KR20230140906A - Non-invasive glucose meter using saliva and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a non-blood glucose measurement sensor using saliva and a method of manufacturing the same. More specifically, it relates to a non-blood glucose measurement sensor using saliva with high sensitivity and fast response and a method of manufacturing the same.
The non-blood glucose measurement sensor using saliva according to the present invention includes a lower plate on which electrodes are formed; An upper plate where a sample inlet is formed through which saliva to be analyzed flows; a spacer laminated between the lower plate and the upper plate and forming a sample inlet channel at a position corresponding to the sample inlet; and an enzyme immobilization layer formed by coating an enzyme immobilization solution on the reaction cell formed by stacking the lower plate and the spacer.

Description

Non-blood glucose measurement sensor using saliva and manufacturing method thereof {NON-INVASIVE GLUCOSE METER USING SALIVA AND MANUFACTURING METHOD THEREOF}

The present invention relates to a non-blood glucose measurement sensor using saliva and a method of manufacturing the same. More specifically, it relates to a non-blood glucose measurement sensor using saliva with high sensitivity and fast response and a method of manufacturing the same.

The blood sugar measurement method used by diabetic patients at home or in hospitals involves directly drawing blood to measure blood sugar levels, which involves considerable pain when used continuously, and there is a risk of infection due to repeated blood collection.

To overcome the limitations of blood glucose measurement through blood collection, non-invasive blood glucose measurement sensors that apply principles such as ultrasound, thermal conductivity, and impedance have been proposed, but they have poor sensitivity, take a long time to measure, and have side effects such as allergies and skin irritation. It is difficult to miniaturize the equipment, so it is not portable, and it is expensive, which is an economic burden.

Among non-invasive blood sugar measurement devices, the continuous blood sugar meter has the advantage of being able to measure blood sugar in real time by attaching it to the abdomen and forearm to measure blood sugar continuously for about 7 days. However, the blood sugar data is inaccurate and the price is high, which poses a significant financial burden. There is.

Meanwhile, there have been efforts to estimate the concentration of glucose in the blood by measuring glucose in saliva. The amount of glucose contained in saliva is small compared to blood, and since it measures a small amount of glucose, the sensor sensitivity is not high and the correlation with actual blood sugar is low.

As part of research to develop a high-performance non-blood glucose measurement sensor, the present inventor developed a non-blood glucose measurement sensor with high sensitivity and a high correlation with actual blood sugar, which led to the present invention.

Domestic Registered Patent No. 10-1436162 Domestic Registered Patent No. 10-1883412

The purpose of the present invention to solve the above problems is to provide a non-blood glucose measurement sensor using saliva with high sensitivity and fast response and a method of manufacturing the same.

The non-blood glucose measurement sensor using saliva of the present invention to solve the above problem includes a lower plate on which electrodes are formed; an upper plate on which a sample inlet through which the saliva to be analyzed is formed; and a lamination between the lower plate and the upper plate, and a sample inlet. It includes a spacer in which a sample inlet channel is formed at a position corresponding to the spacer; and an enzyme immobilization layer formed by coating an enzyme immobilization solution on the reaction cell formed by stacking the lower plate and the spacer.

The electrode is characterized as a nickel-gold plated electrode.

The enzyme immobilization layer is characterized in that it is formed using an inkjet dispenser.

The enzyme immobilization layer is characterized in that it is formed by spraying 0.1 to 10 pℓ of the enzyme immobilization solution with an inkjet dispenser and then drying it.

The method of manufacturing a non-blood glucose measurement sensor using saliva of the present invention to solve the above problem includes a lower plate preparation step (S100) of preparing a lower plate with electrodes; and preparing an upper plate with a sample inlet through which saliva to be analyzed flows. An upper plate preparation step (S200); and a spacer stacking step (S300) of stacking a spacer on the upper part of the lower plate and having a sample inlet channel formed at a position corresponding to the sample inlet; and a reaction formed by stacking the lower plate and the spacer. It includes an enzyme immobilization layer forming step (S400) of coating the cell with an enzyme immobilization solution to form an enzyme immobilization layer (S400); and a top plate stacking step (S500) of stacking the prepared top plate on the spacer.

In the lower plate preparation step (S100), the electrode is characterized as a nickel-gold plating electrode.

The enzyme immobilization layer forming step (S400) is characterized by forming an enzyme immobilization layer using an inkjet dispenser.

The enzyme immobilization layer forming step (S400) is characterized in that it is formed by spraying 0.1 to 10 pℓ of the enzyme immobilization solution using an inkjet dispenser and then drying it.

As described above, according to the non-blood glucose measurement sensor using saliva and its manufacturing method according to the present invention, it has high sensitivity and fast response, and has the effect of easily measuring blood sugar through a non-blood collection method.

Figure 1 is a schematic diagram showing the layered structure of a non-blood glucose measurement sensor using saliva according to the present invention.
Figure 2 is a cross-sectional view showing the layered structure of the non-blood glucose measurement sensor using saliva of the present invention.
Figure 3 is a flow chart of the manufacturing method of the non-blood glucose measurement sensor using saliva of the present invention.
Figure 4 is an example of a non-blood glucose measurement sensor using saliva applied to a measuring device.
Figure 5 is an IT test result of a non-blood glucose measurement sensor using saliva according to the present invention.
Figure 6 shows the current linearity test results for each concentration of saliva in an example of a non-blood glucose measurement sensor using saliva according to the present invention.
Figure 7 shows the measurement reliability according to the electrode material applied to the non-blood glucose measurement sensor using saliva according to the present invention, where A) is a nickel-gold plated electrode and (B) is a conventional carbon paste electrode. Measurement reliability.

Specific features and advantages of the present invention will be described in detail below with reference to the accompanying drawings. Prior to this, if it is determined that a detailed description of the functions and configuration related to the present invention may unnecessarily obscure the gist of the present invention, the detailed description will be omitted.

The present invention relates to a non-blood glucose measurement sensor using saliva and a method of manufacturing the same. More specifically, it relates to a non-blood glucose measurement sensor using saliva with high sensitivity and fast response and a method of manufacturing the same.

Figures 1 and 2 show the layered structure of a non-blood glucose measurement sensor using saliva according to the present invention.

The non-blood glucose measurement sensor using saliva according to the present invention has a lower plate 100 on which electrodes 50 are formed, an upper plate 300 on which a sample inlet 310 through which the saliva to be analyzed is formed, and between the lower plate and the upper plate. A spacer 200 that is stacked and has a sample inlet channel 210 formed at a position corresponding to the sample inlet, and an enzyme immobilization layer 410 formed by coating an enzyme immobilization solution on the reaction cell 400 formed by stacking the lower plate and the spacer. ) includes.

The lower plate 100 is formed with electrodes 50 to generate an electrical signal formed by a redox reaction between the saliva to be analyzed and the enzyme immobilization material.

The lower plate 100 has an electrode 50 formed on a base substrate. The electrode 50 generates an electrical signal by a redox reaction between the saliva to be analyzed and the enzyme immobilization material, and the electrode is connected to a lead terminal (not shown). ) is connected to transmit an electrical signal to the measuring device, and the measuring device measures the concentration of sugar contained in the saliva to be analyzed based on the detected electrical signal value, and applies the correlation coefficient between the measured salivary sugar and blood sugar to determine the salivary sugar The result is converted to a blood sugar value and the blood sugar value is displayed.

The mechanism for measuring blood sugar levels through salivary sugar is as follows.

Glucose contained in the saliva to be analyzed is oxidized to gluconic acid through an enzymatic reaction with glucose oxidase, and glucose oxidase is reduced. Reduced glucose oxidase is oxidized again through reaction with an electron transfer mediator, and oxidized glucose oxidase reacts with other glucose. At the same time, the reduced electron transfer medium moves to the surface of the electrode to which voltage is applied, loses electrons, and is electrochemically oxidized again to continue participating in the reaction. The current generated during the oxidation process of the electron transfer mediator is proportional to the glucose concentration in saliva, and a voltage of 250 to 400 mV is applied to the electrode to measure the amount of current between the working electrode and the reference electrode to measure the glucose concentration contained in the saliva to be analyzed. By quantitatively measuring and converting the blood sugar value by applying the correlation coefficient between actual blood sugar and saliva sugar, it is possible to measure the blood sugar value through a non-blood sampling method.

The electrode 50 includes a reference electrode, a working electrode, and an auxiliary electrode, and the electrode 50 is made of nickel, gold, copper, titanium, silver, aluminum, germanium, tungsten, tin, antimony, indium, and cadmium. , may contain at least one of palladium, lead, zinc, and platinum, and preferably, a nickel-gold plated electrode may be used.

The base material is not limited as long as it is a polymer compound film with insulating properties, but specifically, polyethylene terephthalate (PET), polyester, polycarbonate, polystyrene, polyimide, polyvinyl. It may contain at least one of polyvinylchloride and polyethylene, and preferably, polyethylene terephthalate (PET) can be used.

The base substrate may have a thickness of 50 to 300 ㎛, preferably 150 to 200 ㎛.

Nickel-gold plated electrodes are formed by first forming a nickel-plated layer on a base substrate, and then forming a second gold-plated layer on the nickel-plated layer after patterning the electrode. The nickel-plated and gold-plated layers are formed using an electroless plating method. It can be.

The nickel plating layer is formed to a thickness of 0.1 to 5 μm, preferably 0.5 to 1.5 μm, by treating the plating solution at 40 to 60°C for 5 to 15 minutes using a nickel plating solution containing a water-soluble nickel salt, reducing agent, complexing agent, and buffer.

Patterning of the electrode can be performed using a photolithography method, and can be formed with a line width of 300 to 500 ㎛ and a line spacing of 80 to 150 ㎛. Preferably, the electrode has a line width of 350 to 450 ㎛ and a line spacing of 90 ㎛. It can be formed from 110㎛ to 110㎛.

The gold plating layer is formed to a thickness of 0.01 to 0.05 μm, preferably 0.02 to 0.03 μm, by treating the gold plating solution at 60 to 80°C for 1 to 5 minutes using a gold plating solution containing a gold salt, a reducing agent, a complexing agent, and an organic acid.

The manufactured nickel-gold plated electrode has a surface resistance of 5Ω or less, specifically 3Ω or less, has high electron mobility generated from the redox reaction of salivary sugar and enzymes, and is insoluble and chemically stable.

The upper plate 300 is formed to protect the electrode and enzyme immobilization layer of the lower plate and to facilitate the inflow of the saliva to be analyzed, and a sample inlet 310 through which the saliva to be analyzed is formed.

On the other hand, the saliva to be analyzed has hydrophilicity. Considering the above characteristics, the sample inlet 310 and the material around the inlet of the upper plate 300 are formed to have hydrophilic properties to improve the suction speed of the saliva to be analyzed, and the remaining parts are formed to have hydrophilic properties. It can be formed to have hydrophobicity to prevent the saliva to be analyzed from flowing or being absorbed along the upper plate.

Additionally, the upper plate 300 may be formed to be inclined toward the sample inlet 310 so that the saliva to be analyzed can be concentrated into the sample inlet.

The spacer 200 is laminated between the lower plate and the upper plate to create a space between the lower plate and the upper plate, and a sample inlet channel 210, which is a flow path through which saliva to be analyzed flows, is located at a position corresponding to the sample inlet of the upper plate. This is formed.

Both sides of the spacer 200 are bonded to the area in contact with the lower plate and the upper plate. Adhesion treatment may include either adhesive application or adhesive film treatment.

By stacking the lower plate 100 and the spacer 200, a reaction cell 400, which is a reaction space, is formed, and in the reaction cell 400, a redox reaction occurs through the reaction between the glucose contained in the saliva to be analyzed and the enzyme immobilization material. This occurs.

An enzyme immobilization solution is applied and dried on the lower surface of the reaction cell 400 to form an enzyme immobilization layer 410. An electrode is in contact with the lower part of the enzyme immobilization layer, and an electron acceptor is formed on the surface of the electrode to which a voltage is applied. By measuring the amount of current generated during the oxidation process, the sugar concentration can be measured.

Meanwhile, in order to improve the measurement sensitivity of glucose contained in the target saliva and have rapid response, the enzyme immobilization material must be applied as a thin film, and in the present invention, an inkjet dispenser can be used to form the enzyme immobilization layer of the thin film.

The enzyme immobilization layer 410 is formed by spraying and drying the enzyme immobilization solution with an inkjet dispenser. Specifically, the inkjet dispenser is driven at 45 to 55°C, the voltage is 0.1 to 27 V, and the driving waveform of the inkjet dispenser is Uni. It is a polar type (trapezoid-shaped waveform), consisting of rising time - dwell time - falling time, with rising and falling times from 0.005 to 0.05 ㎲ and dwell times from 0.1 to 11. It can be designed as ㎲.

Additionally, the jetting volume is controlled at 0.1 to 10 pℓ, and after spraying the enzyme immobilization solution, it is dried at 45 to 55°C for 10 to 20 minutes to form an enzyme immobilization layer.

Under the operating conditions of the inkjet dispenser, the enzyme immobilization solution is smoothly discharged to form a thin enzyme immobilization layer evenly without clumping the edges.

The enzyme immobilization solution contains glucose enzyme, electron transfer mediator (redox mediator), and polymer support.

The glucose enzyme is an enzyme that oxidizes glucose, and can be specifically selected from the group consisting of glucose oxidase, glucose dehydrogenase, glucose hexokinase, glutamic oxalacetic transminase, and glutamic pyruvic transminase. However, it is not limited to this. Preferably, flavin adenine dinucleotide-glucose dehydrogenase (FAD-GDH) can be used.

The electron transfer mediator is Ru(NH ₃ ) ₆ ³⁺ , Ru(NH ₃ ) ₆ ²⁺ , ,Ru(NH ₃ )(pyridine) ₅ ³⁺ ,Ru(NH ₃ )(pyridine) ₅ ²⁺ , Fe( Any one selected from the group consisting of CN) ₆ ^3- , Fe(CN) ₆ ^4- , and combinations thereof may be used, and preferably, Ru(NH ₃ ) ₆ ³⁺ or Ru(NH ₃ ) ₆ ²⁺ can be used.

The polymer support is added to disperse and stabilize the glucose enzyme, and includes polyvinyl alcohol, polyvinylidone, polyfluorosulfonate, cellulose acetate, polyamide, hydroxyethyl cellulose, hydroxypropyl cellulose, , carboxymethyl Any one or more of cellulose may be used. Preferably, polyvinyl alcohol can be used.

The enzyme immobilization solution may further add at least one additive selected from the group consisting of a negatively charged compound, a positively charged compound, a hydroxyl compound, a basic amino acid, and an acidic amino acid.

Specifically, negatively charged compounds include 2-thiomalic acid and 2-aminoethylphosphonic acid, positively charged compounds include amines, hydroxylated compounds include 4-mercaptohexane, and basic amino acids include It contains lysine and arginine, and acidic amino acids may include glutamic acid.

Hereinafter, a method of manufacturing a non-blood glucose measurement sensor using saliva according to the present invention will be described.

Figure 3 shows a flowchart of the manufacturing method of the non-blood glucose measurement sensor using saliva of the present invention.

The method of manufacturing a non-blood glucose measurement sensor using saliva of the present invention includes a lower plate preparation step (S100) of preparing a lower plate with electrodes and an upper plate preparation step (S200) of preparing an upper plate with a sample inlet through which the saliva to be analyzed flows. and a spacer stacking step (S300) of stacking a spacer on the upper part of the lower plate and having a sample inlet channel formed at a position corresponding to the sample inlet, and coating the enzyme immobilization solution on the reaction cell formed by stacking the lower plate and the spacer to immobilize the enzyme. It includes an enzyme immobilization layer forming step (S400) of forming an immobilization layer and a top plate stacking step (S500) of stacking the prepared top plate on the spacer.

In the lower plate preparation step (S100), the lower plate 100 on which the electrodes 50 are formed on the base substrate is manufactured and prepared.

The lower plate 100 is formed with electrodes 50 to generate an electrical signal formed by a redox reaction between the saliva to be analyzed and the enzyme immobilization material.

The electrode 50 generates an electrical signal by the oxidation-reduction reaction of the saliva to be analyzed and the enzyme immobilization material, and the electrode is connected to a lead terminal (not shown) to transmit the electrical signal to the measuring device, where the detected Based on the electrical signal value, the concentration of sugar contained in the saliva to be analyzed is measured, and the correlation coefficient between the measured saliva sugar and blood sugar is applied to convert the saliva sugar result into a blood sugar value and display the blood sugar value.

The mechanism for measuring blood sugar levels through salivary sugar is as follows.

Glucose contained in the saliva to be analyzed is oxidized to gluconic acid through an enzymatic reaction with glucose oxidase, and glucose oxidase is reduced. Reduced glucose oxidase is oxidized again through reaction with an electron transfer mediator, and oxidized glucose oxidase reacts with other glucose. At the same time, the reduced electron transfer medium moves to the surface of the electrode to which voltage is applied, loses electrons, and is electrochemically oxidized again to continue participating in the reaction. The current generated during the oxidation process of the electron transfer mediator is proportional to the glucose concentration in saliva, and a voltage of 250 to 400 mV is applied to the electrode to measure the amount of current between the working electrode and the reference electrode to measure the glucose concentration contained in the saliva to be analyzed. By quantitatively measuring and converting the blood sugar value by applying the correlation coefficient between actual blood sugar and saliva sugar, it is possible to measure the blood sugar value through a non-blood sampling method.

The electrode 50 includes a reference electrode, a working electrode, and an auxiliary electrode, and the electrode 50 is made of nickel, gold, copper, titanium, silver, aluminum, germanium, tungsten, tin, antimony, indium, and cadmium. , may contain at least one of palladium, lead, zinc, and platinum, and preferably, a nickel-gold plated electrode may be used.

The base material is not limited as long as it is a polymer compound film with insulating properties, but specifically, polyethylene terephthalate (PET), polyester, polycarbonate, polystyrene, polyimide, polyvinyl. It may contain at least one of polyvinylchloride and polyethylene, and preferably, polyethylene terephthalate (PET) can be used.

The base substrate may have a thickness of 50 to 300 ㎛, preferably 150 to 200 ㎛.

Nickel-gold plated electrodes are formed by first forming a nickel-plated layer on a base substrate, and then forming a second gold-plated layer on the nickel-plated layer after patterning the electrode. The nickel-plated and gold-plated layers are formed using an electroless plating method. It can be.

The nickel plating layer is formed to a thickness of 0.1 to 5 μm, preferably 0.5 to 1.5 μm, by treating the plating solution at 40 to 60°C for 5 to 15 minutes using a nickel plating solution containing a water-soluble nickel salt, reducing agent, complexing agent, and buffer.

Patterning of the electrode can be performed using a photolithography method, and can be formed with a line width of 300 to 500 ㎛ and a line spacing of 80 to 150 ㎛. Preferably, the electrode has a line width of 350 to 450 ㎛ and a line spacing of 90 ㎛. It can be formed from 110㎛ to 110㎛.

The gold plating layer is formed to a thickness of 0.01 to 0.05 μm, preferably 0.02 to 0.03 μm, by treating the gold plating solution at 60 to 80°C for 1 to 5 minutes using a gold plating solution containing a gold salt, a reducing agent, a complexing agent, and an organic acid.

The manufactured nickel-gold plated electrode has a surface resistance of 5Ω or less, specifically 3Ω or less, has high electron mobility generated from the redox reaction of salivary sugar and enzymes, and is insoluble and chemically stable.

In the upper plate preparation step (S200), a upper plate with a sample inlet through which saliva to be analyzed is introduced is prepared.

The upper plate 300 is formed to protect the electrode and enzyme immobilization layer of the lower plate and to facilitate the inflow of the saliva to be analyzed, and a sample inlet 310 through which the saliva to be analyzed is formed.

On the other hand, the saliva to be analyzed has hydrophilicity. Considering the above characteristics, the sample inlet 310 and the material around the inlet of the upper plate 300 are formed to have hydrophilic properties to improve the suction speed of the saliva to be analyzed, and the remaining parts are formed to have hydrophilic properties. It can be formed to have hydrophobicity to prevent the saliva to be analyzed from flowing or being absorbed along the upper plate.

Additionally, the upper plate 300 may be formed to be inclined toward the sample inlet 310 so that the saliva to be analyzed can be concentrated into the sample inlet.

In the spacer stacking step (S300), a spacer 200 having a sample inlet channel formed at a position corresponding to the sample inlet is stacked.

The spacer 200 is laminated between the lower plate and the upper plate to create a space between the lower plate and the upper plate, and a sample inlet channel 210, which is a flow path through which saliva to be analyzed flows, is located at a position corresponding to the sample inlet of the upper plate. This is formed.

Both sides of the spacer are bonded to the area in contact with the lower plate and the upper plate. Adhesion treatment may include either adhesive application or adhesive film treatment.

In the enzyme immobilization layer forming step (S400), an enzyme immobilization solution is coated on the reaction cell formed by stacking the lower plate and the spacer to form an enzyme immobilization layer.

By stacking the lower plate 100 and the spacer 200, a reaction cell 400, which is a reaction space, is formed, and in the reaction cell 400, a redox reaction occurs through the reaction between the glucose contained in the saliva to be analyzed and the enzyme immobilization material. This occurs.

An enzyme immobilization solution is applied and dried on the lower surface of the reaction cell 400 to form an enzyme immobilization layer 410. An electrode is in contact with the lower part of the enzyme immobilization layer, and an electron acceptor is formed on the surface of the electrode to which a voltage is applied. By measuring the amount of current generated during the oxidation process, the sugar concentration can be measured.

Meanwhile, in order to improve the measurement sensitivity of glucose contained in the target saliva and have rapid response, the enzyme immobilization material must be applied as a thin film, and in the present invention, an inkjet dispenser can be used to form the enzyme immobilization layer of the thin film.

The enzyme immobilization layer 410 is formed by spraying and drying the enzyme immobilization solution with an inkjet dispenser. Specifically, the inkjet dispenser is driven at 45 to 55°C, the voltage is 0.1 to 27 V, and the driving waveform of the inkjet dispenser is Uni. It is a polar type (trapezoid-shaped waveform), consisting of rising time - dwell time - falling time, with rising and falling times from 0.005 to 0.05 ㎲ and dwell times from 0.1 to 11. It can be designed as ㎲.

Additionally, the jetting volume is controlled at 0.1 to 10 pℓ, and after spraying the enzyme immobilization solution, it is dried at 45 to 55°C for 10 to 20 minutes to form an enzyme immobilization layer.

Under the operating conditions of the inkjet dispenser, the enzyme immobilization solution is smoothly discharged to form a thin enzyme immobilization layer evenly without clumping the edges.

The enzyme immobilization solution contains glucose enzyme, electron transfer mediator (redox mediator), and polymer support.

The glucose enzyme is an enzyme that oxidizes glucose, and can be specifically selected from the group consisting of glucose oxidase, glucose dehydrogenase, glucose hexokinase, glutamic oxalacetic transminase, and glutamic pyruvic transminase. However, it is not limited to this. Preferably, flavin adenine dinucleotide-glucose dehydrogenase (FAD-GDH) can be used.

The electron transfer mediator is Ru(NH ₃ ) ₆ ³⁺ , Ru(NH ₃ ) ₆ ²⁺ , ,Ru(NH ₃ )(pyridine) ₅ ³⁺ ,Ru(NH ₃ )(pyridine) ₅ ²⁺ , Fe( Any one selected from the group consisting of CN) ₆ ^3- , Fe(CN) ₆ ^4- , and combinations thereof may be used, and preferably, Ru(NH ₃ ) ₆ ³⁺ or Ru(NH ₃ ) ₆ ²⁺ can be used.

The polymer support is added to disperse and stabilize the glucose enzyme, and includes polyvinyl alcohol, polyvinylidone, polyfluorosulfonate, cellulose acetate, polyamide, hydroxyethyl cellulose, hydroxypropyl cellulose, , carboxymethyl Any one or more of cellulose may be used. Preferably, polyvinyl alcohol can be used.

The enzyme immobilization solution may further add at least one additive selected from the group consisting of a negatively charged compound, a positively charged compound, a hydroxyl compound, a basic amino acid, and an acidic amino acid.

Specifically, negatively charged compounds include 2-thiomalic acid and 2-aminoethylphosphonic acid, positively charged compounds include amines, hydroxylated compounds include 4-mercaptohexane, and basic amino acids include It contains lysine and arginine, and acidic amino acids may include glutamic acid.

In the top plate stacking step (S500), the prepared top plate is stacked on the spacer.

The method of manufacturing a non-blood glucose measurement sensor using saliva according to the present invention may further include a post-processing step of trimming the outer peripheral surface of the blood glucose measurement sensor using a slitter or cutting it into a predetermined shape and size after lamination. .

The non-blood glucose measurement sensor using saliva according to the present invention is connected and combined with a measuring device to measure blood sugar. The measuring device quantifies the electrical signal by the redox reaction of the saliva to be analyzed and the enzyme immobilization material, Based on the quantified electrical signal value, the concentration of sugar contained in the saliva to be analyzed is measured, and the correlation coefficient between the measured saliva sugar and blood sugar is applied to convert the saliva sugar result into a blood sugar value and display the blood sugar value.

When a blood sugar measurement is required, the user connects and combines the above measuring device, gargles the mouth 1 to 3 times, collects saliva with a sterilized dropper after 3 to 10 seconds, injects it into the sample inlet, and the measuring device is activated within 1 to 10 seconds. The blood sugar value is displayed on the display. Figure 4 shows an example of a non-blood glucose measurement sensor using saliva applied to a measuring device.

1. Electrode preparation

In order to transmit the nanoampere (50~600nA) level current generated by a saliva-based blood sugar measurement sensor to the measuring device without leakage, an insoluble noble metal electrode with a surface resistance of 5Ω or less is required, and a lower plate with a nickel-gold plated electrode is used as shown below. Manufactured.

Fabrication of the nickel-gold plated electrodes was performed in a clean room, and nickel plating and gold plating were performed using electroless plating methods. Nickel plating was performed at 45°C for 10 minutes, and gold plating was performed at 70°C for 3 minutes.

Prepare a 188㎛ PET (polyethylene terephthalate) film, electroless nickel plating (1㎛) on the film, form an electrode with a line width of 400㎛ and a line spacing of 100㎛ using a photolithography method, and then place the nickel plating on the top. An electroless gold plating (0.025㎛) film was formed.

As a result of testing the surface resistance and corrosion resistance of the manufactured nickel-gold plated electrode, the surface resistance was measured to be less than 3Ω, satisfying the target value of less than 5Ω, and the corrosion resistance was excellent as no change was observed even under continuous salt spray conditions for 72 hours. showed corrosion resistance.

2. Preparation of enzyme immobilization layer

2-1. Preparation of enzyme immobilization solution

An enzyme immobilization solution was prepared by mixing PVA as a polymer support, GDH as a glucose enzyme, hexamine ruthenium as an electron transfer mediator, and additives.

The additives included 2-thiomalic acid, 2-aminoethylphosphonic acid, amine, 4-mercaptohexane, lysine, arginine, and glutamic acid.

2-2. Formation of a thin film enzyme immobilization layer using an inkjet dispenser

A double-sided adhesive middle plate (spacer) was laminated on the manufactured lower plate to form a space (reaction cell) for dispensing the enzyme immobilization solution, and then the enzyme immobilization solution was sprayed and dried using an inkjet dispenser to form an enzyme immobilization layer.

The inkjet dispenser used a voltage of 9 V, the head type (driving waveform) of the inkjet dispenser was a magnetic pass type (unipolar type), the rise and fall times were 0.01 ㎲, and the pause time was 5 ㎲. The jetting volume was 0.5 pℓ, and after spraying the enzyme immobilization solution, it was dried at 50°C for 15 minutes to form an enzyme immobilization layer. Afterwards, the top plate film was laminated with the middle plate (spacer).

Conventional pneumatic (AirJet) dispensers spray at a jetting volume of about 0.5uL/cm, causing problems with enzyme agglomeration at the edges and enzyme immobilization of the thin film. However, when using an inkjet dispenser, the formation of an enzyme immobilization layer and corner aggregation of the thin film occur. Due to the uniform distribution of enzymes, it was confirmed that the reactivity and sensitivity between glucose and enzymes were excellent, making it very suitable for detecting trace amounts of salivary glucose.

3. Sensor performance test

3-1. Current change test per saliva according to time change (I-T test)

The change in current per saliva according to time change was confirmed. As a result of two I-T tests with the same sample per saliva under the conditions of an applied voltage of 350 mV and a measurement time of 5 seconds, discriminatory test results with overlapping measurement curves in the 1.2 to 2.3 second range were confirmed, and the reliability of the measured values was primarily proven. Figure 5 shows an I-T test graph as an example of a non-blood glucose measurement sensor using saliva according to the present invention.

3-2. Sensor linearity test per saliva

In the initial test of linearity by salivary sugar concentration, the slope R² = 0.9576, so the increase in current value may appear uneven as the salivary sugar concentration increases. However, it was judged that this unevenness could be resolved by optimizing the enzyme dispensing amount and incubation time. . Figure 6 shows the current linearity test results for each concentration of saliva as an example of a non-blood glucose measurement sensor using saliva according to the present invention.

3-3. Correlation test between saliva sugar and blood sugar

With the consent of the volunteers, as a result of comparing salivary sugar measurement (equipment: I-T probe) and blood sugar measurement (Roche blood glucose meter) before and after a meal, it was found that salivary sugar changes according to the change in blood sugar before and after a meal, and salivary sugar is converted to blood sugar. When applied, it showed approximately 93% accuracy in blood sugar values. Table 1 below shows the results of the correlation test between salivary sugar and blood sugar.

NO. tester age gender saliva blood sugar blood sugar meter Saliva → blood sugar conversion ceremony After meal ceremony After meal ceremony After meal One Park○○ 57 other 9 13 108 150 100 135 2 Oh○○ 22 female 7 8 85 105 80 98 3 Kim ○○ 27 other 8 10 98 120 91 110

3-4. Evaluation of measurement accuracy by concentration range of prepared saliva

Prepared saliva in the 5, 10, and 15 mg/dl ranges was prepared, measured 10 times, and the average value was shown. Tests were performed at 24-25°C. As a result, it was confirmed that the ISO15197 standard value (CV% 5 or less) was satisfied with CV% of 5 or less in all sections. Table 2 below shows the measurement accuracy test results for each prepared saliva concentration range.

Level 5 mg/dl 10 mg/dl 15 mg/dl Lot. No. #One #2 #3 #One #2 #3 #One #2 #3 One 5.2 5.2 5.2 10 9.7 9.8 15 14 16 2 4.8 4.9 4.8 10 10 9.6 14 15 15 3 5.3 5.1 5.3 9.8 10 10 15 14 15 4 5.4 5.5 5.4 10 9 10.5 14 14 14 5 5.1 5.1 5.1 11 9 10 14 15 14 6 5.2 5.2 5.2 9.7 10 9.8 15 14 14 7 4.7 4.9 4.7 10 10 11 14 15 14 8 4.8 4.8 4.8 9.6 10 9.9 15 14 15 9 5.2 5.1 5.2 10 9 9.6 14 16 15 10 5.2 5.3 5.2 9.7 10 10 15 15 14 AVE
(mg/dL) 5.09 5.11 5.09 9.98 9.67 10.02 14.5 14.6 14.6 VA 0.06 0.04 0.06 0.15 0.22 0.18 0.28 0.49 0.49 SD 0.24 0.21 0.24 0.39 0.47 0.43 0.53 0.70 0.70 CV 4.67 4.07 4.67 3.92 4.88 4.28 3.63 4.79 4.79

4. Reliability by electrode material

A nickel-gold plated electrode manufactured according to an example of the present invention and a conventional carbon paste electrode were prepared, and measurement reliability according to electrode material was confirmed.

Figure 7 shows the measurement reliability according to the electrode material applied to the non-blood glucose measurement sensor using saliva according to the present invention, where A) is a nickel-gold plated electrode and (B) is a conventional carbon paste electrode. Shows measurement reliability.

Table 3 below shows slope and R² values according to electrode material.

Electrode material Nickel + gold plated electrode carbon paste electrode Slope 0.0248 0.0298 R² 0.9984 0.9829

After measuring five times using prepared saliva in the range of 5 to 20 mg/dl, linearity was confirmed with the average value. The carbon paste electrode showed poor accuracy in the 5 mg/dl range, while the nickel+gold plated electrode showed excellent accuracy across the entire prepared saliva range.

As described above, the present invention has been described with a focus on preferred embodiments with reference to the accompanying drawings, but within the scope of those skilled in the art in the technical field to which the present invention pertains without departing from the technical spirit and scope described in the claims of the present invention. The present invention can be implemented with various modifications or modifications. Accordingly, the scope of the present invention should be construed by the appended claims to include examples of many such modifications.

50: electrode
100: lower plate
200: spacer
210: Sample inflow channel
300: top plate
310: sample inlet
400: reaction cell
410: Enzyme immobilization layer

Claims (8)

Lower plate with electrodes formed; and
A top plate where a sample inlet is formed through which saliva to be analyzed flows; and
A spacer laminated between the lower plate and the upper plate and forming a sample inlet channel at a position corresponding to the sample inlet; and
Characterized by comprising; an enzyme immobilization layer formed by coating an enzyme immobilization solution on the reaction cell formed by stacking the lower plate and the spacer.
Non-blood glucose measurement sensor using saliva.
According to clause 1,
The electrode is
Characterized by being a nickel-gold plated electrode.
Non-blood glucose measurement sensor using saliva.
According to clause 1,
The enzyme immobilization layer is
Characterized in that it is formed using an inkjet dispenser.
Non-blood glucose measurement sensor using saliva.
According to clause 3,
The enzyme immobilization layer is
Characterized by spraying 0.1 to 10 pℓ of enzyme immobilization solution with an inkjet dispenser and then drying.
Non-blood glucose measurement sensor using saliva.
A lower plate preparation step (S100) of preparing a lower plate on which electrodes are formed; and
A top plate preparation step (S200) of preparing a top plate with a sample inlet through which the saliva to be analyzed flows;
A spacer stacking step (S300) of stacking a spacer on the upper part of the lower plate and having a sample inlet channel formed at a position corresponding to the sample inlet;
An enzyme immobilization layer forming step (S400) of forming an enzyme immobilization layer by coating an enzyme immobilization solution on the reaction cell formed by stacking the lower plate and the spacer; and
Characterized by comprising a top plate stacking step (S500) of stacking the prepared top plate on the spacer.
Manufacturing method of non-blood glucose measurement sensor using saliva.
According to clause 5,
In the lower plate preparation step (S100)
The electrode is characterized in that it is a nickel-gold plated electrode.
Manufacturing method of non-blood glucose measurement sensor using saliva.
According to clause 5,
The enzyme immobilization layer forming step (S400) is
Characterized by forming an enzyme immobilization layer using an inkjet dispenser.
Manufacturing method of non-blood glucose measurement sensor using saliva.
According to clause 7,
The enzyme immobilization layer forming step (S400) is
Characterized by spraying 0.1 to 10 pℓ of enzyme immobilization solution with an inkjet dispenser and then drying.
Manufacturing method of non-blood glucose measurement sensor using saliva.