KR20170125204A - 1,5-anhydroglucitol measurement means and a method of manufacturing the same - Google Patents

Abstract

The present invention relates to a means for measuring 1,5-anhydroglucitol, and a method of preparing the same, wherein the method comprises the step of forming a glucose removal layer which is coated with a glucose removing reagent that converts glucose in a whole blood sample into glucose-6-phosphate, and forming a reaction layer which is coated with a glucitol reaction reagent that develops a color from an oxide formed by a peroxidation reaction from hydrogen peroxide (H_2O_2) generated through an enzyme reaction with 1,5-anhydroglucitol contained in the sample from which glucose has been removed. Accordingly, a color is developed in the visible range through the enzyme reaction with 1,5-anhydroglucitol, and the concentration of 1,5-anhydroglucitol can be measured in an inexpensive and convenient manner by using a visible light meter. Additionally, since glucose, which generates the same enzyme reaction as 1,5-anhydroglucitol, is removed from the whole blood, the whole blood as is can be used as a sample to measure 1,5-anhydroglucitol.

Description

1,5-anhydroglucitol measurement means and a method for manufacturing the same

The present invention relates to 1,5-anhydroglucitol measurement means and a method for preparing the same, and more particularly, to a method for measuring 1,5-anhydroglucitol by means of an enzyme reaction with 1,5 -anhydroglycitol, Anhydroglucitol measuring means and a method for producing 1,5-anhydroglucitol measuring means capable of measuring the concentration of 1,5-anhydroglucitol inexpensively and conveniently through a measuring instrument.

1,5-anhydroglucitol (1,5-AG) is a kind of glucose derivatives that is the second most abundant of glucose in the blood of a healthy person. , It is a chemical and biochemically very stable substance. Such 1,5-AG is unclear in physiological activity, stable in vivo, rarely metabolized, and excreted in the urine. In addition, there is little supply, excretion, and metabolism of the 1,5-AG value in blood, and there is almost no fluctuation in the daily value of blood and it is hardly influenced by diet. The 1,5-AG in the body is derived from food, and 1,5-AG in the blood is reabsorbed in the kidney, and only a small part is excreted in the urine. In normal people, the amount of orally administered in the daily life and the amount excreted in the urine are balanced, and the 1,5-AG value in the blood is almost constant for a long time. However, since 1,5-AG is structurally similar to glucose, it is competitively reabsorbed in the renal proximal tubule, so the excretion of 1,5-AG is relatively increased in diabetic patients, and the serum concentration of 1,5-AG is decreased .

Recently, the number of diabetic patients has been increasing as the diet has become more abundant. In order to prevent the complication of diabetic patients, it is necessary to keep the blood glucose level close to the blood sugar level of the healthy person, and blood glucose self-measuring apparatus is widely used so that the patient himself can measure the blood glucose level. However, since blood glucose is fluctuated by eating, it is necessary to measure glucose frequently, which is a great burden on the patient. In addition, lack of knowledge makes it difficult to accurately interpret the glucose level measured by the patient, and it is not easy to strictly control blood glucose levels.

On the other hand, 1,5-AG is less susceptible to diets, so it is easier to measure blood glucose levels than glucose measurement, and this 1,5-AG test has the following clinical usefulness. First, the blood concentration is stable. 1,5-AG has little or no effect on new production or metabolism, has no daily or daily variation, and has other external factors such as sex, age, race, liver function, hormone, diet, exercise, duration of diabetes, There is almost no effect, and it changes at a certain rate depending on the degree of excretion per urine. Second, there are few sample constraints. While HbA1c is influenced by haemolysis and fructosamine is influenced by albumin or bilirubin concentration, 1,5-AG is not affected by hemolysis or ischemia and is not influenced by dietary, exercise, or daily fluctuations. Sample collection is possible. Third, 1,5-AG is highly correlated with other plasma control indicators and is sensitive to changes in blood glucose. Therefore, it can be used as an early warning of deterioration of blood glucose and as a reliable test for monitoring blood glucose control. Fourth, the treatment effect can be evaluated quickly by reflecting blood glucose control within a relatively short period of time. In other words, 1,5-AG is more sensitive and rapid decrease than the existing ones according to the increase of urinary glucose excretion. Therefore, it reacts sensitively to the current or immediately preceding blood glucose control by real time, So that appropriate measures can be taken. Fifth, 1,5-AG may suggest an increase in intermittent hyperglycemia and urinary excretion. In diabetic patients, reducing the number of hyperglycemia and maintaining stable blood glucose is very important for the treatment and prognosis. In case of HbA1c or fructosamine, 1,5-AG Measurement is more useful. Sixth, the range of measured values is wide. 1,5-AG has a wide range of variability and can easily detect subtle changes in blood glucose control in patients with hyperglycemia in areas with a near-normal blood glucose level. Seventh, the sensitivity and specificity in the diagnosis of diabetes are high, which is an ideal test.

Conventional methods for measuring 1,5-AG include gas chromatography or liquid chromatography, but these methods are so cumbersome that they are not commonly used in routine laboratory tests in hospital laboratories. Recently, a urine test kit has been introduced which can measure glucose by eliminating interference with an enzyme. The urinalysis kit reflects the blood glucose control status of the diabetic patients in the past one week, and is a self-monitoring kit in which the diabetic patient can accurately grasp his / her blood glucose control state by measuring the patient once per week. Although the 1,5-AG self-monitoring kit provides a great benefit to the patient, the conventional 1,5-AG measurement method is a method of sampling serum or plasma requiring hemocyte separation, It is not suitable for self-measurement. Therefore, a 1,5-AG self-monitoring kit using a trace amount of whole blood as a sample is not realized.

Korea Patent Office Registration No. 10-1470661

Therefore, an object of the present invention is to provide a method for producing 1,5-anhydroglucitol by coloring in the visible light region through an enzyme reaction with 1,5-anhydroglucitol, measuring the concentration of 1,5-anhydroglucitol inexpensively and easily Anhydroglucitol measurement means and a method for producing the same.

In addition, glucose, which causes the same enzyme reaction as 1,5-anhydroglucitol, can be removed from whole blood. Therefore, 1,5-anhydroglycitol, which can measure 1,5- Hydroglycitol and a process for producing the same.

The above-mentioned object is attained by a method for producing glucose-6-phosphate, comprising: a glucose removing layer coated with a glucose removing reagent for converting glucose in a whole blood sample to glucose-6-phosphate; Forming a reaction layer coated with a glucitol reaction reagent which forms an oxide by peroxidation reaction from hydrogen peroxide (H ₂ O ₂ ) generated through an enzyme reaction with glucitol to form a reaction layer , And 5-anhydroglucitol measurement means.

The whole blood sample is preferably moved in the horizontal direction along the glucose removing layer and the reaction layer to measure the amount of 1,5-anhydroglucitol.

Here, the glucose removing agent may be immersed in the glucose removing reagent to form the glucose removing layer; Immersing the glucitol reaction body in the glucitol reaction reagent to form the reaction layer; Wherein the step of bonding the glucose removing layer and the reaction layer comprises the step of bonding one end of the glucose removing layer formed along the length direction and one end of the reaction layer to each other, As shown in FIG.

The glucose-depleting reagent includes a buffer, an accelerator, a glucose-converting enzyme, and a reverse-reaction eliminating reagent. The glucose-converting enzyme for converting glucose to glucose-6- phosphate is selected from the group consisting of hexokinase, glucokinase, And the reverse reaction elimination reagent for eliminating the reverse reaction of glucose-6-phosphate to glucose is selected from the group consisting of pyruvate kinase, adenosine 5'-triphosphate, It is preferable to include phospho (enol) pyruvic acid.

The glucitol reaction reagent includes a buffer, a coloring agent, a surfactant, an oxidase, a peroxidase and a 4-aminoantipyrine, and reacts with 1,5-anhydroglucitol The oxidation reaction enzymes for generating hydrogen peroxide include pyranose oxidase, L-sorbose oxidase, L-sorbose dehydrogenase, 1,5-anhydrous hydrogenase, 1,5-anhydroglucitol dehydrogenase and a mixture thereof, and is preferably selected from the group consisting of hydrogen peroxide and the 4-aminoantipyrine oxidized through the enzymatic reaction of the peroxidase, Wherein the coloring agent is selected from the group consisting of N-Ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethylaniline (MAOS) TOOS), N-Ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAO S), 3,3'-, 5,5'-Tetramethylbenzidine, 3,3'-, 5,5'-Tetramethylbenzidine dihydrochloride, o-Toluidine, 3-methyl-2-benzothlazolinone hydrazone hydrochloride (MBTH), 8- -1-naphthalenesulfonate (ANS) and mixtures thereof.

The above-mentioned object is also achieved by a glucose-depleting reagent comprising a glucose-removing layer coated with a glucose-removing reagent for converting glucose in a whole blood sample to glucose-6-phosphate; The glucitol reaction reagent, which is formed by oxidation of 1,5-anhydroglucitol contained in the sample from which the glucose is removed and hydrogen peroxide (H ₂ O ₂ ) generated through the enzyme reaction, Anhydroglucitol measurement means characterized by comprising a reaction layer.

Here, one end of the glucose removing layer and one end of the reaction layer may be laminated with 20 to 40 length portions among 100 length portions which are the entire length of the reaction layer.

According to the above-described constitution of the present invention, 1,5-anhydroglucitol is reacted with an enzyme to cause color development in the visible light region, and the visible light measuring apparatus is capable of easily and inexpensively controlling the concentration of 1,5-anhydroglucitol Can be measured.

Since glucose causing the same enzyme reaction as 1,5-anhydroglucitol can be removed from whole blood, 1,5-anhydroglucitol can be measured using whole blood as a sample as it is.

1 is a cross-sectional view of 1,5-anhydroglucitol measuring means according to an embodiment of the present invention,
FIG. 2 is a flow chart showing a method for producing 1,5-anhydroglucitol measuring means.

Hereinafter, the 1,5-anhydroglucitol measuring means and the preparation method thereof according to the present invention will be described in detail with reference to the drawings.

As shown in FIG. 1, 1,5-anhydroglucitol (1,5-AG) measuring means 10 includes a glucose removing layer 11 and a reaction layer 13 . The glucose removing layer 11 is a layer which exists to remove glucose existing in the whole blood sample 1 as a spreading region where the whole blood sample 1 is dropped. Glucose interferes with accurate measurement in the measurement of 1,5-anhydroglucitol because it undergoes the same enzymatic reaction as 1,5-anhydroglucitol. Therefore, the glucose is removed from the whole blood sample (1), so that the 1,5-anhydroglucitol value can be accurately measured. The whole blood sample 1 dropped on the glucose removing layer 11 is transferred to the reaction layer 13 in a state in which glucose is removed from the glucose removing layer 11. In other words, the glucose removal layer 11 and the reaction layer 13 can be moved to the reaction layer 13 in which the ends are bonded to each other and the glucose-free whole blood sample 1 is transferred to the reaction layer 13, An oxide is formed and developed. The color developed oxide can be measured through a visible light meter to finally confirm the amount of 1,5-anhydroglucitol. Here, the whole blood sample moves horizontally through the glucose removing layer 11 and the reaction layer 13 to measure the amount of 1,5-anhydroglucitol.

As shown in FIG. 2, the glucose-removing layer 11 is prepared (S1) by the method of manufacturing the 1,5-anhydroglucitol measuring means.

Since the oxidation reaction enzyme used for 1,5-anhydroglucitol measurement also reacts with the glucose in the whole blood sample (1), the glucose elimination reaction must be preceded. Therefore, a glucose removal reagent for converting glucose to glucose-6-phosphate is prepared for removal of glucose, and then the glucose removal body, which is a solid matrix, is immersed in a glucose removal reagent to form a glucose- (11).

The solid matrix constituting the glucose removal body uses a matrix made of a glass fiber material. In the case of the glass fiber matrix, there is an advantage that the sample 1 is kept in a state having a uniform component when the whole blood sample 1 is developed, and the blood cell can be removed when the sample 1 is developed. It is preferable that the glucose removing body use a glass fiber matrix having a thickness of 240 to 400 mu m for rapid development of the whole blood sample 1. [ When the thickness of the glucose removing body is less than 240 탆, the thickness of the glucose removing agent is too small to sufficiently apply the glucose removing reagent. As a result, the glucose may not be completely removed and may remain. On the other hand, when the thickness exceeds 400 탆, the thickness becomes thicker than necessary and the production cost increases. The size of the glucose removal body is suitably 5 mm in width and 20 to 40 mm in length so that the time for the entire blood sample 1 to reach the reaction layer 13 from 35 μL is 30 to 90 seconds.

Glucose removal Glucose removal reagents that are applied to the body to convert glucose to glucose-6-phosphate include buffers, promoters, glucose converting enzymes, and reverse reaction reversal reagents. In some cases, the promoter may not be included in the glucose removal reagent.

The buffer is used not only to optimally condition the enzyme reaction properly, but also to prevent the pH of the coloring agent from being affected by the measurement of samples having pH values different from the pH of the reagent. The pH of the preferred buffer of the present invention is in the range of 6.5 to 8.3. When the pH is less than 6.5, the color sensitivity is lowered and the amount of 1,5-anhydroglucitol can not be measured properly. When the pH is more than 8.3, There is a problem. The pH of the more preferred buffer is 6.1 to 7.0.

The buffer may be selected from the group consisting of HEPES (4- (2-Hydroxyethyl) piperazine-1-ethanesulfonic acid), PIPES (1,4-Piperazinediethanesulfonic acid), Tris (hydroxymethyl) aminomethane, MES (2-Morpholinoethanesulfonic acid) hydrate, And mixtures thereof. Among them, HEPES buffer is most suitable in the present invention, and it is preferable that the concentration is 15 to 50 mM. The concentration of the buffer will have the same effect as the pH of the buffer. That is, when the concentration of the buffer is less than 15 mM, the enzyme reaction is not smoothly performed and the glucose is interfered with in the reaction layer. Also, when the concentration of the buffer is more than 50 mM, the enzyme reaction is not smoothly performed so that the glucose is not converted to glucose-6-phosphate and the glucose remains. The most preferred concentration is 20 mM.

Magnesium sulfate is used as an accelerator, and magnesium sulfate is preferably added at a concentration of 10 to 40 mM. If the concentration of magnesium sulfate is less than 10 mM, the promoter does not act properly. If the concentration of magnesium sulfate is more than 40 mM, the reaction speed becomes excessively high, and a side reaction may occur.

Glucose converting enzyme means an enzyme that converts glucose to glucose-6-phosphate, and hexokinase or glucokinase having high specificity to glucose can be used. In the present invention, a reagent containing a hexokinase is used, but a glucokinase may be used. In this case, the enzyme is preferably contained in an amount of 50 to 500 kU / L. If the kinetics is less than 50 kU / L, the glucose removal efficiency is lowered. If it exceeds 500 kU / L, the enzyme is wasted. The optimum concentration is 400ku / L.

Reaction with hexokinase or glucokinase is a reversible reaction, and reverse reaction elimination is necessary. If the reverse reaction is not removed, glucose-6-phosphate returns to glucose and glucose removal is not easy. Therefore, in order to remove the reverse reaction, the glucose elimination reagent further includes a reverse reaction eliminating reagent. The reverse reaction eliminating reagent includes pyruvate kinase, adenosine 5'-triphosphate and phosphoenolpyruvate (enol) pyruvic acid).

The concentration of pyruvate kinase in the reverse reaction elimination reagent is in the range of 50 to 300 kN / L, and the optimum concentration is 150 kN / L. If the concentration of pyruvate kinase is less than 50 kN / L, the reverse reaction of glucose-6-phosphate can not be controlled properly. If the concentration of pyruvate kinase exceeds 300 kN / L, the concentration of other components is lowered. Adenosine 5'-triphosphate is included to be between 5 and 40 mM, with the most preferred concentration being 20 mM. When the concentration of adenosine 5'-triphosphate is less than 5 mM, it can not perform the role of the reverse reaction elimination reagent. If the concentration of adenosine 5'-triphosphate is more than 40 mM, there is no further increase of the effect. The concentration of phosphoenolpyruvic acid is 10 to 50 mM, and the concentration of concern is 20 mM. When the concentration of phosphoenolpyruvic acid is less than 10 mM, it is difficult to completely control the reverse reaction of glucose even if other components of the reverse reaction elimination reagent are increased. When the concentration exceeds 50 mM, the effect is not increased similarly to adenosine 5'-triphosphate, do.

In some cases, the glucose removing layer 11 may further include a diffusion layer 15 on the upper portion thereof to facilitate the diffusion of the whole blood sample 1. The diffusion layer 15 is formed at the end of the glucose removing layer 11 that is not bonded to the reaction layer 13 and the entire sample 1 is dropped on the diffusion layer 15 to spread the sample 1 evenly within a short period of time The glucose can be more effectively removed. In the case of such a diffusion layer 15, it is preferable that it is made of a mesh, and the size of the mesh is 200 to 300 mu m. When the mesh size is less than 200 탆, the whole blood sample 1 does not spread quickly but is rolled up at a dropped position, so that the glucose removal rate is slow. When the mesh size is more than 300 탆, . The size of such a diffusion layer is suitably 5 mm in width and 5 to 10 mm in length.

1,5-anhydroglucitol reaction layer (S1 ').

Anhydroglucitol reaction layer is prepared separately from the step S1 in which the glucose removing layer 11 is prepared. That is, the glucose removing layer 11 and the 1,5-anhydroglucitol reaction layer 13 may be disposed together in one matrix, but they are preferably separately prepared for ease of manufacture. In the case of the reaction layer 13, the glucose-removed sample 1 is moved through the glucose removing layer 11 to confirm the amount of 1,5-anhydroglucitol. An example of a method for confirming the amount of 1,5-anhydroglucitol through the reaction layer 13 is as follows. That is, 1,5-anhydroglucitol present in the sample 1 is oxidized in the reaction layer 13 It reacts with the reaction enzyme and oxidizes to produce hydrogen peroxide. The hydrogen peroxide thus obtained reacts with the coloring agent to form a purple colored matter. The coloring matter is measured by a visible light meter to determine the amount of 1,5-anhydroglucitol.

The glucitol reaction reagent capable of oxidizing and identifying 1,5-anhydroglucitol is prepared so that 1,5-anhydroglucitol can be measured through such a method, and a glucitol reaction reagent The reaction solution is applied through a method of immersing the body in a reaction reagent for glucitol to form a reaction layer 13.

The solid matrix constituting the glucitol reaction body is preferably a nylon 6,6 matrix with excellent sample application and durability. The nylon 6,6 matrix exhibits fast sample development rates even at low pressures and is applicable to small sample volumes, which is advantageous in that even small amounts of 1,5-anhydroglucitol can be accurately identified. If the thickness is less than 140 탆, the glucitol reaction reagent can not be sufficiently applied. If the thickness exceeds 190 탆, the thickness of the glucitol reaction body is thicker than necessary. There is a disadvantage in that the amount of the reagent reagent to be applied is increased, thereby increasing the manufacturing cost. The size of the glucitol reaction body is suitably 5 mm in width and 5 to 10 mm in length, and the reaction is completed within 60 to 180 seconds in the sample (1) developed through the glucose removal reaction layer (11).

Glucitol Reaction The glucitol reaction reagent, which is applied to the reaction body and generates hydrogen peroxide through oxidation reaction with 1,5-anhydroglucitol, is composed of a buffer, a colorant, a surfactant, an oxidase, a peroxidase and 4 - 4-aminoantipyrine.

In this case, the buffer does not only provide an optimal condition for the enzymatic reaction to be properly performed, but also the pH of the coloring agent (1) when measuring the sample (1) having a pH value different from that of the reagent So that it is not influenced by the user. It is preferable to use the buffering agent of the same kind and concentration as the glucose removal reagent, but in some cases, different buffering agents may be used. It is preferable to use a buffer having a concentration of 50 to 300 mM. When the concentration is less than 50 mM, the enzyme reaction is not smoothly performed, and it is difficult to confirm the exact amount of 1,5-anhydroglucitol. There is a problem that the enzyme reaction is not smoothly performed. A more preferred concentration is 100 to 150 mM. The kind of such a buffering agent can be selected from the kind described in the glucose elimination reagent.

The coloring agent is added so that 1,5-anhydroglucitol is reacted with the oxidation reaction enzyme and can be confirmed in the visible light region. As a coloring agent related to the measurement of hydrogen peroxide, aniline-based reagent desirable. The reagents of the aniline series are N-Ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethylaniline (MAOS), N-Ethyl-N- TOOS), N-Ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAOS), 3,3 ', 5,5'-Tetramethylbenzidine, 3,3' It is preferably selected from the group consisting of -tetramethylbenzidine dihydrochloride, o-Toluidine, 3-methyl-2-benzothlazolinone hydrazone hydrochloride (MBTH), 8-anilino-1-naphthalenesulfonate (ANS) and mixtures thereof. Among them, it is most preferable to use TOOS which is a violet series coloring agent having a wide range measurement range at a concentration of 1,5-anhydroglucitol of 0 to 100 μmol / L. The concentration of the coloring agent is 5 to 20 mM, and the optimum concentration thereof is 10 to 15 mM. When the concentration of the coloring agent is less than 5 mM, the coloring sensitivity of the measuring means is lowered. When the concentration exceeds 20 mM, the coloring sensitivity of the hydrogen peroxide is lowered and the sensitivity is lowered.

The surfactant is a dispersion reagent, which serves to keep the constituents of the measuring means 10 in a dry state, to function as a stabilizer, to keep the color change of the measuring means uniform, and to increase the intensity of color change of the measuring means. Here, 3 - [(3-Cholamidopropyl) dimethylammonio] -1-propanesulfonate (CHAPS), which can increase the color strength of various surfactants, is used as a surfactant. The concentration of CHAPS is in the range of 5 to 10 mM. When the concentration is less than 5 mM, the increase in color development is insignificant. When the concentration is more than 10 mM, there is no further increase in color development, and a surfactant is wasted when the concentration is higher.

The glucitol reaction reagent includes an oxidation reaction enzyme for the oxidation reaction of 1,5-anhydroglucitol. Hydrogen peroxide is generated from 1,5-anhydroglucitol by an enzyme reaction with an oxidation reaction enzyme This hydrogen peroxide reacts with the coloring agent to confirm the amount of 1,5-anhydroglucitol. As described above, hydrogen peroxide is generated from 1,5-anhydroglucitol, and oxidation reaction enzymes, peroxidase, and 4-aminoantipyrine are required for the hydrogen peroxide to develop color.

The oxidation reaction enzymes for oxidizing 1,5-anhydroglucitol include pyranose oxidase, L-sorbose oxidase, L-sorbose dehydrogenase, , 1,5-anhydroglucitol dehydrogenase, which can be used alone or in combination. In this case, the oxidation reaction enzyme preferably contains a concentration of 300 to 500 kU / L. When the total amount of 1,5-anhydroglucitol is less than 300 kU / L, it is difficult to accurately measure the amount of 1,5-anhydroglucitol. There is a disadvantage that the oxidation reaction enzyme is wasted because the oxidation reaction enzyme is excessively added.

1,5-Anhydroglucitol is oxidized through an oxidation reaction enzyme to obtain hydrogen peroxide as a product. Glucitol reaction reagents include peroxidase and 4-aminoantipyrine, which react with peroxidase enzymes to oxidize 4-aminoantipyrine. The oxidized 4-aminoantipyrine reacts with TOOS, which is a coloring agent, to produce a purple oxide. Thus, the amount of purple oxide can be confirmed by a visible light meter, and the amount of 1,5-anhydroglucitol is measured. Here, the concentration of the peroxidase enzyme is preferably 10 to 200 kN / L. When the concentration is less than 10 kN / L, the reaction does not occur properly, and when 200 kN / L, the ratio of the glucitol reaction reagent is affected.

The glucose removing layer 11 and the reaction layer 13 are bonded (S2).

As shown in FIG. 2, the glucose removing layer 11 prepared in the step S1 is combined with the reaction layer 13 formed in the step S1 '. The glucose removing layer 11 and the reaction layer 13 may be formed in one matrix. However, considering the ease of application of the reagent, the glucose removing layer 11 and the reaction layer 13 may be separately prepared, . However, the glucose removing layer 11 and the reaction layer 13 may be formed together in one matrix.

One end of the glucose removing layer 11 and the one end of the reaction layer 13, which are elongated along the longitudinal direction, are joined. Thereafter, when the whole blood sample 1 is dropped on the other end of the glucose removing layer 11, the whole blood sample 1 moves along the longitudinal direction of the glucose removing layer 11 and the glucose is removed in the sample 1, Is transferred to the reaction layer (13) bonded to one end of the glucose removing layer (11), and color development occurs through the reaction in the reaction layer (13). The reaction layer 13 is bonded to one longitudinal end of the glucose removing layer 11 and the entire blood sample 1 is dropped on the other end of the glucose removing layer 11 not bonded to the reaction layer 13 .

At this time, one end of the glucose removing layer 11 and one end of the reaction layer 13 are bonded to the reaction layer 13 such that the whole blood sample 1 can be transferred to the reaction layer 13, It is preferable to combine them so that they are stacked about the length. If the area to be laminated is less than 20 parts long, the sample (1) is not easily transferred to the reaction layer, so it takes a long time to check the amount of 1,5-anhydroglucitol, And the length of the reaction layer 13 for coloring 1,5-anhydroglucitol is short and it is difficult to measure the exact amount of 1,5-anhydroglucitol.

The supporting table 17 may further comprise a lower portion of the bonded glucose removing layer 11 and the reaction layer 13 as the case may be. The supporting table is most preferably made of polyvinyl chloride (PVC) But is not limited to.

Hereinafter, embodiments of the present invention will be described more clearly.

<Examples>

A glucose depletion reagent including a buffer, an accelerator, a glucose conversion enzyme and a reverse reaction elimination reagent, and a glue containing a buffer, a coloring agent, a surfactant, an oxidizing enzyme, a peroxidase and a 4-aminoantipyrine Prepare the Toll Reagent. The glucose removal reagent and the glucitol reaction reagent can be found in the following Tables 1 and 2.

Glucose removal reagent HEOES 20mM hexokinase 400ku / L ATP 20mM pyruvate kinase 300ku / L phospho (enol) pyruvic acid 20mM 마그 마그 사 20mM

Glucitol Reaction Reagent HEPES 120mM 피라노ose oxidase 60ku / L peroxidase 120ku / L TOOS 15mM 4-aminoantipyrine 15mM CHAPS 10 mM sodium azide 10 mM

The porous matrix prepared in each of the glucose removal reagent and the glucitol reaction reagent prepared in such components and concentrations is immersed to sufficiently impregnate each component. After removing the matrix from the glucose removal reagent and the glucitol reaction reagent, the excess reagent is removed with a glass rod and dried in a 40 ° C hot air drier for 10 minutes to obtain a glucose removal layer and a reaction layer. The obtained glucose-removing layer was cut into a size of 5 mm in width and 30 mm in length, and the reaction layer was cut into a size of 5 mm in width and 5 mm in length. The glucose removing layer and the reaction layer are arranged horizontally such that the development of the measurement sample is horizontal flow, and then the glucose removing layer and the reaction layer are laminated so as to overlap each other by 1 mm, Tall measurement means are manufactured.

The results of the 1,5-anhydroglucitol measurement means of the present invention manufactured through such an embodiment and the results of the Lara 1,5-AG Auto Liquid product already established and used in hospitals are shown in Table 3 and It can be seen from FIG. As shown in the graph of FIG. 3, it can be seen that there is a good correlation between the two products with correlation y = 0.9821x + 1.5938 and correlation coefficient r = 0.952.

Sample The present invention (μg / mL) Lara 1,5-AG (μg / mL) One One 2 2 4 3 3 10 12 4 15 18 5 23 26 6 15 17 7 30 33 8 31 27 9 21 23 10 14 16

In the case of the conventional 1,5-anhydroglucitol measuring means, serum or plasma which requires hemocyte separation is used as a sample. Since the amount of sample required for measurement is large, it is not suitable for self-measurement. On the other hand, the 1,5-anhydroglucitol measuring means of the present invention includes a layer for removing glucose in the measuring means, so that the amount of 1,5-anhydroglucitol can be measured even if a whole blood sample is dropped as it is It is very convenient for users to use. Also, in the conventional case, it is impossible to measure the amount of 1,5-anhydroglucitol in the visible light region, so expensive equipment for measuring 1,5-anhydroglucitol is required. In contrast, in the present invention, since the reaction between 1,5-anhydroglucitol and enzyme is performed so as to be measurable in the visible light region, the absorbance can be measured through a small-volume and inexpensive visible light source. Therefore, it is easy for the user to carry the device, and the measurement cost is low, which makes it easy to measure 1,5-anhydroglucitol.

1: Whole blood sample
10: Measuring means
11: Glucose removal layer
13: Reaction layer
15: diffusion layer
17: Support

Claims (12)

A glucose removing layer coated with a glucose removing reagent for converting glucose in a whole blood sample to glucose-6-phosphate, and a glucose removing layer coated with 1,5-anhydroglucitol and enzyme Anhydrous hydrogen peroxide (H ₂ O ₂ ) generated through the reaction to form a reaction layer coated with a glucitol reaction reagent which forms an oxide by peroxidation reaction to form a color; and A method for producing a glucitol measuring means. The method according to claim 1,
Wherein an amount of 1,5-anhydroglucitol is measured by moving the whole blood sample in the horizontal direction along the glucose removing layer and the reaction layer. The method according to claim 1,
Forming a glucose removing layer by immersing the glucose removing body in the glucose removing reagent;
Immersing the glucitol reaction body in the glucitol reaction reagent to form the reaction layer;
And combining the glucose-removing layer and the reaction layer. The method for preparing 1,5-anhydroglycitol measuring means according to claim 1, The method of claim 3,
Wherein the step of combining the glucose removal layer and the reaction layer comprises:
Wherein one end of the glucose-removing layer formed along the length direction and one end of the reaction layer are laminated so as to be laminated. The method according to claim 1,
The glucose-
Anhydroglucitol, a buffer, an accelerator, a glucose-converting enzyme, and a reverse reaction-eliminating reagent. 6. The method of claim 5,
The glucose-converting enzyme for converting glucose to glucose-6-phosphate is selected from the group consisting of hexokinase, glucokinase, and a mixture thereof. The 1,5-anhydroglucitol measurement &Lt; / RTI &gt; 6. The method of claim 5,
The reverse reaction-eliminating reagent for eliminating the reverse reaction of glucose-6-phosphate to glucose is selected from the group consisting of pyruvate kinase, adenosine 5'-triphosphate and phospho (enol ) pyruvic acid). &lt; / RTI &gt; The method according to claim 1,
The glucitol reaction reagent may be,
Anhydroglucitol measuring method according to any one of claims 1 to 3, wherein the method comprises the steps of: (1) adding a buffer solution containing a buffering agent, a coloring agent, a surfactant, an oxidizing enzyme, a peroxidase and a 4-aminoantipyrine. 9. The method of claim 8,
The oxidation reaction enzyme that reacts with 1,5-anhydroglucitol to produce hydrogen peroxide is pyranose oxidase, L-sorbose oxidase, L-sorbose dihydrogenase Anhydroglucitol dehydrogenase, 1,5-anhydroglucitol dehydrogenase, and a mixture thereof. 2. The method according to claim 1, wherein the anhydroglucose is selected from the group consisting of L-sorbose dehydrogenase, 1,5-anhydroglucitol dehydrogenase, Method of manufacturing measuring means. 9. The method of claim 8,
(2-hydroxy-3-sulfopropyl) -3,5-dimethylaniline (MAOS), which reacts with hydrogen peroxide and the 4-aminoantipyrine oxidized through the enzymatic reaction of the peroxidase, , N-Ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAOS), 3-methylaniline , 3'-, 5,5'-Tetramethylbenzidine, 3,3'-, 5,5'-Tetramethylbenzidine dihydrochloride, o-Toluidine, 3-methyl-2- benzothlazolinone hydrazone hydrochloride (MBTH), 8-anilino-1-naphthalenesulfonate (ANS), and mixtures thereof. &Lt; Desc / Clms Page number 24 &gt; A glucose removal layer coated with a glucose removal reagent for converting glucose in a whole blood sample to glucose-6-phosphate;
The glucitol reaction reagent, which is formed by oxidation of 1,5-anhydroglucitol contained in the sample from which the glucose is removed and hydrogen peroxide (H ₂ O ₂ ) generated through the enzyme reaction, A method for measuring 1,5-anhydroglucitol, which comprises a reaction layer. 12. The method of claim 11,
Wherein one end of the glucose removing layer and one end of the reaction layer are laminated with 20 to 40 lengths of 100 lengths of the reaction layer, which is the total length of the reaction layer.

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