KR101749904B1 - Redox reagent composition for biosensor - Google Patents

Abstract

The present invention relates to a redox reaction reagent composition for an electrochemical biosensor comprising a ruthenium-containing complex as an electron transfer mediator and naphthoquinone or a derivative thereof, wherein the ruthenium-containing complex as an electrotransport mediator according to the present invention, The redox reaction reagent composition comprising quinone or a derivative thereof exhibits remarkable glucose detection performance due to the rapid reaction rate between the redox enzyme-naphthoquinone (or derivative thereof) -ruthenium-containing complex and the electrochemical biotin for detecting glucose in blood It is useful for the manufacture of sensors.

Description

Technical Field [0001] The present invention relates to a redox reagent composition for a biosensor,

The present invention relates to a reagent composition for an electrochemical biosensor, which is one of the components of a system for measuring blood glucose, and relates to an electron transfer mediator comprising a ruthenium-containing complex, and naphthoquinone or a derivative thereof.

Since Anton Clemens developed the world's first portable glucose measurement device, efforts to improve the accuracy and convenience of the glucose measuring device have been going on for the last 40 years. The measurement methods include brightness measurement and electrochemical measurement . Although the photometric method has been developed first, this method is disadvantageous in that the accuracy of the measurement device becomes unstable due to contamination of the blood. On the other hand, the electrochemical measurement method is less likely to be contaminated by the blood when compared with the photometric method, and the amount of blood required for the measurement is small, thereby occupying most of the present commercialized measurement methods.

Efforts to improve accuracy are closely related to the development of enzymes. For example, FAD-GOx (enzyme code 1.1.3.4), a glucose oxidase reductase used in electrochemical sensors, is a thermostable, glucose- Although selectivity is excellent, it is difficult to apply all the blood collected from venous, arterial, or capillary blood vessels because the measurement value is influenced by the concentration of oxygen dissolved in the blood.

On the other hand, sensors made using PQQ-GDH (enzyme Classification No.1.5.5.2) enzyme are not affected by oxygen, but various kinds of monosaccharides or disaccharides such as mannose, maltose or lactose, . In particular, high concentrations of maltose, which is injected into diabetic patients, have attracted attention due to their effect on blood glucose levels to be much higher than they actually are (Mehmet, S., Quan, G., Thomas, S ., and Goldsmith, D., Important causes of hypoglycemia in patients with diabetes on peritoneal dialysis. Diabetes Med., 18, 679-682 (2001)). To solve this problem, Roche SA developed a mutant enzyme of PQQ-GDH to solve the inaccuracies of maltose measurement.

The NAD-GDH enzyme has an excellent selectivity for reacting only with glucose, but it is inconvenient to further include NAD ⁺ or NADP ⁺ in the reagent composition component. FAD-GDH (enzyme code 1.1.99.10), an enzyme that complements the above-mentioned disadvantages, has recently been developed by Amano and Toyobo in Japan, and the electrochemical sensor using this enzyme is known to have oxygen And thus can be effectively applied to all blood in the body. Although there is a disadvantage of reacting with xylose, it does not react with mannose, maltose and lactose, and thus has a relatively high selectivity to react with glucose singly.

The substance that affects the accuracy of the sensor along with the enzyme is an electron carrier. The electron donor should be able to measure glucose at high concentration because of its rapid reaction with the enzyme, and it should not be altered for a long time, so the accuracy should be maintained during the distribution period. The electron transfer mediators of FAD-GDH include potassium ferricyanide [K ₃ Fe (CN) ₆ ], phenazine-methosulfate, methoxyphenazine-methosulfate, phenazine methyl sulfate phenazine methylsulfate) or dichloroindophenol are known. However, all of them are susceptible to change due to temperature and humidity, so the accuracy tends to be low when the sensor is stored for a long period of time. Bayer developed a hydrophilic derivative of phenothiazine (US Pat. No. 9039878), which has been supplemented with such disadvantages.

Compared with the use of hexaamine ruthenium chloride [Ru (NH ₃ ) ₆ Cl ₃ ] as an electron transporting agent, potassium ferricyanide is used as an inhibitor, such as uric acid or gentisic acid Hexamyl ruthenium chloride has been used in electrochemical sensors using FAD-GOx (U.S. Patent No. 7288174), because the degree of influence is low and the accuracy is slowed down by humidity. However, the electron donor is not useful for sensor fabrication due to the slow reaction rate with FAD-GDH (EC No. 1.1.99.10).

Examples using two kinds of electron carriers are as follows.

European Patent Publication No. 0238322 discloses a method for significantly increasing the electron transfer rate between bacteria and an electrode using benzoquinone with potassium ferricyanide.

U.S. Patent No. 8057659 discloses a method for applying osmium (Os) and potassium ferricyanide to a glucose sensor.

Korean Patent Publication No. 10-1355127 discloses a method of applying a thionine and a ruthenium complex to a glucose sensor.

Korean Patent Registration No. 10-1531384 discloses a method of applying a naphthol green ratio and a ruthenium complex to a glucose sensor.

U.S. Patent No. 6,858,011 discloses a method of applying to a glucose sensor using a combination of phenazine methosulfate and a ruthenium complex.

A. Amine and colleagues disclose methods for applying phenazine methosulfate and potassium ferricyanide to glucose oxidase and glutamate dehydrogenase.

The present inventors have found that when developing an electron carrier having excellent reactivity with FAD-GDH, a reagent composition comprising a ruthenium-containing complex and a naphthoquinone or a derivative thereof has a rapid reaction rate with FAD-GDH, And completed the present invention.

U.S. Patent Publication No. 9039878 U.S. Patent No. 7288174 U.S. Patent No. 8057659 U.S. Patent Publication No. 8658011 European Patent Registration No. 0238322 Korean Patent Registration No. 10-1355127 Korean Registered Patent No. 10-1531384

The present inventor has developed a novel reagent composition in an electrochemical biosensor for measuring blood glucose using an oxidation-reduction reaction. Specifically, in an electrochemical sensor using FAD-GDH enzyme, it is excellent in selective reactivity with glucose, quick in reaction with enzyme, capable of measuring blood glucose at high concentration, and stable with long-term deterioration during product distribution and use And tried to develop an electron carrier.

The present inventors have experimentally found that when a ruthenium-containing complex as an electron transfer mediator is combined with naphthoquinone or a derivative thereof, the synergistic action of the mutual components exhibits a useful effect as an electron transporting agent although the precise mechanism is unknown Thereby completing the present invention. The present inventor has solved the above-mentioned problems through the following means.

1. A reagent composition for a biosensor comprising an oxidoreductase and an electron transfer mediator, wherein the electron transfer mediator comprises a ruthenium-containing complex, and naphthoquinone or a derivative thereof.

2. The method according to 1 above, wherein the oxidoreductase is a flavin adenine dinucleotide-glucose dehydrogenase, a nicotinamide adenine dinucleotide-glucose dehydrogenase, a glucose dehydrogenase Glucose dehydrogenase, Glutamate dehydrogenase, Cholesterol oxidase, Lactate oxidase, Ascorbic acid oxidase, Alcohol oxidase, Alcohol dehydrogenase, Wherein the reagent composition is one selected from the group consisting of an enzyme (alcohol dehydrogenase) and a bilirubin oxidase.

3. In the above 1 or 2, containing a ruthenium complex _{[Ru (NH 3) 6]} Br 3, [Ru (NH 3) 5 Cl] Cl 2, K 4 [Ru (CN) 6], Ru (NH 3 _{) 6 Cl 3, [Ru (} 2,2 ', 2''- terpyridine) (1,10-phenanthroline) (OH 2)] 2+, trans- [Ru (2,2'-bipyridine) 2 (OH 2 ^{) (OH)] 2 +,} [(2,2'-bipyridine) 2 (OH) RuORu (OH) (2,2'bpy) 2] 4+ and [Ru (4,4'-bipyridine) ( NH 3 ) ₅ ] ²⁺ . ≪ / RTI >

4. The compound according to any one of items 1 to 3, wherein the naphthoquinone and its derivative are 2-methoxy-1,4-naphthoquinone, 5,8-dihydroxy- Methyl-1,4-naphthoquinone, 2-methyl-1,4-naphthoquinone sodium bicarbonate, 1,2-naphthoquinone, 1,4-naphthoquinone, -Naphthoquinone, 2-hydroxy-1,4-naphthoquinone, and 1,2-naphthoquinone-4-sulphonate.

5. The reagent composition according to any one of 1 to 4 above, wherein the redox enzyme is a flavin adenine dinucleotide-glucose dehydrogenase (FAD-GDH).

6. The reagent composition according to any one of 1 to 5 above, wherein the ruthenium-containing complex is 35-95 parts by weight based on 100 parts by weight of the redox enzyme.

7. The reagent composition according to any one of 1 to 6 above, wherein the naphthoquinone or a derivative thereof is 5-31 parts by weight based on 100 parts by weight of the redox enzyme.

8. The reagent composition according to any one of 1 to 7, wherein the reagent composition further comprises, if necessary, additives such as a surfactant, a water-soluble polymer, an amino acid, a dihydrate or a thickening agent.

9. The pharmaceutical composition according to any one of 1 to 8 above, which comprises 100 parts by weight of an oxidoreductase, 0.1-80 parts by weight (preferably 5-31 parts by weight) of naphthoquinone or a derivative thereof, 35 to 95 parts by weight of a ruthenium-containing complex, 1-120 parts by weight of a surfactant, 0.3-12 parts by weight of a water-soluble polymer, 4-51 parts by weight of an amino acid and 38-227 parts by weight of a diacid.

The redox reaction reagent composition comprising the ruthenium-containing complex as an electron transfer mediator according to the present invention and the naphthoquinone or a derivative thereof has a rapid reaction rate between the redox enzyme-naphthoquinone (or derivative thereof) The glucose detection performance is remarkably improved and is useful for the production of an electrochemical biosensor for detecting glucose in blood. In addition, storage stability after long-term storage is excellent.

Particularly, the reagent composition for biosensor containing Naphthol Green B described in Korean Patent Registration No. 10-1531384 has a problem that the composition needs to be filtered due to the presence of a large amount of fine particles precipitated in the composition, However, since the reagent composition developed in the present invention has a very small and small amount of such fine particles and does not require filtration, there is an effect that the mass productivity is increased and the uniformity of the reagent is improved.

Also, the reagent composition described in Korean Patent Publication No. 10-1355127 can maintain the linearity with respect to blood glucose only when the ruthenium-containing complex is contained in an amount of 250 to 340 parts by weight based on 100 parts by weight of the redox enzyme, The linearity can be maintained even when the ruthenium-containing complex is contained in an amount of 35 to 95 parts by weight based on 100 parts by weight of the redox enzyme, thereby reducing the cost of ruthenium, which is relatively expensive.

Also, the reagent composition comprising the ruthenium-containing complex as the electron transfer mediator and the naphthoquinone or the derivative thereof has a fast reaction rate between the redox enzyme and the naphthoquinone or its derivative and the ruthenium-containing complex so that the glucose detection performance in the blood is improved, It is hardly affected by oxygen and interfering substances and is useful for manufacturing a biosensor for glucose detection.

1 is an exploded perspective view of an electrochemical biosensor used in an embodiment of the present invention.
Figures 2 and 3 show the structures of naphthoquinone and its derivatives used in one embodiment of the present invention.
FIG. 4 is a graph showing a change in voltage at the working electrode of the biosensor according to an embodiment of the present invention. In FIG. 4, 0 mV is applied to the instantaneous working electrode covering the auxiliary electrode and the working electrode, After waiting, 200 mV is applied to the working electrode and the current is read in 5 seconds.
FIG. 5 is a graph showing a change in current when a sample with different glucose concentrations is applied using the electrochemical biosensor according to Example 1 and Comparative Examples 1 and 2. FIG.
FIG. 6 is a graph showing a change in current when a sample with different glucose concentrations is applied using the electrochemical biosensor according to Examples 1 to 6. FIG.

Hereinafter, the present invention will be described in detail.

The present invention provides an oxidation-reduction reaction reagent composition for an electrochemical biosensor, which comprises an oxidoreductase and an electron transfer mediator, wherein the electron transfer mediator comprises a ruthenium-containing complex, and naphthoquinone or a derivative thereof .

In the redox reaction reagent composition according to the present invention, the oxidoreductase is reduced by reacting with various metabolites to be measured. Then, the oxidoreductase is reacted with the reduced enzyme and the transfer medium to quantify the metabolite.

Although the present invention describes a glucose measuring biosensor as an example, various metabolic substances such as cholesterol, lactate, creatinine, hydrogen peroxide, glucose, and the like can be obtained by introducing the electron transfer mediator of the present invention into a specific enzyme having a mechanism similar to that of glucose oxidation. The concentration of an organic substance or an inorganic substance such as alcohol, amino acid or glutamate can be quantified.

Therefore, the present invention can be used without limitation in the quantitative determination of various metabolites by changing the kinds of enzymes contained in the reagent composition.

For example, flavin adenine dinucleotide-glucose dehydrogenase (FAD-GDH), nicotinamide adenine dinucleotide-glucose dehydrogenase (NAD-GDH), glutamate dehydrogenase (such as glutamate dehydrogenase, cholesterol oxidase, lactate oxidase, ascorbic acid oxidase, alcohol oxidase, alcohol dehydrogenase or bilirubin oxidase, A quantitative determination of glucose, glutamate, cholesterol, lactate, ascorbic acid, alcohol, or bilirubin can be performed using an enzyme (bilirubin oxidase) or the like.

In the redox reaction reagent composition according to the present invention, the electron transfer mediator reacts with a metabolite to be reduced by an oxidation-reduction reaction with the reduced enzyme, and the reduced electron transfer mediator is formed on the electrode surface Thereby generating a current.

At this time, ruthenium-containing complexes and naphthoquinone or derivatives thereof may be mixed together as the electron transfer mediator.

As in the electron transfer mediator, ruthenium-containing complexes are _{[Ru (NH 3) 6]} Br 3, [Ru (NH 3) 5 Cl] Cl 2, K 4 [Ru (CN) 6], Ru (NH 3) 6 _{Cl 3, [Ru (2,2 '} , 2''- terpyridine) (1,10-phenanthroline) (OH 2)] 2+, trans- [Ru (2,2'-bipyridine) 2 (OH 2) ( ^{OH)] 2 +, [(} 2,2'-bipyridine) 2 (OH) RuORu (OH) (2,2'bpy) 2] 4+ or [Ru (4,4'-bipyridine) ( NH 3) 5 ] ²⁺ can be used.

In the electron transfer mediator, examples of the naphthoquinone derivative include 2-methoxy-1,4-naphthoquinone, 5,8-dihydroxy-1,4-naphthoquinone, Toquinone, 2-methyl-1,4-naphthoquinone sodium bicarbonate, 1,2-naphthoquinone, 1,4-naphthoquinone, 5-hydroxy- Hydroxy-1,4-naphthoquinone or 1,2-naphthoquinone-4-sulphonate (see Figs. 2 and 3).

The most preferred ruthenium complexes in the present invention are hexamyl ruthenium chloride which is stable and reversible in the oxidation-reduction state in an aqueous solution, the reduced electron transport mediator does not react with oxygen, and the oxidation of the reduced electron transport mediator is sensitive to pH .

The most preferred naphthoquinone derivative in the present invention is 1,2-naphthoquinone-4-sulphonate.

The electron transfer mediator according to the present invention significantly improves glucose detection performance by using ruthenium complex and naphthoquinone together.

For example, in Comparative Examples 1 and 2 of the present invention, when either naphthoquinone or hexaamine ruthenium chloride [Ru (NH ₃ ) ₆ Cl ₃ ] is used alone as an electron transfer mediator, It is difficult to apply it as a biosensor because it is slow.

However, when the ruthenium-containing complex and the naphthoquinone or the derivative thereof are used together as the electron transfer mediator according to the present invention, the reaction rate between the redox enzyme and the naphthoquinone or its derivative is fast and the naphthoquinone and ruthenium- Can be used for the production of a biosensor (see Experimental Example 1).

Korean Patent Publication No. 10-1531384 discloses a reagent composition for a biosensor containing Naphthol Green B, which requires a large amount of fine particles due to precipitation in the composition, which requires filtration of the composition. Resulting in a difference in composition ratio. However, the reagent composition developed in the present invention using a ruthenium-containing complex and naphthoquinone or a derivative thereof in combination with the ruthenium-containing complex is very small and small in size and does not require filtration, so that the effect of increasing the mass productivity and improving the uniformity of the reagent have.

Korean Patent Publication No. 10-1355127 discloses an invention capable of maintaining the linearity with respect to blood glucose by containing 250-340 parts by weight of ruthenium-containing complex based on 100 parts by weight of redox enzyme. The reagent composition of the present invention comprises ruthenium Containing complex is contained in an amount of 35 to 95 parts by weight based on 100 parts by weight of the redox enzyme, it is possible to maintain the linearity and reduce the cost of ruthenium, which is relatively expensive.

At this time, the redox reaction reagent composition according to the present invention preferably contains 35-95 parts by weight of a complex containing ruthenium based on 100 parts by weight of the redox enzyme. More preferably, the ruthenium-containing complex is contained in an amount of 50 to 70 parts by weight. One embodiment according to the present invention may contain 56 parts by weight of a ruthenium-containing complex. If the ruthenium-containing complex is contained in an amount of less than 35 parts by weight, the reaction of the sensor becomes insensitive at a high blood glucose concentration. If the ruthenium-containing complex is contained in an amount exceeding 95 parts by weight, .

Also, the redox reaction reagent composition according to the present invention preferably contains 5-31 parts by weight of naphthoquinone or a derivative thereof based on 100 parts by weight of the redox enzyme. More preferably 10-30 parts by weight of naphthoquinone or a derivative thereof. One embodiment according to the present invention may contain 16 parts by weight of naphthoquinone or a derivative thereof. If naphthoquinone or a derivative thereof is contained in an amount of less than 5 parts by weight, the reaction of the sensor becomes insensitive at a high blood glucose concentration. If the naphthoquinone or its derivative is contained in an amount of more than 31 parts by weight, the problem that the reagent does not melt quickly due to blood Can be.

Meanwhile, the reagent composition according to the present invention may be optionally added with additives such as a surfactant, a water-soluble polymer, an amino acid, a disaccharide, and a thickening agent.

In the additive, the surfactant serves to spread the reagent evenly over the electrode when the reagent is dispensed so that the reagent is dispensed at a uniform thickness. Examples of the surfactant include, but are not limited to, Triton X-100, CHAPS (3-cholamidopropyl) dimethylammonio] -1-propanesulfonate, sodium dodecyl sulfate, Perfluorooctane sulfonate, sodium stearate, and the like can be used. The redox reaction reagent composition according to the present invention preferably contains 1-120 parts by weight, preferably about 10-30 parts by weight, of a surfactant based on 100 parts by weight of the redox enzyme.

In this additive, the water-soluble polymer serves as a polymer scaffold of the reagent composition to help stabilize and disperse the enzyme. Examples of the water soluble polymer include polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), perfluoro sulfonate, hydroxyethyl cellulose (PVP), and the like. HEC), hydroxypropyl cellulose (HPC), carboxy methyl cellulose (CMC), cellulose acetate or polyamide can be used. The redox reaction reagent composition according to the present invention preferably contains the water-soluble polymer in an amount of 0.3 to 12 parts by weight, preferably about 2 parts by weight, based on 100 parts by weight of the redox enzyme.

In the additive, the amino acid plays a role of preventing the enzyme and the enzyme from aggregating together and maintaining the activity of the enzyme. The redox reaction reagent composition according to the present invention preferably contains 4-51 parts by weight, preferably about 17 parts by weight, of amino acid based on 100 parts by weight of the redox enzyme.

In the additive, the disaccharide is used to prevent the enzymes from being excessively dried to slow the activity of the enzyme. The redox reaction reagent composition according to the present invention preferably contains 38-227 parts by weight, preferably about 76 parts by weight, of the disaccharide based on 100 parts by weight of the redox enzyme.

The electrochemical biosensor according to the present invention is characterized in that the working electrode, the auxiliary electrode and the confirmation electrode are provided on one plane, and the redox reaction reagent composition according to the present invention is coated on the electrode. Sensor.

Further, the present invention provides an electrochemical biosensor, wherein the working electrode and the auxiliary electrode face each other on a plane, and the oxidation reduction reagent composition according to the present invention is coated on the working electrode, Thereby providing a biosensor.

The planar and face-to-face electrochemical biosensors according to the present invention can be applied to a variety of electrochemical biosensors, such as Korean Patent Application No. 10-2003-0036804, Korean Patent Application No. 10-2005-0010720, Korean Patent Application No. 10-2007-0020447, 2007-0021086, Korean Patent Application No. 10-2007-0025106, Korean Patent Application No. 10-2007-0030346, [E. K. Bauman et al., Analytical Chemistry, vol. 37, p 1378, 1965; K. B. Oldham in "Microelectrodes: Theory and Applications," Kluwer Academic Publishers, 1991.).

Hereinafter, the configuration of the planar electrochemical biosensor will be described with reference to FIG.

First, the planar electrochemical biosensor shown in FIG. 1 includes an upper plate 8 provided with a working electrode, an auxiliary electrode, and a check electrode on one plane, and having a vent 9 for allowing blood to permeate into the sensor; An insert plate (7) for adhering the upper plate and the lower plate, the plate being coated with an adhesive on both sides and allowing the blood to permeate toward the electrode by capillary action; A redox reaction reagent composition (6) according to the present invention coated on the following electrode; An insulating plate 5 provided with a passage portion for defining the area of the following electrode; An operating electrode 3, an auxiliary electrode 2 and a confirmation electrode 4 printed on the lower plate; And a lower plate 1 on which the working electrode, the auxiliary electrode and the check electrode are formed.

As described above, the redox reaction reagent composition comprising a metal-containing complex as an electron transfer mediator according to the present invention and a naphthoquinone or a derivative thereof is prepared by reacting a redox enzyme-naphthoquinone (or derivative thereof) The reaction rate is increased and the glucose detection performance in the blood is remarkably improved and little influence of oxygen and interfering substances in the blood is obtained and it is useful for the production of an electrochemical biosensor for detecting glucose in blood.

Hereinafter, the present invention will be described in more detail with reference to the following examples. The following examples are illustrative of the present invention only and are not intended to limit the scope of the present invention.

Example 1: Preparation of a redox reaction reagent composition containing hexaamine ruthenium chloride and 1,2-naphthoquinone-4-sulphonate as electron transfer mediators

To 100 parts by weight of a flavin adenine dinucleotide-glucose dehydrogenase (FAD-GDH) as an oxidoreductase; 56 parts by weight of hexaamine ruthenium chloride [Ru (NH ₃ ) ₆ Cl ₃ ] as a metal-containing complex; 16 parts by weight (15 mM) of 1,2-naphthoquinone-4-isophonate; 2 parts by weight of polyvinyl pyrrolidone (PVP) as a water-soluble polymer; And 2 parts by weight of Triton X-100; 8 parts by weight of CHAPS; 76 parts trehalose were dissolved in citric phosphate buffer (pH 5.8, 500 ml of 0.3 M concentration) and deionized water (500.0 ml), and the fine particles remaining in the solution were removed by filtration.

≪ Example 2 > Preparation of a planar biosensor containing a reagent composition

As an example of the planar type biosensor, a planar type biosensor having a sample introduction portion with an average value of 0.5 ㎕ was produced as shown in Fig. Detailed methods for manufacturing this planar type biosensor are described in Korean Patent Application No. 10-2003-0036804, Korean Patent Application No. 10-2005-0010720, Korean Patent Application No. 10-2007-0020447, Korean Patent Application No. 10-2007-0021086, Korean Patent Application No. 10-2007-0025106, Korean Patent Application No. 10-2007-0030346, [EK Bauman et al., Analytical Chemistry, vol. 37, p 1378, 1965; KB Oldham in "Microelectrodes: Theory and Applications," Kluwer Academic Publishers, 1991.). In FIG. 1, reference numeral 1 denotes a lower plate made of polyester having a working electrode (area: 0.9 mm ² ) and an auxiliary electrode and a check electrode formed thereon, 2 to 4 are electrodes made by screen printing carbon graphite, 3 is the working electrode, and 4 is the confirmation electrode. Reference numeral 5 designates the area of the upper electrode as an insulator, 6 designates the redox reaction reagent prepared in Example 1 coated on the electrode, 7 designates a 0.10 mm thick fitting plate in which blood is allowed to permeate toward the electrode by capillary action And adhesive on both sides thereof to adhere the upper plate and the lower plate. Numeral 9 is an air outlet for allowing the blood to permeate into the sensor, and 8 is a top plate plastic made of polyester.

Example 3: Preparation of a redox reaction reagent composition containing hexaamine ruthenium chloride and a naphthoquinone derivative as an electron transfer mediator

Among the naphthoquinone derivatives shown in FIG. 2, 2-hydroxy-1,4-naphthoquinone (hereinafter, referred to as naphthoquinone derivative) was used in place of 1,2-naphthoquinone- a) (60 mM) was used as the starting material, to prepare a reagent composition.

Example 4: Preparation of a redox reaction reagent composition containing hexaamine ruthenium chloride and a naphthoquinone derivative as an electron transfer mediator

Among the naphthoquinone derivatives shown in Fig. 2 instead of 1,2-naphthoquinone-4-sulphonate in the reagent composition of Example 1, 5-hydroxy-1,4-naphthoquinone (hereinafter referred to as naphthoquinone derivative b) 10 mM was used as the starting material, to prepare a reagent composition.

Example 5: Preparation of a redox reaction reagent composition comprising hexaamine ruthenium chloride and a naphthoquinone derivative as an electron transfer mediator

In the reagent composition of Example 1, 1,2-naphthoquinone (hereinafter referred to as naphthoquinone derivative c) 10 (hereinafter referred to as " naphthoquinone derivative c " mM was used in place of < RTI ID = 0.0 >

Example 6: Preparation of a redox reaction reagent composition comprising hexaamine ruthenium chloride and a naphthoquinone derivative as an electron transfer mediator

Among the naphthoquinone derivatives shown in Fig. 2, 1,4-naphthoquinone (hereinafter referred to as naphthoquinone derivative d) 10 (hereinafter referred to as naphthoquinone derivative d) was used in place of 1,2- mM was used in place of < RTI ID = 0.0 >

≪ Comparative Example 1 > Preparation of a redox reaction reagent composition containing hexaamine ruthenium chloride as an electron transfer mediator

The reagent composition was prepared in the same manner as in Example 1, except that 1,2-naphthoquinone-4-sulphonate was not used.

≪ Comparative Example 2 > Preparation of a redox reaction reagent composition containing 1,2-naphthoquinone-4-sulphonate as an electron transfer mediator

A reagent composition was prepared in the same manner as in Example 1, except that hexamine-ruthenium chloride was not used.

<Experimental Example 1> Current measurement for a glucose standard solution of a planar biosensor

Current measurement for the glucose standard solution was performed using the planar biosensor prepared in Examples and Comparative Examples. Herein, the glucose standard solution refers to blood prepared by preparing vein-extracted blood so that the hematocrit is 42% and having various glucose concentrations using a glucose analyzer (manufacturer: YSI, model name: 2900D) .

Specifically, a standard solution with glucose concentrations of 46, 138, 226, 365, 458 and 571 mg / dL was applied at 0 mV to the working electrode at the same time as the working electrode and the working electrode were simultaneously covered, After 200 mV was applied to the working electrode, a current of 5 seconds was measured (see FIG. 6). All measurements were performed 10 times for each concentration, and the average value was shown in FIG.

FIG. 4 is a graph showing a change in voltage at the working electrode of the biosensor according to an embodiment of the present invention. When the sample simultaneously covers the auxiliary electrode, the working electrode, and the verify electrode, 0 mV is applied to the working electrode, After 200 mV is applied to the working electrode, the current is read in 5 seconds.

FIG. 5 is a graph showing a change in current when a sample having a different glucose concentration is applied using the planar biosensor according to Example 1, Comparative Example 1, and Comparative Example 2. FIG.

As shown in Fig. 5, the biosensor of Example 1 including hexaamine ruthenium chloride and 1,2-naphthoquinone-4-sulphonate as electron transfer mediators increased the current measured according to the glucose concentration gradually Whereas the biosensor of Comparative Example 1 and Comparative Example 2 which did not contain any one of 1,2-naphthoquinone-4-reductonate or hexaamine ruthenium chloride as an electron transfer agent alone and showed good linearity, Showed that the linearity according to the glucose concentration was not good. At this time, the slope of the current measured by the biosensor according to Example 1 is 20.2 nA / mm ² / (mg / dL) per unit area of the working electrode, indicating that the reaction rate to glucose is very fast.

Therefore, the redox reaction reagent composition according to the present invention reacts very quickly to glucose when applied to a biosensor, and thus is useful for manufacturing a biosensor for detecting glucose in blood.

EXPERIMENTAL EXAMPLE 2 Current Measurement for Glucose Standard Solution of Planar Biosensor According to Examples 3 to 6

Current measurements were performed on the glucose standard solution in the same manner as in Experimental Example 1 using the biosensor prepared in Examples 3 to 6 and the biosensor prepared in Example 2 using the naphthoquinone derivatives shown in FIG.

, 1,2-naphthoquinone-4-For sseolpo carbonate glucose levels 50 ~ 600 mg / dL in the slope of the current per unit area of the working electrode 21.1 nA / mm ² / as shown in Fig. 6 (mg / dL) (YSI 2900D), and for a biosensor using derivative a, the slope of the current at a glucose concentration of 50 to 600 mg / dL is 20.0 nA / mm ² / (mg / dL ), The correlation with the reference device is 0.992, and for a biosensor using derivative b, the slope of the current at a glucose concentration of 50 to 350 mg / dL is 27.8 nA / mm ² / (mg / dL) For the biosensor using derivative c, the slope of the current is 11.1 nA / mm ² / (mg / dL) per unit working electrode at a glucose concentration of 50-200 mg / dL, The relation is 0.844, and in the case of the biosensor using the derivative d, the glucose concentration is 50 to 200 the slope of the current in mg / dL is per unit area of 4.4 nA / mm for a working electrode ² / (mg / dL), the reference device and the correlation is indicated by the biosensor 0.678 with 1,2-naphthoquinone-4-sseolpo carbonate Showed that the reaction rate to glucose was very fast and correlated well with the reference instrument.

Therefore, the redox reaction reagent composition according to the present invention reacts very quickly to glucose when applied to an electrochemical biosensor, and thus is useful for manufacturing a biosensor for detecting glucose in blood.

In the electrochemical biosensor according to the embodiment of the present invention, the working electrode, the auxiliary electrode, and the confirmation electrode are provided on one plane of the lower substrate. However, the working electrode, the auxiliary electrode, For example, an upper substrate).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art.

Claims (4)

A reagent composition for a biosensor comprising an oxidoreductase and an electron transfer mediator, wherein the electron transport mediator comprises a ruthenium-containing complex, and naphthoquinone or a derivative thereof, wherein the oxidoreductase comprises a flavin adenine dinucleotide-glucose dehydrogenase flavin adenine dinucleotide-glucose dehydrogenase). delete According to claim 1, wherein the ruthenium-containing complexes _{[Ru (NH 3) 6]} Br 3, [Ru (NH 3) 5 Cl] Cl 2, K 4 [Ru (CN) 6], Ru (NH 3) 6 Cl _{3, [Ru (2,2 ',} 2''- terpyridine) (1,10-phenanthroline) (OH 2)] 2+, trans- [Ru (2,2'-bipyridine) 2 (OH 2) (OH ^{)] 2 +, [(2,2'} -bipyridine) 2 (OH) RuORu (OH) (2,2'bpy) 2] 4+ and [Ru (4,4'-bipyridine) ( NH 3) 5] ²⁺ . &Lt; / RTI &gt; 3. The compound according to claim 1, wherein the naphthoquinone and its derivative are selected from the group consisting of 2-methoxy-1,4-naphthoquinone, 5,8-dihydroxy-1,4-naphthoquinone, 1,2-naphthoquinone, 1,4-naphthoquinone, 5-hydroxy-1,4-naphthoquinone, 2-methyl-1,4-naphthoquinone, sodium bisulfite, -Hydroxy-1,4-naphthoquinone, and 1,2-naphthoquinone-4-sulphonate.

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