KR20020091473A - A self-sampling-and-flow biosensor comprising parallel microporous electrodes. - Google Patents

Abstract

PURPOSE: A self-sampling flow type biosensor is provided to achieve use convenience and eliminate space restriction, while allowing for a continuous measurement over a long period of time. CONSTITUTION: A self-sampling flow type biosensor(1) comprises an electrode connection part(10); an insulation film(12); a microporous working electrode having a sensor material attached thereto; a microporous auxiliary/reference electrode parallel to the microporous working electrode; a microporous thin film(11) attached to the microporous working electrode; and an absorption pad(16). Selectively, a microporous thin film(15) for introduction of a test sample(20) is interposed between the microporous working electrode and the microporous auxiliary/reference electrode.

Description

A self-sampling-and-flow biosensor comprising parallel microporous electrodes.

The present invention relates to a self-sampling flow-type biosensor comprising a face-to-face porous electrode, specifically, an electrode connector; Insulating film; A porous working electrode to which the sensor material is fixed; A porous auxiliary / reference electrode positioned on the working electrode; A porous thin film attached to the porous working electrode; It relates to a biosensor comprising an absorbent pad, and optionally further comprising a porous thin film for the introduction of a sample inserted into the diaphragm between the porous working electrode and the porous auxiliary / reference electrode.

Electrochemical biomaterial measuring method has high selectivity and sensitivity, and even if the sample is cloudy, the sample can be used without any pretreatment and has the advantage of measuring a small amount of sample in a short time. For example, an invasive method that uses a tool such as a lancet to pierce a portion of the body to collect and measure blood. Recently, in order to replace the invasion type measurement method involving the pain of the patient, methods have been developed to allow long-term measurement by continuously collecting fluid from the skin epidermis instead of blood samples. In addition, there is a growing demand for biosensors capable of continuous measurement for skin adhesion suitable for such measurement methods.

However, the conventional biosensors, which are used to continuously measure large amounts of clinical and general samples, have to transfer the sample to be measured by using a separate device such as a peristaltic pump to the site where the sensor is installed. There is a disadvantage that the operation is inconvenient and expensive.

Accordingly, the present inventors have made an effort to solve the above problems, and as a result, the present invention forms a porous porous electrode by forming a porous electrode on the front and back of the porous thin film, or inserting another porous thin film between the porous electrodes as a diaphragm or A self-sampling-and-flow biosensor comprising a face-to-face porous electrode formed on a ceramic / plastic substrate instead of a porous thin film was fabricated. The present invention was completed by finding out that the material to be measured can be continuously quantitatively analyzed by capillary or chromatographic movement, and the sample can be collected by itself without using a peristaltic pump by using a large porous electrode. .

An object of the present invention is an electrode connector; Insulating film; A porous working electrode to which the sensor material is fixed; A porous auxiliary / reference electrode positioned on the working electrode; A porous thin film attached to the porous working electrode; It includes an absorbent pad, and optionally to provide a biosensor that can further include a porous thin film for sample introduction inserted into the diaphragm between the porous working electrode and the porous auxiliary / reference electrode.

It is an object of the present invention to provide a disposable biosensor capable of continuously measuring the concentration of metabolites contained in a bodily fluid sample drawn out in small amounts from the skin epidermis. In addition, the present invention provides a disposable biosensor that can be used when a limited continuous observation is required.

Figure 1 shows a self-sampling flow-type biosensor comprising a face-to-face porous electrode of the present invention,

FIG. 2A illustrates a facing porous electrode used in the biosensor of FIG. 1 ,

FIG. 2B is a cross sectional view taken along the line a-a 'of the biosensor of FIG. 2A ;

FIG. 2C is a cross- sectional view of the b-b ′ straight line shown in the biosensor of FIG. 2A ;

Figure 3a is a structure of a self-sampling flow-type biosensor comprising a porous porous electrode in which another porous thin film is inserted into the diaphragm between the porous electrode of the present invention,

FIG. 3B is a cross sectional view taken along the line a-a 'of the biosensor of FIG. 3A ;

3c is a cross-sectional view of the b-b 'straight line shown in the biosensor of FIG. 3a ,

Figure 4a is a structure of a self-sampling flow-type biosensor comprising a face-to-face porous electrode formed with a porous working electrode on the ceramic / plastic substrate of the present invention,

FIG. 4B is a cross sectional view taken along the line a-a 'of the biosensor of FIG. 4A ;

4C is a cross- sectional view of the b-b ′ straight line shown in the biosensor of FIG. 4A ;

5 is a state diagram of use of the self-sampling flow-type biosensor comprising a face-to-face porous electrode of the present invention,

FIG. 6A is a dynamic response curve for glucose standard solution of a self-sampled flow-type biosensor comprising a face-to-face porous electrode immobilized with glucose oxidase,

Figure 6b is a calibration curve for glucose standard solution in the biosensor of Figure 6a ,

FIG. 7A is a dynamic response curve for glucose standard solution of a self-sampling flow-type biosensor comprising a face-to-face porous electrode on which glucose oxidase and potassium parsiaside are immobilized, FIG.

FIG. 7b is a calibration curve for glucose standard solution in the biosensor of FIG .

FIG. 8 is a dynamic response curve of glucose standard solution of a self-sampled flow type biosensor comprising a face-to-face porous electrode to which glucose oxidase and a self-assembled monolayer are fixed.

<Explanation of symbols on main parts of the drawings>

1: Biosensor 10: electrode connection

11: porous thin film 12: insulating film

13: porous working electrode 14: porous auxiliary / reference electrode

15: porous thin film 16: absorbent pad for sample introduction

17: adhesive 18: ceramic / plastic substrate

19: Sample 20: Sample

In order to achieve the above object, the present invention is an electrode connecting portion; Insulating film; A porous working electrode to which the sensor material is fixed; A porous auxiliary / reference electrode positioned on the working electrode; A porous thin film attached to the porous working electrode; Provided is a self-sampling flow-type biosensor comprising an absorption pad, and optionally further comprising a porous thin film for introduction of a sample inserted into the diaphragm between the porous working electrode and the porous auxiliary / reference electrode.

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

1 illustrates an example of a biosensor according to the present invention, wherein the biosensor 1 includes an electrode connection part 10; A porous thin film 15 for introducing a sample; Insulating film 12; A porous thin film 11; Porous electrode system; And an absorption pad 16.

It is responsible for introducing the sample 20 into the biosensor 1 by chromatographically moving the sample 20 from the sample 19 to be tested by the osmotic pressure generated by the porous thin film 15 or the absorption pad 16 for sample introduction. These may be measured by a detector (not shown) by a chemical or biochemical reaction with a sensor material immobilized on the surface of the porous working electrode 13 (e.g., detection of electric current by electrons or electrical conductivity). Measurement) occurs in proportion to the concentration of the substrate. Meanwhile, sample by-products generated by a chemical or biochemical reaction with the sensor material are transferred to the absorbent pad 16 through the porous working electrode 13, the porous thin film 11, and the porous auxiliary / reference electrode 14. . Absorption pad 16 not only generates an osmotic pressure for sample introduction, but also absorbs the transferred sample 20 at a constant rate after a series of chemical reactions so that the sample can be continuously measured by controlling the flow of the sample. do.

2Shows a large-area porous electrode used in the self-sampling flow-type biosensor according to the present invention, in which a porous working electrode 13 and a porous auxiliary / reference electrode 14 are stacked on the front and rear surfaces of the porous thin film 11. In more detail, when the configuration is described, The electrode connecting portion 10 and the porous thin film 11 are disposed at the center of the electrode body, and the porous auxiliary / reference electrode 14 and the insulating film 12 are disposed on the surface of the porous working electrode 13 stacked on one surface of the porous thin film 11. ).

In this case, a sensor material such as an oxidase, an antibody, a chemical molecule recognition material, and / or an electron transfer medium is fixed to the surface of the porous working electrode 13. The porous thin film 11 has a mesh-like structure, and when the electrode forming material such as silver, platinum, gold, palladium, indium oxide, etc. is deposited using physical vapor deposition, the porous working electrode 13 and the porous auxiliary / reference electrode (14) can be formed.

Figure 3 shows another example of the biosensor of the present invention, the biosensor comprises an electrode connection (10); A porous thin film 11a serving as a support of the porous electrode; A porous working electrode 13 and an insulating film 12 stacked on one surface of the porous thin film 11a; A porous thin film 15 and an adhesive 17 for sample introduction; A porous auxiliary / reference electrode 14 and an insulating film 12 stacked on one surface of the other porous thin film 11b; Absorption pads 16 are sequentially stacked and formed, more specifically, a porous working electrode 13 having a sensor material fixed thereto is formed on one surface of the porous thin film 11a, and the other porous thin film 11b is formed on the other surface. After the porous auxiliary / reference electrode 14 is formed, another porous thin film 15 is inserted to introduce a sample in the form of a diaphragm between the two electrodes 13 and 14, and an adhesive ( Is fixed by 17). ( FIGS. 3A, 3B and 3C )

In the biosensor of FIG. 3 , the sample is introduced into the biosensor through the porous thin film 15 for introducing the sample inserted into the diaphragm, and the introduced sample is combined with the sensor material fixed to the surface of the porous working electrode 13. A signal that can be measured by a detector (not shown) by a chemical or biochemical reaction (eg, detection of electrical current by an electron or measurement of electrical conductivity) is generated in proportion to the concentration of the substrate.

On the other hand, the sample by-product generated by the chemical or biochemical reaction with the sensor material is absorbed by the absorbent pad 16 can be measured continuously. In addition, by introducing a colloidal gold particle or a conductive polymer coupled with an immunoglobulin at the end of the porous thin film 15 for the introduction of the sample inserted into the diaphragm, the electrical conductivity may be increased to quantify the antigen antibody by measuring the conductivity.

Furthermore, since the porous thin film 11a on which the porous working electrodes are stacked is not related to the movement path of the by-product, the porous thin film 11a may be replaced with a ceramic / plastic substrate, and an example thereof is illustrated in FIG. 4 .

Figure 4 shows another example of the biosensor of the present invention, the biosensor comprises an electrode connection 10; An auxiliary / reference electrode 14 stacked on one surface on the ceramic / plastic substrate 18; A porous thin film 15 and an adhesive 17 for sample introduction; A working electrode 13 and an insulating film 12 stacked on one surface of the other porous thin film 11b; Absorption pads 16 are sequentially stacked and formed, and a sensor material forms a porous working electrode 13 stacked on a porous thin film, and between the porous working electrode 13 and the porous auxiliary / reference electrode 14. It is characterized in that the porous thin film 15 for sample introduction is inserted, and the insulating film 12 is fixed by the adhesive 17. ( FIGS. 4A, 4B and 4C )

Formation of the electrode on the surface of the ceramic / plastic substrate 18 may be a variety of general deposition methods such as screen printing, physical vapor deposition and chemical vapor deposition.

The porous working electrode and the porous auxiliary electrode introduced various porous electrode materials such as silver, platinum, gold, palladium, and indium oxide, and a method of manufacturing the metal was released by applying a high potential in a vacuum to release metal particles. Physical vapor deposition was used to deposit the pattern.

The material of the porous thin film 15 and the absorbent pad 16 for the sample introduction is preferably selected from the group consisting of nitrocellulose, filter paper, glass fiber membranes and inorganic polymers composed of organic polymers of nylon and absorbent ceramics. However, the present invention is not limited thereto, and is not particularly limited as long as it introduces a sample chromatographically by osmotic pressure and absorbs it.

The insulating film 12 may be formed by using a polyvinyl chloride film or by heat treatment after screen printing an insulating polymer paste, and the insulating film 12 may maintain a constant electrode area to improve reproducibility.

As the adhesive 17 for fixing the porous working electrode and the porous auxiliary / reference electrode, a double-sided tape or a conventional strong adhesive may be used. Preferably, a double-sided tape having the same thickness as that of the porous thin film for sample introduction may be used. Is to use.

SUMMARY OF THE INVENTION An object of the present invention is to provide a disposable biosensor capable of continuously measuring the concentration of metabolites contained in a bodily fluid sample drawn from a small amount of skin epidermis, and to provide a biosensor that can be used when continuous observation is required for a limited time. It is.

Examples of the sensor material for generating a signal that can be detected by the detector by a chemical and / or biochemical reaction with a biosensor comprising the large-surface porous electrode of the present invention include oxidases, antibodies, chemical molecule recognition materials, and And / or electron transfer mediators, but may vary depending on the type of substance being measured (eg, glucose, cholesterol, lactate, creatinine, GOT, GTP, etc.).

In the present invention, in order to measure the concentration of glucose in a small amount of tissue fluid (body fluid) continuously flowing out of the skin epidermis, glucose oxidase, glucose oxidase and delivery media, preferably glucose oxidase and electron transfer media, ferrocene And self-assembled monolayers can be produced in combination.

As shown in FIG . 5 , a small amount of tissue fluid (body fluid) samples continuously flowing from the skin epidermis may be used. In this case, the working electrode, the auxiliary electrode, and the thin film may be made porous, and the by-products pass through them. It is possible to continuously measure the metabolic substances contained in the body fluids by allowing them to be moved and by installing absorbent pads so that the body fluids can flow out of the skin surface for a long time without chemical samples. have. It is usually measurable continuously for at least 4 hours, preferably for about 4 to 20 hours.

In addition, in the case of measuring the concentration of cholesterol in the tissue fluid (body fluid) flowing out continuously can be measured by a similar method using cholesterol oxidase.

The above is based on the measurement of glucose. Likewise, by introducing various enzymes on the porous electrode, biological samples such as cholesterol, lactate, creatinine, protein, amino acid, urine, environmental samples, agriculture and industry Various organic or inorganic concentrations in samples or food samples can be quantified.

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

However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the present invention.

Preparation Example 1 Fabrication of Large-Scale Porous Gold Electrode

Fabrication of the large-sized porous gold electrode was made by deposition technique using the LVC-76 SEM Sputtering System. After placing a microporous nylon membrane (15 × 30 ㎜) under the electrode engraved membrane, gold was deposited by injecting argon gas under vacuum to release gold particles. At this time, the pressure of the reaction vessel was 75 mTorr, the plasma current was 25 to 30 mA, and the operating potential was set to 350 to 500 V. In the same manner, gold was also deposited on the back side to form a large porous gold electrode. The formed porous gold electrode was treated with a polyvinyl chloride (PVC) membrane except for the working part (4 mm in diameter) to block the pores so that the sample or substrate was absorbed only by the porous gold electrode part.

Example 1 Fabrication of a Glucose Sensor Using a Large-Scale Porous Gold Electrode Fixing Glucose Oxidase

10 mg / mL of glucose oxidase was dissolved in a buffer solution (140 mM NaCl, pH 7.4 PBS) on the porous gold electrode prepared in Preparation Example 1, and 10 μl of the glucose oxidase solution was immobilized on the working electrode. Thereafter, a porous thin film and an absorption pad were formed on the front and back of the porous gold electrode to complete the glucose sensor.

Example 2 Fabrication of a Glucose Sensor of a Large-Scale Porous Gold Electrode Fixing Glucose Oxidase and Potassium Parsiase

After dissolving 10 mg / mL of glucose oxidase and 200 mM of potassium parianide in a buffer solution (140 mM NaCl, pH 7.4 PBS) on the porous gold electrode prepared by the method of Preparation Example 1, 10 μl of the mixed solution was added thereto. After immobilization on the porous working electrode, a porous thin film and an absorption pad were formed on the front and back of the porous gold electrode to complete the glucose sensor.

Example 3 Fabrication of a Glucose Sensor of a Large-Scale Porous Gold Electrode Fixing Glucose Oxidase and Self-Assembly Monolayer

The following self-assembled monolayer was immobilized on the porous gold electrode manufactured by the method of Preparation Example 1. More specifically, 1.5% 2-mercaptoethylamine is dissolved in ethanol to make 10 mL, and 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide mesaoda in 10 mL of distilled water. 2 mM of 1 (1-ethyl-3 (3-dimethylaminopropyl) carbodiimide methiodide (hereinafter referred to as "EDC") was prepared. After the two solutions were mixed 1: 1, 10 μl was dropped onto the porous working electrode, and 10 μl of the glucose oxidase solution prepared in the above example was immobilized on the porous working electrode. The enzyme was immobilized by dropping 10 μl of a mixed solution of 2 mM EDC and 50 mM ferroceneacetic acid into a 10 mg / mL glucose oxidase solution to a porous working electrode. Thereafter, a porous thin film and an absorption pad were formed on the front and back of the porous gold electrode to complete the glucose sensor.

Hereinafter, the characteristics of the biosensor according to the present invention were tested.

The inventors examined the effects of continuous glucose sensors on the large porous gold electrodes produced by the following examples, respectively.

Experimental Example 1 Glucose Sensor Using a Large-Scale Porous Gold Electrode Fixing Glucose Oxidase

In order to determine whether the glucose glucose-enzyme-fixed glucose sensor obtained in Example 1 was capable of measuring glucose continuously in a glucose standard solution, a current value with respect to time was measured, and the results are shown in FIG. 6.

Figure 6a is a graph showing the results of continuous measurement of the current value with respect to time for glucose standard solution for 30 minutes at the application potential 0.8V. As can be seen in FIG. 6B, the calibration curve for FIG. 6A, the linear response was up to 200 mg / dL glucose with a slope of 2.60 × 10 ⁻³ nA / (mg / dL) and a linearity of 0.988. From these results, it was found that continuous glucose measurement using a large-face porous gold electrode was possible.

<Experiment 2> Glucose sensor using a large surface porous gold electrode fixed with glucose oxidase and potassium parisian ide

In order to determine whether the glucose sensor prepared in Example 2 was capable of measuring glucose continuously in a glucose standard solution, a current value with respect to time was measured, and the results are shown in FIG. 7.

FIG. 7A is a graph showing the results of continuous measurement of a current value of glucose standard solution mixed with potassium ferricyanide 200 mM at a potential of 0.38 V for 4 hours or more. The concentration of glucose (50 mg / dL, 100 mg / dL, 200 mg / dL) shows a constant current value for more than 1 hour. FIG. 7B shows a linear response to glucose 200 mg / dL as a calibration curve for FIG. 7A, where the slope was 85.8 nA / (mg / dL) and the sensitivity was improved and the linearity was 0.998. Therefore, from the results of FIGS. 7A and 7B, the glucose concentration (50 mg / dL, 100 mg / dL, 200) at a relatively low type potential of 0.38 V using a large-area porous electrode immobilized with potassium ferricyanide as an electron transfer medium mg / dL), it can be seen that it can be measured continuously.

Experimental Example 3 Glucose Sensor Using a Face-porous Porous Gold Electrode Fixing Glucose Oxidase and Self-Assembly Monolayer

In order to determine whether the glucose sensor prepared in Example 3 was capable of measuring glucose continuously in a glucose standard solution, a current value against time was measured, and the results are shown in FIG. 8.

8 is a result of continuously measuring the current value of the glucose standard solution for 2 hours or more at an applied potential of 0.45 V, and responding to a constant current value for glucose concentration (100 mg / dL, 200 mg / dL, 400 mg / dL) Shows. Therefore, the glucose concentration (100 mg / dL, 200 mg / dL, 400 mg / dL) at the relatively low type potential of 0.45 V was observed for the large-area porous gold electrode on which the glucose redox enzyme and the self-assembled monolayer were fixed. It was found that can be measured continuously.

As described in detail above, the self-sampling flow-type biosensor including the porous porous electrode of the present invention is easy to manufacture and manipulate, so that it can be conveniently used without restriction of a measuring place without the help of a professional person, and a small amount By introducing a body fluid sample at a constant rate for a long time without pretreatment, the measurement target material can be continuously measured for a long time. In addition, the disposable glucose sensor of the present invention can continuously measure the amount of glucose in the blood for 4 hours or more, compared to the existing glucose sensor, and has the advantage of enabling precise diagnosis by wearing once.

Claims (20)

An electrode connector 10; Insulating film 12; A porous working electrode 13 to which the sensor material is fixed; A porous auxiliary / reference electrode 14 positioned on the working electrode face; A porous thin film 11 attached to the porous working electrode; Self-sampling flow-type biosensor comprising an absorption pad (16). The method of claim 1, further comprising: an electrode connector (10); A porous thin film 15 for introducing a sample; Insulating film 12; A porous thin film 11; A porous working electrode 13 stacked on one surface of the porous thin film 11; A porous auxiliary / reference electrode 14 and an insulating film 12 facing the working electrode; Biosensor comprising an absorption pad 16, the porous electrode is formed on the front and back of the porous thin film. The method of claim 1, wherein the biosensor comprises an electrode connection (10); A porous thin film 11a serving as a support of the porous electrode; A porous working electrode 13 and an insulating film 12 stacked on one surface of the porous thin film 11a; A porous thin film 15 and an adhesive 17 for sample introduction; A porous auxiliary / reference electrode 14 and an insulating film 12 stacked on one surface of the other porous thin film 11b and fixed at positions of the working electrode and the facing surface; Biosensor comprising an absorbent pad 16, further comprising a porous thin film 15 for introducing a sample inserted into the diaphragm between the porous working electrode 13 and the porous auxiliary / reference electrode 14. . The method of claim 1, wherein the biosensor comprises an electrode connection (10); An auxiliary / reference electrode 14 stacked on one surface on the ceramic / plastic substrate 18; A porous thin film 15 and an adhesive 17 for sample introduction; A porous working electrode 13 and an insulating film 12 laminated on one surface of the other porous thin film 11b and fixed to a position opposite to the auxiliary / reference electrode; Biosensor comprising an absorbent pad (16), wherein a porous working electrode (13) having a sensor material laminated on a porous thin film is fixed. The biosensor according to claim 1, wherein the porous thin film (15) for introducing the sample is made of a material capable of introducing the sample chromatographically. The biosensor according to claim 1, wherein the absorbent pad (16) is made of a material capable of introducing a sample chromatographically by osmotic pressure. 7. The material according to any one of claims 5 to 6, wherein the material used for the porous thin film 15 and the absorbent pad 16 for introducing the sample is selected from the group consisting of organic polymers, inorganic polymers and mixtures thereof. Biosensor, characterized in that. The biosensor according to claim 7, wherein the organic polymer is selected from the group consisting of nitrocellulose, manure paper, glass fiber membranes and nylon. 8. The biosensor of claim 7, wherein the inorganic polymer is selected from the group consisting of absorbent ceramics. The biomaterial according to claim 1, wherein the electrode material forming the porous working electrode 13 and the porous auxiliary / reference electrode 14 is selected from the group consisting of silver, platinum, gold, palladium and indium oxide. sensor. The biosensor according to claim 10, wherein the porous working electrode (13) and the porous auxiliary / reference electrode (14) are formed by physical vapor deposition. The biosensor of claim 1, wherein the blood sample is separated into plasma and blood cells by chromatographic movement by a porous thin film and a porous electrode. The biosensor according to claim 1, wherein the porous thin film and the porous electrode can continuously measure a sample for 4 hours or more by chromatographic movement. The biosensor of claim 1, wherein the sensor material is at least one selected from the group consisting of oxidases, antibodies, chemical molecule recognition materials, and electron transfer mediators. 15. The biosensor of claim 14, wherein the sensor material is glucose oxidase. 15. The biosensor of claim 14, wherein the sensor material is a combination of glucose oxidase and potassium parisianide. 15. The biosensor of claim 14, wherein the sensor material is a combination of glucose oxidase and a self-assembled monolayer. 5. A biosensor according to claim 4, wherein the formation of electrodes on the surface of the ceramic / plastic substrate is by screen printing, physical vapor deposition and chemical vapor deposition. The disposable biosensor of claim 1, wherein the biosensor can continuously measure the concentration of the metabolite contained in the bodily fluid sample drawn out from the skin epidermis. The biosensor according to claim 1, wherein the biosensor induces an antigen antibody reaction coupled with colloidal gold particles or a conductive polymer to measure conductivity at an electrode.