KR102314267B1 - Impurity blocking layer containing porous nano-silica complex for penetrating selectively glucose and method for preparing the same - Google Patents

Abstract

The present invention relates to a glucose biosensor having an impurity blocking layer that selectively transmits glucose.

Description

Glucose selective permeation impurity blocking layer comprising nano-porous silica-fixed composite and manufacturing method thereof

The present invention relates to a glucose biosensor having an impurity blocking layer that selectively transmits glucose.

Diabetes mellitus is a disease that occurs due to inappropriate carbohydrate metabolism that does not properly use glucose absorbed into the body, and has excessive blood sugar in the blood, which can cause various complications. Diabetic patients must periodically administer insulin, and in order to accurately administer insulin, blood glucose levels must be checked using a self-diagnosing device such as a glucose sensor.

Glucose detection sensor has been continuously developed based on the first sensor made by enclosing and immobilizing GOx (glucose oxidase), which promotes glucose oxidation, to a polyacrylamide gel membrane and attaching the membrane to the diaphragm sensor electrode.

In order to measure glucose, there is a method of measuring oxygen consumed in relation to the reaction of GOx, pH change due to gluconic acid production, and H ₂ O ₂ . GOx, an enzyme used in blood glucose sensors, is easily and inexpensively obtained, and compared to other enzymes, it is stable to pH, ionic strength, and temperature. .

Blood glucose measurement technology can be largely divided into an electrochemical method and a photometric method. Both the electrochemical method and the photometric method basically use an oxidase capable of oxidizing glucose by reacting with glucose.

In the photometric method, the degree of color change is quantified by measuring the reflectance or transmittance of light using a photometer using a dye source that causes a color change when glucose is oxidized. The biosensor using the photometric method requires a relatively large amount of blood compared to the electrochemical method, and thus the competitiveness is declining compared to the electrochemical method.

The electrochemical method quantifies the glucose concentration by measuring a change in current caused by electrons generated during the electrolysis _{of H 2} O ₂ , a product of the process of being oxidized and decomposed by glucose GOx, by an electrode. Compared to the photometric method, most of them are focusing on the electrochemical method as it has high added value and has the advantage of measuring blood sugar with a small amount of blood.

However, the electrochemical sensor mainly detects the concentration of glucose present in blood, and there is a problem in that blood collection is required. Accordingly, a sensor for detecting glucose in urine is used, but it is difficult to accurately measure the concentration of glucose due to impurities such as vitamins, ascorbic acid, and acetaminophen in the urine. act as a factor in shortening the lifespan of To block these impurities, film films such as silicone, acrylic, and urethane are used, but due to the thickness of the film film itself, the thin film coating required for glucose permeation has to be coated thinly, resulting in reduced durability and reduced measurement sensitivity. have.

Republic of Korea Patent No. 10-1467299

It is an object of the present invention to provide a glucose biosensor including an impurity blocking layer that blocks impurities and selectively transmits glucose.

In order to achieve the above object, one aspect of the present invention is a substrate; an enzyme layer positioned on the substrate and reacting with glucose present in the biomaterial; And located on the enzyme layer, it provides a glucose biosensor comprising an impurity blocking layer comprising a complex of spherical porous nano silica particles.

In one embodiment of the present invention, the biological material may be saliva, tears, blood, sweat, urine, etc., for example, urine.

In one embodiment of the present invention, the impurity blocking layer may be prepared by mixing the nano silica dispersion and the binder solution.

In one embodiment of the present invention, the spherical porous nano silica particles may have a diameter of 30 nm to 1000 nm.

In one embodiment of the present invention, the nano-silica dispersion may be prepared by mixing spherical nano-silica with an organic solvent.

In one embodiment of the present invention, the binder solution is reacted by mixing a hydroxy acyl monomer and an isocyanate; and adding and mixing an inert prepolymer having two or more functional groups, an organic solvent, and a radical reaction initiator to the reaction solution.

In one embodiment of the present invention, the hydroxy acyl monomer may be at least one selected from the group consisting of hydroxy propyl acrylate, hydroxy propyl methacrylate, hydroxy ethyl acrylate and hydroxy ethyl methacrylate.

In one embodiment of the present invention, the isocyanate may be at least one selected from the group consisting of hexamethylene diisocyanate trimer, toluene diisocyanate trimer and methylene diphenyl diisocyanate trimer.

In one embodiment of the present invention, the radical reaction initiator may be at least one selected from the group consisting of acetophenone-based compounds, benzyl ketone-based compounds, benzoin ester-based compounds, ketone-based compounds, azo-based compounds, and peroxide-based compounds. .

In one embodiment of the present invention, the inert prepolymer having two or more functional groups is selected from the group consisting of bifunctional urethane acrylate, tetrafunctional urethane acrylate, hexafunctional urethane acrylate, bifunctional epoxy acrylate and tetrafunctional epoxy acrylate. There may be more than one.

The impurity blocking layer including the composite formed of the spherical porous nano silica particles of the present invention can block impurities and selectively transmit glucose because the silica particles are tightly agglomerated with each other. In addition, the durability of the impurity blocking layer can be improved by applying the binder of the multi-layered complex cross-linking reaction mechanism, and the coating film can be formed thickly, thereby increasing the durability of the sensor.

1 shows the structure of a conventional glucose biosensor.
2 shows the structure of a glucose biosensor according to an embodiment of the present invention.
3 is a graph showing the results of measuring the durability of the glucose biosensor according to an embodiment of the present invention.
4 shows a standard solution used for measurement of urine sugar by a glucose biosensor according to an embodiment of the present invention.
5 is a graph showing results of measurement of an animal urine sample by a glucose biosensor according to an embodiment of the present invention.
6 is a graph showing a measurement reproducibility test result of a glucose biosensor according to an embodiment of the present invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description, detailed descriptions of well-known techniques known to those skilled in the art may be omitted. In addition, in describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. In addition, terms used in this specification are terms used to properly express preferred embodiments of the present invention, which may vary depending on the intention of a user or operator or customs in the field to which the present invention belongs.

Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

Hereinafter, the present invention will be described in more detail with reference to the drawings.

1 shows a conventional glucose biosensor 100, and includes a substrate 101, an electrode 102, an enzyme layer 103, an immobilization layer 104, and an impurity blocking layer 105. The impurity blocking layer of the conventional glucose biosensor is composed of components such as silicone, acrylic, urethane, and fluorine-based compounds, and a thin film coating is required for glucose permeation. However, since the conventional glucose biosensor consists of an enzyme layer, an immobilization layer, and an impurity blocking layer, a thin film coating has to be applied, and thus the durability of the sensor is deteriorated.

The present invention is to solve these conventional problems, and the glucose biosensor 200 according to the embodiment of the present invention shown in FIG. 2 is a substrate 201, an electrode 202, and an enzyme layer 203 without an immobilization layer. and a structure of the impurity blocking layer 204 .

The impurity blocking layer 204 of the present invention is made of a composite of spherical porous nano silica particles. The spherical porous nano-silica particles are combined with each other to form a structure by tightly clumping together in a mesh shape, so that the thin film coating can be coated inside and outside the structure, thereby increasing the thickness of the thin film to more effectively transmit glucose and block impurities. Also, as the thickness of the thin coating increases, the durability of the sensor can be increased.

The impurity blocking layer 204 of the present invention applies a binder having a complex cross-linking reaction mechanism of a spherical multi-layer structure to spherical porous nano silica, and by closely bonding with the enzyme layer, the enzymes of the enzyme layer can be effectively immobilized without an immobilization layer. .

The impurity blocking layer 204 of the present invention may be prepared by mixing the nano silica dispersion and the binder solution.

Specifically, the nano silica dispersion may be prepared by mixing and dispersing 20 to 50 parts by weight of spherical nano silica with 50 to 80 parts by weight of an organic solvent. The nano silica may have a diameter of 30 nm to 1000 nm, for example, may be spherical nano silica particles having a diameter of 500 nm. The organic solvent may be alcohols, ketones, or acetates, but is not limited thereto. The dispersion may be used without limitation as long as it is a spraying device used in this field, for example, a dynomill, a ball mill, a homomixer, an ultrasonic disperser, etc. may be used, but is not limited thereto.

reacting the binder solution by mixing 5 to 15 parts by weight of a hydroxy acyl monomer and 5 to 20 parts by weight of an isocyanate; and 40 to 75 parts by weight of an inert prepolymer having a difunctional or higher group, 40 to 60 parts by weight of an organic solvent, and 1 to 10 parts by weight of a radical reaction initiator to the reaction solution.

The hydroxy acyl monomer may be at least one selected from the group consisting of hydroxy propyl acrylate, hydroxy propyl methacrylate, hydroxy ethyl acrylate and hydroxy ethyl methacrylate.

The isocyanate may be at least one selected from the group consisting of hexamethylene diisocyanate trimer, toluene diisocyanate trimer, and methylene diphenyl diisocyanate trimer.

The radical reaction initiator may be at least one selected from the group consisting of an acetophenone-based compound, a benzyl ketone-based compound, a benzoin ester-based compound, a ketone-based compound, an azo-based compound, and a peroxide-based compound.

The bifunctional or more inert prepolymer may be at least one selected from the group consisting of bifunctional urethane acrylate, tetrafunctional urethane acrylate, hexafunctional urethane acrylate, bifunctional epoxy acrylate and tetrafunctional epoxy acrylate, for example, Polyethylene glycol Diacrylate, Polypropylene glycol Diacrylate, Polycarbonate Diacrylate, Bisphenol A (EO)n Diacrylate ), ditrimethylol propane tetraacrylate, pentaerythritol tetraacrylate, dipentaerythritol pentaacrylate, dipentaerythritol hexaacrylate, etc. It is not limited thereto.

The organic solvent may be used without limitation as long as it is ketones or acetates.

The electrode 202 may be formed on the glass substrate 101 by a semiconductor process. The glass substrate 101 can be replaced with a substrate of another material, and there is no limitation on the material and shape of the electrode, for example, Pt, Ag, Au, carbon, etc. may be used.

The enzyme layer 203 is disposed on the substrate 201, and includes glucose oxidase (GOx), glucose dehydrogenase (GDH), glucose hexokinase, cholesterol oxidase, and glutarase. It may include an enzyme such as Mick Oxaloacetic Transaminase (GOT; Glutamic Oxaloacetic Transaminase) or Glutamic Pyruvic Transaminase (GPT), and furthermore, pyroquinolinequinone which is a coenzyme for each enzyme (PQQ; pyrroquinoline quinone) may be configured to further include, and if an enzyme using glucose as a substrate, it may be included without limitation.

Hereinafter, the present invention will be described in detail by way of Examples. However, these Examples are for explaining the present invention in more detail, and the scope of the present invention is not limited to these Examples.

[Example]

[Example 1]

Preparation of impurity blocking layer solution

Ethanol and nano-silica (500 nm) were added to a ball mill with ethanol (80 parts by weight) and nano-silica (20 parts by weight), and then dispersed at room temperature for 3 hours to prepare a nano-silica dispersion.

After mounting a thermometer and a condenser in a four-necked round flask, hydroxypropyl acrylate (10 parts by weight) and hexamethylene diisocyanate trimer (15 parts by weight) were added under a nitrogen atmosphere, and then the temperature was raised to 50° C. and maintained for 5 hours. while reacting. Then, dipentaerythritol hexaacrylate (70 parts by weight), ethanol (20-50 parts by weight) and hydroxycyclohexyl phenyl ketone (5 parts by weight) were added thereto, and the temperature condition was 20 to 35 ° C. A binder solution was prepared by sufficiently mixing at a stirring speed of 30 to 100 rpm.

Then, the nano-silica dispersion and the binder solution were added to a dispersing device in a weight ratio of 3:0.3 and dispersed at room temperature for 1 hour .

[Example 2]

Manufacturing of biosensors

Glucose Oxidase, BSA, and glutaraldehyde were mixed in a weight ratio of 1:1:1, and 20ul was loaded on the electrode, and then spin-coated (3000RP / 30sec) and naturally cured at room temperature for 3 hours. After loading 20ul of the impurity blocking layer solution prepared as in Example 1 on the cured enzyme layer, spin coating (3000RP / 30sec) and natural curing at room temperature for 3 hours to prepare a biosensor.

[Experimental Example 1]

durability test

After burying 10ul of a 250mg/dL glucose solution in the biosensor prepared as in Example 2, the current value was measured. The concentration of 250 mg/dL glucose solution was repeated 200 times or more, and the measurement deviation was confirmed graphically (FIG. 3). As can be seen in FIG. 3 , it can be confirmed that the durability is maintained without showing a large measurement deviation even after 200 measurements.

[Experimental Example 2]

Precision and Accuracy Measurement Test

The urine glucose measurement precision and accuracy of the biosensor prepared as in Example 2 were measured.

20-25°C and 20-30% R.H. After measuring the standard solution (Level 1, Level 2) (FIG. 4) with the biosensor using COBAS U411 under the conditions, the correlation between the measured concentration of the biosensor and the concentration of the standard solution was compared. Standard solution Level 1 has a value of 0 mg/dL, and Level 2 has a value of (300 - 1000) mg/dL. Each measurement was repeated 20 times using three measuring instruments, and the coefficient of dispersion (CV %) between the standard concentration and the measured concentration was 4.86%, which was detected within 5%.

As a result, as can be seen in Table 1 below, negative and positive were measured according to standard material Level 1 (negative) and Level 2 (positive), and the accuracy was 100%.

[Experimental Example 3]

Correlation test with animal urine samples

The correlation in the animal urine sample of the biosensor prepared as in Example 2 was measured.

20-25°C and 20-30% R.H. Animal samples obtained from normal animals and diabetes-induced animals were measured using a biosensor under the conditions. The correlation between the measured concentration and the concentration measured by a standard instrument (Toshiba, TBA 120FR) was compared, and the results are shown in Table 2 and FIG. 5 . The correlation with the urine sugar concentration measured in animal urine was detected as 0.99.

[Experimental Example 4]

Measurement reproducibility test

The measurement reproducibility of the biosensor prepared as in Example 2 was tested.

20-25°C and 20-30% R.H. Under the conditions, an animal sample (446.8 mg/dL) of known urine sugar concentration was measured by repeating 100 times using a biosensor. As can be seen in Table 3 and FIG. 6, it was confirmed that the measurement was possible by repeating 100 times, and the coefficient of variance (CV %) for the measurement repeated 100 times was confirmed to be 5.02%.

So far, preferred embodiments of the present invention have been mainly looked at. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than in the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

100: biosensor 101: substrate
102: electrode 103: enzyme layer
104: immobilization layer 105: impurity blocking layer
200: biosensor 201: substrate
202: electrode 203: enzyme layer
204: impurity blocking layer

Claims (10)

Board;
an enzyme layer positioned on the substrate and reacting with glucose present in the biomaterial; and
It is located on the enzyme layer and contains a complex of spherical porous nano silica particles,
The composite of the spherical porous nano-silica particles may include 20 to 50 parts by weight of spherical nano-silica particles dispersed in 50 to 80 parts by weight of an organic solvent; and a binder solution; prepared by mixing,
Glucose biosensor, characterized in that the spherical nano-silica particles included in the composite of the spherical porous nano-silica particles bind to each other and are positioned in a mesh shape to selectively transmit glucose while blocking impurities present in the biological material. . delete According to claim 1,
The nano silica particles will have a diameter of 30nm to 1000nm, glucose biosensor. According to claim 1,
The biomaterial is blood, saliva, tears, sweat or urine, glucose biosensor. delete According to claim 1,
The binder solution is
reacting by mixing a hydroxy acyl monomer and an isocyanate; and
Mixing the reaction solution by adding an inert prepolymer having more than two functional groups, an organic solvent, and a radical reaction initiator
Which is produced by, glucose biosensor. 7. The method of claim 6,
The hydroxy acyl monomer is at least one selected from the group consisting of hydroxy propyl acrylate, hydroxy propyl methacrylate, hydroxy ethyl acrylate and hydroxy ethyl methacrylate, a glucose biosensor. 7. The method of claim 6,
The isocyanate is at least one selected from the group consisting of hexamethylene diisocyanate trimer, toluene diisocyanate trimer and methylene diphenyl diisocyanate trimer, glucose biosensor. 7. The method of claim 6,
The difunctional or more inert prepolymer is at least one selected from the group consisting of bifunctional urethane acrylate, tetrafunctional urethane acrylate, hexafunctional urethane acrylate, bifunctional epoxy acrylate and tetrafunctional epoxy acrylate, glucose biosensor. 7. The method of claim 6,
The radical reaction initiator is at least one selected from the group consisting of an acetophenone-based compound, a benzyl ketone-based compound, a benzoin ester-based compound, a ketone-based compound, an azo-based compound, and a peroxide-based compound, a glucose biosensor.

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