JP2009294037A - Enzyme sensor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an enzyme sensor having long-term stability. <P>SOLUTION: The enzyme sensor 1 is equipped with a detection part 10 containing enzyme 100 and coenzyme 300. The enzyme 100 is contained in the detection part 10, in a state of being fixed in the pores of a silica-based mesoporous body 110 and a biopolymer with a molecular weight of 1,000 or higher, a synthetic polymer with a molecular weight of 1,000 or higher and/or an electrolyte are added to the detection part 10. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an enzyme sensor.

Conventionally, research and development of enzyme sensors for detecting a detection target substance in a sample using an enzyme have been actively performed (for example, see Patent Documents 1 to 4).
By the way, in such an enzyme sensor, the enzyme is often used in a state of being fixed to various carriers. Specifically, as an enzyme immobilization method, for example, a carrier binding method in which an enzyme (protein) is bound to an insoluble carrier, a comprehensive immobilization method in which the enzyme is wrapped in a fine lattice of a gel, or a semipermeable polymer coating Inclusive methods such as a microcapsule method for coating with a coating are known.
Japanese Patent No. 3805442 JP 2006-131893 A Japanese Patent No. 2624236 Special table 2003-513230 gazette

However, in the carrier binding method, the enzyme is directly immobilized on a resin or the like by a covalent bond, so the enzyme is not easily detached, but the immobilization operation is complicated, and the enzyme is degraded by a proteolytic enzyme. Or the three-dimensional structure of the enzyme changes due to changes in the external environment.
In addition, in the inclusion methods such as the inclusion fixation method and the microcapsule method, the structural stability of the gel lattice and capsule is insufficient, resulting in a change in the three-dimensional structure of the enzyme accompanying a change in the external environment, resulting in a decrease in enzyme activity. Resulting in.
Therefore, there is a problem that enzyme sensors using enzymes immobilized by these immobilization methods lack long-term stability.

  An object of the present invention is to provide an enzyme sensor having long-term stability.

In order to solve the above-mentioned problem, the invention described in claim 1
In enzyme sensor,
A detection unit containing an enzyme and a coenzyme;
The enzyme is contained in the detection unit in a state of being fixed inside the pores of the silica-based mesoporous material,
A biopolymer having a molecular weight of 1000 or more is added to the detection unit.

The invention described in claim 2
In enzyme sensor,
A detection unit containing an enzyme and a coenzyme;
The enzyme is contained in the detection unit in a state of being fixed inside the pores of the silica-based mesoporous material,
A synthetic polymer having a molecular weight of 1000 or more is added to the detection unit.

The invention according to claim 3
In enzyme sensor,
A detection unit containing an enzyme and a coenzyme;
The enzyme is contained in the detection unit in a state of being fixed inside the pores of the silica-based mesoporous material,
An electrolyte is added to the detection unit.

The invention according to claim 4
In the enzyme sensor as described in any one of Claims 1-3,
Among the compounds represented by the following general formula (1), the compound represented by the following general formula (2), and the compound represented by the following general formula (3) as an electron carrier in the detection unit At least one of the above is added.

(In the formula, R1, R2, and R3 each represents an alkyl group. (Wherein Me represents a metal atom.) In the formula, X ⁻ represents an anion.)

(In the formula, Me represents a metal atom.)

(In the formula, Me represents a metal atom.)

The invention described in claim 5
In the enzyme sensor as described in any one of Claims 1-4,
An electron carrier adsorbed by a predetermined porous body is added to the detection unit as an electron carrier.

According to the present invention, a detection unit comprising an enzyme and a coenzyme is provided, and the enzyme is contained in the detection unit in a state of being fixed inside the pores of the silica-based mesoporous material. A biopolymer having a molecular weight of 1000 or more is added.
In other words, by fixing the enzyme inside the pores of the silica-based mesoporous material, it is possible to prevent a change in the three-dimensional structure of the enzyme that accompanies a change in the external environment. it can.
Further, it has been found that when a coenzyme coexists with a silica-based mesoporous material, the coenzyme is adsorbed on the surface of the silica-based mesoporous material and causes a decrease in the activity of the enzyme. By adding a biopolymer, a biopolymer having a molecular weight of 1000 or more is adsorbed on the surface of the silica-based mesoporous material, and the adsorption of the coenzyme to the silica-based mesoporous material is suppressed. Can be suppressed.
Therefore, an enzyme sensor having excellent long-term stability can be provided.

In addition, according to the present invention, it is provided with a detection unit containing an enzyme and a coenzyme, the enzyme is contained in the detection unit in a state of being fixed inside the pores of the silica-based mesoporous material, and the detection unit Is added with a synthetic polymer having a molecular weight of 1000 or more.
In other words, by fixing the enzyme inside the pores of the silica-based mesoporous material, it is possible to prevent a change in the three-dimensional structure of the enzyme that accompanies a change in the external environment. it can.
Further, it has been found that when a coenzyme coexists with a silica-based mesoporous material, the coenzyme is adsorbed on the surface of the silica-based mesoporous material and causes a decrease in the activity of the enzyme. By adding a synthetic polymer, a synthetic polymer having a molecular weight of 1000 or more is adsorbed on the surface of the silica-based mesoporous material, and the adsorption of the coenzyme to the silica-based mesoporous material is suppressed. Can be suppressed.
Therefore, an enzyme sensor having excellent long-term stability can be provided.

In addition, according to the present invention, it is provided with a detection unit containing an enzyme and a coenzyme, the enzyme is contained in the detection unit in a state of being fixed inside the pores of the silica-based mesoporous material, and the detection unit An electrolyte is added to the electrolyte.
In other words, by fixing the enzyme inside the pores of the silica-based mesoporous material, it is possible to prevent a change in the three-dimensional structure of the enzyme that accompanies a change in the external environment. it can.
Furthermore, it is known that coenzyme coexists with the silica-based mesoporous material and the coenzyme is adsorbed on the surface of the silica-based mesoporous material, causing a decrease in the activity of the enzyme. As a result, ions generated by ionization of the electrolyte are adsorbed on the surface of the silica-based mesoporous material, and adsorption of the coenzyme to the silica-based mesoporous material is suppressed, so that a decrease in the activity of the enzyme can be suppressed. .
Therefore, an enzyme sensor having excellent long-term stability can be provided.

  Hereinafter, the best mode for carrying out the present invention will be described in detail with reference to the drawings. The scope of the invention is not limited to the illustrated example.

The enzyme sensor 1 of the present invention uses, for example, an electrochemical measurement method using an electrode 20 using an enzyme 100 that selectively reacts a detection target substance in a gas sample or a liquid sample with the detection target substance. It is a sensor to detect.
The enzyme sensor 1 includes, for example, a detection unit 10 made of a predetermined electrolyte introduced into the reactor 10a, the electrode 20 is disposed in the detection unit 10, and the enzyme 100 is contained in the detection unit 10. Yes.
In the enzyme sensor 1, for example, a detection target substance in the gas sample or liquid sample is detected by dissolving the gas sample or liquid sample in a predetermined electrolyte introduced into the reactor 10a. .

Specifically, for example, as shown in FIG. 1, the enzyme sensor 1 includes a detection unit 10, a working electrode (electrode 20) disposed in the detection unit 10, a reference electrode 30 and a reference electrode 40, and a working electrode. A potentiostat 50 for measuring a current value generated in the (electrode 20), and the like.
The detection unit 10 is made of, for example, a predetermined electrolytic solution introduced into the reactor 10a. The detection unit 10 includes, for example, the enzyme 100, a silica-based mesoporous material 110 on which the enzyme 100 is fixed, and an electron carrier. 200, coenzyme 300, and the like.
The potentiostat 50 includes, for example, a constant voltmeter 500a, a current measuring instrument 500b, and the like, and is connected to leads connected to the working electrode (electrode 20), the reference electrode 30, and the reference electrode 40. .

(enzyme)
For example, the enzyme 100 is contained in the detection unit 10 in a state of being fixed inside the pores of the silica-based mesoporous material 110.
The enzyme 100 fixed inside the pores of the silica-based mesoporous material 110 is optional as long as it is contained in the detection unit 10. That is, for example, the silica-based mesoporous material 110 on which the enzyme 100 is immobilized may be contained in the detection unit 10 by dispersing it in a predetermined electrolyte introduced into the reactor 10a. The fixed silica-based mesoporous material 110 may be included in the detection unit 10 by fixing it to the electrode 20 or the like. However, from the viewpoint of increasing the sensitivity of the enzyme sensor 1 or the like, the silica-based mesoporous material to which the enzyme 100 is fixed is used. The porous body 110 is preferably contained in the detection unit 10 by being fixed to the electrode 20.

As a method for fixing the enzyme 100 to the silica-based mesoporous material 110, for example, a dipping method in which a solution containing the enzyme 100 is dropped into the silica-based mesoporous material 110, or the silica-based mesoporous material 110 is immersed in a solution containing the enzyme 100. Although the immersion method which invades is mentioned, it is not specifically limited. Thereby, the enzyme 100 can be fixed to the silica-based mesoporous material 110 while maintaining the higher-order structure and activity.
Furthermore, if necessary, it can be used in combination with a known enzyme immobilization method (for example, an immobilization method using a conductive polymer, glutaraldehyde, a photocrosslinkable resin or the like).

The enzyme 100 is arbitrary as long as it is an enzyme that selectively reacts with the detection target substance, and can be appropriately changed depending on the type of the detection target substance.
Specifically, the enzyme 100 is an enzyme (enzyme protein) such as an oxidoreductase, a hydrolase, a transferase, and an isomerase.
The enzyme 100 may be, for example, a natural enzyme molecule or an enzyme fragment including an active site. The enzyme molecule or the fragment of the enzyme containing the active site may be, for example, extracted from animals or plants or microorganisms, or may be cleaved if desired, or may be genetic engineering or chemical. It may also be synthesized.

  Examples of the oxidoreductase include glucose oxidase, lactate oxidase, cholesterol oxidase, alcohol oxidase, formaldehyde oxidase, sorbitol oxidase, fructose oxidase, sarcosine oxidase, fructosylamine oxidase, pyruvate oxidase, xanthine oxidase, ascorbate oxidase, sarcosine. Oxidase, choline oxidase, amine oxidase, glucose dehydrogenase, lactate dehydrogenase, cholesterol dehydrogenase, alcohol dehydrogenase, formaldehyde dehydrogenase, sorbitol dehydrogenase, fructose dehydrogenase, hydroxybutyrate dehydrogenase, glycerol dehydrogenase Glutamate dehydrogenase, pyruvate dehydrogenase, malate dehydrogenase, glutamate dehydrogenase, can be used catalase, peroxidase, uricase, and the like. In addition, cholesterol esterase, acetylcholinesterase, butyrylcholinesterase, creatininase, creatinase, DNA polymerase, mutants of these enzymes, and the like can be used.

  As the hydrolase, for example, protease, lipase, amylase, invertase, maltase, β-galactosidase, lysozyme, urease, esterase, nuclease group, phosphatase group and the like can be used.

  As the transferase, for example, various acyltransferases, kinase groups, aminotransferase groups and the like can be used.

  As the isomerase, for example, racemase group, phosphoglycerate phosphomutase, glucose 6-phosphate isomerase and the like can be used.

  Here, as the particularly preferable enzyme 100, for example, various dehydrogenases can be mentioned.

The enzyme 100 contained in the detection unit 10 may be one type of enzyme or two or more types of enzymes.
Specifically, the molecular weight of the enzyme 100 contained in the detection unit 10 may be, for example, one kind of enzyme or two or more kinds of enzymes having substantially the same molecular weight and / or size (diameter). And / or two or more enzymes having different sizes. In addition, when there are two or more types of enzymes 100 contained in the detection unit 10, the enzymes 100 may be different types of specific substances even if they are two or more types of enzymes that act on the same type of specific substance (substrate). It may be two or more kinds of enzymes that act, or two or more kinds of enzymes that act on the same kind and / or different kinds of specific substances.

Further, when there are two or more types of enzymes 100 contained in the detection unit 10, the two or more types of enzymes are the same even if they are fixed inside separate pores in the silica-based mesoporous material 110. It may be fixed inside the hole.
Here, in particular, when there are two or more types of enzymes 100 contained in the detection unit 10 and the two or more types of enzymes act on different types of specific substances, the enzyme sensor 1 may use the different types of specific substances (2 More than one type of specific substance) can be detected simultaneously.

(Silica-based mesoporous material)
The silica-based mesoporous material 110 can be composed of, for example, various metal oxides such as silicic acid and alumina, complex oxides of silicic acid and other types of metals, and the like.
For example, in the production of the silica-based mesoporous material 110 composed of silicic acid, for example, layered silicate such as kanemite, alkoxysilane, silica gel, water glass, sodium silicate, and the like can be preferably used.

  Specifically, the silica-based mesoporous material 110 includes, for example, a surfactant / inorganic composite in which an inorganic material is mixed with a surfactant to form an inorganic skeleton around the surfactant micelles. After the formation, the surfactant is removed by, for example, baking at 400 ° C. to 600 ° C. or extracting with an organic solvent. As a result, the silica-based mesoporous material 110 has mesopore pores having the same shape as the micelles of the surfactant in the inorganic skeleton.

In the production of the silica-based mesoporous material 110, when a silicon-containing compound such as silicic acid is used as a starting material, for example, a layered silicate such as kanemite is formed, and micelles are inserted between the layers. The pores can be formed by connecting the silicate molecules between the layers in which no is present, and then removing the micelles.
Further, in the production of the silica-based mesoporous material 110, when a silicon-containing substance such as water glass is used as a starting material, for example, silica is formed by collecting and polymerizing silicate molecules around the micelle, and thereafter The pores can be formed by removing the micelles. In this case, the shape of the micelle is usually a columnar shape, and as a result, columnar pores are formed in the silica-based mesoporous material 110.

In the silica-based mesoporous material 110, the inner diameter of the pores can be controlled by changing the micelle diameter by changing the length of the alkyl chain of the surfactant in the production stage. In addition, by adding relatively hydrophobic molecules such as trimethylbenzene and tripropylbenzene together with the surfactant, the micelles can be swollen to form pores with a larger inner diameter.
The pore size (pore diameter) of the silica-based mesoporous material 110 is determined according to the size of the enzyme 100 to be immobilized (diameter of the enzyme 100). That is, for example, by producing the silica-based mesoporous material 110 using a surfactant having a micelle size (micelle diameter) of 0.5 to 2.0 times the size of the enzyme 100, A silica-based mesoporous material 110 whose size is 0.5 to 2.0 times the size of the enzyme 100 to be immobilized can be obtained.

Examples of the shape of the silica-based mesoporous material 110 include powder, granule, sheet, bulk, and film.
In addition, the orientation of the pores in the silica-based mesoporous material 110 may be random or may have a controlled orientation, such as an aggregate of one-dimensional silica nanochannels.

As the type of the silica-based mesoporous material 110, a known type such as KSW, FSM, SBA, MCM, HOM, etc., which has the characteristics that the pore size is uniform and has a large porosity, is adopted. can do.
Further, as a kind of the silica-based mesoporous material 110, for example, CTAB-M, P123 has the characteristics that the pore size is uniform and the direction of the pore (channel) is in one direction. Known types such as -M and F127-M can be employed. Specifically, CTAB-M, P123-M, F127-M, etc., for example, have the same channel direction as the direction of the alumina pores produced using a surfactant as a template in cylindrical alumina pores. This is a membranous silica-based mesoporous material 110 filled with mesoporous silica nanochannel aggregates (one-dimensional silica nanochannel aggregates).

The size of the pores of the silica-based mesoporous material 110 is set to such an extent that a change in the three-dimensional structure of the immobilized enzyme 100 can be prevented. Accordingly, the three-dimensional structure of the immobilized enzyme 100 can be maintained, and the enzyme 100 immobilized in the pores of the silica-based mesoporous material 110 can be stabilized.
Specifically, the size of the pores of the silica-based mesoporous material 110 is, for example, about 0.5 to 2.0 times the size of the immobilized enzyme 100 (enzyme molecule or enzyme fragment containing an active site). It is preferable that the size is about 0.7 to 1.4 times the size of the enzyme 100 to be immobilized, and most preferably about the same as the size of the enzyme 100 to be immobilized. That is, the pore diameter (center pore diameter) in the silica-based mesoporous material 110 is preferably about 0.5 to 2.0 times the diameter of the enzyme 100 to be immobilized. The diameter is more preferably about 0.7 to 1.4 times the diameter, and most preferably about the same as the diameter of the enzyme 100 to be immobilized. In addition, since the specific value of the center pore diameter is determined in relation to the diameter of the enzyme 100 and cannot be defined uniformly, it can be set to about 1 to 50 nm, for example.
Here, when the enzyme 100 forms a multimer, the size (diameter) of the enzyme 100 to be immobilized can be the size (diameter) of the multimer. Here, the multimer refers to a compound in which two or more enzymes (proteins) are bonded directly or via a small molecule such as water, and the bond includes a covalent bond, an ionic bond, a hydrogen bond, Coordination bonds are included. However, the type of these bonds is not particularly limited.

The specific surface area of the silica-based mesoporous material 110 is, for example, about 200 to 1500 m ² .
The pore depth of the silica-based mesoporous material 110 is 2 nm or more. Specifically, the preferable depth range is 20 to 1000 μm, the more preferable depth range is 50 to 500 nm, and the most preferable depth range is 50 to 150 nm.
As for the pitch of the pores of the silica-based mesoporous material 110, when the pitch of the pores is defined as the distance between the centers of the pores, the preferred pitch is 2 to 500 nm, the more preferred pitch is 2 to 100 nm, and the most preferred. The pitch is 2 to 50 nm.

  The size of the pores of the silica-based mesoporous material 110 is about 0.5 to 2.0 times (more preferably about 0.7 to 1.4 times, most preferably about the same) as the size of the enzyme 100 to be immobilized. By doing so, the three-dimensional structure of the enzyme 100 to be immobilized can be easily maintained, so that the enzyme 100 immobilized in the pores of the silica-based mesoporous material 110 can be stabilized.

  Specifically, for example, when formaldehyde dehydrogenase (diameter: about 8 nm) is employed as the enzyme 100, the size of the pores of the silica-based mesoporous material 110 is preferably set to a diameter of 0. 0 of the diameter of the formaldehyde dehydrogenase. It is about 5-2.0 times, more preferably about 0.7-1.4 times the diameter of formaldehyde dehydrogenase, most preferably about the same as the diameter of formaldehyde dehydrogenase. Thereby, for example, as shown in FIG. 2, the formaldehyde dehydrogenase is adsorbed and immobilized on the inner walls of the pores of the silica-based mesoporous material 110 while maintaining the three-dimensional structure.

  Also, for example, when using a membranous silica-based mesoporous material 110 as shown in FIG. 3 filled with a one-dimensional silica nanochannel aggregate, the pore size is preferably About 0.5 to 2.0 times the diameter of formaldehyde dehydrogenase, more preferably about 0.7 to 1.4 times the diameter of formaldehyde dehydrogenase, most preferably about the same as the diameter of formaldehyde dehydrogenase Set. Thereby, formaldehyde dehydrogenase is adsorbed and immobilized on the inner walls of the pores (channels) of the silica-based mesoporous material 110 while maintaining the three-dimensional structure.

(Electronic carrier)
The electron carrier 200 is for facilitating the delivery of electrons between the enzyme 100 and the electrode 20.
Note that the electron carrier 200 is optional as long as it is contained in the detection unit 10, and may be contained in the detection unit 10 in a state of being dissolved in a predetermined electrolyte introduced into the reactor 10a, for example. For example, the details of the silica-based mesoporous material 110 (silica-based mesoporous material 110 dispersed in a predetermined electrolyte introduced into the reactor 10a, or the silica-based mesoporous material 110 fixed to the electrode 20, etc.) It may be contained in the detection unit 10 in a state of being introduced into the hole, or may be contained in the detection unit 10 in a state of being directly fixed to the working electrode (electrode 20), for example.

Since the enzyme 100 is stably fixed inside the pores of the silica-based mesoporous material 110 whose pore size is about 0.5 to 2.0 times the size of the enzyme 100, the product is directly Except when oxidized or reduced at the electrode 20, it is difficult for the active center in the enzyme molecule to transfer electrons with the electrode 20. Further, when the enzyme 100 is fixed inside the pores of the silica-based mesoporous material 110 having pores with a large aspect ratio whose directionality is controlled, such as an aggregate of one-dimensional silica nanochannels, Even when the product is directly oxidized or reduced at the electrode 20, the response is very slow. Therefore, it is preferable to add the electron carrier 200 to the detection unit 10.
In addition, when an oxygen electrode, a hydrogen peroxide electrode, or the like is used as the electrode 20, the reaction is limited by the dissolved oxygen concentration and only a low concentration sample can be measured. The selectivity is poor due to the influence of an oxidizing substance such as ascorbic acid. Such problems arise. Also in this case, it is effective to use the electron carrier 200 together with the enzyme 100 for the purpose of expanding the detection range and improving the selectivity.

Here, it is known that the activity of the enzyme 100 decreases when a quinone compound such as naphthoquinone is used as an electron carrier. This is considered to be because a quinone compound such as naphthoquinone adsorbs to the active center of the enzyme 100.
Therefore, for example, the electron carrier 200 is less likely to be adsorbed to the active center of the enzyme 100 than the quinone-based compound such as naphthoquinone, and can suppress the decrease in the activity of the enzyme 100. The body is preferred.
Specific examples of the activity lowering suppression electron carrier include a part of electron carriers having a metallocene skeleton and an electron carrier adsorbed on a predetermined porous body.

Specific examples of the electron carrier having a metallocene skeleton include, for example, a compound represented by the above general formula (1), a compound represented by the above general formula (2), and the above general formula. The compound represented by (3) is mentioned.
Hereinafter, the compound represented by the above general formula (1), the compound represented by the above general formula (2), and the compound represented by the above general formula (3) are referred to as “a part of the metallocene skeleton. Sometimes called an "electron carrier". Of the electron carriers having a metallocene skeleton, the compound represented by the above general formula (1), the compound represented by the above general formula (2), and the compound represented by the above general formula (3) Other compounds may be referred to as “other electron carriers having a metallocene skeleton”.

Here, as a partly preferable electron carrier having a metallocene skeleton, for example, a ferrocene-based compound in which Me is Fe in the above general formulas (1), (2), and (3) can be given.
Ferrocene compounds in which Me is Fe in the above general formula (1) include ferrocenylmethyldodecyldimethylammonium bromide, ferrocenylmethyldodecyldimethylammonium iodide, ferrocenylmethyltrimethylammonium bromide, ferrocenylmethyltrimethyl. And ferrocenylmethyl ammonium salts such as ammonium iodide.
In addition, examples of the ferrocene-based compound in which Me is Fe in the general formula (2) include ferrocenecarboxylic acid.
Moreover, 1-1 'ferrocene dicarboxylic acid is mentioned as a ferrocene type compound whose Me is Fe in said general formula (3).

  That is, some electron carriers having a particularly preferred metallocene skeleton include ferrocenylmethyldodecyldimethylammonium bromide, ferrocenylmethyldodecyldimethylammonium iodide, ferrocenylmethyltrimethylammonium bromide, ferrocenylmethyltrimethylammonium. Examples thereof include ferrocenylmethylammonium salts such as iodide, ferrocenecarboxylic acid, and 1-1 ′ ferrocenedicarboxylic acid.

The electron carrier adsorbed on the predetermined porous body is specifically an electron carrier adsorbed on an arbitrary porous body such as a silica-based porous body or a carbon porous body.
Here, the electron carrier to be adsorbed to the predetermined porous body is arbitrary as long as it is an electron carrier. Specifically, for example, conductive polymers such as polyaniline, quinone and quinone derivatives (naphthoquinone, benzoquinone, etc.) ), Ferrocene compounds such as ferrocene and ferrocene derivatives, potassium ferricyanide, osmium complexes, and the like.

In addition, in the detection part 10, as the electron mediator 200, some electron mediators having a metallocene skeleton (a compound represented by the above general formula (1) or the above general formula (2) are represented. A compound, a compound represented by the above general formula (3), etc.) and / or an electron carrier adsorbed on a predetermined porous body may be added.
In addition, the type of some of the electron carriers having a metallocene skeleton added to the detection unit 10 may be one type or a plurality of types. The type of the electron carrier adsorbed on the predetermined porous body added to the detection unit 10 may be one type or plural types.

(Coenzyme)
The coenzyme 300 is for catalyzing the expression of the activity of the enzyme 100.
The coenzyme 300 is optional as long as it is contained in the detection unit 10, for example, it may be contained in the detection unit 10 in a state dissolved in a predetermined electrolytic solution introduced into the reactor 10a. For example, the pores of the silica-based mesoporous material 110 (the silica-based mesoporous material 110 dispersed in a predetermined electrolyte introduced into the reactor 10a, or the silica-based mesoporous material 110 fixed to the electrode 20, etc.) May be contained in the detection unit 10 in a state of being introduced into the inside of the electrode, or may be contained in the detection unit 10 in a state of being directly fixed to the working electrode (electrode 20), for example.
In addition, the coenzyme 300 is included in the detection unit 10 together with, for example, various metal atoms, metal ions, metal complexes, various dyes and the like (eg, Fe ²⁺ , Mn ²⁺ , Cu ²⁺ , Zn ²⁺ , Co ^3+, etc.) as cofactors. It may be contained.

  For example, in the case of a reaction that does not proceed easily by the catalysis of the amino acid side chain of enzyme 100, such as a reaction via an anxious intermediate, a low molecular weight organic small molecule that has an appropriate structure and is involved in the expression of the enzyme reaction And metal ions and metal complexes are often used as cofactors. Among the cofactors, small organic molecules and metal complexes are called coenzymes. In particular, when a coenzyme-dependent enzyme is used as the enzyme 100, the enzyme reaction can be efficiently performed by introducing the coenzyme 300 into the detection unit 10.

The coenzyme 300 can be appropriately selected depending on the type of the enzyme 100 (coenzyme-dependent enzyme). Specifically, examples of the coenzyme 300 include nicotinamide adenine dinucleotide (NAD ⁺ ), nicotinamide adenine dinucleotide phosphate (NADP ⁺ ), coenzyme I, coenzyme II, flavin mononucleotide (FMN), Flavin adenine dinucleotide (FAD), lipoic acid, adenosine triphosphate (ATP), thiamine pyrophosphate (TPP), pyridoxal phosphate (PALP), tetrahydrofolate (THF, Coenzyme F), UDP glucose (UDPG), coenzyme A , coenzyme Q, biotin, coenzyme B ₁₂ (cobalamin), one or more combinations of such S- adenosyl methionine.

Here, it is known that when a coenzyme 300 such as NAD ⁺ is used, the activity of the enzyme 100 immobilized in the pores of the silica-based mesoporous material 110 is reduced. This is presumably because the coenzyme 300 such as NAD ⁺ reacts with the silanol group on the surface of the silica-based mesoporous material 110 and affects the activity of the enzyme 100.
Therefore, it is preferable to add to the detection unit 10 a coenzyme activity decrease inhibiting substance for suppressing a decrease in the activity of the enzyme 100 that occurs when the coenzyme 300 is used.
Examples of the coenzyme activity decrease inhibitor include biopolymers, synthetic polymers, electrolytes, and the like.
Note that the coenzyme activity decrease inhibiting substance is optional as long as it is contained in the detection unit 10, for example, it is contained in the detection unit 10 in a state dissolved in a predetermined electrolytic solution introduced into the reactor 10a. Alternatively, for example, it may be contained in the detection unit 10 in a state of being introduced into the pores of the silica-based mesoporous material 110.

  The biopolymer may be any biopolymer having a molecular weight of 1000 or more, and specifically, for example, serum albumin, gelatin, cellulose, starch and the like.

  The synthetic polymer is arbitrary as long as it is a synthetic polymer having a molecular weight of 1000 or more, and specifically, for example, polyethylene glycol and dextran.

  The electrolyte may be any material as long as it dissolves in a predetermined electrolytic solution introduced into the reactor 10a and ionizes into a cation and an anion, and specifically, for example, potassium chloride. .

The detection unit 10 may be added with a biopolymer having a molecular weight of 1000 or more, a synthetic polymer having a molecular weight of 1000 or more, and / or an electrolyte as a coenzyme activity decrease inhibitor.
Moreover, the kind of biopolymer having a molecular weight of 1000 or more added to the detection unit 10 may be one kind or plural kinds. The type of synthetic polymer having a molecular weight of 1000 or more added to the detection unit 10 may be one type or plural types. The type of electrolyte added to the detection unit 10 may be one type or a plurality of types.

(electrode)
The electrode system of the enzyme sensor 1 may employ either a bipolar system with a working electrode and a reference electrode or a tripolar system with a working electrode, a control electrode, and a reference electrode.
As the electrode 20 that is a working electrode, for example, a noble metal such as gold, platinum, copper, and aluminum, SnO ₂ , In ₂ O ₃ , WO ₃ , TiO ₂ , graphite, glassy carbon, or the like can be used.
As the reference electrode 30, for example, silver can be used in the case of the bipolar system, and silver or other metals can be used in the case of the tripolar system.
As the reference electrode 40, for example, a calomel electrode, silver / silver chloride, or the like can be used.

Examples of the working electrode (electrode 20), the reference electrode 30, and the reference electrode 40 include, for example, electrodes used in an electrolysis cell as shown in FIG. 1, but these electrodes have a size and a shape. The configuration is not particularly limited.
Specifically, for example, these electrodes may be large electrodes used in commercially available electrolytic cells, measurement cells, etc., or may be disk electrodes, rotating ring disk electrodes, fiber electrodes, etc. For example, microelectrodes (disc electrodes, cylindrical electrodes, strip electrodes, array strip electrodes, array disc electrodes, ring electrodes, spherical electrodes, comb electrodes, pair electrodes, etc.) manufactured by known microfabrication techniques such as photolithography. May be.

Further, these electrodes can be provided on a predetermined insulating substrate.
Specifically, each electrode can be formed on an insulating substrate by a known method, for example, a screen printing method, a vapor deposition method, a sputtering method, or the like. In addition, all the electrodes may be formed on the same substrate and manufactured as an integrated enzyme sensor 1, or may be formed on a plurality of substrates to constitute the enzyme sensor 1, or separate electrodes. And the enzyme sensor 1 may be configured.
As the insulating substrate, for example, ceramics, glass, plastic, paper, biodegradable material (for example, microorganism-produced polyester) and the like can be used.

(Sensing method)
As a sensing method using the enzyme sensor 1, for example, an electrochemical measurement method can be used. That is, a known measurement method such as a chronoamperometry method, a coulometry method, or a cyclic voltammetry method for measuring an oxidation current or a reduction current can be applied. As a measurement method, any of a disposable method, a batch method, a flow injection method, and the like may be used.

In the electrochemical measurement method, it is also possible to apply a light detection measurement method that detects a color change obtained by oxidizing or reducing a color source.
Specifically, for example, in a glucose sensor, two types of enzymes, glucose oxidase and peroxidase, are immobilized, and an enzyme reaction (“glucose + O _2- (glucose oxidase) → gluconic acid + H ₂ O ₂ ”, “H ₂ O”). ₂ + color source-(peroxidase-> magenta dye)) and the hydrogen source and color source (for example, 4-aminoantipyrine and N-ethyl-N (2-hydroxy-3-sulfo). Glucose concentration can be measured by detecting a color change in which a mixture of propyl) -m-toluidine) becomes reddish purple with peroxidase as a catalyst.

  The measuring instrument main body to which the enzyme sensor 1 is attached at the time of measurement preferably has a function capable of transmitting data to a personal computer by wire or wireless, and preferably can confirm the measured value in real time. In addition, it is desirable that a plurality of types of enzyme sensors 1 can be attached, and that the detection results of the plurality of types of enzyme sensors 1 can be simultaneously measured, and data can be compared and examined.

  Hereinafter, the present invention will be described with reference to specific examples, but the present invention is not limited thereto. In Example 1, an experiment for evaluating the activity of the enzyme 100 was performed.

(1) Synthesis of silica-based mesoporous material 110 First, the silica-based mesoporous material 110 was synthesized.
Specifically, 271.59 g of water glass 1 was mixed with 828.41 g of water, and then heated to 80 ° C. Separately, 80 g of docosyltrimethylammonium chloride (DTMA-Cl) was added to 1 L of water at 70 ° C., and after becoming a completely transparent liquid, 70 mL of triisopyrbenzene was further added and vigorously stirred with a homomixer for 30 minutes. This emulsion was immediately added to the water glass solution and further stirred for 5 minutes. To this was added 2N hydrochloric acid over about 1 hour, and the mixture was stirred at pH 8.5 for about 3 hours. This was subjected to suction filtration, and then redispersed and filtered in hot water at 70 ° C. repeatedly. This was dried at 45 ° C. for 3 days, and then fired in an electric furnace at 550 ° C. for 6 hours to obtain a white powdery silica-based mesoporous material 110.
The structure of this white powder was measured using a powder X-ray diffractometer (Science RAD-B apparatus), and the pore diameter, surface area, and total pore volume were measured using a nitrogen adsorption device. The powder was confirmed to be a porous mesoporous silica material having a two-dimensional hexagonal pore arrangement structure with a diameter of about 8.2 nm. The obtained silica-based mesoporous material 110 may be hereinafter referred to as “large-diameter FSM”.

(2) Immobilization of enzyme 100 Next, the enzyme 100 was immobilized on the silica-based mesoporous material 110. Here, formaldehyde dehydrogenase was used as the enzyme 100.
Specifically, 50 mg of large-diameter FSM powder and 5 mL of an aqueous solution of formaldehyde dehydrogenase (phosphate buffer pH 6.9) having a concentration of formaldehyde dehydrogenase of 4.2 mg / mL were mixed, and a rotating device ( The mixture was gently stirred at 4 ° C. for 24 hours. Then, this liquid mixture was centrifuged at 7000 rpm for 3 minutes, solid content was collect | recovered, and washing | cleaning by 5 mL of deionized water, and the centrifugation on the same conditions were repeated 3 times. As a result, formaldehyde dehydrogenase was immobilized on the large-diameter FSM. The obtained substance, that is, the large-diameter FSM on which formaldehyde dehydrogenase is immobilized may be hereinafter referred to as “FDH-immobilized FSM”.
Furthermore, when the amount of formaldehyde dehydrogenase adsorbed on the large-diameter FSM was measured using the supernatant obtained by the above centrifugation, the amount adsorbed on the large-diameter FSM was 132 mg / g.

(3) Activity test of enzyme 100 Next, the activity of the enzyme 100 was measured.
Here, a reaction in which formaldehyde dehydrogenase oxidizes formaldehyde as a substrate to formic acid in the presence of a coenzyme (NAD ⁺ ) (HCHO + NAD ⁺ + 3H ₂ O − (formaldehyde dehydrogenase) → HCOO ⁻ + NADH + 2H ₃ O ⁺ ) was used to measure the activity of enzyme 100. That is, a wavelength 340 nm (ε ₃₄₀ = 6222 M ⁻¹ cm ⁻¹ ) characteristic of NADH (reduced type) generated by reduction of NAD ⁺ (oxidized type) is selected, and the generated NADH concentration is obtained from the change in absorbance. Was used to measure the activity of enzyme 100.

(3-1) Activity of Immobilized Enzyme First, the activity of the enzyme 100 immobilized inside the pores of the silica-based mesoporous material 110 was measured.
Specifically, 0.64 mg of the FDH-immobilized FSM prepared above was added and dispersed in 2.5 mL of phosphate buffer (pH = 7.41), and 1.2 mg of coenzyme (NAD ⁺ ) and 300 μL. A substrate aqueous solution (formaldehyde aqueous solution having a formaldehyde concentration of 0.3%) was added and reacted at 30 ° C. Then, the absorbance at 340 nm was measured from when the coenzyme and the aqueous substrate solution were added until the reaction reached equilibrium. For comparison, the same measurement was performed for free enzyme (free formaldehyde dehydrogenase). The enzyme amounts of the enzyme 100 fixed to the large-diameter FSM used for the measurement and the free enzyme were set to be the same. The result is shown in FIG.

  In FIG. 4, the horizontal axis represents the reaction time, and the vertical axis represents the absorbance at 340 nm. The solid line indicates the enzyme 100 (immobilized enzyme) immobilized on the large-diameter FSM, and the broken line indicates the free enzyme.

According to FIG. 4, it was found that the enzyme 100 fixed to the large-diameter FSM proceeds to the same extent as the free enzyme.
Thereby, it was found that the enzyme 100 fixed inside the pores of the large-diameter FSM maintained the activity without being denatured.

(3-2) Time-dependent change in activity of immobilized enzyme Next, the time-dependent change in activity of the enzyme 100 immobilized in the pores of the silica-based mesoporous material 110 was measured.

  Specifically, first, samples [1] to [4] were prepared.

  Sample [1] was dispersed in 5 mL of phosphate buffer solution (pH = 7.41) by adding 56.6 mg of the FDH-immobilized FSM prepared above and gently applied to a rotating device (about 100 rpm) at 4 ° C. for 1 hour. Created by stirring.

Sample [2] was dispersed by adding 56.6 mg of the FDH-immobilized FSM prepared above to 5 mL of phosphate buffer (pH = 7.41), adding 1.8 mg of NAD ⁺ , and rotating the apparatus (about 100 rpm) and gently stirred at 4 ° C. for 1 hour.

  Sample [3] was dispersed by adding 56.6 mg of the FDH-immobilized FSM prepared above to 5 mL of phosphate buffer (pH = 7.41), adding 1.3 mg of naphthoquinone, and rotating the apparatus (about 100 rpm). ) And gently stirring at 4 ° C. for 1 hour.

Sample [4] was prepared by adding 56.6 mg of the FDH-immobilized FSM prepared above to 5 mL of phosphate buffer (pH = 7.41) and dispersing 1.8 mg of NAD ⁺ and 1.3 mg of naphthoquinone. It was made by adding and gently stirring at 4 ° C. for 1 hour on a rotating device (about 100 rpm).

Next, 1.2 mg of coenzyme (NAD ⁺ ) and 300 μL of a substrate aqueous solution (formaldehyde aqueous solution having a formaldehyde concentration of 0.3%) were added to each of the prepared samples and reacted at 30 ° C. . Then, the absorbance at 340 nm was measured from when the coenzyme and the aqueous substrate solution were added until the reaction reached equilibrium. Thereafter, each of the samples used for the absorbance measurement was stored at 30 ° C., and the same measurement was performed after 3, 7, 9, 13, 16, and 22 days. The result is shown in FIG.

  In FIG. 5, the horizontal axis represents the number of days, and the vertical axis represents the relative activity when the enzyme activity on the first day (day 0) is 100%, that is, the absorbance at 340 nm in the equilibrium state on the first day is 100%. Relative absorbance is shown. Circle plot (○) shows the result of sample [1], triangle plot (△) shows the result of sample [2], square plot (□) shows the result of sample [3], diamond plot (◇) shows the sample [4] The results are shown.

According to FIG. 5, it was found that the sample [1], that is, the enzyme 100 immobilized inside the pores of the silica-based mesoporous material 110, maintained an enzyme activity of 80% or more even after 20 days.
Thereby, it turned out that the fall of the activity of the enzyme 100 can be suppressed by fixing the enzyme 100 inside the pore of the silica-type mesoporous material 110. FIG.

Further, according to FIG. 5, the sample with the NAD ⁺ added (sample [2]) has a greater degree of decrease in enzyme activity than the sample without the NAD ⁺ added (sample [1]). I understood.
This is presumably because NAD ⁺ reacts with silanol groups on the surface of the large-diameter FSM to affect the enzyme activity.

Further, according to FIG. 5, it is found that the sample (sample [3]) to which naphthoquinone is added has a greater degree of decrease in enzyme activity than the sample to which naphthoquinone is not added (sample [1]). It was. Furthermore, the sample (sample [3]) to which naphthoquinone was added was found to have a greater degree of decrease in enzyme activity than the sample to which NAD ⁺ was added (sample [2]).
This is probably because naphthoquinone is adsorbed on the active center of the enzyme 100 in the FDH-immobilized FSM to inhibit the activity.

In addition, according to FIG. 5, the samples with added NAD ⁺ and naphthoquinone (Sample [4]), as compared to the sample without the addition of NAD ⁺ and naphthoquinone (Sample [1]), the decrease in enzyme activity It turns out that the degree is large. In addition, the sample to which NAD ⁺ and naphthoquinone were added (sample [4]) had a greater degree of decrease in enzyme activity than the sample to which only NAD ⁺ was added (sample [2]), and only naphthoquinone was added. It was found that the degree of decrease in enzyme activity was small compared to the sample (Sample [3]).
This is considered to be because the adsorption of naphthoquinone to the active center of the enzyme 100 was suppressed by the presence of NAD ⁺ near the active center of the enzyme 100.

(3-3) NAD ⁺ and time course of the activity of the immobilized enzyme with the addition of coenzyme for activity reduction inhibitor Then, NAD ⁺ and the coenzyme for activity reduction suppressing agent (electrolyte, a molecular weight of 1,000 or more biopolymers The time course of the activity of the enzyme 100 immobilized in the pores of the silica-based mesoporous material 110 with the addition of a synthetic polymer having a molecular weight of 1000 or more was measured.

  Specifically, first, samples [5] to [7] were prepared. Sample [8] was prepared for comparison.

Sample [5] was dispersed by adding 56.6 mg of the FDH-immobilized FSM prepared above to 5 mL of phosphate buffer (pH = 7.41), adding 1.8 mg of NAD ⁺ and electrolyte (12 mg). Of potassium chloride) was added, and the mixture was gently stirred for 1 hour at 4 ° C. on a rotating apparatus (about 100 rpm).

Sample [6] was dispersed by adding 56.6 mg of the FDH-immobilized FSM prepared above to 5 mL of phosphate buffer (pH = 7.41), adding 1.8 mg of NAD ^+, and having a molecular weight of 1000 or more. The biopolymer (20 mg of bovine serum albumin) was added, and the mixture was gently stirred at 4 ° C. for 1 hour on a rotating device (about 100 rpm).

Sample [7] was dispersed by adding 56.6 mg of the FDH-immobilized FSM prepared above in 5 mL of phosphate buffer (pH = 7.41), adding 1.8 mg of NAD ^+, and having a molecular weight of 1000 or more. The synthetic polymer (20 mg of dextran) was added, and the mixture was gently stirred at 4 ° C. for 1 hour on a rotating device (about 100 rpm).

For sample [8], an amino group-modified FSM in which formaldehyde dehydrogenase was immobilized was prepared, and the prepared amino group-modified FSM was added to 5 mL of phosphate buffer (pH = 7.41) and dispersed. It was prepared by adding 1.8 mg of NAD ⁺ and gently stirring at 4 ° C. for 1 hour on a rotator (about 100 rpm).
Specifically, 2 mL of 3-APTES and 18 mL of ethanol were added to 50 mg of the large-diameter FSM prepared above and gently stirred for 3 hours to modify the amino group on the silanol group on the surface of the large-diameter FSM. Subsequently, it was washed with dehydrated ethanol and dried overnight at 45 ° C. to prepare an amino group-modified FSM. Then, the prepared amino group-modified FSM powder was mixed with 5 mL of an aqueous formaldehyde dehydrogenase solution (phosphate buffer pH 6.9) having a concentration of formaldehyde dehydrogenase of 4.2 mg / mL, and a rotating device (about The mixture was gently stirred at 4 ° C. for 24 hours. Then, this liquid mixture was centrifuged at 7000 rpm for 3 minutes, solid content was collect | recovered, and washing | cleaning by 5 mL of deionized water, and the centrifugation on the same conditions were repeated 3 times. As a result, an amino group-modified FSM in which formaldehyde dehydrogenase was immobilized was prepared.

Next, 1.2 mg of coenzyme (NAD ⁺ ) and 300 μL of a substrate aqueous solution (formaldehyde aqueous solution having a formaldehyde concentration of 0.3%) were added to each of the prepared samples and reacted at 30 ° C. . Then, the absorbance at 340 nm was measured from when the coenzyme and the aqueous substrate solution were added until the reaction reached equilibrium. Then, each sample used for absorbance measurement was stored at 30 ° C., and the same measurement was performed after 1 day, 2 days, 3 days, 7 days, 10 days, 21 days, and 34 days. The result is shown in FIG.

  In FIG. 6, the horizontal axis represents the number of days, the vertical axis represents the relative activity when the enzyme activity on the first day (day 0) is 100%, that is, the absorbance at 340 nm in the equilibrium state on the first day is 100%. Relative absorbance is shown. Circle plot (○) shows the result of sample [5], triangle plot (△) shows the result of sample [6], square plot (□) shows the result of sample [7], diamond plot (◇) shows the sample [8] The results are shown.

According to FIG. 6, in the sample (sample [6]) to which a biopolymer (bovine serum albumin) having a molecular weight of 1000 or more is added, the enzyme activity does not decrease with the addition of NAD ⁺ during the measurement period. I understood.
Further, according to FIG. 6, NAD ⁺ is added to the sample (sample [5]) to which the electrolyte (potassium chloride) is added and the sample to which the synthetic polymer (dextran) having a molecular weight of 1000 or more (sample [7]) is added. It was found that the decrease in enzyme activity associated with was suppressed.
As a result, cations generated by ionization of the electrolyte, biopolymers having a molecular weight of 1000 or more, and synthetic polymers having a molecular weight of 1000 or more are adsorbed on the surface of the large-diameter FSM, and NAD ⁺ reacts with silanol groups on the surface of the large-diameter FSM. It was found that the decrease in enzyme activity accompanying the addition of NAD ⁺ is suppressed. Among them, it was found that a biopolymer (bovine serum albumin) having a molecular weight of 1000 or more can suitably suppress NAD ⁺ from reacting with a silanol group on the surface of a large-diameter FSM.

(3-4) Time-dependent change in the activity of the immobilized enzyme accompanying the addition of the activity-reduction-suppressing electron carrier Next, the activity-reduction-suppressing electron carrier (some electron carriers having a metallocene skeleton (above-mentioned) A compound represented by the general formula (1), a compound represented by the above general formula (2), a compound represented by the above general formula (3), and the like. The time course of the activity of the enzyme 100 immobilized inside the pores of the silica-based mesoporous material 110 was measured.

  Specifically, first, samples [9] to [13], [15], and [16] were prepared. Samples [14], [17], and [18] were prepared for comparison.

  Sample [9] was prepared by adding 56.6 mg of the FDH-immobilized FSM prepared above to 5 mL of phosphate buffer (pH = 7.41) and dispersing the sample to disperse some electron carriers having a metallocene skeleton (1.2 mg). Of ferrocenecarboxylic acid) was added, and the mixture was gently stirred at 4 ° C. for 1 hour on a rotating apparatus (about 100 rpm).

  Sample [10] was prepared by adding 56.6 mg of the FDH-immobilized FSM prepared above to 5 mL of phosphate buffer (pH = 7.41) and dispersing it, and then transferring another electron carrier (1.1 mg of metallocene skeleton). Hydroxymethylferrocene) was added, and the mixture was gently stirred for 1 hour at 4 ° C. on a rotating apparatus (about 100 rpm).

  Sample [11] was prepared by adding 56.6 mg of the FDH-immobilized FSM prepared above to 5 mL of a phosphate buffer (pH = 7.41) and dispersing it, and dispersing another electron carrier (1.2 mg of metallocene skeleton). Dimethylaminomethylferrocene) was added and the mixture was gently stirred for 1 hour at 4 ° C. on a rotating device (about 100 rpm).

  Sample [12] was prepared by dispersing 56.6 mg of the FDH-immobilized FSM prepared above in 5 mL of phosphate buffer (pH = 7.41), and dispersing the other electron carrier (1.7 mg of metallocene skeleton). (η-benzenehexafluorophosphate) was added and the mixture was gently stirred for 1 hour at 4 ° C. on a rotating apparatus (about 100 rpm).

  Sample [13] was prepared by adding 56.6 mg of the FDH-immobilized FSM prepared above to 5 mL of phosphate buffer (pH = 7.41) and dispersing the sample, and transferring a part of the electron carrier (1.7 mg) having a metallocene skeleton. Of ferrocenylmethyltrimethylammonium bromide) was added to the rotator (about 100 rpm) and gently stirred at 4 ° C. for 1 hour.

  Sample [14] was prepared by adding 56.6 mg of the FDH-immobilized FSM prepared above to 5 mL of a phosphate buffer (pH = 7.41) and dispersing it, and using a metal complex (3.5 mg of ruthenium red) as an electron carrier. Was added to the rotator (about 100 rpm) and stirred gently at 4 ° C. for 1 hour.

  Sample [15] is a residue obtained by adsorbing a quinone compound (1.3 mg of naphthoquinone) as an electron carrier to a predetermined porous body (10 mg of molecular sieve (pore diameter: 1 nm)) and coloring with quinone by centrifugation. By extracting the product, a naphthoquinone adsorption molecular sieve was prepared. Then, 56.6 mg of the FDH-immobilized FSM prepared above is added to 5 mL of phosphate buffer (pH = 7.41), and all of the prepared naphthoquinone adsorption molecular sieve is added and dispersed, and then applied to a rotating device (about 100 rpm). It was prepared by gently stirring at 4 ° C. for 1 hour.

  Sample [16] was prepared by adsorbing a conductive polymer (2.0 mg of polyaniline) as an electron carrier to a predetermined porous body (8.0 mg of carbon porous body), thereby preparing a polyarinin-adsorbing carbon porous body. . Then, 56.6 mg of the FDH-immobilized FSM prepared above was added to 5 mL of phosphate buffer (pH = 7.41), and all of the prepared polyarinin-adsorbed carbon porous material was added and dispersed, and a rotating device (about 100 rpm) And then gently stirring at 4 ° C. for 1 hour.

  Sample [17] was prepared in the same manner as Sample [8] to prepare an amino group-modified FSM in which formaldehyde dehydrogenase was immobilized, and the prepared amino group was added to 5 mL of phosphate buffer (pH = 7.41). The modified FSM was added and dispersed, and 1.3 mg of naphthoquinone was added, and the mixture was gently stirred at 4 ° C. for 1 hour on a rotating device (about 100 rpm).

  Sample [18] was dispersed by adding 56.6 mg of the FDH-immobilized FSM prepared above to 5 mL of phosphate buffer (pH = 7.41), adding 1.3 mg of naphthoquinone, and rotating the apparatus (about 100 rpm). ) And gently stirring at 4 ° C. for 1 hour.

Next, 1.2 mg of coenzyme (NAD ⁺ ) and 300 μL of a substrate aqueous solution (formaldehyde aqueous solution having a formaldehyde concentration of 0.3%) were added to each of the prepared samples and reacted at 30 ° C. . Then, the absorbance at 340 nm was measured from when the coenzyme and the aqueous substrate solution were added until the reaction reached equilibrium. Then, each sample used for absorbance measurement was stored at 30 ° C., and the same measurement was performed after 1 day, 2 days, 3 days, 7 days, 10 days, 21 days, and 34 days. The result is shown in FIG.

  In FIG. 7, the horizontal axis represents the number of days and the vertical axis represents the relative activity when the enzyme activity on the first day (day 0) is 100%, that is, the absorbance at 340 nm in the equilibrium state on the first day is 100%. Relative absorbance is shown. The white circle plot (◯) is the result of sample [9], the white triangle plot (Δ) is the result of sample [10], the white square plot (□) is the result of sample [11], and the white diamond plot (◇) is the sample [9]. 12] As a result, the white inverted triangle plot (▽) is the result of sample [13], the black circle plot (●) is the result of sample [14], the black triangle plot (▲) is the result of sample [15], and the black square plot (■) shows the result of sample [16], black diamond-shaped plot (♦) shows the result of sample [17], and black inverted triangle plot (▼) shows the result of sample [18].

According to FIG. 7, a sample to which an electron carrier (quinone compound) adsorbed on a predetermined porous body is added as the electron carrier 200, that is, a sample to which a naphthoquinone adsorption molecular sieve is added (sample [15]), In a sample to which an electron carrier (conductive polymer) adsorbed on a predetermined porous body is added as the electron carrier 200, that is, a sample to which a polyarinin-adsorbed carbon porous body is added (sample [16]), the enzyme activity decreases. It was found that the sample [16] maintained about 90% of the enzyme activity even after 30 days, and the sample [15] maintained about 80% of the enzyme activity after 30 days.
Thus, an electron carrier such as a quinone compound or a conductive polymer is adsorbed to a predetermined porous body, so that an electron carrier such as a quinone compound or a conductive polymer is difficult to adsorb on the active center of the enzyme 100. Thus, it was found that the decrease in enzyme activity was suppressed.

Further, according to FIG. 7, among ferrocene compounds, a sample to which a part of an electron carrier having a metallocene skeleton was added (that is, a sample to which ferrocenecarboxylic acid was added (sample [9]), ferrocenylmethyltrimethyl, In the sample to which ammonium bromide was added (sample [13]), it was found that the decrease in enzyme activity was suppressed. In particular, it was found that sample [13] retained about 70% enzyme activity even after 20 days, and sample [9] retained about 50% enzyme activity after 20 days.
Thereby, it was found that some electron carriers having a metallocene skeleton are less likely to be adsorbed to the active center of the enzyme 100 and less likely to cause a decrease in enzyme activity than quinone compounds such as naphthaquinone.
On the other hand, among ferrocene compounds, a sample to which another electron carrier having a metallocene skeleton is added (that is, a sample to which hydroxymethylferrocene is added (sample [10]), a sample to which dimethylaminomethylferrocene is added (sample [11] ]), A sample to which η-benzenehexafluorophosphate was added (sample [12])) was found to cause a decrease in enzyme activity and long-term stability was not expected.

(3-5) NAD ⁺ , Coenzyme Activity Decrease Inhibitor, and Change in Activity of Immobilized Enzyme Accompanying Addition of Activity Decrease Inhibition Type Electron Carrier Next, NAD ⁺ A biopolymer having a molecular weight of 1000 or more, a synthetic polymer having a molecular weight of 1000 or more) and an activity-reduction-suppressing electron carrier (some electron carriers having a metallocene skeleton, an electron carrier adsorbed on a predetermined porous body) The change over time of the activity of the enzyme 100 immobilized inside the pores of the silica-based mesoporous material 110 accompanying the addition was measured.

  Specifically, first, samples [19] to [22] were prepared. A sample [23] was prepared for comparison.

Sample [19] was dispersed by adding 56.6 mg of the FDH-immobilized FSM prepared above to 5 mL of phosphate buffer (pH = 7.41), adding 1.8 mg of NAD ^+, and having a molecular weight of 1000 or more. Of a biopolymer (20 mg bovine serum albumin) and a part of an electron carrier having a metallocene skeleton (1.2 mg of ferrocenecarboxylic acid) were added and gently stirred at 4 ° C. for 1 hour on a rotating device (about 100 rpm). Created by.

Sample [20] was dispersed by adding 56.6 mg of the FDH-immobilized FSM prepared above to 5 mL of phosphate buffer (pH = 7.41), adding 1.8 mg of NAD ^+, and having a molecular weight of 1000 or more. Of biopolymer (20 mg bovine serum albumin) and a part of electron carrier with metallocene skeleton (1.7 mg ferrocenylmethyltrimethylammonium bromide) are added to a rotating device (about 100 rpm) at 4 ° C. It was created by stirring gently for a time.

Sample [21] was prepared by adsorbing a conductive polymer (2.0 mg of polyaniline) as an electron carrier to a predetermined porous body (8.0 mg of carbon porous body), thereby preparing a polyarinin-adsorbing carbon porous body. . Then, 56.6 mg of the FDH-immobilized FSM prepared above was added to 5 mL of phosphate buffer (pH = 7.41), 1.8 mg of NAD ⁺ was added, and a biopolymer having a molecular weight of 1000 or more (20 mg of 20 mg Bovine serum albumin) and all of the prepared polyarinin-adsorbed carbon porous material were added and dispersed, and the mixture was prepared by gently stirring at 4 ° C. for 1 hour on a rotating device (about 100 rpm).

Sample [22] was dispersed by adding 56.6 mg of the FDH-immobilized FSM prepared above to 5 mL of phosphate buffer (pH = 7.41), adding 1.8 mg of NAD ^+, and having a molecular weight of 1000 or more. Of a synthetic polymer (20 mg of polyethylene glycol) and a part of an electron carrier having a metallocene skeleton (1.8 mg of ferrocenylmethyltrimethylammonium bromide) were added to a rotating apparatus (about 100 rpm) at 4 ° C. for 1 hour. It was created by gently stirring.

Sample [23] was dispersed by adding 56.6 mg of the FDH-immobilized FSM prepared above in 5 mL of phosphate buffer (pH = 7.41), adding 1.8 mg of NAD ⁺ and 1.3 mg Of naphthoquinone was added, and the mixture was gently stirred for 1 hour at 4 ° C. on a rotating device (about 100 rpm).

Next, 1.2 mg of coenzyme (NAD ⁺ ) and 300 μL of a substrate aqueous solution (formaldehyde aqueous solution having a formaldehyde concentration of 0.3%) were added to each of the prepared samples and reacted at 30 ° C. . Then, the absorbance at 340 nm was measured from when the coenzyme and the aqueous substrate solution were added until the reaction reached equilibrium. Thereafter, each sample used for absorbance measurement was stored at 30 ° C., and the same measurement was performed after 1 day, 2 days, 3 days, 6 days, 15 days, 22 days and 28 days. The result is shown in FIG.

  In FIG. 8, the horizontal axis represents the number of days, the vertical axis represents the relative activity when the enzyme activity on the first day (day 0) is 100%, that is, the absorbance at 340 nm in the equilibrium state on the first day is 100%. Relative absorbance is shown. Circle plot (◯) shows the result of sample [19], triangle plot (△) shows the result of sample [20], square plot (□) shows the result of sample [21], diamond plot (◇) shows the sample [22] As a result, the inverted triangle plot (▽) shows the result of the sample [23].

  According to FIG. 8, the coenzyme activity decrease inhibitory substance (molecular weight 1000 or more) compared to the sample not added with the coenzyme activity decrease inhibitory substance and the activity decrease inhibitory electron carrier (sample [23]). Of biopolymers, synthetic polymers having a molecular weight of 1000 or more) and activity-suppressing electron carriers (some electron carriers having a metallocene skeleton, electron carriers adsorbed on a predetermined porous body) That is, adsorbed to samples (samples [19] and [20]) to which a part of an electron carrier having a molecular weight of 1000 or more and a metallocene skeleton is added, a biopolymer having a molecular weight of 1000 or more, and a predetermined porous body In the sample (sample [21]) with the added electron carrier added, the sample with the synthetic polymer having a molecular weight of 1000 or more and a part of the electron carrier having the metallocene skeleton (sample [22]), the enzyme activity decreased. Is depressed It was found that 90% or more of the enzyme activity was maintained after 20 days.

According to the enzyme sensor 1 of the present invention described above, the detection unit 10 containing the enzyme 100 and the electron carrier 200 is provided, and the working electrode (electrode 20) is arranged in the detection unit 10 to provide an electron. The transmission body 200 is an electron transmission body adsorbed on a predetermined porous body.
Therefore, by adsorbing the electron carrier to a predetermined porous body, it is possible to suppress the adsorption of the electron carrier to the active center of the enzyme 100. Therefore, even when the electron carrier 200 is used, Even when stored in a buffer solution in which the electron carrier 200 is dissolved, the enzyme sensor 1 having excellent long-term stability can be provided.
Here, the electron carrier adsorbed by the predetermined porous body is fixed in the vicinity of the electrode 20 together with the silica-based mesoporous body 110 to which the enzyme 100 is fixed, from the viewpoint of increasing the sensitivity of the enzyme sensor 1 and the like. It is preferable to contain in the detection part 10. Specifically, for example, the porous body on which the electron carrier is adsorbed and the silica-based mesoporous body 110 on which the enzyme 100 is fixed are mixed or stacked to be fixed to the electrode 20. preferable. Any immobilization method may be used. For example, a nylon mesh and an O-ring may be used to tightly fix them on the electrode 20, or a known immobilization method may be used.

Moreover, according to the enzyme sensor 1 of the present invention described above, the detection unit 10 including the enzyme 100 and the electron carrier 200 is provided, and the working electrode (electrode 20) is disposed in the detection unit 10. The electron carrier 200 is a part of an electron carrier having a metallocene skeleton (a compound represented by the above general formula (1), a compound represented by the above general formula (2), the above general formula (3 ).
Therefore, a compound having a metallocene skeleton having a structure having a quaternary ammonium base as a side chain (a compound represented by the above general formula (1)) or a compound having a metallocene skeleton having a structure having a carboxyl group as a side chain (Compound represented by the above general formula (2), compound represented by the above general formula (3)) adsorbs to the active center of the enzyme 100 as compared with an electron carrier such as a quinone compound. Even if it is a case where the electron carrier 200 is used and it is the case where it preserve | saves in the buffer solution in which the electron carrier 200 melt | dissolves, the enzyme sensor 1 which has the outstanding long-term stability is provided. be able to.

In addition, according to the enzyme sensor 1 of the present invention described above, the detection unit 10 containing the enzyme 100 and the coenzyme 300 is provided, and the enzyme 100 is fixed inside the pores of the silica-based mesoporous material 110. The detection unit 10 is added with a biopolymer having a molecular weight of 1000 or more, a synthetic polymer having a molecular weight of 1000 or more, and / or an electrolyte.
That is, by fixing the enzyme 100 inside the pores of the silica-based mesoporous material 110, it is possible to prevent a change in the three-dimensional structure of the enzyme 100 accompanying a change in the external environment. Can be suppressed.
Furthermore, it has been found that when the coenzyme 300 coexists with the silica-based mesoporous material 110, the coenzyme 300 is adsorbed on the surface of the silica-based mesoporous material 110 and causes a decrease in the activity of the enzyme 100. A bioenzyme having a molecular weight of 1000 or more and a molecular weight of 1000 or more are added to the detection unit 10 by adding a substance for inhibiting the decrease in coenzyme activity (a biopolymer having a molecular weight of 1000 or more, a synthetic polymer and / or an electrolyte having a molecular weight of 1000 or more) Ions generated by ionization of the above synthetic polymer and / or electrolyte are adsorbed on the surface of the silica-based mesoporous material 110 and the adsorption of the coenzyme 300 to the surface of the silica-based mesoporous material 110 is suppressed. Even when coenzyme 300 is used by adding an enzyme activity decrease inhibitor, it is also stored in a buffer solution in which coenzyme 300 is dissolved. Even when an, it is possible to suppress the reduction of the activity of the enzyme 100.
Therefore, the enzyme sensor 1 having excellent long-term stability can be provided.
The coenzyme activity decrease inhibitor was adsorbed on the surface of the silica-based mesoporous material 110, thereby suppressing the adsorption of the coenzyme 300 to the surface of the silica-based mesoporous material 110. The adsorption of the coenzyme 300 to the surface of the silica mesoporous material 110 may be suppressed by modifying the surface of the silica mesoporous material 110 with, for example.

In addition, according to the enzyme sensor 1 of the present invention described above, the silica mesoporous material 110 and the enzyme 100 fixed inside the pores of the silica mesoporous material 110 are provided, and the silica mesoporous material. The size of the 110 pores is set to 0.5 to 2.0 times the size of the enzyme 100.
Therefore, the enzyme 100 can be firmly fixed inside the pores of the silica-based mesoporous material 110, and the enzyme 100 can be fixed with a high degree of freedom, thereby preventing a change in the three-dimensional structure of the enzyme 100. Thus, a decrease in the activity of the enzyme 100 can be suppressed.
Further, the silica-based mesoporous material 110 is porous and has a very large specific surface area. Therefore, when the silica-based mesoporous material 110 is used as a carrier, the enzyme 100 is immobilized at a higher concentration with a larger adsorption amount than when a carrier having a specific surface area smaller than that of the silica-based mesoporous material 110 is used. Can do. Furthermore, since the enzyme 100 is maintained in a moderately dispersed state by firmly fixing the enzyme 100 inside the pores of the silica-based mesoporous material 110, the enzyme 100 is agglomerated and deactivated. Can be avoided. That is, by using the silica-based mesoporous material 110 whose pore size is set to 0.5 to 2.0 times the size of the enzyme 100 as a carrier, the enzyme 100 can be concentrated at a higher concentration with a larger adsorption amount. Since the enzyme 100 can be agglomerated and inactivated due to aggregation, the enzyme sensor 1 having excellent sensitivity can be provided.
In addition, since the enzyme 100 is fixed in a moderately dispersed state, the permeation diffusibility of the substance is improved and the enzyme reaction proceeds efficiently.

  In addition, according to the enzyme sensor 1 of the present invention described above, the silica-based mesoporous material 110 has a concentration action of the detection target substance, so that a very small amount of the detection target substance in the gas phase or the liquid phase can be made extremely sensitive. Can be detected. In addition, by using the electron carrier 200, the electron transfer between the active center of the enzyme 100 fixed inside the pores of the silica-based mesoporous material 110 and the electrode 20 is mediated and promoted, with high sensitivity. A high-speed response can be obtained. Due to these actions, it becomes possible to detect a detection target substance with extremely high speed and high sensitivity by electrochemical measurement, and it is possible to configure the enzyme sensor 1 having high speed, high sensitivity, long life and excellent stability.

  Moreover, according to the enzyme sensor 1 of the present invention described above, a sensor for detecting a specific odor or gas by detecting a very small amount of a detection target substance in a gas phase or a liquid phase with extremely high sensitivity. Can be used as It can also be used to detect a specific taste in the liquid phase or to detect the production and / or production amount of a specific substance in a bioreactor.

  Further, according to the enzyme sensor 1 of the present invention described above, in measuring, the voltage of the working electrode (electrode 20) with respect to the reference electrode 40 is set to a specific voltage, thereby avoiding the influence of the measurement interfering substance and detecting. The target substance can be measured with high sensitivity and selectivity. In particular, by using the electron carrier 200, the set voltage can be lowered and the selectivity can be improved.

  Further, according to the enzyme sensor 1 of the present invention described above, it is necessary to change the type of the enzyme 100 as the molecular identification element depending on the substance to be detected. For example, as the enzyme 100, glucose oxidase or glucose dehydrogenase is used when the detection target substance (measurement target) is glucose, alcohol oxidase or alcohol dehydrogenase is used when the measurement target is ethanol, and formaldehyde when the measurement target is formaldehyde. For oxidase or formaldehyde dehydrogenase, a mixture of cholesterol esterase and cholesterol oxidase can be used when the measurement target is total cholesterol.

  The present invention is not limited to the above-described embodiment, and can be appropriately changed without departing from the gist thereof.

  Although the enzyme sensor 1 is configured to include the potentiostat 50, the present invention is not limited to this, and the enzyme sensor 1 and the potentiostat 50 may be provided separately.

  The electrochemical measurement by the enzyme sensor 1 is performed by using a bipolar structure (working electrode (electrode 20) and reference electrode 30) or a tripolar structure (working electrode (electrode 20), reference electrode 30 and reference electrode) formed on one substrate. 40) may be used, or independent electrodes (working electrode (electrode 20), reference electrode 30, reference electrode 40) may be used in combination.

  When each electrode of bipolar structure (working electrode (electrode 20) and reference electrode 30) or tripolar structure (working electrode (electrode 20), reference electrode 30 and reference electrode 40) is formed on a predetermined insulating substrate For example, a predetermined electrolyte may be dropped on a region including the region where the electrode 20 is formed on the insulating substrate (hereinafter referred to as “analysis region”), and the detection region 10 may be formed on the analysis region. For example, a liquid phase chamber filled with a predetermined electrolytic solution may be provided on the analysis region, and the detection unit 10 may be provided on the analysis region (liquid phase chamber).

  The enzyme 100 is optional as long as it is contained in the detection unit 10. For example, the enzyme 100 may be contained in the detection unit 10 in a state of a free enzyme dissolved in a predetermined electrolyte introduced into the reactor 10 a. For example, it may be contained in the detection unit 10 in a state of being fixed to a carrier other than the silica-based mesoporous material 110, or may be contained in the detection unit 10 in a state of being directly fixed to the electrode 20 or the like, for example. Also good.

It is a schematic diagram which shows an example of the enzyme sensor of this invention. It is a figure which shows typically the 1st example of the silica type mesoporous body by which the enzyme was fix | immobilized inside the pore. It is a figure which shows typically the 2nd example of the silica type mesoporous body by which the enzyme was fix | immobilized inside the pore. It is the result obtained by measuring the enzyme activity fixed inside the pores of the silica-based mesoporous material. It is the result obtained by measuring the time-dependent change of the activity of the enzyme fixed inside the pores of the silica-based mesoporous material. It is the result obtained by measuring the time-dependent change of the activity of the enzyme immobilized inside the pores of the silica-based mesoporous material with the addition of NAD ⁺ and a coenzyme activity decrease inhibiting substance. It is the result obtained by measuring the time-dependent change of the activity of the enzyme fixed inside the pores of the silica-based mesoporous material with the addition of the activity lowering suppression type electron carrier. Obtained by measuring time-dependent changes in the activity of the enzyme immobilized in the pores of the silica-based mesoporous material with the addition of NAD ⁺ , a coenzyme activity-reducing inhibitor and an activity-reducing inhibitory electron carrier It is a result.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Enzyme sensor 10 Detection part 100 Enzyme 110 Silica mesoporous body 200 Electron carrier 300 Coenzyme

Claims (5)

A detection unit containing an enzyme and a coenzyme;
The enzyme is contained in the detection unit in a state of being fixed inside the pores of the silica-based mesoporous material,
An enzyme sensor, wherein a biopolymer having a molecular weight of 1000 or more is added to the detection unit. A detection unit containing an enzyme and a coenzyme;
The enzyme is contained in the detection unit in a state of being fixed inside the pores of the silica-based mesoporous material,
A synthetic polymer having a molecular weight of 1000 or more is added to the detection unit. A detection unit containing an enzyme and a coenzyme;
The enzyme is contained in the detection unit in a state of being fixed inside the pores of the silica-based mesoporous material,
An enzyme sensor, wherein an electrolyte is added to the detection unit. In the enzyme sensor as described in any one of Claims 1-3,
Among the compounds represented by the following general formula (1), the compound represented by the following general formula (2), and the compound represented by the following general formula (3) as an electron carrier in the detection unit An enzyme sensor characterized in that at least one of the above is added.
(Wherein R1, R2, R3 are, during the formula Me represents an alkyl group, a metal atom in formula X ^-.. Represents an anion.)
(In the formula, Me represents a metal atom.)
(In the formula, Me represents a metal atom.) In the enzyme sensor as described in any one of Claims 1-4,
An enzyme sensor, wherein an electron carrier adsorbed by a predetermined porous body is added as an electron carrier to the detection unit.