KR102300186B1 - Two electrode system and biosensor including the same - Google Patents

Abstract

The present application relates to a two-electrode system and a biosensor including the same, and more particularly, to a two-electrode system including only a plurality of working electrodes without including a reference electrode and a counter electrode, and a biosensor including the same .

Description

Two-electrode system and biosensor including same

The present application relates to a two-electrode system and a biosensor including the same, and more particularly, to a two-electrode system including only a plurality of working electrodes without including a reference electrode and a counter electrode, and a biosensor including the same .

An electrochemical sensor is an analytical device that acts as a transducer that converts a signal generated as a result of a specific biological recognition phenomenon into a detectable and measurable electrical signal. These electrochemical sensors have been known as the most commonly used biosensors for measurement and diagnostic tests in clinical analysis, and have overcome most of the disadvantages and problems limiting the use of other types of biosensors.

Compared to most other biosensors, electrochemical biosensors have high sensitivity, specificity for target analytes, fast response time, ability to operate in turbid or colored solutions, ease of use, and the possibility of making low-cost portable devices. It offers several advantages. Moreover, the continuous response of the electrode system enables on-line control and makes it applicable to a wide range of specimens. Based on these advantages, the field of electrochemical biosensors continues to grow at a rapid pace.

In electrochemical analysis, a reference electrode is essential for measuring and controlling the electrochemical potential of a solution. Various types of reference electrodes have been used in microfluidic chip-based electrochemical biosensors. One type is the fabrication of microfluidic thin films such as metals or Ag/AgCl as pseudo-reference electrodes. When a pseudo reference electrode is used as a metal mounted on a substrate by a fine thin film process, the electrode manufacturing procedure is simple, but the electrochemical potential is not fixed and unstable, so it is difficult to apply the intended potential to the working electrode. This is because all electrochemically active substances in solution affect the potential of the reference electrode. In addition, when a similar reference electrode is fabricated and used with an Ag/AgCl film fabricated in a fine process, the electrochemical potential is more stable than in the previous case, but the process is complicated because a chlorination process to make the Ag layer into AgCl is additionally required. Alternatively, Ag/AgCl mixtures may be screen-printed, in which similar screen-printed reference electrodes can be washed out in microfluidics. Therefore, it is very difficult to maintain a constant electrochemical potential for a long period of time. The smaller the pseudo reference electrode is, the more difficult it is to maintain the electrochemical potential. Another type of reference electrode is to place the reference electrode outside the microchannel. Its electrochemical potential is more stable than that of a similar reference electrode, but a significant resistance, i. The counter electrode is problematic in a similar way. When the surface area of the counter electrode is larger than the surface area of the working electrode, the counter electrode can have the intended exact translocation potential. Therefore, integrating the counter electrode into the microfluid is cumbersome.

Patent Document 1: Korean Patent Application No. 10-2011-0129528

According to an embodiment of the present application, an object of the present application is to provide a two-electrode system including only a working electrode without a reference electrode and a counter electrode, and a biosensor including the same.

One aspect of the present application is a glass substrate; a pair of working electrodes formed on the glass substrate and spaced apart from each other by a predetermined interval; a siloxane layer including an amine end group formed on the glass substrate; an antibody layer bound to an amine-terminal group of the siloxane layer by a crosslinking agent; and an organic material layer formed on each of the pair of working electrodes.

Another aspect of the present application is a biosensor including the two-electrode system described above.

Another aspect of the present application comprises the steps of preparing a glass substrate; forming a pair of working electrodes on the glass substrate; treating the glass substrate on which the working electrode is formed with a silane-based compound solution to form an amine-terminated layer on the glass substrate, and heating at 100 to 150° C. for 15 to 45 minutes to form a siloxane layer; forming an organic material layer on the working electrode by electropolymerization; and treating an amine-terminal group of the siloxane layer with a crosslinking agent to attach an antibody.

According to an embodiment of the present application, the potential of the working electrode is determined by fixing a medium material having a fast electron transfer to the working electrode, thereby driving the biosensor with two working electrodes without a separate reference electrode manufacturing process. .

According to an embodiment of the present application, since the electrochemical potential is determined by the fixed organic material layer, the biosensor can be constantly and stably driven.

According to an embodiment of the present application, since there is no reference electrode, it is possible to provide a chip-based electrochemical biosensor that is simple to manufacture and portable.

According to an embodiment of the present application, the sensor can be operated simply by using a battery, and thus the sensor can be used while carrying it, thereby greatly improving the convenience of the biosensor user.

According to an embodiment of the present application, by fabricating a plurality of working electrode pairs on one substrate, the concentration of several detection target substances in a group represented by CK-MB, cTnI, myoglobin, etc. can be simultaneously measured with only one sample injection. can

1 is a schematic diagram of a conventional four-electrode system.
2 is a schematic diagram of a two-electrode system according to an embodiment of the present application.
3 is a schematic diagram of a two-electrode system according to an embodiment of the present application.
4 is a schematic diagram of a two-electrode system according to an embodiment of the present application.
5 is a view for explaining the mechanism of the two-electrode system according to an embodiment of the present application.
6 is an image of a three-dimensional two-electrode system according to an embodiment of the present application.
7 is a schematic diagram of a three-dimensional two-electrode system according to an embodiment of the present application.
8 is a schematic diagram of a three-dimensional two-electrode system according to an embodiment of the present application.
9 is a result graph for Experimental Example 1.
10 is a graph showing the result of measuring the current with respect to the DA concentration for an embodiment of the present application.
11 is a cyclic voltammetry graph for an embodiment of the present application.
12 is a cyclic voltammetry graph for an embodiment of the present application.
13 is a graph of the amount of charge versus time for each concentration of CK-MB and a graph of the amount of charge versus the concentration of CK-MB for an embodiment of the present application.

The terms used in the present application are only used to describe specific embodiments, and are not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly dictates otherwise. In the present application, terms such as “comprise” or “have” are intended to designate that the features, components, etc. described in the specification are present, and one or more other features or components may not be present or may be added. Doesn't mean there isn't.

Unless defined otherwise, all terms used herein, including technical and scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not

Hereinafter, the two-electrode system of the present application will be described in detail with reference to the accompanying drawings. However, the accompanying drawings are exemplary, and the two-electrode system of the present application is not limited by the accompanying drawings.

1 is a schematic diagram of a conventional four-electrode system, and FIG. 2 is a schematic diagram of a two-electrode system according to an embodiment of the present application. As shown in FIG. 1, the conventional two-electrode system of the present application includes the working electrodes WE1 and WE2, the counter electrode CE, and the reference electrode RE, whereas the working electrode as shown in FIG. It has a distinction that includes only (WE1, WE2).

As shown in FIG. 3 , the two-electrode system of the present application forms an ITO electrode on a glass substrate, includes a siloxane layer to form an amine end group on the glass substrate, and uses a crosslinking agent to bind the antibody layer And, an organic material layer is formed on the ITO electrode, and the organic material layer on the ITO electrode serves as a working electrode, a counter electrode, and a reference electrode, and the electrochemical potential can be constantly maintained with two working electrodes without a reference electrode.

Hereinafter, the two-electrode system will be described in more detail with reference to FIG. 4 .

As shown in FIG. 4 , the two-electrode system 1 includes a glass substrate 11 . Specific components and thickness of the glass substrate are not particularly limited, and any glass substrate may be applied as long as it can be applied in the technical field to which the present application belongs.

The two-electrode system 10 includes a pair of working electrodes 12 formed on a glass substrate 11 and spaced apart from each other at predetermined intervals.

Each of the pair of working electrodes is preferably made of, but not limited to, Indium Tin Oxide (ITO). Through this, the effect of increasing the sensitivity of the sensor may be exhibited.

Preferably, the size of each of the working electrodes is 15 μm or less. Through this magnitude, the effect of amplifying the signal may be exhibited.

It is preferable that the predetermined distance between the working electrodes be 30 μm or less. It may exhibit the effect of amplifying the signal.

A method of forming the working electrode on the glass substrate is not particularly limited, and any method may be applied as long as it can be applied in the technical field to which the present application pertains.

In addition, the two-electrode system 1 includes a siloxane layer 14 including amine end groups formed on the glass substrate 11 .

After cleaning the glass substrate on which the above-described working electrode is formed, 5% N-(6-aminohexyl)aminomethyltriethoxysilane (N-(6-aminohexyl)aminomethyltriethoxysilane, AHAMTES) or aminopropyltriethoxysilane, which is an anhydrous toluene solvent By immersing in a solution of a silane group represented by silane (aminopropyltriethoxysilane) at room temperature for 10 to 30 hours, a layer including an amine-terminated group is formed on a glass substrate. The amine-terminal group-containing layer prevents the antibody, which will be described later, from being attached to and falling off the glass substrate.

Thereafter, the glass substrate is removed from the solution, rinsed with ethanol, sonicated in ethanol for 3 to 7 minutes, and heated on a high-temperature plate at 100° C. to 150° C. for 15 minutes to 45 minutes, the siloxane layer to form The ammonium ion can be changed to a neutral amine to provide a more reactive amine functional group.

In addition, the two-electrode system 1 includes an organic material layer 15 formed on each of the pair of working electrodes 12 .

In the present application, the organic layer is a mediating layer, so that electron transfer between the working electrode and the label represented by AP occurs rapidly, and the potential of the working electrode is determined.

5 is a view for explaining a mechanism of a two-electrode system according to an embodiment of the present application. As shown in FIG. 5 , an organic material layer (M _OX + e- ↔ M _red ) having a fast electron transfer is adsorbed on the two working electrodes. Since the two working electrodes are covered with this organic layer, the redox reaction of this organic layer occurs before other redox-capable materials in solution. And the electrochemical potential of the two working electrodes is determined by the formal potential of the organic material.

The organic material of the organic material layer is a material capable of electropolymerization and electrochemically repeatable redox. Any organic material having these characteristics can be applied to the present application. As an example, but not limited thereto, poly(methylene green) (poly(methylene green), PMG) and poly(toluidine blue) may include at least one selected from the group consisting of (poly(toluidine blue, PTB). This organic layer is redox before other electrochemically active substances in solution.

Hereinafter, although PMG among organic materials will be mainly described, the organic material of the present application is not limited to PMG.

The method of stacking PMG in organic materials is a phosphate buffer solution (pH 7.0) containing 0.1 M KNO3, 0.5 mM methylene green monomer (MG) and 0.1 mM dopamine (DA) by exposing a working electrode to a solution of dopamine (DA). Electropolymerization can be done.

When the electropolymerization method is used, the redox species can be easily polymerized to form a layer, and can be selectively adsorbed on the electrode surface. The thickness of the layer can be easily controlled by controlling the electropolymerization conditions. In addition, various materials can be laminated using an electropolymerization method. In particular, in the case of PMG and PTB, it can serve as an excellent electron transport mediator for 4-aminophenol (AP).

In addition, by mixing the DA solution with the MG solution and then performing electropolymerization together, the PMG on the electrode can be stably fixed. This allows the electropolymerized PDA to impart adhesion to various substrates. Poly(dopamine) (PDA) may also enhance the absorption capacity of conductive polymer layers such as polypyrrole.

However, the PDA may have a similar action to the above-mentioned action on the material for the other organic layer.

In the case of PDA, since it is not an electrically conductive material, electron transfer resistance may increase, so it is preferable to appropriately control the ratio of DA and MG.

Here, in the oxidation reaction of AP, it is preferable that PMG and PDA are included in a ratio of 1: 0.1 to 0.3. More preferably, it is contained in a ratio of 0.5 mM: 0.1 mM. When the concentration of DA is higher than 0.3, the AP oxidation signal is weakened. This is because PDAs inhibit electron transport due to their electrical resistivity. On the other hand, when the concentration of DA is less than 0.1, a sufficient PMG layer is not formed, so that the surface of the working electrode is not sufficiently covered.

The PMG formed by the above-described method may serve as a counter electrode and a reference electrode in a two-electrode system.

In addition, the two-electrode system 1 includes an antibody layer 14 bonded to an amine-terminal group of the siloxane layer 13 by a crosslinking agent.

The antibody of the antibody layer is a material corresponding to a detection target material for a disease to be detected in the two-electrode system. In the present application, once a detection target material is determined, an antibody that can be applied thereto can be selected. Therefore, there is no need to specifically limit the antibody applied to the present application. However, for convenience of explanation, the following antibodies are listed. Illustratively, but not limited thereto, it may include at least one selected from the group consisting of anti CK-MB, anti cTnI and anti myoglobin.

Here, when the aforementioned pair of working electrodes are included in a plurality of pairs, a plurality of detection target materials may be detected. For example, one pair of working electrodes may detect CK-MB, and the other pair of working electrodes may detect cTnI.

Hereinafter, CK-MB (Creatine Kinase-MB) will be mainly described among the antibodies, but the antibody of the present application is not limited to CK-MB.

In addition, the crosslinking agent is not particularly limited, and any crosslinking agent that can be used in the technical field to which the present application belongs may be appropriately used, and among them, glutaraldehyde is preferably used.

The glass substrate on which the organic layer is formed is rinsed with deionized water and then dried with N2 gas. After that, it is treated with a 5% glutaraldehyde solution for 30 minutes to 1.5 hours, and rinsed with a phosphate buffer solution. Incubate for 1 hour in PBS solution containing 50 μg/mL mouse anti-creatine kinase-MB, and wash with phosphate buffer solution. In addition, by exposure to 1% bovine serum albumin solution for 30 hours, excessive active sites were restricted and non-specific binding was inhibited.

As described above, the two-electrode system of the present application may be two-dimensional, or three-dimensional, as will be described later.

6 is an image of a three-dimensional two-electrode system according to an embodiment of the present application, and FIG. 7 is a schematic diagram of a three-dimensional two-electrode system according to an embodiment of the present application. 6 and 7, it includes a lower substrate and an upper substrate, and the lower substrate and the upper substrate are spaced apart from each other by a predetermined distance and may be coupled to face each other. These bonds can be joined by connections in the gallium indium eutectic.

Hereinafter, such a three-dimensional two-electrode system will be described in more detail with reference to FIG. 8 . However, the description of the two-dimensional two-electrode system described above will be omitted in the description of the three-dimensional two-electrode system for clarity of invention.

As shown in FIG. 8 , in the three-dimensional two-electrode system 2 , the lower substrate 20 and the upper substrate 30 are spaced apart at regular intervals and disposed to face each other.

The lower substrate 20 includes a first glass substrate 21; a pair of first working electrodes 22 formed on the first glass substrate 21 and spaced apart from each other by a predetermined interval; a first siloxane layer 23 including an amine end group formed on the first glass substrate 21; a first antibody layer 24 bonded to an amine-terminal group of the siloxane layer 23 by a crosslinking agent; and a first organic material layer 25 formed on each of the pair of first working electrodes 22 .

The upper substrate 30 is spaced apart from the lower substrate 20 at a predetermined distance and faces, a second glass substrate 31; a pair of second working electrodes 32 formed on the second glass substrate 31 and spaced apart from each other by a predetermined interval; a second siloxane layer 33 including an amine end group formed on the second glass substrate 31; a second antibody layer 34 bonded to an amine-terminal group of the siloxane layer 33 by a crosslinking agent; and a lower substrate including a second organic material layer 35 formed on each of the pair of second working electrodes 32 .

Here, the distance between the first working electrode 22 and the second working electrode 33 is 30 μm or less. In addition, one side of the upper substrate and one side of the lower substrate may be connected by a gallium indium eutectic connection part.

The first organic material layer or the second organic material layer is a material capable of electropolymerization and electrochemically repeatable redox, poly(methylene green), PMG) and poly(toluidine blue) (poly(toluidine blue, PTB) may include at least one selected from the group consisting of.

Even in the case of the three-dimensional two-electrode, each of a pair of first working electrodes and a pair of second working electrodes is included in a plurality of pairs to detect a plurality of detection target substances.

Another aspect of the present application is a biosensor including the aforementioned two-electrode system. Any configuration applicable to a biosensor in the technical field to which the present application belongs may be included in the present application.

The detection target material of the biosensor may be selected from the group consisting of CK-MB, cTnI and myoglobin.

Another aspect of the present application is a method of manufacturing a two-electrode system, the method comprising: preparing a glass substrate; forming a pair of working electrodes on the glass substrate; treating the glass substrate on which the working electrode is formed with a silane-based compound solution to form an amine-terminated layer on the glass substrate, and heating at 100 to 150° C. for 15 to 45 minutes to form a siloxane layer; forming an organic material layer on the working electrode by electropolymerization; and treating an amine-terminal group of the siloxane layer with a crosslinking agent to attach the antibody.

Here, as a solution of a silane-based compound or a solution of a compound containing silane, the content or concentration is not particularly limited, and various amounts or concentrations may be applied. Silane-based compounds include, but are not limited to, by way of example, N- (6-aminohexyl) aminomethyltriethoxysilane (N- (6-aminohexyl) aminomethyltriethoxysilane) or aminopropyltriethoxysilane (aminopropyltriethoxysilane) can do.

Here, the working electrode may include indium tin oxide (ITO).

The crosslinking agent is not particularly limited, and any crosslinking agent applicable in the technical field to which the present application belongs may be applied, and glutaraldehyde is preferably applied.

The parts described in the two-electrode system described above will be omitted in the description of the method for manufacturing the two-electrode system for clarity of the invention.

Hereinafter, the present application will be described in more detail through experimental examples.

[ Experimental example ]

1. 3D IDA in the electrode 2 electrode Check the shape of the system

에서 고온의 플레이트에서 가열하여, 실록산층을 형성하였다 (도 4 및 도 8에서 도면 부호 13, 23, 33로 표시). 이 후, 0.1 M KNO3를 함유하는 포스페이트 버퍼 용액(pH 7.0)에 0.5 mM의 MG와 0.1 mM의 DA가 혼합된 용액에 이들을 노출하여, 유기물층을 형성하였다(도 4 및 도 8에서 도면 부호 15, 25, 35로 도시함). 이하, 다른 설명이 없는 한 전술한 방법에 의하여 제작된 2전극 시스템을 사용하였다.After preparing the glass substrate, the ITO electrode was formed in the shape of an interdigitated electrode array (IDA) on the glass substrate, washed, and then immersed in a siloxane solution represented by 5% AHAMTES at room temperature for 24 hours. An end group layer was formed. It was rinsed with ethanol and then sonicated in ethanol for 5 minutes. After this, 120 for 30

was heated on a high-temperature plate at , to form a siloxane layer (indicated by reference numerals 13, 23, and 33 in FIGS. 4 and 8). Thereafter, they were exposed to a solution in which 0.5 mM MG and 0.1 mM DA were mixed in a phosphate buffer solution (pH 7.0) containing 0.1 M KNO 3 to form an organic layer (reference numeral 15 in FIGS. 4 and 8 , 25 and 35). Hereinafter, unless otherwise described, a two-electrode system manufactured by the above method was used.

The working electrode was fabricated in a flat structure and in an IDA structure. The experiment was carried out as follows, and the results are shown in FIG. 9 .

Specifically, after exposing the electrode to a solution in which the ferricyanide redox pair is dissolved, the current density magnitude of the ferricyanide redox reaction was compared through simulation and electrochemical experiments. At this time, the electrochemical experiment was performed with two working electrodes without a reference electrode. As a result, the current density was significantly measured in the working electrode fabricated with the IDA structure. Through this, the effect of amplifying the current signal due to the IDA structure was confirmed. Additionally, it was confirmed that the electrochemical potential of the two working electrodes was determined by the formal potential of ferricyanide when the oxidation-reduction of ferricyanide was observed only with two working electrodes having a flat structure. Similarly, in the case of the working electrode of the IDA structure, it was confirmed that the electrochemical potential of the working electrode was determined by the formal potential of ferricyanide when the electrochemical system was configured with only two working electrodes without a reference electrode.

2. PMG's electropolymerization ( electropolymerization )

In order to stably laminate the PMG by the electropolymerization method, and to confirm the relationship between the PDA and the PMG, the following experiment was performed.

A solution containing 0.5, 0.25, 0.1, 0.01, and 0 mM of DA was prepared in a 0.5 mM MG solution, and electropolymerization was performed with MG in a three-electrode system so that PMG/PDA was adsorbed to the electrode. After putting the PMG/PDA-adsorbed electrode into Tris buffer (indicated by a black circle in FIG. 10) and Tris buffer solution in which AP is dissolved (indicated by a black triangle in FIG. 10), respectively, a cyclic voltammetry diagram was obtained in a three-electrode system. . The value at the time when the observed oxidation current is the largest was extracted and shown in FIG. 10 . Through this experiment, it was confirmed that the DA concentration of 0.1 mM is most suitable. When the concentration of DA exceeds 0.1 mM, DA is electropolymerized to generate PDA, which prevents the redox of PMG because it is not conductive, and when the concentration of DA is less than 0.1 mM, PMG is stably fixed to the ITO electrode. was observed to be insufficient to help.

In addition, after exposing the ITO electrode to a mixed solution of MG and DA, MG and DA were continuously oxidized using cyclic voltammetry, and PMG and PDA were adsorbed on the electrode. A demonstration of this process is shown in Fig. 11 (A). As confirmed in FIG. 11(B), two oxidation peaks are observed in the first cycle, which is caused by two electron reactions appearing as MG becomes PMG. And as confirmed in FIG. 11(C), the second oxidation peak in the second cycle increased more than twice as compared to that observed in the first cycle. This is because the reduced form of PMG formed after the first cycle remains in the electrode, and the current magnitude increases due to the overlap between oxidation of this and the oxidation of MG in solution.

3. with PMG AP redox evaluation of the cycle

Cyclic voltammogram obtained after exposing the working electrode of IDA structure without PMG to Tris buffer solution in which AP is dissolved (Fig. 12(A)), obtained after exposing the IDA working electrode adsorbed by PMG/PDA to Tris buffer The cyclic voltammetry (Fig. 12(B)) and the cyclic voltammetry (Fig. 12(C)) obtained after exposing the IDA working electrode adsorbed by the PMG/PDA to the Tris buffer solution in which the AP was dissolved were compared. As a result, in the absence of PMG/PDA, AP oxidation-reduction is irreversible and it was difficult to determine the formal potential of AP. there was. In addition, the AP formal potential at that time was almost identical to that of the PMG. Through this, it was confirmed that PMG mediates electron transfer between the working electrode and AP. In addition, when an electrochemical system is configured with only two working electrodes to which PMG/PDA is fixed without a reference electrode, it is possible to determine where the electrochemical potential of the working electrode is.

4. 2 electrode Detection of human CK-MB in 3D IDA electrodes in the system

In order to evaluate the performance of the PMG/PDA 3D IDA chip, an immunosensor for detecting CK-MB was manufactured as follows.

The two-electrode system prepared in Experimental Example 1 was used, and an immune sensor was manufactured using the time-charge (quantity) method.

A graph of the amount of charge versus time for each concentration of CK-MB and a graph of the amount of charge versus the concentration of CK-MB are shown in FIG. 13 . PMG/PDA is adsorbed on the IDA working electrode of the first substrate, and the antibody is fixed on the glass surface, exposed to an antigen solution having a concentration of 100 ng/mL and incubated for 1 hour, and then the excess amount is added to the phosphate buffer solution. After rinsing with , antibody incubation. After that, while the labeling enzyme is attached, the antibody that catches the previously attached antibody is incubated. A second substrate is prepared in the same manner. After manufacturing a microfluidic channel by attaching the working electrodes of the first and second substrates to face each other, alkaline phosphatate, a substrate for the enzyme, is injected into the channel. After 10 minutes, apply a constant voltage and read the current level at that time. These immunoassays were also performed at antigen concentrations of 10, 1 ng/mL, 100, 10, and 1 pg/mL. And at least three sensors were manufactured for each concentration, and FIG. 13(A) is an example of them, and FIG. 13(B) shows the average value and deviation for each antigen concentration. Through this, it was confirmed that the 3D IDA on which the PMG/PDA was fixed was sensitively driven as an immune sensor in the two-electrode system.

Through these experiments, it was confirmed that by forming an appropriate media layer on the working electrode, a two-electrode system capable of exhibiting an excellent sensing effect without a reference electrode or a counter electrode and a biosensor including the same could be manufactured. In addition, it was confirmed that the Faraday current signal was amplified in the 3D IDA driven by the two-electrode system. In addition, it was confirmed that this two-electrode system can detect human Ck-MB with very high sensitivity and reliability. In addition, it is not limited to simply CK-MB, but can be applied to various detection materials including proteins or nucleic acids. Since the manufacturing process of such a chip is very simple, it was confirmed that it is suitable for mass production at an appropriate cost.

The above-described Examples and Experimental Examples are only described for the purpose of explaining the present application, but the present application is not limited thereto.

1, 2: Two-electrode system
20: lower substrate
30: upper substrate
11, 21, 31: glass substrate
12, 22, 32: working electrode
13. 23, 33: siloxane layer
14, 24, 34: antibody layer
15, 25, 35: organic layer

Claims (15)

glass substrate;
a pair of working electrodes formed on the glass substrate and spaced apart from each other by a predetermined interval;
a siloxane layer including an amine end group formed on the glass substrate;
an antibody layer bound to an amine-terminal group of the siloxane layer by a crosslinking agent; and
A two-electrode system comprising an organic material layer formed on each of the pair of working electrodes and including a material capable of electrochemically repeatable redox. The method of claim 1,
A size of each of the pair of working electrodes is 15 μm or less, and the predetermined interval is 30 μm or less. The method of claim 1,
wherein the antibody is an antibody corresponding to a detection target material for a disease to be detected in the two-electrode system. The method of claim 1,
The two-electrode system wherein the antibody is at least one selected from the group consisting of anti CK-MB, anti cTnI and anti myoglobin. The method of claim 1,
A two-electrode system in which the siloxane layer including the amine end group and the antibody layer bound to the amine-terminal group of the siloxane layer by a crosslinking agent are further formed on the pair of working electrodes. The method of claim 1,
The organic material of the organic material layer includes at least one selected from the group consisting of poly(methylene green), PMG) and poly(toluidine blue, PTB). The method of claim 1,
A two-electrode system in which the pair of working electrodes are included in a plurality of pairs to detect a plurality of detection target substances. The method of claim 1,
The two-electrode system includes a lower substrate; and an upper substrate spaced apart from the lower substrate at a predetermined interval and disposed facing each other,
The lower substrate is:
a first glass substrate;
a pair of first working electrodes formed on the first glass substrate and spaced apart from each other by a predetermined interval;
a first siloxane layer including an amine end group formed on the first glass substrate;
an antibody layer bound to an amine-terminal group of the siloxane layer by a crosslinking agent; and
a first organic material layer formed on each of the pair of first working electrodes;
The upper substrate is:
a second glass substrate;
a pair of second working electrodes formed on the second glass substrate and spaced apart from each other by a predetermined interval;
a second siloxane layer including an amine end group formed on the substrate;
an antibody layer bound to an amine-terminal group of the siloxane layer by a crosslinking agent; and
An upper substrate including a second organic material layer formed on each of the pair of second working electrodes,
A two-electrode system wherein a distance between the first working electrode and the second working electrode is 30 μm or less. 9. The method of claim 8,
Each of the organic materials of the first organic layer and the second organic layer is an electropolymerizable and electrochemically repeatable redox material, and poly(methylene green) (poly(methylene green), PMG) and poly(toluidine blue) (poly A two-electrode system comprising at least one selected from the group consisting of (toluidine blue, PTB). 9. The method of claim 8,
A two-electrode system in which one side of the working electrode of the upper substrate and one side of the working electrode of the lower substrate are electrically connected through a eutectic process so that the distance between the first working electrode and the second working electrode is 30 μm or less. 9. The method of claim 8,
A two-electrode system in which each of the pair of first working electrodes and the pair of second working electrodes is included in a plurality of pairs to detect a plurality of detection target substances. A biosensor comprising the two-electrode system of any one of claims 1 to 11. 13. The method of claim 12,
The detection target material of the biosensor includes at least one selected from the group consisting of CK-MB, cTnI, and myoglobin. preparing a glass substrate;
forming a pair of working electrodes on the glass substrate;
treating the glass substrate on which the working electrode is formed with a silane-based compound solution to form an amine-terminated layer on the glass substrate, and heating at 100 to 150° C. for 15 to 45 minutes to form a siloxane layer;
forming an organic material layer on the working electrode by electropolymerization; and
and treating an amine-terminal group of the siloxane layer with a crosslinking agent to attach an antibody. 15. The method of claim 14,
A method of manufacturing a two-electrode system wherein a siloxane layer is also formed on the pair of working electrodes by the step of forming the siloxane layer.

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