Electrode for electrochemical sensor, electrochemical sensor comprising same, and method for manufacturing electrochemical sensor

Abstract

The present invention relates to an electrode for an electrochemical sensor and an electrochemical sensor employing the electrode as a working electrode, the electrode comprising a working electrode layer, an enzyme reaction layer disposed on the upper surface of the working electrode layer, and a crosslinking agent layer disposed on the upper surface of the enzyme reaction layer, wherein: the enzyme reaction layer is made of an enzyme reaction layer composition including an enzyme, a mediator, and a first crosslinking agent; the crosslinking agent layer includes a second crosslinking agent; and the enzyme reaction layer composition includes less than 1.5% v / v of the first crosslinking agent. The electrode for an electrochemical sensor, according to the present invention, allows for an electrochemical sensor that exhibits excellent repeatability over multiple measurements.

Description

Electrode for electrochemical sensor, electrochemical sensor including same, and method for manufacturing electrochemical sensor

[0001] The present invention relates to an electrode for an electrochemical sensor used as a biosensor, an electrochemical sensor including the same, and a method for manufacturing an electrochemical sensor.

[0002]

[0003] Technology that detects human pathogens with high sensitivity and specificity is crucial for early diagnosis and treatment. Early diagnosis can significantly reduce the financial burden on patients due to worsening conditions and enhance the effectiveness of treatment.

[0004] For these reasons, diagnostic devices are already widely used, but they suffer from limitations such as slow response times and significant costs, costing the healthcare industry billions of dollars annually. Therefore, the development of cheaper, more efficient, and more reproducible devices is needed.

[0005] A biosensor is a device that analyzes target molecules using a biological recognition system. A bioreceptor is designed to interact with a specific analyte, producing a measurable effect via a transducer. A key requirement for a biosensor is high selectivity for the analyte within a matrix of chemical or biological components. Based on this selectivity, bioreceptors can be categorized into antigens / antibodies, enzymes, nucleic acids / DNA, and cellular structures.

[0006] Among these, biosensors utilizing enzyme interactions utilize the specific binding and catalytic capabilities of enzymes. Specifically, they recognize the measured substance through mechanisms such as the enzyme's ability to convert the analyte into a substance detectable by the sensor, the analyte's ability to inhibit or activate the enzyme's function, or the enzyme's ability to change its properties upon interaction with the analyte.

[0007] In general, enzyme-based biosensors that measure lactate, glucose, etc. using blood are targeted for one-time measurements, but in order to evaluate exercise performance during physical activity, continuous and continuous monitoring of lactate in the human body, which changes in real time according to muscle fatigue, is required.

[0008] Korean Patent Publication No. 10-1624769 discloses a sensor that measures lactic acid concentration with high precision in a short period of time. The biosensor comprises an electrode system and a reagent layer sequentially laminated on a substrate, and the reagent layer contains lactate oxidase, a mediator, and N-(2-acetamide)-2-aminoethanesulfonic acid. However, the sensitivity range of lactic acid is low, making measurement using body fluids other than blood or plasma, such as sweat, impossible. In addition, it is practically difficult to collect blood multiple times to continuously monitor the target substance, making continuous measurement difficult using the above method. In addition, the continuous measurement sensor has a problem in that the deviation of the result value is large for each measurement, making repeated measurements difficult.

[0009] Furthermore, Korean Patent Publication No. 10-0814193 discloses a method for manufacturing a biosensor containing a layered inorganic compound. However, this method has the problem that the sensor is affected by humidity due to hydrogen bonding with external water molecules, thereby reducing the reliability of the electrical signal. Furthermore, to accurately measure the concentration of a target substance, linearity of the electrical signal according to the concentration of the target substance must be secured.

[0010] Therefore, there is a need to develop a non-invasive electrochemical sensor that has a small deviation in the measurement results, enables continuous body fluid diagnosis, has excellent linearity of the electrical signal according to the concentration of the target substance, and has reduced humidity influence.

[0011]

[0012] The present invention aims to solve the above-described problems by providing an electrode for an electrochemical sensor with reduced humidity sensitivity and improved repeatability. Furthermore, the present invention aims to provide an electrochemical sensor with improved linearity of an electrical signal depending on the concentration of a target substance.

[0013] In addition, the present invention aims to provide a high-precision continuous measurement electrochemical sensor and a method for manufacturing the electrochemical sensor by applying the electrode for the electrochemical sensor to a working electrode.

[0014] However, the problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0015]

[0016] To solve the above problem,

[0017] The present invention provides an electrode for an electrochemical sensor, comprising: a working electrode layer; an enzyme reaction layer disposed on an upper surface of the working electrode layer; and a crosslinking agent layer disposed on an upper surface of the enzyme reaction layer, wherein the enzyme reaction layer is formed of an enzyme reaction layer composition comprising an enzyme, a mediator, and a first crosslinking agent, the crosslinking agent layer comprises a second crosslinking agent, and the enzyme reaction layer composition comprises less than 1.5% v / v of the first crosslinking agent.

[0018] In the present invention, the enzyme may include at least one selected from the group consisting of oxidase and dehydrogenase.

[0019] In the present invention, the enzyme may include lactate oxidase.

[0020] In the present invention, the mediator may include at least one selected from the group consisting of a ruthenium compound and phenazine methosulfate and derivatives thereof.

[0021] In the present invention, the first cross-linking agent may include (3-aminopropyl)triethoxysilane.

[0022] In the present invention, the second cross-linking agent may include glutaraldehyde.

[0023] In the present invention, the electrode for the electrochemical sensor may be washed with a buffer solution containing phosphate buffered saline.

[0024] In the present invention, the electrode for the electrochemical sensor may be washed with a buffer solution containing phosphate buffered saline for 3 minutes or more.

[0025] In addition, the present invention provides an electrochemical sensor including: a substrate; a working electrode disposed on an upper surface of the substrate; and a reference electrode disposed on an upper surface of the working electrode and spaced apart from the working electrode, wherein the working electrode is any one of the electrodes for the electrochemical sensor.

[0026] The present invention may further include an electrode protection member formed on the upper surface of the substrate and positioned spaced apart from the working electrode and the reference electrode.

[0027] The present invention may further include at least one of a metal electrode layer and a wiring section on the upper side of the substrate of the electrochemical sensor.

[0028]

[0029] In the present invention, the distance between the working electrode and the electrode protection part may be 50 μm to 200 μm.

[0030] The present invention may further include an inlet portion and an exhaust portion in the electrochemical sensor.

[0031]

[0032] In addition, the present invention provides a method for manufacturing an electrochemical sensor, comprising the steps of: (a) forming an electrode portion including a working electrode and a reference electrode on a substrate; (b) forming an enzyme reaction layer on at least a portion of the working electrode; (c) forming a cross-linking agent layer on the enzyme reaction layer; and (d) dropping a buffer solution on the cross-linking agent layer, wherein the enzyme reaction layer is formed of an enzyme reaction layer composition including an enzyme, a mediator, and a first cross-linking agent, the cross-linking agent layer includes a second cross-linking agent, and the enzyme reaction layer composition includes less than 1.5% v / v of the first cross-linking agent.

[0033] In the method for manufacturing an electrochemical sensor of the present invention, the buffer solution may include phosphate buffered saline.

[0034] The present invention may further include a step (e) of forming an electrode protection portion on the upper surface of the substrate, spaced apart from the working electrode and the reference electrode.

[0035] In the present invention, the step (a) may be characterized in that it is performed by any one selected from the group consisting of screen printing, letterpress printing, intaglio printing, flatbed printing, and photolithography.

[0036] In the present invention, the steps (b) and (c) may be characterized in that they are performed by any one selected from the group consisting of flow coating, inkjet, and drop casting.

[0037] In addition, the present invention provides an electrochemical sensor manufactured by any one of the above electrochemical sensor manufacturing methods.

[0038]

[0039] The electrode for an electrochemical sensor according to the present invention can increase the crosslinking rate of an enzyme and reduce the influence of humidity on the electrochemical sensor by combining two types of crosslinking agents and controlling the concentration of the crosslinking agents within a specific range. Furthermore, by removing uncrosslinked enzyme and externally exposed mediators through a cleaning process, when the electrode for an electrochemical sensor is applied as a working electrode, an electrochemical sensor with improved repeatability can be provided.

[0040] Therefore, the electrochemical sensor according to the present invention can be usefully applied to a non-invasive biosensor because it is capable of continuous measurement of a detection target substance using a detection target sample such as sweat.

[0041] In addition, when the electrochemical sensor according to the present invention includes an electrode protection part that is positioned spaced apart from the working electrode and the reference electrode on the upper surface of the substrate, the linearity of the electrical signal according to the concentration of the target substance can be improved.

[0042]

[0043] Figure 1 illustrates a laminated structure of an electrode for an electrochemical sensor, i.e., a working electrode, according to one embodiment of the present invention.

[0044] Figure 2 illustrates a laminated structure of an electrochemical sensor according to one embodiment of the present invention.

[0045] Figure 3 is a plan view schematically illustrating an electrochemical sensor according to one embodiment of the present invention.

[0046] Figure 4 is a graph showing the current value of an electrochemical sensor according to the concentration of lactate in the electrochemical sensors of Examples 1, 2 and Comparative Example 1.

[0047] Figures 5a to 5c are graphs showing the current values ​​of the electrochemical sensor repeatedly measured according to the concentration of lactate in the electrochemical sensors of Examples 1 and 2 and Comparative Example 1, respectively.

[0048] Figure 6 is a graph measuring the current value of an electrochemical sensor depending on whether the cleaning process of Examples 1 and 3 was performed.

[0049] Figures 7a and 7b are comparative evaluations of whether or not an appearance defect occurs in an electrochemical sensor by changing the stacking order of the first cross-linking agent and the second cross-linking agent.

[0050] Figure 8 is a front view showing a laminated structure of an electrochemical sensor including an electrode protection unit.

[0051] Figure 9a is a side view illustrating a laminated structure of an electrochemical sensor including an electrode protection unit.

[0052] FIG. 9b is a side view illustrating a laminated structure of an electrochemical sensor including an electrode protection unit according to another embodiment of the present invention.

[0053] Figure 10a is a plan view illustrating a laminated structure of an electrochemical sensor including an electrode protection unit.

[0054] FIG. 10b is a plan view illustrating a laminated structure of an electrochemical sensor including an electrode protection unit according to another embodiment of the present invention.

[0055] Figure 11 is a graph showing the current value of an electrochemical sensor measured according to the concentration of lactate in Example 1 and Examples 4 to 6.

[0056] Figure 12 is a graph showing the current value of an electrochemical sensor measured according to the concentration of lactate in Example 5 and Examples 7 to 9.

[0057]

[0058] What each symbol represents is as follows:

[0059] 100: Electrochemical sensor

[0060] 110: Substrate

[0061] 110-1: Metal electrode layer

[0062] 120: Working electrode

[0063] 120-1: Working electrode layer

[0064] 120-2: Enzyme reaction layer

[0065] 120-3: Crosslinking layer

[0066] 130: Reference electrode

[0067] 140: Insulating layer

[0068] 150: Wiring section

[0069] 200: Electrode protection unit

[0070] 210: Inlet

[0071] 220: Exhaust

[0072]

[0073] The present invention relates to an electrode for an electrochemical sensor, an electrochemical sensor including the same, and a method for manufacturing the same, wherein by combining two types of cross-linking agents and controlling the concentration of the cross-linking agent within a specific range, the cross-linking rate of an enzyme is increased, and the influence of humidity on the electrochemical sensor is reduced, and by removing uncross-linked enzyme and mediator exposed to the outside through a washing process, when the electrode for the electrochemical sensor is applied as a working electrode, an electrochemical sensor having improved repeat measurement performance is provided.

[0074] In addition, the electrochemical sensor according to the present invention may further include an electrode protection portion on the upper portion of the working electrode, thereby preventing damage to the enzyme in the enzyme reaction layer from the outside and improving the linearity of the electrical signal according to the concentration of the target substance to be detected.

[0075]

[0076] Specifically, the present invention relates to an electrochemical sensor electrode comprising a working electrode layer; an enzyme reaction layer disposed on an upper surface of the working electrode layer; and a cross-linking agent layer disposed on an upper surface of the enzyme reaction layer, wherein the enzyme reaction layer is formed of an enzyme reaction layer composition comprising an enzyme, a mediator, and a first cross-linking agent, and particularly, by including the first cross-linking agent at a specific concentration, the technical feature is that the repeated measurement performance of a continuous measurement type electrochemical sensor including the electrochemical sensor electrode as a working electrode is improved.

[0077] In an electrochemical sensor including the electrode for an electrochemical sensor of the present invention as a working electrode, the detection target sample may be a biological sample such as blood, body fluid, urine, tears, sweat, etc., and may be sweat, which is suitable for a sensor for continuous measurement. The detection target sample may include an analyte whose concentration is to be analyzed, for example, glucose, cholesterol, lactate, ascorbic acid, glutamate, cortisol, etc., and the electrochemical sensor according to the present invention may be one that analyzes the concentration of lactate.

[0078] The present invention is characterized in that the repeat measurement performance of an electrochemical sensor is improved by controlling the composition and concentration of a cross-linking agent in the structure of a conventional sensor. Preferably, the electrode for an electrochemical sensor and the electrochemical sensor of the present invention reduce the standard deviation by 50% or more, preferably 80% or more, in multiple measurements, preferably 2 or more times, and most preferably 3 or more times, compared to a conventional sensor, and also reduce the standard deviation according to concentration by preferably 80% or more. Accordingly, compared to a conventional sensor, it has the effect of significantly reducing the deviation according to concentration in repeated measurements and also reducing measurement dispersion.

[0079]

[0080] Hereinafter, embodiments of the present invention will be described in more detail with reference to the drawings. However, the following drawings attached to this specification illustrate preferred embodiments of the present invention and, together with the contents of the invention described above, serve to further understand the technical concept of the present invention. Therefore, the present invention should not be interpreted as being limited to the matters described in such drawings.

[0081] As used herein, the terms "comprises" and / or "comprising" are used to mean that they do not exclude the presence or addition of one or more other components, steps, operations, and / or elements other than the mentioned components, steps, operations, and / or elements. Like reference numerals refer to like elements throughout the specification.

[0082] Spatially relative terms such as "above," "top," and "upper" can be used to easily describe the relationship between one component and other components, as illustrated in the drawings. Spatially relative terms should be understood to include different orientations of the components during use or operation, in addition to the orientation depicted in the drawings. For example, if a component depicted in the drawings were flipped over, a component described as "below" or "lower" of another component could actually be placed "above" the other component. Thus, the exemplary term "below" can encompass both above and below. Components can also be oriented in other directions, and thus spatially relative terms can be interpreted based on their orientation.

[0083] The term “oxidizing enzyme” as used herein can be used interchangeably with “oxidase” and “oxidase”.

[0084]

[0085] Electrodes for electrochemical sensors

[0086] The electrode for an electrochemical sensor of the present invention may include a working electrode layer; an enzyme reaction layer; and a cross-linking agent layer, wherein the enzyme reaction layer is formed of an enzyme reaction layer composition including an enzyme, a mediator, and a first cross-linking agent, the cross-linking agent layer includes a second cross-linking agent, and the enzyme reaction layer composition is characterized in that it includes less than 1.5% v / v of the first cross-linking agent. Referring to FIG. 1, the electrode (120) for an electrochemical sensor according to one embodiment of the present invention includes a structure in which a working electrode layer (120-1); an enzyme reaction layer (120-2); and a cross-linking agent layer (120-3) are sequentially laminated.

[0087]

[0088] Working electrode layer (120-1)

[0089] The working electrode layer (120-1) detects an electrical signal generated by the transport of electrons derived from a detection target substance. In one embodiment of the present invention, the working electrode layer (120-1) may include at least one selected from the group consisting of carbon (C), gold (Au), silver (Ag), copper (Cu), platinum (Pt), titanium (Ti), nickel (Ni), tin (Sn), molybdenum (Mo), cobalt (Co), Ag-Pd-Cu alloy, pyrolytic graphite, glassy carbon, carbon paste, perfluorocarbon (PFC), and carbon nanotubes (CNT), or may be an alloy thereof, but is not limited thereto as long as it is a material capable of electrical signal detection and current flow. In one or more embodiments, the materials used in the working electrode may be used alone, or two or more materials may be used as a multilayer film. It is preferable to use carbon paste in terms of process ease, versatility, and economy.

[0090] The above working electrode layer may be performed by including one or more processes selected from the group consisting of screen printing, letterpress printing, intaglio printing, planar printing, and photolithography, and screen printing is preferable in terms of ease and reliability of the process.

[0091]

[0092] Enzyme reaction layer (120-2)

[0093] The electrochemical sensor of the present invention is characterized by including a cross-linking agent in an enzyme reaction layer without including a separate fixing agent, thereby simplifying the process by eliminating unnecessary processes due to including a separate fixing agent, and has the advantage of reducing the influence of the sensor on humidity.

[0094] The enzyme reaction layer (120-2) may be formed from an enzyme reaction layer composition including an enzyme, a mediator, and a first cross-linking agent, and may be a layer in which an electrical signal detected by the working electrode layer (120-1) is generated as electrons included in the detection target substance are transferred to the mediator. Accordingly, as illustrated in FIG. 1, the enzyme reaction layer (120-2) may be formed by directly contacting the upper surface of the working electrode layer (120-1), but is not limited thereto.

[0095]

[0096] The enzyme may be provided to increase or decrease the rate of metabolism by forming an enzyme-substrate complex by binding to a detection target substance included in a detection target sample and controlling the activation energy of a chemical reaction. In one or more embodiments, the enzyme may be selected depending on the type of the detection target substance, and may include one or more types selected from the group consisting of oxidases and dehydrogenases. For example, the oxidizing enzyme may include at least one of glucose oxidase, cholesterol oxidase, lactate oxidase, ascorbic acid oxidase, or alcohol oxidase, and the dehydrogenase may include at least one of glucose dehydrogenase, glutamate dehydrogenase, lactate dehydronase, or alcohol dehydrogenase. According to one embodiment of the present invention, the enzyme may be lactate oxidase and / or lactate dehydronase, and may be for detecting lactate as a substrate.

[0097] For example, the enzyme may be included in an amount of 1 IU or more and 30 IU or less, and preferably 8 IU or more and 20 IU or less. The IU is an international unit of enzyme, and may refer to the amount of enzyme required for the enzyme to convert 1 μmol of substrate into a product per minute at 30°C under optimal conditions. If the enzyme is included in too little amount, less than the above range, the current value cannot be measured due to limitations in the reaction speed with the target substance, and if it is out of the above range, the current value may increase significantly, which may cause problems in the performance of the sensor, and is also not desirable from an economic perspective.

[0098] The above enzyme can be used by dissolving it in a buffer solution. The above buffer is not particularly limited as long as it maintains a constant pH, and may include at least one selected from the group consisting of tris(hydroxymethyl)aminomethane (Tris), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), phosphate buffered saline (PBS), 2-[N-morpholino]ethanesulfonic acid (2-(N-morpholino)ethanesulfonic acid; MES), citrate buffer (Citric acid / Citrate PH), and citric acid / phosphate buffer, which are similar to the pH of body fluids, and may preferably be phosphate buffered saline (PBS) or 2-[N-morpholino]ethanesulfonic acid (MES).

[0099]

[0100] The above mediator may be provided to generate an electrical signal by transporting electrons derived from a reduced detection target substance. In one or more embodiments, the mediator is Prussian blue, hexaammineruthenium(III) chloride, 1-Methoxy-5-methylphenaziniummethylsulfate (1-Methoxy PMS) of the Phenazine methosulfate series, potassium ferricyanide, potassium ferrocyanide, dimethylferrocene (DMF), ferricinium, ferrocene monocarboxylic acid (FCOOH), 7,7,8,8-tetracyanoquino-dimethane (TCNQ), Tetrathia fulvalene (TTF), nickelocene (Nc), N-methylacidinium (NMA+), tetrathiatetracene (TTT), N-methylphenazinium (NMP+), hydroquinone, 3-dimethylaminobenzoic acid (MBTHDMAB), 3-methyl2-benzothiozolinone hydrazone, 2-methoxy-4-allylphenol, 4-aminoantipyrin (AAP), dimethylaniline, 4-aminoantipyrene, 4-methoxynaphthol, 3,3',It may include at least one selected from the group consisting of 5,5'-tetramethylbenzidine (3,3',5,5'-tetramethyl benzidine (TMB), 2,2-azino-di-[3-ethyl-benzthiazoline sulfonate], o-dianisidine, o-toluidine, 2,4-dichlorophenol, 4-amino phenazone, benzidine, and bipyridine-osmium complex compounds, and in terms of improving the electrical sensitivity of the working electrode layer (120-1), Prussian blue, and / or hexaammineruthenium(III) chloride and It is preferred to include 1-Methoxy-5-methylphenaziniummethylsulfate (1-Methoxy PMS).

[0101]

[0102] Since the mediator is mixed with the enzyme in a single layer, the freedom in material selection can be increased and the process can be simplified. The mediator is preferably included in the enzyme reaction layer composition in an amount of more than 20 nmol and less than 60 nmol. If the amount exceeds the above range, even when lactic acid is included in a low concentration, the current intensity may increase, reducing the resolution and thus the precision of the measured current value may be reduced.

[0103]

[0104] The cross-linking agent according to the present invention comprises two types of cross-linking agents, and is characterized in that the two types of cross-linking agents are respectively included as separate layers in the enzyme reaction layer and the cross-linking agent layer. The two types of cross-linking agents are essential for protecting the enzyme, mixing and fixing the mediator and the enzyme, etc., and are not limited as long as they have a substituent that can chemically bond to the amino group of the enzyme at the same time or physically bond to it. For example, (3-aminopropyl)triethoxysilane can be mentioned as the first cross-linking agent included in the enzyme reaction layer, and glutaraldehyde can be mentioned as the second cross-linking agent included in the cross-linking agent layer. When (3-aminopropyl)triethoxysilane is included as the first cross-linking agent included in the enzyme reaction layer, it is preferable because it includes both an amino group and a silane group in the molecule, thereby forming a strong cross-linking bond with the amino group of the enzyme, thereby physically fixing the enzyme, thereby improving reactivity and stability. The first cross-linking agent may be included in a small amount of less than 1.5% v / v, and preferably less than 1.0% v / v, based on the total volume of the enzyme reaction layer composition solution. If the amount exceeds the above range, the binding ratio with the second cross-linking agent becomes excessively high, thereby reducing the enzyme cross-linking rate, while unnecessarily increasing the thickness of the sensor, which reduces the sensitivity range of the sensor.

[0105] In the case of the second crosslinking agent, the composition of the crosslinking agent layer (120-3) described later will be described in detail.

[0106]

[0107] Crosslinking layer (120-3)

[0108] The crosslinking layer of the present invention can be included as a separate layer external to the enzyme reaction layer and play a protective role by preventing the penetration of impurities and ionic components into the enzyme reaction layer.

[0109] The electrode for an electrochemical sensor according to the present invention is characterized in that it includes a cross-linking agent layer as a separate layer on the upper surface of the enzyme reaction layer. Accordingly, it has the effect of protecting the enzyme from external temperature changes, while at low humidity it dries and shrinks, and at high humidity it expands, giving tension to the exterior of the enzyme and blocking the active site of the enzyme, thereby facilitating the storage of the electrochemical sensor, especially under normal atmospheric humidity conditions, and reducing the effect of humidity influence. If the cross-linking agent is included together with the mediator and / or enzyme in the enzyme reaction layer instead of including the cross-linking agent as a separate layer, a phenomenon in which the enzyme reaction layer partially aggregates due to a problem in the process may occur, thereby rendering the electrochemical sensor inoperable.

[0110] In addition, the electrode for an electrochemical sensor according to the present invention is characterized by not including a separate fixing agent such as chitosan. In the case of a conventional electrode for an electrochemical sensor, it includes an amine-based hygroscopic polymer such as chitosan, but in this case, there is a problem that the reliability of the electric signal is reduced due to hydrogen bonding with external water molecules. Therefore, in the case of a conventional electrode for an electrochemical sensor, an additional process such as a filtration process for the fixing agent is required to reduce the influence of the sensor due to humidity, but the electrode for an electrochemical sensor according to the present invention is characterized by not including a separate fixing agent such as chitosan, thereby simplifying the process and having the effect of reducing the influence of humidity.

[0111]

[0112] The crosslinking agent layer (120-3) may be manufactured from a crosslinking agent composition. Specifically, the crosslinking agent layer of the present invention may be formed by applying the crosslinking agent composition to the upper surface of the enzyme reaction layer (120-2). In one embodiment of the present invention, the crosslinking agent layer composition may include at least one selected from the group consisting of a solvent and a second crosslinking agent.

[0113] The second cross-linking agent is not particularly limited, but preferably includes glutaraldehyde. When glutaraldehyde is included as the second cross-linking agent included in the cross-linking agent layer, it is preferable in that it forms a strong cross-linking bond with the amino group of the enzyme, thereby physically fixing the enzyme, thereby improving usability and stability.

[0114] The second cross-linking agent may be included preferably at 1.0% v / v or less based on the total volume of the cross-linking agent layer composition solution, and more preferably, the first cross-linking agent included in the enzyme reaction layer (120-2) and the second cross-linking agent included in the cross-linking agent layer (120-3) may be included at a molar ratio of 1:1 to 1:4. If the second cross-linking agent exceeds the above range, a problem may arise in which current noise increases, resulting in a decrease in the reliability of the measured current value.

[0115]

[0116] The solvent is not particularly limited as long as it dissolves the crosslinking agent, but may preferably be phosphate buffered saline (PBS). The solvent may be included in an amount of 60 to 99.5 wt% based on the total weight of the crosslinking agent layer composition.

[0117]

[0118]

[0119] The signal detection principle of the above electrochemical sensor electrode (120) is exemplarily explained as follows. When sweat, which is a sample to be detected, is injected into the electrochemical sensor electrode, lactic acid, which is a substance to be detected in the sweat, binds to lactate oxidase and / or lactate dehydrogenase contained in the enzyme reaction layer (120-2), and accordingly, the bound enzyme acquires a charge. Subsequently, the reduced enzyme is oxidized by a mediator, and the mediator is reduced. The reduced mediator loses electrons at the surface of the working electrode layer (120-1) to which a certain voltage is applied and is oxidized again. In this process, supply (desorption) of electrons to the working electrode occurs for charge balance, and voltage and / or current are generated depending on the increase or decrease in the relative voltage between the working electrode and the reference electrode. The concentration of lactate contained in sweat is proportional to the amount of current generated during the oxidation of the mediator, so the concentration of lactate can be calculated from this.

[0120]

[0121] Electrochemical sensor

[0122] Referring to FIG. 2, an electrochemical sensor (100) according to one embodiment of the present invention includes a substrate (110); a working electrode (120) disposed on an upper surface of the substrate; and a reference electrode (130) disposed on an upper surface of the working electrode and spaced apart from the working electrode, wherein the working electrode is the <electrode for electrochemical sensor> described above. In addition, referring to FIG. 3, the electrochemical sensor according to the present invention may further include a wiring portion (150) and an insulating layer (140).

[0123] In addition, the electrochemical sensor formed according to the present invention can be used as a non-invasive biosensor capable of continuous measurement of lactate. If the electrochemical sensor is implemented in the form of a substrate film having flexible characteristics, it can be attached to the skin, and can be used to continuously measure the concentration of lactate contained in sweat, which is a target sample, and observe changes in the concentration.

[0124]

[0125] substrate (110)

[0126] The substrate (110) serves to structurally support components included in an electrode for an electrochemical sensor.

[0127] In one or more embodiments, the substrate (110) may be implemented in the form of a base film having a rigid material such as glass or having flexible properties. When the substrate (110) is implemented to be flexible, specific examples of materials that can be applied to the base film include polyester resins such as polyethylene terephthalate, polyethylene isophthalate, polyethylene naphthalate, and polybutylene terephthalate; cellulose resins such as diacetyl cellulose and triacetyl cellulose; polycarbonate resins; acrylic resins such as polymethyl (meth) acrylate and polyethyl (meth) acrylate; styrene resins such as polystyrene and acrylonitrile-styrene copolymers; polyolefin resins such as polyethylene, polypropylene, polyolefins having a cyclo- or norbornene structure, and ethylene-propylene copolymers; vinyl chloride resins; amide resins such as nylon and aromatic polyamides; imide resins; Examples of films that include thermoplastic resins such as polyethersulfone resins; sulfone resins; polyetheretherketone resins; sulfated polyphenylene resins; vinyl alcohol resins; vinylidene chloride resins; vinyl butyral resins; allylate resins; polyoxymethylene resins; and epoxy resins, and films made of blends of the above thermoplastic resins can also be used. In addition, films made of thermosetting resins such as (meth)acrylic, urethane, acrylic urethane, epoxy, silicone, or ultraviolet-curable resins can also be used. The thickness of such transparent optical films can be appropriately determined, but generally, it can be determined to be 1 ㎛ to 500 ㎛ in consideration of workability such as strength and handleability, thin layer property, etc., and 1 ㎛ to 300 ㎛ is preferable, and 5 ㎛ to 200 ㎛ is more preferable.

[0128] Such a base film may contain one or more suitable additives. Examples of the additives include ultraviolet absorbers, antioxidants, lubricants, plasticizers, release agents, anti-coloring agents, flame retardants, nucleating agents, antistatic agents, pigments, and colorants. The base film may have a structure including various functional layers, such as a hard coating layer, an antireflection layer, and a gas barrier layer, on one or both sides of the film. The functional layers are not limited to those described above, and may include various functional layers known to be usable in the art depending on the intended use.

[0129] Additionally, the substrate film may be surface-treated, if necessary. Such surface treatments include dry treatments such as plasma treatment, corona treatment, and primer treatment, and chemical treatments such as alkaline treatment including saponification treatment.

[0130]

[0131] Metal electrode layer (110-1)

[0132] According to one embodiment of the present invention, the substrate (110) may further include a metal electrode layer (110-1) in a multi-layer form on the upper side of the substrate.

[0133] For example, the metal electrode layer (110-1) may be provided as a passage through which electrons or holes generated in the oxidation-reduction reaction of the detection target substance are transmitted, and may be provided on the upper surface of the substrate (110) using a photolithography method, or may be provided integrally with the working electrode layer (120-1) of the working electrode.

[0134] Since the above metal electrode layer (110-1) can be applied with the contents of the working electrode layer of the above <electrode for electrochemical sensor>, its description will be omitted.

[0135]

[0136] Working electrode (120)

[0137] The working electrode (120) may be provided as a passage through which electrons or holes generated in the oxidation-reduction reaction of the detection target substance are transferred. In the present invention, the area of ​​the working electrode (120) in the plane direction is not particularly limited, but may be 1 to 10 mm. 2 In particular, when the electrochemical sensor of the present invention further includes an electrode protection part described later, the area in the plane direction of the working electrode (120) is 5 to 10 mm 2 This is desirable in terms of improving the reactivity of the working electrode.

[0138] In addition, the configuration and function of the above working electrode can be applied as is to the contents of the above-described <Electrochemical Sensor Electrode>, so description thereof is omitted.

[0139]

[0140] Reference electrode (130)

[0141] The reference electrode (130) is not particularly limited as long as it has a constant potential and serves as a reference for the potential for obtaining the generated potential of the working electrode. In one or more embodiments, the reference electrode may be an electrode that has been treated to prevent it from being affected by external environments such as temperature, humidity, and the composition of the target substance or sample, and if the external environment is fixed, a metal that has not been separately treated may be used. For example, the reference electrode may include at least one selected from the group consisting of a silver-silver chloride (Ag / AgCl) electrode, a calomel electrode, a mercury-mercury sulfate electrode, and a mercury-oxide mercury electrode, and the like, but is not limited thereto. However, considering that the hysteresis of the potential for a temperature cycle is less and the potential is stable up to a high temperature, it is preferable that it is a silver-silver chloride (Ag / AgCl) electrode that has little potential change. The above-described reference electrode can be provided on a substrate through a screen printing method, such as the working electrode layer (120-1) of the above-described working electrode. In the present invention, the area of ​​the reference electrode (130) in the plane direction is not particularly limited, but is 1 to 5 mm. 2 It could be.

[0142]

[0143] Wiring section (150)

[0144] The wiring unit (150) may be provided to perform a role of transmitting an electrical signal measured from the working electrode (120) and the reference electrode (130) to the display device. The wiring unit may be arranged in contact with the upper surface of the substrate (110) and may include wiring connected to the working electrode (120) and / or the reference electrode (130). The wiring connected to the working electrode (120) and the wiring connected to the reference electrode (130) may be formed to be spaced apart from each other. The wiring unit may be formed of the same material as at least a portion of the working electrode (120) and / or the reference electrode (130), and for example, the wiring unit may be formed integrally by forming an electrode layer on the substrate (110) and patterning it. An electrical signal measured through a working electrode (120) and a reference electrode (130) can be transmitted to a driving IC chip through a wiring section, and the driving IC chip can calculate the concentration of a substance to be measured.

[0145]

[0146] Insulating layer (140)

[0147] The insulating layer (140) can be formed by directly contacting the substrate (110), and can limit the size of the area where the working electrode (120), the reference electrode (130), and / or the wiring portion (150) are formed, separate the working electrode and the reference electrode to prevent current interference, and serve to protect from external substances. In addition, due to the oxidation-reduction reaction of the working electrode (120), the metal electrode layer (110-1) on the substrate (110) can be prevented from being oxidized and the surface damaged. The insulating layer (140) can be made of any insulating material without limitation, but preferably, an oxide-based or nitride-based inorganic insulating material, or an organic polymer material of a UV-curable or thermally curable type using a photoinitiator can be used. Specifically, the material of the insulating layer (140) may be silicon oxide, acrylic resin, polyester, polyimide, polytetrafluoroethene (PTFE), poly(p-xylylene), indium tin oxide (ITO), or indium zinc oxide (IZO), which may be used alone or in combination of two or more. For example, the insulating layer may be formed of only indium tin oxide (ITO) or indium zinc oxide (IZO). The ITO and IZO are electrically conductive and chemically stable, so that they can effectively protect the metal electrode layer from oxidation-reduction reactions by preventing the metal electrode layer from direct contact with the atmosphere. Therefore, the reliability of the electrical signal detected by the metal layer can be improved.

[0148] The above insulating layer (140) may be provided on the upper surface of the metal electrode layer (110-1) on the substrate (110) using photolithography or screen printing. The thickness of the insulating layer (140) may be appropriately determined, but according to an embodiment of the present invention, it may be manufactured before the creation of the working electrode and the reference electrode, and thus may have a lower thickness than the working electrode and the reference electrode (see FIG. 2).

[0149]

[0150] Electrode protection unit (200)

[0151] Referring to FIG. 9, the electrochemical sensor according to the present invention may further include an electrode protection unit (200), and the electrode protection unit (200) may be disposed on the substrate (110) to be spaced apart from the working electrode (120) and the reference electrode (130). The electrode protection unit (200) may serve to prevent damage to the enzyme in the enzyme reaction layer from the outside. In addition, the electrode protection unit (200) prevents a decrease in the slope of the concentration-current value of the detection target substance due to a decrease in the enzyme content in the enzyme reaction layer, and serves to improve the linearity of the electrical signal according to the concentration of the detection target substance.

[0152] The electrode protection unit (200) may be disposed at a certain distance from the working electrode (120) and the reference electrode (130), and may be disposed at a distance from the working electrode (120) and / or the reference electrode (130), preferably at a distance of 30 μm to 300 μm, more preferably at a distance of 50 μm to 200 μm from the working electrode (120). In the present invention, the distance may mean the shortest distance between the two layers. If the distance from the working electrode (120) and the reference electrode (130) is too small, the detection target sample or detection target substance does not sufficiently come into contact with the working electrode, which is the reaction unit, so that the difference in current value according to the increase in the concentration of the detection target sample or detection target substance is not large, and accordingly, there is a problem in that the concentration and current value slopes are not sufficiently secured. In addition, there is a problem that the linearity improvement effect due to the inclusion of an electrode protection unit may be somewhat reduced when the distance between the working electrode (120) and the reference electrode (130) is excessively large.

[0153] In the present invention, a barrier rib can be formed by laminating an optical adhesive such as an optically clear adhesive (OCA) between the working electrode (120) and / or the reference electrode (130) and the electrode protection member (200) so that the electrode protection member (200) is formed spaced apart from the working electrode (120) and / or the reference electrode (130).

[0154] The above electrode protection part (200) can be used without limitation as long as it is a hydrophilic material, but preferably, polyethylene terephthalate (PET) that has been treated to be hydrophilic can be used.

[0155] In addition, it is preferable that the electrode protection part (200) be formed with a thickness of 50 ㎛ to 200 ㎛ in order to improve the linearity of the electric signal.

[0156] Referring to FIGS. 9A and 9B, in the case of the electrochemical sensor structure including the electrode protection unit (200), the electrode protection unit (200) or a portion of the substrate (110) may further include an inlet (210) through which a detection target sample or detection target substance can be introduced, and an exhaust unit (220) through which internal air is discharged to allow the detection target sample or detection target substance to be introduced into the electrochemical sensor. The inlet unit (210) and the exhaust unit (220) form a flow path within the electrochemical sensor together with the electrode protection unit (200), and the detection target sample or detection target substance introduced through the inlet unit (210) flows along the flow path within the electrochemical sensor and reaches the reaction units, i.e., the working electrode (120) and the reference electrode (130), where a reaction occurs. The shape, width, and depth of the flow path are not particularly limited, and may be appropriately changed depending on the type of the detection target sample or detection target substance, or the shape and size of the electrochemical sensor.

[0157]

[0158] <Method for manufacturing electrochemical sensors>

[0159] The present invention provides a method for manufacturing an electrochemical sensor, comprising the steps of: (a) forming an electrode portion including a working electrode and a reference electrode on a substrate; (b) forming an enzyme reaction layer on at least a portion of the working electrode; (c) forming a cross-linking agent layer on the enzyme reaction layer; and (d) dropping a buffer solution on the cross-linking agent layer, wherein the enzyme reaction layer is formed of an enzyme reaction layer composition including an enzyme, a mediator, and a first cross-linking agent, the cross-linking agent layer includes a second cross-linking agent, and the enzyme reaction layer composition includes less than 1.5% v / v of the first cross-linking agent.

[0160] In the method for manufacturing an electrochemical sensor of the present invention, it is preferable that the buffer solution is phosphate buffered saline.

[0161] In the method for manufacturing an electrochemical sensor of the present invention, a step (e) of forming an electrode protection portion on the upper surface of the substrate, spaced apart from the working electrode and the reference electrode, may be further included. Specifically, the step (e) of forming the electrode protection portion is not particularly limited as long as the distance from the working electrode is maintained constant, but may be formed by laminating an optical adhesive such as an optically clear adhesive (OCA) on the upper surface of the working electrode to form a partition wall, and then laminating a hydrophilic material such as polyethylene terephthalate (PET).

[0162] The above step (a) can be performed by any one selected from the group consisting of screen printing, letterpress printing, intaglio printing, planar printing and photolithography, and screen printing is more preferable.

[0163] The above screen printing can be performed using a screen printing mesh of 100 to 450 mesh, but is not limited thereto.

[0164] The above photolithography may be a method for integrally forming wiring by forming a carbon paste and / or metal film on a substrate and patterning it through a mask.

[0165] Steps (b) and (c) above may be performed by any one selected from the group consisting of flow coating, ink jet, and drop casting, and drop casting is more preferred. The drop casting may use 0.5 to 5 uL of solution in each step, and may be dried for 10 to 60 minutes or more to remove all remaining solvents.

[0166] The cleaning process according to the present invention may include a step (d) of dropping a buffer solution on a crosslinking layer, and more specifically, may include a step of dropping the buffer solution on a working electrode region including an enzyme reaction layer and a crosslinking layer. The buffer solution is not particularly limited as long as it maintains a constant pH, but may include at least one selected from the group consisting of tris(hydroxymethyl)aminomethane (Tris), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), phosphate buffered saline (PBS), 2-[N-morpholino]ethanesulfonic acid (MES), citrate buffer (Citric acid / Citrate PH), and citric acid / phosphate buffer, which are similar to the pH of body fluids. For process reasons, phosphate buffered saline (PBS) or 2-[N-morpholino]ethanesulfonic acid (MES) is preferable.

[0167]

[0168] In addition, the present invention provides an electrochemical sensor manufactured by the above manufacturing method, and may exhibit all the characteristics described in the above item <Electrochemical Sensor>.

[0169]

[0170] Hereinafter, experimental examples including specific examples and comparative examples are presented to help understand the present invention, but these are only illustrative of the present invention and do not limit the scope of the appended claims. It is obvious to those skilled in the art that various changes and modifications to the examples are possible within the scope and technical idea of ​​the present invention, and it is also natural that such changes and modifications fall within the scope of the appended claims.

[0171]

[0172] Manufacturing Example 1: Manufacturing of enzyme reaction layer composition

[0173] Manufacturing Example 1-1: Manufacturing of enzyme reaction layer composition 1

[0174] Lox, 1-m-PMS, Ru, and APTES were each diluted in phosphate buffered saline (PBS) by stirring to obtain concentrations as shown in Table 1 below, and stock solutions 1 to 4 were prepared.

[0175] Original solution 1 Original solution 2 Original solution 3 Original solution 4 Composition LOx1-m-PMSRuAPTES Concentration 20IU / mL 100mM 48.4mM 5%

[0176] Lox: Lactate Oxidase (LCO-301, TOYOBO)1-m-PMS: 1-Methoxy-5-methylphenaziniummethylsulfate (1-m-PMS (M8640, Sigma-Aldrich))

[0177] Ru: Hexaammineruthenium(III) chloride, 98% (C36334, ACROS)

[0178] APTES: (3-Aminopropyl)triethoxysilane (440140, Sigma-Aldrich)

[0179]

[0180] After this, the above-mentioned original solution 1, original solution 2, original solution 3, original solution 4, and PBS were mixed in a ratio of 4:2:1:1:2 and left at room temperature for 30 minutes to prepare an enzyme reaction layer composition 1.

[0181]

[0182] Manufacturing Example 1-2: Manufacturing of Enzyme Reaction Layer Composition 2

[0183] Enzyme reaction layer composition 2 was prepared in the same manner as in Manufacturing Example 1-1, except that the above-mentioned original solution 1, original solution 2, original solution 3, original solution 4, and PBS were mixed in a ratio of 4:2:1:2:1.

[0184]

[0185] Manufacturing Example 1-3: Manufacturing of enzyme reaction layer composition 3

[0186] Enzyme reaction layer composition 3 was prepared in the same manner as in Manufacturing Example 1-1, except that the above-mentioned original solution 1, original solution 2, original solution 3, and original solution 4 were mixed in a ratio of 4:2:1:3.

[0187]

[0188] Manufacturing Example 1-4: Manufacturing of enzyme reaction layer composition 4

[0189] Enzyme reaction layer composition 4 was prepared in the same manner as in Manufacturing Example 1-1, except that the following solution 5 was used instead of the above solution 4 and mixed in the following ratio.

[0190] -Original solution 5: Glutaraldehyde (G5882, Sigma-Aldrich) was diluted with purified water (DI) by stirring until the concentration became 0.6%, and then original solution 5 was prepared.

[0191] -Mixing ratio: The above original solution 1, original solution 2, original solution 3, original solution 5, and PBS were mixed in a ratio of 8:4:2:2:4.

[0192]

[0193] The contents and concentrations of the enzyme, mediator, and cross-linking agent of the enzyme reaction layer compositions 1 to 4 finally manufactured according to the above Manufacturing Examples 1-1 to 1-4 are converted and shown in Table 2 below. In the case of the enzyme reaction layer compositions 1 to 3, (3-aminopropyl)triethoxysilane, which is the first cross-linking agent, was used as the cross-linking agent, and in the case of the enzyme reaction layer composition 4, glutaraldehyde, which is the second cross-linking agent, was used as the cross-linking agent. The enzyme content is expressed in international enzyme units (IU) by calculating the final content of the enzyme contained in each composition, the mediator content is calculated by adding the moles of two mediators (1-methoxy-5-methylphenazinium methyl sulfate and hexaamineruthenium(III) chloride) contained in each composition, and the crosslinking agent concentration is expressed in volume percentage by calculating the final concentration of (3-aminopropyl)triethoxysilane or glutaraldehyde contained in each composition.

[0194]

[0195] Enzyme reaction layer composition Enzyme (IU) Mediator (nmol) Crosslinker (% v / v) Enzyme reaction layer composition 11649.70.5 Enzyme reaction layer composition 21649.71.0 Enzyme reaction layer composition 31649.71.5 Enzyme reaction layer composition 41649.70.2

[0196] -Enzyme: Lactate Oxidase (LCO-301, TOYOBO) -Mediator: 1-Methoxy-5-methylphenaziniummethylsulfate (1-m-PMS (M8640, Sigma-Aldrich)), Hexaammineruthenium(III) chloride, 98% (C36334, ACROS)

[0197] -First cross-linking agent: (3-Aminopropyl)triethoxysilane (440140, Sigma-Aldrich)

[0198] -Second cross-linking agent: glutaraldehyde (G5882, Sigma-Aldrich)

[0199]

[0200] Manufacturing Example 2-1: Manufacturing of crosslinking agent layer composition A

[0201] A crosslinking layer composition A was prepared by diluting glutaraldehyde (G5882, Sigma-Aldrich) in purified water (DI) by stirring until the concentration became 0.6%.

[0202]

[0203] Manufacturing Example 2-2: Manufacturing of crosslinking agent layer composition B

[0204] (3-Aminopropyl)triethoxysilane (440140, Sigma-Aldrich) was diluted with phosphate buffered saline (PBS) by stirring to a concentration of 0.6%, and then a crosslinking layer composition B was prepared.

[0205]

[0206] Examples 1 to 3 and Comparative Examples 1 to 2: Preparation of electrochemical sensors

[0207] Example 1: Preparation of electrochemical sensor 1

[0208] A 180㎛ thick PET substrate is prepared, and APC alloy (Ag-Pd-Cu alloy) and IZO (Indium Zinc Oxide) are printed on the upper surface of the substrate using photolithography to form a metal electrode layer and a wiring portion.

[0209] Except for the areas where the wiring section is not formed and the reference electrode and working electrode areas, an insulating layer is formed by printing using a photolithography method.

[0210] In the working electrode region where the IZO layer is not formed, a working electrode layer is first formed by screen-printing carbon paste. 2 μl of the enzyme reaction layer composition 1 manufactured through the above Manufacturing Example 1-1 is dropped onto the upper surface of the working electrode layer, and then the enzyme reaction layer is formed by drop-casting by drying at room temperature. 2 μl of the crosslinking layer composition A according to the above Manufacturing Example 2-1 is dropped onto the upper surface of the working electrode layer by drop-casting, and then dried to form a crosslinking layer.

[0211] Afterwards, PBS was dropped onto the working electrode area, and after 3 minutes, the working electrode was cleaned through an air-blowing process.

[0212]

[0213] Example 2: Preparation of electrochemical sensor 2

[0214] Electrochemical sensor 2 was manufactured in the same manner as in Example 1, except that enzyme reaction layer composition 2 was used instead of enzyme reaction layer composition 1.

[0215]

[0216] Example 3: Preparation of electrochemical sensor 3

[0217] A metal electrode layer, a wiring portion, an insulating layer, a working electrode layer, an enzyme reaction layer, and a crosslinking agent layer were formed in the same manner as in Example 1, but no separate cleaning process was performed.

[0218]

[0219] Example 4: Preparation of electrochemical sensor A-1

[0220] A 180㎛ thick PET substrate is prepared, and APC alloy (Ag-Pd-Cu alloy) and IZO (Indium Zinc Oxide) are printed on the upper surface of the substrate using photolithography to form a metal electrode layer and a wiring portion.

[0221] Except for the areas where the wiring section is not formed and the reference electrode and working electrode areas, an insulating layer is formed by printing using a photolithography method.

[0222] In the working electrode region where the IZO layer is not formed, a working electrode layer is first formed by screen-printing carbon paste. 2 μl of the enzyme reaction layer composition 1 manufactured through the above Manufacturing Example 1-1 is dropped onto the upper surface of the working electrode layer, and then the enzyme reaction layer is formed by drop-casting by drying at room temperature. 2 μl of the cross-linking layer composition A according to the above Manufacturing Example 2-1 is dropped onto the upper surface of the working electrode layer by drop-casting, and then dried to form a cross-linking layer. (Working electrode area in the planar direction: 5.72 mm) 2 , reference electrode area 2.83mm 2 )

[0223] After sufficiently drying the formed cross-linking layer under low humidity conditions at room temperature, PBS is dropped on the working electrode area, and after 3 minutes, the working electrode is cleaned through an air-blowing process. To create a gap on the upper surface of the cleaned working electrode, a 50㎛ optically clear adhesive (OCA, 3M 8146-4) is laminated to form a 50㎛ barrier. Afterwards, a PET film (AMTE AFG-C0307) with a hydrophilic treatment is laminated on the upper surface of the OCA to form an electrode protection part.

[0224]

[0225] Example 5: Preparation of electrochemical sensor A-2

[0226] Electrochemical sensor A-2 was manufactured in the same manner as in Example 4, except that a 100 μm optically transparent adhesive was laminated to form a 100 μm barrier to create a gap on the upper surface of the cleaned working electrode.

[0227]

[0228] Example 6: Preparation of electrochemical sensor A-3

[0229] Electrochemical sensor A-3 was manufactured in the same manner as in Example 4, except that a 200 μm optically transparent adhesive was laminated to form a 200 μm barrier to create a gap on the upper surface of the cleaned working electrode.

[0230]

[0231] Example 7: Preparation of electrochemical sensor A-4

[0232] An electrochemical sensor A-4 was manufactured in the same manner as in Example 4, except that a 25 μm optically transparent adhesive was laminated to form a 25 μm barrier to create a gap on the upper surface of the cleaned working electrode.

[0233]

[0234] Example 8: Preparation of electrochemical sensor A-5

[0235] An electrochemical sensor A-5 was manufactured in the same manner as in Example 4, except that a 500 μm optically transparent adhesive was laminated to form a 500 μm barrier to create a gap on the upper surface of the cleaned working electrode.

[0236]

[0237] Example 9: Preparation of electrochemical sensor A-6

[0238] An electrochemical sensor A-6 was manufactured in the same manner as in Example 4, except that a 1,000 μm thick optically transparent adhesive was laminated to form a 1,000 μm barrier to create a gap on the upper surface of the cleaned working electrode.

[0239]

[0240]

[0241] Comparative Example 1: Manufacturing of Electrochemical Sensor 4

[0242] An electrochemical sensor 4 was manufactured in the same manner as in Example 1, except that enzyme reaction layer composition 3 was used instead of enzyme reaction layer composition 1.

[0243]

[0244] Comparative Example 2: Manufacturing of electrochemical sensor 5

[0245] An electrochemical sensor 5 was manufactured in the same manner as in Example 1, except that enzyme reaction layer composition 4 was used instead of enzyme reaction layer composition 1, and crosslinking agent layer composition B was used instead of crosslinking agent layer composition A.

[0246]

[0247]

[0248] <Experimental Example>

[0249] Experimental Example 1: Evaluation of the sensitivity range according to the content of the enzyme reaction layer composition

[0250] In the electrochemical sensors of Examples 1 and 2 and Comparative Example 1, aqueous solutions containing lactic acid at concentrations of 1, 5, 10, 20 and 30 mM were evenly added to the working electrode and the reference electrode, respectively, and the current flowing was measured for each concentration. The number of samples was 4, and the average current value of each sample was calculated and shown in Table 3 below, and the result graph of Table 3 is depicted in Fig. 4. The unit of the displayed current value is μA.

[0251] - Measuring device: CHI630E

[0252] - Measurement method: Amperometric it curve

[0253] - Measurement voltage: 0.1V, 200sec

[0254] Concentration (mM) Example 1 Example 2 Comparative Example 1 11.25 1.07 1.28 54.89 5.2 14.7 110.5 26.87 8.86 207.05 7.9 59.12 308.7 19.34 8.32

[0255] Experimental Example 2: Evaluation of Repeated Measurement Performance by Content of Enzyme Reaction Layer Composition

[0256] In the electrochemical sensors of Examples 1 and 2 and Comparative Example 1, aqueous solutions containing lactic acid at concentrations of 1, 5, 10, 20 and 30 mM were evenly added to the working electrode and the reference electrode, respectively, and the current flowing was repeatedly measured twice for each concentration. The number of samples was 4, and the average current value of each sample was calculated and shown in Table 4 below, and the result graphs of Table 4 are depicted in Figs. 5a to 5c. The unit of the displayed current value is μA.

[0257] - Measuring device: CHI630E

[0258] - Measurement method: Amperometric it curve

[0259] - Measurement voltage: 0.1V, 200sec

[0260] Concentration (mM) Example 1 Example 2 Comparative Example 11 times 2 times 1 time 2 times 1 time 2 times 11.25 1.18 1.07 1.07 1.28 1.23 54.89 5.125.215.09 4.7 14.78 105.5 25.5 86.87 6.74 8.86 7.49 207.05 6.807.9 57.779 128.41 308.718.479.348.79 8.327.41

[0261]

[0262] Experimental Example 3: Evaluation of repeatability according to the presence or absence of a cleaning process

[0263] In each of the electrochemical sensors of Examples 1 and 3, aqueous solutions containing lactic acid at concentrations of 1, 5, 10, 20, and 30 mM were evenly added to the working and reference electrodes, respectively, and the flowing current was measured. The number of samples was 4, and the average current value of each sample was calculated and shown in Table 5 below, and the result graph of Table 5 is depicted in Fig. 6. The unit of the displayed current value is μA.

[0264] - Measuring device: CHI630E

[0265] - Measurement method: Amperometric it curve

[0266] - Measurement voltage: 0.1V, 200sec

[0267] Concentration (mM) Example 1 Example 3 12.821.2556.734.89108.905.522011.777.053014.278.71

[0268] Experimental Example 4: Confirmation of Appearance Defects According to Changes in the Composition of the First and Second Cross-linking Agents

[0269] The appearance of each of the electrochemical sensors of Example 1 and Comparative Example 2 was photographed, and the results are shown in FIGS. 7a and 7b.

[0270]

[0271] Experimental Example 5: Linearity Evaluation of Electrical Signals According to the Distance Between Electrode Protection Units

[0272] In the electrochemical sensors of Examples 1, 5, 7, and 8, aqueous solutions containing lactic acid at concentrations of 1, 5, 10, 20, and 30 mM were evenly added to the working and reference electrodes, respectively, and the current flowing was measured for each concentration. The number of samples was 4, and the average current value of each sample was calculated and shown in Table 6 below, and the result graph of Table 6 is depicted in Fig. 11. The unit of the displayed current value is μA.

[0273] - Measuring device: CHI630E

[0274] - Measurement method: Amperometric it curve

[0275] - Measurement voltage: 0.1V, 200sec

[0276] Concentration (mM) Example 1 Example 4 Example 5 Example 6 Example 7 Example 8 Example 9 11.25 1.25 1.25 1.25 1.11 1.74 1.79 54.89 2.93 3.08 4.00 1.45 4.20 6.35 105.5 25.7 25.05 6.00 1.68 5.46 7.33 207.05 7.45 8.25 7.00 1.616 567.80 308.719 5110.918.00 1.66 7.19 7.90

[0277]

[0278]

[0279] Referring to the experimental data in Tables 3 and 4 and the graphs applying the same, FIGS. 4 and 5a to 5c, in the case of Examples 1 and 2 according to the present invention, the deviation (dispersion) of the current value according to the concentration of lactic acid was small due to the inclusion of a specific concentration of the first cross-linking agent according to the present invention in the enzyme reaction layer, and thus, it was confirmed that the repeated measurement performance of the electrochemical sensor was improved.

[0280] Specifically, referring to the graphs illustrated in Table 3 and FIG. 4, in the case of Examples 1 and 2 including a specific concentration of the first cross-linking agent according to the present invention, the resolution is in the range of 1 to 30 mM, but in the case of Comparative Example 1, which is outside the specific concentration range of the present invention, the resolution is in a narrower sensitivity range of 1 to 10 mM, so that it can be seen that the electrochemical sensor of the example having a first cross-linking agent concentration of less than 1.5% has excellent measurement performance. In addition, referring to the graphs illustrated in Table 4 and FIGS. 5a to 5c, in the case of Examples 1 and 2 including a specific concentration of the first cross-linking agent according to the present invention, the variation in the electric signal measurement value according to repeated measurements is small, whereas in the case of Comparative Example 1, which is outside the specific concentration range of the present invention, the variation in the electric signal measurement value according to repeated measurements is large, so that it can be seen that the electrochemical sensor of the example having a first cross-linking agent concentration of less than 1.5% has excellent measurement performance.

[0281] In addition, referring to the graphs shown in Table 5 and Figure 6, the electrochemical sensor of Example 1 that underwent the cleaning process had less deviation (dispersion) than the electrochemical sensor of Example 3 that did not undergo the cleaning process, and it was confirmed that the repeat measurement performance of the electrochemical sensor improved as the cleaning process was performed.

[0282] Meanwhile, referring to the results of FIGS. 7a and 7b, it was confirmed that when the stacking order of the first cross-linking agent and the second cross-linking agent according to the present invention was changed, a phenomenon of poor appearance of the sensor occurred.

[0283] In summary, in the case of the electrochemical sensor of the present invention, when two types of cross-linking agents are included in the enzyme reaction layer and the cross-linking agent layer, respectively, and the content of the first cross-linking agent included in the enzyme reaction layer is controlled and the cross-linking rate of the enzyme is improved through a washing process, it was confirmed that the deviation (dispersion) of the measured value during repeated measurements is reduced, thereby exhibiting an excellent effect in terms of the repeated measurement performance of the sensor.

[0284] In addition, through the results of Experimental Example 5, it was possible to confirm the linearity of the electric signal according to the separation distance of the electrode protection part.

[0285] Specifically, referring to Table 6 and the graphs of FIGS. 11 and 12, in the case of an electrochemical sensor including an electrode protection unit according to the present invention, it was confirmed that the linearity of the electric signal changed depending on the distance between the working electrode and the reference electrode and the electrode protection unit. In particular, in the case of Examples 4 and 5, the measured value of the electric signal also increased steadily as the concentration of lactic acid increased, and accordingly, it was confirmed that the linearity of the electric signal was greatly improved compared to Example 1 which did not include the electrode protection unit. In the case of Example 7, where the distance between the working electrode and the reference electrode was less than 50 μm, it was confirmed that the difference in the current value according to the increase in the lactic acid concentration was not large because the detection target substance did not sufficiently come into contact with the working electrode, which is the reaction unit. In addition, in the case of Examples 8 and 9, where the distance exceeds 200 μm, it was confirmed that the linearity improvement effect due to the inclusion of the electrode protection unit was somewhat lower than in the cases of Examples 4 to 6.

[0286]

[0287] The electrode for an electrochemical sensor according to the present invention can increase the crosslinking rate of an enzyme and reduce the influence of humidity on the electrochemical sensor by combining two types of crosslinking agents and controlling the concentration of the crosslinking agents within a specific range. Furthermore, by removing uncrosslinked enzyme and externally exposed mediators through a cleaning process, when the electrode for an electrochemical sensor is applied as a working electrode, an electrochemical sensor with improved repeatability can be provided.

[0288] Therefore, the electrochemical sensor according to the present invention can be usefully applied to a non-invasive biosensor because it is capable of continuous measurement of a detection target substance using a detection target sample such as sweat.

[0289] In addition, when the electrochemical sensor according to the present invention includes an electrode protection part that is positioned spaced apart from the working electrode and the reference electrode on the upper surface of the substrate, the linearity of the electrical signal according to the concentration of the target substance can be improved.

Claims

1. Working electrode layer; An enzyme reaction layer disposed on the upper surface of the working electrode layer; and It includes a crosslinking agent layer disposed on the upper surface of the enzyme reaction layer, The above enzyme reaction layer is formed of an enzyme reaction layer composition including an enzyme, a mediator, and a first cross-linking agent, The crosslinking agent layer comprises a second crosslinking agent, An electrode for an electrochemical sensor, wherein the enzyme reaction layer composition comprises a first cross-linking agent of less than 1.5% v / v.

2. In claim 1, An electrode for an electrochemical sensor, wherein the enzyme comprises at least one selected from the group consisting of oxidases and dehydrogenases.

3. In claim 1, An electrode for an electrochemical sensor, wherein the enzyme comprises lactate oxidase.

4. In claim 1, An electrode for an electrochemical sensor, wherein the mediator comprises at least one selected from the group consisting of a ruthenium compound and phenazine methosulfate and its derivatives.

5. In claim 1, An electrode for an electrochemical sensor, wherein the first cross-linking agent comprises (3-aminopropyl)triethoxysilane.

6. In claim 1, An electrode for an electrochemical sensor, wherein the second cross-linking agent comprises glutaraldehyde.

7. In claim 1, The above-mentioned electrochemical sensor electrode is an electrochemical sensor electrode washed with a buffer solution containing phosphate buffered saline.

8. In claim 7, The above-mentioned electrode for an electrochemical sensor is an electrode for an electrochemical sensor, which is washed with a buffer solution containing phosphate buffered saline for 3 minutes or more.

9. Substrate; a working electrode disposed on the upper surface of the substrate; and A reference electrode is disposed on the upper surface of the substrate and spaced apart from the working electrode, An electrochemical sensor, wherein the working electrode is an electrode for an electrochemical sensor according to any one of claims 1 to 8.

10. In claim 9, An electrochemical sensor further comprising an electrode protection member formed on the upper surface of the substrate and positioned spaced apart from the working electrode and the reference electrode.

11. In claim 9, An electrochemical sensor further comprising at least one of a metal electrode layer and a wiring portion on the upper side of the substrate.

12. In claim 10, An electrochemical sensor wherein the distance between the working electrode and the electrode protection part is 50 μm to 200 μm.

13. In claim 10, An electrochemical sensor further comprising an inlet portion and an exhaust portion.

14. Step (a) of forming an electrode portion including a working electrode and a reference electrode on a substrate; Step (b) of forming an enzyme reaction layer on at least a portion of the working electrode; Step (c) of forming a crosslinking agent layer on the enzyme reaction layer; and A method for manufacturing an electrochemical sensor, comprising a step (d) of dropping a buffer solution on the crosslinking layer, The above enzyme reaction layer is formed of an enzyme reaction layer composition including an enzyme, a mediator, and a first cross-linking agent, The crosslinking agent layer comprises a second crosslinking agent, A method for manufacturing an electrochemical sensor, characterized in that the enzyme reaction layer composition comprises a first cross-linking agent of less than 1.5% v / v.

15. A method for manufacturing an electrochemical sensor according to claim 14, wherein the buffer solution comprises phosphate buffered saline.

16. In claim 14, A method for manufacturing an electrochemical sensor, further comprising a step (e) of forming an electrode protection portion on the upper surface of the substrate, spaced apart from the working electrode and the reference electrode.

17. In claim 14, A method for manufacturing an electrochemical sensor, characterized in that the above step (a) is performed by any one selected from the group consisting of screen printing, letterpress printing, intaglio printing, flat panel printing, and photolithography.

18. In claim 14, A method for manufacturing an electrochemical sensor, characterized in that the above steps (b) and (c) are performed by any one selected from the group consisting of flow coating, inkjet, and drop casting.

19. An electrochemical sensor manufactured by any one of the electrochemical sensor manufacturing methods of claims 14 to 18.

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