Electrochemical sensor and manufacturing method therefor

Abstract

The present application relates to an electrochemical sensor and, more specifically, to an electrochemical sensor and a manufacturing method therefor, the sensor being capable of stably fixing as many fixing materials as possible to the surface of an operation electrode of the electrochemical sensor. To solve the problem, a first aspect of the present application can provide the electrochemical sensor comprising: a housing which contains an electrode part including the operation electrode and which is vertically bonded to a channel part positioned thereunder; a support which contains the electrode part including the operation electrode and which is vertically bonded to the housing positioned thereon; and the electrode part including the operation electrode interpolated between the housing and the support, wherein the support has a first channel part having a recess formed such that the operation electrode is correspondingly positioned therein and a second channel part having a recess formed such that a counter electrode or a reference electrode as an electrode other than the operation electrode is correspondingly positioned therein, and a fluid trap part including a partition wall part and a slot part is formed on one side of the outer periphery of the first channel part of the support, the fluid trap part trapping a fluid therein when external pressure (P2) of the fluid trap part is greater than or equal to internal pressure (P1) and discharging the fluid therein to the outside thereof when the external pressure (P2) of the fluid trap part is less than the internal pressure (P1). According to the present application, fixing materials can be effectively and stably fixed only to the operation electrode of the electrochemical sensor, and thus an electrochemical sensor that is very sensitive and accurate to bio-marker detection can be provided and the electrochemical sensor can be economically manufactured by reducing unnecessary processes and costs.

Description

Electrochemical sensor and method for manufacturing the same

[0001] The present invention relates to an electrochemical sensor, and more specifically, to an electrochemical sensor capable of stably fixing a maximum amount of a fixing substance to the surface of a working electrode of the electrochemical sensor, and a method for manufacturing the same.

[0002] This invention is a research project conducted by the Korea Electronics Technology Institute (ETRI) with the support of the Ministry of Trade, Industry and Energy, under the title of "Development of Korea-led K-Sensor Technology for Market Leadership" under the research project titled "Development of an Optical Sensor System for Early Diagnosis of Adult Diseases Based on Urine Biomarkers" (Project No. 00256247, Project Unique No. 1415187356).

[0003] In general, a biosensor is a device that can selectively detect trace amounts of biochemical substances to be analyzed by converting biological interactions and recognition reactions into electrical or optical signals by combining a biological receptor with a recognition function for a specific substance with an electrical or optical transducer.

[0004] As an example of a conventional biosensor, there is a method that attaches a separate fluorescent label to a target biological substance and then detects the biological substance using an optical scanner, etc. However, this method has the problem of requiring a large analytical device with an expensive optical system for signal detection, and there is a problem of having to uniformly label various target substances with fluorescent substances. In addition, there was a concern that the labeling would cause structural changes in the biological substance, real-time monitoring was impossible, the various preparation steps for labeling were cumbersome, and there was even a problem of contamination.

[0005] To address these issues, a biosensor has recently been provided as an electrochemical sensor using optical components. This consists of a sensor head that transmits an optical signal and an antibody that binds to the sensor head. After the antibody captures the target antigen to be detected, an optical signal for measuring the concentration of the biomarker is transmitted through the sensor head, and the target antigen is quantitatively detected through changes in the optical signal.

[0006] Such electrochemical sensors are divided into a two-electrode system consisting of a working electrode and a reference electrode, and a three-electrode system including a counter electrode, and by using these, the potential, current, charge, etc. change in response to the concentration of a specific component of the measurement solution, which is related to the concentration of the specific component. Various methods are used to fix the detection substance to the working electrode for biomarker quantification, such as spin coating, dip coating, roll coating, screen coating, spray coating, spin casting, flow coating, ink jet, and drop casting, and drop casting is particularly widely used.

[0007] However, in the process of fixing the fixing material to fix the detection material to the electrode, various methods including the drop casting above have a problem in that the fixing material is fixed to an electrode other than the working electrode, and there is a problem in that there is a deviation in the amount of detection sample, which is limited by the skill of the manufacturer.

[0008] In addition, there is a need for a technical proposal for a method for manufacturing an electrochemical sensor capable of detecting a biomarker in a very economical range without wasting an excessive amount of sample by confirming the value of the amount of fixing material to effectively and effectively fix the fixing material to the working electrode through the surface treatment process of the electrode.

[0009] In this regard, Korean Patent Publication No. 10-2021-0113799 presents a technology for 'electrochemical sensor and manufacturing method thereof', but this technology is only a technology for providing an electrochemical sensor that minimizes the influence of changes in external chlorine concentration by forming an organic polymer film on a reference electrode, and thus does not present a technical proposal for a manufacturing method of an electrochemical sensor that efficiently fixes a fixing substance on a working electrode, and thus fails to solve the existing problems.

[0010] The present invention was derived to solve the above problems, and aims to provide an electrochemical sensor and a method for manufacturing the same, which can most efficiently and maximally fix a fixed substance to the surface of a working electrode while preventing a fixed substance from being fixed to an electrode other than a working electrode.

[0011] In order to achieve the above task, a first aspect of the present invention provides an electrochemical sensor, comprising: a housing including an electrode portion including a working electrode and vertically connected to a channel portion located at a lower portion; a support including an electrode portion including a working electrode and vertically connected to the housing located at an upper portion; and an electrode portion including an working electrode inserted between the housing and the support; wherein the support is formed with a first channel portion having a groove formed so that the working electrode is positioned correspondingly and a second channel portion having a groove formed so that a counter electrode or a reference electrode as an electrode other than the working electrode is positioned correspondingly; and a fluid trap portion including a partition wall portion and a slot portion is formed on one side of an outer periphery of the first channel portion of the support, wherein the fluid trap portion is characterized in that when an external pressure (P2) of the fluid trap portion is greater than or equal to an internal pressure (P1), a fluid is trapped in the fluid trap portion, and when the external pressure (P2) of the fluid trap portion is less than the internal pressure (P1), a fluid in the fluid trap portion is discharged to the outside of the fluid trap portion.

[0012] In one embodiment of the present invention, the bulkhead portion may be a triangular pillar, and the slot portion may be formed between the bulkhead portions, but is not limited thereto.

[0013] In one embodiment of the present invention, the triangular prism is an equilateral triangular prism, and the ratio of the length of one side of the base of the equilateral triangular prism to the length of the slot portion may be 15 to 20:1, but is not limited thereto.

[0014] In one embodiment of the present invention, the triangular prism is an equilateral triangular prism, and the ratio of the length of one side of the base to the height of the equilateral triangular prism may be 17:20 to 30, but is not limited thereto.

[0015] In one embodiment of the present invention, the housing may include, but is not limited to, a first injection port formed on one side corresponding to the working electrode; a second injection port formed on one side of the counter electrode to form a side flow at the electrode; and an outlet port formed on one side of the reference electrode so that the solution injected into the second injection port passes through the electrode section and is discharged.

[0016] In one embodiment of the present invention, the support may further include, but is not limited to, a second injection hole connected to the second channel portion and connected to the second injection port and a discharge hole connected to the discharge port.

[0017] The second aspect of the present invention provides a method for manufacturing an electrochemical sensor, comprising: S1) a step of treating an electrode surface with sulfuric acid, wherein the electrode part including the working electrode of the electrochemical sensor according to any one of claims 1 to 6 is subjected to sulfuric acid treatment while applying voltage while increasing the voltage from -0.4 V to +1.4 V in preset units at a scan rate of 100 mV / s and, conversely, while decreasing the voltage from +1.4 V to -0.4 V in preset units; S2) a step of treating a protein G conjugate for fixing a protein G conjugate to the working electrode after the step of treating the electrode surface with sulfuric acid; and S3) a step of treating an antibody for fixing an antibody, which is an electrochemical signal material, to the working electrode after the step of treating the protein G conjugate.

[0018] In one embodiment of the present invention, in the step S2) of processing the protein G conjugate, the protein G conjugate is a thiolated protein G (ProteinG-SH), and the concentration of the thiolated protein G may be 40 μg / ml or more, but is not limited thereto.

[0019] In one embodiment of the present invention, the antibody treatment step S3) may have an antibody concentration of 2 ㎍ / ㎖ or more, but is not limited thereto.

[0020] In one embodiment of the present invention, the method for manufacturing the electrochemical sensor may additionally include, but is not limited to, a binding inhibition treatment step (S4) of treating with a protein non-specific binding inhibitor after the S3) antibody treatment step.

[0021] According to the present invention, a fixed substance can be effectively and stably fixed only to the working electrode of an electrochemical sensor.

[0022] This allows for the provision of highly sensitive and accurate electrochemical sensors for biomarker detection.

[0023] In addition, electrochemical sensors can be manufactured economically by reducing unnecessary processing steps and costs.

[0024] Figure 1 is an exploded perspective view of the electrochemical sensor of the present invention.

[0025] Figure 2 is a perspective view of the combination of Figure 1.

[0026] Figure 3 is an enlarged view of a portion of the support and fluid trap section.

[0027] Figure 4 is a conceptual diagram of the action of fluid pressure in the fluid trap section.

[0028] Figure 5 is a flow diagram of the fluid flowing into the first inlet.

[0029] Figure 6 is a fluid flow diagram of the process in which the fluid introduced into the second inlet is discharged through the outlet.

[0030] Figure 7 is a block diagram of a surface treatment process of an electrochemical sensor in one embodiment of the present invention.

[0031] Figure 8 is a block diagram of a bio-marker detection process using an electrochemical sensor in one embodiment of the present invention.

[0032] Figure 9 is a block diagram showing the flow of a surface treatment method, which is one of the methods for manufacturing an electrochemical sensor of the present invention.

[0033] Figure 10 is a graph confirming the change in surface condition according to the number of times the electrode is treated with sulfuric acid.

[0034] Figure 11 is a graph showing the state in which a protein G conjugate is fixed to a working electrode after sulfuric acid treatment of the electrode in one embodiment of the present invention and the subsequent process.

[0035] Figure 12 is a graph showing the state value of the protein G complex immobilized on the operating electrode according to the change in the concentration of thiolated protein G (ProteinG-SH) in one embodiment of the present invention.

[0036] Figure 13 is a graph showing the signal difference according to the incubation time of thiolated protein G (ProteinG-SH) on the working electrode in one embodiment of the present invention.

[0037] Figure 14 is a graph showing the signal difference between antibody-treated and thiolated protein G (ProteinG-SH)-treated states in one embodiment of the present invention.

[0038] Figure 15 is a block diagram showing a surface treatment method in which a step of treating a protein non-specific binding inhibitor is added in a method for manufacturing an electrochemical sensor according to one embodiment of the present invention.

[0039] Figure 16 shows the results of confirming the signal processing of the target antigen after drop casting bovine serum albumin (BSA) at different concentrations onto the working electrode.

[0040] Figure 17 is a graph of electrical signal values ​​for target antigen processing by concentration.

[0041] Hereinafter, the present invention will be described in detail with reference to the attached drawings and implementation examples or embodiments so that a person having average knowledge in the technical field pertaining to the present invention can easily reproduce the present invention.

[0042] The present invention may be implemented in many different forms and is not limited to the implementation examples and embodiments described herein.

[0043] Throughout this specification, whenever a part is said to “include” a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise specifically stated.

[0044] Throughout this specification, when a part is said to be “connected” with a component, this includes the meaning that the problem-solving principle may be substantially the same as that of the combined part, even if there are parts to which other components may be added, or that the part is indirectly connected.

[0045] Throughout this specification, when a step is said to be located “before” or “after” another step, this includes not only cases where the step is directly connected to the other step, but also cases where another step exists between the two steps.

[0046] The terms “about,” “substantially,” etc., used throughout this specification are used in a meaning that is at or close to the numerical value when manufacturing and material tolerances inherent to the meaning referred to are presented, and are used to prevent unscrupulous infringers from unfairly exploiting the disclosure in which exact or absolute values ​​are mentioned to aid understanding of the present invention.

[0047] The terms “step of” or “step of” used throughout this specification do not mean “step for”.

[0048]

[0049] FIG. 1 is an exploded perspective view of the electrochemical sensor of the present invention, FIG. 2 is a combined perspective view of FIG. 1, FIG. 3 is an enlarged view of a portion of a support and a fluid trap section, and FIG. 4 is a conceptual diagram of the action of fluid pressure in the fluid trap section. Referring to FIGS. 1 to 4, the present invention will be described in detail. A first aspect of the present invention includes: a housing (200) including an electrode section (100) including an operating electrode (110) and vertically connected to a channel section (300) located at the bottom; a support (300) including an electrode section (100) including an operating electrode (110) and vertically connected to the housing (200) located at the top; An electrode part (100) including an operating electrode (110) inserted between the housing (200) and the support (300); wherein the support (300) is formed with a first channel part (310) having a groove formed so that the operating electrode (110) is positioned correspondingly and a second channel part (320) having a groove formed so that a counter electrode (120) or a reference electrode (130) as an electrode other than the operating electrode is positioned correspondingly, and a fluid trap part (330) including a partition wall part (331) and a slot part (332) is formed on one side of the outer periphery of the first channel part (310) of the support, and when the external pressure (P2) of the fluid trap part is greater than or equal to the internal pressure (P1), the fluid is trapped in the fluid trap part (330), and when the external pressure (P2) of the fluid trap part is less than the internal pressure (P1), the fluid is trapped in the fluid trap part (330). An electrochemical sensor can be provided, characterized in that the fluid is discharged outside the fluid trap section.

[0050]

[0051] The above housing (200) is connected vertically with the support (300) and includes an electrode part (100) inside. The housing may be made of glass, for example, but is not particularly limited thereto.

[0052]

[0053] The housing (200) may include an inlet for injecting a sample or a treatment solution and an outlet for discharging the injected sample or treatment solution. In one embodiment of the present invention, the housing (200) may include, but is not limited to, a first inlet (210) formed on one side of a position corresponding to the working electrode (110), as shown in FIG. 2; a second inlet (220) formed on one side of the counter electrode (120) to form a side flow at the electrode; and an outlet (230) formed on one side of the reference electrode (130) so that the solution injected into the second inlet (220) passes through the electrode unit (100) and is discharged.

[0054]

[0055] FIG. 5 is a flow diagram of a fluid introduced into a first inlet, and FIG. 6 is a fluid flow diagram of a process in which a fluid introduced into a second inlet is discharged through an outlet. Referring additionally to FIGS. 5 and 6, the first inlet (210) is a place where a fixing substance for the electrode surface, such as a thiolated protein (ProteinG-SH), an antibody, or bovine serum albumin (BSA), or other treatment solution, is injected into the working electrode (110). The first inlet may be formed as a hole on one side of a position corresponding to the working electrode (110), and the fixing substance or treatment solution reaches the first channel portion (310) described below through the first inlet and comes into contact with the working electrode (110). The injected solution is primarily trapped in the fluid trap portion (330) described in detail below, so that only the working electrode can be effectively fixed with the fixing substance.

[0056]

[0057] The second injection port (220) is where a body fluid sample or treatment solution is injected, and as shown in FIG. 6, the body fluid sample or treatment solution injected through the second injection port (220) reaches the second channel section (320), passes through the fluid trap section (330), forms a lateral flow passing through the first channel section (310), reacts with the operating electrode (110), and the treatment solution is discharged through the discharge port (230).

[0058]

[0059] The mutual positions of the second inlet (220) and the outlet (230) may be formed symmetrically with the first inlet (210) connected to the first channel portion as an axis to form a side flow and may be a structure that passes through the fluid trap portion (330). For example, as shown in FIGS. 2 and 5, the second inlet (220) may be formed on one side of the counter electrode (120), and the outlet (230) may be formed on one side of the reference electrode (130) at a position symmetrical to the second inlet with the first inlet as an axis, but is not limited thereto. Any structure in which the solution injected into the second inlet (220) passes through the fluid trap portion (330), passes through the working electrode (110) located in the first channel portion, and then passes through the fluid trap portion (330) again to discharge the fluid to the outlet (230) is possible and is not particularly limited.

[0060]

[0061] Referring to FIGS. 1 to 4, the present invention will be described in detail. The support (300) includes an electrode portion (100) including a working electrode (110), and is vertically connected to the housing (200) located at the top. The support (300) may be made of a material selected from the group consisting of silicon (Si), polyester (PET) film, glass, quartz, alumina (Al2O3), PMMA (polymethylmethacrylate), PS (polystyrene), and plastics such as COC (cyclic olefin copolymer), but is not limited thereto.

[0062]

[0063] Referring to FIGS. 1 and 3, a first channel portion (310) and a second channel portion (320) are formed in the support (300) to allow a fluid to be fixed or flowed, and the first channel portion (310) has a groove formed so that the operating electrode (110) is positioned correspondingly, and the second channel portion (320) has a groove formed so that the counter electrode (120) or the reference electrode (130) is positioned correspondingly, and the electrodes are positioned in the grooves, and the fluid is fixed or flows through the grooves.

[0064]

[0065] Referring to FIG. 4, as one feature of the present invention, the first channel portion (310) is formed with a fluid trap portion (330) including a partition portion (331) and a slot portion (332) on one side of its outer periphery. The fluid trap portion (330) is characterized in that when the external pressure (P2) of the fluid trap portion is greater than or equal to the internal pressure (P1), the fluid is trapped in the fluid trap portion (330), and when the external pressure (P2) of the fluid trap portion is less than the internal pressure (P1), the fluid within the fluid trap portion is discharged outside the fluid trap portion.

[0066]

[0067] The internal pressure (P1) and external pressure (P2) formed by the partition wall (331) and slot portion (332) of the fluid trap portion (330) can be explained according to (b) of Fig. 4 and the following <Equation 1>.

[0068]

[0069] <Formula 1>

[0070]

[0071]

[0072] Here, each parameter value is as follows.

[0073]

[0074]

[0075] E tot : total interfacial energy

[0076] A sl,sg,lg : interface area (solid-liquid, solid-gas, liquid-gas)

[0077] : surface tension (solid-liquid, solid-gas, liquid-gas)

[0078] : equilibrium contact angle

[0079]

[0080] Referring to FIG. 4, FIG. 5 and the above <Formula 1>, the pressure ΔP=P1-P2 in the slot portion (332) formed according to the structure of the partition wall portion of the fluid trap portion (330) is explained. When comparing the area value in the direction of the second channel portion (320) in the slot portion (332), A sl Compared to the area value of A lg Since the area value of increases significantly, the value of ΔP becomes negative (P2 〉P1), so the fluid that enters the first inlet (210) is temporarily trapped inside the fluid trap section (330).

[0081]

[0082] Meanwhile, referring to FIG. 4, FIG. 6 and the above <Formula 1>, the flow of the fluid injected into the second injection port is explained. As shown in FIG. 6, the fluid injected into the second injection port (220) passes through the fluid trap portion (330) in the injection direction from the second channel portion (320-1) and flows into the first channel portion (310), and then passes through the fluid trap portion to the second channel portion (320-2) in the discharge direction to form a side flow, and the fluid can flow into the discharge port (230). Referring to FIG. 4 and the above <Formula 1>, the fluid injected into the second channel portion can cause A in the slot portion to flow. lg As the area value of decreases, the value of ΔP becomes positive (P1 〉P2), so that fluid can flow into the fluid trap section through the slot section, and the introduced fluid can be discharged through the outlet (230) while forming a side flow as the fluid flows out of the fluid trap section again.

[0083]

[0084] In one embodiment of the present invention utilizing the principle of surface tension of a fluid, the partition wall portion (331) may be a triangular pillar, and the slot portion (332) may be formed between the partition walls, but is not limited thereto.

[0085]

[0086] For example, the triangular prism is an equilateral triangular prism, and the ratio of the length of one side of the base (see L1 in FIG. 3) to the length of the slot portion (see L2 in FIG. 3) in the equilateral triangular prism may be 15 to 20:1, but is not limited thereto. Preferably, the ratio of the length of one side of the base to the length of the slot portion in the equilateral triangular prism may be 17:1, but is not limited thereto.

[0087]

[0088] In one embodiment of the present invention, the triangular prism is an equilateral triangular prism, and the ratio of the length of one side of the base (see L1 of FIG. 3) to the height (see H of FIG. 3) of the equilateral triangular prism may be 17:20 to 30, but is not limited thereto. Preferably, the ratio of the length of one side of the base to the height of the equilateral triangular prism may be 17:25, but is not limited thereto.

[0089]

[0090] Due to these characteristics of the present invention, it can provide the advantage of stably fixing a fixed substance only to the working electrode of an electrochemical sensor, and in the process of detecting a bio-marker through the electrochemical sensor of the present invention, it can provide the advantage of being applicable to the entire electrode section including the working electrode.

[0091]

[0092] FIG. 7 is a block diagram of a surface treatment process of an electrochemical sensor according to an embodiment of the present invention, and FIG. 8 is a block diagram of a biomarker detection process using an electrochemical sensor according to an embodiment of the present invention. First, referring to FIG. 7, an embodiment of a surface treatment process of the electrochemical sensor according to the present invention will be described. In a process for manufacturing an electrochemical sensor, if a process for fixing a fixing material such as thiolated protein G (ProteinG-SH) to the working electrode is required, that is, if surface modification of the sensor is required, the fixing material such as thiolated protein is first injected through the first inlet. The fixing material injected through the first inlet is trapped only in the fluid trap part included in the first channel part, and the fixing material is fixed only to the working electrode without affecting the counter electrode or the reference electrode at all. And after a certain incubation time, through a washing process of injecting phosphate-buffered saline (PBS) through the second inlet, the washing solution flows from the second channel section through the fluid trap section into the first channel section, washes away unfixed substances, forms a side flow, and then flows back out to the second channel section and is discharged through the outlet. For example, in the case of fixing an antibody, the above process can be easily repeated to stably fix it to the working electrode.

[0093]

[0094] And referring to FIG. 8, an embodiment of the process of detecting a biomarker of the present invention will be described. When a body fluid sample is injected into the second inlet, the body fluid sample passes through the fluid trap from the second channel section and reacts with antibodies fixed to the working electrode in the first channel section (antigen-antibody binding), and after a certain period of incubation, a washing process is performed by injecting phosphate-buffered saline (PBS) through the second inlet. The washing solution flows from the second channel section through the fluid trap section into the first channel section to wash away unfixed substances, form a side flow, and then flows back out to the second channel section and is discharged through the outlet. In this manner, when an oxidation-reduction aqueous solution is subsequently injected into the second inlet, the reaction substance at the working electrode undergoes an oxidation-reduction reaction, and by measuring the electric signal thereof, the concentration of the biomarker can be easily and stably analyzed.

[0095]

[0096] Referring to FIGS. 1 and 2, the present invention will be described in detail. In one embodiment of the present invention, the support (300) may additionally include, but is not limited to, a second injection hole (321) connected to the second inlet (220) and connected to the second channel portion, and a discharge hole (322) connected to the discharge portion (230). The second injection hole (321) buffers the fluid flowing into the second inlet (220) before moving it to the second channel portion, captures the solution, and then moves the fluid to the second channel portion (320) through the second channel inlet (323), thereby forming a smooth side flow and contributing to the stability of the fluid reaction. In addition, the above discharge hole (322) can perform the function of collecting the solution in which the reaction has been completed through the discharge path (324) by the side flow and stably discharging the solution through the discharge port (230).

[0097]

[0098] Referring to FIGS. 1 and 2, the present invention will be described in detail. The electrode unit (100) is positioned between the housing (200) and the support (300), and includes a working electrode (110). It may be a three-electrode structure including a counter electrode (120) and a reference electrode (130) as an example herein, but is not limited thereto. It may also be a two-electrode structure composed of only a working electrode and a reference electrode.

[0099]

[0100] The electrode part (100) can be inserted and fixed between the housing (200) and the support (300), and for example, after depositing the electrode part (100) on the housing (200), the housing (200) including the electrode can be covered on top of the support (300) to form an exemplary structure of an electrochemical sensor, but is not particularly limited thereto.

[0101]

[0102] FIG. 9 is a block diagram showing the flow of a surface treatment method, which is one of the methods for manufacturing an electrochemical sensor of the present invention, and the present invention will be described in detail with reference to FIG. 9, and a second aspect of the present invention can provide a method for manufacturing an electrochemical sensor, including: S1) an electrode surface sulfuric acid treatment step of applying a voltage while increasing the voltage in preset units from -0.4 V to +1.4 V at a scan rate of 100 mV / s and, conversely, applying the voltage while decreasing it in preset units from +1.4 V to -0.4 V, while treating the electrode part including the working electrode of the electrochemical sensor according to any one of claims 1 to 6 with sulfuric acid; S2) a protein G conjugate treatment step of fixing a protein G conjugate to the working electrode after the electrode surface sulfuric acid treatment step; and, S3) an antibody treatment step of fixing an antibody, which is an electrochemical signal material, to the working electrode after the protein G conjugate treatment step.

[0103] And, applying voltage while increasing it in preset units from -0.4 V to +1.4 V and conversely applying voltage while decreasing it in preset units from +1.4 V to -0.4 V while performing sulfuric acid treatment is defined as one time or one cycle.

[0104] For example, the preset unit may be 0.01 V, and accordingly, the sulfuric acid is processed while applying voltage to the electrode including the working electrode at a scan rate of 100 mV / s and increasing the voltage from -0.4 V to +1.4 V in steps of 0.01 V, and then decreasing the voltage again from +1.4 V to -0.4 V in steps of 0.01 V.

[0105] That is, the voltage is applied in the order of -0.4V to -0.39V, -0.38V, -0.37V… to +1.4V, and then again in the order of +1.4V to +1.39V, +1.38V, +1.37V… to -0.4V while processing sulfuric acid.

[0106] Of course, the preset unit is not limited to 0.01V and can be set differently.

[0107]

[0108] Fig. 10 is a graph confirming the change in surface condition according to the number of times the electrode is treated with sulfuric acid, and Fig. 11 is a graph confirming the state value of the protein G conjugate fixed to the electrode by performing a process thereafter after the sulfuric acid treatment of the electrode in one embodiment of the present invention.

[0109]

[0110] Referring to FIGS. 10 and 11, when the working electrode (110), the counter electrode (120), and the reference electrode (130) are treated with sulfuric acid while applying voltage at a scan rate of 100 mV / s and increasing the voltage from -0.4 V to +1.4 V in preset units, and conversely, decreasing the voltage from +1.4 V to -0.4 V in preset units, a current value of a significant numerical value can be confirmed as in FIG. 11. At this time, it is preferable to use sulfuric acid at a concentration of 30 to 70 mM, and it can be performed by drop casting on the electrode part (100) at about 50 to 90 μl, but is not limited thereto.

[0111]

[0112] In particular, as shown in Fig. 10, when the voltage was applied sequentially from the first time while increasing the voltage from -0.4 V to +1.4 V in preset units at a scan rate of 100 mV / s and conversely decreasing the voltage from +1.4 V to -0.4 V in preset units, and when sulfuric acid was applied, it can be confirmed that the shape of the graph shows similar values ​​from the 15th time or more, and through this, it can be confirmed that a significant value can be obtained in a process of treating the electrode part with sulfuric acid 15 times or more.

[0113]

[0114] Referring to FIG. 9, the present invention will be described in detail. The S2) protein G conjugate treatment step is a step of fixing the protein G conjugate to the operating electrode (110) after the electrode surface sulfuric acid treatment step (S1).

[0115] The protein G conjugate may be one in which the cysteine ​​tag is directly covalently linked to protein G, or may be linked via a linker, wherein the linker is inserted between protein G and the cysteine, but is not particularly limited. For example, the linker may be a peptide consisting of 2 to 10 amino acids.

[0116]

[0117] FIG. 12 is a graph showing the state value of the protein G conjugate fixed to the working electrode according to the change in the concentration of thiolated protein G (ProteinG-SH) in one embodiment of the present invention. Referring to FIG. 12, in the step S2) of processing the protein G conjugate, the concentration of the thiolated protein G (ProteinG-SH) may be 40 μg / ml or more, but is not limited thereto. Since it can be confirmed that a similar signal difference is shown when the concentration of the thiolated protein G (ProteinG-SH) is 40 μg / ml or more, the numerical significance lies in the fact that the fixing material can be efficiently fixed to the electrode by selecting the most appropriate concentration.

[0118]

[0119] Fig. 13 is a graph showing a signal difference according to the incubation time of thiolated protein G (ProteinG-SH) on an operating electrode in one embodiment of the present invention. In one embodiment of the present invention, in the step of treating the protein G conjugate S2), it is preferable to incubate at room temperature for 4 hours or more after treating with 40 ㎍ / ㎖ or more of thiolated protein G. As can be seen in Fig. 13, although the difference in average signal value is similar depending on the incubation time, the significance of numerical limitation lies in the fact that the error range is the smallest at 4 hours.

[0120]

[0121] FIG. 14 is a graph showing the signal difference after treatment with thiolated protein G (ProteinG-SH) and after treatment with an antibody in one embodiment of the present invention. Referring to FIG. 14, the present invention will be described. In one embodiment of the present invention, the S3) antibody treatment step can confirm a similar signal value when the antibody concentration is 2 ㎍ / ㎖ or higher, and thus, the numerical significance lies in the fact that the antibody can be efficiently fixed by selecting the most appropriate concentration.

[0122]

[0123] FIG. 15 is a block diagram showing a surface treatment method in which a step of treating a protein non-specific binding inhibitor is added in a method for manufacturing an electrochemical sensor according to one embodiment of the present invention. Referring to FIG. 15, the present invention will be described. The method for manufacturing an electrochemical sensor according to the present invention may additionally include a binding inhibition treatment step of treating with a protein non-specific binding inhibitor after the S3) antibody treatment step, but is not limited thereto.

[0124]

[0125] The above protein nonspecific binding inhibitor is necessary to suppress distortion of the electric signal value when the target antigen nonspecifically binds to the electrode site where the antibody is not fixed, and in one embodiment of the present invention, the protein nonspecific binding inhibitor is bovine serum albumin (BSA), and may have a concentration of 0.01% or more, but is not limited thereto.

[0126]

[0127] Meanwhile, Fig. 16 is a result of confirming the signal processing of the target antigen after drop casting bovine serum albumin (BSA) on the working electrode according to concentration. As shown in Fig. 16, it can be confirmed that the target antigen signal processing is possible when the concentration of bovine serum albumin (BSA) is 0.01% or higher.

[0128]

[0129] Meanwhile, the present invention can provide a method for detecting a biomarker using an electrochemical sensor, including a target antigen processing step of introducing an antigen into an operating electrode and measuring an electric signal using the electrochemical sensor.

[0130]

[0131] In one embodiment of the present invention, in the target antigen processing step, an antigen at a concentration of 2 to 10 ng / ml may be injected into the working electrode, but is not limited thereto, and FIG. 17 is a graph of electric signal values ​​for target antigen processing by concentration. Referring to FIG. 17, linearity of the electric signal value was confirmed in the range of antigen concentrations of 2 to 10 ng / ml.

[0132]

[0133] Below, specific examples are presented.

[0134]

[0135] [Example 1]

[0136] <Manufacturing of housing and electrode parts>

[0137] (1) After photoresist coating (PR coating) on ​​a glass wafer as a housing and patterning, Ti / Au (30 / 200 nm) was deposited using an E-beam evaporator, and the photoresist was removed to deposit the working electrode and counter electrode, respectively.

[0138] (2) After photoresist coating again, Ti / Ag (50 / 1200 nm) was deposited using an E-beam evaporator, and the photoresist was removed to deposit the reference electrode.

[0139]

[0140] <Support: Manufacturing of the first channel section and the second channel section>

[0141] After photoresist coating on a silicon wafer, a first channel portion and a fluid trap portion were patterned to include a working electrode, and a counter electrode and a reference electrode were patterned to include a side flow, and then etched using a deep RIE method and the photoresist was removed to manufacture a support having a first channel portion and a second channel portion formed thereon.

[0142]

[0143] <Housing and Support Joint>

[0144] An electrochemical sensor was manufactured by combining a housing and a support on which electrodes were deposited using anodic bonding.

[0145]

[0146] [Example 2]

[0147] <Sulfuric acid treatment of electrode surface>

[0148] (1) Electrochemical sensor: Model 'DropSens C220AT Gold Electrode'

[0149] (Working electrode (WE) and counter electrode (CE): Au, reference electrode (RE): Ag / AgCl)

[0150] (2) Sulfuric acid (H2SO4): Prepared at a concentration of 50 mM

[0151] (3) After covering the entire electrode section with 70 μL, surface treatment was sequentially performed from 1 to 16 times at a scan rate of 100 mV / s and a voltage range of -0.4 V to +1.4 V, and the electric signal value was measured using a measuring device (PalmSens Emstat Pico development kit) and shown in Fig. 11. It was confirmed that it is desirable to perform sulfuric acid treatment of the electrode surface at least 15 times at a scan rate of 100 mV / s and a voltage range of -0.4 V to +1.4 V.

[0152]

[0153] [Example 3]

[0154] <Protein G conjugate treatment>

[0155] (1) The working electrode of the sensor cleaned by Example 2 was treated with 70 μL of thiolated protein G (ProteinG-SH) at various concentrations (20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL) and then incubated.

[0156] (2) After incubation, the electrode was washed with 1 ml of phosphate buffered saline (PBS), and then the electrochemical signal material (5 mM [Fe(CN)6] in 0.1 M KCl solution) 3- / 4- After placing the solution) on the working / relative / reference electrode, DPV (differential pulse voltammetry) was measured to measure ΔIp (peak current difference value) for each concentration, which is shown in Fig. 12. It was confirmed that similar signal values ​​were generated above 40 ㎍ / ㎖, and the ΔIp value at 40 ㎍ / ㎖ is shown in Fig. 10.

[0157] (3) After the concentration of thiolated protein G (ProteinG-SH) was set to 40 ㎍ / ㎖, the ΔIp value was measured at room temperature at regular intervals (1 hour, 2 hours, 3 hours, and 4 hours), and the results are shown in Fig. 13. It was confirmed that it is desirable to perform the incubation for approximately 4 hours.

[0158]

[0159]

[0160] [Example 4]

[0161] Antibody treatment

[0162] (1) In Example 3, the working electrode treated with 40 μg / ml thiolated protein G (ProteinG-SH) was treated with antibodies at different concentrations (1 μg / ml, 2 μg / ml, 3 μg / ml, 4 μg / ml) and then incubated.

[0163] (2) After incubation, the electrode was washed with 1 ml of phosphate buffered saline (PBS), and then the electrochemical signal material (5 mM [Fe(CN)6] in 0.1 M KCl solution) 3- / 4- After placing the solution) on the working / relative / reference electrode, DPV (differential pulse voltammetry) was measured to measure ΔIp by concentration, which is shown in Fig. 14. It was confirmed that a similar signal difference was observed after 2 ㎍ / ㎖, and it was confirmed that it is desirable to process the antibody at a minimum of 2 ㎍ / ㎖.

[0164]

[0165] [Example 5]

[0166] <Binding inhibition treatment>

[0167] (1) In Example 4, 7 μL of bovine serum albumin (BSA) in phosphate buffered saline (PBS) was drop casted onto the working electrode treated with an antibody concentration of 2 μg / mL, incubated at room temperature for about 30 minutes, washed with 1 mL of PBS, and then electrochemical signal material (5 mM [Fe(CN)6] in 0.1 M KCl solution) was added. 3- / 4- After drop casting 70㎕ of the solution onto the working / relative / reference electrode, measure DPV (differential pulse voltammetry) to confirm the electrode fixation results.

[0168] (2) Afterwards, 7 μl is drop-casted on the working electrode according to the antigen concentration (in PBS), incubated at room temperature for about 30 minutes, and the signal change is confirmed.

[0169] (3) 7 ㎕ of bovine serum albumin (BSA in PBS) was drop-casted onto the working electrode at various concentrations (0.01 wt%, 0.1 wt%, 1 wt%), incubated at room temperature for about 30 minutes, washed with 1 ㎖ of PBS, and then added to the electrochemical signal material (5 mM [Fe(CN)6] in 0.1 M KCl solution). 3- / 4- After drop-casting 70 ㎕ of the solution onto the working / relative / reference electrode, DPV (differential pulse voltammetry) was measured and the ΔIp value is shown in Fig. 16. It was confirmed that there was a signal change at a level where signal processing of the target antigen was possible at a concentration of 0.01 wt% bovine serum albumin (BSA) or higher.

[0170]

[0171] (Example 6)

[0172] <Target antigen treatment>

[0173] (1) In Example 5, an electrochemical sensor was obtained by surface-treating a working electrode treated with an antibody concentration of 2 μg / ml with bovine serum albumin (BSA in PBS) of 0.01 wt or more.

[0174] (2) After drop-casting 7 ㎕ of antigen concentration (in PBS) on the working electrode of the electrochemical sensor, it was incubated at room temperature for about 30 minutes. After that, it was washed with 1 ㎖ of PBS, and then the electrochemical signal material (5 mM [Fe(CN)6] in 0.1 M KCl solution) was added. 3- / 4- After drop casting 70㎕ of the solution onto the working / relative / reference electrode, DPV (differential pulse voltammetry) was measured and the ΔIp value is shown in Figure 17. It was confirmed that a linear signal was formed in the antigen concentration range of 2 to 10 ng / ㎖.

[0175]

[0176] While the present invention has been described above with reference to implementation examples or embodiments, these are merely illustrative. Those skilled in the art will understand that various modifications and equivalent alternative embodiments are possible. Therefore, the true scope of technical protection of the present invention should be determined by the technical spirit of the appended claims.

Claims

1. A housing that includes an electrode portion including an operating electrode and is connected to a channel portion located at the bottom; A support body that includes an electrode section including an operating electrode and is connected to the housing located at the top; An electrode part including an operating electrode interposed between the housing and the support; The support body is formed with a first channel portion having a groove formed so that the operating electrode is positioned correspondingly, and a second channel portion having a groove formed so that an electrode other than the operating electrode, such as a counter electrode or a reference electrode, is positioned correspondingly. On one side of the outer periphery of the first channel portion of the above support, a fluid trap portion including a partition portion and a slot portion is formed, The fluid trap part is characterized in that when the external pressure (P2) of the fluid trap part is greater than or equal to the internal pressure (P1), the fluid is trapped in the fluid trap part, and when the external pressure (P2) of the fluid trap part is less than the internal pressure, the fluid inside the fluid trap part is discharged outside the fluid trap part. Electrochemical sensor.

2. In paragraph 1, The above bulkhead is a triangular pillar, and the slot is formed between the bulkheads. Electrochemical sensor.

3. In paragraph 2, The above triangular prism is an equilateral triangular prism, and the ratio of the length of one side of the base to the length of the slot portion in the equilateral triangular prism is 15 to 20:

1. Electrochemical sensor.

4. In paragraph 2, The above triangular prism is an equilateral triangular prism, and the ratio of the length of one side of the base to the height of the equilateral triangular prism is 17:20 to 30. Electrochemical sensor.

5. In paragraph 1, The housing has a first injection port formed on one side at a position corresponding to the operating electrode; A second inlet formed on one side of the counter electrode to form a side flow at the electrode; and, It includes an outlet formed on one side of the reference electrode so that the solution injected into the second inlet passes through the electrode section and is discharged; Electrochemical sensor.

6. In paragraph 1, The support body further includes a second injection hole connected to the second channel section and connected to the second injection port, and an exhaust hole connected to the exhaust port. Electrochemical sensor. 7.S1) An electrode surface sulfuric acid treatment step in which the electrode part including the operating electrode of the electrochemical sensor according to any one of claims 1 to 6 is treated with sulfuric acid while applying voltage while increasing the voltage in preset units from -0.4 V to +1.4 V at a scan rate of 100 mV / s and, conversely, while decreasing the voltage in preset units from +1.4 V to -0.4 V; S2) After the electrode surface sulfuric acid treatment step, a protein G conjugate treatment step for fixing the protein G conjugate to the working electrode; and, S3) After the protein G conjugate processing step, an antibody processing step for fixing an antibody, which is an electrochemical signal material, to the working electrode is included. Method for manufacturing an electrochemical sensor.

8. In paragraph 7, In the above S2) protein G conjugate processing step, the protein G conjugate is thiolated protein G (ProteinG-SH), and the concentration of the thiolated protein G is 40 ㎍ / ㎖ or more. Method for manufacturing an electrochemical sensor.

9. In paragraph 7, The above S3) antibody treatment step is one in which the antibody concentration is 2㎍ / ㎖ or more. Method for manufacturing an electrochemical sensor.

10. In paragraph 7, After the above S3) antibody treatment step, a binding inhibition treatment step (S4) is additionally included, in which the protein non-specific binding inhibitor is treated; Method for manufacturing an electrochemical sensor.

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