KR100887632B1 - Biosensor - Google Patents

Abstract

The biosensor according to the present invention includes a first substrate, an adhesive cover disposed on the first substrate with a sample inlet, a second substrate covering the adhesive cover, a first substrate, an adhesive cover and a second substrate, and a sample inlet It includes a through-hole extending in a direction intersecting with the.

Biosensor, small sample, back hole, adjusting reagent, multi function

Description

Biosensor {BIOSENSOR}

1 is a perspective view of a biosensor according to a first embodiment of the present invention.

2 is an exploded perspective view of a biosensor according to a first embodiment of the present invention.

3 and 4 are plan views illustrating cutting a biosensor according to a first embodiment of the present invention.

5 to 7 are schematic perspective views showing a method of cutting a biosensor according to a first embodiment of the present invention.

8 is an exploded perspective view of a biosensor according to a second embodiment of the present invention.

9 is an exploded perspective view of a biosensor according to a third embodiment of the present invention.

10 is an exploded perspective view of a biosensor according to a fourth embodiment of the present invention.

11 is an exploded perspective view of a biosensor according to a fifth embodiment of the present invention.

12 is a dynamic response curve showing a measurement result using the constant voltage vs. current method in the experimental example of the present invention.

13 is a graph showing the measuring principle using the constant voltage vs. current method of the biosensor according to the experimental example of the present invention.

FIG. 14 is a graph showing the results of different measurements of erythrocyte volume and glucose concentration before fitting the adjustment reagent. FIG.

FIG. 15 is a graph showing the results obtained by varying erythrocyte volume and glucose concentration after fitting the adjustment reagent. FIG.

The present invention relates to a biosensor, and more particularly, to a biosensor for measuring a target substance in a blood sample.

In general, biosensors such as blood glucose meters mainly use electrochemical methods. In general, in the electrochemical measurement method, the enzyme and the adjusting reagent are fixed in a cell composed of a positive electrode and a negative electrode. When the sample is introduced into such a biosensor, oxygen or an electron transfer medium is reduced when the target substance in the sample is oxidized by the catalytic action of the enzyme, and the reduced oxygen or electron transfer medium is forcibly oxidized by the voltage of the electrode. To cause a change in electrons. Electrochemical measurement is a method of quantifying the change of the former and indirectly measuring the amount of the target substance.

Since such biosensors use blood as a sample, interference is caused by various types of blood. In addition, biosensors need to be developed to enable faster, more convenient and accurate measurements using less blood samples.

An attempt is made to provide a biosensor using a smaller amount of sample. In addition, to avoid interference with various blood types, to provide a biosensor that can be measured accurately and conveniently.

The biosensor penetrates through the first substrate, the adhesive cover disposed with the sample inlet on the first substrate, the second substrate covering the adhesive cover, the first substrate, the adhesive cover and the second substrate, and intersect with the sample inlet. It includes at least one through hole formed to extend.

The sample inlet may include a first sample inlet formed on one side of the adhesive cover and a second sample inlet formed on the other side of the adhesive cover, and the first sample inlet and the second sample inlet may be formed at positions shifted from each other.

The biosensor may further include a first electrode formed along a circumference of the first substrate between the first substrate and the adhesive cover, and a second electrode formed surrounded by the first electrode. In this case, the second electrode may be formed to surround the through hole.

The biosensor may further include a non-conductive material layer disposed between the first electrode and the second electrode and the adhesive cover to expose a portion of the first electrode and the second electrode.

The biosensor may further include a third electrode disposed between the adhesive cover and the second substrate. In this case, the first electrode, the second electrode and the third electrode may be formed of a metal, a metal salt or a paste of carbon (C), gold (Au), silver (Ag), platinum (Pt), copper (Cu), titanium (Ti), and the like. It may be formed of a material in the form.

On the other hand, the biosensor is a first electrode formed between the first substrate and the adhesive cover covering the entire surface of the first substrate, and a second electrode formed covering the entire surface of the second substrate between the adhesive cover and the second substrate It may further include.

In addition, the one or more through holes may include a plurality of through holes, and the plurality of through holes may be formed to extend in parallel to each other. At this time, the first electrode formed by exposing the through hole between the first substrate and the adhesive cover, and the second electrode formed by exposing the through hole between the adhesive cover and the second substrate may be further included.

The biosensor may further include a first electrode formed on the first substrate, an insulating layer formed on the first electrode, and a second electrode formed on the insulating layer. The first electrode includes a first sample measuring unit extending in a direction parallel to the through holes between the through holes, and the second electrode is spaced apart from the first sample measuring unit by a predetermined distance and formed in parallel with the second sample. It may include a measuring unit.

On the other hand, the biosensor further comprises an adjustment reagent disposed at the sample inlet, the adjustment reagent is an enzyme that reacts to the material contained in the sample injected into the sample inlet, an electron transfer medium for delivering electrons generated from the enzyme, and Dispersion stabilizers for dispersing and stabilizing electron transfer mediators may be included.

Enzymes include glucose oxidase, glucose dehydrogenase, alcohol oxidase, alcohol dehydrogenase, PQQ (pyrroloquinone) and nicotinamide adenine dinucleotide / hydrogen, nicotine Amide adenine dinucleotides).

Electron transfer mediators are ferrocene, quinone, cobalt, nickel, ruthenium, ferricane compounds, rhodium, palladium, osmium, iridium, platinum, and hexaamineruthenium (III). ) Chloride, derivatives containing them, and transition metals.

Dispersion stabilizers include polyvinylalcohol, polyethyleneoxide, polyethyleneglycol, carboxymethyl cellulose, hydroxyethyl cellulose, 2-hydroxyethyl cellulose ethyl cellulose, hydroxypropyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, polyvinylidenefluoride, polymethylmethacrylate, It may include at least one of styrene butyl rubber (stylene butyl rubber).

The modulating reagent may further include a surfactant, and the surfactant may include at least one of an anionic surfactant, a cationic surfactant, an amphoteric surfactant, and a nonionic surfactant. The anionic surfactant may include at least one of soap and alkylbenzene sulfonate.

The modulating reagent further comprises a phase transfer catalyst, wherein the phase transfer catalyst is one of phosphonium based, crown ethers based, ammonium based and polyethylene glycol based (PEG) reagents. It may include at least one.

On the other hand, the adjustment reagents include glucose oxidase, hexaamineruthenium (III) chloride, carbonyl methyl cellulose, microcrystalline cellulose, tricaprylmethyl ammonium chloride, t-octylphenoxypolyethoxyethanol (t-Octylphenoxypolyethoxyethanol) and soap (soap) may be included.

Hereinafter, with reference to the drawings will be described an embodiment of the present invention. These embodiments of the present invention are merely for illustrating the present invention and the present invention is not limited thereto.

1 is a perspective view of a biosensor 100 according to a first embodiment of the present invention. As shown in FIG. 1, the biosensor 100 includes a first substrate 10, a first electrode 50, a second electrode 52, and a first substrate 10 formed on the first substrate 10. And a second substrate 30 covering the adhesive cover 20 and the adhesive cover 20 disposed on the sample inlet 22. In addition, the biosensor 100 includes a through hole 40 formed to penetrate the first substrate 10, the adhesive cover 20, and the second substrate 30. The sample inlet 22 includes a first sample inlet 22a provided on one side of the biosensor 100 and a second sample inlet 22b provided on the other side, and the through hole 40 includes It is formed in the direction which cross | intersects the 1st sample injection port 22a and the 2nd sample injection port 22b.

2 is an exploded perspective view of the biosensor 100 according to the first embodiment of the present invention. Hereinafter, each component of the biosensor 100 will be described in more detail with reference to FIG. 2.

As shown in FIG. 2, the biosensor 100 includes an adjustment reagent 62 introduced over a defined electrode at a specific portion of the electrode. In the present embodiment, the adjusting reagent 62 is fixed on a portion of the first electrode 50. The adjusting reagent 62 includes an enzyme that reacts with a substance contained in the sample injected into the sample inlet 22, an electron transfer medium for delivering electrons generated from the enzyme, and a dispersion stabilizer for dispersing and stabilizing the enzyme and the electron transfer medium. Include. The components of the adjusting reagent 62 will be described later.

Holes 40a and 40e forming a part of the through hole 40 are formed in the first substrate 10 and the second substrate 30, respectively. The first substrate 10 and the second substrate 30 may be made of plastic, polyester, polypropylene, polycarbonate-based polymer material, ceramic, glass, and the like, and preferably, a polyester-based polyethylene terephthalate (PET) film. Can be used.

The adhesive cover 20 disposed between the first substrate 10 and the second substrate 30 forms the sample inlet 22 while bonding the first substrate 10 and the second substrate to each other. At this time, a hole 40c is formed in the adhesive cover 20 to form a part of the through hole 40. The adhesive cover 20 may be made of a tape coated with an adhesive on both or one side of the film substrate. The adhesive of the tape forming the adhesive cover 20 may be acrylic, urethane, epoxy, rubber, polyvinyl ether, silicone system, PET film may be used as the film substrate.

The biosensor 100 further includes a third electrode 54 disposed between the adhesive cover 20 and the second substrate 30. In addition, the first electrode 50 is formed along the circumference of the first substrate 10 on the first substrate 10, the second electrode 52 is disposed to be wrapped by the first electrode 50, It is formed to surround the hole formed in the first substrate 10. These electrodes transfer the flow of electrons by the reaction after introduction of the sample. In this case, each of the electrodes acts as a working electrode, a counter electrode, and a recognition electrode, and may further form three or more electrodes as necessary. In this embodiment, as an example, the first electrode 50 serves as a working electrode, the second electrode 52 serves as a recognition electrode, and the third electrode 54 serves as a counter electrode. On the other hand, the hole 40d for forming a part of the through hole 40 is also formed in the third electrode 54.

The first electrode 50, the second electrode 52, and the third electrode 54 may be pasted or plated using various electrode materials such as gold, platinum, silver, carbon, tungsten, nickel, and copper. It may be formed in the form of a plate, preferably carbon dough is used. The electrodes 50, 52, and 54 may be formed on a substrate using a method such as screen printing, photolithography, adhesion, or deposition, and may be formed such that only measurement sites are separated by an insulator film or an adhesive. have.

The biosensor 100 further includes a non-conductive material layer 60 between the first electrode 50, the second electrode 52, and the adhesive cover 20, so that the first electrode 50 and the second electrode 52. Use only a portion of the) as the measurement site. The non-conductive material layer 60 is also provided with a hole 40b forming a part of the through hole 40.

As described above, the through hole 40 is positioned at the same position on the first substrate 10, the non-conductive material layer 60, the adhesive cover 20, the third electrode 54 and the second substrate 30, respectively. The holes 40a, 40b, 40c, 40d, and 40e are formed to penetrate the entire biosensor 100. The through hole 40 is formed in the direction crossing the sample inlet 22. In one example, the through hole 40 extends in a direction orthogonal to the first sample inlet 22a and the second sample inlet 22b formed in parallel to each other.

As the through hole 40 is provided, the samples injected into the first sample inlet 22a and the second sample inlet 22b are prevented from entering the different sample inlets 22. Therefore, by using each sample inlet 22 separately, it is possible to reduce the amount of the sample by half than when the through-hole 40 is not provided. Therefore, the biosensor 100 having such a structure can be easily applied to an electrochemical biosensor requiring a small amount of a sample.

In this embodiment, the first sample inlet 22a and the second sample inlet 22b are arranged to be offset from each other. Therefore, the sample is injected into the second sample inlet 22b, the sample is measured, and the portion in which the second sample inlet 22b is formed is cut and measured again using the first sample inlet 22a. Meanwhile, the shapes of the sample inlet 22 and the through hole 40 shown in FIGS. 1 and 2 are for illustrating the present invention, and may be formed by modifying the shape, tilt, width, and width according to various conditions. .

3 and 4 are plan views of the biosensor according to the first embodiment of the present invention, and show the biosensors before cutting 100 and after cutting 100a, respectively. As shown, the biosensor 100 cuts the portion where the second sample inlet 22b is formed along the line indicated by A-A 'and reuses the portion where the remaining first sample inlet 22a is formed.

5 to 7 is a schematic diagram sequentially illustrating a cutting process of the biosensor 100 of the above-described structure. As shown in the drawing, a part of the biosensor is inserted into the cutting device 70, and the biosensor 100 is cut using the cutter 70a provided in the cutting device 70. In this case, the cutting device 70 may be disposed inside or outside the measurement display device 72 or may be provided separately from the measurement display device 72. 5 to 7 illustrate, for example, being disposed in the measurement display device 72.

Hereinafter, the configuration of the adjustment reagent 62 will be described in detail. As described above, the modulating reagent 62 includes an enzyme, an electron transfer mediator, and a binding stabilizer.

One or more enzymes can be used depending on the target substance to be measured, such as glucose, lactate, alcohol, cholesterol, creatinine, proteins, amino acids, environmental substances, or industrial substances. Do. In one example, the enzyme may be glucose oxidase (glucose oxidase), glucose dehydrogenase to measure glucose, alcohol oxidase (alcohol oxidase) and dehydrogenase (alcohol dehydrogenase), PQQ ( pyrroloquinone), NAD / NADH (nicotinamide adenine dinucleotide / hydrogen, nicotinamide adenine dinucleotide). The specificity of the biosensor 100 is determined by these enzymes at the end.

Examples of electron transfer mediators include ferrocene, quinone, cobalt, nickel, ruthenium, persian compounds, rhodium, palladium, osmium, iridium, platinum and derivatives thereof. In addition, organic or inorganic compounds including transition metals may be used. Preferably hexaamineruthenium (III) chloride is used. The use of these electron transfer mediators reduces the potential of the species, reducing the action of the interfering species, compared to the case of using oxygen, resulting in more accurate results.

As a binding stabilizer, polyvinylalcohol, polyethyleneoxide, polyethyleneglycol, carboxymethyl cellulose, hydroxyethyl cellulose, 2-hydroxyethyl cellulose hydroxy ethyl cellulose, hydroxypropyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, polyvinylidenefluoride, polymethylmethacrylate , Styrene butyl rubber or the like can be used. Preferably, carboxy methylcellulose and microcrystalline cellulose are used.

In addition, the conditioning reagent 62 may further include one or more surfactants. Surfactants are used to improve the homogeneous dispersion capacity, solubility and reaction rate of the conditioning reagent 62. The surfactant has both a nonpolar terminal group (Hydrophobic or Lipophilic) and a polar terminal group (Hydrohphilic or water soluble) in the molecule. In addition, in the surfactant, the end group having affinity with the organic material wraps the organic material having no affinity with water from the inside, and the terminal structure of the polar group toward the outside has a dissolving power.

The surfactant may comprise at least one of anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants. Anionic surfactants are ionized in an aqueous solution so that the main agent of the active agent becomes an anion. Anionic surfactants include soap or alkylbenzene sulfonate. Cationic surfactants ionize to become cations. Examples of the cationic surfactants include higher amine halides, quaternary ammonium salts, and alkylpyridinium salts. Amphoteric surfactants may be ionized to be either cations or anions. Amphoteric surfactants include amino acids. Nonionic surfactants are not ionized. Examples of nonionic surfactants include polyethylene glycols.

Surfactants have properties such as washing power, emulsification, and dispersing power. Therefore, it can be widely used as a detergent, an emulsifying agent, a lubricant, a fungicide, a dispersant and the like according to its characteristics. Compounds and auxiliaries may be used with surfactants for improved function.

In addition, in an embodiment of the present invention, the use of a phase transfer catalyst may be parallel. The phase transfer catalyst increases the reaction rate of the adjusting reagent 62, enables use even at low temperatures, and is effective in reactions used with organic compounds. Phase transfer catalysts are used for the synthesis of inorganic compounds and organic compounds that are not soluble in organic solvents.The phase transfer reactions between water and solvents can be easily used because they occur uniformly in aqueous solutions, at room temperature, at atmospheric pressure, and in open systems. have. It can also be used effectively in liquid-liquid heterogeneous systems. In addition, the improved flow through phase transitions is less susceptible to changes in blood type, resulting in improved electrical transport. As the phase transfer catalyst, reagents such as phosphonium based, crown ethers based, ammonium based, and polyethylene glycol based (PEG) may be used.

The amount of these main compositions and adjuvants may be from 0.01 to about 20 weight percent of the total composition. As an example, 0.01-20% soap (detergent) can be incorporated. Soap molecules have both polar and non-polar properties, which are products of nonionic surfactants such as aliphatic alcohols, acids, amide phenols, alkyl phenols and their oxides. The cationic detergent may be an ammonium compound such as alkylmethylammonium halide. Free fatty acids (C _6-20 ) can be mixed in the compositions of the present invention, and these free fatty acids are present for their function as protein adsorbents and as dissolution enhancers.

8 is an exploded perspective view of the biosensor 200 according to the second embodiment of the present invention. As shown, in the biosensor 200 of the present embodiment, an electrode is formed only between the first substrate 210 and the adhesive cover 220. That is, in this embodiment, only the first electrode 250 and the second electrode 252 are formed on the first substrate 210 to act as the working electrode and the counter electrode, respectively. Since the rest of the configuration of this embodiment is the same as the first embodiment described above, a description thereof will be omitted.

9 is an exploded perspective view of the biosensor 300 according to the third embodiment of the present invention. As shown, the biosensor 300 according to the third embodiment of the present invention includes one electrode 350 covering the entire front surface of the first substrate 310 except for the through hole 340 on the first substrate 310. ) Is formed, and the other electrode 354 covering the entire front surface of the adhesive cover 320 except for the through hole 340 is formed between the adhesive cover 320 and the second substrate 330. At this time, any one of the two electrodes to act as a working electrode, the other one to act as a counter electrode to form a large-faced electrode.

In this case, even when the entire width of the electrodes 350 and 354 is 0.5 cm or more, only half or less of the samples can be introduced by adjusting the left and right widths of the through holes 340.

In addition, two or more target substances may be detected by introducing adjustment reagents for different uses into sample injection holes 322 provided on both sides of the biosensor 300. In the present embodiment, the measurement site sensor made in any form may be introduced by directly modifying or modifying an enzyme, an antibody, or a molecular cognitive substance on an electrode, and may also introduce various membranes including an adjustment reagent. Since the rest of the configuration of this embodiment is the same as the first embodiment described above, a description thereof will be omitted.

10 is an exploded perspective view of the biosensor 400 according to the fourth embodiment of the present invention. As shown, the biosensor 400 according to the present embodiment is provided with a plurality of through holes 440, the plurality of through holes 440 are formed to extend in parallel to each other. Therefore, a first electrode 450 having a shape extending from one branch to several branches is disposed between the first substrate 410 and the adhesive cover 420, and the adhesive cover 420 and the second substrate 430 are disposed. The second electrode 454 having a shape corresponding to the first electrode 450 is disposed between the second electrodes 454. In this case, the first electrode 450 and the second electrode 454 may act as a working electrode and a counter electrode, respectively.

In the present exemplary embodiment, the electrode cells 450a and 454a formed of several branches in the first electrode 450 and the second electrode 454 may be used as measurement cells of the sample. In this case, the electrode cells 450a and 454a may be further formed in parallel, and the position, path or shape of the sample inlet 422 may be changed, and the portion of the electrode cells 450a and 454a that are limited to the top or the bottom thereof. It can also be adjusted to form a separation.

11 is an exploded perspective view of the biosensor 500 according to the fifth embodiment of the present invention. In the biosensor 500 according to the present embodiment, both electrodes are formed on the first substrate 510. That is, the biosensor 500 of the present embodiment includes a first electrode 550 formed on the first substrate 510, an insulating layer (not shown) formed on the first electrode 550, and a second formed on the insulating layer. Electrode 552. In addition, the first electrode 550 includes a first sample measuring unit 550a which extends in a direction parallel to the through holes 540 between the through holes 540, and the second electrode 552 includes And a second sample measuring unit 552a which is formed to be parallel to the first sample measuring unit 550a by being spaced apart by a predetermined distance. Accordingly, each of the first electrode 550 and the second electrode 552 acts as a working electrode and a counter electrode.

The present invention will be described in detail by the following experimental examples. The following experimental examples illustrate the present invention, but the present invention is not limited thereto.

Experimental Example

A biosensor as in the first embodiment of the present invention was produced. A working electrode and a recognition electrode were formed on the first substrate by using conductive carbon paste, and the counter electrode was simultaneously formed on the second substrate by screen printing along with the electrode connection. Next, it was dried for 20 minutes at 100 ℃ using a dry oven. After that, only a specific portion to be used as an electrode cell was left, and the rest was applied with an insulating dough.

In addition, a pre-processed mold was press-pressed to the center portion of the electrode and the adhesive cover to form a through hole having a size of 0.8 mm × 1 mm. After fixing the adjusting reagent on the electrode cell, leaving the contacting portion, and attaching the remaining portion with an adhesive cover to form a sample inlet. Next, crude solution A and solution B having a compounding ratio as shown in Table 1 below were prepared.

First, 100 mM phosphate buffered solution (PBS: phosphate buffered saline) was prepared. Then, in 1 ml of PBS, enzyme (glucose oxidase), electron transfer medium (Ruthenium (III) hexaamine), dispersion stabilizer carboxymethyl cellulose, t-octylphenoxypolyethoxyethanol (t-Octylphenoxypolyethoxyethanol) 10 The mg was mixed in sequence.

To solution B, microcrystalline cellulose, tricaprylmethyl ammonium chloride, and soap were further added.

1.5 mg of each of the prepared solution A and solution B were applied to the electrodes on the first substrate, and then dried at 50 ° C. for 20 minutes to fix them. Thereafter, a molded adhesive cover was attached and the second substrate was covered and pressed again.

First, the sensitivity of the biosensor to the chronoamperometry was measured using a glucose standard solution. FIG. 12 is a dynamic response curve obtained when the solution B is immobilized on an electrode, and the electrochemical measurement method of the constant voltage vs. current method is performed using the biosensor prepared as described above. At this time, the measurement range was standard glucose solution 0, 100, 200, 300, 400 mg / dL. After the sample was filled in the sample inlet, the constant voltage was applied at 300 mV of the applied potential, and the amount of change in the current over time was measured. At this time, the slope was 0.06 [dl / (mg / dL)], and the linearity was 0.99, which showed excellent linearity sensitivity at each concentration.

Next, the sensitivity of the biosensor to the blood sample by the optimization of the adjusting reagent was measured. Using the biosensor prepared as described above was measured by the method of Figure 13 by changing the glucose concentration and blood red blood cell volume (Hct: hematocrit).

First, when the blood sample comes into contact with the sample inlet, it is naturally filled in the entire passage in a short time, and the catalysis of the following Chemical Formula 1 is developed.

Glucose (glucose) + GO _x -FAD → Gluconic acid + GO _x -FADH ₂

GO _x -FADH ₂ + mediator (ox) → GO _x -FAD + mediator (red)

In Formula 1, GO _X -FADH ₂ and GO _X -FAD represents the reduction state and oxidation state of FAD (flavin adeninedin nucleotide) which is an active site of glucose oxidase (GOx), respectively.

When the sample touches the recognition electrode, the voltage is cut off between the electrodes, and the reaction time is 6 seconds. After 6 seconds, a constant voltage of 300 mV flows between the working electrode and the counter electrode for 1 second. At this time, the concentration of glucose was measured as the amount of change in the amount of current flowing between each other.

14 is a graph showing the measurement result according to the glucose concentration by changing the red blood cell volume ratio (Hct) of the blood to 37, 43, 61% after applying the solution A, Figure 15 is a solution B is applied to the biosensor It is a graph showing the measurement result value according to the glucose concentration by changing the red blood cell volume ratio of 32, 43, 57%.

At this time, the average amount input to 100 biosensors was 0.6, and the result of the graph shows that the influence of blood type was small due to the interaction of the adjusting reagents. In FIG. 14, the average coefficient of variation (CV) of each concentration was 5% or more, and FIG. 15 was 3% or less. As such, it can be seen that the precision of the measurement was improved by further adding microcrystalline cellulose, tricaprylmethyl ammonium chloride and soap.

Although the preferred embodiments of the present invention have been described above, the present invention is not limited thereto, and various modifications and changes can be made within the scope of the claims and the detailed description of the invention and the accompanying drawings. Naturally, it belongs to the range of.

As described above, the biosensor significantly reduces the amount of sample, can be measured two or more times, and has the effect that can be used for the measurement of two or more target substances. In addition, accurate measurement is possible using a suitable calibration reagent.

Claims (20)

First substrate, An adhesive cover provided with a sample inlet on the first substrate, A second substrate covering the adhesive cover; At least one through hole penetrating the first substrate, the adhesive cover, and the second substrate and extending in a direction crossing the sample injection hole; Biosensor comprising a. The method of claim 1, The sample inlet, A first sample inlet formed on one side of the adhesive cover and a second sample inlet formed on the other side of the adhesive cover; And the first sample inlet and the second sample inlet are formed at positions shifted from each other. The method of claim 2, A first electrode formed between the first substrate and the adhesive cover along a circumference of the first substrate, and A second electrode surrounded by the first electrode and formed between the first substrate and the adhesive cover; Biosensor further comprising a. The method of claim 3, The second electrode is formed to surround the through hole. The method of claim 4, wherein And a nonconductive material layer disposed between the first electrode and the second electrode and the adhesive cover while exposing a portion of the first electrode and the second electrode. The method of claim 3, And a third electrode disposed between the adhesive cover and the second substrate. The method of claim 6, At least one element selected from the group consisting of carbon (C), gold (Au), silver (Ag), platinum (Pt), copper (Cu), and titanium (Ti) Biosensor comprising a. The method of claim 1, A first electrode formed between the first substrate and the adhesive cover to cover the entire surface except for the through hole of the first substrate, and A second electrode formed between the adhesive cover and the second substrate to cover the entire surface except for the through hole of the adhesive cover; Biosensor further comprising a. The method of claim 1, The at least one through hole includes a plurality of through holes, wherein the through holes are formed to extend in parallel to each other. The method of claim 9, The biosensor further comprises a first electrode formed between the first substrate and the adhesive cover while exposing the through hole, and a second electrode formed by exposing the through hole between the adhesive cover and the second substrate. . The method of claim 9, A first electrode formed on the first substrate, An insulating layer formed on the first electrode, and A second electrode formed on the insulating layer Biosensor further comprising a. The method of claim 11, The first electrode includes a first sample measuring unit formed between the through holes extending in a direction parallel to the through holes, The second electrode is a biosensor comprising a second sample measuring unit formed in parallel with a predetermined distance spaced apart from the first sample measuring unit. The method of claim 1, Further comprising an adjustment reagent disposed in the sample inlet, The adjustment reagent is, An enzyme that reacts with a substance contained in a sample injected into the sample inlet, An electron transfer medium for transferring electrons generated from the enzyme, and Dispersion stabilizer for dispersing and stabilizing the enzyme and the electron transfer medium Biosensor comprising a. The method of claim 13, The enzyme is glucose oxidase (glucose oxidase), glucose dehydrogenase (alcohol oxidase), alcohol dehydrogenase (alcohol dehydrogenase), PQQ (pyrroloquinone) and NAD / NADH (nicotinamide adenine dinucleotide / hydrogen, A biosensor comprising at least one of nicotinamide adenine dinucleotide). The method of claim 13, The electron transport medium is ferrocene (ferrocene), quinone (quinone), cobalt (cobalt), nickel (nickel), ruthenium (ruthenium), perisian compound, rhodium, palladium, osmium, iridium, platinum, hexaamine ruthenium (III) ) A biosensor comprising at least one of chloride, derivatives thereof, and transition elements. The method of claim 13, The dispersion stabilizer is polyvinylalcohol, polyethyleneoxide, polyethyleneglycol, carboxymethyl cellulose, hydroxymethyl cellulose, 2-hydroxyethyl cellulose hydroxy ethyl cellulose, hydroxypropyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, polyvinylidenefluoride, polymethylmethacrylate Biosensor comprising at least one of styrene butyl rubber. The method of claim 13, The adjusting reagent further comprises a surfactant, Wherein said surfactant comprises at least one of anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants. The method of claim 17, The anionic surfactant comprises at least one of a soap (soap) and alkylbenzene sulfonate (alkylbenzene sulfonate). The method of claim 13, The adjusting reagent further includes a phase transfer catalyst, The phase transfer catalyst includes at least one of phosphonium based, crown ethers based, ammonium based and polyethylene glycol based reagents. The method of claim 13, The adjusting reagent is glucose oxidase, hexaamineruthenium (III) chloride, carboxy methyl cellulose, microcrystalline cellulose, tricaprylmethyl ammonium chloride, t A biosensor comprising at least one substance selected from the group consisting of octylphenoxypolyethoxyethanol and soap.

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