017/038134 | 10/23/2017 | WO | 00 |
