KR102481839B1 - Biosensor - Google Patents

Abstract

The present invention, a substrate; an electrode unit formed on the substrate; and an electrode protection/electron transport part formed on the electrode part, wherein the electrode protection/electron transport part contains Prussian blue, and measures current density with respect to the concentration of an analyte. It relates to a biosensor in which the slope represented by the calibration curve of satisfies Equation 1 below.
[Equation 1]
-60.728μ ² +341.56μ-77.801 ≤ ω ≤ -60.728μ ² +341.56μ+22.199
ω is the slope of the calibration curve represented by nA/mm ² ·mM;
wherein μ is the content of Prussian blue expressed as a weight percent with respect to the total weight of the electrode protection/electron transport part;
The minimum value of ω is a number greater than zero.

Description

Biosensor {Biosensor}

The present invention relates to biosensors.

A biosensor is a biosensor in which a biological receptor having a function of recognizing biological substances such as pathogens, DNA, or blood sugar is combined with an electrical or optical transducer to convert biological interactions and recognition responses into electrical or optical signals, thereby detecting substances to be analyzed. It is a device that can be selectively detected.

Depending on this selectivity, there are biosensors using various interactions such as antigen/antibody, enzyme, nucleic acid/DNA, and cellular structure. Research and development on biosensors using enzyme interactions are actively progressing in terms of the ability to detect a group of (e.g., etc.) and the ability to use various types of transduction methods for the recognition of an analyte.

Since such a biosensor aims to measure an analyte, it is said that accurately measuring the concentration of an analyte in a specific output result while reducing the measurement time is an important factor in determining the performance of the biosensor. can see.

In particular, it is common to measure the concentration of the analyte contained in the sample by injecting the biological sample itself, rather than directly extracting the analyte contained in the sample and injecting it into the biosensor. The medical industry is paying billions of dollars every year for high-accuracy measurement of samples containing objects, such as saliva, sweat, etc., and it is necessary to develop more efficient and reproducible devices. Do.

In order to solve this technical problem, Patent Publication No. 10-2019-0101203 discloses a glucose sensor capable of quickly measuring low-concentration glucose, but the content of Prussian blue is high, It can cause corrosion of metallic electrode parts or substrates, and it is disclosed that low-concentration glucose can be measured according to the content of Prussian blue. In order to shorten the measurement time, a separate device, For example, there is a problem of having to have a wiring connection, etc., so it is urgent to introduce a biosensor capable of rapidly measuring low-concentration analytes with high accuracy without such a device.

Publication Patent No. 10-2019-0101203

An object of the present invention is to provide a biosensor with improved accuracy with respect to a low-concentration analyte included in a sample.

An object of the present invention is to provide a biosensor in which the required time for measurement of a low-concentration analyte contained in a sample is shortened.

An object of the present invention is to provide a biosensor capable of responding to various concentration ranges of an analyte.

The present invention, a substrate; an electrode unit formed on the substrate; and an electrode protection/electron transport part formed on the electrode part, wherein the electrode protection/electron transport part contains Prussian blue, and measures current density with respect to the concentration of an analyte. It relates to a biosensor in which the slope represented by the calibration curve of satisfies Equation 1 below.

[Equation 1]

-60.728μ ² +341.56μ-77.801 ≤ ω ≤ -60.728μ ² +341.56μ+22.199

ω is the slope of the calibration curve represented by nA/mm ² ·mM;

wherein μ is the content of Prussian blue expressed as a weight percent with respect to the total weight of the electrode protection/electron transport part;

The minimum value of ω is a number greater than zero.

In the present invention, in the first aspect, the value of the coefficient of determination (R ² ) of the calibration curve may be 0.8 or more.

In the second aspect of the present invention, the Prussian blue content (μ) with respect to the total weight of the electrode protection/electron transport part may be 0.1% by weight or more and less than 2.0% by weight.

In the third aspect of the present invention, the concentration of the analyte may be 0.1 mM to 0.3 mM.

The present invention, in the fourth aspect, the electrode portion, gold (Au); silver (Ag); copper (Cu); platinum (Pt); titanium (Ti); nickel (Ni); tin (Sn); molybdenum (Mo); palladium (Pd); cobalt (Co); and alloys thereof; pyrolytic graphite; glassy carbon; carbon paste; perfluorocarbons (PFCs); And it may include one or more selected from the group consisting of carbon nanotubes (CNT).

In the fifth aspect, the present invention may further include an enzyme layer on the electrode protection/electron transport layer.

In the sixth aspect of the present invention, the enzyme layer may include glucose oxidase or glucose dehydronase.

In the seventh aspect, the present invention may further include an ionic polymer film on the enzyme layer.

In the eighth aspect of the present invention, the ionic polymer membrane may include a fluoro copolymer containing a functional group derived from sulfonic acid in its structure.

According to the biosensor according to the present invention, by designing the slope of the calibration curve of the measurement current density according to the concentration of the analyte to be limited to a region within a specific range, it is possible to measure with high accuracy even for a low-concentration analyte included in the sample. and shorten the time required for measurement.

In addition, according to the biosensor according to the present invention, it is possible to measure various concentration ranges of analytes included in a sample with high accuracy in a short time through a parameter range for a limited slope.

In addition, according to the biosensor according to the present invention, it is possible to manufacture a biosensor exhibiting high accuracy in a short time while reducing the content of Prussian blue compared to the prior art.

1 is a diagram showing a limited slope range of a calibration curve represented by a biosensor of the present invention.
2 is a diagram showing a laminated structure of a biosensor according to an embodiment of the present invention.
3 is a diagram illustrating a biosensor according to an embodiment of the present invention.
FIG. 4 is a cross-sectional view of the biosensor shown in FIG. 3 taken along A-A'.
5 and 6 are diagrams showing the results measured by the biosensors of Example 2 and Comparative Example 1.

The present invention relates to a biosensor, and more particularly, to a substrate; an electrode unit formed on the substrate; and an electrode protection/electron transport part formed on the electrode part, wherein the electrode protection/electron transport part contains Prussian blue, and , characterized in that the slope indicated by the calibration curve of the measured current density for the concentration of the analyte is included in a certain region.

As described above, by manufacturing the biosensor according to the optimal design of the electron medium, it is possible to measure with high accuracy even for a sample containing a low concentration of the analyte, and at the same time, it is possible to effectively reduce the required measurement time.

Hereinafter, with reference to the drawings, embodiments of the present invention will be described in more detail. However, the following drawings attached to this specification illustrate preferred embodiments of the present invention, and serve to further understand the technical idea of the present invention together with the contents of the above-described invention, so the present invention is described in such drawings should not be construed as limited to

Terms used in this specification are for describing embodiments, and are not intended to limit the present invention. In this specification, singular forms also include plural forms unless specifically stated otherwise in a phrase.

As used herein, "comprises" and/or "comprising" refers to the presence or addition of one or more other elements, steps, operations and/or elements other than the mentioned elements, steps, operations and/or elements. It is used in the sense of not excluding. Like reference numerals designate like elements throughout the specification.

<Biosensor>

1 is a diagram showing a limited slope range of a calibration curve represented by a biosensor of the present invention, FIG. 2 is a diagram showing a laminated structure of a biosensor according to an embodiment of the present invention, and FIG. 3 is a diagram showing the present invention. is a diagram illustrating a biosensor according to an embodiment, and FIG. 4 is a cross-sectional view of the biosensor shown in FIG. 3 taken along A-A'.

1 to 4, the biosensor of the present invention includes a substrate 10; an electrode part 20 formed on the substrate 10; and an electrode protection/electron transport unit 30 formed on the electrode unit 20, wherein the electrode protection/electron transport unit 30 includes Prussian blue, and the analyte ( It may be a biosensor in which the slope of the calibration curve of the measured current density for the analyte) concentration satisfies Equation 1 below.

[Equation 1]

-60.728μ ² +341.56μ-77.801 ≤ ω ≤ -60.728μ ² +341.56μ+22.199

ω is the slope of the calibration curve represented by nA/mm ² ·mM; wherein μ is the content of Prussian blue expressed as a weight percent with respect to the total weight of the electrode protection/electron transport part; The minimum value of ω is a number greater than zero.

The substrate 10 serves to provide a structural base for components constituting the biosensor.

The substrate 10 may be made of a rigid material such as glass or implemented in the form of a film having flexible properties, and may be conventionally or later developed, and in one or more embodiments, silicon, polyester resins such as glass, glass epoxy, ceramics, polyethylene naphthalate (PET), and polybutylene terephthalate; cellulosic resins such as diacetyl cellulose and triacetyl cellulose; polycarbonate-based resin; acrylic resins such as polymethyl (meth)acrylate and polyethyl (meth)acrylate; styrenic resins such as polystyrene and acrylonitrile-styrene copolymer; polyolefin-based resins such as polyethylene, polypropylene, polyolefins having a cyclo-based or norbornene structure, and ethylene-propylene copolymers; vinyl chloride-based resins; amide resins such as nylon and aromatic polyamide; imide-based resins; polyethersulfone-based resins; sulfone-based resins; polyether ether ketone-based resins; sulfurized polyphenylene-based resins; vinyl alcohol-based resin; vinylidene chloride-based resins; vinyl butyral-based resins; allylate-based resins; polyoxymethylene-based resin; A film composed of a thermoplastic resin such as an epoxy resin may be used, and a film composed of a blend of the thermoplastic resin may also be used. In addition, a film made of a thermosetting resin or an ultraviolet curable resin such as (meth)acrylic, urethane, acrylurethane, epoxy, or silicone may be used, but is not limited thereto.

The thickness of the substrate 10 is not particularly limited, but generally may be 1 to 500 μm, preferably 1 to 300 μm, and 5 to 300 μm, in consideration of workability such as strength and handling, and thin layer properties. 200 μm is more preferable.

In one embodiment, the substrate 10 may contain one or more appropriate additives, and the additives include, for example, an ultraviolet absorber, an antioxidant, a lubricant, a plasticizer, a release agent, an anti-coloring agent, a flame retardant, a nucleating agent, An antistatic agent, a pigment, a colorant, etc. are mentioned.

In one embodiment, the substrate 10 may have a structure including various functional layers such as a hard coating layer, an antireflection layer, and a gas barrier layer on one or both surfaces of the substrate, and the functional layer is not limited to the above. , It may include various functional layers depending on the use.

In one embodiment, the substrate 10 may be surface-treated as needed, and such surface treatment includes, for example, plasma treatment, corona treatment, primer treatment, and the like. dry treatment, chemical treatment such as alkali treatment including saponification treatment, and the like.

The biosensor of the present invention may include the electrode unit 20 on the substrate 10 .

The electrode unit 20 may be provided to sense an electrical signal generated by a reaction of an analyte included in a sample.

The electrode unit 20, in one or more embodiments, is gold (Au); silver (Ag); copper (Cu); platinum (Pt); titanium (Ti); nickel (Ni); tin (Sn); molybdenum (Mo); palladium (Pd); cobalt (Co); and alloys thereof; pyrolytic graphite; glassy carbon; carbon paste; perfluorocarbons (PFCs); And it may include one or more selected from the group consisting of carbon nanotubes (CNT). The materials used for the electrode part 20 may be used alone or two or more materials may be used as a multilayer film.

The electrode unit 20 may preferably be provided as a working electrode 20-1, which is an electrode for a reaction to occur. In one embodiment, the biosensor of the present invention includes a reference electrode 20-1. 2) and/or a counter electrode (not shown) may be further included.

The reference electrode 20 - 2 has a constant potential and may be provided to serve as a reference electrode for obtaining a generated potential of the working electrode.

The reference electrode 20-2, in one or more embodiments, is a silver-silver chloride (Ag/AgCl) electrode, a calomel electrode, a mercury-mercury sulfate electrode, and a mercury-mercury oxide At least one selected from the group consisting of -oxide mercury) electrodes can be used, and considering that the hysteresis of the potential with respect to the temperature cycle is less and the potential is stable up to high temperature, silver-silver chloride (Ag / AgCl) It is preferable that it is an electrode.

The counter electrode may be provided to send or receive current so that a reaction occurs on the surface of the working electrode. That is, the flow of current occurs mainly through exchange between the counter electrode and the working electrode, and oxidation and reduction reactions occur. At this time, the reference electrode can serve as a feedback sensor to measure and monitor the potential of the counter electrode and maintain it in a constant state. there is.

The counter electrode is the working electrode and All materials used in the reference electrode may be used, and it is preferable to use the same material as the working electrode and/or the reference electrode in order to simplify the process and improve manufacturing cost.

The biosensor of the present invention may include an electrode protection/electron transport unit 30 formed on the electrode unit 20 .

The electrode protection/electron transport unit 30 may be provided to protect the electrodes constituting the electrode unit 20 and to improve electrical sensitivity of the electrode unit 20 through electron transport. there is.

In one embodiment, the electrode protection/electron transport unit 30 may be formed on the electrode unit 20, and in another embodiment, it may be formed on the electrode unit and a partial area of the substrate outside the electrode unit. it could be

The electrode protection/electron transport unit 30 includes Prussian blue for an electron transport function, and the content of the Prussian blue is based on the total weight of the electrode protection/electron transport unit 30. It may be included in 0.1% by weight or more and less than 2.0% by weight relative to. When the content of Prussian blue is in the above range, an effect of improving sensitivity by improving electron transport function can be expected, and there are advantages in terms of corrosion prevention and reliability due to oxidation of metallic electrode parts.

Prussian blue included in the electrode protection/electron transport unit 30 can perform an electron transport function, but is a blue pigment whose main component is potassium hexacyanoferrous (II) oxide iron (III). Due to its oxidative properties, the sensitivity of the biosensor can be improved, and at the same time, the metallic electrode part 20 located at the lower side can be oxidized and corroded. Therefore, in order to prevent oxidation of the electrode part by Prussian blue, the electrode protection/electron transport unit 30 may include carbon or the like, and in one embodiment, the electrode protection/electron transport unit 30 The electron transport unit 30 may be a carbon paste containing Prussian blue.

The biosensor of the present invention may further include an enzyme layer 40 on the electrode protection/electron transport unit 30 .

The enzyme layer 40 may be provided to include an enzyme that specifically reacts with the analyte (substance to be detected) included in the sample.

The enzyme included in the enzyme layer 40 is not particularly limited as long as it is a biocatalyst that increases the rate of metabolism by lowering the activation energy of a chemical reaction by forming an enzyme-substrate complex by combining with an analyte included in the sample. The enzymes may be changed according to the analyte to be measured by the biosensor, and include lactate oxidase, glucose oxidase, cholesterol oxidase, and ascorbic acid oxidase. , uric acid oxidase (Urate oxidase), and alcohol oxidase (Alcohol oxidase) an oxidase that may include one or more selected from the group consisting of; Or a dehydrogenase that may include at least one selected from the group consisting of lactate dehydronase, glucose dehydronase, glutamaate dehydronase, and alcohol dehydronase It may be, preferably, may include glucose oxidase or glucose dehydronase.

Taking the glucose sensor as an embodiment of the present invention as an example, the reaction in the enzyme layer 40 and the signal detection principle of the electrode unit 20 will be described as follows.

When a sample to be measured is injected into the glucose sensor, glucose contained in the sample is oxidized by glucose oxidase or glucose dehydrogenase, and glucose oxidase or glucose dehydrogenase is reduced. At this time, the electron transfer mediator oxidizes glucose oxidase or glucose dehydrogenase, and itself is reduced. The reduced electron transfer mediator loses electrons on the electrode surface to which a certain voltage is applied and is oxidized again electrochemically. Since the glucose concentration in the sample is proportional to the current amount or current density generated in the course of oxidation of the electron transfer mediator, the glucose concentration can be measured by measuring the current amount or current density.

The biosensor of the present invention may further include an ionic polymer membrane 50 on the enzyme layer 40 .

The ionic polymer membrane 50 passes only the substance to be measured (analyte) among the substances included in the sample, thereby preventing the permeation of external impurity ions, thereby reducing the oxidase or dehydrogenase of the enzyme layer 40. It may be provided to perform a role of protection.

The ionic polymer membrane 50 may include a fluorocopolymer having a functional group derived from sulfonic acid in its structure, and may preferably include Nafion.

The biosensor of the present invention, in one embodiment, includes a working electrode 20-1 and a wiring part 60 branching from one side of the reference electrode 20-2 and extending toward the distal end of the biosensor; It may further include an insulating layer 70 for electrically insulating.

The wiring part 60 may be provided to perform a role of connecting a means for applying a voltage to the biosensor and a means for measuring current density in the electrode part.

The wiring part 60 may include gold (Au); silver (Ag); copper (Cu); platinum (Pt); titanium (Ti); nickel (Ni); tin (Sn); molybdenum (Mo); palladium (Pd); cobalt (Co); And one or more selected from the group consisting of alloys thereof may be used, for example, it may be a silver-palladium-copper alloy (APC), but it is not particularly limited as long as it enables transmission of electrical signals.

In the biosensor of the present invention, the sample may be a biological sample such as blood, bodily fluid (saliva, sweat, tears, etc.), urine, or other liquid sample, but is not limited thereto.

In the biosensor of the present invention, the analyte (substance to be detected) may be glucose, lactic acid, uric acid, etc., but is not limited thereto.

<Slope of calibration curve>

In the biosensor of the present invention, the slope of the calibration curve of the measured current density with respect to the concentration of the analyte may satisfy Equation 1 below.

[Equation 1]

-60.728μ ² +341.56μ-77.801 ≤ ω ≤ -60.728μ ² +341.56μ+22.199

ω is the slope of the calibration curve represented by nA/mm ² ·mM; wherein μ is the content of Prussian blue expressed as a weight percent with respect to the total weight of the electrode protection/electron transport part; The minimum value of ω is a number greater than zero.

The slope of the calibration curve represented by “ω” is the concentration (mM) of the analyte contained in the sample injected into the electrode part of the biosensor and the measured current density (nA/mm ² ) for that concentration. Indicates the slope of the calibration curve expressed by a linear equation for a specific concentration range.

The concentration of the analyte included in the sample is preferably 0.5 mM or less, and more preferably, in terms of reliability, may be 0.1 mM to 0.3 mM.

In an embodiment, the measured current density may be a current density measured by a method described in <Method of Measuring a Biosensor Signal> , and is preferably a current density in a saturation state in terms of reliability.

The coefficient of determination (R ² ) of the calibration curve represented by the measurement current for the concentration of the analyte is preferably 0.8 or more, more preferably 0.9 or more, from the viewpoint of reliability.

As described above, the content of Prussian blue represented by "μ" is preferably 0.1% by weight or more and less than 2.0% by weight with respect to the total weight of the electrode protection/electron transport part.

In order for those skilled in the art to clearly understand and easily implement the present invention in the technical field to which the corresponding invention pertains, parameters represented by Equation 1 will be described as an example.

A biosensor containing 0.3% by weight of Prussian blue based on the total weight of the electrode protection/electron transport unit was prepared.

The lower limit of the slope of the calibration curve of the biosensor, calculated by Equation 1, is 19.20148 as “-60.728·(0.3) ² +341.56·(0.3)-77.801”, and the upper limit is “-60.728·(0.3) ² +341.56·(0.3)+22.199”, which is 119.20148.

Samples with concentrations of 0.1mM, 0.15mM, 0.2mM, 0.25mM, and 0.3mM of the analyte were injected into the biosensor, and the current density (nA/mm ² ) value measured in the saturation state was plotted to prepare a calibration curve. do.

When the slope of the calibration curve prepared above is 19.20148 to 119.20148, it corresponds to a biosensor that satisfies Equation 1 above.

If the slope of the calibration curve prepared above is continuously displayed according to the content of Prussian blue included in the electrode protection/electron transport unit, it can be displayed as a constant area (S) as shown in FIG.

In another embodiment of the present invention, in order to further improve the accuracy of the biosensor, the slope of the calibration curve of the measured current density with respect to the concentration of the analyte may satisfy Equation 2 below.

[Equation 2]

-60.728μ ² +341.56μ-52.801 ≤ ω ≤ -60.728μ ² +341.56μ-2.801

The ω and μ are as defined in [Equation 1] above.

According to the present invention, as shown in Equations 1 and 2 above, the slope of the calibration curve represented by the concentration of the analyte contained in the sample and the measured current density is the Prussian blue ), it is possible to manufacture a biosensor capable of measuring with high accuracy and precision in a short time even for a low-concentration sample by designing the biosensor to be included in the function range for the content of.

<Method of manufacturing biosensor>

The biosensor manufacturing method of the present invention includes the steps of forming an electrode unit on a substrate (a); Forming an electrode protection/electron transport part on the electrode part (b); Forming an enzyme layer on the electrode protection/electron transport unit (c); A step (d) of forming an ionic polymer film on the enzyme layer may be included.

The step (a) may be performed by including one or more processes selected from the group consisting of screen printing, letterpress printing, intaglio printing, lithography, and photolithography.

In one or more embodiments, in step (a), it is preferable to perform a photolithography process to form a wiring part, and each electrode is formed by screen printing, letterpress printing, intaglio printing, and lithographic printing. It may be performed by any one selected from the group, and preferably may be screen printing.

The photolithography may be a method of integrally forming a wire by forming a carbon paste and/or a metal film on a substrate and patterning the film through a mask.

The steps (b), (c) and (d) may be performed by any one selected from the group consisting of flow coating, ink jet, and drop casting, It is more preferable that it is drop casting.

The biosensor manufactured by the above manufacturing method may exhibit all the characteristics described in the above item <biosensor> .

<Method of measuring biosensor signal>

The present invention includes a method for measuring an electrochemical signal of an analyte using a biosensor manufactured by the biosensor manufacturing method.

In this specification, “electrochemical measurement” refers to measurement by applying an electrochemical measurement method, and in one or more embodiments, an amperometric method, a potentiometric method, a coulometric method, etc. are mentioned, and preferably Preferably, it may be an amperometric method.

In an embodiment, the method for measuring a biosensor signal of the present disclosure includes applying a voltage to an electrode unit including the working electrode and a reference electrode after contact with a sample, and measuring a response current value emitted when the voltage is applied. and calculating an electrochemical signal of the substance to be detected in the sample based on the response current value. The applied voltage is not particularly limited, but in one or more embodiments, it may be -500 to +500 mV, preferably -200 to +200 mV based on the silver-silver chloride electrode (Ag / AgCl electrode) .

In the method for measuring a biosensor signal of the present disclosure, in an embodiment, a voltage may be applied to the electrode part after contacting the sample and maintaining it in an unapplied state for a predetermined time, and the electrode part simultaneously with the contact with the reagent. A voltage may be applied to

According to the biosensor signal measuring method of the present disclosure, even for a low-concentration analyte included in a sample, it is possible to reduce the measurement time with high accuracy.

<Biosensor signal measurement system>

In one embodiment, the present invention provides an electrochemical analysis of an analyte in a sample, including the biosensor, a means for applying a voltage to an electrode part of the biosensor, and a means for measuring a current at the electrode part. It relates to an electrochemical signal measuring system of a biosensor for measuring a signal.

According to the biosensor signal measurement system of the present invention, even low-concentration analytes included in a sample can be measured with high accuracy and in a short time.

The applying means is not particularly limited as long as it is conductive to the electrode part of the biosensor and can apply a voltage, and a known applying means can be used. As the applying means, in one or more embodiments, a contactor capable of contacting the electrode portion of the biosensor, a power source such as a DC power supply, and the like can be included.

The measuring means is for measuring a plurality of currents in the electrode portion generated when voltage is applied, and in one or more embodiments, a response current value correlated with the amount of electrons emitted from the electrode portion of the biosensor is measured. Anything that is possible can be used, and those used in biosensors conventionally or later developed can be used.

Hereinafter, specific embodiments of the present invention will be described. However, the present invention is not limited to the embodiments disclosed below and may be implemented in various different forms, only the present embodiments make the disclosure of the present invention complete, and common knowledge in the art to which the present invention pertains. It is provided to completely inform the person who has the scope of the invention, and the present invention is only defined by the scope of the claims.

<Examples and Comparative Examples>

Example 1

On the substrate, a Ag alloy layer with a thickness of about 2000 Å and a protective layer of IZO metal with a thickness of about 500 Å were patterned using photolithography, and then a carbon paste electrode layer (including Prussian blue) with a thickness of about 10 μm was screen-printed. , The content of the Prussian blue is 0.3% by weight based on the total weight of the carbon paste. On this, an enzyme layer in which glucose oxidase was immobilized with chitosan was sequentially stacked to form a working electrode.

The biosensor of Example 1 was fabricated by forming an Ag/AgCl reference electrode on a substrate spaced apart from the working electrode.

Example 2

The biosensor of Example 2 was manufactured in the same manner as Example 1, except that the content of Prussian blue was 0.5% by weight based on the total weight of the carbon paste.

Example 3

The biosensor of Example 3 was manufactured in the same manner as Example 1, except that the content of Prussian blue was 1.0% by weight based on the total weight of the carbon paste.

Comparative Example 1

A biosensor of Comparative Example 1 was manufactured in the same manner as in Example 1, except that the content of Prussian Blue was 3.0% by weight based on the total weight of the carbon paste.

Comparative Example 2

A biosensor of Comparative Example 2 was manufactured in the same manner as in Example 1, except that the content of Prussian Blue was 5.0% by weight based on the total weight of the carbon paste.

<Experimental Example 1>: Evaluation of slope, accuracy and precision of linear regression line

The biosensors of Examples and Comparative Examples were measured by the following method.

As a sample, a sample in which 0.1 to 0.3 mM glucose was dissolved in phosphate buffered saline (PBS) was made and measured. The volume of the sample used at this time is 30 μl, but it is applied on the reference electrode and the working electrode in a sufficient amount applied to the electrode surface. The quantity is not limited. A constant voltage was applied to this using an electrochemical measuring instrument CHI630E, and a corresponding change in current was measured. The slope, accuracy, and precision evaluation results of the linear regression line according to the Prussian blue content are shown in Table 1 below.

<Experimental Example 2>: Evaluation of current value and stabilization index according to measurement time

The biosensors of Example 2 and Comparative Example 1 were measured by the following method.

As a sample, five samples were prepared and measured by dissolving 0.1 mM glucose in phosphate buffered saline (PBS). Any amount applied on the electrode is not limited. A constant voltage was applied using an electrochemical measuring device CHI630E, and the corresponding change in current was measured for each time period, and is shown in FIG. 5 .

In addition, the value obtained by subtracting the current value (I _s ) measured in the stabilization state from the current value (I _t ) measured at a specific time is divided by the current value (I _s ) measured in the stabilization state, and the stabilization index calculated by multiplying by 100 is measured. It was calculated for each time period and is shown in FIG. 6 .

The smaller the stabilization index, the higher the accuracy of the biosensor. Therefore, among different biosensors measured at the same time, a biosensor with a smaller stabilization index value shows that the measurement time is further shortened and the precision is improved. .

Stability index = {(I _t - I _s ) / I _s } * 100

Referring to Table 1, when the glucose concentration was measured using the biosensor of Examples 1 to 3, the slope of the calibration curve corresponding to the Prussian blue content corresponding to the range disclosed in the present invention, the coefficient of determination (coefficient of determination; R ² ) is 0.84 or more, up to 0.98, it can be seen that the accuracy is improved compared to the biosensors of Comparative Example 1 and Comparative Example 2, in which the slope of the calibration curve is outside the range disclosed in the present invention. Looking at the percentage of relative standard deviation (RSD), it can be seen that the biosensors of Examples 1 to 3 exhibit superior precision compared to the biosensors of Comparative Examples 1 and 2.

In addition, referring to FIGS. 5 and 6, the stabilization index of the current value measured at the same time is about 10 times greater than the biosensor of Example 2 (0.5% PB) in the biosensor of Comparative Example 1 (PB 3%). Comparing the spread between the data of the 60sec time range, it can be seen that the biosensor of Comparative Example 1 (PB 3%) is not completely stabilized, so the measured current spread between the samples in the 60sec range is larger than that of the biosensor of Example 2 (PB 0.5%). can That is, in the case of the biosensor of Example 2 (PB 0.5%), it can be seen that the measurement time is relatively shortened and the accuracy is improved compared to the biosensor of Comparative Example 1 (PB 3%).

10: substrate 20: electrode part
30: electrode protection / electron transport unit 40: enzyme layer
50: ionic polymer film 60: wiring part
70: insulating film

Claims (9)

Board;
an electrode unit formed on the substrate; and
A biosensor including an electrode protection/electron transport unit formed on the electrode unit,
The electrode protection/electron transport unit includes Prussian blue,
A biosensor in which the slope of the calibration curve of the measured current density for the concentration of the analyte satisfies Equation 1 below.
[Equation 1]
-60.728μ ² +341.56μ-77.801 ≤ ω ≤ -60.728μ ² +341.56μ+22.199
ω is the slope of the calibration curve represented by nA/mm ² ·mM;
wherein μ is the content of Prussian blue expressed as a weight percent with respect to the total weight of the electrode protection/electron transport part;
The minimum value of ω is a number greater than zero.
The biosensor according to claim 1, wherein the value of the coefficient of determination (R ² ) of the calibration curve is 0.8 or more.
The biosensor according to claim 1, wherein the Prussian blue content (μ) is greater than or equal to 0.1% by weight and less than 2.0% by weight relative to the total weight of the electrode protection/electron transport unit.
The biosensor of claim 1, wherein the concentration of the analyte is 0.1 mM to 0.3 mM.
The method according to claim 1, wherein the electrode unit, gold (Au); silver (Ag); copper (Cu); platinum (Pt); titanium (Ti); nickel (Ni); tin (Sn); molybdenum (Mo); palladium (Pd); cobalt (Co); and alloys thereof; pyrolytic graphite; glassy carbon; carbon paste; perfluorocarbons (PFCs); And a biosensor comprising at least one member selected from the group consisting of carbon nanotubes (CNTs).
The biosensor according to claim 1, further comprising an enzyme layer on the electrode protection/electron transport unit.
The biosensor of claim 6, wherein the enzyme layer includes glucose oxidase or glucose dehydronase.
The biosensor according to claim 6, further comprising an ionic polymer film on the enzyme layer.
9. The biosensor according to claim 8, wherein the ionic polymer membrane comprises a fluoro copolymer comprising in its structure a functional group derived from sulfonic acid.

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