KR20210042486A - Electrochemical biosensor and manufacturing method thereof - Google Patents

Abstract

The present invention relates to an electrochemical blood glucose sensor and to a manufacturing method thereof. According to one embodiment of the present invention, the electrochemical blood glucose sensor includes a working electrode in which an electrochemical phenomenon occurs in contact with blood. The working electrode may include: a metal substrate including stainless steel; a reaction layer comprising glucose oxidase and a first immobilizing material for immobilizing glucose oxidase by cross-linking with the glucose oxidase, respectively, and provided on the metal substrate; and a second fixing material for additionally fixing the glucose oxidase in the reaction layer, and comprising a fixing layer provided to cover the reaction layer.

Description

Electrochemical biosensor and manufacturing method thereof TECHNICAL FIELD

The present invention relates to an electrochemical blood sugar sensor and a method for manufacturing the same, and more particularly, to a blood sugar sensor for measuring blood sugar using an electrochemical method, and a method for manufacturing the same.

Health abnormalities caused by various diseases such as diabetes can be confirmed through blood sugar measurement. In particular, it can be said that it is essential to frequently and accurately measure and monitor a subject's blood sugar in order to manage diabetes and complications, which are one of the major causes of disease death in adults. For this blood glucose measurement, spectroscopic and electrochemical methods are mainly used.

Spectroscopic measurement is a method using the oxidation reaction of glucose by glucose oxidase and peroxidase, and the amount of blood glucose is determined by using the change in absorption or fluorescence that appears after the reaction. The electrochemical method induces a redox reaction of blood sugar by means of a glucose-specific enzyme, and determines the blood sugar concentration by measuring the flow of electric charges resulting from this with an electrode provided in a blood sugar sensor. In particular, compared to spectroscopic measurement, the electrochemical method has less interference effect caused by other substances in the blood, can shorten the time to measure blood glucose, and allows the device itself to be miniaturized, simplified, and inexpensive. Due to this, it is a method that is mainly used recently.

In the case of a blood glucose sensor using a conventional electrochemical method (hereinafter, referred to as “conventional sensor”), the noise signal is very large compared to the signal generated by glucose, which is the substance to be measured (hereinafter, “Problem 1”). ”). That is, when the blood glucose concentration is measured using a conventional sensor, the noise increase degree of the current signal itself is greater than the increase degree of the current generated according to the glucose concentration.

In addition, in the conventional sensor, as the concentration of glucose increases, the error range of the detected signal increases proportionally (hereinafter, referred to as “problem 2”). That is, when the blood sugar concentration is measured using a conventional sensor, a larger error range occurs as a signal detected at a high blood sugar level.

In accordance with these problems 1 and 2, the conventional sensor had to have a very large performance deviation of about 15%. Moreover, since all of the conventional sensors are made of precious metals such as platinum (Pt), there is a problem (hereinafter referred to as “problem 3”) that the manufacturing cost is inevitably increased.

In order to solve the problems of the prior art as described above, the present invention provides an electrochemical blood glucose sensor having a smaller noise of the current signal, that is, a large signal-to-noise ratio (SNR) compared to the current signal according to the glucose concentration, and a method of manufacturing the same. Its purpose is to provide.

In addition, an object of the present invention is to provide an electrochemical blood glucose sensor and a method of manufacturing the same, which can reduce an error range of a current signal that increases with an increase in glucose concentration.

In addition, an object of the present invention is to provide an electrochemical blood glucose sensor and a method of manufacturing the same, which can reduce the manufacturing cost by using an inexpensive metal as an electrode.

In addition, an object of the present invention is to provide an electrochemical blood glucose sensor capable of effectively detecting glucose, which is a substance to be measured, by reducing the influence on the concentration of an interfering substance, and a method of manufacturing the same.

However, the problem to be solved by the present invention is not limited to the problems mentioned above, and other problems that are not mentioned can be clearly understood by those of ordinary skill in the art from the following description. There will be.

An electrochemical blood glucose sensor according to an embodiment of the present invention for solving the above problems includes a working electrode in which an electrochemical phenomenon occurs in contact with blood, wherein the working electrode is stainless steel. steel); A reaction layer each comprising a glucose oxidase and a first immobilizing material for immobilizing glucose oxidase by cross-linking with glucose oxidase, and provided on a metal substrate; And a second fixing material for additionally fixing glucose oxidase in the reaction layer, and a fixing layer provided to cover the reaction layer.

The reaction layer and the fixed layer may be formed on a portion of the metal substrate in contact with blood.

The electrochemical phenomenon may be a phenomenon in which hydrogen peroxide generated by a reaction between glucose oxidase in a reaction layer and glucose in blood reacts with ions supplied from a metal substrate.

The second fixing material may prevent penetration of at least one type of interfering material among interfering materials reacting with hydrogen peroxide in addition to the metal substrate, or may reduce the penetration rate of the interfering material.

The working electrode may include a needle-shaped portion, and a reaction layer and a fixed layer may be provided on the needle-shaped portion.

The first fixing material may include chitosan, and the second fixing material may include nafion.

An electrochemical blood glucose sensor according to an embodiment of the present invention includes: a counter electrode provided for measuring a current generated according to an electrochemical phenomenon in a working electrode; And a reference electrode, which is a counter electrode provided for applying a voltage to the working electrode, and may be implemented to be inserted into a blood glucose meter.

In the blood glucose meter, a voltage of a predetermined size is applied between the metal substrate and the reference electrode, but a lower voltage may be applied to the metal substrate.

The predetermined size may be from 0.07 V to 0.13 V.

A method of manufacturing an electrochemical blood glucose sensor according to an embodiment of the present invention is a method of manufacturing an electrochemical blood glucose sensor including a working electrode in which an electrochemical phenomenon occurs in contact with blood. A preparation step of preparing a metal substrate that is a first configuration of the working electrode including ); The reaction layer, which is the second configuration of the working electrode, each comprising a glucose oxidase and a first immobilizing material for immobilizing glucose oxidase by cross-linking with glucose oxidase, is formed on a metal substrate. A step of forming a reaction layer to form a reaction layer; And a fixed layer forming step of forming a fixed layer, which is a third component of the working electrode including a second fixing material for additionally fixing glucose oxidase in the reaction layer, but covering the reaction layer.

The step of forming the reaction layer may include soaking a portion of the metal substrate in contact with blood in a first solution in which the first fixing material and glucose oxidase are mixed.

The first solution may be a solution in which glucose oxidase is mixed with a solution containing a first fixing material having an amount of 0.12% (v/v) to 0.13% (v/v).

The forming of the fixed layer may include immersing the portion of the metal substrate on which the reaction layer is formed in a second solution containing a second fixing material.

The second solution may be a solution containing a second fixing material in an amount of 1.4% (v/v) to 1.6% (v/v).

The electrochemical blood glucose sensor includes a counter electrode provided for measuring a current generated by an electrochemical phenomenon in the working electrode, and a reference electrode that is a counter electrode provided for applying a voltage to the working electrode. ), but is implemented to be inserted into a blood glucose meter, and a voltage of a predetermined size is applied between the metal substrate and the reference electrode in the blood glucose meter, but a lower voltage may be applied to the metal substrate.

The predetermined size may be from 0.07 V to 0.13 V.

The present invention configured as described above has the advantage of having less noise of the current signal itself, that is, having a large signal-to-noise ratio (SNR) compared to the current signal according to the glucose concentration.

In addition, the present invention has the advantage of reducing the error range of the current signal, which increases as the glucose concentration increases.

In addition, the present invention has the advantage of reducing the manufacturing cost by using stainless steel, which is an inexpensive metal, as an electrode.

In addition, the present invention has the advantage of being able to more effectively detect glucose by reducing the influence on the concentration of the interfering substance and increasing the selectivity of glucose, which is a substance to be measured.

The effects that can be obtained in the present invention are not limited to the above-mentioned effects, and other effects not mentioned can be clearly understood by those of ordinary skill in the art from the following description. will be.

1 shows an electrochemical blood glucose sensor 100 and a blood glucose meter 200 according to an embodiment of the present invention.
2 shows the configuration of an electrochemical blood glucose sensor 100 according to an embodiment of the present invention.
3 shows various examples of a cross section cut along the dotted line shown in the blood contact portion 11 of the working electrode 10 in FIG. 2(b).
4 shows an electrochemical phenomenon occurring in the blood contact portion 11 of the working electrode 10.
5 shows a manufacturing process sequence of the working electrode 10 of the electrochemical blood glucose sensor 100 according to an embodiment of the present invention.
6 shows a graph of chronoamperometry according to the concentration of hydrogen peroxide (0 M, 10 μM, 20 μM, and 30 μM) for each of the first and second cases.
7 is a graph of chronoamperometry according to the concentration of hydrogen peroxide (0 M, 0.2 μM, 0.6 μM, 1.2 mM, 2 mM, 3 mM, 4 mM) for each of the first and second cases, and hydrogen peroxide. Shows the graph of current change by concentration of.
8 shows an open circuit potential graph over time for each of the first and second cases.
9 shows a cyclic voltammetry graph comparing the presence and absence of hydrogen peroxide for the second case.
10 shows a process of preparing a third needle.
11 shows a process of preparing a fourth needle.
12 and 13 are graphs of chronoamperometry according to the concentration of glucose (0 M, 1 mM, 3 mM, 6 mM, 10 mM, 15 mM, 20 mM) for each of the third and fourth cases. , Shows a graph of current change by glucose concentration.
14 and 15 show graphs of changes in current according to a change in concentration of an interfering substance at different constant applied voltages for each of the third and fourth cases.
16 to 20 show graphs of changes in current according to a change in concentration of an interfering substance at a constant applied voltage for each of the fifth to seventh cases, the fourth case, and the eighth case.
21 and 22 show graphs of current change according to a change in concentration of an interfering substance at a constant applied voltage for each of the ninth and fourth cases.
23 shows an experimental apparatus used for an experiment of performance analysis of the electrochemical blood glucose sensor 100 according to an embodiment of the present invention.

The above objects and means of the present invention and effects thereof will become more apparent through the following detailed description in connection with the accompanying drawings, and accordingly, those of ordinary skill in the technical field to which the present invention pertains to facilitate the technical idea of the present invention. I will be able to do it. In addition, in describing the present invention, when it is determined that a detailed description of a known technology related to the present invention may unnecessarily obscure the subject matter of the present invention, a detailed description thereof will be omitted.

The terms used in the present specification are for describing exemplary embodiments, and are not intended to limit the present invention. In the present specification, the singular form also includes the plural form in some cases unless specifically stated in the phrase. In the present specification, terms such as "include", "include", "to prepare" or "have" do not exclude the presence or addition of one or more other elements other than the mentioned elements.

In the present specification, terms such as “or” and “at least one” may represent one of words listed together, or a combination of two or more. For example, “or B” “at least one of “and B” may include only one of A or B, and may include both A and B.

In this specification, the description following “for example” may not exactly match the information presented, such as the recited characteristic, variable, or value, and tolerances, measurement errors, limitations of measurement accuracy, and other commonly known factors. It should not be limited to the embodiments of the invention according to the various embodiments of the present invention to effects such as modifications, including.

In the present specification, when a component is described as being'connected' or'connected' to another component, it may be directly connected or connected to the other component, but other components exist in the middle. It should be understood that it may be possible. On the other hand, when a component is referred to as being'directly connected' or'directly connected' to another component, it should be understood that there is no other component in the middle.

In the present specification, when a component is described as being'on' or'adjacent' of another component, it may be directly in contact with or connected to another component, but another component exists in the middle. It should be understood that it is possible. On the other hand, when a component is described as being'directly above' or'directly' of another component, it may be understood that another component does not exist in the middle. Other expressions describing the relationship between elements, for example,'between' and'directly,' can be interpreted as well.

In the present specification, terms such as'first' and'second' may be used to describe various elements, but the corresponding elements should not be limited by the above terms. In addition, the terms above should not be interpreted as limiting the order of each component, and may be used for the purpose of distinguishing one component from another component. For example, the'first element' may be named'second element', and similarly, the'second element' may also be named'first element'.

Unless otherwise defined, all terms used in the present specification may be used with meanings that can be commonly understood by those of ordinary skill in the art to which the present invention belongs. In addition, terms defined in a commonly used dictionary are not interpreted ideally or excessively unless explicitly defined specifically.

Hereinafter, a preferred embodiment according to the present invention will be described in detail with reference to the accompanying drawings.

1 shows an electrochemical blood glucose sensor 100 and a blood glucose meter 200 according to an embodiment of the present invention. In addition, Figure 2 shows the configuration of the electrochemical blood glucose sensor 100 according to an embodiment of the present invention. That is, Figure 2 (a) briefly shows the outside of the electrochemical blood glucose sensor 100 according to an embodiment of the present invention, Figure 2 (b) is an electrochemical blood glucose sensor 100 according to an embodiment of the present invention. Briefly show the inside of.

The electrochemical blood sugar sensor 100 according to an embodiment of the present invention is a sensor that measures blood sugar using an electrochemical method, and may be variously referred to as a strip, a test strip, etc., and may be used for disposable or continuous blood sugar measurement. I can. The electrochemical blood glucose sensor 100 according to an embodiment of the present invention may be configured to be inserted into the insertion part 210 of the blood glucose meter 200 as shown in FIG. 1.

At this time, the blood glucose meter 200 may measure blood glucose according to an electrochemical method by contacting various electrodes 10, 20, 30 of the blood glucose sensor 100 inserted in the insertion part 210, and the result It can be displayed through the display unit 220. Such a blood glucose meter 200 may be of various types, and may be a continuous blood glucose meter.

Referring to FIG. 2, the electrochemical blood glucose sensor 100 according to an embodiment of the present invention includes a working electrode 10, a counter electrode 20, and a reference electrode, which are various electrodes. ) 30, and a blood input unit 40, which is a part into which blood is introduced, may be included. At this time, each of the electrodes 10, 20, 30 may be disposed to be spaced apart from each other. However, each of the electrodes 10, 20, and 30 is not limited to the shape shown in FIG. 2 or the like, and may have various shapes. In particular, as shown in FIG. 2, the counter electrode 20 may have a larger area than the working electrode 10.

The working electrode 10 is an electrode in which an electrochemical phenomenon occurs due to blood contact. That is, the working electrode 10 includes a blood contact portion 11 (indicated by a checkered pattern in Fig. 2(b)) in contact with blood. However, unlike FIG. 2, in addition to the working electrode 10, the counter electrode 20 and the reference electrode 30 may also include a blood contact part.

3 shows various examples of a cross section cut along the dotted line shown in the blood contact portion 11 of the working electrode 10 in FIG. 2(b).

Referring to FIG. 3, the blood contact part 11 may include a structure in which a metal substrate 12, a reaction layer 13, and a fixed layer 14 are sequentially stacked.

Specifically, the metal substrate 12 is made of a conductive metal material, but includes stainless steel. The effect of stainless steel will be described later.

The reaction layer 13 is a layer in which a chemical reaction occurs by having a glucose oxidase that reacts with blood sugar, and is provided on a metal substrate. In particular, the reaction layer 13 includes, in addition to glucose oxidase, a first immobilizing material that binds to glucose oxidase to immobilize glucose oxidase. In this case, the first immobilizing material is a material that primarily immobilizes glucose oxidase, and may be preferably a material that cross-links with glucose oxidase. For example, the first fixing material may include chitosan, but is not limited thereto.

The fixing layer 14 is a layer including a second fixing material that further fixes the glucose oxidase of the reaction layer 13 and is provided on the reaction layer 13 to cover the reaction layer 13. In this case, the pinned layer 14 may correspond to the uppermost layer in the blood contact portion 11. In particular, the second immobilizing material is a material that secondaryly immobilizes glucose oxidase, and may be preferably a material having a predetermined adhesiveness. For example, the second fixing material may include Nafion, but is not limited thereto.

That is, in the present invention, in addition to the primary fixing by cross-linking of the first fixing material of the reaction layer 13, the structure in which the secondary fixing by the second fixing material of the fixing layer 14 covering the reaction layer 13 is additionally possible. By having a, the degree of fixation to glucose oxidase can be greatly improved.

4 shows an electrochemical phenomenon occurring in the blood contact portion 11 of the working electrode 10.

On the other hand, referring to FIG. 4, when blood flows through the blood input unit 40, the introduced blood is positioned on the blood contact portions of the working electrode 10, the counter electrode 20, and the reference electrode 30. , Electrochemical phenomenon occurs. _{At this time, in the electrochemical phenomenon, hydrogen peroxide (H 2} O ₂ ) is generated by a reaction between glucose oxidase in the reaction layer 13 and glucose in the blood, and the generated hydrogen peroxide (H ₂ O ₂ ) is supplied from the metal substrate 12. which ions, that is, e (2e ^-) refers to a phenomenon in which the reaction. That is, the glucose of the blood introduced through the blood input unit 40 penetrates the reaction layer 13 through the fixed layer 14 and reacts with the glucose oxidase of the reaction layer 13, and the hydrogen peroxide generated as a result of the reaction ( H ₂ O ₂ ) reacts with the ions of the metal substrate 12.

At this time, the second fixing material of the pinned layer 14 allows glucose to penetrate, but it is possible to prevent the penetration of one type of interfering material among the interfering materials or reduce the penetration rate of the interfering material. In this case, the interfering substance refers to a substance that affects the electrochemical detection of hydrogen peroxide. For example, the interfering substance may be ascorbic acid, acetaminophen, or the like.

The counter electrode 20 is an electrode provided for measuring a current generated according to an electrochemical phenomenon, and includes a conductive material. That is, in the case where the electrochemical blood glucose sensor 100 according to an embodiment of the present invention is inserted into the blood glucose meter 200, the current generated by the electrochemical phenomenon in the working electrode 10 is applied to the working electrode 10. ) And the counter electrode 20. In this case, the measured current may have a value proportional to the glucose concentration of blood injected through the blood input unit 40. However, since such a current measurement principle is well known in the art, a more detailed description thereof will be omitted.

The reference electrode 30 is an electrode provided for applying a voltage to the working electrode 10 and includes a conductive material. That is, when the electrochemical blood glucose sensor 100 according to an embodiment of the present invention is inserted into the blood glucose meter 200, the voltage between the working electrode 10 and the reference electrode 30 by the blood glucose meter 200 Is applied. Electric charges may be supplied to the metal substrate 12 of the working electrode 10 by this applied voltage. The ions of the above-described metal substrate 12 reacting with hydrogen peroxide (H ₂ O _{2) may be continuously supplied by the supplied electric charge.} To this end, a voltage of a certain amount is applied between the metal substrate 12 of the working electrode 10 and the reference electrode 30, but a lower voltage is applied to the metal substrate 12, and a higher voltage is applied to the reference electrode 30. It may be desirable for a voltage to be applied.

In this case, the magnitude (absolute value) of the applied voltage may be preferably 0.07 V to 0.13 V, but is not limited thereto. However, the above-described range of the applied voltage may correspond to a range capable of reducing the error range of the current measurement signal, which increases as the glucose concentration increases, and this will be described in more detail through an experiment to be described later.

In addition, although not shown in detail in the drawings, the electrochemical blood glucose sensor 100 according to an embodiment of the present invention is a substrate supporting each of the electrodes 10, 20, 30, etc., in addition to the above-described configuration, and has a pointed one end. It may further include a needle (needle) to allow blood collection by piercing the stratum corneum of the skin. In this case, a plurality of needles may be provided, and the diameter of the tip may be formed in a micrometer range to enable painless blood collection, but is not limited thereto. In addition, the working electrode 10 may have a needle-shaped portion to perform the function of a needle. In this case, it may be preferable that the blood contact portion 11 is formed on the needle-shaped portion of the working electrode 10.

Hereinafter, a method of manufacturing the electrochemical blood glucose sensor 100 according to an embodiment of the present invention will be described. However, among the configurations of the electrochemical blood glucose sensor 100 according to an embodiment of the present invention, since the configuration of the working electrode 10 has the greatest difference from the prior art, hereinafter, only the manufacturing method of the working electrode 10 is described. It will be described in more detail. That is, the remaining components other than the working electrode 10 can be manufactured using a conventional technique.

5 shows a manufacturing process sequence of the working electrode 10 of the electrochemical blood glucose sensor 100 according to an embodiment of the present invention.

5, in order to manufacture the working electrode 10 of the electrochemical blood glucose sensor 100 according to an embodiment of the present invention, S10 to S30 may be performed.

That is, a metal substrate 12 including stainless steel is prepared (S10). At this time, after washing the metal substrate 12 with ethanol or the like, drying at high temperature for a certain period of time (eg, drying at 75° C. or higher for 1 hour or more) may be performed.

Thereafter, the reaction layer 13 is formed on the metal substrate 12 (S20). At this time, a first solution in which the first fixing material and glucose oxidase are mixed is first prepared. Next, after soaking (soaking) the blood contact part 11 of the metal substrate 12 in the prepared first solution for a certain time (for example, immersion for 30 minutes or more), the metal substrate 12 is dried for a certain time. The reaction layer 13 can be formed by repeating the process of (for example, drying at room temperature for 1 hour or more) 3 times. However, in order to form the reaction layer 13 on the blood contact 11, after coating the region of the metal substrate 12 excluding the blood contact 11, the above-described immersion and drying process will be performed. I can.

In particular, the first solution may be preferably a solution in which glucose oxidase is mixed with a solution containing a first fixing material having an amount of 0.12% (v/v) to 0.13% (v/v), but is not limited thereto. . However, the above-described content range of the first fixed material may correspond to a range capable of reducing the error range of the current measurement signal, which increases as the glucose concentration increases, and this will be described in more detail through an experiment to be described later.

Thereafter, a fixed layer 14 covering the reaction layer 13 is formed (S30). At this time, a second solution containing a second fixing material is prepared. Next, after immersing the portion of the metal substrate 12 on which the reaction layer 13 is formed in the prepared second solution for a certain period of time (for example, immersion for 30 minutes or more), the metal substrate 12 is dried for a certain period of time. By (for example, drying at room temperature for 1 hour or more), the fixed layer 14 can be formed. Next, when the area of the metal substrate 12 other than the blood contact part 11 is coated in S20, the coating on at least a part of the area may be removed.

In particular, the second solution may be preferably a solution containing the second fixing material in an amount of 1.4% (v/v) to 1.6% (v/v), but is not limited thereto. However, the range of the second fixing material described above may correspond to a range capable of reducing the error range of the current measurement signal, which increases as the glucose concentration increases, and this will be described in more detail through an experiment to be described later.

Meanwhile, since the metal substrate 12, the reaction layer 13, the fixed layer 14, and the like have already been described above with reference to FIGS. 1 to 4, detailed descriptions thereof will be omitted.

Hereinafter, an experiment performed to analyze the performance of the electrochemical blood glucose sensor 100 according to an embodiment of the present invention will be described.

23 shows an experimental apparatus used for an experiment of performance analysis of the electrochemical blood glucose sensor 100 according to an embodiment of the present invention.

For the convenience of the experiment, the working electrode WE used a needle shape, not a rod shape, and the counter electrode CE and the reference electrode RE were wire and rod, respectively. Form was used. In addition, as shown in FIG. 23, each experiment was performed by placing each electrode in a glass cell and connecting each electrode to a measuring device using metal tongs connected to a conducting wire. At this time, if necessary, each experiment was performed by putting a solution (blood, etc.) to be tested into a glass cell.

Specifically, in each experiment, a saturated calomel electrode was used as a reference electrode, a platinum wire was used as a counter electrode, and a stainless 304 needle or a processed product thereof was operated according to each experiment. It was used as an electrode.

<Performance comparison experiment between platinum and stainless steel>

This experiment is an experiment for comparing the performance between platinum used as a working electrode in the prior art and stainless steel used as a metal substrate for a working electrode according to the present invention. Specifically, a stainless 304 needle formed of a platinum nanostructure (hereinafter referred to as “first needle”) and a Stainless 304 needle (hereinafter referred to as “second needle”) are prepared respectively.

At this time, the process of preparing the first needle is as follows. In other words, after washing the Stainless 304 needle with ethanol for 1 hour or more using an ultrasonic cleaner, it is dried in an oven at 75° C. for 1 hour. Then, about 3 cm of the end of the Stainless 304 needle is immersed in 3 mL of 10 mM cysteamine solution for 3 hours to form a monolayer of cysteamine on the surface, and then washed with distilled water. Then, a Stainless 304 needle with a monolayer of cysteamine was immersed in a mixed solution of 8 mL of distilled water, 12 μL of chloroplatinic acid, and 1 mL of 0.1 M ascorbic acid, and stirred at 70° C. for 4 hours to form a platinum nanostructure on the surface.

Meanwhile, in this experiment, a 1X PBS (Phosphate Buffer Saline) solution of pH 6.8 containing 0.1 M KCl was used.

-Chronoamperometry experiment-

First, PBS with different concentrations of hydrogen peroxide for the case where the first needle is the working electrode (hereinafter, referred to as “first case”) and the second needle as the working electrode (hereinafter referred to as “second case”). A constant applied voltage (about -0.15 V) was applied from the solution, and the current change over time was measured, that is, a chronoamperometry experiment was performed.

6 shows a graph of chronoamperometry according to the concentration of hydrogen peroxide (0 M, 10 μM, 20 μM, and 30 μM) for each of the first and second cases. That is, FIG. 6(a) is a chronoamperometry graph according to the concentration of hydrogen peroxide (0 M, 10 μM, 20 μM, 30 μM) in the first case, and FIG. 6(b) shows the concentration of hydrogen peroxide (0 M, 10 μM, 20 μM, 30 μM) according to the chronoamperometry graphs are shown, respectively.

Referring to Fig. 6, for each case, an increase in current was observed. This is judged as a current generated when hydrogen peroxide is reduced. However, as shown in FIG. 6(a), in the first case, not only did not the tendency according to the concentration appear, but also the noise was large. Therefore, it can be seen that the working electrode including platinum, such as the first needle, is not suitable as a sensor for hydrogen peroxide.

On the other hand, as shown in FIG. 6(b), in the second case, as the concentration of hydrogen peroxide increases, the current increases and the signal change tends to be constant, as well as very little noise. Therefore, it can be seen that the working electrode including stainless steel, such as the second needle, is very suitable as a sensor for hydrogen peroxide.

In addition, in order to compare the linearity of the current according to the concentration of hydrogen peroxide for each of the first and second cases, a chronoamperometry experiment at a concentration of 0 to 4 mM of hydrogen peroxide in a PBS solution was additionally performed.

7 is a graph of chronoamperometry according to the concentration of hydrogen peroxide (0 M, 0.2 μM, 0.6 μM, 1.2 mM, 2 mM, 3 mM, 4 mM) for each of the first and second cases, and hydrogen peroxide. Shows the graph of current change by concentration of. That is, FIG. 7(a) is a chronoamperometry graph according to the concentration of hydrogen peroxide (0 M, 0.2 μM, 0.6 μM, 1.2 mM, 2 mM, 3 mM, 4 mM) in the first case, and FIG. 7(b) is the second In the case, a chronoamperometry graph according to the concentration of hydrogen peroxide (0 M, 0.2 μM, 0.6 μM, 1.2 mM, 2 mM, 3 mM, 4 mM), FIG. 7(c) is a graph of current change by concentration of hydrogen peroxide in the first case, 7(d) shows graphs of current changes for each concentration of hydrogen peroxide in the second case.

Referring to FIG. 7, the first case shows a high current, but does not show linearity depending on the concentration of hydrogen peroxide, whereas the second case shows a relatively small current, but shows a high reliability of 99.27%, which is obvious according to the hydrogen peroxide concentration. Shows linearity. Therefore, it can be confirmed again that the working electrode including stainless steel, such as the second needle, is very suitable as a sensor for hydrogen peroxide.

-Open Circuit Potential Experiment-

In the second case, an open circuit potential experiment was performed to confirm whether the current change was observed by etching by hydrogen peroxide. That is, when 0.2 mM hydrogen peroxide was added in 200 seconds in a PBS solution, the open circuit potential for each of the first and second cases was measured.

8 shows an open circuit potential graph over time for each of the first and second cases.

As a result of the experiment, when 0.2 mM hydrogen peroxide was added in 200 seconds in a PBS solution, the second case reacted very quickly compared to the first case, resulting in a positive potential value. This indicates that in the second case the metal is oxidized at its working electrode. That is, in the second case, it can be confirmed that current is generated due to electron exchange generated when stainless steel is oxidized by hydrogen peroxide during the corrosion process of the working electrode. Therefore, it was confirmed that hydrogen peroxide can be detected through a change in current as the working electrode including stainless steel is etched by the hydrogen peroxide, like the second needle.

or

However, in the previous chronoamperometry experiment, the current change occurring in the second case is judged to be the result of the reaction when ^{Fe 2+ transfers electrons supplied by negative voltage to hydrogen peroxide, not electron exchange due to corrosion of stainless steel.} .

-Cyclic voltammetry experiment-

In order to examine the electrochemical behavior according to the hydrogen peroxide concentration in the second case, cyclic voltammetry was measured for the case where hydrogen peroxide was not present in the PBS solution and in the case where 3.5 mM and 7 mM hydrogen peroxide were present.

9 shows a cyclic voltammetry graph comparing the presence and absence of hydrogen peroxide for the second case.

As a result of the experiment, it was confirmed that the reduction current increased according to the concentration of hydrogen peroxide in the vicinity of -0.1 V and -0.15 V. That is, when measuring the current by applying an applied voltage in the vicinity of these, it can be expected that the current increases according to the hydrogen peroxide concentration.

<Performance comparison experiment of various working electrodes including stainless steel>

This experiment is an experiment to compare the performance between various working electrodes including stainless steel. Specifically, two needles (hereinafter referred to as "third needle" and "fourth needle" respectively) having a metal base made of stainless steel and having different layers formed on the metal base are prepared respectively. , The fourth needle may correspond to the working electrode according to the present invention.

10 shows a process of preparing a third needle.

Referring to FIG. 10, the process of preparing the third needle is as follows. In other words, after washing the Stainless 304 needle with ethanol for 1 hour or more using an ultrasonic cleaner, it is dried in an oven at 75° C. for 1 hour. Then, a solution obtained by dissolving 3 mg of glucose oxidase in 100 μL of 0.25% (v/v) Nafion aqueous solution is prepared. Then, the end of the Stainless 304 needle is immersed in the solution for about 30 minutes, and then dried at room temperature for 1 hour. As a result, a third needle having a mixed layer of Nafion and glucose oxidase formed on the end of the Stainless 304 needle may be prepared. However, after coating the Stainless 304 needle part outside the region where the mixed layer of Nafion and glucose oxidase is formed, the process of forming the mixed layer (such as immersion and drying) may be performed.

11 shows a process of preparing a fourth needle.

Referring to FIG. 11, the process of preparing the fourth needle is as follows. In other words, after washing the Stainless 304 needle with ethanol for 1 hour or more using an ultrasonic cleaner, it is dried in an oven at 75° C. for 1 hour. Then, a solution in which 3 mg of glucose oxidase is dissolved in 200 μL of 0.125% (v/v) chitosan aqueous solution, that is, a first solution, is prepared. Then, the end of the Stainless 304 needle is immersed in the first solution for about 30 minutes, and then dried at room temperature for 1 hour. The process of immersing and drying the chitosan aqueous solution is repeated two more times. As a result, a reaction layer, which is a mixed layer of chitosan and glucose oxidase, is formed on the end of the Stainless 304 needle. Then, a 1.5% (v/v) Nafion aqueous solution, that is, a second solution is prepared. Then, the end of the needle having the reaction layer is immersed in the second solution for about 30 minutes, and then dried at room temperature for 1 hour. As a result, a reaction layer on the end of the stainless 304 needle, and a fourth needle having a fixed layer including Nafion covering the reaction layer may be prepared. However, after coating the stainless 304 needle portion outside the region in which the reaction layer and the fixing layer are formed, the reaction layer and the fixing layer may be formed.

Meanwhile, in this experiment, a serum solution (actual blood environment) in which 1 mL of human serum was mixed with 9 mL of a 1X PBS solution of pH 6.8 containing 0.1 M KCl was used.

-Chronoamperometry experiment-

First, serum in which the concentration of glucose is different for the case where the third needle is the working electrode (hereinafter referred to as “third case”) and the fourth needle is the working electrode (hereinafter referred to as “the fourth case”). A constant applied voltage (about -0.15 V) was applied from the solution, and the current change over time was measured, that is, a chronoamperometry experiment was performed.

12 and 13 are graphs of chronoamperometry according to the concentration of glucose (0 M, 1 mM, 3 mM, 6 mM, 10 mM, 15 mM, 20 mM) for each of the third and fourth cases. , Shows a graph of current change by glucose concentration. That is, FIG. 12(a) is a chronoamperometry graph according to the concentration of glucose (0 M, 1 mM, 3 mM, 6 mM, 10 mM, 15 mM, 20 mM) in the third case, and FIG. 12(b) is the third case. In the graph of current change by glucose concentration, FIG. 13(a) is a chronoamperometry graph according to the glucose concentration (0 M, 1 mM, 3 mM, 6 mM, 10 mM, 15 mM, 20 mM) in the fourth case, FIG. 13 (b) shows graphs of current change by glucose concentration in the fourth case, respectively.

That is, the change of current according to the glucose concentration in the third and fourth cases was observed by using that glucose is oxidized by glucose oxidase in the presence of oxygen and hydrogen peroxide is generated. Referring to FIGS. 12 and 13, in both the third and fourth cases, the current constantly increased with a high reliability of 99.5% or more as the glucose concentration increased within the glucose concentration range of 1 to 20 mM, and the noise was also very low. appear. However, in the third case, the coefficient of variation is in the range of 0.1 to 0.84%, and in the fourth case, the coefficient of variation is in the range of 0.063 to 0.71%. That is, the fourth case showed less current fluctuation than the third case. Therefore, when the third needle is the working electrode and the fourth needle is the working electrode, it is suitable as a glucose detection sensor in an actual blood environment, but considering the current fluctuation, the fourth needle is a glucose detection sensor as in the present invention. You can see that it is suitable.

-Interfering substance effect comparison experiment-

First, the average lowest concentration, average concentration, and average highest concentration in blood of Ascorbic acid and Acetaminophen, which are abundantly present in the blood and are well known as interfering substances for hydrogen peroxide, were confirmed, respectively. As a result, the concentrations of the interfering substances actually present in the blood are shown in Table 1.

Type of interfering substance
(Interfering species) Concentration [mM] Reference Ieast Average maximum glucose
(Glucose) 7 The minimum value for diabetics on an empty stomach Ascorbic acid
(Ascorbic acid) 0.15 0.25 0.035 Mean value of human serum Acetaminophen
(Acetaminophen) 0.06 0.13 0.2 Mean value of human serum

Next, for each of the third and fourth cases, it was attempted to confirm the selectivity for glucose by checking the effects of these interfering substances present in the blood. That is, by increasing the concentrations of these interfering substances (Ascorbic acid and Acetaminophen), we tried to find out whether these interfering substances act as an interfering factor in the detection of 7 mM glucose, the lowest blood glucose in fasting diabetic patients. Specifically, the concentration of these interfering substances in the serum solution is gradually increased by increasing the concentration of the interfering substances in the blood with the average minimum concentration, average concentration, average maximum concentration, and twice the average maximum concentration (2x Maximum). The current was measured while increasing. 14 and 15 show graphs of changes in current according to a change in concentration of an interfering substance at different constant applied voltages for each of the third and fourth cases. That is, FIG. 14 shows a case of an applied voltage of -0.15 V in the third case, and FIG. 15 shows a case of an applied voltage of -0.1 V in the fourth case, respectively.

As a result, as shown in FIGS. 14 and 15, in both the third case and the fourth case, under the condition that glucose was not included, it was found that the current change according to the concentration of the interfering substance was small, but the fourth case was the first. It was found that the current change was less than that of the 3 case. In addition, in the condition including glucose, the current gradually increased according to the concentration of the interfering substance in the third case, but the current change according to the concentration of the interfering substance was small in the fourth case. In this case, the third and fourth cases have applied voltages of -0.15 V and -0.1 V, respectively. Accordingly, it can be seen that a case where the fourth needle is the working electrode and the applied voltage is -0.1 V is suitable as a glucose detection sensor having the best glucose selectivity.

<Performance comparison experiment of the working electrode according to the concentration of the first fixed substance or the second fixed substance>

This experiment is an experiment for comparing the performance of each working electrode, which is a working electrode including stainless steel according to the present invention, but manufactured with different concentrations of the first fixed material or the second fixed material. Specifically, in addition to the fourth needle, four needles (hereinafter, “the fifth needle”, “the sixth needle”, “the seventh needle” and (Referred to as “the eighth needle” respectively.) In addition, one needle (hereinafter referred to as “the ninth needle”), which is the working electrode according to the present invention manufactured by varying the concentration of the second fixing material, is prepared. Prepare each.

At this time, the preparation process of the fifth needle to the ninth needle is the same as the preparation process of the fourth needle described above, except that only the chitosan concentration of the first solution or the nipion concentration of the second solution is different. That is, the preparation process of the fifth needle to the eighth needle differs only in the chitosan concentration of the first solution in the preparation process of the fourth needle described above. That is, the chitosan concentration is 0.05% (v/v), 0.075% (v/v), 0.1% (v/v), and 0.15% (v/v), respectively. In addition, the preparation process of the ninth needle differs only in the nifion concentration of the second solution in the preparation process of the fourth needle described above. That is, the nifion concentration is 0.25% (v/v).

First, when the fifth needle is a working electrode (hereinafter, referred to as “the fifth case”), the sixth needle is a working electrode (hereinafter, referred to as “the sixth case”), and when the seventh needle is a working electrode (hereinafter, referred to as “the sixth case”) "Case 7"), the case where the 8th needle is the working electrode (hereinafter referred to as the "Case 8"), the case where the ninth needle is the working electrode (hereinafter referred to as "the ninth case") and the fourth case For each, it was attempted to confirm the selectivity to glucose by checking the effects of the interfering substances (Ascorbic acid and Acetaminophen) present in the blood. That is, by increasing the concentration of these interfering substances, we tried to find out whether these interfering substances act as an interfering factor in the detection of 7 mM glucose, the lowest blood glucose in fasting diabetic patients. Specifically, the concentration of these interfering substances in the serum solution is gradually increased by increasing the concentration of the interfering substances in the blood with the average minimum concentration, average concentration, average maximum concentration, and twice the average maximum concentration (2x Maximum). The current was measured while increasing.

16 to 20 show graphs of changes in current according to a change in concentration of an interfering substance at a constant applied voltage for each of the fifth to seventh cases, the fourth case, and the eighth case. That is, FIG. 16 shows a current change graph in a fifth case, FIG. 17 in a sixth case, FIG. 18 in a seventh case, FIG. 19 in a fourth case, and FIG. 20 in an eighth case.

In addition, FIGS. 21 and 22 show graphs of changes in current according to a change in concentration of an interfering substance at a constant applied voltage for each of the ninth and fourth cases. That is, FIG. 21 shows current change graphs in the ninth case and FIG. 22 in the fourth case, respectively.

As a result, as shown in FIGS. 16 to 20, it was found that the current change was the least in the fourth case prepared using the first solution having a chitosan concentration of 0.125% (v/v). In addition, as shown in Figs. 21 and 22, the fourth case prepared using the first solution having a Nafion concentration of 1.5% (v/v) showed less current change than the ninth case. Therefore, it can be seen that the case where the fourth needle is the working electrode is suitable as a glucose detection sensor having better glucose selectivity than the case where the fifth to ninth needles are working electrodes.

As shown through an experiment on the configuration and effects of the present invention described above, the present invention not only has less noise of the current signal itself, that is, the signal-to-noise ratio (SNR) is greater than that of the current signal according to the glucose concentration, There is an advantage of reducing the error range of the current signal, which increases as the glucose concentration increases. In addition, the present invention can reduce the manufacturing cost by using stainless steel, which is an inexpensive metal, as an electrode, reduce the influence on the concentration of the interfering substance, and increase the selectivity of the substance to be measured, thereby more effectively detecting glucose. There is this.

Although specific embodiments have been described in the detailed description of the present invention, various modifications are possible without departing from the scope of the present invention. Therefore, the scope of the present invention is not limited to the described embodiments, and should be defined by the claims to be described later and equivalents to the claims.

10: working electrode 11: blood contact
20: counter electrode 30: reference electrode
100: blood glucose sensor 200: blood meter
210: insertion unit 220: display unit

Claims (16)

Including a working electrode (working electrode) in contact with blood to generate an electrochemical phenomenon,
The working electrode,
A metal substrate including stainless steel;
A reaction layer each comprising a glucose oxidase and a first immobilizing material for immobilizing glucose oxidase by cross-linking with glucose oxidase, and provided on a metal substrate; And
A fixing layer including a second fixing material for additionally fixing glucose oxidase in the reaction layer and covering the reaction layer;
Electrochemical blood sugar sensor comprising a. The method of claim 1,
The reaction layer and the fixed layer are electrochemical blood glucose sensor, characterized in that formed on a portion of the metal substrate in contact with blood. The method of claim 2,
The electrochemical phenomenon is a phenomenon in which hydrogen peroxide generated by a reaction between glucose oxidase in the reaction layer and glucose in the blood reacts with ions supplied from a metal substrate. The method of claim 3,
The second fixing material is an electrochemical blood sugar sensor, characterized in that the penetration of at least one of the interference substances reacting with the hydrogen peroxide in addition to the metal substrate to prevent or reduce the penetration rate of the interference. The method of claim 2,
The working electrode includes a needle-shaped portion, and an electrochemical blood glucose sensor comprising a reaction layer and a fixed layer on the needle-shaped portion. The method of claim 1,
The first fixing material includes chitosan, and the second fixing material includes nafion. The method of claim 1,
A counter electrode provided for measuring a current generated according to an electrochemical phenomenon in the working electrode; And
Further comprising a reference electrode (reference electrode) is a counter electrode provided for voltage application to the working electrode,
Electrochemical blood glucose sensor, characterized in that implemented to be inserted into the blood glucose meter. The method of claim 7,
In the blood glucose meter, a voltage of a predetermined size is applied between the metal substrate and the reference electrode, but a lower voltage is applied to the metal substrate. The method of claim 8,
The predetermined size is an electrochemical blood glucose sensor, characterized in that 0.07 V to 0.13 V. As a method of manufacturing an electrochemical blood glucose sensor comprising a working electrode in contact with blood to generate an electrochemical phenomenon,
A preparation step of preparing a metal substrate that is a first configuration of a working electrode including stainless steel;
The reaction layer, which is the second configuration of the working electrode, each comprising a glucose oxidase and a first immobilizing material for immobilizing glucose oxidase by cross-linking with glucose oxidase, is formed on a metal substrate. A step of forming a reaction layer to form a reaction layer;
A fixed layer forming step of forming a fixed layer, which is a third configuration of the working electrode including a second fixing material for additionally fixing glucose oxidase in the reaction layer, but covering the reaction layer;
Method of manufacturing an electrochemical blood glucose sensor comprising a. The method of claim 10,
The step of forming the reaction layer,
A method of manufacturing an electrochemical blood glucose sensor comprising the step of immersing a portion of a metal substrate in contact with blood in a first solution in which a first fixing material and a glucose oxidase are mixed. The method of claim 11,
The first solution is a method of manufacturing an electrochemical blood glucose sensor, characterized in that the solution containing a first fixing material of 0.12% (v/v) to 0.13% (v/v) is mixed with glucose oxidase. The method of claim 12,
The forming step of forming the fixed layer,
A method of manufacturing an electrochemical blood glucose sensor comprising the step of immersing a portion of the metal substrate on which the reaction layer is formed in a second solution containing a second fixing material. The method of claim 13,
The second solution is a method of manufacturing an electrochemical blood glucose sensor, characterized in that it is a solution containing a second fixing material in an amount of 1.4% (v/v) to 1.6% (v/v). The method according to any one of claims 10 to 14,
The electrochemical blood glucose sensor,
Further comprising a counter electrode provided for measuring the current generated according to the electrochemical phenomenon in the working electrode, and a reference electrode provided for applying a voltage to the working electrode, wherein the blood glucose meter The method of manufacturing an electrochemical blood glucose sensor, characterized in that a voltage of a predetermined size is applied between the metal substrate and the reference electrode in a blood glucose meter and a lower voltage is applied to the metal substrate. The method of claim 15,
The predetermined size is an electrochemical blood glucose sensor, characterized in that 0.07 V to 0.13 V.

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