阅读说明：本技术 用于nadh测量的生物传感器用电极及其制造方法 (Biosensor electrode for NADH measurement and method for manufacturing the same ) 是由 李圭弘 于 2018-12-28 设计创作，主要内容包括：本发明涉及用于NADH测量用生物传感器的电极及其制造方法,在通过根据本发明的方法制造的电极中,在电聚合反应时电流流动稳定,改性后的物质的接触角显着减小,从而具有能够提高表面改性效率并且电极能够重复使用多次的优点。此外,在本发明的所述电极用于NADH测量用生物传感器的情况下,在没有干扰现象的情况下保持高水平的灵敏度和选择性,从而在存在极少量的测量物质而必须进行预处理过程的血液或血清中也能够容易地测量目标对象。此外,在所述电极用于NADH测量用生物传感器的情况下,能够连续且实时地测量细胞活度而能够应用于细胞毒性评估领域,并且能够测量丧失了线粒体功能的死细胞中的细胞活度。(The present invention relates to an electrode for a biosensor for measuring NADH and a method for manufacturing the same, in which current flow is stable at the time of electropolymerization reaction, and a contact angle of a modified substance is remarkably reduced, thereby having advantages that surface modification efficiency can be improved and the electrode can be repeatedly used several times. Further, in the case where the electrode of the present invention is used for a biosensor for measuring NADH, a high level of sensitivity and selectivity is maintained without interference phenomenon, so that a target object can be easily measured also in blood or serum in which a pretreatment process is necessary in the presence of an extremely small amount of a measuring substance. Further, in the case where the electrode is used for a biosensor for measuring NADH, the cell activity can be continuously measured in real time, and can be applied to the field of cytotoxicity assessment, and the cell activity in dead cells that have lost mitochondrial function can be measured.)

1. A method of manufacturing an electrode for a biosensor, comprising:

step a): washing the electrode with sulfuric acid;

step b): placing the electrode of step a) into 4-aminothiophenol and culturing, then immersing into the first solution, and then applying a voltage; and

step c): immersing the electrode of step b) in a second solution and then applying a voltage.

2. The method for manufacturing an electrode according to claim 1, wherein the first solution of step b) is a phosphate buffer having a molarity of 90mM to 100 mM.

3. The method for manufacturing an electrode according to claim 1, wherein the second solution of the step c) is a phosphate buffer having a molarity of 5mM to 15 mM.

4. The electrode manufacturing method according to claim 1, wherein in the step b) and the step c), the voltage is applied by cyclic voltammetry.

5. The electrode manufacturing method according to claim 4, wherein, in the step b) and the step c), the cyclic voltammetry scans at a potential of 0.8V to-0.4V.

6. The electrode manufacturing method according to claim 1, wherein the electrode for biosensor is used for measuring NADH (reduced state of nicotinamide adenine dinucleotide).

7. The electrode manufacturing method according to claim 1, wherein the electrode is one or more selected from the group consisting of gold, aluminum, platinum, nickel, graphene, a silver nanowire film, a metal grid, carbon, and indium tin oxide.

8. An electrode for biosensor, which is manufactured by the electrode manufacturing method according to any one of claims 1 to 7 and is used for measuring NADH.

9. A biosensor comprising an electrode according to any one of claims 1 to 7.

Technical Field

The present invention relates to an electrode for a biosensor and a method for manufacturing the same, and more particularly, to an electrode for a biosensor for measuring NADH (reduced state of NAD, reduced state of nicotinamide adenine dinucleotide) and a method for manufacturing the same.

Background

With the development of biotechnology, the demand and demand for faster and more accurate analysis techniques is increasing. Among these analytical techniques, many studies on biosensors have recently been conducted. In particular, the demand for a biosensor, which is a biological device capable of sensing physical or chemical stimuli from the outside, is increasing by applying electronic engineering by simulating biological functions. These biosensors are attracting much attention because of their very high selectivity or sensitivity to a measurement object.

Various studies have been made on biosensors so far, and these biosensors can be classified into biomaterials using enzymes, fungi, and tissues such as animals and plants. It can be divided into a moiety that recognizes a specific molecule and causes a physical or chemical change proportional to its concentration and a moiety that converts the chemical change of these substances into a site of an electrical signal. The biosensor is particularly capable of selectively identifying a specific molecule in a sample and does not require separate purification, and thus has advantages of very short detection time and very high accuracy.

Electrochemical-based biosensors combine the analytical capabilities of electrochemical methods with the specificity of biological recognition. That is, the biorecognition phenomenon is detected by a change in current or potential by immobilizing or including a biospecific reagent such as an enzyme, an antigen, an antibody, or a biochemical substance on the surface of an electrode. In such electrochemical-based biosensors, the resistance of the electrode itself and the surface characteristics of the electrochemical reaction taking place are very important.

In addition, maintenance of the homeostasis of mitochondria, which is a small organ of a cell responsible for ATP synthesis, which is essential in life activities, is very important in life activities, and when an abnormality occurs, it may cause simultaneous multiple diseases such as metabolic disorders and cerebral stroke. In order to confirm the function of mitochondria, coenzyme generated during respiration in mitochondria is commonly used. In particular, Nicotinamide Adenine Dinucleotide (NAD) is one of important coenzymes found in cells, and is widely used in glycolysis and TCA cycle in cell respiration, and a reduction potential stored in nadh (reduced form of Nicotinamide Adenine Dinucleotide) corresponding to a reduction state of the NAD is converted into ATP by an electron transport system or used for assimilation.

Therefore, although many studies are focused on the basic scientific application of NADH reaction, in the case of the conventional light absorption method, NADH measurement in blood has a very serious interference phenomenon, and sensitivity is low and selectivity is significantly reduced, sample consumption is very much, and a pretreatment process is complicated, so that it is difficult to easily perform measurement. Furthermore, the related art WST-1 and MTT (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) assay methods correspond to the following modes: after the chemical substance is applied, NADH factor and color reaction are induced to measure absorbance at a specific wavelength, but this method requires a large amount of sample, has a long reaction time, and is not suitable for continuous and real-time monitoring due to poor decomposition ability. In addition, in order to measure NADH by an electrochemical method, the redox current of NADH is measured after an enzymatic reaction is induced or a mediator of ruthenium or cyanide ions, which is a catalytic material actively performing electron transport, is put in, but in this case, since oxidation and reduction generally occur at a voltage value of 1000mV or more, the electrode surface is damaged and repeated measurement is impossible, and the decomposition ability is remarkably decreased.

Disclosure of Invention

Technical problem

The present inventors have found the following fact to complete the present invention: in the modification of the surface of an electrode for biosensors using 4-aminothiophenol, it is prepared by a step of changing the concentration of an immersion solution at the time of applying a voltage among the steps of applying cyclic voltammetry, thereby not only remarkably improving the modification efficiency but also having high sensitivity and selectivity and being capable of being reused many times.

In the present invention, there is provided an electrode manufacturing method for a biosensor, comprising:

step a): washing the electrode with sulfuric acid;

step b): placing the electrode of step a) into 4-aminothiophenol and culturing, then immersing into the first solution and applying a voltage; and

step c): immersing the electrode of step b) in a second solution and applying a voltage.

In the present invention, the first solution of step b) may be a phosphate buffer having a molarity of 90mM to 100 mM.

Further, in the present invention, the second solution of step c) may be a phosphate buffer having a molarity of 5mM to 15 mM.

In the present invention, in the step b) and the step c), a voltage may be applied by cyclic voltammetry.

Further, in the present invention, in the step b) and the step c), the scanning may be performed at a potential of 0.8V to-0.4V by cyclic voltammetry.

In addition, in the present invention, the electrode may be one or more selected from the group consisting of gold, aluminum, platinum, nickel, graphene, a silver nanowire film, a metal grid, and indium tin oxide.

In the present invention, the electrode for biosensor can be used for measuring NADH (reduced state of nicotinamide adenine dinucleotide).

There is provided an electrode for biosensors, which is manufactured by the manufacturing method according to the present invention and is used to measure NADH.

There is provided a biosensor comprising an electrode according to the invention.

Hereinafter, the electrode for biosensors for measuring the NADH according to the present invention will be described in detail.

In an embodiment of the present invention, there is provided an electrode manufacturing method for a biosensor, including: step a): washing the electrode with sulfuric acid; step b): placing the electrode of step a) in 4-aminothiophenol (4-ATP) and culturing, then immersing in the first solution, and then applying a voltage; and step c): immersing the electrode of step b) in a second solution and then applying a voltage.

In the present invention, the step of washing the electrode with sulfuric acid in the step a) may be 1 hour to 3 hours, preferably 2 hours, but is not limited thereto. When the electrode is washed for less than 1 hour and more than 3 hours, etching of the electrode surface cannot be sufficiently performed.

In the present invention, the electrode for biosensor can be used for measuring NADH.

In the present invention, the "Biosensor (Biosensor)" refers to an analysis device that combines a physicochemical detector with a substance capable of reacting with a bio-element or an analyte, and is used to search for the analyte. By engineering sensitive biological elements, biologically isolated samples, etc., signals can be converted so that signals generated by interaction with the analyte substance can be more easily measured and quantified. For the purposes of the present invention, the substance which can react with the analyte can be N-phenyl quinone diimine which is used for measuring NADH.

Further, in the present invention, in the step a), the molar concentration of the sulfuric acid may be 5mM to 15mM, preferably 10mM, but is not limited thereto. In the case where the molar concentration of sulfuric acid is less than 5mM and more than 15mM, the surface of the electrode may not be sufficiently etched or the electrode may be corroded.

In the present invention, the "4-aminothiophenol" is an organic substance capable of forming a self-assembled monolayer as a regular ordered organic molecular film spontaneously coated on the surface of an electrode, and NH of the 4-aminothiophenol can be changed by electrons generated during the oxidation and reduction of NADH₂To measure the NADH content of the sample.

In the present invention, the first solution and the second solution may be phosphate bufferedLiquid (PBS). For the purpose of the present invention, the first solution may be a phosphate buffer solution having a molarity of 90mM to 100mM, and the second solution may be a phosphate buffer solution having a molarity of 5mM to 15mM, but is not limited thereto. As described above, when the electropolymerization reaction is performed by changing the molar concentrations of the first solution and the second solution, the contact angle between N-phenyl quinone diimine and the electrode can be from 48 by stabilizing the current flowIs reduced to 39And the electrode can be reused up to 20 times. In addition, in the present invention, in the case where the molar concentration of the first solution is less than 90mM and more than 100mM, imine cannot be sufficiently produced, and in the case where the molar concentration of the second solution is less than 5mM and more than 15mM, the contact angle cannot be sufficiently reduced as desired in the present invention.

In the present invention, when the contact angle of N-phenyl quinone diimine is decreased as described above, N-phenyl quinone diimine that can be bound to the surface of the electrode is significantly increased, so that the reaction with NADH becomes active, which results in the sensitivity of the electrode, thereby enabling detection also in the case where a very small amount of NADH is present in the sample.

In the present invention, the "cyclic voltammetry" is one of methods capable of directly grasping a reaction occurring on the electrode surface, and when the potential is changed in proportion to time, a method of recording a flowing current as a current-potential curve is repeated a plurality of times to perform potential scanning. For the purpose of the present invention, the step of applying a voltage in the steps b) and c) may be performed by cyclic voltammetry, preferably scanning at a potential of 0.8V to-0.4V, but is not limited thereto.

In one embodiment of the present invention, the electrode according to the present invention may be one or more selected from the group consisting of gold, aluminum, platinum, nickel, graphene, silver nanowire film, carbon, metal grid, and indium tin oxide. Preferably, the electrode may be gold, but is not limited thereto.

In another embodiment of the present invention, there is provided an electrode for a biosensor, which is manufactured by the manufacturing method according to the present invention and is used to measure NADH.

In the electrode for a biosensor of the present invention, contents related to the electrode, the biosensor, the cyclic voltammetry, the first solution and the second solution are repeated as described in the manufacturing method, and detailed description is omitted below.

In a further embodiment of the invention, a biosensor is provided comprising an electrode according to the invention.

In the biosensor of the present invention, the contents related to the electrode, the biosensor, the cyclic voltammetry, the first solution and the second solution are repeated as described in the manufacturing method, and detailed description is omitted below.

In the present invention, the biosensor may be more suitable for measuring NADH for the purposes of the present invention.

The biosensor of the present invention may include a biosensor, which may be generally included in biosensors, an amplifier, a processing machine, an electronic system including a screen, and the like, in addition to the electrode according to the present invention.

In one embodiment of the present invention, the biosensor measures the NADH value in a sample by converting the imine of N-phenyl quinone diimine bound to the surface of an electrode into amine and then converting it into imine again by electrons generated during the oxidation of NADH contained in the sample, and measuring the generated electrons and changing to a quantifiable value. In the case of the related art in which the redox reaction of NADH is measured by a reaction with a dehydratase in mitochondria through a color reaction such as wst-1 or MTT assay, it is necessary to presuppose the complete function of mitochondria, but by the above-described measuring method, in the electrode manufactured by the manufacturing method according to the present invention, the measuring method of the biosensor corresponds to a main reaction rather than an enzymatic reaction, and thus no dehydratase is required, and thus the cell activity can be measured by quantitative measurement of NADH released into a culture solution upon cell death.

FIG. 7 is a schematic view of a process of measuring NADH in a sample using the biosensor according to the present invention, and will be described in detail with reference to the same.

As shown in (a) of fig. 7, in order to measure the presence or absence of NADH in blood, a process of measuring and quantifying the amount of emitted electrons may be performed by a biosensor including the electrode according to the present invention without performing a separate sample pre-treatment process after extracting a sample of a target object.

In addition, as shown in (b) of fig. 7, in order to measure the amount of NADH generated in response to cell death, after a culture solution of a target cell is applied to a biosensor including an electrode according to the present invention, the amount of electrons emitted is measured and quantified, so that the degree of cell death can be measured.

The electrode manufactured by the above-described manufacturing method according to the present invention has a significantly higher degree of binding of N-phenyl quinone diimine compared to the electrode of the prior art, and thus has high sensitivity to be able to measure a small amount of NADH contained in a sample, as in the above-described process, even without performing a separate sample pretreatment.

Advantageous effects

In the electrode manufactured by the method according to the present invention, not only the current flow is stabilized at the time of the electropolymerization reaction, but also the contact angle is remarkably reduced, so that the surface modification efficiency can be improved. In addition, the electrode used in the biosensor may be repeatedly used several times through this process.

In addition, in the case where the electrode according to the present invention is used for a biosensor, sensitivity and selectivity are maintained without interference phenomenon, so that a target object can be measured also in blood or serum without a pretreatment process corresponding to a very small amount, and cell activity can be continuously and real-timely measured, and can be applied to the field of cytotoxicity. In addition, cell activity can be measured by measuring NADH secreted into serum in dead cells that have lost mitochondrial function.

Drawings

Fig. 1 shows a schematic view of a modified surface of an electrode according to an embodiment of the present invention and a measurement result of a degree of hydrophilicity.

Fig. 2 shows a graph measuring the contact angle of imine occurring at the time of surface modification according to an embodiment of the present invention.

Fig. 3 shows the results of cyclic voltammetry measurements according to the concentration of phosphate buffer according to an embodiment of the present invention.

Fig. 4 shows a result of performing image analysis on the surface of the electrode by SEM according to an embodiment of the present invention.

Fig. 5 shows the result of performing image analysis on the surface of the electrode by SEM according to an embodiment of the present invention.

FIG. 6 shows NADH measurements for various concentrations using electrodes in accordance with an embodiment of the present invention.

Fig. 7 (a) and (b) are schematic diagrams showing a NADH measuring process using a biosensor for NADH measurement according to an embodiment of the present invention.

Detailed Description

Hereinafter, the present invention will be described in more detail by examples. These examples are intended only to illustrate the present invention more specifically, and it is obvious to those skilled in the art that the scope of the present invention is not limited by these examples in light of the spirit of the present invention.