KR102411089B1 - A composition for bio-sensor and biosensor including the same - Google Patents

Abstract

According to an embodiment of the present application, a composition for a biosensor using an electrochemical reaction, capable of oxidizing a biotarget included in a biological sample - When the biotarget is oxidized, a predetermined electron is provided from the biotarget - Enzyme , When the predetermined electrons are provided, the first electron transport mediator that can be reduced and the second electron transport mediator that can oxidize the first electron transport mediator and are reduced when the first electron transport mediator is oxidized; Including, wherein the first electron transport mediator includes at least one of a ruthenium complex or a ferricyanide complex, wherein the chemical formula of the second electron transport mediator is 3-(1-methoxyphena) Zin-5-ium-5-yl) propane-1-sulfonate (3-(1-methoxyphenazin-5-ium-5-yl)propane-1-sulfonate), a composition for a biosensor may be provided.

Description

A composition for a biosensor and a biosensor comprising the same

The present invention relates to a composition for a biosensor for obtaining biometric information and a biosensor including the same, and more particularly, to a composition for a biosensor with improved stability and measurement precision for obtaining biometric information, and a biosensor comprising the same it's about

Recently, as interest in health increases, bio-sensors are widely used. A biosensor is a device capable of measuring information about an analyte in the body by integrating a biological element and a physicochemical element, and various measurement methods such as an electrochemical method, an optical method, and a heat sensing method may be applied to the biosensor.

In particular, the biosensor to which the electrochemical method is applied is used more frequently because of its relatively easy miniaturization and low cost compared to other methods. However, the biosensor to which the electrochemical method is applied has disadvantages in that the stability of the mixture of the measurement object and the composition for measurement and the selectivity of the measurement object are rather low. demand is increasing.

One object of the present invention is to provide a composition for a biosensor capable of acquiring biometric information with improved measurement accuracy for a measurement target of a biological sample and a biosensor including the same.

The problem to be solved by the present invention is not limited to the above-mentioned problems, and the problems not mentioned will be clearly understood by those of ordinary skill in the art to which the present invention belongs from the present specification and the accompanying drawings. .

According to an aspect of the present application, a composition for a biosensor using an electrochemical reaction, capable of oxidizing a biotarget included in a biological sample - When the biotarget is oxidized, a predetermined electron is provided from the biotarget - Enzyme , a second electron transport mediator capable of oxidizing the first electron transport mediator, which can be reduced when the predetermined electrons are provided, and a second electron transport mediator that is reduced when the first electron transport mediator is oxidized Including, wherein the first electron transport mediator includes at least one of a ruthenium complex or a ferricyanide complex, wherein the chemical formula of the second electron transport mediator is 3-(1-methoxyphena) Zin-5-ium-5-yl) propane-1-sulfonate (3-(1-methoxyphenazin-5-ium-5-yl)propane-1-sulfonate), a composition for a biosensor may be provided.

According to another aspect of the present application, a biosensor including the composition for a biosensor may be provided.

The solutions to the problems of the present invention are not limited to the above-described solutions, and solutions not mentioned will be clearly understood by those of ordinary skill in the art to which the present invention belongs from the present specification and the accompanying drawings. will be able

According to the present invention, it is possible to provide a composition for a biosensor capable of acquiring biometric information with improved stability in the external environment, improved stability and improved selectivity for a biotarget, and improved measurement accuracy, and a biosensor including the same. have.

The effects of the present invention are not limited to the above-described effects, and the effects not mentioned will be clearly understood by those of ordinary skill in the art to which the present invention pertains from the present specification and accompanying drawings.

1 is a flowchart illustrating a diagnostic protocol of an enzyme electrode method.
2 is a view showing the structure of a biosensor to which a composition for a biosensor according to an embodiment of the present specification is applied.
3 is a graph showing the intensity of a measured current over time for each glucose concentration contained in a biological sample in a biosensor to which the composition for a biosensor according to an embodiment of the present specification is applied.
4 and 5 are graphs showing the intensity of the measured current over time for each glucose concentration contained in the biological sample in the biosensor to which the reagent composition according to the comparative example is applied.
6 is a graph showing the intensity of the measured current according to the glucose concentration contained in the biological sample in the biosensor to which the composition for a biosensor according to an embodiment of the present specification is applied.
7 and 8 are graphs of the intensity of the measured current according to the glucose concentration contained in the biological sample in the biosensor to which the reagent composition according to the comparative example is applied.
9 is a view for confirming whether sediment is generated as time elapses in the composition for a biosensor and the conventional reagent composition according to an embodiment of the present specification.
10 is a view showing changes in glucose concentration over time in a biosensor composition and a conventional reagent composition according to an embodiment of the present specification.

The embodiments described in this specification are for clearly explaining the spirit of the present invention to those of ordinary skill in the art to which the present invention pertains, so the present invention is not limited by the embodiments described herein, and the present invention It should be construed as including modifications or variations that do not depart from the spirit of the present invention.

The terms used in this specification have been selected as widely used general terms as possible in consideration of the functions in the present invention, but they may vary depending on the intention, custom, or emergence of new technology of those of ordinary skill in the art to which the present invention belongs. can However, if a specific term is defined and used in an arbitrary sense, the meaning of the term will be separately described. Therefore, the terms used in this specification should be interpreted based on the actual meaning of the terms and the contents of the entire specification, rather than the names of simple terms.

The drawings attached to this specification are for easily explaining the present invention, and the shapes shown in the drawings may be exaggerated as necessary to help understand the present invention, so the present invention is not limited by the drawings.

In the present specification, when it is determined that a detailed description of a known configuration or function related to the present invention may obscure the gist of the present invention, a detailed description thereof will be omitted if necessary.

According to an aspect of the present application, a composition for a biosensor using an electrochemical reaction, capable of oxidizing a biotarget included in a biological sample - When the biotarget is oxidized, a predetermined electron is provided from the biotarget - Enzyme and a second electron transport mediator capable of oxidizing the first electron transport mediator, which can be reduced when the predetermined electron is provided, and a second electron transport mediator that is reduced when the first electron transport mediator is oxidized. and, the first electron transport mediator includes at least one of a ruthenium complex or a ferricyanide complex, wherein the chemical formula of the second electron transport mediator is 3-(1-methoxyphenazine) -5-ium-5-yl) propane-1-sulfonate (3-(1-methoxyphenazin-5-ium-5-yl)propane-1-sulfonate), a composition for a biosensor may be provided.

In this case, the second electron transfer mediator may be provided with a composition for a biosensor having the following structural formula.

(In the above structural formula, OMe represents a methoxy group)

At this time, the enzyme is flavin adenine dinucleotide-glucose dehydrogenase, fructosamine oxidases (FAOX), fructosyl peptide oxidase (Fructosyl peptide oxidase), keto Ketoamine oxidase, nicotinamide adenine dinucleotideglucose dehydrogenase, glucose oxidase, cholesterol oxidase, cholesterol esterase, lactate A composition for a biosensor comprising at least one of the group consisting of lactate oxidase and urate oxidase may be provided.

The ratio of the weight of the first electron transfer medium to the weight of the enzyme is 3 to 6, and the ratio of the weight of the second electron transfer medium to the weight of the enzyme is 0.001 to 0.1, the composition for a biosensor is provided can be

A composition for a biosensor may be provided in which a ratio of the weight of the first electron transfer medium to the weight of the second electron transfer medium is 200 to 600.

According to another aspect of the present application, a biosensor including the composition for a biosensor may be provided.

The present invention relates to a composition for a biosensor for obtaining biometric information and a biosensor including the same, and more particularly, to a composition for a biosensor with improved stability and measurement precision for obtaining biometric information and a biosensor using the same will be.

Hereinafter, a composition for a biosensor according to an embodiment of the present specification will be described later.

1. Biosensor Overview

A bio-sensor receives a biological sample from a user, and includes a biological analyte included in the biological sample and biological elements (eg, enzyme, antigen, antibody, biochemical substance, hormone) included in the bio-sensor. It is an analytical sensor for acquiring the user's biometric information based on the user's biometric information. The analyte may be glucose (or a substance to which glucose is bound), cholesterol, protein, ketone, creatine, and the like.

However, the electrochemical biosensor is currently widely used because of its easy miniaturization and low cost compared to the optical biosensor. In particular, an enzyme-linked electrode method is widely used for an electrochemical biosensor.

On the other hand, in the enzyme electrode method, in order to improve the electron transfer efficiency for electrons due to the redox reaction occurring in the biosensor, a method of using two kinds of electron transfer mediators in a detection reagent has been proposed.

The biosensor disclosed by the present invention relates to a biosensor to which the above-described enzyme electrode method is applied.

1 is a flowchart illustrating a diagnostic protocol for an enzyme electrode method.

Referring to FIG. 1 , the protocol for the enzyme electrode method includes the steps of injecting a biological sample into the biosensor (S20), reacting the biotarget of the biological sample with a detection reagent (S40), and applying a voltage to the biosensor ( S60) and obtaining biometric information (S80).

The biosensor may include an electrode unit and a sample providing unit. A detection reagent may be applied to at least a portion of the electrode part. The detailed structure, function, etc. of the biosensor will be described later with reference to FIG. 2 , so the detailed content will be omitted.

In the step (S20) of providing a biological sample to the biosensor, the biological sample may be provided to the biosensor. The biological sample may include a biotarget to be measured. The biological sample may be different depending on the type of the biotarget to be measured. For example, the biological sample may be blood, urine, body fluid, or the like. When a specific biotarget is specified, a sample particularly suitable for detecting the biotarget can be determined. In particular, when the target to be measured is a protein in blood cells, such as glucose, glycated hemoglobin, or glycated albumin, the biological sample may be blood.

After the injection (S20), the bio-target may react with a reactant included in the detection reagent. The detection reagent may include an enzyme, a first electron transfer mediator and a second electron transfer mediator. In this case, the enzyme may specifically mediate the reaction to the bio-target.

In the step S40 of reacting the biotarget of the biological sample with the detection reagent, the biotarget may be oxidized by the enzyme. The biotarget may be a reactant of a redox reaction mediated by the enzyme.

When the biotarget is oxidized, predetermined electrons may be provided.

The given electrons can bind to the enzyme. In this case, the enzyme may be reduced as the biotarget is oxidized. The reduced enzyme may be oxidized by the first electron transfer mediator included in the detection reagent. In this case, the reduced enzyme may be oxidized to provide electrons, and the first electron transfer mediator may be reduced.

Alternatively, the provided electrons may be bound to the first electron transfer mediator included in the detection reagent. In this case, the first electron transfer mediator may be reduced as the biotarget is oxidized.

The reduced first electron transport mediator may be oxidized by the second electron transport mediator. In this case, the second electron transport mediator may be reduced as the reduced first electron transport mediator is oxidized.

In the step S40 of reacting the biotarget of the biological sample with the detection reagent, the above-described reaction occurrence scale may be different depending on the amount of the biotarget included in the biological sample. For example, when the biological sample is blood and the biotarget is glucose, the magnitude of the reaction described above may be greater as the concentration of glucose in the blood increases. That is, the glucose concentration in the blood may be determined to be higher as the scale of the reduced first electron transfer mediator increases.

In the step of applying a voltage to the electrode part ( S60 ), a current flow may be detected in at least a part of the electrode part. In the applying step ( S60 ), the biosensor may be provided to an external device capable of applying a predetermined potential to the biosensor.

At this time, the reduction potential of the reduced second electron transfer mediator may be applied. When a reducing potential is applied, the reduced second electron transport mediator may be oxidized. When the reduced second electron transport mediator is oxidized, the reduced second electron transport mediator may donate electrons. The provided electrons may move through the electrically connected region of the electrode part.

As a result, the information on the amount of the bio-target may correspond to information on electrons (which may be measured by current flow) moving on the electrode unit.

In the step of acquiring the biometric information ( S80 ), the external device provided with the biotarget may acquire the biometric information based on current information obtained from the electrode unit. As described above, the current information on the electrode part may correspond to the information about the amount of the bio-target. The external device may calculate biometric information based on current information according to a predetermined criterion.

In general, the first electron transport mediator or the second electron transport mediator includes potassium ferricyanide [K3Fe(CN)6], ferrocene, ferrocene derivatives, quinone derivatives, phenazine-methosulfate (phenazine-) Methosulfate), methoxyphenazine-methosulfate, phenazine methyl sulfate, dichloroindophenol, etc. are widely used.

However, the above-described conventional electron transport medium is very much affected by light, temperature, and humidity, and thus there is a problem that the reliability of the biosensor may be damaged because there is a risk of deterioration when stored for a long period of time.

The present invention is intended to solve the above-mentioned conventional problems, and hereinafter, a composition for a biosensor according to an embodiment of the present specification will be described later.

2. Biosensors and compositions for biosensors

2-1 Structure and Function of Biosensor

Hereinafter, a biosensor to which the composition for a biosensor according to an embodiment of the present specification is applied will be described with reference to FIG. 2 .

2 is a view showing the structure of a biosensor to which a composition for a biosensor according to an embodiment of the present specification is applied.

Referring to FIG. 2 , the biosensor 100 to which the composition for a biosensor according to an embodiment of the present specification is applied has a first cover part 110 , a second cover part 120 , a spacer 130 , and a sample providing part. 135 , an insulating part 140 , a detection reagent part 150 , and an electrode part 160 may be included.

The spacer 130 , the sample providing part 135 , the insulating member 140 , and the electrode part 160 may be temporarily positioned between the first cover part 110 and the second cover part 120 . can

The insulating part 140 may be positioned between the spacer 130 and the electrode part 160 .

The first cover unit 110 may be an outer cover of one surface of the biosensor 100 .

The first cover part 110 may include an air exhaust part 112 . The air discharge unit 112 may be fluidically connected to the sample providing unit 135 .

Although not shown in the drawings, the air discharge unit 112 may not be included in the first cover unit 110 . For example, at least a portion of the first cover part 110 may be spaced apart from the spacer 130 to form a flow path fluidly connected to the outside.

The second cover part 120 may be an outer cover of the other surface of the biosensor 100 . The second cover unit 120 may be a base substrate on which individual components of the biosensor 100 may be disposed.

The spacer 130 may be disposed between the first cover part 110 and the second cover part 120 . At least a portion of the spacer 130 may have a retracted shape.

At least a portion of the sample providing unit 135 may be positioned at the same height as the spacer 130 with respect to the thickness direction of the biosensor 100 .

The sample providing part 135 may be formed in the retracted region of the spacer 130 . The sample providing part 135 may be a space formed by the spacer 130 , the first cover part 110 , and the second cover part 120 .

When a biological sample (eg, blood, urine, etc.) is provided from the user, the sample providing unit 135 may have a shape extending in the longitudinal direction so as to be moved inside by a capillary phenomenon. For example, the sample providing unit 135 may have a cylindrical shape or a rectangular shape in which a vertical ratio to a horizontal length is greater than or equal to a predetermined ratio.

In this case, the biological sample provided by the user may include a bio-target. According to an example, the bio-target may be glucose (or a substance to which glucose is bound), cholesterol, protein, ketone, creatine, and the like. According to a specific example, the biological sample used for measuring glucose is a blood sample, and the blood sample is provided to the sample providing unit 135 .

When a user's biological sample is injected into the sample providing unit 135 , at least a portion of the air included in the sample providing unit 135 may be discharged to the outside through the air discharge unit 112 . The position of the air discharge unit 112 may correspond to the position of the sample providing unit 135 when viewed from the top.

The insulating unit 140 may block an electrical connection between the detection reagent unit 150 and at least a partial region of the electrode unit 160 .

The insulating unit 140 blocks at least a part of the electrical connection between the individual components of the biosensor 100 and the electrode unit 160 to attenuate an error with respect to the current signal generated in the electrode unit 160 . can

The insulating part 140 may include a region into which at least a partial region is introduced. According to an example, the detection reagent unit 150 may be connected to a partial region of the electrode unit 160 through the retracted region.

The insulating part 140 may include at least an insulating material. According to an example, the insulating material includes polyethylene terephthalate, polyethylene, polypropylene, polyimide, polystyrene, acrylic resin, or polyester. ), but is not limited thereto.

The detection reagent unit 150 may be located at a position corresponding to the sample providing unit 135 . The detection reagent unit 150 may physically contact at least a portion of the biological sample when the biological sample is provided to the sample providing unit 135 .

The detection reagent unit 150 may be located at a position corresponding to the retracted region of the insulating unit 140 .

The detection reagent unit 150 may be applied to at least a partial region of the electrode unit 160 .

The detection reagent unit 150 may include the composition for a biosensor according to an embodiment of the present specification. In the present specification, the composition for a biosensor may be referred to as a reagent composition. The details of the composition for the biosensor will be described later, and thus the content that may be overlapped will be omitted.

The electrode part 160 may be arranged on the second cover part 120 .

Referring to FIG. 2 , the electrode unit 160 includes a first lead wire 162 , a second lead wire 163 , a third lead wire 164 , a first electrode 166 , a second electrode 167 , and a third An electrode 168 may be included.

The first lead wire 162 , the second lead wire 163 , the third lead wire 164 , the first electrode 166 , the second electrode 167 and the third electrode 168 are conductive on the base substrate of the electrode part. It may include a material, and may be implemented by a conventional method including screen printing, etching, attaching a conductor tape, and physical vapor deposition on the base substrate. The conductive material is not limited to specific types such as conductive polymers, carbon, metal particles, graphite, and platinum.

The first lead wire 162 may be electrically connected to the first electrode 166 . The second lead wire 163 may be electrically connected to the second electrode 167 . The third lead wire 164 may be electrically connected to the third electrode 168 .

The first lead wire 162 , the second lead wire 163 , and the third lead wire 164 may be electrically connected to a device in which the biosensor 100 can be mounted.

Information regarding currents flowing through the first lead wire 162 , the second lead wire 163 , and the third lead wire 164 may be provided to the device. When a predetermined potential is applied to the electrode unit 160 , current may be generated in at least a partial region of the electrode unit 160 . In this case, the generated current may be due to a redox reaction between the biotarget included in the user's biological sample and the biosensor composition included in the detection reagent unit 150 .

Information on the current generated in at least a partial region of the electrode part 160 is transmitted to the device through at least one of the first lead wire 162 , the second lead wire 163 , and the third lead wire 164 . can be provided.

The device may obtain information about the user's biosignal based on an electrical signal provided from at least one of the first lead wire 162 , the second lead wire 163 , and the third lead wire 164 . . For example, when the target to be measured is blood sugar information of the user, the device may calculate blood sugar data by correcting the provided electrical signal.

The first electrode 166 may be a working electrode. The first electrode 166 may be electrically connected to the first lead wire 162 .

The second electrode 167 may be a counter electrode. The second electrode 167 may be electrically connected to the second lead wire 163 .

The third electrode 168 may be a reference electrode. The third electrode 168 may be electrically connected to the third lead wire 164 .

The biosensor composition may not be applied on the third electrode 168 .

The first electrode 166 and the second electrode 167 may be connected by the biosensor composition. The biosensor composition may be applied on the first electrode 166 . The composition for the biosensor may be applied on the second electrode 167 .

The third electrode 168 may not be physically directly connected to the first electrode 166 . The third electrode 168 may not be physically directly connected to the second electrode 167 .

The device may acquire first current information based on a current flowing through the first electrode 166 and the second electrode 167 .

For example, when a blood sample is provided to the sample providing unit 135 , the first electrode 166 and the second electrode 167 may be connected through the detection reagent unit 150 and the blood sample. have. At this time, when a predetermined voltage is applied to the first electrode 166 and the second electrode 167 , first current information is obtained through the first lead wire 162 and the second lead wire 163 . can

In this case, the first current information may relate to the intensity of a current flowing between the electrode connected through the biosensor composition and the blood sample.

The device may acquire second current information based on a current flowing through the third electrode 168 and the second electrode 167 .

For example, when a blood sample is provided to the sample providing unit 135 , the third electrode 168 and the second electrode 167 may be connected through the blood sample. At this time, when a predetermined voltage is applied to the third electrode 168 and the second electrode 167 , second current information is acquired through the second lead wire 163 and the third lead wire 164 . can be

In this case, the second current information may relate to an intensity of a current flowing between electrodes connected through a blood sample that does not contain the biosensor composition.

The device may calculate the user's biometric information based on the first current information and the second current information. For example, when a blood sample is provided to the biosensor, the device based on the first current information and the second current information, but reflecting an error correction factor, a glucose concentration (blood glucose-related information) can be calculated.

Hereinafter, the composition and effects of the biosensor composition according to the embodiment of the present specification included in the detection reagent unit 150 will be described later.

2-2 Composition and characteristics of composition for biosensor

The detection reagent unit 150 may include the composition for a biosensor according to an embodiment of the present specification.

The composition for a biosensor may include a biological element capable of specifically reacting with an analyte (which may be referred to as a biotarget) included in a biological sample.

The composition for a biosensor may include an enzyme, at least two or more electron transport mediators, a surfactant, a water-soluble polymer, and a stabilizer.

The surfactant can prevent the enzyme included in the biosensor composition from adhering to the electrode, and the detection reagent unit 150 is the first electrode 166 , the reference electrode 167 and/or the It can be applied evenly on the confirmation electrode 168 . The amount of the surfactant relative to the detection reagent unit 150 is not limited to a specific numerical range.

According to one example, the surfactant is from the group consisting of Triton X-100, sodium dodecyl sulfate, sodium stearate, and polysorbate 20. At least one may be selected.

The water-soluble polymer may help stabilize and disperse the enzyme included in the detection reagent unit 150 . According to one embodiment, the water-soluble polymer is polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polyethylene glycol (PEG), polyethylene oxide (PEO), hydroxypropyl cellulose (HPC), carboxymethyl cellulose (CMC) ), at least one type may be selected from the group consisting of cellulose acetate (CA).

The stabilizer may help stabilize the enzyme included in the detection reagent unit 150 . According to an example, the stabilizer may be at least one selected from the group consisting of trehalose, L-arginine, and citric acid.

The enzyme may mediate a redox reaction with respect to a biotarget of a biological sample provided from a user.

According to one embodiment, the enzyme is flavin adenine dinucleotide-glucose dehydrogenase, fructosamine oxidase (FAOX), fructosyl peptide oxidase (Fructosyl peptide oxidase) , Ketoamine oxidase, nicotinamide adenine dinucleotideglucose dehydrogenase, glucose oxidase, cholesterol oxidase, cholesterol esterase, cholesterol esterase It may include at least one selected from the group consisting of lactate oxidase and urate oxidase.

The at least two electron transport mediators may include a first electron transport mediator and a second electron transport mediator.

The first electron transport medium may be a metal-containing complex. According to an example, the first electron transport mediator may include at least one of a ruthenium complex and a ferricyanide complex. According to an example, the ruthenium complex includes 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+　and [Ru(4) At least one of the group consisting of ,4'-bipyridine)(NH3)5]2+ may be used. According to an example, the ferrocyanide complex may be K3Fe(CN)6. However, the first electron transport mediator may utilize a conventionally known electron transport mediator, and is not limited to the above-described example.

Preferably, the biosensor composition may contain 300 to 600 parts by weight of the first electron transfer medium based on 100 parts by weight of the enzyme.

The second electron transport mediator is 3-(1-methoxyphenazin-5-ium-5-yl) propane-1-sulfonate (3-(1-methoxyphenazin-5-ium-5-yl)propane-1 -sulfonate).

The second electron transport medium may be represented by Structural Formula 1 below.

[Structural Formula 1]

Preferably, the composition for a biosensor may contain 0.1 to 10 parts by weight of the second electron transfer mediator of Formula 1 based on 100 parts by weight of the enzyme.

Preferably, the composition for the biosensor may contain 200 to 600 times the amount of the second electron transport mediator of Formula 1 based on the weight ratio compared to the first electron transport mediator.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples of the composition for a biosensor.

3. Effects of compositions for biosensors

3-1 Example

The composition for a biosensor according to the first embodiment contains a predetermined enzyme, and based on 100 parts by weight of the predetermined enzyme, PVP in 10 parts by weight or more and 30 parts by weight or less, 10 parts by weight or more and 30 parts by weight or less of Triton x-100, 100 parts by weight or more and 300 parts by weight trehalose, 100 parts by weight or more and 300 parts by weight or less L-arginine, 200 parts by weight or more and 600 parts by weight hexaamine ruthenium chloride and 1 weight It was prepared to contain more than 100 parts by weight and not more than 100 parts by weight and 3-(1-methoxyphenazin-5-ium-5-yl)propane-1-sulfonate.

3-2 Comparative Example

<Comparative Example 1>

To demonstrate the effect of a second electron transport mediator (3-(1-methoxyphenazin-5-ium-5-yl) propane-1-sulfonate) included in the reagent composition, the second electron transport mediator was A reagent composition that was not included was prepared.

The reagent composition not containing the second electron transfer medium contains 10 parts by weight or more and 30 parts by weight or less of PVP, 10 parts by weight or more and 30 parts by weight of Triton x-100 based on 100 parts by weight of the predetermined enzyme. , 100 parts by weight or more and 300 parts by weight of trehalose, 100 parts by weight or more and 300 parts by weight or less of L-arginine, and 200 parts by weight or more and 600 parts by weight of hexaamine ruthenium chloride.

<Second Comparative Example>

In order to demonstrate the effect of the first electron transfer mediator (described in relation to hexaamine ruthenium chloride in this comparative example) included in the reagent composition, a reagent composition not including the first electron transport mediator was prepared.

The composition for a biosensor that does not contain the first electron transfer medium includes a predetermined enzyme, but based on 100 parts by weight of the predetermined enzyme, 10 parts by weight or more and 30 parts by weight or less of PVP, 10 parts by weight or more 30 parts by weight or less of Triton x-100, 100 parts by weight or more and 300 parts by weight trehalose, 100 parts by weight or more and 300 parts by weight or less L-arginine, and 1 part by weight to 100 parts by weight and 3 -(1-Methoxyphenazin-5-ium-5-yl) It was prepared to contain propane-1-sulfonate.

<Comparative Example 3>

Proof of reagent stability of electron transport mediators according to Formula 1 compared to PMS (phenazine methosulfate), 1-Methoxy PMS, and 1-Methoxy Phenazine ethosulfate, which were previously used electron transport mediators In order to do this, a reagent composition containing an electron transport medium that has been conventionally used was prepared.

The reagent composition containing PMS contains a predetermined enzyme, and based on 100 parts by weight of the predetermined enzyme, PVP in 10 parts by weight or more and 30 parts by weight or less, Triton x- in 10 parts by weight or more and 30 parts by weight or less 100, 100 parts by weight or more and 300 parts by weight or less of trehalose, 100 parts by weight or more and 300 parts by weight or less L-arginine, 200 parts by weight or more and 600 parts by weight hexaamine ruthenium chloride, and 1 part by weight or more 100 parts by weight It was prepared to contain up to 1 part by weight and not less than 1 part by weight and not more than 100 parts by weight of PMS.

The reagent composition containing 1-methoxy PMS contains a predetermined enzyme, but based on 100 parts by weight of the predetermined enzyme, 10 parts by weight or more and 30 parts by weight or less of PVP, 10 parts by weight or more and 30 parts by weight or less of Triton x-100, 100 parts by weight or more and 300 parts by weight trehalose, 100 parts by weight or more and 300 parts by weight or less L-arginine, 200 parts by weight or more and 600 parts by weight hexaamine ruthenium chloride and 1 weight It was prepared to contain more than 100 parts by weight and not more than 1 part by weight and not more than 100 parts by weight of 1-methoxy PMS.

The reagent composition containing PES contains a predetermined enzyme, but based on 100 parts by weight of the predetermined enzyme, PVP in 10 parts by weight or more and 30 parts by weight or less, Triton x- in 10 parts by weight or more and 30 parts by weight or less 100, 100 parts by weight or more and 300 parts by weight trehalose, 100 parts by weight or more and 300 parts by weight or less L-arginine, 200 parts by weight or more and 600 parts by weight hexaamine ruthenium chloride, and 1 part by weight or more 100 parts by weight It was prepared to contain up to 1 part by weight and not less than 1 part by weight and not more than 100 parts by weight of PMS.

3-3 Experiment result

<Experimental Example 1> - Current data

Experimental results regarding current data according to the type of reagent composition are shown in FIGS. 3 to 8 .

: 마이크로암페어)은 연산 증폭기(OP-AMP: Operational amplifier)를 이용하여 증폭되었고, 증폭된 데이터를 1/100을 곱하여 최종 데이터를 산출하였다. (도 3 내지 도 8에 나타난 y축의 전류 데이터는 실제 데이터에 대해 1/100이 곱연산된 데이터임) 글루코스 농도는 0.2초 간격으로 측정되었다. 시간에 따른 증폭된 최종 데이터는 도 3 내지 도 5에 도시되었고, 전입 인가 구간에 대한 엔드 포인트(end point) 시점에서의 전류 데이터는 상술된 방법에 따라 최종 데이터로 산출되어 도 6 내지 도 8에 도시되었다. 실험예 1에 사용된 글루코스 용액의 농도는 0, 20, 100, 250, 400, 600mg/dL로 하였다. In Experimental Example 1, a biosensor was prepared by applying the reagent compositions according to Example 1, Comparative Example 1, and Comparative Example 2 to electrodes, respectively. After providing a glucose solution to the sample providing unit of the biosensor, the intensity of the current obtained from the electrode unit was measured as a predetermined voltage was applied to the electrode unit. Specifically, it is generated by inserting the biosensor into a measuring meter capable of applying a voltage, and applying a voltage of 200mV for 2 seconds (4 seconds to 5 seconds) without applying a voltage to the biosensor for 3 seconds. The strength of the current was measured. Measured current value (

: microampere) was amplified using an operational amplifier (OP-AMP), and final data was calculated by multiplying the amplified data by 1/100. (The y-axis current data shown in FIGS. 3 to 8 is data obtained by multiplying the actual data by 1/100) The glucose concentration was measured at 0.2 second intervals. The final data amplified over time is shown in FIGS. 3 to 5, and the current data at the end point of the transfer application period is calculated as final data according to the above-described method and shown in FIGS. 6 to 8 was shown The concentration of the glucose solution used in Experimental Example 1 was 0, 20, 100, 250, 400, 600 mg/dL.

3 shows the results of the above experiment using the biosensor to which the detection reagent according to Example 1 is applied. As a result of the experiment, in the biosensor according to Example 1, it was confirmed that the measured current data for each time period was clearly divided.

4 shows the results of the above experiment through the biosensor to which the detection reagent according to Comparative Example 1 is applied. As a result of the experiment, in the biosensor according to Example 1, it was confirmed that the measured current data for each time period was not clearly divided.

5 shows the results of the above experiment through the biosensor to which the detection reagent according to Comparative Example 2 is applied. As a result of the experiment, in the biosensor according to Comparative Example 2, it was confirmed that the measured current data for each time period was not clearly divided.

As a result, according to the biosensor to which the detection reagent according to Example 1 is applied, even when time elapses, the current data for each concentration of the biotarget is clearly separated, so that a biosensor having further improved reliability can be provided.

Experimental results confirming the straightness of the current data according to the type of reagent composition are shown in FIGS. 6 to 8 . As described above, the current data shown in FIGS. 6 to 8 is a time point when a voltage of 200 mV is applied to each biosensor when 2 seconds have elapsed (that is, when considering a time when no voltage is applied for 3 seconds) , when 5 seconds have elapsed).

6 shows the results of the above experiment using the biosensor to which the detection reagent according to Example 1 is applied. As a result of the experiment, it was confirmed that the measured current data linearly amplified according to the concentration of glucose in the biosensor according to Example 1 was changed.

7 shows the results of the above experiment through the biosensor to which the reagent composition according to Comparative Example 1 is applied. 8 shows the results of the above experiment through the biosensor to which the reagent composition according to Comparative Example 2 is applied.

As a result of the experiment, it was confirmed that the measured current data linearly amplified according to the concentration of glucose in the biosensors according to Comparative Examples 1 and 2 did not change. That is, according to the biosensor to which the detection reagent according to Example 1 is applied, a biosensor with improved measurement reliability and measurement accuracy for a biotarget to be detected can be provided.

<Experimental Example 2> - Stability ①

The experimental results for confirming the stability according to the type of reagent composition are shown in FIG. 9 .

In Experimental Example 2, the reagent compositions according to Example 1, Comparative Example 1, and Comparative Example 2 were centrifuged at 1000 rpm for 20 minutes to determine whether precipitates were generated.

9 is a view confirming whether a precipitate is generated in the reagent compositions according to Example 1 and Comparative Example 3;

When a precipitate is generated for the reagent composition, a separate filtering process may be required, and it is difficult to maintain a uniform concentration of the analyte with respect to the reagent composition, so the reliability of the measurement data for the analyte may be damaged. .

Referring to FIG. 9 , no separate precipitate was generated in the detection reagents 310 and 320 according to Example 1, but separate filtering is required in the plurality of reagent compositions 330, 340, 350 and 360 according to Comparative Example 3 It is confirmed that precipitates that become

As a result, it is confirmed that the chemical for a biosensor according to an embodiment of the present specification has higher stability than a conventional reagent composition (that is, a solution containing an electron transfer medium used in the prior art).

<Experimental Example 3> - Stability ②

In the reagent compositions according to Examples and Comparative Examples, the experimental results for confirming the change in the concentration of the biotarget over time is shown in FIG. 10 .

In Experimental Example 1, after applying the reagent compositions according to Example 1 and Comparative Example 3 to the electrode, a biosensor was manufactured. Thereafter, the glucose solution was provided to the sample providing unit of the biosensor and stored in a chamber at 30 degrees Celsius for 3 months to observe the change. The concentration of the glucose solution provided at this time was 100 mg/dL.

10 is a view showing changes in glucose concentration with time for reagent compositions according to Example 1 and Comparative Example 3; 10 illustrates a first graph 420 , a second graph 440 , a third graph 460 , and a fourth graph 480 .

The first graph 420 is a graph showing the change in glucose concentration with time of the reagent composition according to Example 1.

The second graph 440 is a graph showing the change in glucose concentration with time of the reagent composition containing 1-Methoxy PES in Comparative Example 3.

The third graph 460 is a graph showing the change in glucose concentration with time of the reagent composition containing 1-Methoxy PMS in Comparative Example 3.

The fourth graph 480 is a graph showing the change in glucose concentration with time of the reagent composition containing PMS in Comparative Example 3.

Referring to FIG. 10 , it is confirmed that the stability of the reagent composition according to Example 1 is maintained for a longer period of time compared to the reagent composition prepared according to Comparative Example 3. In the reagent composition according to Example 1, substantially the same amount of glucose concentration was maintained for 3 months1, and in particular, it was confirmed to have higher stability than the reagent composition containing 1-Methoxy PMS and the reagent composition containing PMS.

The methods according to the embodiments of the present invention described above may be used alone or in combination with each other. Since each step described in the method according to the embodiment of the present invention is not essential, each method may be performed including all of the steps as well as only some of the steps. Also, since the order in which each step is described is only for convenience of description, each step in the method described in the present invention does not necessarily have to be performed in the described order.

The above description is merely illustrative of the technical spirit of the present invention, and various modifications and variations will be possible without departing from the essential characteristics of the present invention by those skilled in the art to which the present invention pertains. Accordingly, the embodiments of the present invention described above may be implemented separately or in combination with each other.

Therefore, the embodiments disclosed in the present invention are not intended to limit the technical spirit of the present invention, but to explain, and the scope of the technical spirit of the present invention is not limited by these embodiments. The protection scope of the present invention should be construed by the following claims, and all technical ideas within the scope equivalent thereto should be construed as being included in the scope of the present invention.

Claims (7)

A composition for a biosensor using an electrochemical reaction, comprising:
an enzyme capable of oxidizing a biotarget included in a biological sample, wherein a predetermined electron is provided from the biotarget when the biotarget is oxidized;
a first electron transfer medium capable of being reduced when the predetermined electrons are provided; and
a second electron transport mediator capable of oxidizing the first electron transport mediator and being reduced when the first electron transport mediator is oxidized; including,
The first electron transport medium includes at least one of a ruthenium complex or a ferricyanide complex,
The chemical formula of the second electron transport mediator is 3-(1-methoxyphenazin-5-ium-5-yl) propane-1-sulfonate (3-(1-methoxyphenazin-5-ium-5-yl)propane -1-sulfonate),
The ratio of the weight of the first electron transport mediator to the weight of the enzyme is 3 to 6, and the ratio of the weight of the second electron transport mediator to the weight of the enzyme is 0.001 to 0.1,
The second electron transport medium has the following structural formula,

(In the above structural formula, OMe represents a methoxy group)
A composition for a biosensor.
According to claim 1,
The composition for the biosensor includes a surfactant, a water-soluble polymer and a stabilizer,
The surfactant is at least one from the group consisting of Triton X-100, sodium dodecyl sulfate, sodium stearate, and polysorbate 20,
A composition for a biosensor.
According to claim 1,
The enzyme is flavin adenine dinucleotide-glucose dehydrogenase, fructosamine oxidases (FAOX), fructosyl peptide oxidase, ketoamine oxidase (Ketoamine oxidase), nicotinamide adenine dinucleotideglucose dehydrogenase, glucose oxidase, cholesterol oxidase, cholesterol esterase, lactate oxidase oxidase), containing at least one or more of the group consisting of urate oxidase
A composition for a biosensor.
3. The method of claim 2,
The water-soluble polymer is polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polyethylene glycol (PEG), polyethylene oxide (PEO), hydroxypropyl cellulose (HPC), carboxymethyl cellulose (CMC), cellulose acetate ( CA) at least one species from the group consisting of,
A composition for a biosensor.
5. The method of claim 4,
the ratio of the weight of the first electron transport medium to the weight of the second electron transport medium is 200 to 600
A composition for a biosensor.
3. The method of claim 2,
The stabilizer is at least one kind from the group consisting of trehalose, L-arginine, and citric acid,
A composition for a biosensor.
According to any one of claims 1 to 6, comprising the composition for a biosensor,
biosensor.

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