KR102695332B1 - Enzyme-substrate reaction-based biosensor for component detection of biological analytes and detection method using the same - Google Patents

Abstract

The present invention relates to a method for quantitatively and qualitatively measuring biological substances or environmental substances using a biosensor, and more specifically, to a method for quantitatively and qualitatively measuring biological substances or environmental substances using a biosensor by directly binding a small-molecular-weight electrochemical signal generator to a substrate used for an enzymatic reaction, and then quantitatively and qualitatively measuring the content of the biological substance by utilizing a competitive reaction or direct substrate decomposition reaction of the metabolic reaction of the enzyme. According to the present invention, by directly binding a small-molecular-weight electrochemical signal generator to a substrate or a metabolic initiating substance, changes in the physical properties of the substrate or the metabolic initiating substance are minimized, and further, by directly binding a spectroscopic and electrochemical signal generator bound to the substrate or the metabolic initiating substance, accurate measurement is possible without introducing an additional reagent for signal detection from the signal generator, and a method for quantitatively measuring biological substances or environmental substances using a 2D or 3D type biosensor can be provided that can conveniently and quickly measure various analytes with only one type of test strip by using various types of signal generators.

Description

{Enzyme-substrate reaction-based biosensor for component detection of biological analytes and detection method using the same}

The present invention relates to a biosensor, and more specifically, to a biosensor based on an enzyme-substrate reaction for detecting components of a biological analyte and a detection method using the same.

In general, the term biosensor refers to a sensor that has biological specificity for a biological substance to be measured, i.e., an analyte. It refers to a device that measures the presence or content of an analyte by fixing biological molecules such as antibodies or enzymes to a support or electrode to selectively detect the analyte, and converting electrons generated or eliminated by binding to a receptor of the analyte into a measurable quantity such as an optical signal or electrical signal.

Measurement methods that utilize the immune reaction between analytes and antibodies are classified into qualitative methods that determine the presence or absence of analytes and quantitative or semi-quantitative methods that measure the amount of analytes. Since quantitative or semi-quantitative methods using spectroscopic detection require expensive analysis equipment such as a fluorescence analyzer, quantitative or semi-quantitative methods using electrochemical reactions are recently preferred because they can be easily measured with simple operation without expensive equipment.

The most notable feature of a biosensor using an immune response according to a conventional technology is that an electron transfer mediator is used. Conventionally, as such electron transfer mediators, ferrocene and ferrocene derivatives; quinone and quinone derivatives; organic and inorganic materials containing transition metals such as hexaamine ruthenium, osmium-containing polymers, and potassium ferricyanide; and electron transfer organic substances such as organic conductive salts or biomass have been used. However, the above-mentioned electron transfer mediators are subject to interference from various interfering substances such as ascorbic acid, acetaminophen, and uric acid among biological measurement substances, and therefore, there is a problem in that accurate measurement is impossible.

And conventional biosensors used fluorescent substances or radioactive isotopes as signal sources. However, expensive measuring equipment was required, and when a signal source such as colloidal metal was attached to the substrate in the enzyme-substrate reaction, the physical properties of the substrate changed, making accurate measurement impossible. In general, when carrier proteins were attached to a substrate, which is a low-molecular substance, errors in the reaction kinetics could occur. In addition, there was a problem that the number of substrates attached to the signal source could not be arbitrarily controlled, which could cause differences between manufacturing batches.

Republic of Korea Patent Publication No. 10-2017-0132861 (Biosensor system for rapid detection of analytes).

Accordingly, the present invention is intended to solve the problems of the above-mentioned prior art, and a first object of the present invention is to provide an enzyme-substrate reaction-based biosensor for detecting components of a biological analyte, which minimizes changes in the physical properties of a substrate or a metabolic initiating substance by directly binding an electrochemical signal generator having a small molecular weight to the substrate or a metabolic initiating substance, and further enables arbitrarily controlling the characteristics of the electrochemical signal generator bound to the substrate or the metabolic initiating substance, and a detection method using the same.

The second object of the present invention is to provide an enzyme-substrate reaction-based biosensor for detection of components of a biological analyte, which enables accurate quantitative measurement without introducing additional reagents for signal detection from a signal generation source, and which can conveniently and quickly measure various analytes using only one type of test sensor strip using various types of signal generation sources, and a detection method using the same.

The third object of the present invention is to provide a biosensor based on an enzyme-substrate reaction for detecting components of biological analytes capable of qualitatively or quantitatively measuring biological or environmental substances by using a sensor surface having a three-dimensional as well as two-dimensional layered structure, and a detection method using the same, by utilizing the role of an electron transfer mediator that directly reacts to an electrode by utilizing the electrochemical properties of the final product of an enzyme-substrate reaction.

However, the technical problems to be achieved in the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

In order to achieve the above technical task, a biosensor based on an enzyme-substrate reaction for detecting components of a biological analyte is provided, comprising: a pair of electrodes installed on a substrate (80), exposed while being spaced apart from each other by a predetermined interval and capable of coming into contact with an analyte; a filter (50) coated on the electrodes and excited as a cathode; and a terminal (90) for outputting an output current (110) generated between the electrodes when a predetermined voltage is applied to an electron transfer mediator (20) that has passed through the filter (50) from the analyte; wherein the electron transfer mediator (20) is characterized in that a positively charged substance based on an enzyme-substrate reaction is covalently bonded to a substrate.

In addition, it further includes a control unit (120) that controls to apply different plural constant voltages for a predetermined voltage.

Additionally, one pair of electrodes is a two-dimensional electrode (75b) exposed on the substrate (80) or a three-dimensional electrode (60a, 60b) protruding on the substrate (80).

The above-described purpose of the present invention can also be achieved by a method for detecting components of a biological analyte using a biosensor based on an enzyme-substrate reaction, which comprises the steps of: (S100) preparing an analyte from a biological sample; (S200) separating the analyte by performing an enzyme-substrate reaction or a metabolic reaction to include a portion to which a positively charged substance, which is an electron transfer mediator (20), is covalently bonded; (S300) passing the electron transfer mediator (20) through a filter (50) excited as a cathode; (S400) generating a current between a pair of electrodes to which a predetermined voltage is applied by the electron transfer mediator (20) passing through the filter (50); and (S500) detecting a component of the analyte based on the output current (110) by outputting the current as an output current (110) through a terminal connected to the electrodes.

Additionally, substances with a net charge of negative are substrates or metabolites, and positively charged substances are positively charged substances with a signal-generating function.

Additionally, the covalent bond between the substrate and the positively charged substance can be performed by an amine-carboxyl coupling method, a thiol coupling method, an avidin-biotin coupling method, or an aldehyde coupling method.

Alternatively, the positively charged material of signaling function is dopamine, quantum dots, ferrocene, ferrocyanide, osmium, europium or ruthenium containing organic/inorganic complexes.

Additionally, the enzyme-substrate reaction can result in the substrate being decomposed by the enzyme reaction alone or by the enzyme activation reaction.

Additionally, the predetermined voltage is generated by controlling the control unit to apply at least one of a plurality of different constant voltages.

Additionally, the positively charged substances of signal generation function are small molecules in the range of 1 to 1000 Dalton.

In addition, an amine group can be attached to the positively charged substance of the above signal generation function, and a covalent reaction can be induced using EDC (N-ethyl-N'-(dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) for a substrate and a metabolic initiator containing a carboxyl group.

According to one embodiment of the present invention, by directly binding a small molecular weight electrochemical signal generator to a substrate or a metabolic initiating substance, changes in the physical properties of the substrate or metabolic initiating substance can be minimized.

In addition, the characteristics of the electrochemical signal generator bound to the substrate or metabolic initiator can be arbitrarily controlled, enabling accurate measurement without introducing additional reagents for signal detection from the signal generator.

Additionally, by using various types of signal sources, various analytes can be conveniently and quickly measured with only one type of test strip.

In addition, by directly coupling a small molecular weight electrochemical signal generator to a substrate or a metabolic starting substance, changes in the physical properties of the substrate or metabolic starting substance are minimized, and accurate measurement is possible without introducing additional reagents.

Additionally, by using various types of signal sources, various analytes can be conveniently and quickly measured with only one type of test strip.

Additionally, different components can be detected by applying different constant voltages to the electrodes.

However, the effects obtainable from the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by a person having ordinary skill in the art to which the present invention belongs from the description below.

The following drawings attached to this specification illustrate preferred embodiments of the present invention, and together with the detailed description of the invention described below, serve to further understand the technical idea of the present invention, and therefore, the present invention should not be interpreted as being limited to matters described in such drawings.
Figure 1 is a schematic diagram of the direct reaction of a typical enzyme-substrate reaction.
Figure 2 is a schematic diagram of a competitive reaction of a typical enzyme-substrate reaction.
Figure 3 is a perspective view of a biosensor (100) having a three-dimensional electrode according to the present invention.
Figure 4a is a perspective view of a pair of three-dimensional electrodes (60a, 60b) among the biosensors (100) illustrated in Figure 3.
Figure 4b is a perspective view showing a three-dimensional electrode (70a, 70b) coated with a filter (50) on the surface of the three-dimensional electrode (60a, 60b) illustrated in Figure 4a.
FIG. 4c is a perspective view of a pair of two-dimensional electrodes (75a, 75b) of the biosensor (100) illustrated in FIG. 3 as another embodiment of the present invention.
Figure 5 is a schematic diagram of an enzyme-substrate reaction according to the present invention and application of an electronic mediator (20).
Figure 6 is a flow chart showing a method for detecting components of a biological analyte using a biosensor based on an enzyme-substrate reaction according to the present invention.

Hereinafter, with reference to the attached drawings, embodiments of the present invention will be described in detail so that a person having ordinary skill in the art to which the present invention pertains can easily implement the present invention. However, since the description of the present invention is merely an embodiment for structural and functional explanation, the scope of the rights of the present invention should not be construed as being limited by the embodiments described in the text. That is, since the embodiments can be variously modified and can have various forms, the scope of the rights of the present invention should be understood to include equivalents that can realize the technical idea. In addition, the purpose or effect presented in the present invention does not mean that a specific embodiment must include all of them or only such effects, and therefore the scope of the rights of the present invention should not be understood as being limited thereby.

The meanings of terms described in the present invention should be understood as follows.

The terms "first", "second", etc. are intended to distinguish one component from another, and the scope of the right should not be limited by these terms. For example, the first component may be referred to as the second component, and similarly, the second component may also be referred to as the first component. When a component is referred to as being "connected" to another component, it should be understood that it may be directly connected to the other component, but there may also be another component in between. On the other hand, when a component is referred to as being "directly connected" to another component, it should be understood that there is no other component in between. Meanwhile, other expressions that describe the relationship between components, such as "between" and "directly between" or "adjacent to" and "directly adjacent to", should be interpreted in the same way.

A singular expression should be understood to include the plural expression unless the context clearly indicates otherwise, and the terms "comprises" or "have" should be understood to specify the presence of a stated feature, number, step, operation, component, part, or combination thereof, but not to exclude the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

All terms used herein, unless otherwise defined, have the same meaning as commonly understood by a person of ordinary skill in the art to which the present invention belongs. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with the contextual meaning of the relevant art, and shall not be interpreted as having an ideal or overly formal meaning unless explicitly defined in the present invention.

Composition of biosensor

The present invention relates to a schematic reaction diagram of a direct reaction of a conventional enzyme-substrate reaction, and a schematic reaction diagram of a competitive reaction of a conventional enzyme-substrate reaction. As shown in FIGS. 1 and 2, a substrate binds to a specific enzyme to form an enzyme-substrate complex, and when separated from the enzyme, a specific product is formed. In the case of a competitive reaction such as that of FIG. 2, a pseudosubstrate instead of a substrate binds to the enzyme to form a specific product.

Fig. 3 is a perspective view of a biosensor (100) having a three-dimensional electrode according to the present invention. As shown in Fig. 3, a substrate (80) has a rectangular parallelepiped shape and is provided with a plurality of terminals (90) for inputting and outputting electric signals at one end. An electrode (70) is provided at the other end of the substrate (80). A pair of electrodes (70) spaced apart from each other is provided, and optionally, two or more pairs may be provided, and different constant voltages are applied to each pair to detect different components.

The control unit (120) may be a microcomputer, CPU or custom semiconductor placed on the back surface of the substrate (80).

FIG. 4A is a perspective view of a pair of three-dimensional electrodes (60a, 60b) of the biosensor (100) illustrated in FIG. 3. As illustrated in FIG. 4A, the pair of three-dimensional electrodes (60a, 60b) protrude from the substrate (80) and are spaced apart from each other. Since the pair of three-dimensional electrodes (60a, 60b) protrude, only the electrodes (60a, 60b) can be immersed in the liquid of the analyte or micro droplets can be dropped only on the electrodes (60a, 60b). The separation distance can be appropriately selected in the range of 1 mm to 10 mm. This is because if they are too close, a short circuit may occur, and if they are too far, the current may be weak. A pair of three-dimensional electrodes (60a, 60b) are applied with a constant constant voltage (e.g., 0.2 V, 0.5 V, 1.0 V, 1.2 V, 1.4 V, ..., 3 V) by the control unit (120). Different components can be detected by different constant voltages. For example, if the analyte is urine, which contains a lot of vitamin C, 0.5 V can be applied to detect only osmium.

FIG. 4b is a perspective view showing a three-dimensional electrode (70a, 70b) coated with a filter (50) on the surface of a three-dimensional electrode (60a, 60b) illustrated in FIG. 4a. As illustrated in FIG. 4b, the coated three-dimensional electrode (70a, 70b) has a surface coated with a filter (50) having fine pores, and the filter (50) is excited as a cathode (-). The filter (50) can be applied or covered. The filter (50) can be a membrane film or a polymer film, and can be made of a conductive catalyst.

FIG. 4c is a perspective view of a pair of two-dimensional electrodes (75a, 75b) of the biosensor (100) illustrated in FIG. 3 as another embodiment of the present invention. The pair of two-dimensional electrodes (75a, 75b) may be flat, like the substrate (80), or may have a slightly protruding shape.

Detection Principle and Method

Hereinafter, a detection method using the aforementioned biosensor (100) will be described in detail with reference to the attached drawings. First, FIG. 5 is a schematic diagram of an enzyme-substrate reaction and an electron transfer mediator (20) applied according to the present invention. As illustrated in FIG. 5, a negative charge (30) can be a substrate or a metabolic starting material according to an enzyme-substrate reaction. For example, it can be a material in which a metal derivative is bound to an amino acid dimer. When an amino acid is cleaved by an enzyme, ACE2, in vitro, a part in which a metal derivative of a positron (10) is bound is generated, and a material in which the net charge of the cleaved part is a positive charge or a neutral charge is generated.

The source of the positive charge (10) is a signal generator which is a metal derivative. The positive charge (10) has a low molecular weight (1 to 1,000 Dalton range) compared to the negative charge (30, 40). This is to minimize changes in the physical properties of the substrate or the starting metabolite and to enable accurate measurement without introducing additional reagents. The positive charge (10) can be an organic/inorganic complex containing dopamine, quantum dots, ferrocene, ferrocyanide, osmium, europium or ruthenium.

In conventional biosensors, the method of attaching a signal generator is to attach the signal generator directly to an antigen in the form of a large molecular weight carrier protein. This has limited the ability to increase the intensity of the electric signal generator by additionally using a method of binding through an antigen-antibody reaction.

At least some of the negatively charged substances (30) and the positively charged substances (10) covalently bond with each other to become substrates for enzyme-substrate reactions. This covalent bond can be performed by an amine-carboxyl coupling method, a thiol coupling method, an avidin-biotin coupling method, or an aldehyde coupling method.

Some substances of the covalently bonded negative charge (30) and positive charge (10) have a positive or neutral charge and pass through the cathode-excited filter (50) to reach the electrodes (60a, 60b). The electron transfer mediator (20) passed in this way transfers electrons between the electrodes (60a, 60b) to which a certain voltage is applied, thereby generating an output current (110). Accordingly, the concentration of the substrate or metabolite in the analyte can be calculated in proportion to the amount of the output current (110), or it can be confirmed that an enzyme-substrate reaction has occurred. In addition, if the voltage of the constant voltage is changed, other components can be detected. This detection is quantitative detection and/or qualitative detection.

Meanwhile, the electron transfer mediator (20) that transfers electrons to the electrode can repeat the electron transfer process if a separate enzyme-substrate that generates electrons exists on the electrode surface and generates electrons.

On the other hand, molecules with negative charges including total charges of negative charges (40) (e.g., amino acids or proteins with total charges of negative charges), their complexes, or electron transfer mediators react with the filter (50) excited as a cathode and thus do not pass through the filter (50). Accordingly, current is generated at the electrodes (60a, 60b) only by the electron transfer mediator (20) that has passed through. By detecting this current, it is possible to confirm that an enzyme-substrate reaction has occurred.

Figure 6 is a flow chart showing a method for detecting components of a biological analyte using a biosensor based on an enzyme-substrate reaction according to the present invention. As shown in Figure 6, first, a target analyte is prepared from a biological sample (S100). The analyte can be collected by exhaled breath, saliva, blood collection, urine collection, swab, etc. The collected analyte is mixed with an enzyme, etc., which has been dispensed into a storage unit.

Next, the analyte performs an enzyme-substrate reaction or a metabolic reaction to separate a portion including a portion to which a positively charged substance, which is an electrical transfer mediator (20), is covalently bonded, thereby generating a positively charged or neutral substance (S200). In addition, the enzyme-substrate reaction may be a substrate decomposition by an enzyme reaction alone or an enzyme activation reaction. That is, a separation reaction occurs between the substrate, the metabolic initiator, and the enzyme, etc., through a reaction with the substrate or the metabolic initiator. At this time, the portion to which the portion including the signal generator is bound can be cut off, and at this time, the value of the total charge of the denatured fragment can be used to convert it into a mediator that passes through the filter. Specifically, the signal generator can attach an amine group to the positively charged substance of the signal generation function, and cause a covalent reaction using EDC (N-ethyl-N'-(dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) on the substrate and metabolic initiator containing a carboxyl group.

Next, the electron transfer medium (20) passes through the cathode-excited filter (50) (S300). On the other hand, the two negative charges (40) or their combinations do not pass through the filter (50) because they exert a repulsive force on the cathode-excited filter (50). Therefore, a current is generated at the electrodes (60a, 60b) only by the electron transfer medium (20) that has passed through.

Next, the electron transfer medium (20) that has passed through the pores of the filter (50) generates a current between a pair of electrodes to which a predetermined voltage is applied (S400).

Next, the current is output as an output current (110) through a terminal connected to the electrode, thereby detecting the component of the analyte based on the output current (110) (S500). By detecting this output current (110), it is possible to confirm that an enzyme-substrate reaction has occurred. Meanwhile, the electron transfer mediator (20) that has transferred electrons can pass through the filter (50) in reverse, and this process is repeated.

If a different component is to be detected, the control unit (120) applies a constant voltage of 1.4 V instead of a constant voltage of 1.0 V to the electrodes (60a, 60b) and then repeats the above-described process.

The first aspect of the present invention provides a method for qualitatively and quantitatively measuring biological or environmental substances by directly introducing a substrate or a metabolic initiator that directly participates in an enzyme-substrate reaction, etc. into a competitive reaction mechanism, and this method comprises the steps of collecting an analyte to be analyzed from a sample; performing a reaction between a substrate portion of the enzyme-substrate reaction in the analyte and an enzyme; performing a decomposition reaction between a substrate including the same reaction site, such as the same amino acid sequence as the substrate portion of the enzyme-substrate reaction in the analyte, and the enzyme; detecting spectroscopic and electrochemical signals from the enzyme-substrate reaction; performing signal detection by optimizing the recognition of the reaction signal by a sensor detecting the signal as a multi-electrode having a three-dimensional structure or a two-dimensional structure; and performing a filter present on the surface of the electrode so that an electron mediator selectively moves, wherein the substrate participating in the direct reaction or the substrate including the same reaction site includes a signal generator attached by a covalent bond.

In addition, the second aspect of the present invention provides a method for qualitatively and quantitatively measuring biological or environmental substances by participating in a measurement reaction in the form of enzyme-activated and reaction-activated substances in enzyme-substrate reactions, etc., and which comprises the steps of collecting an analyte to be analyzed from a sample; the step of binding one component of the analyte to an activated portion of an enzyme or a reaction-initiating substance; the step of performing a reaction between the substrate portion of the enzyme-substrate reaction in the analyte and the enzyme; the step of performing a decomposition reaction between the enzyme and the substrate including the same reaction site, such as the same amino acid sequence as the substrate portion of the enzyme-substrate reaction in the analyte; the step of detecting spectroscopic and electrochemical signals from the enzyme-substrate reaction; the step of performing signal detection by optimizing the recognition of the reaction signal by forming a multi-electrode having a three-dimensional structure or a two-dimensional structure for the sensor detecting the signal; and the step of having a filter on the surface of the electrode so that an electron medium selectively moves, wherein the substrate participating in the reaction or the substrate including the same reaction site includes a signal generator attached by a covalent bond.

In addition, the third aspect of the present invention provides a method for qualitatively and quantitatively measuring a biological or environmental substance using a competitive reaction mechanism in which a component that serves as a substrate or a metabolic initiator for an enzyme is used, comprising the steps of: collecting an analyte to be analyzed from a sample; performing a reaction between a substrate portion of an enzyme-substrate reaction in the analyte and an enzyme; performing a decomposition reaction between a substrate including the same reaction site, such as the same amino acid sequence as the substrate portion of the enzyme-substrate reaction in the analyte, and the enzyme; detecting spectroscopic and electrochemical signals from the enzyme-substrate reaction; performing signal detection by optimizing the recognition of a reaction signal by a sensor that detects the signal as a multi-electrode having a three-dimensional structure or a two-dimensional structure; and a step in which a filter exists on the surface of the electrode so that an electron mediator selectively moves, wherein the substrate participating in the reaction or the substrate including the same reaction site includes a signal generator attached by a covalent bond.

The method for qualitative and quantitative measurement of biological or environmental substances using a biosensor according to the present invention has the characteristic of directly attaching a low molecular weight signal generator to a substrate or a component containing the same reaction site as the substrate by covalent bonding in the attachment reaction of the signal generator.

The covalent bonding of a signal generator with a substrate and a metabolite or a component including the same reaction site as the substrate and metabolite can be performed by a chemical bonding reaction used in a covalent bonding reaction, such as an amine-carboxyl coupling method, a thiol coupling method, an avidin-biotin coupling method, or an aldehyde coupling method. For example, in the amine-carboxyl coupling method, an amine group is attached to a signal generator, and a substrate and a metabolite (or a substance including the same reaction site) in which a carboxyl group is present or attached are prepared, and then a coupling reaction using EDC (N-ethyl-N'-(dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) is performed. At this time, a method of performing the reaction using EDC alone is also possible. At this time, by controlling the number of amine groups of the substrate and metabolite, the number of bonds between the signal generator and the substrate can be controlled, and the signal contribution amount of the signal generator can be confirmed equivalently compared to the enzyme-substrate reaction. Additionally, by changing the reduction potential value and emission wavelength depending on the characteristics of the signal source, it is possible to measure two or more signals from one sensor strip.

As the signal generator, a small molecule of 1000 Daltons or less is preferable in order to minimize changes in accessibility to substrates or metabolic initiators or in kinetic physical properties, and a substance that can directly generate an electrical signal or act as an electron transfer mediator is used.

Specific examples of signal generators satisfying the above-described molecular weight ranges include, but are not limited to, inorganic/organic complexes containing dopamine, iron, ferrocyanide, osmium, europium, or ruthenium. The inorganic/organic complexes include, in addition to the organic or metal moiety that functions as the signal generator for electrical transmission, an organic functional group moiety for covalent bonding with a substrate or a metabolite.

A substrate or metabolic initiator attached to a signal generator is dispensed in a certain amount in the form of freeze-drying, etc., into the storage unit, and when analytes such as exhaled breath, saliva, blood, urine, and swabs from the sample are introduced through analyte collection, enzymes, etc. dispensed into the storage unit are mixed with the analytes. Then, in the case of a competitive reaction and an activated reaction, a reaction is performed with the substrate and metabolic initiator.

Then, the part that is combined with the signal generator can be cut off by the separation reaction between the substrate or metabolic initiator and the enzyme, etc., and at this time, the value of the total charge of the denatured fragment can be used to convert it into a medium that passes through the filter. This is the principle that contributes to the generation of an electrical signal or spectroscopic signal because the electron transfer medium can finally reach the electrode.

In the present invention, by using a three-dimensional electrode, the shortcoming of the existing two-dimensional electrode in which signals are transmitted only at the contact surface where the solution and the electrode come into contact can be overcome by manufacturing the electrode in three dimensions. In addition, even in the case of two dimensions, there is an advantage in that a system capable of multiple inspections as well as highly efficient signal detection can be created by designing the results of signal generation sources to be recognized separately at multiple locations.

For example, as explained in Fig. 5, if a negatively charged polymer is directly bound to the electrode surface in the form of a filter, molecules including a negatively charged electron transfer mediator cannot pass through, but after separation through an enzyme-substrate reaction, a reactant to which an electron transfer mediator is bound can move to the electrode surface when it becomes positively or neutrally charged, thereby generating an electrical signal or a spectroscopic signal.

The detailed description of the preferred embodiments of the present invention disclosed above has been provided to enable those skilled in the art to implement and practice the present invention. While the above has been described with reference to preferred embodiments of the present invention, it will be understood by those skilled in the art that various modifications and changes can be made to the present invention without departing from the scope of the present invention. For example, those skilled in the art can utilize each of the configurations described in the above-described embodiments in a manner that combines them. Accordingly, the present invention is not intended to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

The present invention may be embodied in other specific forms without departing from the spirit and essential characteristics of the present invention. Accordingly, the above detailed description should not be construed in all aspects as restrictive but rather as illustrative. The scope of the present invention should be determined by a reasonable interpretation of the appended claims, and all changes coming within the equivalent scope of the present invention are intended to be embraced therein. The present invention is not intended to be limited to the embodiments set forth herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein. In addition, claims that do not have an explicit citation relationship in the claims may be combined to constitute an embodiment or may be included as a new claim by post-application amendment.

10 : positive charge,
20: Transmitting electrons through the medium,
30: Negative charge (or some substance with negative charge),
40: Second negative charge,
50 : Filter,
60, 60a, 60b: (Uncoated) 3D electrodes,
70, 70a, 70b: Coated 3D electrodes,
75a, 75b: Coated 2D electrodes,
80 : substrate,
90 : terminal,
100 : Biosensor,
110 : Output current,
120: Control unit.

Claims (11)

Two or more pairs of electrodes installed on the other side of the substrate (80) and exposed while being spaced apart from each other by a predetermined interval so as to be in contact with the analyte;
A filter (50) coated with pores on the above electrode and made of a conductive catalyst and excited as a cathode;
A terminal (90) provided on one side of the substrate (80) to output an output current (110) generated between the electrodes or to receive an electric signal when a predetermined voltage is applied to the electron transfer medium (20) that has passed through the filter (50) from the above-mentioned analysis material; and
It includes a control unit (120) that controls to apply different multiple constant voltages to each pair of the above electrodes,
The above electron transfer mediator (20) is a covalent bond of a negatively charged substance and a positively charged substance based on an enzyme-substrate reaction.
The above negatively charged substance is a substrate or metabolite, and the above positively charged substance is a positively charged substance having a signal generating function.
The covalent bond between the substrate and the positively charged substance is performed by an amine-carboxyl coupling method, a thiol coupling method, an avidin-biotin coupling method, or an aldehyde coupling method,
The positively charged substance of the above signal generation function is a low molecule in the range of 1 to 1,000 Dalton.
The above two or more pairs of electrodes are three-dimensional electrodes (60a, 60b) protruding on the substrate (80), and
The above enzyme-substrate reaction is a biosensor based on an enzyme-substrate reaction for detecting components of a biological analyte, characterized in that the substrate is decomposed in a solution including a filter surface by an enzyme reaction alone or an enzyme activation reaction. delete delete In a method for detecting components of a biological analyte using a biosensor according to Article 1,
Step of preparing an analyte from a biological sample (S100);
A step (S200) in which the above-mentioned analyte is separated by performing an enzyme-substrate reaction or metabolic reaction, including a portion to which a positively charged substance, which is an electrical transfer mediator (20), is covalently bonded;
Step (S300) in which the above electron transfer medium (20) passes through a filter (50) coated with pores on an electrode and made of a conductive catalyst and excited as a cathode;
Step (S400) in which the electron transfer medium (20) passing through the above filter (50) generates a current between two or more pairs of electrodes to which a predetermined voltage is applied; and
A step (S500) of detecting a component of the analyte based on the output current (110) by outputting the current as an output current (110) through a terminal connected to the electrode;
The above two or more pairs of electrodes are three-dimensional electrodes (60a, 60b) protruding on the substrate (80).
The covalent bond between the substrate and the positively charged substance is performed by an amine-carboxyl coupling method, a thiol coupling method, an avidin-biotin coupling method, or an aldehyde coupling method,
The positively charged substances having the above signal generating function are low-molecular weight substances in the range of 1 to 1,000 Dalton, and are inorganic and organic complexes containing dopamine, quantum dots, ferrocene, ferrocyanide, osmium, europium or ruthenium.
The above-mentioned predetermined voltage is generated by controlling the control unit to apply at least one of a plurality of different constant voltages,
Attaching an amine group to the positively charged substance of the above signal generating function,
Covalent reactions are performed using EDC (N-ethyl-N'-(dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) on substrates and metabolic starting materials containing carboxyl groups, and
The above enzyme-substrate reaction is a method for detecting components of a biological analyte using a biosensor based on an enzyme-substrate reaction, characterized in that the substrate is decomposed in a solution including a filter surface by the enzyme reaction alone or by an enzyme activation reaction. delete delete delete delete delete delete delete

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