KR20160107464A - Enzyme-based glucose sensor using potentiometric detection and method for preparing the same - Google Patents

Abstract

The present invention relates to an enzyme-based glucose sensor using potentiometric detection and a method for preparing the same. The glucose sensor comprises: an electrode of which a surface is reformed with a gold-zinc alloy dendrite oxide; a hydrogen ion-sensitive type conductive polymer formed on the oxide; and a glucose degradation enzyme covalently bonded to the conductive polymer. The glucose sensor detects glucose by measuring an electric potential difference based on an enzyme reaction without applying the external voltage.

Description

TECHNICAL FIELD [0001] The present invention relates to an enzyme-based potentiometric glucose detection sensor and a method for producing the same,

More particularly, the present invention relates to a sensor for detecting an enzyme-based potentiometric glucose and a method for producing the same, and more particularly to a sensor for detecting glucose by a potentiometric glucose Based glucose sensor and a method for producing the same.

A sensor is a device that selectively captures the physical quantity or stoichiometry of a measurement object and converts it into a useful signal. BACKGROUND ART An electrochemical biosensor is used as a device for detecting various analytes in the fields of environment, medicine, and food by using a bioreceptor such as an enzyme, a microorganism, and an immunity. Especially, in the field of medicine, Due to its high reaction specificity and rapid response, it is capable of selectively, rapidly, and accurately measuring the target substance.

A blood glucose sensor is a type of electrochemical biosensor that measures the concentration of glucose in the blood or urine. The conventional commercially available blood glucose sensor is a second-generation biosensor using an electrochemical method (time-current method).

Conventional blood glucose sensors using the current method have high sensitivity, high selectivity to monosaccharide, and ease of portability. However, it has been found that ascorbic acid, dopamine, acetaminophen having an oxidation potential similar to the glucose detection potential, (such as acetaminophen) reacts, resulting in interference (so-called interference effect) and low sensitivity.

On the other hand, the enzyme-based potentiometric glucose sensor detects the substance generated by the reaction between glucose and enzyme, and it is not necessary to apply a specific electric potential, so that the above-mentioned interference substances can be avoided. However, the blood sugar sensor using the potentiometric method has not been studied much as compared with the glucose sensor using the current measurement method.

On the other hand, metal oxides having various nanostructures (nanowires, nanorods, nanotubes, nanoparticles, nanofibers, etc.) have been widely applied to glucose sensors with no or no effect. Initial studies on metal oxides for glucose detection have focused on the use of universal electrode materials such as Cu, Ni, Fe, Pt and Au. However, these materials have brought about fundamental disadvantages such as selectivity, low efficiency and contamination of metal electrode surfaces due to chemisorbed intermediates.

(Dendrite structure) single and multi-metal alloy oxides with a new structure using various metals (Au, Cu, Pt, Pd-Pt, Pd-Ag, Cu-Co, etc.) Research has been carried out, but research on multi-metal alloy dendritic materials has not yet been conducted, and there is little research into practical use thereof.

In addition, in order to solve the problem of contamination on the surface of the metal oxide material and selectivity of the monosaccharide, studies on a functional electroconductive polymer suitable for biological materials (enzyme, protein, DNA, etc.) As a representative example, studies on chemical and biosensor devices using polypyrrole and polyaniline have already been reported, but these materials have a serious problem that they are easily oxidized and decomposed in the air in spite of excellent electrical conductivity, resulting in a remarkable decrease in stability.

Therefore, the present inventors have made intensive efforts to invent a stable glucose sensor having high selectivity and high sensitivity which is not influenced by interfering substances in blood glucose detection, and as a result, it has been found that the electrode surface is modified with an oxidizing material of a multi metal alloy having a dendritic structure In order to solve the problem of contamination of the surface of the oxide material and to improve the stability of the enzyme by preventing direct contact with the metal of the enzyme, a sensor having a hydrogen ion sensitive electroconductive polymer introduced between the oxidizing substance and the glucose degrading enzyme was prepared, In this sensor, the alloy dendrite oxide and the polymer act as probes and the concentration is measured only by the potential difference without applying the external voltage. Therefore, the influence of the interfering substance is blocked, the sensitivity of the sensor is remarkably increased, and the stability of the sensor due to the stabilization of the enzyme due to the polymer And the present invention is completed Respectively.

Korean Patent Publication No. 10-2014-0006437 Korean Patent Publication No. 10-2010-0059577

An object of the present invention is to provide a sensor for detecting glucose having a simple and high sensitivity by measuring a potential difference according to an enzyme reaction without external voltage application.

Another object of the present invention is to provide a method for producing an enzyme-based potentiometric glucose detection sensor.

It is still another object of the present invention to provide a method for measuring glucose concentration easily by measuring a potential difference according to an enzyme reaction without external voltage application.

According to an aspect of the present invention, there is provided an electrode comprising: a surface-modified electrode made of a multi-metal alloy dendritic oxide; A hydrogen ion-sensitive conductive polymer formed on the oxide; And a glucose degrading enzyme bound to the conductive polymer through a covalent bond, wherein the glucose is detected by measuring a potential difference according to an enzyme reaction without application of an external voltage, wherein the glucose is detected by an enzyme-based potentiometric glucose A sensor for detection is provided.

The sensor of the present invention is a two-electrode sensor and the electrode can be fabricated using a carbon-based material as the working electrode. For example, as the material of the working electrode, carbon ink, graphite, glassy carbon, graphene, or the like may be used, but it is not limited thereto. The reference electrode may be, but is not limited to, a silver electrode (Ag / AgCl).

In one embodiment of the present invention, a carbon ink can be used as the material of the working electrode and a silver ink can be used as the material of the reference electrode in the case of a disposable sensor.

In the present invention, the working electrode may be a multi-metal alloy dendritic oxide and the electrode surface may be modified. The term "multimetal" in the multi-metal alloy dendritic oxide refers to an alloy of two or more metals, preferably an alloy of two or three metals, more preferably a copper-cobalt alloy or a gold- Lt; / RTI >

In one embodiment of the present invention, the working electrode may be a surface-modified electrode made of gold-zinc alloy dendritic oxide.

The Au-Zn alloy dendrite of the present invention has a dendritic structure, and thus can be applied as a preferable electrode in an electrochemical device due to a unique hierarchical structure having a plurality of active sites and an extremely high surface area.

The gold-zinc alloy dendrite may have an atomic metal content ratio of Au and Zn of 70-90: 30-10.

Electrolyte Potentiometric Method The principle of blood glucose detection is to detect H ⁺ produced by the reaction between glucose and glucose oxidase, and the reaction mechanism is as follows.

Glucose + FAD-GOx → Gluconolactone + FADH ₂ -GOx ------------ (1)

FADH ₂ -GOx + O _{2 -} > FAD-GOx + H ₂ O ₂ ----------------------------- (2)

H ₂ O _{2 -} > 2H ⁺ + O ₂ + 2e ^- ------------------------------------- - (3)

In the present invention, the gold-zinc alloy dendritic oxide film acts as an electrode for sensing hydrogen ions to detect glucose. When hydrogen ions are generated by oxidizing glucose in blood, which is a specimen of glucose degrading enzyme, the gold-zinc alloy dendritic oxide film functions as a probe of hydrogen ion. This is because the reaction of oxygen and hydrogen ion in the dendritic oxide film is used, Is as follows.

The sensor of the present invention can measure the concentration of glucose by measuring the potential difference between the reference electrode and the dendritic oxide electrode according to the reaction. As described above, since the sensor of the present invention uses the effect potential difference method, it is not necessary to apply a specific potential from the outside, so that it is possible to measure without using an external direct current voltmeter and a current measuring system. More importantly, since there is no need to apply a specific electric potential from the outside, it is possible to avoid the interference of the interfering substances reacting to these electric potentials, thereby blocking the interference effect.

The hydrogen ion-sensitive conductive polymer may be a conductive polymer containing -COOH or -NH ₂ functional groups, and a conductive polymer containing a carboxyl group is preferable. More preferably, it may be a conductive polymer containing terthiophene having excellent physical, chemical, mechanical and electrical properties. Most preferably, the hydrogen ion-sensitive conductive polymer is selected from the group consisting of terthiophene benzoic acid (TTBA), terthiophene carboxylic acid (TTCA), and dithienopyrrole benzoic acid dithienyl pyrrole benzoic acid, DTPBA).

In the present invention, a gold-zinc alloy dendritic oxide film is used as a main material for sensing hydrogen ions, and a hydrogen ion-sensitive conductive polymer is introduced therein to improve sensitivity to hydrogen ions of the dendritic oxide film, And also to stabilize the glucose degrading enzyme through peptide bonds.

Specifically, the hydrogen ion-sensitive conductive polymer is in an aqueous solution having a -COO-carboxylic ^acid-zinc alloy oxide dendrites on the hydrogen ion-sensitive polymer - it dissociates or associated with the H ⁺ acts as a receptor of the H ⁺ gold The sensitivity to the potential change with respect to the hydrogen ion concentration can be improved much more than when the dendrite oxide film alone is present. In addition, in the present invention, by disposing the conductive polymer between the metal and the enzyme, not only the surface of the electrode is protected, but also the enzyme is protected to enhance the stability of the enzyme. .

In one embodiment of the present invention, the enzyme can be bound to the polymer through the covalent bond between the carboxyl group of the conductive polymer and the amine group of the enzyme, thereby enhancing the stability of the enzyme in the sensor, thereby maintaining high selectivity .

In the present invention, the glucose degrading enzyme is selected from the group consisting of glucose oxidase, glucose dehydrogenase, glucose hexokinase, glutamic acid acecetic transaminase and glutamic pyruvic transaminase Can be selected from the group consisting of.

As known, since glucose oxidase requires a flavin adenine dinucleotide (FAD) to act as a catalyst for the glucose oxidation reaction, the glucose oxidase in the present invention is a sugar oxidized enzyme Enzyme (FAD-GOx) can be used in the form. In the catalytic reaction by glucose oxidase, FAD acts as an electron acceptor. In addition, a glucose dehydrogenase, glucose hexokinase, glutamic acid aceetic transaminase, glutamic pyruvic transaminase and the like, which are involved in glucose metabolism, can be used as a glucose degrading enzyme.

The sensor for detecting glucose according to the present invention may further comprise a Nafion membrane coated on the layer on which the glucosease is formed.

The Nafion membrane serves to protect the surface of the sensor, for example, by protecting the surface from elements that affect the pH, thereby increasing the stability of the sensor. Sensors protected by Nafion membrane have the advantage of being able to be stored for a long time.

According to another aspect of the present invention, there is provided a method of manufacturing a sensor for detecting glucose by measuring a potential difference according to an enzyme reaction, comprising the steps of: modifying a gold-zinc alloy dendrite on a surface of an electrode through voltage application; Forming the gold-zinc alloy dendrite in an alkali solution to form an oxide; (H ⁺ ) -sensitive conductive polymer on the gold-zinc alloy dendritic oxide through dislocation scanning; And binding the glucose polymer to the conductive polymer through a covalent bond. The present invention also provides a method of manufacturing a sensor for detecting potentiometric glucose.

In one embodiment of the present invention, the dendrites can be formed by applying a voltage at -0.2 V to -0.7 V for 150 to 250 seconds.

The alkali solution preferably may be used without limitation, if the solution containing an _{-OH NaOH, KOH, NH 4 OH} , LiOH, Mg (OH) 2, Ca (OH) 2, Ba (OH) 2, Al (OH) ₃ may be used.

The hydrogen ion-sensitive conductive polymer and the glucose degrading enzyme used in the sensor manufacturing method of the present invention are as described above, and the method of producing the polymer membrane of TTCA and DTPBA on the metal dendrite oxide film is the same as that of TTBA.

The sensor manufacturing method of the present invention may further include a step of activating the carboxyl group of the conductive polymer by treating the electrode modified with the polymer with a catalyst before the glucose degrading enzyme binding step after the step of modifying the polymer.

The catalyst can be used without limitation as a catalyst for making a peptide bond, and is used as a crosslinking agent. Preferably, at least one of EDC (1-Ethyl-3- [3- (dimethylamino) propyl] carbodiimide hydrochloride, NHS (N-hydroxysuccinimide), and glutaraldehyde is used. no.

In one embodiment of the present invention, the step of binding the glucose degrading enzyme to the conductive polymer via a covalent bond comprises reacting the carboxyl group-activated electrode with a saccharide oxidizing enzyme to convert the carboxyl group of the conductive polymer and the amine group To form a covalent bond between them.

The method for producing a glucose detection sensor may further include the step of coating a Nafion membrane on the glucose-decomposing enzyme-forming layer.

By using the thus-prepared potentiometric glucose detection sensor, it is possible to easily detect glucose in the blood.

According to another aspect of the present invention, there is provided a method for measuring a glucose concentration by measuring a potential difference due to an enzyme reaction without application of an external voltage, wherein the enzyme is a glucose oxidase, - a zinc alloy dendritic oxide and a hydrogen ion-sensitive conductive polymer are used.

When the glucose concentration measuring method is used, it is unnecessary to apply an external voltage, so that it is possible to eliminate the influence of an obstructive substance that reacts to the same voltage. The potential difference is measured using a natural oxidation method through an enzyme reaction It is possible to measure more easily and precisely.

The glucose detection sensor of the present invention uses the potential difference method to overcome the disadvantage that the external constant voltage meter and the current measurement meter employed in the conventional sensor are lowered in sensitivity due to the interfering substance, Zinc alloy dendritic oxide film is used as a hydrogen ion probe and the polymer membrane is formed between the oxide film and the saccharide oxidizing enzyme to improve the stability of the enzyme reaction so that the stable and improved sensitivity as well as excellent lifetime and durability There is an effect that can be secured.

Fig. 1 is a schematic diagram of a potentiometric glucose sensor construction.
2 shows electrochemical polymerization curves of pTTBA by (A) Au-Zn alloy dendrite electrodeposition curve through voltage application and (B) circulating voltage-current method.
FIG. 3 is a graph showing the results of (A) Au - Zn DOx, (B) Au - Zn DOx / pTTBA, (C) Au - Zn DOx / pTTBA / FAD - GOx, and Au - Zn DOx / pTTBA / SEM surface photograph.
FIG. 4 shows a calibration curve for the change in glucose concentration using the potential difference method.
Figure 5 shows a graph of the effect on temperature and relative humidity.
Figure 6 shows a glucose detection sensitivity graph for interferents.
FIG. 7 shows a calibration curve according to the addition of glucose to a specific mammalian medium.
Figure 8 shows calibration curves using three concentrations of low, medium, and high concentrations for glucose detection in real blood.

Hereinafter, the constitution and effects of the present invention will be described in more detail with reference to test examples and examples. However, these test examples and examples are provided for illustrative purposes only in order to facilitate understanding of the present invention, and the scope and scope of the present invention are not limited by the following examples.

[ Example  1] The multi-metal oxide and H ⁺  Application of Conductive Polymer Potential difference method  Manufacture of glucose sensor

FIG. 1 shows a process of manufacturing a potentiometric blood glucose sensor. On screen print carbon electrode To form a gold-zinc alloy dendrite, 30 mM Gold (III) chloride trihydrate and 30 mM zinc chloride are dissolved in 0.1 M sodium sulfate, and the solution is adjusted to pH 4.0 with 0.1 M sodium hydroxide solution. Zinc alloy dendrite was formed on the screen print carbon electrode by applying voltage for-200V for 200 seconds (FIG. 2A), and the surface of the electrode was washed with ethanol and tertiary distilled water. The method of forming the gold-zinc alloy dendritic oxide film is a method of forming the gold-zinc alloy dendritic oxide film by the linear current-voltage method and scanning seven times from + 0.0V to + 1.5V, and then using the distilled water and ethanol, And washed. 1 mM TTBA, a H ⁺ -sensitive polymer monomer, was dissolved in a mixed solvent of di (propylene glycol) methyl ether and tripropylene glycol methyl ether (1: 1) After dissolving, 5 μL was dropped on the gold-zinc alloy dendritic oxide, dried at 40 ° C., and then electrochemically polymerized in a 0.1 M phosphate buffer solution (pH 7.4) by cyclic current-voltage method. The scanning potential range was 0.0 V to +1.6 V, and the scanning speed was swept three times at 100 mV / s to form a polymer membrane (FIG. 2B).

The electrodes of the potentiometric blood glucose sensor used a screen print electrode composed of a working electrode and a reference electrode. The working electrode was made of carbon ink, and the reference electrode was made of silver ink.

The electrode on which pTTBA was formed was placed in a solution of 10.0 mM EDC (1-Ethyl-3- [3- (dimethylamino) propyl] carbodiimide hydrochloride) and 10.0 mM NHS (N-hydroxysuccinimide) dissolved in 0.1 M phosphate buffer solution And reacted at 30 DEG C for 6 hours to activate the carboxyl acid group of pTTBA. 10 μL of FAD-GOx (6 mg / mL) solution was added to the surface of pTTBA and reacted at 4 ° C. for 12 hours to form a covalent bond between the carboxyl acid group of pTTBA and the amine group of FAD-GOx. Enzyme adsorption on the surface of the sensor was also increased by the enzyme clustering method. The solution to be used for the enzyme clustering was prepared by adding ammonium sulfate (0.55 mg / mL) and enzyme (FAD-GOx) as a precipitant of the enzyme to a solution of FAD-GOx (6 mg / mL) as glutaraldehyde , 0.5%). Screen-printed transformed to FAD-GOx 10 μL of the enzyme clustering solution was dropped on the surface of the carbon electrode and reacted at 4 ° C. for 12 hours. The amount of enzyme adsorption was increased through covalent bonding between amine groups between FAD-GOx. Finally, 1 μL of Nafion (1.0%) was coated on the enzyme layer to prepare a glucose sensor.

[ Test Example  1] Each of the degeneration stages of the sensor SEM  Surface image

SEM analysis was performed to identify the surface of each denaturation step of the novel potentiometric blood glucose sensor according to the present invention.

3 is a SEM surface photograph of each of the degenerating steps of the sensor. FIG. 3 (A) shows a gold-zinc dendritic structure having a multidirectional tree structure of gold-zinc. Figure 3 (B) is a gold-zinc dendrites on the cyclic voltammetry to the surface of the H ⁺ sensitive polymer electrodeposition picture using a gold-to H ⁺ sensitive polymer between the zinc of the tree of the dendrite is formed dendrite And the porosity was decreased. 3 (C) is a photograph of a surface on which FAD-GDH is immobilized on the H ⁺ sensitive polymer. As shown in FIG. 3 (B), FAD-GDH was immobilized on the H ⁺ -sensitive polymer, so that no distinct dendritic multibrin type morphology was observed, and the surface was very rough. FIG. 3 (D) is a photograph of a surface of 0.5% Nafion coated with a FAD-GDH layer in the form of a net film, and the surface of the film is softer than the surface of FIG. 3 (C).

From the above results, the surface state according to the denaturation step of the potentiometric glucose sensor of the present invention was confirmed.

[ Test Example  2] According to the present invention Potential difference method  Blood glucose sensor Glucose  Performance evaluation of potential change by concentration

The blood glucose sensor prepared in Example 1 was dissolved in a glucose solution of 30 mg / dL, 100 mg / dL, 200 mg / dL, 300 mg / dL, 400 mg / dL and 500 mg / dL dissolved in 0.1 M phosphate buffer And the change in the potential according to the glucose concentration was measured. The results are shown in Fig.

10 sensors were used for each concentration interval. Showed excellent linearity at a sugar concentration of 30 to 500 mg / dL. The correlation equation of this calibration curve is E (mv) = (1.80 + 0.485) + (0.15 + 0.002) [C] (mg / dL) and the correlation coefficient is 0.998. Relative standard deviation was 5.15% ( n = 10) at the sugar concentration of 200mg / dL, showing excellent reproducibility. In addition, the relative standard deviation range with respect to the average potential difference value of each concentration interval is within ± 5 to 13%.

[ Test Example  3] Glucose  Measurements of temperature and relative humidity Influence  evaluation

The glucose sensor prepared according to Example 1 was treated with a glucose solution having a concentration of 200 mg / dL dissolved in 0.1 M phosphate buffer solution (pH 7.4) to measure the glucose potential at various temperatures (15, 16, 20, 25, 30, 40, 50, 60, 70, 80%) and relative humidity (20, 30, 40, 50, 60, 70, 80%

Temperature and relative humidity were measured with three blood glucose sensors at each interval. In FIG. 5 (A), the detection sensitivity was constant at 20 to 40 ° C., but the detection sensitivity at 15 ° C. was reduced by 24% as compared with 25 ° C. This indicates that the activity of the enzyme is lowered at a temperature of 20 ° C or less, and as a result, the glucose sensitivity is lowered. In FIG. 5 (A), it was confirmed that glucose sensitivity was kept constant in the range of 20 to 80% relative humidity.

[ Test Example  4] Glucose  Of interfering substances Influence  evaluation

Figure 6 is a graph comparing glucose detection sensitivity when interfering substances are present. The inhibitors used in this study were ascorbic acid (AA, 5mg / dL), dopamine (DA, 5mg / dL), acetaminophen (AP, 15mg / dL), four monosaccharides (mannose dL), lactose (10 mg / dL), xylose (10 mg / dL), fructose (30 mg / dL)).

Investigation of the interfering substances was carried out in a glucose solution with a concentration of 200 mg / dL dissolved in 0.1 M phosphate buffer (pH 7.4) containing each of the above inhibitors. In the case of the monosaccharide, 200 mg / dL of the glucose solution containing the above four monosaccharides was measured and found to be 1.2% lower than the average potential difference value of glucose. In the case of dopamine, acetaminophen and ascorbic acid, glucose sensitivity was decreased by 5.2%, 5.5% and 4.8%, respectively. These inhibitors (monosaccharides, DA, AP and AA) It does not become a problem.

[ Test Example  5] Moth Glucose  Concentration measurement

The concentration of hematocrit was equally diluted to 10% by using physiological saline (0.9% NaCl), and glucose was added to each of 6 different concentrations (29, 119, 159, 250, 348 and 434 mg / dL) < / RTI > was prepared.

FIG. 7 is a calibration curve for the glucose concentration (29, 119, 159, 250, 348, 434 mg / dL) of six blood samples using the potential difference method. Three sensors were used for each concentration interval. The linearity of the blood glucose concentration was 29 ~ 434 mg / dL. The correlation equation of this calibration curve is E (mV) = (1.178 ± 0.615) + (0.137 ± 0.004) [C] (mg / dL) and the correlation coefficient is 0.99.

[ Test Example  6] In real blood Glucose  Concentration measurement

FIG. 8 is a calibration curve obtained using mouse blood (low, medium, high) having three different glucose concentrations for detecting glucose in actual blood. Three sensors were used for each concentration interval. The correlation equation of this calibration curve is E (mV) = (19.45 + 0.626) + (0.137 + 0.012) [C] (mg / dL). Glucose was detected from the blood of actual human using the above correlation and its concentration was 99 ± 8.6. In addition, the measurement result using a commercially available blood glucose sensor (OneTouch Ultra) is 105 ± 1.5. Using two sensors, it was confirmed that the measurement results of glucose concentration in real blood were in good agreement. Therefore, the sensor developed through the present invention can be used as a sensor for detecting glucose in actual blood.

Claims (14)

An electrode whose surface is modified with a gold-zinc alloy dendritic oxide;
A hydrogen ion-sensitive conductive polymer formed on the oxide; And
And a glucose degrading enzyme bound to the conductive polymer through a covalent bond,
An enzyme-based potentiometric glucose sensor for detecting glucose by measuring a potential difference according to an enzyme reaction without external voltage application. The sensor according to claim 1, wherein the electrode is a carbon-based electrode as the working electrode. The method of claim 1, wherein the hydrogen ion-sensitive conductive polymer is selected from the group consisting of tthiophene benzoic acid (TTBA), tthiophenecarboxylic acid (TTCA), and dithienylpyrrole benzoic acid (DTPBA) And a sensor for detecting a potential difference glucose. The method according to claim 1, wherein the glucose degrading enzyme is selected from the group consisting of glucose oxidase, glucose dehydrogenase, glucose hexokinase, glutamicum acecetic transaminase and glutamic pyruvic trans- Wherein the sensor is selected from the group consisting of glucose, dextrose, fructose, fructose, fructose, and fructose. The sensor for detecting potential difference glucose according to claim 1, further comprising a Nafion membrane coated on the glucose degrading enzyme. A method for producing a sensor for detecting glucose by measuring a potential difference according to an enzyme reaction,
Modifying the gold-zinc alloy dendrites on the electrode surface through voltage application;
Forming the gold-zinc alloy dendrite in an alkali solution to form an oxide;
(H ⁺ ) -sensitive conductive polymer on the gold-zinc alloy dendritic oxide through dislocation scanning;
And bonding a glucose degrading enzyme to the conductive polymer through a covalent bond. The method of claim 6, wherein the alkali solution is _{NaOH, KOH, NH 4 OH,} LiOH, Mg (OH) 2, Ca (OH) 2, Ba (OH) 2, and Al (OH) is selected from the group consisting of ₃ And a sensor for detecting a potential difference glucose. The method of claim 6, wherein the hydrogen ion-sensitive conductive polymer is at least one selected from the group consisting of tthiophene benzoic acid (TTBA), tthiophenecarboxylic acid (TTCA), and dithienylpyrrolbenzoic acid (DTPBA) Wherein the method comprises the steps of: 7. The method of claim 6, wherein the glucosease is glucose oxidase, glucose dehydrogenase, glucose hexokinase, glutamic acid acecetic transaminase, glutamic pyruvic transgene, Wherein the enzyme is selected from the group consisting of glucose, dextrose, fructose, fructose, fructose, fructose, and minia. The method according to claim 6, further comprising a step of activating the carboxyl group of the conductive polymer by treating the electrode modified with the polymer before the glucose degrading enzyme binding step . 11. The method according to claim 10, wherein the catalyst is a catalyst for making a peptide bond. [7] The method of claim 6, further comprising coating a Nafion membrane on the glucose degrading enzyme. A method for detecting glucose in blood using a potentiometric glucose detecting sensor produced according to any one of claims 6 to 12. A method for measuring glucose concentration by measuring a potential difference according to an enzyme reaction without applying an external voltage,
The enzyme is a glucose oxidase,
Zinc alloy dendritic oxide and a hydrogen ion-sensitive conductive polymer modified on the electrode surface are used as probes of hydrogen ions.

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