JP7032906B2 - Composition for glucose redox reaction and glucose measurement using flavin compound - Google Patents

Description

The present invention relates to a glucose redox reaction using a flavin compound and a composition containing the flavin compound. More specifically, the present invention relates to a method for measuring glucose using a flavin compound as a mediator of glucose oxidase or glucose dehydrogenase. The present invention also relates to a composition for measuring glucose containing a flavin compound which can be advantageously used as a diagnostic reagent for diabetes, as a glucose sensor, and in a glucose measuring kit.

Glucose measurement is used for blood glucose monitoring of diabetic patients and the like. Glucose oxidase (hereinafter, also referred to as GOD) or glucose dehydrogenase (hereinafter, also referred to as GDH) is usually used for the quantification of glucose. These are used in self-measuring (SMBG) devices for blood glucose concentration and continuous blood glucose measuring (also referred to as CGM or FGM) devices that can be used at home.

Glucose oxidase is an oxidoreductase that catalyzes the reaction of oxidizing β-D-glucose to D-glucono-1,5-lactone (gluconolactone). Glucose oxidase uses oxygen as an electron acceptor and flavin adenine dinucleotide (FAD) as a cofactor.

Glucose dehydrogenase is classified as an oxidoreductase and uses glucose and electron acceptors as substrates to catalyze reactions that produce gluconolactones and reduced acceptors. Examples of the glucose dehydrogenase include nicotine amide dinucleotide-dependent GDH, nicotine amide dinucleotide phosphate-dependent GDH, pyrroloquinoline quinone (PQQ) -dependent GDH, and FAD-dependent GDH (flavin-bound GDH).

When using GOD for glucose measurement, a hydrogen peroxide electrode can be used. In this method, a voltage of +0.6 V (vs. Ag / AgCl) to + 0.9 V (vs. Ag / AgCl) is applied to the hydrogen peroxide electrode to generate peroxidation when gluconolactone is generated from glucose. Measure hydrogen. This method is mainly used for SMBG and CGM. However, since this method applies a relatively high voltage, there is a problem that it is affected by contaminants such as ascorbic acid contained in the measurement solution.

Therefore, a method has been developed in which an artificial electron mediator (hereinafter, also simply referred to as a mediator) is used instead of hydrogen peroxide. In this method, the oxidized mediator becomes reduced when glucose is enzymatically converted to gluconolactone. Then, the reduced mediator transfers an electron to the electrode and returns to the oxidized form. As an advantage of the method using a mediator, the applied voltage can be made lower than that in the case of the hydrogen peroxide electrode. However, conventional mediators have the problem of being expensive and toxic. For example, metal complexes such as potassium ferricyanide, which is a typical mediator, are harmful when they enter the human body.

Other conventional mediators known include quinones, phenazines, viologens, cytochromes, phenoxazines, phenothiazines, ferricyanides such as potassium ferricyanide, ferredoxins, ferrocene or ferrocene derivatives, osmium complexes and the like. rice field.

In GOD and GDH, the cofactor FAD is often embedded inside the enzyme molecule, so when these are used for glucose measurement, electrons cannot usually be transferred to the electrode without a mediator. However, the mediator is expensive and difficult to immobilize on the electrode surface. For example, in glucose measurement of diabetic patients, if a mediator is used for a CGM device or an FGM device without being immobilized on an electrode, the mediator present in the measurement solution may flow into the body, but many mediators are used. If it is not biocompatible or toxic, it is not desirable for it to flow into the body. Therefore, the conventional glucose measurement method using a mediator is not suitable for CGM and FGM devices, and it is necessary to take a separate measure to prevent the mediator from flowing into the body.

Patent Document 1 describes a mediator compound in which a change in current value is suppressed before and after the storage period of a reagent.
Patent Document 2 describes a biosensor using an oxidoreductase. As the mediator compound, a ferrocyanide compound or the like is used.
Patent Document 3 describes a redox reaction using an electron transfer agent.

Although interest in fuel cells is increasing, conventional fuel cells often use platinum, and artificial compounds such as osmium complexes are often used as electron transfer mediators. Platinum is expensive, and artificial compounds are toxic and have disposal costs. Under such circumstances, a fuel cell using an enzyme electrode instead of platinum has been reported (for example, Patent Document 4). The mediator used in Patent Document 4 is a metal complex.

FAD-dependent GDH (flavin-bound GDH) is known to incorporate FAD into the FAD binding site when a polypeptide is synthesized from the mRNA encoding it and folded. Therefore, the active FAD-dependent GDH is non-covalently bound to FAD (holoenzyme). Further, it is known that FAD-dependent GDH that is not bound to FAD does not exist stably (apoenzyme). On the other hand, NAD-dependent GDH can also exist as an apoenzyme, and it is necessary to add NAD from the outside in order to exhibit its activity. FAD-dependent GOD is generally known to incorporate FAD into the FAD binding site during protein expression. However, there may be some that exist as an apo type.

International Publication No. 2012/042903 Special Table 2007-509355 (Patent No. 4839219) Japanese Patent Application Laid-Open No. 1-317425 (Patent No. 3071790) Japanese Patent Application Laid-Open No. 2008-71584

In certain embodiments, it is an object of the present invention to provide a method for electrochemically measuring glucose and a composition for measuring glucose, which can solve the above-mentioned problems. In another embodiment, it is an object of the present invention to provide an improved glucose fuel cell and a power generation method.

As a result of diligent efforts to solve the above problems, the present inventor has found that a flavin compound can surprisingly function as a mediator in an enzyme-catalyzed redox reaction of glucose. Completed.

Although not bound by any particular theory, each compound has its own redox potential, and in general the redox potential of a free flavin compound is the flavin compound in the flavoprotein. It is on the minus side of the redox potential of (the redox potential is low). For example, the redox potential of free FAD (-0.219 V (vs. NHE)) is lower than the redox potential of FAD in flavoproteins (~ 0 V (vs. NHE)) (Vote biochemistry). (Top) See Chapter 15, 2nd Edition). In such a case, it is theoretically considered that electrons do not move from the compound having a high redox potential to the compound having a low redox potential. However, in the present invention, when an electrochemical measurement was performed on a system in which a flavoprotein and a free flavin compound were mixed, an electric current was observed. This is a surprising finding in light of the above.

［式中、Ｒ^１は存在しないか又は－ＣＨ_３、若しくは

であり、Ｒ^１が存在しない場合には、Ｒ^１が連結して描かれている窒素原子は隣接炭素原子との間に二重結合を形成しておりかつＲ^２はＨであり、
Ｒ^２は存在しないか、又はＨであり、Ｒ^２が存在しない場合には、Ｒ^２が連結して描かれている窒素原子は隣接炭素原子との間に二重結合を形成しており、
Ｒ^３は、Ｈ、リン酸基、又は

である］
で表される化合物である、１に記載の組成物、２、５若しくは６に記載の電極、３、５若しくは６に記載の電池又は４、５若しくは６に記載のセンサー。
[８] フラビン化合物が、フラビンモノヌクレオチド、リボフラビン、ルミクロム、フラビンアデニンジヌクレオチド又はルミフラビンである、１若しくは７に記載の組成物、２、５、６若しくは７に記載の電極、３、５、６若しくは７に記載の電池又は４、５、６若しくは７に記載のセンサー。
[９] 前記(i)において、人工電子メディエーターーが、キノン類、フェナジン類、ビオロゲン類、シトクロム類、チオレドキシン類、フェノキサジン類、フェノチアジン類、フェリシアン化物、フェレドキシン類、フェロセン、フェロセン誘導体、及び金属錯体である、
前記(ii)において、非水溶性の電子メディエーターーがフェロセン、非水溶性のキノン類、非水溶性のフェナジン類、非水溶性のビオロゲン類、非水溶性のチオレドキシン類、非水溶性のフェノキサジン類、非水溶性のフェノチアジン類、非水溶性のフェレドキシン類、及び非水溶性のフェロセン誘導体である、又は
前記(iii)において、さらに、酸化還元電位が０．２Ｖ以上の電子メディエーターを含まない、
１、７若しくは８に記載の組成物、２、５、６、７若しくは８に記載の電極、３、５、６、７若しくは８に記載の電池又は４、５、６、７若しくは８に記載のセンサー。
[１０] グルコースを含みうる試料と、遊離型のフラビン化合物と、精製されたＦＡＤ依存性グルコースオキシダーゼ又は精製されたＦＡＤ依存性グルコースデヒドロゲナーゼとを接触させる工程、及び電流を測定する工程を含む、グルコースの電気化学的測定方法、
ただし該方法は、
(i) 単独で電子をＦＡＤ依存性グルコースオキシダーゼ又はＦＡＤ依存性グルコースデヒドロゲナーゼから受け取ることのできる人工電子メディエーターを外部から添加しない、
(ii) 単独で電子をＦＡＤ依存性グルコースオキシダーゼ又はＦＡＤ依存性グルコースデヒドロゲナーゼから受け取ることのできる非水溶性の電子メディエーターを外部から添加しない、又は
(iii) 単独で電子をＦＡＤ依存性グルコースオキシダーゼ又はＦＡＤ依存性グルコースデヒドロゲナーゼから受け取ることのできる、酸化還元電位が０．３Ｖ以上の電子メディエーターを外部から添加しない、
前記方法。
[１１] 遊離型のフラビン化合物を電子メディエーターとして用いる発電方法であって、アノード及びカソードを使用し、
電解質に接触しているアノードにフラビン化合物を供給し、グルコース燃料を供給する工程を有し、
該アノードが、精製されたＦＡＤ依存性グルコースデヒドロゲナーゼ又は精製されたＦＡＤ依存性グルコースオキシダーゼを有する電極である、該発電方法、
ただし該方法は、
(i) 単独で電子をＦＡＤ依存性グルコースオキシダーゼ又はＦＡＤ依存性グルコースデヒドロゲナーゼから受け取ることのできる人工電子メディエーターを外部から添加しない、
(ii) 単独で電子をＦＡＤ依存性グルコースオキシダーゼ又はＦＡＤ依存性グルコースデヒドロゲナーゼから受け取ることのできる非水溶性の電子メディエーターを外部から添加しない、又は
(iii) 単独で電子をＦＡＤ依存性グルコースオキシダーゼ又はＦＡＤ依存性グルコースデヒドロゲナーゼから受け取ることのできる、酸化還元電位が０．３Ｖ以上の電子メディエーターを外部から添加しない、
前記方法。
[１２] 該フラビン化合物が固相中に固定化されている非酵素型のフラビン化合物である、１０又は１１に記載の方法。
[１３] 精製されたＦＡＤ依存性グルコースオキシダーゼ又は精製されたＦＡＤ依存性グルコースデヒドロゲナーゼが固相中に固定化されている、１０～１２のいずれかに記載の方法。
[１４] フラビン化合物が以下の化学式（Ｉ）

［式中、Ｒ^１は存在しないか又は－ＣＨ_３、若しくは

であり、Ｒ^１が存在しない場合には、Ｒ^１が連結して描かれている窒素原子は隣接炭素原子との間に二重結合を形成しておりかつＲ^２はＨであり、
Ｒ^２は存在しないか、又はＨであり、Ｒ^２が存在しない場合には、Ｒ^２が連結して描かれている窒素原子は隣接炭素原子との間に二重結合を形成しており、
Ｒ^３は、Ｈ、リン酸基、又は

である］
で表される化合物である、１０～１３のいずれかに記載の方法。
[１５] フラビン化合物が、フラビンモノヌクレオチド、リボフラビン、ルミクロム、フラビンアデニンジヌクレオチド又はルミフラビンである、１０～１４のいずれかに記載の方法。
[１６] 前記(i)において、人工電子メディエーターが、キノン類、フェナジン類、ビオロゲン類、シトクロム類、チオレドキシン類、フェノキサジン類、フェノチアジン類、フェリシアン化物、フェレドキシン類、フェロセン、フェロセン誘導体、及び金属錯体である、
前記(ii)において、非水溶性の電子メディエーターがフェロセン、非水溶性のキノン類、非水溶性のフェナジン類、非水溶性のビオロゲン類、非水溶性のチオレドキシン類、非水溶性のフェノキサジン類、非水溶性のフェノチアジン類、非水溶性のフェレドキシン類、及び非水溶性のフェロセン誘導体である、又は
前記(iii)において、さらに、酸化還元電位が０．２Ｖ以上の電子メディエーターを外部から添加しない、
１０～１５のいずれかに記載の方法。 The present invention includes the following.
[1] A composition for measuring glucose, which comprises a free flavin compound and contains a purified FAD-dependent glucose oxidase or a purified FAD-dependent glucose dehydrogenase.
However, the composition is
(i) does not contain artificial electron mediators, which are externally added and can receive electrons alone from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase.
(ii) Does not contain or contain water-insoluble electron mediators that are externally added and can receive electrons alone from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase.
(iii) Does not contain an electron mediator having an oxidation-reduction potential of 0.3 V or higher, which is externally added and can receive electrons alone from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase.
The composition for measuring glucose.
[2] An anode electrode comprising a free flavin compound and containing purified FAD-dependent glucose oxidase or purified FAD-dependent glucose dehydrogenase.
However, the electrode is
(i) does not contain artificial electron mediators, which are externally added and can receive electrons alone from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase.
(ii) Does not contain or contain water-insoluble electron mediators that are externally added and can receive electrons alone from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase.
(iii) Does not contain an electron mediator having an oxidation-reduction potential of 0.3 V or higher, which is externally added and can receive electrons alone from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase.
The anode electrode.
[3] A glucose fuel cell having the anode electrode, the cathode electrode, and the fuel tank containing glucose according to 2.
[4] A glucose sensor having a free flavin compound and a purified FAD-dependent glucose oxidase or a purified FAD-dependent glucose dehydrogenase.
However, the sensor is
(i) does not contain artificial electron mediators, which are externally added and can receive electrons alone from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase.
(ii) Does not contain or contain water-insoluble electron mediators that are externally added and can receive electrons alone from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase.
(iii) The sensor, which is externally added and does not contain an electron mediator having a redox potential of 0.3 V or higher, which can receive electrons alone from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase.
[5] The electrode according to 2, the battery according to 3, or the sensor according to 4, wherein the flavin compound is a non-enzymatic flavin compound immobilized in a solid phase.
[6] The electrode according to 2 or 5 or the battery or 4 or 5 according to 3 or 5 in which purified FAD-dependent glucose oxidase or purified FAD-dependent glucose dehydrogenase is immobilized in a solid phase. The sensor described in.
[7] The flavin compound has the following chemical formula (I).

[In the formula, R ¹ does not exist or -CH ₃ or

In the absence of R ¹ , the nitrogen atom depicted with R ¹ linked forms a double bond with the adjacent carbon atom and R ² is H.
If R ² is absent or H and R ² is absent, the nitrogen atom depicted with R ² linked forms a double bond with the adjacent carbon atom.
R3 is H ^, a phosphate group, or

Is]
The composition according to 1, the electrode according to 2, 5 or 6, the battery according to 3, 5 or 6, or the sensor according to 4, 5 or 6, which is a compound represented by 1.
[8] The composition according to 1 or 7, the electrode according to 2, 5, 6 or 7, wherein the flavin compound is a flavin mononucleotide, riboflavin, lumichrome, flavin adenine dinucleotide or lumiflavin. Or the battery according to 7 or the sensor according to 4, 5, 6 or 7.
[9] In (i) above, the artificial electronic mediator is a quinone, a phenazine, a viologen, a cytochrome, a thioredoxin, a phenoxazine, a phenothiazine, a ferricianide, a ferredoxin, a ferrocene, a ferrocene derivative, and It is a metal complex,
In (ii) above, the water-insoluble electronic mediators are ferrosene, water-insoluble quinones, water-insoluble phenazines, water-insoluble viologens, water-insoluble thioredoxins, and water-insoluble phenoxazine. , Water-insoluble phenothiazines, water-insoluble ferredoxins, and water-insoluble ferrocene derivatives, or in (iii) above, further free of electron mediators with a redox potential of 0.2 V or higher.
The composition according to 1, 7 or 8, the electrode according to 2, 5, 6, 7 or 8, the battery according to 3, 5, 6, 7 or 8 or the battery according to 4, 5, 6, 7 or 8. Sensor.
[10] Glucose comprising a step of contacting a sample which may contain glucose with a free flavin compound and a purified FAD-dependent glucose oxidase or a purified FAD-dependent glucose dehydrogenase, and a step of measuring a current. Electrochemical measurement method,
However, this method is
(i) No external addition of artificial electron mediators capable of receiving electrons from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase alone.
(ii) No external addition of water-insoluble electron mediators capable of receiving electrons from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase alone, or
(iii) No external addition of an electron mediator having a redox potential of 0.3 V or higher, which can receive electrons from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase alone.
The method.
[11] A power generation method using a free flavin compound as an electron mediator, using an anode and a cathode.
It has a step of supplying a flavin compound to an anode in contact with an electrolyte and supplying glucose fuel.
The power generation method, wherein the anode is an electrode having purified FAD-dependent glucose dehydrogenase or purified FAD-dependent glucose oxidase.
However, this method is
(i) No external addition of artificial electron mediators capable of receiving electrons from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase alone.
(ii) No external addition of water-insoluble electron mediators capable of receiving electrons from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase alone, or
(iii) No external addition of an electron mediator having a redox potential of 0.3 V or higher, which can receive electrons from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase alone.
The method.
[12] The method according to 10 or 11, wherein the flavin compound is a non-enzymatic flavin compound immobilized in a solid phase.
[13] The method according to any of 10 to 12, wherein purified FAD-dependent glucose oxidase or purified FAD-dependent glucose dehydrogenase is immobilized in a solid phase.
[14] The flavin compound has the following chemical formula (I).

[In the formula, R ¹ does not exist or -CH ₃ or

In the absence of R ¹ , the nitrogen atom depicted with R ¹ linked forms a double bond with the adjacent carbon atom and R ² is H.
If R ² is absent or H and R ² is absent, the nitrogen atom depicted with R ² linked forms a double bond with the adjacent carbon atom.
R3 is H ^, a phosphate group, or

Is]
The method according to any one of 10 to 13, which is a compound represented by.
[15] The method according to any of 10 to 14, wherein the flavin compound is flavin mononucleotide, riboflavin, lumichrome, flavin adenine dinucleotide or lumiflavin.
[16] In (i) above, the artificial electron mediator is a quinone, a phenazine, a viologen, a cytochrome, a thioredoxin, a phenoxazine, a phenothiazine, a ferricianide, a ferredoxin, a ferrocene, a ferrocene derivative, and a metal. It is a complex,
In (ii) above, the water-insoluble electron mediator is ferrosene, water-insoluble quinones, water-insoluble phenazines, water-insoluble viologens, water-insoluble thioredoxins, water-insoluble phenoxazines. , Water-insoluble phenothiazines, water-insoluble ferredoxins, and water-insoluble ferrosene derivatives, or in (iii) above, further, do not add an electron mediator having a redox potential of 0.2 V or more from the outside. ,
The method according to any one of 10 to 15.

This specification includes the disclosure content of Japanese Patent Application No. 2016-205889, which is the basis of the priority of the present application.

Unlike conventional mediators such as potassium ferricyanide, the flavin compound of the present invention is generally present in the living body, has high biocompatibility, and is considered to have no toxicity to the human body or low toxicity. Therefore, the composition containing the flavin compound of the present invention and the measuring method using the flavin compound can be used for the continuous glucose measurement of the implantable type in the body. Further, the flavin compound of the present invention can be applied to a glucose fuel cell.

The response current when AtGLD and various flavin compounds are used is shown. The response current when GLD1 and various flavin compounds are used is shown. The response current when GOD and various flavin compounds are used is shown. The response current when MpGDH and various flavin compounds are used is shown. The response currents when GLD1 and FMN are used for samples containing various concentrations of glucose are shown. The response currents when GDH-M2 and FMN are used for samples containing various concentrations of glucose are shown. The response currents when GLD1 and FAD are used for samples containing various concentrations of glucose are shown. The response currents when GDH-M2 and RF are used for samples containing various concentrations of glucose are shown. The response currents when GLD1 and LC are used for samples containing various concentrations of glucose are shown.

(Flavin compound and composition containing flavin compound)
In certain embodiments, the present invention provides flavin compounds. In another embodiment, the present invention provides a composition for measuring glucose containing a flavin compound. The flavin compound of the present invention functions as a mediator in a glucose redox reaction catalyzed by glucose oxidase or glucose dehydrogenase. To function as a mediator means that the flavin compound contributes to electron transfer. For example, in a system using an electrode, the flavin compound of the present invention receives an electron from GOD or GDH, becomes a reduced form, and transfers an electron to the electrode. Return to oxidized form. From this point of view, the flavin compound of the present invention can also be referred to as an electron transfer mediator. These terms are synonymous herein.

FAD-dependent GOD and GDH form a holoenzyme together with the flavin-based compound FAD, which is a coenzyme. Unless otherwise specified, the term GOD as used herein refers to FAD-dependent GOD. Unless otherwise specified, the term GDH as used herein refers to FAD-dependent GDH. In holoenzyme, FAD forms a complex with the enzyme by non-covalent bond and is usually buried in the enzyme. That is, the FAD contained in the holoenzyme is not released in the solution. GOD and GDH without coenzymes are called apoenzymes and do not show activity against glucose. As used herein, the flavin compound of the present invention does not include the coenzyme contained in the holoenzyme. That is, the flavin compound of the present invention exists in a state of being free from the enzyme. Unless otherwise specified, the flavin compound of the present invention is referred to as a free flavin compound or a free flavin compound. On the flip side, the coenzyme FAD contained in the holoenzymes of GOD and GDH does not fall under the "flavin compound of the present invention" as used herein.

In a certain embodiment, the flavin compound of the present invention can be used by being immobilized in a solid phase such as an electrode surface. Even in such a case, the immobilized flavin compound is not contained in the holoenzyme and exists independently of the GOD and GDH enzymes. Therefore, such an immobilized flavin compound is also included in the present specification by the term "free flavin compound" for convenience. Further, such flavin compounds existing independently of GOD and GDH enzymes may be referred to as non-enzyme type flavin compounds and non-enzyme-bound flavin compounds. Non-enzyme-type flavin compounds and non-enzyme-bound flavin compounds may also be immobilized in the solid phase.

［式中、Ｒ^１は存在しないか又は－ＣＨ_３、若しくは

であり、Ｒ^１が存在しない場合には、Ｒ^１が連結して描かれている窒素原子は隣接炭素原子との間に二重結合を形成しておりかつＲ^２はＨであり、
Ｒ^２は存在しないか、又はＨであり、Ｒ^２が存在しない場合には、Ｒ^２が連結して描かれている窒素原子は隣接炭素原子との間に二重結合を形成しており、
Ｒ^３は、Ｈ、リン酸基、又は

である］。 In certain embodiments, the flavin compounds of the invention can be compounds of the following general formula I:

[In the formula, R ¹ does not exist or -CH ₃ or

In the absence of R ¹ , the nitrogen atom depicted with R ¹ linked forms a double bond with the adjacent carbon atom and R ² is H.
If R ² is absent or H and R ² is absent, the nitrogen atom depicted with R ² linked forms a double bond with the adjacent carbon atom.
R3 is H ^, a phosphate group, or

Is].

In certain embodiments, the flavin compound of the invention is riboflavin (RF) (CAS 83-88-5).

In certain embodiments, the flavin compound of the invention is a flavin mononucleotide (FMN) (CAS 146-17-8).

In certain embodiments, the flavin compound of the invention is lumichrome (LC) (CAS 1086-80-2).

In certain embodiments, the flavin compound of the invention is flavin adenine dinucleotide (FAD) (CAS 146-14-5).

In certain embodiments, the flavin compound of the invention is lumiflavin (LF) (CAS 1088-56-8).

It is generally known that flavin compounds have three types of redox states and three types of ionization states. In the above chemical formula, the flavin compound of the present invention is described in a neutral and oxidized form. However, not only in this form, the flavin compound of the present invention can be an oxidized type (quinone type), a semi-quinone type, or a reduced type (hydroquinone type). Further, the flavin compound of the present invention may be a neutral type or an anion type. For convenience, the flavin compounds of the invention, eg, the flavin compounds of the invention represented by the above chemical formulas, include neutral or anionic, oxidized, semiquinone or reduced forms. It shall be. For example, after adding a neutral and oxidized compound as the flavin compound of the present invention to the measurement system, it may be changed to an oxidized and anionic compound by the pH of the solution or proton transfer. Such compounds are also included in the flavin compounds of the present invention.

The flavin compound of the present invention may be artificially synthesized or a natural product may be obtained. It may also be commercially available.

In certain embodiments, the flavin compounds of the invention can be used with glucose dehydrogenase (FAD-dependent GDH). In another embodiment, the flavin compounds of the invention can be used with glucose oxidase (FAD-dependent GOD). In another embodiment, the composition comprising the flavin compound of the present invention further comprises glucose dehydrogenase or glucose oxidase. GDH or GOD may be free in the solution or may be immobilized on a solid phase. GDH or GOD may be present alone or linked to another compound or protein, such as an enzyme or antibody.

In certain embodiments, the composition comprising the flavin compound of the present invention is:
(i) does not contain an externally added artificial electron mediator that can alone receive electrons from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase.
(ii) Excludes externally added, water-insoluble electron mediators capable of receiving electrons from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase alone, or
(iii) It does not contain an electron mediator having an oxidation-reduction potential of 0.3 V or more, which is externally added and can receive electrons alone from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase. The same applies to the electrodes of the present invention and the sensors of the present invention in certain embodiments. Unless otherwise specified in the present specification, the redox potential is based on the silver-silver chloride electrode.

Further, in a certain embodiment, the method for electrochemically measuring glucose and the method for generating electricity using the flavin compound of the present invention are used.
(i) No external addition of artificial electron mediators capable of receiving electrons from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase alone.
(ii) No external addition of water-insoluble electron mediators capable of receiving electrons from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase alone, or
(iii) No external electron mediator having a redox potential of 0.3 V or higher, which can receive electrons from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase alone, is not added. The term “external addition” as used herein includes addition immediately before and during measurement, and addition in preparation of measurement reagents.

In certain embodiments, the compositions, electrodes, and sensors containing the flavin compounds of the invention are externally added, redox, capable of receiving electrons alone from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase. Does not include electron mediators with potentials of 0.3 V or higher, 0.2 V or higher, 0.1 V or higher, or 0 V or higher, 0.8 V or lower, 0.7 V or lower, and 0.6 V or lower. In another embodiment, the composition, electrode, and sensor containing the flavin compound of the present invention are externally added and can receive electrons alone from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase, oxidation. The reduction potential is 0 to 0.8V, 0 to 0.7V, 0 to 0.6V, 0.1 to 0.7V, 0.2 to 0.6V, 0.3 to 0.8V, 0.3 to 0. Does not contain 0.7V, 0.3-0.6V electronic mediators. Similarly, the methods using the flavin compounds of the present invention in certain embodiments are excluded.

In certain embodiments, the compositions, electrodes, and sensors containing the flavin compounds of the invention are water-insoluble, externally added, capable of receiving electrons alone from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase. Does not include sex electronic mediators. Water-insoluble electronic mediators include ferrocene, water-insoluble quinones such as phenanthroline quinones, water-insoluble phenazines such as phenazine, water-insoluble viologens, water-insoluble thioredoxins, and water-insoluble. Examples thereof include, but are not limited to, phenoxazines, water-insoluble phenothiazines, water-insoluble ferredoxins, and water-insoluble ferrocene derivatives. Similarly, the methods using the flavin compounds of the present invention in certain embodiments are excluded.

In certain embodiments, the compositions, electrodes, and sensors containing the flavin compounds of the invention alone do not include conventional artificial electron mediators capable of receiving electrons from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase alone. Similarly, in certain embodiments, the methods using the flavin compounds of the present invention are excluded. Conventional artificial electron mediators capable of receiving electrons alone from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase include potassium ferricyanide, quinones, anthraquinones, and phenazines that are artificially added to the system from the outside. , Viologens, cytochromes, thioredoxins, phenoxazines, phenothiazines, ferricyanides such as potassium ferricyanide, ferredoxins, ferrocene or ferrocene derivatives, osmium complexes, cobalt complexes, ruthenium complexes, iron complexes, vanadium complexes and the like. Examples thereof include complexes that can independently receive electrons from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase. These compounds and derivatives include a sulfonyl group, a sulfo group, a carbonyl group, a carboxyl group, an amino group, an imino group, a nitro group, a nitroso group, a hydroxy group, an alkyl group (for example, C1-C16 alkyl, for example, a methyl group), and an alkoxyl group. , It may have one or more functional groups such as an acyl group, a cyano group and a halogen group. Specific examples of the electron mediator compound include 1,1'-dimethylferrocene, ferrocenecarboxylic acid, ferrocenecarboxyaldehyde, benzoquinone, hydroquinone, ferricyan / ferrocyanide (K or Na salt, for example, potassium ferricyanide), and ferricinium / ferrocene. , Phenazine methsulfate, 1-methoxy-5-methylphenadium methylsulfate, 2,6-dichlorophenol indophenol, chloranil, Bromanyl, phenanthrolinquinone, anthraquinone, octacyanotantistate ion, octacyano Molybdenate ion, [Ni (PQ ₃ )] ²⁺ ion, eg [Ni (PQ ₃ )] Cl ₂ , [Ni (PQ) ₃ ] Br ₂ , [Os (dmo) ₂ (1-vinylimidazole) X] X, [Os (dmo) ₂ (1-vinylimidazole) X] X ₂ , [Os (dmo) ₂ (imidazole) X] X, [Os (dmo) ₂ (imidazole) X] X ₂ , [Os (dmo) ) ₂ (1-methylimidazole) X] X ₂ and [Os (dmo) ₂ (methylimidazole) X] X ₂ (dmo in these compounds is 4,4'-dimethoxy-2,2'-bipyridine. , X is a halogen), and examples thereof include those capable of receiving electrons from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase alone.

In certain embodiments, artificial electron mediators capable of receiving electrons from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase alone are potassium ferricyanide, ferrocene, [Ni (PQ ₃ )] Cl ₂ , and [Ni (PQ 3)]. ) ₃ ] Br ₂ . Thus, in certain embodiments, the composition, electrode, and sensor containing the flavin compound of the invention comprises potassium ferricyanide, ferrocene, [Ni (PQ ₃ )] Cl ₂ , and [Ni (PQ) ₃ ] Br ₂ . do not have. Also, in certain embodiments, they are not added by the method of the invention.

It is known that when the ruthenium complex is used as a single electron mediator, the FAD-dependent GDH does not receive electrons from the ruthenium complex even when a predetermined potential is applied. On the other hand, it is known that when 1-mPMS is added to a system containing a ruthenium complex and a FAD-dependent GDH, the ruthenium complex can receive electrons when a predetermined potential is applied (Japanese Patent Laid-Open No. See 2013-083634). Although not bound by any particular theory, the interaction between the ruthenium complex and the FAD-dependent GDH is sufficient to allow the ruthenium complex to receive electrons in the FAD contained in the GDH. It is considered that they cannot be in close proximity. Therefore, when the ruthenium complex is used together with FAD-dependent GDH as a single electron mediator, it is considered that no response current is observed even if a predetermined potential is applied. On the other hand, if there is another artificial electron mediator 1-mPMS in this system that can alone receive electrons directly from FAD-dependent GDH, then 1-mPMS first receives electrons from FAD-dependent GDH and then It is thought that electrons are transferred to the ruthenium complex. At this time, in a system containing a ruthenium complex as a single electron mediator and a FAD-dependent GDH, the ruthenium complex refers to "alone electrons from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase" as used herein. It does not fall under the category of "artificial electronic mediators that can be received". However, the additional presence of 1-mPMS in the system allows the ruthenium complex to receive electrons. Therefore, in a system containing 1-mPMS and a ruthenium complex as an electron mediator and containing a FAD-dependent GDH, the combination of 1-mPMS and a ruthenium complex can transfer electrons from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase. You can receive it. At this time, when the flavin compound of the present invention is used instead of 1-mPMS, the free flavin compound present in the solution receives an electron from the FAD-dependent GDH, and then the flavin compound receives the electron in the ruthenium complex. It is thought that it will be possible to pass it. In certain embodiments, the invention includes a combination of such an electron mediator and a flavin compound of the invention. That is, in certain embodiments, the composition for measuring glucose of the present invention contains a free flavin compound, contains purified FAD-dependent glucose oxidase or purified FAD-dependent glucose dehydrogenase, and alone FAD electrons. It comprises a ruthenium complex that cannot be received from dependent glucose oxidase or FAD dependent glucose dehydrogenase, but is capable of receiving electrons from the flavin compounds of the invention in the coexistence with the flavin compounds of the invention, such as hexaammine ruthenium. Conversely, such a combination of an electron mediator and a flavin compound of the present invention is not excluded from the present invention. Thus, the compositions of the invention exclude artificial electron mediators capable of receiving electrons alone from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase, but from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase. Conventional artificial electron mediators capable of further receiving electrons from the free flavin compound of the present invention that has received the electrons are not excluded (included in the present invention). In other words, the compositions of the invention exclude artificial electron mediators that can alone receive electrons directly from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase. The same applies to the electrodes, sensors, and methods of the present invention.

In the above, "added to the composition or the measuring system from the outside" means addition at a concentration that can affect the response current in the glucose concentration measurement (addition in an amount effective for glucose measurement). For example, in glucose concentration measurement, even if a very small amount of artificial electron mediator that does not affect the response current is externally added to the composition of the present invention, it has no effect on the glucose concentration measurement using the composition of the present invention. And therefore is included in the present invention. Further, the fact that the composition of the present invention does not contain an externally added artificial electron mediator means that the composition does not contain an externally added artificial electron mediator, that is, it does not affect the response current. It may contain a small amount of artificial electron mediator, but does not contain a substantial amount of artificial electron mediator that affects the response current. Thus, in certain embodiments, compositions comprising an artificial electron mediator capable of receiving electrons from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase alone in a substantial amount affecting the response current are excluded from the invention. Be taken. In addition, in the measurement of glucose concentration related to the human body, a very small amount of electrons that are acceptable without adversely affecting the human body and do not affect the response current alone are FAD-dependent glucose oxidase or FAD-dependent. Even if an artificial electron mediator that can be received from sex glucose dehydrogenase is added to the composition of the present invention from the outside, it does not affect the measurement of glucose concentration using the composition of the present invention, and therefore, the present invention has no effect. Be included. The same applies to the electrodes, sensors, and methods of the present invention.

The present inventor previously reported on a fusion protein in which FAD-dependent GDH was linked to cytochrome (International Publication No. 2017/094776). In this document, fusion proteins are referred to as Cytb-GDH and GDH-Cytb. In certain embodiments, such fusion proteins can be used as FAD-dependent GDH. At this time, the cytochrome moiety included in the fusion protein (Cytb-GDH or the like) is a part of the fusion protein and does not correspond to the cytochrome added from the outside as referred to in the present specification. Thus, in certain embodiments, a glucose measurement composition comprising a free flavin compound and a purified glucose dehydrogenase-cytochrome fusion protein is provided, provided that the composition does not contain externally added cytochromes. ..

(Method of immobilizing GDH or GOD)
GDH and GOD can be immobilized on the solid phase by any known method. For example, GDH or GOD may be immobilized on a bead, a film, or an electrode surface. Immobilization methods include 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, etc. It may be adsorbed and fixed on the top, or these may be used in combination. Typically, GOD or GDH is immobilized on a carbon electrode with glutaraldehyde and then treated with a reagent having an amine group to block glutaraldehyde. The amount of GDH or GOD to be immobilized can be an amount capable of generating a current required for electrochemical measurement or fuel cell power generation, and can be appropriately determined.

(Method for immobilizing flavin compound of the present invention)
In certain embodiments, the flavin compound of the present invention may be present alone in a solution, or may be immobilized on a solid phase such as a bead, a film, or an electrode surface. Immobilization methods include 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, etc. It may be adsorbed and fixed on the top, or these may be used in combination. The amount of the flavin compound to be immobilized can be an amount that can generate a current required for electrochemical measurement or fuel cell power generation, and can be appropriately determined.

In certain embodiments, the flavin compounds of the invention may be immobilized and further GDH or GOD may be immobilized. In this case, they may be immobilized by the same or different methods.

The final concentration of the flavin compound of the present invention added to the sample solution is not particularly limited, and is, for example, 0.01 to 200 mM, 0.02 to 150 mM, 0.03 to 100 mM, 0.04 to 80 mM, 0.05 to 60 mM. , 0.06 to 50 mM, 0.08 to 40 mM, 0.1 mM to 30 mM, 0.15 to 20 mM, 0.2 to 10 mM, 0.3 to 5 mM, 0.4 to 3 mM, for example 0.05 to 10 mM. It can be a range. The final concentration of the flavin compound of the present invention added to the sample solution is not particularly limited, and is, for example, 0.001 to 5% (w / v), 0.003 to 3% (w / v), 0.005 to 1%. (W / v), 0.01 to 0.5% (w / v), 0.02 to 0.3% (w / v), 0.03 to 0.1% (w / v). .. The order of addition of the flavin compound and other enzymes or reagents is not limited, and the flavin compound may be added simultaneously or sequentially.

When the flavin compound is used with GOD, the concentration of each component of the electrochemical measurement reagent can be adjusted according to the concentration range of the reduced mediator contained in the sample or estimated to be produced in the sample. can.

Unless otherwise specified, the glucose dehydrogenase or glucose oxidase contained in the composition of the present invention is a purified enzyme. The cell extract containing GOD or GDH, the cell disruption solution, and the crude enzyme extract contain various contaminants other than GOD and GDH. For example, in the case of microorganisms, it has been reported that the amount of riboflavin in the crude enzyme extract is about 53 to 133 μM (J Indust Micro Biotech 1999, 22, pp. 8-18). If such a crude enzyme extract or the like is subjected to electrochemical measurement as it is, the transfer of electrons to the electrode is hindered by the contaminating substance receiving electrons, so that the riboflavin concentration in the crude enzyme extract is used. Accurate electrochemical measurements are difficult. Therefore, in the electrochemical glucose measurement method of the present invention, GOD or GDH from which contaminants have been removed is used. As used herein, GOD or GDH is purified or is a purified enzyme, which means that the protein does not necessarily have to be a pure product, and is a contaminant from the enzyme preparation to the extent that an electrochemical measurement of glucose is possible. Is removed.

(Glucose dehydrogenase)
Glucose dehydrogenase is widespread in nature and can be obtained by searching for enzymes of microbial and animal and plant origin. In the case of microorganisms, it can be obtained from, for example, filamentous fungi, yeast, bacteria and the like. The glucose dehydrogenase of the present invention is, for example, a microorganism classified into the subphylum Mucor, Mucorales, Mucorales, Mucoraceae, for example, Mucor, Absidia, Actinomucor, Circinella. It can be GDH or a variant thereof derived from a species such as a genus.

Microorganisms of the genus Mucor include Mucor plainii, Mucor cilcinelloides, Mucor hiemalis, Mucor mucor mucor mucor mucor mucor mucor mucor mucor mucor mucor mucor mucor mucor mucor mucor mucor mucor mucor mucor mucor ), Mucor javanicus, Mucor dimorphosporus, and the like. Examples of the microorganism belonging to the genus Absidia include Absidia cylindrospora and Absidia hyalospora. Examples of Actinomucor microorganisms include Actinomucor elegance. Circinella genus Circinella includes circinella simplex, circinella sp., circinella angalensis, circinella angalensis, circinella cinella cinella cinella cinella , Circinella minor, Circinella mucoroides, Circinella rigida, Circinella circinella can be circinella circinella and circinella circinella. In certain embodiments, GDH can be derived from Aspergillus oryzae. In certain embodiments, GDH can be GDH of BBI product code GLD1 (derived from Aspergillus oryzae). In certain embodiments, the GDH can be an Aspergillus terreus-derived GDH (see Patent No. 5020070).

(Glucose oxidase)
Glucose oxidase is widespread in nature and can be obtained by searching for enzymes of microbial and animal-plant origin. In the case of microorganisms, it can be obtained from, for example, filamentous fungi, yeast, bacteria and the like. The glucose oxidase of the present invention can be, for example, a GOD derived from a species such as Aspergillus, Penicillium, Apis, Mucor, Fuzarium, Streptomyces, Talaromyces, or a variant thereof. In certain embodiments, the GOD can be of Toyobo's catalog number GLO-201 (derived from the genus Aspergillus). In addition, in the column of the structure (Structure) of the product catalog of GLO-201, there is a glycoprotein (Glycoprotein with 2 moles of FAD) with 2 moles of FAD, which is a holoenzyme.

(GOD or GDH gene)
To obtain a gene encoding GOD or GDH, a commonly used gene cloning method is usually used. For example, chromosomal DNA or mRNA can be extracted from microbial cells or various cells capable of producing GOD or GDH by a conventional method, for example, by the method described in Current Protocols in Molecular Biology (WILEY Interscience, 1989). Furthermore, cDNA can be synthesized using mRNA as a template. Using the chromosomal DNA or cDNA thus obtained, a library of chromosomal DNA or cDNA can be prepared.

Then, a method of synthesizing an appropriate probe DNA based on the amino acid sequence of GOD or GDH and selecting the GDH gene from a library of chromosomal DNA or cDNA using this, or an appropriate primer DNA based on the amino acid sequence. The DNA containing the target gene fragment is amplified by an appropriate polymerase chain reaction (PCR method) such as the 5'RACE method or the 3'RACE method, and these DNA fragments are ligated to form the full length of the target gene. DNA containing the above can be obtained.

Any known gene can be used as the gene encoding GDH, and examples thereof include the GDH gene derived from the genus Mucor (described in Japanese Patent No. 4648993). A modified version of this gene may be used, and examples thereof include the modified genes described in International Publication No. 2012/169512 and International Publication No. 2015/099112. Further, a variant in which the T387A / I545T mutation is introduced into GDH (also referred to as MpGDH) derived from Mucor plainii described in Japanese Patent No. 4648993 may be used. Further, as an example of the gene encoding GDH, the GDH gene (AtGLD) derived from Aspergillus terreus can be mentioned (Patent No. 5020070).

Any known gene can be used as the gene encoding GOD, and examples thereof include the GOD gene derived from the genus Aspergillus. The gene may be modified.

These genes may be ligated or inserted into various vectors, or may be integrated into chromosomes or genomes. When using a vector, for cloning into the vector, commercially available kits such as TA Cloning Kit (Invitrogen) and In-Fusion HD Cloning Kit (Clontech); pUC119 (Takarabio), pUC18 (Takarabio), Commercially available plasmid vector DNAs such as pBR322 (TakaraBio), pBluescript SK + (Stratagene), pYES2 / CT (Invitrogen); and commercially available bacterial plunger vector DNAs such as λEMBL3 (Stratagene) can be used. The recombinant DNA is used to transform a host organism such as Escherichia coli, preferably Escherichia coli JM109 strain (Takara Bio Inc.) or Escherichia coli DH5α strain (Takara Bio Inc.). The recombinant DNA contained in the obtained transformant is purified using QIAGEN Plasmamid Mini Kit (Qiagen) or the like.

(Manufacturing of GOD or GDH)
GOD or GDH may be produced by culturing the transformed cells obtained as described above, expressing the GOD or GDH gene contained in the cells, and then isolating the expressed protein from the culture. ..

Any of a synthetic medium and a natural medium can be used as long as it contains a normal medium for culturing filamentous fungi, that is, a carbon source, a nitrogen source, an inorganic substance, and other nutrients in an appropriate ratio. The medium for culturing the above-mentioned microbial host cells is, for example, yeast extract, tripton, peptone, meat extract, corn steep liquor, or one or more nitrogen sources such as soybean or wheat bran leachate, and sodium chloride and phosphoric acid. Add one or more of inorganic salts such as primary potassium, secondary potassium phosphate, magnesium sulfate, magnesium chloride, ferric chloride, ferrous sulfate or manganese sulfate, and if necessary, add sugar raw materials, vitamins, etc. Those added as appropriate are used.

As the culturing conditions, the culturing conditions for filamentous fungi usually known by those skilled in the art may be adopted. For example, the initial pH of the medium is adjusted to 5 to 10, the culturing temperature is 20 to 40 ° C., and the culturing time is several hours to several hours. It can be appropriately set for days, preferably 1 to 7 days, more preferably 2 to 5 days, and the like. The culturing means is not particularly limited, and aeration-stirred deep culture, shaking culture, static culture and the like can be adopted, but culturing is preferably performed under conditions such that the dissolved oxygen is sufficient. For example, as an example of the medium and culture conditions for culturing Aspergillus microorganisms, shaking culture at 30 ° C. and 160 rpm for 3 to 5 days using the DPY medium described in Examples described later can be mentioned. When culturing microbial host cells, a deep culture with aeration stirring is performed at a culture temperature of 10 to 42 ° C., preferably a culture temperature of about 25 ° C. for 4 to 24 hours, and more preferably a culture temperature of about 25 ° C. for 4 to 8 hours. , Shaking culture, static culture, etc. may be carried out.

After the culture is completed, GOD or GDH is collected from the culture. For this, a usual known enzyme collecting means may be used. For example, the supernatant fraction of the medium is collected, the cells are subjected to ultrasonic destruction treatment, grinding treatment, etc. by a conventional method, or the enzyme is extracted using a lytic enzyme such as lysozyme. The enzyme can be excreted from the cells by shaking or leaving it to lyse in the presence of toluene or the like. Then, this solution is filtered, centrifuged, etc. to remove the solid portion, and if necessary, the nucleic acid is removed with streptomycin sulfate, protamine sulfate, manganese sulfate, etc., and then ammonium sulfate, alcohol, acetone, etc. are added thereto. Fractionate, and the precipitate is collected to obtain a crude enzyme of GOD or GDH.

The crude enzyme can also be further purified using any known means. To obtain a purified enzyme preparation, for example, a gel filtration method using cefadex, ultrogel, biogel, etc .; an adsorption elution method using an ion exchanger; an electrophoresis method using a polyacrylamide gel, etc .; an adsorption method using hydroxyapatite. Elution method; Precipitation method such as vine sugar density gradient centrifugation method; Hydrophobic chromatography method; Affinity chromatography method; Fractionation method using molecular sieve membrane or hollow yarn membrane, etc. is appropriately selected, or a combination thereof is carried out. Thereby, a purified enzyme preparation can be obtained.

Whether or not the enzyme has been purified may be determined by polyacrylamide gel electrophoresis and staining, and a glucose sample having a known concentration should be used to test whether glucose can be appropriately measured electrochemically. May be done by.

(Measurement of GDH activity)
GDH (EC 1.1.99.10) catalyzes the reaction of oxidizing the hydroxyl groups of glucose to produce glucono-δ-lactone. At this time, the electron acceptor receives an electron and becomes a reduced electron acceptor. The activity of GDH can be measured by utilizing this principle of action, for example, using the following measurement system using phenazinemethsulfate (PMS) and 2,6-dichloroindophenol (DCIP) as electron acceptors. ..
(Reaction 1) D-glucose + PMS (oxidized type)
→ D-glucono-δ-lactone + PMS (reduced form)
(Reaction 2) PMS (reduced type) + DCIP (oxidized type)
→ PMS (oxidized type) + DCIP (reduced type)

Specifically, first, in (Reaction 1), PMS (reduced form) is produced with the oxidation of D-glucose. By the subsequent progress (reaction 2), DCIP is reduced as PMS (reduced form) is oxidized. The degree of disappearance of this "DCIP (oxidized type)" can be detected as the amount of change in absorbance at a wavelength of 600 nm, and the enzyme activity can be determined based on this amount of change.

The activity of GDH can be measured according to the following procedure. Mix 2.05 mL of 100 mM phosphate buffer (pH 7.0), 0.6 mL of 1MD-glucose solution and 0.15 mL of 2 mM DCIP solution, and incubate at 37 ° C. for 5 minutes. Then 0.1 mL of 15 mM PMS solution and 0.1 mL of enzyme sample solution are added to initiate the reaction. The absorbance at the start of the reaction and over time is measured to determine the amount of decrease in absorbance (ΔA600) at 600 nm with the progress of the enzymatic reaction per minute, and the GDH activity is calculated according to the following formula. At this time, GDH activity defines 1 U as the amount of enzyme that reduces 1 μmol of DCIP per minute in the presence of D-glucose at a concentration of 200 mM at 37 ° C.

In the formula, 3.0 is the liquid volume of the reaction reagent + enzyme reagent (mL), 16.3 is the mmol molecular extinction coefficient (cm ² / μmol) under the present activity measurement conditions, and 0.1 is the liquid volume of the enzyme solution. (ML), 1.0 is the optical path length (cm) of the cell, ΔA600 _blank is 1 minute of absorbance at 600 nm when a 100 mM phosphate buffer (pH 7.0) was added instead of the enzyme sample solution and the reaction was started. The amount of decrease per unit, df, represents the dilution factor.

(Measurement of GOD activity)
GOD (EC 11.3.4) catalyzes the reaction of oxidizing the hydroxyl groups of glucose to produce glucono-δ-lactone and hydrogen peroxide.
The activity of GOD can be measured by reacting the generated hydrogen peroxide with peroxidase to develop a color-developing agent by utilizing this principle of action.

Method for measuring glucose oxidase activity <Reagent>
100 mM MES-Na buffer pH 5.7
0.5% 4-aminoantipyrine (manufactured by Tokyo Kasei Kogyo Co., Ltd., 4AA) solution 5.0 g / L TOOS (N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine sodium, Dojin Kagaku Co., Ltd. MES-Na buffer solution 30.0 mL, 4AA solution 0.3 mL, TOOS solution 0.3 m
L, POD solution 0.3 mL, and D-glucose solution 6.0 mL are mixed to prepare a reaction reagent.

<Measurement conditions>
Preheat 3 mL of the reaction reagent at 37 ° C. for 5 minutes. After adding 0.1 mL of GOD solution and mixing gently, the absorbance at 555 nm is measured with a spectrophotometer controlled at 37 ° C. using water as a control. The measured value is the change in absorbance per minute after 1 minute to 3 minutes at 555 nm. The number of micromoles of hydrogen peroxide produced per minute at 37 ° C. is defined as the active unit (U) in the enzyme solution, and the calculation is performed according to the following formula.
Activity (U / ml) = {(ΔAs-ΔA0) × 3.1 × df} ÷ (32.8 × 0.5 × 0.1)
ΔAs: Change in absorbance per minute of the reaction solution ΔA0: Change in absorbance of the control solution per minute 32.8: Of the quinoneimine dye produced by the reaction
Millimole absorption coefficient (mM ^-1 · cm ^-1 )
0.5: Mol number of quinoneimine dye produced by 1 mol of hydrogen peroxide df: Dilution coefficient

(Glucose assay kit)
In certain embodiments, the present invention provides a glucose assay kit comprising the flavin compound of the present invention. In certain embodiments, the kit comprises GDH. In another embodiment the kit comprises GOD. By using this kit, blood glucose (blood glucose level) can be measured using GOD or GDH. The measurement may be performed continuously.

The glucose assay kit of the present invention contains a sufficient amount of the flavin compound of the present invention for at least one assay. Typically, the glucose assay kit of the invention includes, in addition to the flavin compounds of the invention, GOD or GDH, the buffer required for the assay, a glucose standard solution for making a calibration curve, and guidelines. In certain embodiments, the glucose assay kit of the invention, eg, a glucose assay kit for SMBG, CGM, or FGM, comprises the flavin compound of the invention and GOD or GDH as the same reagent. In another embodiment, the glucose assay kit of the invention comprises a flavin compound and GOD or GDH as separate reagents. The flavin compounds of the invention are provided in various forms, for example, as lyophilized reagents, as reagents immobilized on the surface of beads or electrodes, or as solutions in suitable storage solutions, preferably shaded solutions. can do.

In the case of the colorimetric glucose assay kit, the glucose concentration can be measured, for example, as follows. GDH is used in the reaction layer of the glucose assay kit, and N- (2-acetamide) imide 2 acetic acid (ADA), bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris), and sodium carbonate are used as reaction promoters. And a liquid or solid composition containing one or more substances selected from the group consisting of imidazole is retained. Here, a pH buffer and a color-developing reagent (color-changing reagent) are added as needed. A sample containing glucose is added thereto, and the mixture is reacted for a certain period of time. During this period, the absorbance corresponding to the maximum absorption wavelength of the dye produced by polymerizing or reduced dye by directly receiving electrons from GDH is monitored. Calibration prepared in advance using a glucose solution of standard concentration from the rate of change in absorbance per hour in the rate method and from the change in absorbance up to the point when all glucose in the sample is oxidized in the endpoint method. The glucose concentration in the sample can be calculated based on the ion curve.

As a color-developing reagent (color-changing reagent) that can be used in this method, glucose can be quantified by adding, for example, 2,6-dichloroindophenol (DCIP) as an electron acceptor and monitoring the decrease in absorbance at 600 nm. .. Further, it is possible to determine the amount of diformazan produced by adding nitrotetrazolium blue (NTB) as a color-developing reagent and measuring the absorbance at 570 nm, and to calculate the glucose concentration. Needless to say, the color-developing reagent (color-changing reagent) used is not limited to these.

(Glucose sensor)
In certain embodiments, the present invention provides a glucose sensor using the flavin compound of the present invention. The glucose sensor of the present invention has the flavin compound of the present invention and GDH or GOD. As the electrode of the glucose sensor, a carbon electrode, a gold electrode, a platinum electrode or the like is used, and GOD or GDH enzyme can be applied or immobilized on the electrode. Further, or independently, the flavin compound of the present invention may be applied or immobilized on the electrode. For the immobilization method, refer to the paragraph "GDH or GOD immobilization method" above.

The flavin compound and GOD or GDH of the present invention can be used in various electrochemical measurement methods by using potentiostat, galvanostat and the like. Electrochemical measurement methods include various methods such as amperometry, such as chronoamperometry, voltammetry, such as cyclic voltammetry, potentiometer, and coulometry. For example, by measuring the current when glucose is reduced by the amperometry method, the glucose concentration in the sample can be calculated. The applied voltage may be, for example, −1000 mV to +1000 mV (vs. Ag / AgCl), although it depends on the conditions and the setting of the apparatus.

(Electrochemical measurement of glucose)
In certain embodiments, the present invention comprises contacting a sample that may contain glucose with a free flavin compound with purified glucose oxidase or purified glucose dehydrogenase and measuring the current of glucose. Provided is a chemical measurement method. The flavin compound and / or enzyme may be immobilized in the solid phase.

For example, the electrochemical measurement of glucose concentration can be performed as follows. Put buffer in a constant temperature cell and keep it at a constant temperature. A GDH or GOD-immobilized 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 or an Ag / Ag + electrode) are used. The flavin compound of the present invention is added to the reaction solution. 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 glucose solution of the standard concentration. The applied potential can be, for example, +800 mV or less, +700 mV or less, +600 mV or less, +500 mV or less, +400 mV or less, +300 mV or less, +200 mV or less, +100 mV or less, +50 mV or less, -200 mV or more, -100 mV or more, -50 mV or more, For example, it can be 0 mV or more, for example, +800 mV to −200 mV, +800 mV to -100 mV, +800 mV to -50 mV, + 600 mV to 0 mV, + 500 mV to 0 mV, + 400 mV to 0 mV, + 300 mV to 0 mV, + 200 mV to 0 mV. (Both against silver chloride reference electrode).

As a specific example, a GDH or GOD of 0.2U to 150U, more preferably 0.5U to 100U is immobilized on a glassy carbon (GC) electrode, and the response current value with respect to the glucose concentration is measured. In the electrolytic cell, 10.0 ml of 100 mM potassium phosphate buffer (pH 6.0) is added. A GC electrode is connected to the potentiostat BAS100B / W (manufactured by BAS), the solution is stirred at 37 ° C., and + 500 mV is applied to the silver chloride reference electrode. A 1MD-glucose solution is added to these systems so that the final concentration is 5, 10, 20, 30, 40, 50 mM, and the steady-state current value is measured for each addition. This current value is plotted against a known glucose concentration (5, 10, 20, 30, 40, 50 mM) to create a calibration curve. This makes it possible to quantify glucose with a GDH or GOD enzyme-immobilized electrode.

Further, a printed electrode can be used for electrochemical measurement. This makes it possible to reduce the amount of solution required for measurement. In this case, the electrodes are preferably formed on an insulating substrate. Specifically, it is desirable to form the electrodes on the substrate by a photolithography technique or a printing technique such as screen printing, gravure printing, or flexographic printing. Examples of the material of the insulating substrate include silicon, glass, ceramic, polyvinyl chloride, polyethylene, polypropylene, polyester and the like, but it is more preferable to use a material having strong resistance to various solvents and chemicals. The area of the working electrode can be set according to the desired response current. For example, in one embodiment, the area of the working electrode is 1 mm ² or more, 1.5 mm ² or more, 2 mm ² or more, 2.5 mm ² or more, 3 mm ² or more, 4 mm ² or more, 5 mm ² or more, 6 mm ² or more, 7 mm ² or more. , 8mm ² or more, 9mm ² or more, 10mm ² or more, 12mm ² or more, 15mm ² or more, 20mm ² or more, 30mm ² or more, 40mm ² or more, 50mm ² or more, 1cm ² or more, 2cm ² or more, 3cm ² or more, 4cm It can be ² or more, 5 cm ² or more, for example, 10 cm ² or more. In certain embodiments, the area of the working electrode can be 10 cm ² or less, 5 cm ² or less, for example 1 cm ² or less. The opposite pole can be similar.

In one embodiment, a continuous blood glucose measuring device having the glucose sensor of the present invention is provided. The continuous blood glucose measuring device may include a CGM (continuous glucose monitoring) device and an FGM (Flash glucose monitoring) device that can grasp the fluctuation of the blood glucose level over 24 hours. Also in certain embodiments, the present invention provides a continuous glucose monitoring method using the compositions, electrodes or sensors of the present invention.

In certain embodiments, the CGM device of the invention may have a glucose sensor for placement under the skin. The sensor can be worn for a period of time, eg 1 day to 3 weeks. The sensor may be disposable or reusable. The sensor may have a sensor membrane layer to prevent direct contact of the tissue with the enzyme. The sensor can be designed to be inserted into the abdomen using an applicator. In certain embodiments, the present invention provides a CGM device further comprising a transmitter and a sensor-to-transmitter connection. The transmitter does not have to be implanted. Preferably the transmitter is capable of communicating with the radio receiver. The CGM device may further have an electrical receiver that continuously displays glucose levels. The CGM device may optionally have a finger stick for calibration. CGM devices may have instructions for use.

(Fuel cell of the present invention)
In certain embodiments, the present invention provides an anode for a fuel cell having the flavin compound of the present invention or a composition containing the flavin compound, and a fuel cell provided with the anode. In certain embodiments, the present invention provides a glucose-fueled power generation method using the flavin compound of the present invention, a composition containing the flavin compound, or the anode.

The fuel cell of the present invention comprises the flavin compound of the present invention, a fuel tank, a cathode, an anode having a GOD or GDH, and an electrolyte. Further, the fuel cell of the present invention can arrange a load resistance between the anode and the cathode, if necessary, and can be provided with wiring for that purpose. In certain embodiments, the load resistance is part of the fuel cell of the present invention. In certain embodiments, the load resistance is not part of the fuel cell of the invention, and the fuel cell of the invention is configured to be connectable to a suitable load resistance. In the fuel cell of the present invention, GOD or GDH constitutes a part of the anode. For example, GOD or GDH may be in close proximity to or in contact with the anode, may be immobilized, or may be adsorbed. The fuel tank contains glucose. That is, when the term "fuel cell" is used in the present specification, it means a fuel cell that uses glucose as fuel unless otherwise specified. Glucose is oxidized by the action of GOD or GDH to generate a reduced mediator, and when this transfers an electron to an electrode, an electric current flows. In certain embodiments, the fuel cell of the invention may have an ion exchange membrane that separates the anode and cathode. The ion exchange membrane may have pores of 1 nm to 20 nm. The anode can be a common electrode such as a carbon electrode. For example, an electrode made of a conductive carbon substance such as carbon black, graphite, or activated carbon, or an electrode made of a metal such as gold or platinum can be used. Specific examples thereof include carbon paper, glassy carbon, and HOPG (highly oriented pyrolytic graphite). As the paired cathode, for example, an electrode catalyst generally used in a fuel cell such as platinum or platinum alloy is used as a carbonaceous material such as carbon black, graphite or activated carbon, or a conductive material made of gold, platinum or the like. It is possible to use an electrode carried on the body or a conductor made of an electrode catalyst itself such as platinum or a platinum alloy as a cathode electrode, and supply an oxidizing agent (catalyst side substrate, oxygen, etc.) to the electrode catalyst. can.

In another embodiment, the substrate reducing enzyme electrode can be used as the cathode paired with the anode composed of the substrate oxidizing enzyme electrode as described above. Examples of the redox enzyme that reduces the oxidizing agent include known enzymes such as laccase and bilirubin oxidase. When an oxidoreductase is used as a catalyst for reducing the oxidant, a known electron transfer mediator may be used, if necessary. The cathode mediator may be the same as or different from the anode mediator. Examples of the oxidizing agent include oxygen and hydrogen peroxide.

In certain embodiments, an oxygen-selective membrane (eg, a dimethylpolysiloxane membrane) is placed around the cathode electrode to avoid the effects of impurities (such as ascorbic acid and uric acid) that interfere with the electrode reaction at the cathode. Can be done.

The power generation method of the present invention includes a step of supplying glucose as a fuel to an anode having GOD or GDH. When glucose is supplied to the anode having GOD or GDH, the glucose is oxidized to D-glucono-δ-lactone, and the simultaneously generated electrons are transferred by GOD or GDH to the electron transfer between the oxidase and the electrode. Is transferred to an electron transfer mediator, for example, a flavine compound, and electrons are transferred to a conductive substrate (anode electrode) by the electron transfer mediator. An electric current is generated when electrons reach the cathode electrode from the anode electrode through wiring (external circuit).

The protons (H ⁺ ) generated in the above process move in the electrolyte solution to the cathode electrode. Then, at the cathode electrode, the protons that have moved from the anode in the electrolyte solution, the electrons that have moved from the anode side through the external circuit, and the oxidizing agent (cathode side substrate) such as oxygen and hydrogen peroxide react with each other. Water is produced. This can be used to generate electricity.

The flavin compound of the present invention and the composition containing the flavin compound can be used for measuring the glucose concentration in a sample, which can be useful for, for example, diagnosis of diabetes and self-monitoring of blood glucose level. In addition, the composition of the present invention can be used together with an enzyme electrode and can be used for various electrochemical measurements. In addition, the composition of the present invention can be used together with an enzyme sensor. In addition, the composition of the present invention can be used for a glucose measurement kit or a glucose sensor. Further, the flavin compound of the present invention and the composition containing the flavin compound can be used for a fuel cell. That is, the flavin compound of the present invention and the composition containing the flavin compound can be used in a power generation method using glucose as fuel. This is an example, and the use of the flavin compound of the present invention or the composition containing the flavin compound is not limited to this.

The invention is further illustrated by the following examples. However, the technical scope of the present invention is not limited to those examples.

[Example 1]
1. 1. Introduction of Mucor-derived GDH gene into a host and confirmation of GDH activity First, a gene encoding a GDH mutant in which each mutation of T387A / I545T was introduced into Mucor-derived GDH (MpGDH) described in Japanese Patent No. 4648993 was obtained. .. The amino acid sequence of the MpGDH mutant is shown in SEQ ID NO: 1, and the base sequence of the gene is shown in SEQ ID NO: 2. A DNA construct was prepared by inserting the MpGDH mutant gene, which is the target gene, into the multi-cloning site of the plasmid pUC19 by a conventional method. Specifically, as pUC19, the pUC19 linearized Vector attached to the In-Fusion HD Cloning Kit (Clontech) was used. At the In-Fusion Cloning Site at the multi-cloning site of pUC19, the MpGDH mutant gene was ligated using the In-Fusion HD Cloning Kit described above according to the protocol attached to the kit, and the plasmid for construction (pUC19). -MpGDH mutant) was obtained.

In addition, a gene encoding modified GDH (MpGDH-M1) was obtained by introducing each mutation of N66Y / N68G / C88A / Q233R / T387C / E554D / L557V / S559K into GDH (MpGDH, SEQ ID NO: 1) derived from the genus Mucor. The amino acid sequence of MpGDH-M1 is shown in SEQ ID NO: 3, and the base sequence of the gene is shown in SEQ ID NO: 4. A DNA construct was prepared by inserting the MpGDH-M1 gene, which is the target gene, into the multi-cloning site of the plasmid pUC19 by a conventional method. Similarly, at the In-Fusion Cloning Site at the multi-cloning site of pUC19, the MpGDH-M1 gene was ligated using the above-mentioned In-Fusion HD Cloning Kit according to the plasmid attached to the kit. A plasmid for construct (pUC19-MpGDH-M1) was obtained.

Using the obtained recombinant plasmid pUC19-MpGDH-M1 as a template and KOD-Plus- (manufactured by Toyobo Co., Ltd.), a synthetic oligonucleotide of SEQ ID NO: 7-12, a PCR reaction was carried out under the following conditions.

That is, 5 μl of 10 × KOD-Plus-buffer solution, 5 μl of dNTPs mixed solution prepared so that dNTP is 2 mM each, 2 μl of 25 mM DDL ₄ solution, and a DNA construct ligated with the template MpGDH-M1 gene. 50 ng, 15 pmol of each of the above synthetic oligonucleotides, and 1 Unit of KOD-Plus- were added, and the total volume was adjusted to 50 μl with sterile water. Incubate the prepared reaction solution at 94 ° C for 2 minutes using a thermal cycler (manufactured by Eppendorf), and then "94 ° C, 15 seconds"-"50 ° C, 30 seconds"-"68 ° C, 8 minutes". The cycle of was repeated 30 times.

A part of the reaction solution was electrophoresed on a 1.0% agarose gel, and it was confirmed that about 8,000 bp of DNA was specifically amplified. The DNA thus obtained was treated with the restriction enzyme DpnI (manufactured by NEW ENGLAND BIOLABS), the remaining template DNA was cleaved, and then Escherichia coli JM109 was transformed and developed on an LB-amp agar medium. Inoculate the grown colonies into LB-amp medium [1% (W / V) bactolipton, 0.5% (W / V) peptone, 0.5% (W / V) NaCl, 50 μg / ml Ampicillin] 2.5 ml. The cells were shake-cultured at 37 ° C. for 20 hours to obtain a culture. The culture was centrifuged at 7,000 rpm for 5 minutes to collect cells and obtain cells. Then, a recombinant plasmid was extracted from this cell using QIAGEN tip-100 (manufactured by Qiagen) and purified to obtain 2.5 μg of DNA. The base sequence of the DNA encoding MpGDH-M1 in the plasmid was determined using a multicapillary DNA analysis system Applied Biosystems 3130xl Genetic Analyzer (manufactured by Life Technologies), and as a result, 175 of the amino acid sequence shown in SEQ ID NO: 1 was determined. A DNA construct encoding MpGDH-M1 / A175C / N214C / G466D (SEQ ID NO: 5), which is a variant in which alanin at the position is replaced with cysteine, asparagine at position 214 is replaced with cysteine, and glycine at position 466 is replaced with aspartic acid. Obtained (pUC19-MpGDH-M2) (SEQ ID NO: 6).

These genes were expressed in Aspergillus sojae and their GDH activity was evaluated.

Specifically, for the purpose of obtaining MpGDH mutants and MpGDH-M2, Double-joint PCR (Fungal Genetics and Biology, 2004, Vol. 41, p973-981) was performed using the GDH gene. A cassette consisting of the 5'arm region-PyrG gene (uracil nutritional requirement marker) -TEF1 promoter gene-flavin-bound GDH gene-3'arm region was constructed, and the pyrG disrupted strain derived from the Aspergillus soya NBRC4239 strain was constructed by the following procedure. It was used for transformation of the pyrG gene upstream 48 bp, coding region 896 bp, downstream 240 bp deficient strain). 100 ml of a polypeptone dextrin liquid medium containing 20 mM uridine in a 500 ml Erlenmeyer flask was inoculated with conidia of a pyrG-disrupted strain derived from Aspergillus soya NBRC4239 strain, and the cells were cultured by shaking at 30 ° C. for about 20 hours. Collected. Protoplasts were prepared from the recovered cells. Using the obtained protoplast and 20 μg of the target gene-inserted DNA construct, transformation was performed by the protoplast PEG method, and then Czapek-Dox minimal medium containing 0.5% (w / v) agar and 1.2 M sorbitol (Difco). Incubated at 30 ° C. for 5 days or more using pH 6) to obtain transformed Aspergillus soya as having the ability to form colonies.

The obtained transformed Aspergillus sawyer can be grown in a uridine-free medium by introducing pyrG, which is a gene that complements the uridine requirement, and is selected as a strain into which the target gene is introduced. did it. From the obtained strains, the target transformant was confirmed by PCR and selected.

Each GDH production was carried out using transformed Aspergillus sawyer transformed with the gene of the MpGDH mutant.

DPY liquid medium in a 200 ml Erlenmeyer flask (1% (w / v) polypeptone, 2% (w / v) dextrin, 0.5% (w / v) yeast extract, 0.5% (w / v) KH ₂ PO4, 0.05% (w / v) yeast _{4.7H 20} ; pH unadjusted) 40 ml was inoculated with the seeds of each strain, and the culture was carried out at 30 ° C. for 4 days at 160 rpm. Then, the cells were filtered from the culture after culturing, and the obtained medium supernatant fraction was concentrated to 10 mL with Amicon Ultra-15,30K NMWL (manufactured by Millipore), and 20 mM potassium phosphate buffer containing 150 mM NaCl was added. Apply to HiRoad 26/60 Superdex 200 pg (manufactured by GE Healthcare) equilibrated with a solution (pH 6.5), elute with the same buffer, collect the fraction showing GDH activity, and collect the MpGDH mutant and MpGDH-. A refined sample of M2 was obtained. This enzyme is in a state of being bound to FAD via its FAD binding site (holoenzyme).

2. 2. Introduction of Aspergillus terreus-derived GDH gene into a host and acquisition of purified preparations The Aspergillus terreus-derived GDH (AtGLD) gene described in Japanese Patent No. 5020070 was expressed in Aspergillus sojae in the same manner as above. Further, GDH was produced in the same manner as described above, and the obtained medium supernatant fraction was purified to obtain a purified sample of AtGLD. This enzyme is in a state of being bound to FAD via its FAD binding site (holoenzyme).

3. 3. Sensor evaluation using flavin compounds Chronoamperometry was performed by print electrode measurement using purified preparations of MpGDH mutants and two flavin compounds (riboflavin (RF) and flavin mononucleotide (FMN)). Specifically, on a DEP Chip electrode (round, with carbon damling; manufactured by Biodevice Technology) on which a glassy carbon working electrode (2.64 mm ² ) and a silver chloride reference electrode are printed. , 35 mg / ml purified enzyme MpGDH variant solution was loaded with 10 μL. After that, it was connected to the ALS electrochemical analyzer 814D (manufactured by BAS) using the DEP Chip dedicated connector. Then, chronoamperometry was performed by applying a voltage of +300 mV (vs. Ag / Ag +). Specifically, a potassium phosphate buffer solution (pH 7.0) having a final concentration of 50 mM, 20 mM glucose, and a 100 μM RF solution were placed on a 10 μL electrode to carry out a reaction, and the current value for 60 seconds was measured. Similarly, measurements were made using 1 mM FMN instead of 100 μM RF. Also, as a control, a similar test was performed using a glucose solution containing no RF or FMN.

The results are shown in FIG. FIG. 4 shows the results of chronoamperometry measurement in which MpGDH mutant enzyme and RF or FMN were mixed. A significantly higher response current was observed with the addition of RF and FMN as compared to the case without the mixture of RF and FMN. At an applied voltage of +300 mV (vs. Ag / Ag ⁺ ), the response current was 30.6 nA when RF or FMN was not added 60 seconds after the start of measurement, but when RF was added 33. It was 5 nA, and when FMN was added, a high value of 53.2 nA was shown.

Subsequently, the same measurement as above was performed using the purified sample of GDH-M2. As a result, a significantly higher response current was observed when RF and FMN were added as compared with the case where RF and FMN were not mixed. At an applied voltage of +300 mV (vs. Ag / Ag +), the response current was 17.5 nA when RF or FMN was not added 60 seconds after the start of measurement, but 19.2 nA when RF was added. When FMN was added, a high value of 19.6 nA was shown. That is, a measurable response current was observed compared to the background.

Subsequently, chronoamperometry by print electrode measurement was performed in the same manner as above using the purified preparation of AtGLD and four flavin compounds (RF, FMN, lumichrome (LC), flavin adenine dinucleotide (FAD)). rice field. However, the applied voltage was +100 mV (vs. Ag / Ag ⁺ ), and 100 μM RF, 1 mM FMN, 100 μM LC, and 1 mM FAD were used, respectively. The results are shown in FIG. FIG. 1 is a chronoamperometry measurement result in which AtGLD enzyme and RF or FMN, LC, and FAD are mixed. A significantly higher response current was observed when RF, FMN, LC, and FAD were added as compared with the case where the flavin compound was not mixed. At an applied voltage of +100 mV (vs. Ag / Ag ⁺ ), the response current was 1.4 nA when the flavin compound was not added 60 seconds after the start of measurement, but 3.3 nA when RF was added. When FMN was added, it was 17.9 nA, when LC was added, it was 10.3 nA, and when FAD was added, it was as high as 2.7 nA. That is, a measurable response current was observed compared to the background. In all cases where the flavin compound was mixed without adding glucose, the response current was lower than 1.4 nA.

Subsequently, printing is performed in the same manner as above using Glucose Dehydrogenase (FAD-dependent) (manufactured by BBI, Product Code: GLD1, hereinafter referred to as GLD1) and four flavin compounds (RF, FMN, LC, FAD). Chronoamperometry by electrode measurement was performed. These GDHs are also in a state of being bound to FAD via the FAD binding site (holoenzyme). However, the applied voltage was +100 mV (vs. Ag / Ag ⁺ ). The results are shown in FIG. FIG. 2 shows the results of chronoamperometry measurement in which GLD1 enzyme and RF or FMN, LC, and FAD are mixed. A significantly higher response current was observed when RF, FMN, LC, and FAD were added as compared with the case where the flavin compound was not mixed. At an applied voltage of +100 mV (vs. Ag / Ag ⁺ ), the response current was 1.1 nA when the flavin compound was not added 60 seconds after the start of measurement, but 2.1 nA when RF was added. When FMN was added, it was 30.8 nA, when LC was added, it was 3.5 nA, and when FAD was added, it was as high as 5.7 nA. That is, a measurable response current was observed compared to the background.

Subsequently, using Glucose Oxidase (manufactured by Toyobo Co., Ltd., Product Code: GLO-201, hereinafter referred to as GOD) and two flavin compounds (RF, FMN), chronoamperometry by print electrode measurement in the same manner as above. Was done. These GDHs are also in a state of being bound to FAD via the FAD binding site (holoenzyme). However, the printed electrode uses SCREEN-PRINTED ELECTRODES (Drop Sense, DRP-110), which has a carbon working electrode (12.6 mm ² ) and a silver reference electrode printed on it, and is a dedicated connector (Drop Sense). , DRP-CAC) was used to connect to the ALS Electrochemical Analyzer 814D (manufactured by BAS). Then, the applied voltage was set to +100 mV (vs. Ag / Ag ⁺ ). The results are shown in FIG. FIG. 3 shows the results of chronoamperometry measurement in which GOD enzyme and RF or FMN are mixed. A significantly higher response current was observed with the addition of RF and FMN as compared to the case without the flavin compound mixed. At an applied voltage of +100 mV (vs. Ag / Ag ⁺ ), the response current was 9.8 nA when the flavin compound was not added 60 seconds after the start of measurement, but 25.0 nA when RF was added. When FMN was added, a high value of 31.6 nA was shown. That is, a measurable response current was observed compared to the background.

As described above, when a flavin-based compound is used, the response current obtained when GDH or GOD reacts with glucose is increased, and it can be said that glucose can be measured with high sensitivity. In addition, since the response current depends on the glucose concentration, the response current based on the glucose-containing sample with a known concentration is measured in advance to create a calibration curve, and the response current of the glucose-containing sample with an unknown concentration is measured. , The glucose concentration can be measured from the calibration curve. The above is just an example, and the present invention is not limited thereto. For example, if a larger response current is preferred, the area of the electrodes, eg the area of the working electrode, can be scaled up.

From the above, since an increase in the response current was observed in the presence of the flavin compound of the present invention as compared with the case of the absence of the flavin compound, the free flavin compound of the present invention functions as a mediator and GOD or It was confirmed that GDH mediates the transfer of electrons when it oxidizes glucose to gluconolactone. The redox potential of the flavin compound is known, and it is known that electrons can be transferred depending on the redox potential of the partner compound, but the flavin compound is actually mixed with GDH or GOD to measure glucose. No examples have been reported so far.

[Example 2]
1. 1. Confirmation of glucose concentration dependence in electrochemical measurement 2 μL of 35 mg / ml GLD1 solution, 3 μL of 20 mM potassium phosphate buffer (pH 7.0) containing 1.5 M potassium chloride, and 5 μL of 1 mM FMN. , The mixture was placed on the DEP Chip electrode. Then, cyclic voltammetry was performed in which the voltage was swept in the range of −400 mV to +200 mV (vs. Ag / AgCl). The sweep speed was 10 mV / sec. Subsequently, 2M glucose solutions were added so as to have various glucose concentrations, and cyclic voltammetry was performed in the same manner. The value obtained by adding the oxidation current value ( _IO ) and the reduction current value ( _IR ) at a specific potential and dividing by 2 was calculated, and it was investigated whether this calculated value increased depending on the glucose concentration. In general, if the value at a constant response current in chronoamperometry applied at a specific potential increases in a substrate concentration-dependent manner, it will be at the same potential as before in cyclic _voltammetry ( _IO + IR). It is known that the value of) / 2 also increases and is linked.

_GLD1 and FMN were mixed and measured, and the results of plotting the glucose concentration and the ( _IO + IR) / 2 value are shown in FIG. As a result, it was confirmed that the ( _IO + _IR ) / 2 value also increased in a glucose concentration-dependent manner at +100 mV (vs. Ag / AgCl), and it was found that FMN functions as a mediator. Furthermore, it was confirmed that the ( _IO + _IR ) / 2 value also increased in a glucose concentration-dependent manner even at -100 mV (vs. Ag / AgCl).

It is considered that no response current is observed even if the response current is measured under the above conditions using a conventional artificial electron mediator instead of the flavin compound of the present invention. This is because the conventional artificial electron mediator has a redox potential different from that of the flavin compound of the present invention, and for example, potassium ferricyanide has a redox potential of about 250 mV. Then, even if potassium ferricyanide is used as a mediator, a response current does not flow at an applied potential of about 100 mV, and it is considered necessary to apply a potential of about 350 mV or more in order to observe the response current. However, in the compound of the present invention, a response current was observed even at a low potential of +100 mV. This is advantageous in various applications.

Subsequently, in the same manner as above, MpGDH-M2 and FMN were mixed, and cyclic voltammetry was performed in which the voltage was swept in the range of −400 mV to +200 mV (vs. Ag / AgCl). The result of plotting the glucose concentration and the ( _IO + _IR ) / 2 value is shown in FIG. As a result, it was confirmed that the ( _IO + _IR ) / 2 value also increased in a glucose concentration-dependent manner at +100 mV (vs. Ag / AgCl), and it was found that FMN functions as a mediator.

Subsequently, 2 μL of 35 mg / ml GLD1 solution, 3 μL of 20 mM potassium phosphate buffer (pH 7.0) containing 1.5 M potassium chloride, and 5 μL of 1 mM FAD were mixed with the DEP Chip electrode. I put it. Then, cyclic voltammetry was performed in which the voltage was swept in the range of 0 mV to +600 mV (vs. Ag / AgCl). The sweep speed was 10 mV / sec. Subsequently, 2M glucose solutions were added so as to have various glucose concentrations, and cyclic voltammetry was performed in the same manner. FIG. 7 shows the results of measuring _GLD1 and FAD in a mixed manner and plotting the glucose concentration and the ( _IO + IR) / 2 value. As a result, it was confirmed that the ( _IO + _IR ) / 2 value also increased in a glucose concentration-dependent manner at +100 mV (vs. Ag / AgCl), and it was found that FAD functions as a mediator.

Subsequently, 2 μL of 35 mg / ml MpGDH-M2 solution, 3 μL of 20 mM potassium phosphate buffer (pH 7.0) containing 1.5 M potassium chloride, and 5 μL of 100 μM RF were mixed with the DEP Chip electrode. And put it on. Then, cyclic voltammetry was performed in which the voltage was swept in the range of 0 mV to +600 mV (vs. Ag / AgCl). The sweep speed was 10 mV / sec. Subsequently, 2M glucose solutions were added so as to have various glucose concentrations, and cyclic voltammetry was performed in the same manner. _MpGDH -M2 and RF were mixed and measured, and the results of plotting the glucose concentration and the ( _IO + IR) / 2 value are shown in FIG. As a result, it was confirmed that the ( _IO + _IR ) / 2 value also increased in a glucose concentration-dependent manner at +500 mV (vs. Ag / AgCl), and it was found that FAD functions as a mediator. Furthermore, glucose concentration dependence was also confirmed at +400 mV (vs. Ag / AgCl).

Subsequently, 2 μL of 35 mg / ml GLD1 solution, 3 μL of 20 mM potassium phosphate buffer (pH 7.0) containing 1.5 M potassium chloride, and 5 μL of 100 μM LC were mixed with the DEP Chip electrode. I put it. Then, cyclic voltammetry was performed in which the voltage was swept in the range of 0 mV to +600 mV (vs. Ag / AgCl). The sweep speed was 10 mV / sec. Subsequently, 2M glucose solutions were added so as to have various glucose concentrations, and cyclic voltammetry was performed in the same manner. _GLD1 and LC were mixed and measured, and the results of plotting the glucose concentration and the ( _IO + IR) / 2 value are shown in FIG. As a result, it was confirmed that the ( _IO + _IR ) / 2 value also increased in a glucose concentration-dependent manner at +500 mV (vs. Ag / AgCl), and it was found that LC functions as a mediator.

In addition, the effect of combining the ruthenium complex and FMN was confirmed. 2 μL of 35 mg / ml GLD1 solution, 3 μL of 20 mM potassium phosphate buffer (pH 7.0) containing 1.5 M potassium chloride, 2.5 μL of 100 μM hexaamminecobaltium, and 1 mM FMN 2. 5 μL was mixed and placed on a DEP Chip electrode. Then, cyclic voltammetry was performed in which the voltage was swept in the range of −400 mV to +200 mV (vs. Ag / AgCl). The sweep speed was 10 mV / sec. Subsequently, a 2M glucose solution was added so as to have a glucose concentration of 18 mM, and cyclic voltammetry was performed in the same manner. For comparison, tests were conducted with hexaammine ruthenium alone and FMN alone at similar concentrations. As a result, GLD1 and hexaammine ruthenium were mixed and measured, and the ( _IO + IR) / 2 value at 0 _V when 18 mM glucose was added was 2 nA. _GLD1 and FMN were mixed and measured, and the ( _IO + IR) / 2 value when 18 mM glucose was added was 3 nA. _GLD1 was measured by mixing hexaammine ruthenium and FMN, and the ( _IO + IR) / 2 value when 18 mM glucose was added was 11 nA, and a synergistic increase in response current could be confirmed. In each case, the ( _IO + _IR ) / 2 value when glucose was not added was in the range of 0 to 1 nA.

Since glucose can be sufficiently detected by an electrochemical measuring device if the sensitivity is several nA, the glucose contained in the sample can be detected if a calibration curve is prepared by preparing a calibration curve. It is possible. Therefore, in Example 1, when the response current is slightly improved as compared with the case where glucose is not added when glucose is added, it can be said that the response current is also improved when glucose is further added.

[Example 3]
1. 1. Construction of fuel cell An anode electrode is manufactured by cross-linking and immobilizing purified GDH on a carbon electrode with glutaraldehyde. In addition, a cathode electrode is produced by immobilizing bilirubin oxidase (BOD, manufactured by Amano Enzyme) on a porous carbon electrode. A biobattery is constructed in which a naphthion membrane is sandwiched between an anode electrode and a cathode electrode as a solid polymer electrolyte membrane. In addition, a constant load resistance and a voltmeter are inserted in series and in parallel between the wiring connecting the anode electrode and the cathode electrode, respectively. On the anode side, an electrolyte solution containing 1 M potassium phosphate buffer (pH 7.0), 10 mM FMN, and 400 mM D-glucose is placed, glucose is supplied, and the mixture is operated under the conditions of 30 ° C. and pH 7, so that a current flows. To.

Although not bound by any particular theory, the fuel cell of the present invention is considered as follows. That is, even when a low potential of 100 mV was applied in Example 1, a response current was observed in the presence of the compound of the present invention. Therefore, for example, in the case of a fuel cell using oxygen, a large electromotive force is expected by subtracting 100 mV from the redox potential of oxygen. This is advantageous in various applications.

By using the flavin compound of the present invention or the composition containing the flavin compound of the present invention, glucose measurement can be performed by a mediator having low toxicity or no toxicity. It can be used, for example, for implantable continuous glucose measurements. It can also be used in glucose fuel cells.

All publications, patents and patent applications mentioned or cited herein are incorporated herein by reference in their entirety.

Brief description of the sequence SEQ ID NO: 1 Amino acid sequence of GDH derived from Mucor prinii SEQ ID NO: 2 Nucleic acid sequence of GDH derived from Mucor prianii SEQ ID NO: 3 MpGDH-M1 (aa)
SEQ ID NO: 4 MpGDH-M1 (DNA)
SEQ ID NO: 5 MpGDH-M2 (aa)
SEQ ID NO: 6 MpGDH-M2 (DNA)
SEQ ID NO: 7-12 Primer

Claims (19)

Glucose Electrochemistry comprising contacting a sample containing glucose with a free flavin compound with purified FAD-dependent glucose oxidase or purified FAD-dependent glucose dehydrogenase, and measuring current. Measurement method,
However, this method is
(i) No external addition of artificial electron mediators capable of receiving electrons from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase alone, or
(ii) A water-insoluble electron mediator capable of receiving electrons from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase alone is not added from the outside, and electrons are alone FAD-dependent glucose oxidase or FAD-dependent. No external addition of electron mediators with a redox potential of 0.2 V or higher, which can be received from glucose dehydrogenase.
The method. A power generation method that uses a free flavin compound as an electron mediator, using an anode and a cathode.
It has a step of supplying a flavin compound to an anode in contact with an electrolyte and supplying glucose fuel.
The power generation method, wherein the anode is an electrode having purified FAD-dependent glucose dehydrogenase or purified FAD-dependent glucose oxidase.
However, this method is
(i) No external addition of artificial electron mediators capable of receiving electrons from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase alone, or
(ii) A water-insoluble electron mediator capable of receiving electrons from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase alone is not added from the outside, and electrons are alone FAD-dependent glucose oxidase or FAD-dependent. No external addition of electron mediators with a redox potential of 0.2 V or higher, which can be received from glucose dehydrogenase.
The method. The method according to claim 1 or 2, wherein the flavin compound is a non-enzymatic flavin compound immobilized in a solid phase. The method according to any one of claims 1 to 3, wherein the purified FAD-dependent glucose oxidase or the purified FAD-dependent glucose dehydrogenase is immobilized in a solid phase. The flavin compound has the following chemical formula (I)

[In the formula, R ¹ does not exist or -CH ₃ or

In the absence of R ¹ , the nitrogen atom depicted with R ¹ linked forms a double bond with the adjacent carbon atom and R ² is H.
If R ² is absent or H and R ² is absent, the nitrogen atom depicted with R ² linked forms a double bond with the adjacent carbon atom.
R3 is H ^, a phosphate group, or

Is]
The method according to any one of claims 1 to 4, which is a compound represented by. The method according to any one of claims 1 to 5, wherein the flavin compound is flavin mononucleotide, riboflavin, lumichrome, flavin adenine dinucleotide or lumiflavin. In (i) above, the artificial electron mediator is a quinone, a phenazine, a viologen, a cytochrome, a thioredoxin, a phenoxazine, a phenothiazine, a ferricianide, a ferredoxin, a ferrosen, a ferrose derivative, and a metal complex. Or, in (ii) above, the water-insoluble electronic mediator is ferrosene, water-insoluble quinones, water-insoluble phenothins, water-insoluble viologens, water-insoluble thioredoxins, water-insoluble phenoki. Saddins, water-insoluble phenothiazines, water-insoluble ferredoxins, and water-insoluble ferrosene derivatives.
The method according to any one of claims 1 to 6. Anode electrode, which contains a free flavin compound and contains a purified FAD-dependent glucose dehydrogenase,
However, the electrode is
(i) Externally added, free of artificial electron mediators capable of receiving electrons alone from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase, or
(ii) Externally added electrons alone, free of water-insoluble electron mediators capable of receiving electrons alone from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase, and externally added electrons alone Does not contain an electron mediator having a redox potential of 0.2 V or higher, which can be received from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase.
The anode electrode. A glucose fuel cell comprising the anode electrode, the cathode electrode, and the fuel tank containing glucose according to claim 8. Anode electrode, which contains a free flavin compound and contains purified FAD-dependent glucose oxidase ,
However, the electrode is
(i) Externally added, free of artificial electron mediators capable of receiving electrons alone from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase, or
(ii) Externally added electrons alone, free of water-insoluble electron mediators capable of receiving electrons alone from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase, and externally added electrons alone Does not contain an electron mediator having a redox potential of 0.2 V or higher, which can be received from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase.
With the anode electrode
A glucose fuel cell having a cathode electrode and a fuel tank containing glucose . A glucose sensor with a free flavin compound and a purified FAD-dependent glucose dehydrogenase,
However, the sensor is
(i) Externally added, free of artificial electron mediators capable of receiving electrons alone from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase, or
(ii) Externally added electrons alone, free of water-insoluble electron mediators capable of receiving electrons alone from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase, and externally added electrons alone The sensor, which does not contain an electron mediator having a redox potential of 0.2 V or higher, which can receive from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase. A glucose sensor with a free flavin compound and purified FAD-dependent glucose oxidase ,
However, the sensor is
(i) Externally added, free of artificial electron mediators capable of receiving electrons alone from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase, or
(ii) Externally added electrons alone, free of water-insoluble electron mediators capable of receiving electrons alone from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase, and externally added electrons alone The sensor, which does not contain an electron mediator having a redox potential of 0.2 V or higher, which can receive from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase. The electrode according to claim 8, the battery according to claim 9 or 10 , or the sensor according to claim 11 or 12 , wherein the flavin compound is a non-enzymatic flavin compound immobilized in a solid phase. The electrode according to claim 8 or 13 , the battery according to claim 9 , 10 or 13 , wherein the purified FAD-dependent glucose oxidase or the purified FAD-dependent glucose dehydrogenase is immobilized in a solid phase. The sensor according to claim 11 , 12 or 13 . A glucose-measuring composition comprising a free flavin compound and a purified FAD-dependent glucose dehydrogenase, wherein the composition is:
(i) Externally added, free of artificial electron mediators capable of receiving electrons alone from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase, or
(ii) Externally added electrons alone, free of water-insoluble electron mediators capable of receiving electrons alone from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase, and externally added electrons alone Does not contain an electron mediator having a redox potential of 0.2 V or higher, which can be received from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase.
The composition for measuring glucose. A composition for measuring glucose containing a free flavin compound and a purified FAD-dependent glucose oxidase , wherein the composition is:
(i) Externally added, free of artificial electron mediators capable of receiving electrons alone from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase, or
(ii) Externally added electrons alone, free of water-insoluble electron mediators capable of receiving electrons alone from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase, and externally added electrons alone Does not contain an electron mediator having a redox potential of 0.2 V or higher, which can be received from FAD-dependent glucose oxidase or FAD-dependent glucose dehydrogenase.
The composition for measuring glucose. The flavin compound has the following chemical formula (I)

[In the formula, R ¹ does not exist or -CH ₃ or

In the absence of R ¹ , the nitrogen atom depicted with R ¹ linked forms a double bond with the adjacent carbon atom and R ² is H.
If R ² is absent or H and R ² is absent, the nitrogen atom depicted with R ² linked forms a double bond with the adjacent carbon atom.
R3 is H ^, a phosphate group, or

Is]
The composition according to claim 15 or 16 , the electrode according to claim 8, 13 or 14 , the battery according to claim 9, 10, 13 or 14 , or the battery according to claim 11 , 12 which is a compound represented by. , 13 or 14 . The composition according to claim 15 , 16 or 17 , wherein the flavin compound is flavin mononucleotide, riboflavin, lumichrome, flavin adenine dinucleotide or lumiflavin, the electrode according to claim 8, 13 , 14 or 17 . The battery according to 9, 10 , 13 , 14 or 17 , or the sensor according to claim 11 , 12, 13 , 14 or 17 . In (i) above, the artificial electron mediator is a quinone, a phenazine, a viologen, a cytochrome, a thioredoxin, a phenoxazine, a phenothiazine, a ferricianide, a ferredoxin, a ferrocene, a ferrocene derivative, and a metal complex. Or, in (ii) above, the water-insoluble electronic mediator is ferrocene, water-insoluble quinones, water-insoluble phenazines, water-insoluble viologens, water-insoluble thioredoxins, water-insoluble phenoki. The composition according to claim 15, 16, 17 or 18 , which is a sagin, a water-insoluble phenothiazine, a water-insoluble ferredoxin, and a water-insoluble ferrocene derivative, claims 8, 13, 14, and 6. 17. The electrode according to 17 or 18 , the battery according to claim 9 , 10, 13, 14, 17 or 18, or the sensor according to claim 11, 12 , 13, 14, 17 or 18.

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