WO2018110828A1 - Redox reagent composition for biosensor - Google Patents

Redox reagent composition for biosensor Download PDF

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WO2018110828A1
WO2018110828A1 PCT/KR2017/012368 KR2017012368W WO2018110828A1 WO 2018110828 A1 WO2018110828 A1 WO 2018110828A1 KR 2017012368 W KR2017012368 W KR 2017012368W WO 2018110828 A1 WO2018110828 A1 WO 2018110828A1
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naphthoquinone
reagent composition
glucose
ruthenium
weight
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PCT/KR2017/012368
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French (fr)
Korean (ko)
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정성권
임승민
이은상
김남혁
김예슬
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환인제약 주식회사
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose

Definitions

  • the present invention relates to a reagent composition for an electrochemical biosensor, which is one component of a system for measuring blood glucose, and relates to an electron transfer medium comprising a ruthenium-containing complex and a naphthoquinone or a derivative thereof.
  • the measuring methods are mainly photometric and electrochemical measuring methods. Separated by. Although photometry was first developed, this method has the disadvantage that the measuring device is contaminated with blood and thus the accuracy becomes unstable. On the other hand, the electrochemical measurement method compared to the photometric method, the measurement device is less contaminated by blood, and the amount of blood required for the measurement is occupied most of the current commercial measurement method.
  • FAD-GOx Enzyme Classification No. 1.1.3.4
  • a glucose redox enzyme used in electrochemical sensors is heat stable and only oxidizes glucose.
  • the selectivity is excellent, the measurement value is affected by the concentration of oxygen dissolved in the blood, so it is difficult to apply it to all blood collected from venous vessels, arterial vessels, or capillaries.
  • sensors made using PQQ-GDH Enzyme Classification No. 1.1.5.2
  • PQQ-GDH Enzyme Classification No. 1.1.5.2
  • enzymes are not affected by oxygen, but can contain several types of monosaccharides or disaccharides such as mannose, maltose or lactose.
  • high concentrations of maltose injected into diabetic patients have received attention because they affect blood glucose levels to be measured 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.Diabet. Med., 18, 679-682 (2001)).
  • Roche developed a mutant enzyme of PQQ-GDH to largely eliminate the inaccuracy of maltose measurements.
  • the NAD-GDH enzyme has good selectivity to react only with glucose, but it is inconvenient to additionally include NAD + or NADP + in the reagent composition.
  • FAD-GDH Enzyme Classification No. 1.1.99.10
  • FAD-GDH Enzyme Classification No. 1.1.99.10
  • xylose it does not react with mannose, maltose and lactose, so that the selectivity of reacting alone with glucose is relatively excellent.
  • the material that affects the accuracy of the sensor is the electron transporter.
  • the electron transporter must be able to measure high blood glucose level due to its rapid reaction with enzymes, and must not be altered for a long time, so the accuracy must be maintained for the distribution period.
  • electron transfer mediators of FAD-GDH include potassium ferricyanide [K 3 Fe (CN) 6 ], phenazine-methosulfate, methoxyphenazine-methosulfate, and phenazinemethyl sulfate ( phenazine methylsulfate or dichloroindophenol are known, and both are susceptible to deterioration due to temperature and humidity. Bayer has developed a hydrophilic derivative of phenothiazine that compensates for this disadvantage (US Patent No. 9039878).
  • hexaamineruthenium chloride [Ru (NH 3 ) 6 Cl 3 ] is used as an electron transporter, compared with the case of using potassium ferricyanide, it may be caused by an interfering substance such as uric acid or gentisic acid. Because of the low degree of influence and the low degree of accuracy degradation due to humidity, hexaamineruthenium chloride has been used in electrochemical sensors using FAD-GOx (US Patent No. 7288174). However, the electron transporter is not useful for fabricating the sensor due to the slow reaction rate with FAD-GDH (EC No. 1.1.99.10).
  • EP 0238322 discloses a method of using benzoquinone with potassium ferricyanide to significantly increase the electron transfer rate between bacteria and electrodes.
  • US Patent No. 8057659 discloses a method of applying osmium (Os) and potassium ferricyanide to a glucose sensor.
  • Korean Patent Publication No. 10-1355127 discloses a method of applying to a glucose sensor using a thionine and ruthenium complex together.
  • Korean Patent Publication No. 10-1531384 discloses a method for applying a glucose sensor using a naphthol green ratio and a ruthenium complex together.
  • US Patent Publication No. 8658011 discloses a method for applying a phenazinemethosulfate and ruthenium complex together to a glucose sensor.
  • the inventors of the present invention have developed an electron transporter having excellent reactivity with FAD-GDH, and a reagent composition including a ruthenium-containing complex and a naphthoquinone or a derivative thereof has a rapid reaction rate with FAD-GDH, thereby significantly improving glucose detection performance. It was found and completed the present invention.
  • the present inventors have attempted to develop a novel reagent composition in an electrochemical biosensor measuring blood glucose using redox reactions. Specifically, in the electrochemical sensor using the FAD-GDH enzyme, it has excellent selective reactivity with glucose, fast reaction with the enzyme, which enables high blood sugar measurement, and stability that does not deteriorate during product distribution and use for a long time. Efforts have been made to develop electron transporters.
  • the present inventors have found through experiments that when ruthenium-containing complexes and naphthoquinones or derivatives thereof are combined as electron transfer mediators, they do not know the exact mechanism, but due to the synergy of mutual components, they show useful effects as electron transfer mediators. Thus, the present invention has been completed. The present inventor has solved the above problem through the following means.
  • a reagent composition for a biosensor comprising an oxidoreductase and an electron transfer medium, wherein the electron transfer medium comprises a ruthenium-containing complex, and naphthoquinone or a derivative thereof.
  • redox enzyme flavin adenine dinucleotide-glucose dehydrogenase, nicotinamide adenine dinucleotide-glucose dehydrogenase, or glucose dehydrogenase.
  • glucose dehydrogenase glutamate dehydrogenase
  • cholesterol oxidase lactate oxidase
  • ascorbic acid oxidase alcohol oxidase
  • alcohol dehydrogenase alcohol dehydrogenase
  • a reagent composition which is one kind selected from the group consisting of an enzyme (alcohol dehydrogenase) and bilirubin oxidase.
  • naphthoquinone and its derivatives are 2-methoxy-1,4-naphthoquinone, 5,8-dihydroxy-1,4-naphthoquinone, 2- Methyl-1,4-naphthoquinone, 2-methyl-1,4-naphthoquinone sodium bisulfite, 1,2-naphthoquinone, 1,4-naphthoquinone, 5-hydroxy-1,4 -A reagent composition, characterized in that it is one kind selected from the group consisting of naphthoquinone, 2-hydroxy-1,4-naphthoquinone and 1,2-naphthoquinone-4-sulfonate.
  • oxidoreductase is flavin adenine dinucleotide-glucose dehydrogenase (FAD-GDH).
  • reagent composition according to any one of 1 to 7, further comprising additives such as surfactants, water-soluble polymers, amino acids, disaccharides, or thickeners as necessary.
  • a reagent composition comprising 35-95 parts by weight of ruthenium-containing complex, 1-120 parts by weight of surfactant, 0.3-12 parts by weight of water-soluble polymer, 4-51 parts by weight of amino acid and 38-227 parts by weight of disaccharide.
  • the redox reagent composition comprising a ruthenium-containing complex, a naphthoquinone or a derivative thereof, according to the present invention, has a fast reaction rate between a redox enzyme-naphthoquinone (or a derivative thereof) -metal complex, Glucose detection performance is significantly improved, which is useful for the manufacture of electrochemical biosensors for the detection of glucose in the blood. In addition, the storage stability of long-term storage is excellent.
  • a reagent composition for a biosensor comprising Naphthol Green B described in Korean Patent Publication No. 10-1531384 requires filtration of the composition due to the presence of a large amount of fine particles due to precipitation in the composition.
  • the reagent composition developed in the present invention has the effect of increasing the mass productivity and improving the uniformity of the reagents because the microparticles are very small and a small amount, so no filtration is required.
  • the reagent composition described in Republic of Korea Patent Publication No. 10-1355127 should maintain the linearity to blood sugar only if the ruthenium-containing complex should contain 250 to 340 parts by weight based on 100 parts by weight of the oxidoreductase, but the reagent composition of the present invention Even if the ruthenium-containing complex contains 35 to 95 parts by weight based on 100 parts by weight of the redox enzyme, the linearity can be maintained, thereby reducing the cost of the relatively expensive ruthenium.
  • the reagent composition comprising a ruthenium-containing complex and naphthoquinone or a derivative thereof as an electron transfer medium has a fast reaction rate between oxidoreductase, naphthoquinone or a derivative thereof and a ruthenium-containing complex, thereby improving glucose detection performance in blood and Since it is hardly affected by oxygen and interfering substances, it is useful in the manufacture of biosensors for glucose detection.
  • FIG. 1 is an exploded perspective view of an electrochemical biosensor used in an embodiment of the present invention.
  • 2 and 3 are structures of naphthoquinone and derivatives thereof used in one embodiment of the present invention.
  • Figure 4 is a graph showing the voltage change in the working electrode of the biosensor according to an embodiment of the present invention, when the sample covers the auxiliary electrode and the working electrode and covered the blood confirmation electrode to apply 0 mV to the working electrode for 4 seconds After waiting, 200 mV is applied to the working electrode and current is read in 5 seconds.
  • FIG. 5 is a graph illustrating changes in current appearing when a sample having a different glucose concentration is applied using an electrochemical biosensor according to Example 1 and Comparative Examples 1 to 2.
  • FIG. 5 is a graph illustrating changes in current appearing when a sample having a different glucose concentration is applied using an electrochemical biosensor according to Example 1 and Comparative Examples 1 to 2.
  • FIG. 6 is a graph illustrating changes in current appearing when a sample having a different glucose concentration is applied using an electrochemical biosensor according to Examples 1 to 6.
  • FIG. 6 is a graph illustrating changes in current appearing when a sample having a different glucose concentration is applied using an electrochemical biosensor according to Examples 1 to 6.
  • the present invention includes an oxidoreductase and an electron transfer mediator, wherein the electron transfer mediator comprises a ruthenium-containing complex and a naphthoquinone or a derivative thereof. .
  • the redox enzyme is reduced by reacting with various metabolites to be measured, and then the metabolites are quantified by reacting with the reduced enzyme and the delivery medium.
  • the present invention describes a glucose measuring biosensor as an example, various metabolites such as cholesterol, lactate, creatinine, hydrogen peroxide, by introducing the electron transfer mediator of the present invention into a specific enzyme having a mechanism similar to the oxidation of glucose, The concentration of organics or inorganics such as alcohols, amino acids or glutamate can be quantified.
  • the present invention can be used without limitation in the quantification of various metabolites by varying the type of enzyme contained in the reagent composition.
  • flavin adenine dinucleotide-glucose dehydrogenase FAD-GDH
  • nicotinamide adenine dinucleotide-glucose dehydrogenase NAD-GDH
  • glutamic acid dehydrogenase Glutamate dehydrogenase
  • cholesterol oxidase lactate oxidase
  • lactate oxidase ascorbic acid oxidase
  • alcohol oxidase alcohol dehydrogenase or bilirubin oxidation
  • enzymes bilirubin oxidase
  • quantification of glucose, glutamate, cholesterol, lactate, ascorbic acid, alcohol or bilirubin can be performed.
  • the electron transfer medium is reduced by redox reaction with the reduced enzyme by reacting with a metabolite, and the electron transfer medium in the reduced state thus formed has an electrode surface to which an oxidation potential is applied. Generates a current in the
  • a ruthenium-containing complex and naphthoquinone or a derivative thereof may be mixed together.
  • ruthenium-containing complexes include [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 ] One or more of 2+ can be used.
  • naphthoquinone derivatives include 2-methoxy-1,4-naphthoquinone, 5,8-dihydroxy-1,4-naphthoquinone, 2-methyl-1,4-naph Toquinone, 2-methyl-1,4-naphthoquinone sodium bisulfite, 1,2-naphthoquinone, 1,4-naphthoquinone, 5-hydroxy-1,4-naphthoquinone, 2- At least one of hydroxy-1,4-naphthoquinone or 1,2-naphthoquinone-4-sulfonate may be used (see FIGS. 2 and 3).
  • the most preferred ruthenium complex in the present invention is hexaamineruthenium chloride, which is stable and reversible in the redox state in aqueous solution, and the reduced electron transfer mediator does not react with oxygen, and oxidation of the reduced electron transfer mediator is sensitive to pH. It does not have the property.
  • the most preferred naphthoquinone derivative in the present invention is 1,2-naphthoquinone-4-sulfonate.
  • glucose detection performance is remarkably improved by using a ruthenium complex and naphthoquinone together.
  • Korean Patent Publication No. 10-1531384 discloses a reagent composition for a biosensor comprising Naphthol Green B, which requires a filtration of the composition due to the presence of a large amount of fine particles due to precipitation in the composition. The difference in composition ratio is caused.
  • the reagent composition developed in the present invention using a combination of a ruthenium-containing complex and a naphthoquinone or a derivative thereof does not require filtration due to the very small and small amount of such particulates, thus increasing the productivity and improving the uniformity of the reagent. have.
  • Republic of Korea Patent Publication No. 10-1355127 discloses an invention that can maintain the linearity to blood sugar by containing a ruthenium-containing complex 250-340 parts by weight based on 100 parts by weight of the oxidoreductase, the reagent composition of the present invention is ruthenium Even if the complex contains only 35-95 parts by weight based on 100 parts by weight of the redox enzyme, the linearity can be maintained, thereby reducing the cost of relatively expensive ruthenium.
  • the redox reagent composition according to the present invention preferably contains 35 to 95 parts by weight of the ruthenium-containing complex based on 100 parts by weight of the redox enzyme. More preferably, it contains 50-70 weight part of ruthenium containing complexes.
  • One embodiment according to the present invention may contain 56 parts by weight of ruthenium containing complex. If the ruthenium-containing complex is less than 35 parts by weight, the reaction of the sensor may be desensitized at a high blood sugar level. If the content is more than 95 parts by weight, the reagent may not be dissolved quickly by the blood. .
  • the redox 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, it contains 10-30 weight part of naphthoquinone or its derivative (s).
  • One embodiment according to the present invention may contain 16 parts by weight of naphthoquinone or a derivative thereof. If it contains less than 5 parts by weight of naphthoquinone or derivatives thereof, there is a problem that the response of the sensor is desensitized at high blood sugar concentration, and if it contains more than 31 parts by weight, the reagent does not dissolve quickly by blood. There may be.
  • the reagent composition according to the present invention may be optionally added by selecting additives such as surfactants, water-soluble polymers, amino acids, disaccharides, and thickeners.
  • the surfactant serves to distribute the reagent evenly over the electrode when dispensing the reagent so that the reagent is dispensed with a uniform thickness.
  • the surfactant include, but are not limited to, Triton X-100, CHAPS ⁇ 3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonate ⁇ , sodium dodecyl sulfate, purple Luorooctane sulfonate or sodium stearate may be used.
  • the redox reagent composition according to the present invention preferably contains 1-120 parts by weight of surfactant, preferably about 10-30 parts by weight, based on 100 parts by weight of redox enzyme.
  • the water-soluble polymer serves as a polymer support of the reagent composition to help stabilize and disperse the enzyme.
  • the water-soluble polymer include, but are not limited to, polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), polyfluoro sulfonate, hydroxyethyl cellulose; HEC), hydroxypropyl cellulose (HPC), carboxy methyl cellulose (CMC), cellulose acetate or polyamide, and the like.
  • the redox reagent composition according to the present invention preferably contains 0.3-12 parts by weight, preferably about 2 parts by weight of the water-soluble polymer, based on 100 parts by weight of the redox enzyme.
  • the amino acid prevents agglomeration between the enzyme and the enzyme to maintain the activity of the enzyme.
  • the redox reagent composition according to the present invention preferably contains 4-51 parts by weight of amino acid, preferably about 17 parts by weight, based on 100 parts by weight of the redox enzyme.
  • the redox reagent composition according to the present invention preferably contains 38-227 parts by weight of disaccharide, preferably about 76 parts by weight, based on 100 parts by weight of the redox enzyme.
  • the electrochemical biosensor, the working electrode, the auxiliary electrode and the confirmation electrode is provided on one plane, the planar electrochemical bio characterized in that the redox reaction reagent composition according to the present invention is coated on the electrode Provide a sensor.
  • the present invention provides an electrochemical biosensor, wherein the working electrode and the auxiliary electrode are provided to face each other on different planes, and the redox reagent composition according to the present invention is coated on the working electrode.
  • the working electrode and the auxiliary electrode are provided to face each other on different planes, and the redox reagent composition according to the present invention is coated on the working electrode.
  • planar and face type electrochemical biosensor according to the present invention, Korean Patent Application No. 10-2003-0036804, Republic of Korea Patent Application 10-2005-0010720, Republic of Korea Patent Application 10-2007-0020447, Republic of Korea Patent Application 10- 2007-0021086, Republic of Korea Patent Application 10-2007-0025106, Republic of Korea Patent Application 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.
  • the planar electrochemical biosensor shown in FIG. 1 includes a top plate 8 having a working electrode, an auxiliary electrode, and a confirming electrode provided on one plane, and having a venting unit 9 for introducing blood into the sensor;
  • An adhesive is coated on both sides to serve to bond the upper plate and the lower plate, and a fitting plate 7 for allowing blood to penetrate into the electrode by capillary action;
  • An insulating plate 5 having a passage portion for defining an area of the following electrode;
  • a redox reagent composition comprising a metal-containing complex and a naphthoquinone or a derivative thereof as the electron transfer medium according to the present invention is provided between an oxidoreductase-naphthoquinone (or a derivative thereof) -metal-containing complex.
  • the reaction rate is fast, and thus the glucose detection performance in the blood is significantly improved, and since it is hardly influenced by oxygen and interfering substances in the blood, it is useful for the preparation of an electrochemical biosensor for detecting glucose in the blood.
  • FAD-GDH flavin adenine dinucleotide-glucose dehydrogenase
  • FAD-GDH flavin adenine dinucleotide-glucose dehydrogenase
  • Ru (NH 3 ) 6 Cl 3 hexaamineruthenium chloride
  • PVP polyvinylpyrrolidone
  • Triton X-100 8 parts by weight of CHAPS
  • trehalose were dissolved in Citrix phosphate buffer (pH 5.8, 500 ml at 0.3 M concentration) and deionized water (500.0 ml), and the remaining particles in the solution were filtered off.
  • planar biosensor As an example of the planar biosensor, a planar biosensor having an average value of 0.5 ⁇ l sample introduction portion was prepared as shown in FIG. 1.
  • Detailed methods for manufacturing the planar biosensors include 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.
  • 1 to 1 is a lower plate plastic made of polyester in which the working electrode (area: 0.9 mm 2 ) and the auxiliary electrode and the confirmation electrode are formed, 2 to 4 are electrodes made by screen printing carbon graphite, 2 is an auxiliary electrode, 3 is a working electrode and 4 is a confirmation electrode.
  • 5 is an insulator defining the area of the upper electrode, 6 is a redox reagent composition prepared in Example 1 coated on the electrode, and 7 is a 0.10 mm thick insert plate which allows blood to penetrate into the electrode by capillary action.
  • the adhesive is coated on both sides thereof to bond the upper and lower plates, 9 is an air outlet for blood to penetrate into the sensor, and 8 is an upper plastic made of polyester.
  • naphthoquinone derivatives shown in FIG. 2 in place of 1,2-naphthoquinone-4-sulfonate in the reagent composition of Example 1, 2-hydroxy-1,4-naphthoquinone (hereafter naphthoquinone derivative)
  • a reagent composition was prepared in the same manner as in Example 1 except that 60 mM was used.
  • naphthoquinone derivatives shown in FIG. 2 in place of 1,2-naphthoquinone-4-sulfonate in the reagent composition of Example 1, 5-hydroxy-1,4-naphthoquinone (hereafter naphthoquinone derivative) b)
  • a reagent composition was prepared in the same manner as in Example 1 except that 10 mM was used.
  • naphthoquinone derivative c 1,2-naphthoquinone (hereinafter referred to as naphthoquinone derivative c) 10
  • a reagent composition was prepared in the same manner as in Example 1 except for using mM.
  • naphthoquinone derivative d 1,4-naphthoquinone (hereinafter referred to as naphthoquinone derivative d) 10
  • a reagent composition was prepared in the same manner as in Example 1 except for using mM.
  • a reagent composition was prepared in the same manner as in Example 1 except that 1,2-naphthoquinone-4-sulfonate was not used.
  • a reagent composition was prepared in the same manner as in Example 1 except that hexaamineruthenium chloride was not used.
  • glucose standard solution is blood prepared from vein blood so that the hematocrit becomes 42% and manufactured using a glucose analyzer (manufacturer: YSI, model name: 2900D) to have various glucose concentrations. .
  • the standard solution with glucose concentrations of 46, 138, 226, 365, 458, and 571 mg / dL simultaneously covered the auxiliary electrode, the working electrode, and the confirmation electrode with 0 mV applied to the working electrode, and waited for 4 seconds. After applying 200 mV to the working electrode was measured for 5 seconds current (see Figure 6). All measurements were made 10 times for each concentration and the average value is shown in FIG. 6.
  • Figure 4 is a graph showing the voltage change in the working electrode of the biosensor according to an embodiment of the present invention, when the sample covers the auxiliary electrode, the working electrode and the confirmation electrode at the same time to apply a 0 mV to the working electrode, waiting for 4 seconds After 200 mV is applied to the working electrode, the current is read in 5 seconds.
  • FIG. 5 is a graph illustrating a change in current appearing 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. 5 is a graph illustrating a change in current appearing 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.
  • the biosensor of Example 1 including hexaamineruthenium chloride and 1,2-naphthoquinone-4-sulfonate as an electron transfer medium gradually increases the current measured according to the glucose concentration. While showing good linearity, the biosensors of Comparative Examples 1 and 2, which were used alone without containing any one of 1,2-naphthoquinone-4-sulfonate and hexaamineruthenium chloride as electron transfer mediums It can be seen that the linearity according to the glucose concentration is not good. At this time, the slope of the current measured in the biosensor according to Example 1 was expressed as 20.2 nA / mm 2 / (mg / dL) per unit area of the working electrode, indicating that the reaction rate against glucose was very fast.
  • the redox reagent composition according to the present invention reacts very quickly to glucose when applied to a biosensor, and thus is useful for preparing a biosensor for detecting glucose in blood.
  • the slope of the current at a glucose concentration of 50 to 600 mg / dL was 21.1 nA / mm 2 / (mg / dL) per unit area of the working electrode.
  • the correlation with the reference equipment (YSI 2900D) is 0.998, and for biosensors 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 per unit area of working electrode).
  • the correlation with the reference equipment is 0.992, and for biosensors with 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) per unit area of the working electrode,
  • the correlation with the equipment is 0.940, and in the case of biosensors using derivative c, the slope of the current at a glucose concentration of 50 to 200 mg / dL is 11.1 nA / mm 2 / (mg / dL) per unit area of the working electrode, which is correlated with the reference equipment.
  • the relationship is 0.844.
  • the glucose concentration is between 50 and 200.
  • the slope of the current at mg / dL was 4.4 nA / mm 2 / (mg / dL) per unit area of the working electrode and the correlation with the reference equipment was 0.678, indicating that the biosensor using 1,2-naphthoquinone-4-sulfonate
  • the reaction rate to glucose was very fast and correlated with the reference equipment.
  • the redox reagent composition according to the present invention is very quickly reacted to glucose when applied to an electrochemical biosensor, and thus is useful for preparing a biosensor for detecting glucose in blood.
  • the electrochemical biosensor according to an embodiment of the present invention is provided that the working electrode, the auxiliary electrode and the confirmation electrode are provided on one plane of the lower substrate, but the working electrode, the auxiliary electrode and the confirmation electrode are on different planes (eg, For example, it may be provided to face to the upper substrate).

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Abstract

The present invention relates to a redox reagent composition for an electrochemical biosensor, containing, as electron transfer mediators, a ruthenium-containing complex and naphthoquinone or a derivative thereof. According to the present invention, the redox reagent composition containing, as electron transfer mediators, a ruthenium-containing complex, and naphthoquinone or a derivative thereof has a remarkable glucose detection performance due to the fast reaction rate between oxidoreductase, naphthoquinone (or a derivative thereof), and the ruthenium-containing complex, and thus is useful in the manufacture of an electrochemical biosensor for the detection of blood glucose.

Description

바이오센서용 산화환원반응 시약조성물Redox Reagent Composition for Biosensor
본 발명은 혈당을 측정하기 위한 시스템의 구성품 중의 하나인 전기화학적 바이오센서용 시약조성물에 관한 것으로서, 루테늄 함유 착물, 및 나프토퀴논 또는 이의 유도체를 포함하는 전자전달매개체에 관한 것이다.The present invention relates to a reagent composition for an electrochemical biosensor, which is one component of a system for measuring blood glucose, and relates to an electron transfer medium comprising a ruthenium-containing complex and a naphthoquinone or a derivative thereof.
안톤 클레멘스(Anton Clemens)가 세계 최초로 휴대용 글루코스 측정 장치를 개발한 이래로 글루코스 측정 장치의 정확도와 편리성을 향상시키려는 노력이 최근 40 여 년 동안 지속되고 있는데, 측정방법으로는 크게 광도측정법과 전기화학적 측정법으로 구분된다. 광도측정법이 비록 먼저 개발되긴 하였으나, 이 방법은 측정 장치가 혈액에 오염되어 정확도가 불안하게 되는 단점이 있다. 반면에 전기화학적 측정법은 광도측정법과 비교하여 볼 때, 측정 장치가 혈액에 의하여 오염되는 경우가 적고, 측정에 필요로 하는 혈액의 양이 적어서 현재의 상용화된 측정법의 대부분을 차지하게 되었다.Since Anton Clemens developed the world's first portable glucose measuring device, efforts to improve the accuracy and convenience of glucose measuring devices have continued for the last 40 years. The measuring methods are mainly photometric and electrochemical measuring methods. Separated by. Although photometry was first developed, this method has the disadvantage that the measuring device is contaminated with blood and thus the accuracy becomes unstable. On the other hand, the electrochemical measurement method compared to the photometric method, the measurement device is less contaminated by blood, and the amount of blood required for the measurement is occupied most of the current commercial measurement method.
정확도를 향상 시키려는 노력은 효소의 발전과 밀접한 관계를 가지고 있는데, 예를 들어 전기화학적 센서에 사용되는 글루코스 산화환원효소인 FAD-GOx(효소분류 번호 1.1.3.4)는 열에 안정하고 글루코스만을 산화시키는 반응선택성은 탁월하지만, 혈액에 녹아 있는 산소의 농도에 따라 측정치가 영향을 받는 단점이 있어서 정맥혈관, 동맥혈관 또는 모세혈관 등에서 채취되는 모든 혈액에 두루두루 적용하기 어렵다.Efforts to improve accuracy are closely related to the development of enzymes. For example, FAD-GOx (Enzyme Classification No. 1.1.3.4), a glucose redox enzyme used in electrochemical sensors, is heat stable and only oxidizes glucose. Although the selectivity is excellent, the measurement value is affected by the concentration of oxygen dissolved in the blood, so it is difficult to apply it to all blood collected from venous vessels, arterial vessels, or capillaries.
반면에 PQQ-GDH(효소분류 번호 1.1.5.2)효소를 이용하여 만든 센서는 산소에 의한 영향은 없지만, 만노스(mannose), 말토스(maltose) 또는 락토스(lactose) 등과 같은 여러 종류의 단당류 또는 이당류에 의하여 영향을 받는 단점이 있는데, 특히 당뇨환자에 주입하는 고농도의 말토스는 혈당농도가 실제보다 훨씬 높게 측정되도록 영향을 주어서 주목을 받아왔다(Mehmet, S., Quan, G., Thomas, S., and Goldsmith, D., Important causes of hypoglycemia in patients with diabetes on peritoneal dialysis. Diabet. Med., 18, 679-682(2001)). 로슈사는 이러한 문제를 해결하기 위하여 PQQ-GDH의 변형효소(mutant enzyme)를 개발하여서 말토스에 의한 측정의 부정확성을 상당 부분 해소하였다.On the other hand, sensors made using PQQ-GDH (Enzyme Classification No. 1.1.5.2) enzymes are not affected by oxygen, but can contain several types of monosaccharides or disaccharides such as mannose, maltose or lactose. In particular, high concentrations of maltose injected into diabetic patients have received attention because they affect blood glucose levels to be measured 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.Diabet. Med., 18, 679-682 (2001)). To solve this problem, Roche developed a mutant enzyme of PQQ-GDH to largely eliminate the inaccuracy of maltose measurements.
NAD-GDH 효소는 글루코스와만 반응하는 선택성은 좋지만 NAD+ 또는 NADP+를 시약조성 성분에 추가로 포함하여야 하는 불편함이 있다. 상기에 열거한 단점들을 보완하는 효소로서 FAD-GDH(효소분류 번호 1.1.99.10)가 상대적으로 최근에 일본의 아마노사와 토요보사에 의하여 개발되었는데, 이 효소를 이용하는 전기화학적 센서는 혈중에 녹아 있는 산소의 양에 영향을 받지 않아서 체내의 모든 혈액에 효과적으로 적용할 수 있다는 장점이 있다. 단 자일로오즈(xylose)와 반응하는 단점이 있기는 하지만, 만노스, 말토스 및 락토스와는 반응하지 않아서 글루코스와 단독으로 반응하는 선택성이 비교적 우수하다.The NAD-GDH enzyme has good selectivity to react only with glucose, but it is inconvenient to additionally include NAD + or NADP + in the reagent composition. FAD-GDH (Enzyme Classification No. 1.1.99.10) has been developed relatively recently by Amano and Toyobo of Japan. It is not affected by the amount of it has the advantage that it can be effectively applied to all the blood in the body. Although it has a disadvantage of reacting with xylose, it does not react with mannose, maltose and lactose, so that the selectivity of reacting alone with glucose is relatively excellent.
효소와 더불어 센서의 정확도에 영향을 주는 물질은 전자전달체이다. 전자전달체는 효소와의 반응이 빨라서 고농도의 혈당 측정이 가능해야 하고, 장기간 변질되지 않아서 정확도가 유통기간 동안 유지되어야 한다. FAD-GDH의 전자전달매개체로는 포타슘페리시아나이드[K3Fe(CN)6], 페나진메토설페이트(phenazine-methosulfate), 메톡시페나진메토설페이트(methoxyphenazine-methosulfate), 페나진메틸설페이트(phenazine methylsulfate) 또는 디클로로인도페놀(dichloroindophenol) 등이 알려져 있는데, 이들 모두 온도와 습도에 의하여 변질되기 쉽기 때문에 센서를 장기간 보관할 시 정확도가 떨어지는 경향이 있다. 바이엘사는 이러한 단점이 보완된 페노티아진(phenothiazine)의 친수성 유도체를 개발하였다(미국 등록특허공보 제 9039878호).In addition to enzymes, the material that affects the accuracy of the sensor is the electron transporter. The electron transporter must be able to measure high blood glucose level due to its rapid reaction with enzymes, and must not be altered for a long time, so the accuracy must be maintained for the distribution period. Examples of electron transfer mediators of FAD-GDH include potassium ferricyanide [K 3 Fe (CN) 6 ], phenazine-methosulfate, methoxyphenazine-methosulfate, and phenazinemethyl sulfate ( phenazine methylsulfate or dichloroindophenol are known, and both are susceptible to deterioration due to temperature and humidity. Bayer has developed a hydrophilic derivative of phenothiazine that compensates for this disadvantage (US Patent No. 9039878).
헥사아민루테늄클로라이드[Ru(NH3)6Cl3]를 전자전달체로 사용할 경우 포타슘페리시아나이드를 사용할 경우와 비교해 볼 때, 요산(uric acid) 또는 겐티식산(gentisic acid)과 같은 방해 물질에 의해 영향을 받는 정도가 적고 습도에 의하여 정확도가 둔화되는 정도가 적어서, 헥사아민루테늄클로라이드가 FAD-GOx를 이용하는 전기화학적 센서에 사용되어 왔다(미국 등록특허공보 제 7288174호). 그러나 상기 전자전달체는 FAD-GDH(EC No. 1.1.99.10)와 반응하는 속도가 느려서 센서 제작에 유용하지 않다.When hexaamineruthenium chloride [Ru (NH 3 ) 6 Cl 3 ] is used as an electron transporter, compared with the case of using potassium ferricyanide, it may be caused by an interfering substance such as uric acid or gentisic acid. Because of the low degree of influence and the low degree of accuracy degradation due to humidity, hexaamineruthenium chloride has been used in electrochemical sensors using FAD-GOx (US Patent No. 7288174). However, the electron transporter is not useful for fabricating the sensor due to the slow reaction rate with FAD-GDH (EC No. 1.1.99.10).
한편 두 가지의 전자전달체를 이용한 예들은 하기와 같다.Meanwhile, examples using two electron transporters are as follows.
유럽 등록특허공보 제 0238322호는 벤조퀴논을 포타슘페리시아나이드와 함께 사용하여 박테리아와 전극간의 전자전달속도를 현저히 증가시키는 방법에 관하여 개시하고 있다. EP 0238322 discloses a method of using benzoquinone with potassium ferricyanide to significantly increase the electron transfer rate between bacteria and electrodes.
미국 등록특허공보 제 8057659호는 오스뮴(Os)과 포타슘페리시아나이드를 글루코스센서에 적용하는 방법에 관하여 개시하고 있다.US Patent No. 8057659 discloses a method of applying osmium (Os) and potassium ferricyanide to a glucose sensor.
대한민국 등록특허공보 제 10-1355127호는 티오닌과 루테늄 착물을 함께 사용하여 글루코스센서에 적용하는 방법에 관하여 개시하고 있다.Korean Patent Publication No. 10-1355127 discloses a method of applying to a glucose sensor using a thionine and ruthenium complex together.
대한민국 등록특허공보 제 10-1531384호는 나프톨그린비와 루테늄 착물을 함께 사용하여 글루코스센서에 적용하는 방법에 관하여 개시하고 있다.Korean Patent Publication No. 10-1531384 discloses a method for applying a glucose sensor using a naphthol green ratio and a ruthenium complex together.
미국 등록특허공보 제 8658011호는 페나진메토설페이트와 루테늄 착물을 함께 사용하여 글루코스센서에 적용하는 방법에 관하여 개시하고 있다.US Patent Publication No. 8658011 discloses a method for applying a phenazinemethosulfate and ruthenium complex together to a glucose sensor.
아민(A. Amine)과 그의 동료들은 페나진메토설페이트와 포타슘페리시아나이드를 글루코스 옥시다아제에와 글루타메이트 디하이드로게나아제에 적용하는 방법에 관하여 개시하고 있다.A. Amine and his colleagues disclose a method of applying phenazinemethosulfate and potassium ferricyanide to glucose oxidase and glutamate dehydrogenase.
본 발명자들은 FAD-GDH와의 반응성이 뛰어난 전자전달체를 개발하던 중, 루테늄 함유 착물, 및 나프토퀴논 또는 이의 유도체를 포함하는 시약조성물이 FAD-GDH와의 반응속도가 빨라져 글루코스 검출 성능이 현저히 향상되는 것을 알아내고 본 발명을 완성하였다.The inventors of the present invention have developed an electron transporter having excellent reactivity with FAD-GDH, and a reagent composition including a ruthenium-containing complex and a naphthoquinone or a derivative thereof has a rapid reaction rate with FAD-GDH, thereby significantly improving glucose detection performance. It was found and completed the present invention.
본 발명자는 산화환원반응을 이용하여 혈당을 측정하는 전기화학적 바이오센서에 있어서, 신규의 시약조성물을 개발하고자 하였다. 구체적으로는 FAD-GDH 효소를 이용하는 전기화학적 센서에 있어서, 글루코스와의 선택적 반응성이 우수하고, 효소와의 반응이 빨라 고농도의 혈당 측정이 가능하며, 제품 유통 및 사용 가운데 장기간 변질되지 않는 안정성을 갖춘 전자전달체의 개발에 노력하였다.The present inventors have attempted to develop a novel reagent composition in an electrochemical biosensor measuring blood glucose using redox reactions. Specifically, in the electrochemical sensor using the FAD-GDH enzyme, it has excellent selective reactivity with glucose, fast reaction with the enzyme, which enables high blood sugar measurement, and stability that does not deteriorate during product distribution and use for a long time. Efforts have been made to develop electron transporters.
본 발명자는 전자전달매개체로서 루테늄 함유 착물과, 나프토퀴논 또는 이의 유도체를 조합했을 때, 정확한 기전은 모르겠으나, 상호 성분의 시너지 작용으로 인해, 전자전달체로서 유용한 효과를 나타냄을 실험을 통해 발견하게 되어 본 발명을 완성하기에 이르렀다. 본 발명자는 전술한 과제를 아래의 수단을 통해 해결하였다.The present inventors have found through experiments that when ruthenium-containing complexes and naphthoquinones or derivatives thereof are combined as electron transfer mediators, they do not know the exact mechanism, but due to the synergy of mutual components, they show useful effects as electron transfer mediators. Thus, the present invention has been completed. The present inventor has solved the above problem through the following means.
1. 산화환원효소 및 전자전달매개체를 포함하는 바이오센서용 시약조성물로서, 전자전달매개체가 루테늄 함유 착물, 및 나프토퀴논 또는 이의 유도체를 포함하는 것을 특징으로 하는 시약조성물.1. A reagent composition for a biosensor comprising an oxidoreductase and an electron transfer medium, wherein the electron transfer medium comprises a ruthenium-containing complex, and naphthoquinone or a derivative thereof.
2. 상기 1 에 있어서, 산화환원효소가 플라빈아데닌디뉴클레오티드-글루코스탈수소효소(flavin adenine dinucleotide-glucose dehydrogenase), 니코틴아미드아데닌디뉴클레오티드-글루코스탈수소효소(nicotinamide adenine dinucleotide-glucose dehydrogenase), 글루코스탈수소효소(glucose dehydrogenase), 글루탐산탈수소효소(glutamate dehydrogenase), 콜레스테롤산화효소(cholesterol oxidase), 락테이트산화효소(lactate oxidase), 아스코빅산산화효소(ascorbic acid oxidase), 알코올산화효소(alcohol oxidase), 알코올탈수소효소(alcohol dehydrogenase) 및 빌리루빈산화효소(bilirubin oxidase)로 이루어지는 군으로부터 선택되는 1 종인 것을 특징으로 하는 시약조성물.2. The redox enzyme according to 1 above, wherein the redox enzyme is flavin adenine dinucleotide-glucose dehydrogenase, nicotinamide adenine dinucleotide-glucose dehydrogenase, or glucose dehydrogenase. (glucose dehydrogenase), glutamate dehydrogenase, cholesterol oxidase, lactate oxidase, ascorbic acid oxidase, alcohol oxidase, alcohol dehydrogenase A reagent composition, which is one kind selected from the group consisting of an enzyme (alcohol dehydrogenase) and bilirubin oxidase.
3. 상기 1 또는 2 에 있어서, 루테늄 함유 착물이 [Ru(NH3)6]Br3, [Ru(NH3)5Cl]Cl2, K4[Ru(CN)6], Ru(NH3)6Cl3, [Ru(2,2',2''-terpyridine)(1,10-phenanthroline)(OH2)]2+, trans-[Ru(2,2'-bipyridine)2(OH2)(OH)]2 +, [(2,2'-bipyridine)2(OH)RuORu(OH)(2,2'bpy)2]4+ 및 [Ru(4,4'-bipyridine)(NH3)5]2+ 로 이루어지는 군으로부터 선택되는 1 종인 것을 특징으로 하는 시약조성물.3. The ruthenium-containing complex according to 1 or 2 above, wherein the ruthenium containing complex is [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 ] A reagent composition, characterized in that one kind selected from the group consisting of 2+ .
4. 상기 1 내지 3 중 어느 하나에 있어서, 나프토퀴논 및 이의 유도체가 2-메톡시-1,4-나프토퀴논, 5,8-디하이드록시-1,4-나프토퀴논, 2-메틸-1,4-나프토퀴논, 2-메틸-1,4-나프토퀴논 소듐바이썰파이트, 1,2-나프토퀴논, 1,4-나프토퀴논, 5-하이드록시-1,4-나프토퀴논, 2-하이드록시-1,4-나프토퀴논 및 1,2-나프토퀴논-4-썰포네이트로 이루어지는 군으로부터 선택되는 1 종인 것을 특징으로 하는 시약조성물.4. The method according to any one of 1 to 3, wherein naphthoquinone and its derivatives are 2-methoxy-1,4-naphthoquinone, 5,8-dihydroxy-1,4-naphthoquinone, 2- Methyl-1,4-naphthoquinone, 2-methyl-1,4-naphthoquinone sodium bisulfite, 1,2-naphthoquinone, 1,4-naphthoquinone, 5-hydroxy-1,4 -A reagent composition, characterized in that it is one kind selected from the group consisting of naphthoquinone, 2-hydroxy-1,4-naphthoquinone and 1,2-naphthoquinone-4-sulfonate.
5. 상기 1 내지 4 중 어느 하나에 있어서, 산화환원효소가 플라빈아데닌디뉴클레오티드-글루코스탈수소효소(FAD-GDH)인 것을 특징으로 하는 시약조성물.5. The reagent composition according to any one of 1 to 4, wherein the oxidoreductase is flavin adenine dinucleotide-glucose dehydrogenase (FAD-GDH).
6. 상기 1 내지 5 중 어느 하나에 있어서, 산화환원효소 100 중량부를 기준으로 루테늄 함유 착물이 35-95 중량부인 것을 특징으로 하는 시약조성물.6. The reagent composition according to any one of 1 to 5, wherein the ruthenium-containing complex is 35-95 parts by weight based on 100 parts by weight of the oxidoreductase.
7. 상기 1 내지 6 중 어느 하나에 있어서, 산화환원효소 100 중량부를 기준으로 나프토퀴논 또는 이의 유도체가 5-31 중량부인 것을 특징으로 하는 시약조성물.7. The reagent composition according to any one of 1 to 6, wherein the naphthoquinone or its derivative is 5-31 parts by weight based on 100 parts by weight of the oxidoreductase.
8. 상기 1 내지 7 중 어느 하나에 있어서, 필요에 따라 계면활성제, 수용성 고분자, 아미노산, 이당체 또는 점증제와 같은 첨가제를 추가로 포함하는 것을 특징으로 하는 시약조성물.8. The reagent composition according to any one of 1 to 7, further comprising additives such as surfactants, water-soluble polymers, amino acids, disaccharides, or thickeners as necessary.
9. 상기 1 내지 8 중 어느 하나에 있어서, 산화환원효소 100 중량부를 기준으로, 산화환원효소 100 중량부, 나프토퀴논 또는 이의 유도체 0.1-80 중량부(바람직하게는 5-31 중량부), 루테늄 함유 착물 35-95 중량부, 계면활성제 1-120 중량부, 수용성 고분자 0.3-12 중량부, 아미노산 4-51 중량부 및 이당체 38-227 중량부를 포함하는 것을 특징으로 하는 시약조성물.9. according to any one of 1 to 8, based on 100 parts by weight of the oxidoreductase, 100 parts by weight of the oxidoreductase, 0.1-80 parts by weight of naphthoquinone or a derivative thereof (preferably 5-31 parts by weight), A reagent composition comprising 35-95 parts by weight of ruthenium-containing complex, 1-120 parts by weight of surfactant, 0.3-12 parts by weight of water-soluble polymer, 4-51 parts by weight of amino acid and 38-227 parts by weight of disaccharide.
본 발명에 따른 전자전달매개체인 루테늄 함유 착물, 및 나프토퀴논 또는 이의 유도체를 포함하는 산화환원반응 시약조성물은 산화환원효소-나프토퀴논(또는 이의 유도체)-금속함유 착물 간의 반응속도가 빨라, 글루코스 검출 성능이 현저히 향상되어, 혈액 내 글루코스 검출을 위한 전기화학적 바이오센서의 제조에 유용하다. 또한 장기간 보관에 따른 저장안정성도 우수하다.The redox reagent composition comprising a ruthenium-containing complex, a naphthoquinone or a derivative thereof, according to the present invention, has a fast reaction rate between a redox enzyme-naphthoquinone (or a derivative thereof) -metal complex, Glucose detection performance is significantly improved, which is useful for the manufacture of electrochemical biosensors for the detection of glucose in the blood. In addition, the storage stability of long-term storage is excellent.
특히 대한민국 등록특허공보 제 10-1531384호에서 기술된 나프톨 그린 B(Naphthol Green B)를 포함하는 바이오센서용 시약조성물은 조성물 내 침전 인한 미립자가 다량 존재하여 조성물의 여과가 필요하며 여과에 따른 조성비의 차이가 유발되나, 본 발명에서 개발한 시약조성물은 이러한 미립자가 매우 작고 소량이어서 여과가 필요하지 않으므로 양산성 증가와 시약의 균일도가 향상되는 효과가 있다.In particular, a reagent composition for a biosensor comprising Naphthol Green B described in Korean Patent Publication No. 10-1531384 requires filtration of the composition due to the presence of a large amount of fine particles due to precipitation in the composition. Although the difference is caused, the reagent composition developed in the present invention has the effect of increasing the mass productivity and improving the uniformity of the reagents because the microparticles are very small and a small amount, so no filtration is required.
또한 대한민국 등록특허공보 제 10-1355127호에서 기술된 시약 조성물은 루테늄함유 착물을 산화환원효소 100 중량부를 기준으로 250 ~ 340 중량부를 함유하여야만 혈당에 대한 직선성을 유지할 수 있으나, 본 발명의 시약조성물은 루테늄함유 착물을 산화환원효소 100 중량부를 기준으로 35 ~ 95 중량부 함유하여도 직선성을 유지할 수 있어 상대적으로 고가인 루테늄의 원가절감 효과가 있다.In addition, the reagent composition described in Republic of Korea Patent Publication No. 10-1355127 should maintain the linearity to blood sugar only if the ruthenium-containing complex should contain 250 to 340 parts by weight based on 100 parts by weight of the oxidoreductase, but the reagent composition of the present invention Even if the ruthenium-containing complex contains 35 to 95 parts by weight based on 100 parts by weight of the redox enzyme, the linearity can be maintained, thereby reducing the cost of the relatively expensive ruthenium.
또한 전자전달매개체로 루테늄함유 착물 및 나프토퀴논 또는 이의 유도체를 포함하는 시약조성물은 산화환원효소와 나프토퀴논 또는 이의 유도체 그리고 루테늄함유 착물 간의 반응속도가 빨라서 혈액 내 글루코스 검출 성능이 향상되고, 혈액 내 산소 및 방해 물질의 영향을 거의 받지 않아, 글루코스 검출을 위한 바이오센서의 제조에 유용하다.In addition, the reagent composition comprising a ruthenium-containing complex and naphthoquinone or a derivative thereof as an electron transfer medium has a fast reaction rate between oxidoreductase, naphthoquinone or a derivative thereof and a ruthenium-containing complex, thereby improving glucose detection performance in blood and Since it is hardly affected by oxygen and interfering substances, it is useful in the manufacture of biosensors for glucose detection.
도 1 은 본 발명의 일 실시예에서 사용한 전기화학적 바이오센서의 분해사시도이다.1 is an exploded perspective view of an electrochemical biosensor used in an embodiment of the present invention.
도 2 및 3 은 본 발명의 일 실시예에서 사용한 나프토퀴논 및 이의 유도체들의 구조이다.2 and 3 are structures of naphthoquinone and derivatives thereof used in one embodiment of the present invention.
도 4 는 본 발명의 일 실시예에 따른 바이오센서의 작동전극에서 전압 변화를 나타낸 그래프로서, 시료가 보조전극과 작동전극을 덮고 혈액 확인전극을 덮는 순간 작동전극에 0 mV 를 걸어주고, 4 초간 대기한 후 작동전극에 200 mV 를 걸어 준 뒤 5 초에 전류를 읽는 것을 나타낸다.Figure 4 is a graph showing the voltage change in the working electrode of the biosensor according to an embodiment of the present invention, when the sample covers the auxiliary electrode and the working electrode and covered the blood confirmation electrode to apply 0 mV to the working electrode for 4 seconds After waiting, 200 mV is applied to the working electrode and current is read in 5 seconds.
도 5 는 실시예 1 과 비교예 1 내지 2 에 따른 전기화학적 바이오센서를 이용하여 글루코스 농도를 달리한 시료를 적용하였을 경우, 나타나는 전류의 변화를 측정한 그래프이다.FIG. 5 is a graph illustrating changes in current appearing when a sample having a different glucose concentration is applied using an electrochemical biosensor according to Example 1 and Comparative Examples 1 to 2. FIG.
도 6 은 실시예 1 내지 6 에 따른 전기화학적 바이오센서를 이용하여 글루코스 농도를 달리한 시료를 적용하였을 경우, 나타나는 전류의 변화를 측정한 그래프이다.FIG. 6 is a graph illustrating changes in current appearing when a sample having a different glucose concentration is applied using an electrochemical biosensor according to Examples 1 to 6. FIG.
이하 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 산화환원효소 및 전자전달매개체를 포함하되,상기 전자전달매개체가 루테늄 함유 착물, 및 나프토퀴논 또는 이의 유도체를 포함하는 것을 특징으로 하는 전기화학적 바이오센서용 산화환원반응 시약조성물을 제공한다.The present invention includes an oxidoreductase and an electron transfer mediator, wherein the electron transfer mediator comprises a ruthenium-containing complex and a naphthoquinone or a derivative thereof. .
본 발명에 따른 산화환원반응 시약조성물에 있어서, 상기 산화환원효소는 측정하고자 하는 다양한 대사물질과 반응하여 환원되는 것으로, 이후 환원된 효소와 전달매개체와 반응하여 대사물질을 정량하게 된다.In the redox reagent composition according to the present invention, the redox enzyme is reduced by reacting with various metabolites to be measured, and then the metabolites are quantified by reacting with the reduced enzyme and the delivery medium.
본 발명은 글루코스 측정 바이오센서를 실시예로 설명하지만, 글루코스의 산화와 비슷한 기작을 가지는 특정효소에 본 발명의 전자전달매개체를 도입함으로써, 다양한 대사물질 예를 들면 콜레스테롤, 락테이트, 크레아티닌, 과산화수소, 알코올, 아미노산 또는 글루타메이트와 같은 유기물 또는 무기물의 농도를 정량할 수 있다.Although the present invention describes a glucose measuring biosensor as an example, various metabolites such as cholesterol, lactate, creatinine, hydrogen peroxide, by introducing the electron transfer mediator of the present invention into a specific enzyme having a mechanism similar to the oxidation of glucose, The concentration of organics or inorganics such as alcohols, amino acids or glutamate can be quantified.
따라서 본 발명은 시약조성물에 포함되는 효소의 종류를 달리함으로써 다양한 대사물질의 정량에 제한 없이 이용될 수 있다.Therefore, the present invention can be used without limitation in the quantification of various metabolites by varying the type of enzyme contained in the reagent composition.
예를 들면 플라빈아데닌디뉴클레오티드-글루코스탈수소효소(flavin adenine dinucleotide-glucose dehydrogenase, FAD-GDH), 니코틴아미드아데닌디뉴클레오티드-글루코스탈수소효소(nicotinamide adenine dinucleotide-glucose dehydrogenase, NAD-GDH), 글루탐산탈수소효소(glutamate dehydrogenase), 콜레스테롤산화효소(cholesterol oxidase), 락테이트산화효소(lactate oxidase), 아스코빅산산화효소(ascorbic acid oxidase), 알코올산화효소(alcohol oxidase), 알코올탈수소효소(alcohol dehydrogenase) 또는 빌리루빈산화효소(bilirubin oxidase) 등을 사용하여, 글루코스, 글루타메이트, 콜레스테롤, 락테이트, 아스코빅산, 알코올 또는 빌리루빈 등의 정량을 수행할 수 있다.For example, flavin adenine dinucleotide-glucose dehydrogenase (FAD-GDH), nicotinamide adenine dinucleotide-glucose dehydrogenase (NAD-GDH), glutamic acid dehydrogenase (glutamate dehydrogenase), cholesterol oxidase, lactate oxidase, ascorbic acid oxidase, alcohol oxidase, alcohol dehydrogenase or bilirubin oxidation Using enzymes (bilirubin oxidase) and the like, quantification of glucose, glutamate, cholesterol, lactate, ascorbic acid, alcohol or bilirubin can be performed.
본 발명에 따른 산화환원반응 시약조성물에 있어서, 상기 전자전달매개체는 대사물질과 반응하여 환원된 효소와 산화환원반응하여 환원되게 되며, 이렇게 형성된 환원상태의 전자전달매개체는 산화전위가 인가된 전극표면에서 전류를 발생시키는 역할을 수행한다.In the redox reagent composition according to the present invention, the electron transfer medium is reduced by redox reaction with the reduced enzyme by reacting with a metabolite, and the electron transfer medium in the reduced state thus formed has an electrode surface to which an oxidation potential is applied. Generates a current in the
이때 상기 전자전달매개체로는 루테늄 함유 착물과, 나프토퀴논 또는 이의 유도체를 함께 혼합하여 사용할 수 있다.In this case, as the electron transfer medium, a ruthenium-containing complex and naphthoquinone or a derivative thereof may be mixed together.
상기 전자전달매개체에서, 루테늄 함유 착물로는 [Ru(NH3)6]Br3, [Ru(NH3)5Cl]Cl2, K4[Ru(CN)6], Ru(NH3)6Cl3, [Ru(2,2',2''-terpyridine)(1,10-phenanthroline)(OH2)]2+, trans-[Ru(2,2'-bipyridine)2(OH2)(OH)]2 +, [(2,2'-bipyridine)2(OH)RuORu(OH)(2,2'bpy)2]4+ 또는 [Ru(4,4'-bipyridine)(NH3)5]2+ 등 가운데 1 종 이상 사용할 수 있다.In the electron transfer medium, ruthenium-containing complexes include [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 ] One or more of 2+ can be used.
상기 전자전달매개체에서, 나프토퀴논 유도체로는 2-메톡시-1,4-나프토퀴논, 5,8-디하이드록시-1,4-나프토퀴논, 2-메틸-1,4-나프토퀴논, 2-메틸-1,4-나프토퀴논 소듐바이썰파이트, 1,2-나프토퀴논, 1,4-나프토퀴논, 5-하이드록시-1,4-나프토퀴논, 2-하이드록시-1,4-나프토퀴논 또는 1,2-나프토퀴논-4-썰포네이트 등 가운데 1 종 이상을 사용할 수 있다(도 2 및 3 참조).In the electron transfer medium, naphthoquinone derivatives include 2-methoxy-1,4-naphthoquinone, 5,8-dihydroxy-1,4-naphthoquinone, 2-methyl-1,4-naph Toquinone, 2-methyl-1,4-naphthoquinone sodium bisulfite, 1,2-naphthoquinone, 1,4-naphthoquinone, 5-hydroxy-1,4-naphthoquinone, 2- At least one of hydroxy-1,4-naphthoquinone or 1,2-naphthoquinone-4-sulfonate may be used (see FIGS. 2 and 3).
본 발명에서 가장 바람직한 루테늄 착물로는 헥사아민루테늄클로라이드로서, 수용액에서 산화-환원 상태가 안정하며 가역적이고, 환원된 전자전달매개체가 산소와 반응하지 않으며, 환원된 전자전달매개체의 산화가 pH 에 민감하지 않는 특성을 갖는다.The most preferred ruthenium complex in the present invention is hexaamineruthenium chloride, which is stable and reversible in the redox state in aqueous solution, and the reduced electron transfer mediator does not react with oxygen, and oxidation of the reduced electron transfer mediator is sensitive to pH. It does not have the property.
또한 본 발명에서 가장 바람직한 나프토퀴논 유도체로는 1,2-나프토퀴논-4-썰포네이트이다.In addition, the most preferred naphthoquinone derivative in the present invention is 1,2-naphthoquinone-4-sulfonate.
본 발명에 따른 상기 전자전달매개체는 루테늄 착물과, 나프토퀴논을 함께 사용함으로써 글루코스 검출 성능이 현저히 향상된다.In the electron transfer medium according to the present invention, glucose detection performance is remarkably improved by using a ruthenium complex and naphthoquinone together.
예를 들어 본 발명의 비교예 1 과 2 에서 전자전달매개체로 나프토퀴논과 헥사아민루테늄클로라이드[Ru(NH3)6Cl3] 중 어느 하나를 각각 단독으로 사용할 경우에는 산화환원효소와 반응속도가 느려서 바이오센서로 적용하기가 어렵다.For example, when one of naphthoquinone and hexaamineruthenium chloride [Ru (NH 3 ) 6 Cl 3 ] is used alone as the electron transfer medium in Comparative Examples 1 and 2 of the present invention, the oxidoreductase and the reaction rate are It is slow and difficult to apply as a biosensor.
하지만 본 발명에 따른 전자전달매개체로 루테늄함유 착물, 및 나프토퀴논 또는 이의 유도체를 함께 사용할 경우에는 산화환원효소와, 나프토퀴논 또는 이의 유도체 사이의 반응속도가 빠르고, 나프토퀴논과 루테늄 함유 착물과의 반응속도가 빨라서 바이오센서의 제조에 유용하게 사용할 수 있다(실험예 1 참조).However, when a ruthenium-containing complex and a naphthoquinone or a derivative thereof are used together as an electron transfer medium according to the present invention, the reaction rate between the oxidoreductase, naphthoquinone or a derivative thereof is fast, and the naphthoquinone and ruthenium-containing complex are Fast reaction rate can be usefully used for the production of biosensors (see Experimental Example 1).
대한민국 등록특허공보 제 10-1531384호에서는 나프톨 그린 B(Naphthol Green B)를 포함하는 바이오센서용 시약조성물을 게시하는데, 이는 조성물 내 침전으로 인한 미립자가 다량 존재하여 조성물의 여과가 필요하며, 여과에 따른 조성비의 차이가 유발된다. 하지만 루테늄함유 착물과, 나프토퀴논 또는 이의 유도체를 조합하여 사용하는 본 발명에서 개발한 시약조성물은 이러한 미립자가 매우 작고 소량이어서 여과가 필요하지 않으므로, 양산성 증가와 시약의 균일도가 향상되는 효과가 있다.Korean Patent Publication No. 10-1531384 discloses a reagent composition for a biosensor comprising Naphthol Green B, which requires a filtration of the composition due to the presence of a large amount of fine particles due to precipitation in the composition. The difference in composition ratio is caused. However, the reagent composition developed in the present invention using a combination of a ruthenium-containing complex and a naphthoquinone or a derivative thereof does not require filtration due to the very small and small amount of such particulates, thus increasing the productivity and improving the uniformity of the reagent. have.
대한민국 등록특허공보 제 10-1355127호에서는 루테늄함유 착물을 산화환원효소 100 중량부를 기준으로 250-340 중량부 함유하여 혈당에 대한 직선성을 유지할 수 있는 발명을 게시하는데, 본 발명의 시약조성물은 루테늄 함유 착물을 산화환원효소 100 중량부를 기준으로 35-95 중량부만 함유하더라도 직선성을 유지할 수 있어, 상대적으로 고가인 루테늄의 원가절감 효과가 있다.Republic of Korea Patent Publication No. 10-1355127 discloses an invention that can maintain the linearity to blood sugar by containing a ruthenium-containing complex 250-340 parts by weight based on 100 parts by weight of the oxidoreductase, the reagent composition of the present invention is ruthenium Even if the complex contains only 35-95 parts by weight based on 100 parts by weight of the redox enzyme, the linearity can be maintained, thereby reducing the cost of relatively expensive ruthenium.
이때 본 발명에 따른 산화환원반응 시약조성물은 산화환원효소 100 중량부를 기준으로 루테늄 함유 착물을 35-95 중량부 함유하는 것이 바람직하다. 보다 바람직하게는 루테늄 함유 착물을 50-70 중량부 함유한다. 본 발명에 따른 일 구현예는 루테늄 함유 착물을 56 중량부 함유할 수 있다. 만약 루테늄 함유 착물을 35 중량부 미만으로 함유할 경우에는 높은 혈당농도에서 센서의 반응이 둔감해지는 문제가 있고, 95 중량부를 초과하여 함유할 경우에는 혈액에 의하여 시약이 빨리 녹지 않는 문제가 있을 수 있다.At this time, the redox reagent composition according to the present invention preferably contains 35 to 95 parts by weight of the ruthenium-containing complex based on 100 parts by weight of the redox enzyme. More preferably, it contains 50-70 weight part of ruthenium containing complexes. One embodiment according to the present invention may contain 56 parts by weight of ruthenium containing complex. If the ruthenium-containing complex is less than 35 parts by weight, the reaction of the sensor may be desensitized at a high blood sugar level. If the content is more than 95 parts by weight, the reagent may not be dissolved quickly by the blood. .
또한 본 발명에 따른 산화환원반응 시약조성물은 산화환원효소 100 중량부를 기준으로 나프토퀴논 또는 이의 유도체를 5-31 중량부 함유하는 것이 바람직하다. 보다 바람직하게는 나프토퀴논 또는 이의 유도체를 10-30 중량부 함유한다. 본 발명에 따른 일 구현예는 나프토퀴논 또는 이의 유도체를 16 중량부 함유할 수 있다. 만약 나프토퀴논 또는 이의 유도체를 5 중량부 미만으로 함유할 경우에는 높은 혈당농도에서 센서의 반응이 둔감해지는 문제가 있고, 31 중량부를 초과하여 함유할 경우에는 혈액에 의하여 시약이 빨리 녹지 않는 문제가 있을 수 있다.In addition, the redox 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, it contains 10-30 weight part of naphthoquinone or its derivative (s). One embodiment according to the present invention may contain 16 parts by weight of naphthoquinone or a derivative thereof. If it contains less than 5 parts by weight of naphthoquinone or derivatives thereof, there is a problem that the response of the sensor is desensitized at high blood sugar concentration, and if it contains more than 31 parts by weight, the reagent does not dissolve quickly by blood. There may be.
한편 본 발명에 따른 시약조성물은 계면활성제, 수용성 고분자, 아미노산, 이당체 및 점증제 등과 같은 첨가제를 선택하여 임의로 추가 할 수 있다.Meanwhile, the reagent composition according to the present invention may be optionally added by selecting additives such as surfactants, water-soluble polymers, amino acids, disaccharides, and thickeners.
상기 첨가제에 있어서, 계면활성제는 시약을 분주할 때 시약이 전극 위에서 골고루 퍼져서 시약이 균일한 두께로 분주되게 하는 역할을 한다. 상기 계면활성제로는 비제한적인 예로써 트리톤 X-100(Triton X-100), CHAPS{3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate}, 소듐도데실설페이트(sodium dodecyl sulfate), 퍼플루오로옥탄설포네이트(perfluorooctane sulfonate) 또는 소듐스테아레이트(sodium stearate) 등을 사용할 수 있다. 본 발명에 따른 산화환원반응 시약조성물은 산화환원효소 100 중량부를 기준으로 계면활성제를 1-120 중량부, 바람직하게는 약 10-30 중량부 함유하는 것이 바람직하다.In the additive, the surfactant serves to distribute the reagent evenly over the electrode when dispensing the reagent so that the reagent is dispensed with a uniform thickness. Examples of the surfactant include, but are not limited to, Triton X-100, CHAPS {3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonate}, sodium dodecyl sulfate, purple Luorooctane sulfonate or sodium stearate may be used. The redox reagent composition according to the present invention preferably contains 1-120 parts by weight of surfactant, preferably about 10-30 parts by weight, based on 100 parts by weight of redox enzyme.
상기 첨가제에 있어서, 수용성 고분자는 시약조성물의 고분자 지지체로서 효소의 안정화 및 분산(dispersing)을 돕는 역할을 수행한다. 상기 수용성 고분자로는 비제한적인 예로써 폴리비닐피롤리돈(polyvinyl pyrrolidone; PVP), 폴리비닐알코올 (polyvinyl alcohol; PVA), 폴리플루오로설포네이트(perfluoro sulfonate), 하이드록시에틸 셀룰로오즈(hydroxyethyl cellulose; HEC), 하이드록시프로필 셀룰로오즈(hydroxypropyl cellulose; HPC), 카르복시메틸셀룰로오즈 (carboxy methyl cellulose; CMC), 셀룰로오즈 아세테이트(cellulose acetate) 또는 폴리아미드(polyamide) 등을 사용할 수 있다. 본 발명에 따른 산화환원반응 시약조성물은 산화환원효소 100 중량부를 기준으로 상기 수용성 고분자를 0.3-12 중량부, 바람직하게는 약 2 중량부 함유하는 것이 바람직하다.In the additive, the water-soluble polymer serves as a polymer support of the reagent composition to help stabilize and disperse the enzyme. Examples of the water-soluble polymer include, but are not limited to, polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), polyfluoro sulfonate, hydroxyethyl cellulose; HEC), hydroxypropyl cellulose (HPC), carboxy methyl cellulose (CMC), cellulose acetate or polyamide, and the like. The redox reagent composition according to the present invention preferably contains 0.3-12 parts by weight, preferably about 2 parts by weight of the water-soluble polymer, based on 100 parts by weight of the redox enzyme.
상기 첨가제에 있어서, 아미노산은 효소와 효소간에 서로 뭉쳐지는 것을 방지하여 효소의 활성을 유지시키는 역할을 한다. 본 발명에 따른 산화환원반응 시약조성물은 산화환원효소 100 중량부를 기준으로 아미노산을 4-51 중량부, 바람직하게는 약 17 중량부 함유하는 것이 바람직하다.In the additive, the amino acid prevents agglomeration between the enzyme and the enzyme to maintain the activity of the enzyme. The redox reagent composition according to the present invention preferably contains 4-51 parts by weight of amino acid, preferably about 17 parts by weight, based on 100 parts by weight of the redox enzyme.
상기 첨가제에 있어서, 이당체는 효소가 지나치게 건조되어 효소의 활성이 둔화되는 것을 막기 위하여 사용된다. 본 발명에 따른 산화환원반응 시약조성물은 산화환원효소 100 중량부를 기준으로 이당체를 38-227 중량부, 바람직하게는 약 76 중량부 함유하는 것이 바람직하다.In the above additives, disaccharides are used to prevent the enzyme from becoming too dry and the activity of the enzyme is slowed down. The redox reagent composition according to the present invention preferably contains 38-227 parts by weight of disaccharide, preferably about 76 parts by weight, based on 100 parts by weight of the redox enzyme.
또한 본 발명은 전기화학적 바이오센서에 있어서, 작동전극, 보조전극 및 확인전극이 한 평면상에 구비되고, 상기 전극 위에 본 발명에 따른 산화환원반응 시약조성물이 코팅된 것을 특징으로 하는 평면형 전기화학적 바이오센서를 제공한다.In another aspect, the present invention, the electrochemical biosensor, the working electrode, the auxiliary electrode and the confirmation electrode is provided on one plane, the planar electrochemical bio characterized in that the redox reaction reagent composition according to the present invention is coated on the electrode Provide a sensor.
나아가 본 발명은 전기화학적 바이오센서에 있어서, 작동전극 및 보조전극이 서로 다른 평면상에서 대면하도록 구비되고, 상기 작동전극 위에 본 발명에 따른 산화환원반응 시약조성물이 코팅된 것을 특징으로 하는 대면형 전기화학적 바이오센서를 제공한다.Furthermore, the present invention provides an electrochemical biosensor, wherein the working electrode and the auxiliary electrode are provided to face each other on different planes, and the redox reagent composition according to the present invention is coated on the working electrode. Provide a biosensor.
본 발명에 따른 상기 평면형 및 대면형 전기화학적 바이오센서는, 대한민국 특허출원 10-2003-0036804호, 대한민국 특허출원 10-2005-0010720호, 대한민국 특허출원 10-2007-0020447호, 대한민국 특허출원 10-2007-0021086호, 대한민국 특허출원 10-2007-0025106호, 대한민국 특허출원 10-2007-0030346호, [E. K. Bauman et al., Analytical Chemistry, vol 37, p 1378, 1965; K. B. Oldham in "Microelectrodes: Theory and Applications," Kluwer Academic Publishers, 1991.] 등에 공지된 방법을 통해 제조할 수 있다.The planar and face type electrochemical biosensor according to the present invention, Korean Patent Application No. 10-2003-0036804, Republic of Korea Patent Application 10-2005-0010720, Republic of Korea Patent Application 10-2007-0020447, Republic of Korea Patent Application 10- 2007-0021086, Republic of Korea Patent Application 10-2007-0025106, Republic of Korea Patent Application 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.
이하 상기 평면형 전기화학적 바이오센서를 도 1 을 참조하여 그 구성을 설명한다.Hereinafter, the configuration of the planar electrochemical biosensor will be described with reference to FIG. 1.
먼저 도 1에 나타낸 평면형 전기화학적 바이오센서는 작동전극과 보조전극과 확인전극이 한 평면상에 구비되는 것으로, 혈액이 센서 안으로 스며들도록 하기 위한 통기부(9)를 구비한 상판(8); 양면에 접착제가 코팅되어 있어 상기 상판과 하기 하판을 접착하는 역할을 하며, 혈액이 모세작용으로 전극 쪽으로 스며들도록 하기 위한 끼움판(7); 하기 전극에 코팅되는 본 발명에 따른 산화환원반응 시약조성물(6); 하기 전극의 면적을 규정하기 위한 통로부가 구비된 절연판(5); 하기 하판 상에 프린트되는 작동전극(3)과 보조전극(2)과 확인전극(4); 및 상기 작동전극과 보조전극과 확인전극이 형성되는 하판(1)이 순차적으로 적층된 구조를 갖는다. First, the planar electrochemical biosensor shown in FIG. 1 includes a top plate 8 having a working electrode, an auxiliary electrode, and a confirming electrode provided on one plane, and having a venting unit 9 for introducing blood into the sensor; An adhesive is coated on both sides to serve to bond the upper plate and the lower plate, and a fitting plate 7 for allowing blood to penetrate into the electrode by capillary action; Redox reagent composition 6 according to the present invention coated on the electrode; An insulating plate 5 having a passage portion for defining an area of the following electrode; A working electrode 3, an auxiliary electrode 2 and a confirmation electrode 4 printed on the lower plate; And a lower plate 1 in which the working electrode, the auxiliary electrode, and the confirmation electrode are formed.
상술한 바와 같이, 본 발명에 따른 전자전달매개체로 금속함유 착물, 및 나프토퀴논 또는 이의 유도체를 포함하는 산화환원반응 시약조성물은 산화환원효소-나프토퀴논(또는 이의 유도체)-금속함유 착물 간의 반응속도가 빨라져 혈액 내 글루코스 검출 성능이 현저히 향상되고, 혈액 내 산소 및 방해 물질의 영향을 거의 받지 않아, 혈액 내 글루코스 검출을 위한 전기화학적 바이오센서의 제조에 유용하다. As described above, a redox reagent composition comprising a metal-containing complex and a naphthoquinone or a derivative thereof as the electron transfer medium according to the present invention is provided between an oxidoreductase-naphthoquinone (or a derivative thereof) -metal-containing complex. The reaction rate is fast, and thus the glucose detection performance in the blood is significantly improved, and since it is hardly influenced by oxygen and interfering substances in the blood, it is useful for the preparation of an electrochemical biosensor for detecting glucose in the blood.
이하 본 발명을 하기의 실시예에 의하여 더욱 상세히 설명한다. 단 하기의 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기의 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.
<실시예 1> 전자전달매개체로 헥사아민루테늄클로라이드 및 1,2-나프토퀴논-4- 썰포네이트를 포함하는 산화환원반응 시약조성물의 제조Example 1 Preparation of Redox Reaction Reagent Composition Comprising Hexaamineruthenium Chloride and 1,2-Naphthoquinone-4-Sulfonate as Electron Transfer Mediators
산화환원효소로 플라빈아데닌디뉴클레오티드-글루코스탈수소효소(flavin adenine dinucleotide-glucose dehydrogenase, FAD-GDH) 100 중량부에 대하여; 금속함유 착물로 헥사아민루테늄클로라이드[Ru(NH3)6Cl3] 56 중량부; 1,2-나프토퀴논-4-썰포네이트를 16 중량부(15 mM); 수용성 고분자로 폴리비닐피롤리돈(polyvinyl pyrrolidone; PVP) 2 중량부; 및 Triton X-100 2 중량부;CHAPS 8 중량부; trehalose 76 중량부를 시트릭포스페이트 완충액(pH 5.8, 0.3 M 농도의 500 ml) 및 탈이온수(500.0 ml)에 녹이고, 용액 내에 남아있는 미립자들은 걸러서 제거하였다. 100 parts by weight of flavin adenine dinucleotide-glucose dehydrogenase (FAD-GDH) as an oxidoreductase; 56 parts by weight of hexaamineruthenium chloride [Ru (NH 3 ) 6 Cl 3 ] as a metal-containing complex; 16 parts by weight (15 mM) of 1,2-naphthoquinone-4-sulfonate; 2 parts by weight of polyvinylpyrrolidone (PVP) as a water-soluble polymer; And 2 parts by weight of Triton X-100; 8 parts by weight of CHAPS; 76 parts by weight of trehalose were dissolved in Citrix phosphate buffer (pH 5.8, 500 ml at 0.3 M concentration) and deionized water (500.0 ml), and the remaining particles in the solution were filtered off.
<실시예 2> 시약조성물을 포함하는 평면형 바이오센서의 제조 Example 2 Preparation of a Planar Biosensor Comprising a Reagent Composition
평면형 바이오센서의 일예로서 도 1 에 나타낸 것과 같이 평균값 0.5 ㎕ 시료도입부를 가진 평면형 바이오센서를 제조하였다. 본 평면형 바이오센서를 제조하는 상세한 방법은 대한민국 특허출원 10-2003-0036804호, 대한민국 특허출원 10-2005-0010720호, 대한민국 특허출원 10-2007-0020447호, 대한민국 특허출원 10-2007-0021086호, 대한민국 특허출원 10-2007-0025106호, 대한민국 특허출원 10-2007-0030346호, [E. K. Bauman et al., Analytical Chemistry, vol 37, p 1378, 1965; K. B. Oldham in "Microelectrodes: Theory and Applications," Kluwer Academic Publishers, 1991.] 등에 공지된 방법을 참조하여 제조하였다. 도 1 에서 1 은 작동전극(면적: 0.9 mm2)과 보조전극 및 확인전극이 형성되는 폴리에스터로 만든 하판 플라스틱이고, 2 내지 4 는 탄소그라파이트를 스크린 프린트하여 만든 전극으로, 2 는 보조전극, 3 은 작동전극이며, 4 는 확인전극이다. 5 는 절연체로써 위 전극의 면적을 규정하고, 6 은 전극에 코팅되는 실시예 1 에서 제조한 산화환원반응 시약조성물이고, 7 은 혈액이 모세작용으로 전극쪽으로 스며들도록 한 0.10 mm 두께의 끼움판으로, 그 양면에 접착제가 코팅되어 있어서 상판과 하판을 접착시키는 역할을 하고,9 는 혈액이 센서안으로 스며들도록 하기 위한 공기배출구이며,8 은 폴리에스터로 만든 상판 플라스틱이다.As an example of the planar biosensor, a planar biosensor having an average value of 0.5 µl sample introduction portion was prepared as shown in FIG. 1. Detailed methods for manufacturing the planar biosensors include 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. 1 to 1 is a lower plate plastic made of polyester in which the working electrode (area: 0.9 mm 2 ) and the auxiliary electrode and the confirmation electrode are formed, 2 to 4 are electrodes made by screen printing carbon graphite, 2 is an auxiliary electrode, 3 is a working electrode and 4 is a confirmation electrode. 5 is an insulator defining the area of the upper electrode, 6 is a redox reagent composition prepared in Example 1 coated on the electrode, and 7 is a 0.10 mm thick insert plate which allows blood to penetrate into the electrode by capillary action. The adhesive is coated on both sides thereof to bond the upper and lower plates, 9 is an air outlet for blood to penetrate into the sensor, and 8 is an upper plastic made of polyester.
<실시예 3> 전자전달매개체로 헥사아민루테늄클로라이드 및 나프토퀴논의 유도체를 포함하는 산화환원반응 시약조성물의 제조 Example 3 Preparation of Redox Reaction Reagent Composition Comprising Derivatives of Hexaamine Ruthenium Chloride and Naphthoquinone as Electron Transfer Mediators
실시예 1 의 시약조성물에서 1,2-나프토퀴논-4-썰포네이트 대신에 도 2 에 제시된 나프토퀴논 유도체 중에서, 2-하이드록시-1,4-나프토퀴논 (이하, 나프토퀴논 유도체 a라 함) 60 mM 을 사용한 것을 제외하고는 실시예 1 과 동일하게 실시하여 시약조성물을 제조하였다.Among the naphthoquinone derivatives shown in FIG. 2 in place of 1,2-naphthoquinone-4-sulfonate in the reagent composition of Example 1, 2-hydroxy-1,4-naphthoquinone (hereafter naphthoquinone derivative) A reagent composition was prepared in the same manner as in Example 1 except that 60 mM was used.
<실시예 4> 전자전달매개체로 헥사아민루테늄클로라이드 및 나프토퀴논의 유도체를 포함하는 산화환원반응 시약조성물의 제조 Example 4 Preparation of a Redox Reaction Reagent Composition Comprising Derivatives of Hexaamineruthenium Chloride and Naphthoquinone as Electron Transfer Mediators
실시예 1 의 시약조성물에서 1,2-나프토퀴논-4-썰포네이트 대신에 도 2 에 제시된 나프토퀴논 유도체 중에서, 5-하이드록시-1,4-나프토퀴논 (이하, 나프토퀴논 유도체 b라 함) 10 mM 을 사용한 것을 제외하고는 실시예 1 과 동일하게 실시하여 시약조성물을 제조하였다.Among the naphthoquinone derivatives shown in FIG. 2 in place of 1,2-naphthoquinone-4-sulfonate in the reagent composition of Example 1, 5-hydroxy-1,4-naphthoquinone (hereafter naphthoquinone derivative) b) A reagent composition was prepared in the same manner as in Example 1 except that 10 mM was used.
<실시예 5> 전자전달매개체로 헥사아민루테늄클로라이드 및 나프토퀴논의 유도체를 포함하는 산화환원반응 시약조성물의 제조 Example 5 Preparation of Redox Reagent Reaction Comprising Derivatives of Hexaamineruthenium Chloride and Naphthoquinone as Electron Transfer Mediators
실시예 1 의 시약조성물에서 1,2-나프토퀴논-4-썰포네이트 대신에 도 2 에 제시된 나프토퀴논 유도체 중에서, 1,2-나프토퀴논 (이하, 나프토퀴논 유도체 c라 함) 10 mM 을 사용한 것을 제외하고는 실시예 1 과 동일하게 실시하여 시약조성물을 제조하였다.Among the naphthoquinone derivatives shown in FIG. 2 in place of 1,2-naphthoquinone-4-sulfonate in the reagent composition of Example 1, 1,2-naphthoquinone (hereinafter referred to as naphthoquinone derivative c) 10 A reagent composition was prepared in the same manner as in Example 1 except for using mM.
<실시예 6> 전자전달매개체로 헥사아민루테늄클로라이드 및 나프토퀴논의 유도체를 포함하는 산화환원반응 시약조성물의 제조 Example 6 Preparation of Redox Reagent Reaction Comprising Derivatives of Hexaamineruthenium Chloride and Naphthoquinone as Electron Transfer Mediators
실시예 1 의 시약조성물에서 1,2-나프토퀴논-4-썰포네이트 대신에 도 2 에 제시된 나프토퀴논 유도체 중에서, 1,4-나프토퀴논 (이하, 나프토퀴논 유도체 d라 함) 10 mM 을 사용한 것을 제외하고는 실시예 1 과 동일하게 실시하여 시약조성물을 제조하였다.Among the naphthoquinone derivatives shown in FIG. 2 instead of 1,2-naphthoquinone-4-sulfonate in the reagent composition of Example 1, 1,4-naphthoquinone (hereinafter referred to as naphthoquinone derivative d) 10 A reagent composition was prepared in the same manner as in Example 1 except for using mM.
<비교예 1> 전자전달매개체로 헥사아민루테늄클로라이드를 포함하는 산화환원반응 시약조성물의 제조 Comparative Example 1 Preparation of Redox Reaction Reagent Composition Containing Hexaamine Ruthenium Chloride as Electron Transfer Media
1,2-나프토퀴논-4-썰포네이트를 사용하지 않은 것을 제외하고는 실시예 1 과 동일하게 실시하여 시약조성물을 제조하였다.A reagent composition was prepared in the same manner as in Example 1 except that 1,2-naphthoquinone-4-sulfonate was not used.
<비교예 2> 전자전달매개체로 1,2-나프토퀴논-4-썰포네이트를 포함하는 산화환원반응 시약조성물의 제조 <Comparative Example 2> Preparation of redox reaction reagent composition containing 1,2-naphthoquinone-4-sulfonate as electron transfer medium
헥사아민루테늄클로라이드을 사용하지 않은 것을 제외하고는 실시예 1 과 동일하게 실시하여 시약조성물을 제조하였다.A reagent composition was prepared in the same manner as in Example 1 except that hexaamineruthenium chloride was not used.
<실험예 1> 평면형 바이오센서의 글루코스 표준용액에 대한 전류측정 Experimental Example 1 Current Measurement of Glucose Standard Solution of a Planar Biosensor
실시예와 비교예에서 제조한 평면형 바이오센서를 이용하여 글루코스 표준용액에 대한 전류측정을 수행하였다. 여기서 상기 글루코스 표준용액은 정맥에서 추출한 혈액을 적혈구용적률(hematocrit)이 42 % 가 되도록 제조하고 글루코스 분석기(제조사:YSI, 모델명: 2900D)를 사용하여 여러 가지의 글루코스 농도를 가지도록 제조한 혈액을 말한다.Current measurements were performed on glucose standard solutions using planar biosensors prepared in Examples and Comparative Examples. Herein, the glucose standard solution is blood prepared from vein blood so that the hematocrit becomes 42% and manufactured using a glucose analyzer (manufacturer: YSI, model name: 2900D) to have various glucose concentrations. .
구체적으로 각각의 글루코스 농도가 46, 138, 226, 365, 458 및 571 mg/dL 인 표준용액이 보조전극과 작동전극과 확인전극을 동시에 덮는 순간 작동전극에 0 mV 를 걸어주고, 4 초간 대기한 후 작동전극에 200 mV 를 걸어준 뒤 5 초 전류를 측정하였다(도 6 참조). 모든 측정은 각 농도당 10 회씩 측정하였고, 그 평균값을 도 6 에 나타내었다.Specifically, the standard solution with glucose concentrations of 46, 138, 226, 365, 458, and 571 mg / dL simultaneously covered the auxiliary electrode, the working electrode, and the confirmation electrode with 0 mV applied to the working electrode, and waited for 4 seconds. After applying 200 mV to the working electrode was measured for 5 seconds current (see Figure 6). All measurements were made 10 times for each concentration and the average value is shown in FIG. 6.
도 4 는 본 발명의 일 실시예에 따른 바이오센서의 작동전극에서 전압 변화를 나타낸 그래프로서, 시료가 보조전극과 작동전극과 확인전극을 동시에 덮는 순간 작동전극에 0 mV 를 걸어주고, 4 초간 대기한 후 작동전극에 200 mV 를 걸어 준 뒤 5 초에 전류를 읽는 것을 나타낸다.Figure 4 is a graph showing the voltage change in the working electrode of the biosensor according to an embodiment of the present invention, when the sample covers the auxiliary electrode, the working electrode and the confirmation electrode at the same time to apply a 0 mV to the working electrode, waiting for 4 seconds After 200 mV is applied to the working electrode, the current is read in 5 seconds.
도 5 는 실시예 1 과 비교예 1 과 비교예 2 에 따른 평면형 바이오센서를 이용하여 글루코스 농도를 달리한 시료를 적용하였을 경우, 나타나는 전류의 변화를 측정한 그래프이다.FIG. 5 is a graph illustrating a change in current appearing 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.
도 5 에 나타난 바와 같이, 전자전달매개체로 헥사아민루테늄클로라이드와 1,2-나프토퀴논-4-썰포네이트를 함께 포함하는 실시예 1 의 바이오센서는 글루코스 농도에 따라서 측정되는 전류가 점진적으로 증가하는 좋은 직선성을 보이는 반면에, 전자전달매게체로 1,2-나프토퀴논-4-썰포네이트이나 헥사아민루테늄클로라이드 중 어느 하나를 포함하지 않고 단독으로 사용한 비교예 1 과 비교예 2 의 바이오센서는 글루코스 농도에 따른 직선성이 좋지 않은 것을 알 수 있었다. 이때 실시예 1 에 따른 바이오센서에서 측정되는 전류의 기울기는 작동전극의 단위면적당 20.2 nA/mm2/(mg/dL) 로 나타나, 글루코스에 대한 반응속도가 매우 신속함을 알 수 있다.As shown in FIG. 5, the biosensor of Example 1 including hexaamineruthenium chloride and 1,2-naphthoquinone-4-sulfonate as an electron transfer medium gradually increases the current measured according to the glucose concentration. While showing good linearity, the biosensors of Comparative Examples 1 and 2, which were used alone without containing any one of 1,2-naphthoquinone-4-sulfonate and hexaamineruthenium chloride as electron transfer mediums It can be seen that the linearity according to the glucose concentration is not good. At this time, the slope of the current measured in the biosensor according to Example 1 was expressed as 20.2 nA / mm 2 / (mg / dL) per unit area of the working electrode, indicating that the reaction rate against glucose was very fast.
따라서 본 발명에 따른 산화환원반응 시약조성물은 바이오센서에 적용하였을 경우 글루코스에 대하여 매우 신속하게 반응하므로, 혈액 내 글루코스 검출을 위한 바이오센서의 제조에 유용하다.Therefore, the redox reagent composition according to the present invention reacts very quickly to glucose when applied to a biosensor, and thus is useful for preparing a biosensor for detecting glucose in blood.
<실험예 2> 실시예 3 내지 6 에 따른 평면형 바이오센서의 글루코오스 표준용액에 대한 전류측정 Experimental Example 2 Current Measurement of Glucose Standard Solution of the Planar Biosensor According to Examples 3 to 6
도 2 에 나타낸 나프토퀴논 유도체를 사용한 실시예 3 내지 6 에서 제작한 바이오센서와 실시예 2 에서 제작한 바이오센서를 이용하여 실험예 1 과 동일한 방법으로 글루코스 표준용액에 대한 전류측정을 수행하였다.Using the biosensors prepared in Examples 3 to 6 and the biosensors prepared in Example 2 using the naphthoquinone derivative shown in Figure 2 was carried out current measurement for the glucose standard solution in the same manner as in Experiment 1.
도 6 에 나타낸 바와 같이, 1,2-나프토퀴논-4-썰포네이트의 경우 글루코스 농도 50 ~ 600 mg/dL 에서 전류의 기울기는 작동전극의 단위면적당 21.1 nA/mm2/(mg/dL), 기준 장비 (YSI 2900D)와 상관관계는 0.998 이고, 유도체 a 를 사용한 바이오센서의 경우 글루코스 농도 50 ~ 600 mg/dL 에서 전류의 기울기는 작동전극의 단위면적당 20.0 nA/mm2/(mg/dL), 기준 장비와 상관관계는 0.992 이며, 유도체 b 를 사용한 바이오센서의 경우 글루코스 농도 50 ~ 350 mg/dL 에서 전류의 기울기는 작동전극의 단위면적당 27.8 nA/mm2/(mg/dL), 기준 장비와 상관관계는 0.940 이며, 유도체 c 를 사용한 바이오센서의 경우 글루코스 농도 50 ~ 200 mg/dL 에서 전류의 기울기는 작동전극의 단위면적당 11.1 nA/mm2/(mg/dL), 기준 장비와 상관관계는 0.844 이며, 유도체 d 를 사용한 바이오센서의 경우 글루코스 농도 50 ~ 200 mg/dL 에서 전류의 기울기는 작동전극의 단위면적당 4.4 nA/mm2/(mg/dL), 기준 장비와 상관관계는 0.678 로 나타나 1,2-나프토퀴논-4-썰포네이트를 사용한 바이오센서가 글루코오스에 대한 반응속도가 매우 신속하고 기준 장비와 상관관계가 좋음을 알 수 있었다.As shown in FIG. 6, in the case of 1,2-naphthoquinone-4-sulfonate, the slope of the current at a glucose concentration of 50 to 600 mg / dL was 21.1 nA / mm 2 / (mg / dL) per unit area of the working electrode. , The correlation with the reference equipment (YSI 2900D) is 0.998, and for biosensors 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 per unit area of working electrode). ), The correlation with the reference equipment is 0.992, and for biosensors with 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) per unit area of the working electrode, The correlation with the equipment is 0.940, and in the case of biosensors using derivative c, the slope of the current at a glucose concentration of 50 to 200 mg / dL is 11.1 nA / mm 2 / (mg / dL) per unit area of the working electrode, which is correlated with the reference equipment. The relationship is 0.844. For biosensors with derivative d, the glucose concentration is between 50 and 200. The slope of the current at mg / dL was 4.4 nA / mm 2 / (mg / dL) per unit area of the working electrode and the correlation with the reference equipment was 0.678, indicating that the biosensor using 1,2-naphthoquinone-4-sulfonate The reaction rate to glucose was very fast and correlated with the reference equipment.
따라서 본 발명에 따른 산화환원반응 시약조성물은 전기화학적 바이오센서에 적용하였을 경우 글루코스에 대하여 매우 신속하게 반응하므로, 혈액 내 글루코오스 검출을 위한 바이오센서의 제조에 유용하다.Therefore, the redox reagent composition according to the present invention is very quickly reacted to glucose when applied to an electrochemical biosensor, and thus is useful for preparing a biosensor for detecting glucose in blood.
한편 본 발명의 실시예에 따른 전기화학적 바이오센서는 작동전극, 보조전극 및 확인전극이 하부기판의 한 평면상에 구비되는 것으로 제공되지만, 작동전극, 보조전극 및 확인전극이 서로 다른 평면상(예를 들어 상부기판)에서 대면하도록 구비되어 제공될 수도 있다.On the other hand, the electrochemical biosensor according to an embodiment of the present invention is provided that the working electrode, the auxiliary electrode and the confirmation electrode are provided on one plane of the lower substrate, but the working electrode, the auxiliary electrode and the confirmation electrode are on different planes (eg, For example, it may be provided to face to the upper substrate).
이상에서 본 발명은 기재된 구체예에 대해서만 상세히 설명되었지만 본 발명의 범위 내에서 다양한 변형 및 수정이 가능함은 통상의 기술자에게 명백한 것으로, 이러한 변형 및 수정이 첨부된 특허청구범위에 속함은 당연한 것이다.Although the present invention has been described in detail only with respect to the described embodiments, it will be apparent to those skilled in the art that various modifications and variations are possible within the scope of the present invention, and such modifications and modifications belong to the appended claims.

Claims (4)

  1. 산화환원효소 및 전자전달매개체를 포함하는 바이오센서용 시약조성물로서, 전자전달매개체가 루테늄 함유 착물, 및 나프토퀴논 또는 이의 유도체를 포함하는 것을 특징으로 하는 시약조성물.A reagent composition for a biosensor comprising a oxidoreductase and an electron transfer medium, wherein the electron transfer medium comprises a ruthenium-containing complex, and naphthoquinone or a derivative thereof.
  2. 제 1 항에 있어서, 산화환원효소가 플라빈아데닌디뉴클레오티드-글루코스탈수소효소(flavin adenine dinucleotide-glucose dehydrogenase), 니코틴아미드아데닌디뉴클레오티드-글루코스탈수소효소(nicotinamide adenine dinucleotide-glucose dehydrogenase), 글루코스탈수소효소(glucose dehydrogenase), 글루탐산탈수소효소(glutamate dehydrogenase), 콜레스테롤산화효소(cholesterol oxidase), 락테이트산화효소(lactate oxidase), 아스코빅산산화효소(ascorbic acid oxidase), 알코올산화효소(alcohol oxidase), 알코올탈수소효소(alcohol dehydrogenase) 및 빌리루빈산화효소(bilirubin oxidase)로 이루어지는 군으로부터 선택되는 1 종인 것을 특징으로 하는 시약조성물.The method according to claim 1, wherein the oxidoreductase is flavin adenine dinucleotide-glucose dehydrogenase, nicotinamide adenine dinucleotide-glucose dehydrogenase, glucose dehydrogenase glucose dehydrogenase, glutamate dehydrogenase, cholesterol oxidase, lactate oxidase, ascorbic acid oxidase, alcohol oxidase, alcohol dehydrogenase (alcohol dehydrogenase) and bilirubin oxidase (bilirubin oxidase) is one kind selected from the group consisting of a reagent composition.
  3. 제 1 항에 있어서, 루테늄 함유 착물이 [Ru(NH3)6]Br3, [Ru(NH3)5Cl]Cl2, K4[Ru(CN)6], Ru(NH3)6Cl3, [Ru(2,2',2''-terpyridine)(1,10-phenanthroline)(OH2)]2+, trans-[Ru(2,2'-bipyridine)2(OH2)(OH)]2 +, [(2,2'-bipyridine)2(OH)RuORu(OH)(2,2'bpy)2]4+ 및 [Ru(4,4'-bipyridine)(NH3)5]2+ 로 이루어지는 군으로부터 선택되는 1 종인 것을 특징으로 하는 시약조성물.The ruthenium-containing complex of claim 1 wherein the ruthenium containing complex is selected from 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 ] A reagent composition, characterized in that one kind selected from the group consisting of 2+ .
  4. 제 1 항에 있어서, 나프토퀴논 및 이의 유도체가 2-메톡시-1,4-나프토퀴논, 5,8-디하이드록시-1,4-나프토퀴논, 2-메틸-1,4-나프토퀴논, 2-메틸-1,4-나프토퀴논 소듐바이썰파이트, 1,2-나프토퀴논, 1,4-나프토퀴논, 5-하이드록시-1,4-나프토퀴논, 2-하이드록시-1,4-나프토퀴논 및 1,2-나프토퀴논-4-썰포네이트로 이루어지는 군으로부터 선택되는 1 종인 것을 특징으로 하는 시약조성물.The method of claim 1, wherein the naphthoquinone and its derivatives are 2-methoxy-1,4-naphthoquinone, 5,8-dihydroxy-1,4-naphthoquinone, 2-methyl-1,4- Naphthoquinone, 2-methyl-1,4-naphthoquinone sodium bisulfite, 1,2-naphthoquinone, 1,4-naphthoquinone, 5-hydroxy-1,4-naphthoquinone, 2 A reagent composition, characterized in that one kind selected from the group consisting of -hydroxy-1,4-naphthoquinone and 1,2-naphthoquinone-4-sulfonate.
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