JP2013102736A - Specimen for biochemical analysis, improved in pigment stability - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a specimen for a biochemical analysis having storage stability by suppressing the self-coloring of a leuco pigment.SOLUTION: The specimen for the biochemical analysis, includes a multilayer structure containing the leuco pigment, and an enzyme producing hydrogen peroxide by oxidation reaction in the presence of a substrate and oxygen, wherein the specimen contains (a) at least the leuco pigment and one or more kinds of antioxidants in one layer, and (b) peroxidase and an antioxidant scavenging enzyme, in a layer separate from the one layer.

Description

  The present invention relates to the stabilization of leuco dyes during dry analysis. More specifically, the present invention relates to a test specimen for multilayer analysis using a leuco dye stabilizer and an enzyme that eliminates it.

In the field of clinical examination, various items are measured by enzymatic methods. For example, a method of quantifying the concentration of a substance by generating hydrogen peroxide using an oxidase such as glucose oxidase and measuring the absorbance after color development in the presence of peroxidase and a dye has been conventionally known.
As a kind of the dye used at this time, a dye called a Trinder reagent or a dye called a leuco dye exists. The Trinder reagent is a dye composed of two kinds of couplers such as 4-aminoantipyrine and various Trinder reagents, and is characterized by a stable dye against light and heat. In terms of sensitivity, it reacts with 2 molecules of hydrogen peroxide. On the other hand, leuco dyes are 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine sodium (DA67) and N- (carboxymethylaminocarbonyl) -4,4-bis (dimethylamino) biphenylamine (DA64). ) And the like, it reacts with one molecule of hydrogen peroxide, so it can measure with high sensitivity, but because it is unstable to light and heat, there are problems such as high blanks before measurement. Various studies have been conducted on the suppression of self-coloration.

  Patent Document 1 discusses suppression of self-color development of leuco dye DA67 with a liquid reagent by allowing cyclodextrin and / or a derivative thereof to coexist in a color-developing reagent. Although short-term effects within 10 days are recognized, there is no description of the behavior when stored for a long time, and the effects of long-term storage are unclear. In Patent Document 2, when the leuco dye DA64 and fructosyl amino acid oxidase or fructosyl amino acid oxidase and peroxidase coexist in the solution, the absorbance of the colored DA64 increases at an absorption wavelength of 727 nm, and self-coloring occurs even when the storage period is prolonged. Reported to have been suppressed. In Patent Document 3, self-coloring of a solution reagent is suppressed by allowing a leuco dye to coexist in a solution with one or more reducing agents comprising reducing thioalcohols and reducing sulfates. Specifically, when DA67 and specific reducing agents such as sodium sulfite and sodium disulfite are used, a high effect is obtained. However, in the above three patent documents, self-development suppression of liquid leuco dyes in solution is studied. For dry analytical test specimens, avoid light and heat during the drying process at the time of specimen preparation and storage after preparation. However, it is very difficult in terms of suppressing self-coloring and has not been studied so far.

Against this background, there are no examples of leuco dyes that are particularly sensitive but difficult to stabilize, and are used in dry biochemical analysis specimens. Tests for dry biochemical analysis with good storage stability of leuco dyes A piece was desired.

Japanese Unexamined Patent Publication No. 01-118768 WO2003 / 033601 JP 2008-201968 A

An object of the present invention is to provide a test piece for biochemical analysis having storage stability by suppressing the self-color development of a leuco dye.

As a result of intensive studies by the present inventors, it was found that the self-color development of the leuco dye can be suppressed over a long period of time by allowing the leuco dye and the antioxidant to coexist. As a result of further investigation, it has been found that ascorbic acid and glutathione have extremely remarkable effects, and the present invention has been completed.

Conventionally, there is a problem that ascorbic acid (reducing agent) contained in blood affects the oxidative coloring reaction and adversely affects the accuracy, and various methods for eliminating the influence of ascorbic acid have been studied. However, the present invention uses ascorbic acid as a reducing agent (antioxidant) that suppresses self-color development of the dye, acts as a dye stabilizer before the oxidative color reaction, and ascorbic acid is oxidized by ascorbate oxidase during the oxidative color reaction. By the erasing method, it is possible to provide a method for stabilizing a leuco dye and a method for reducing non-specific color development when performing an oxidative color reaction. The present invention provides a test strip for biochemical analysis using these methods.

That is, this invention consists of the following structures.
(1) A test piece for biochemical analysis comprising a leuco dye and a multilayer structure comprising a substrate and an enzyme that generates hydrogen peroxide by an oxidation reaction in the presence of oxygen,
(A) at least one layer containing a leuco dye and one or more antioxidants;
(B) A test piece for biochemical analysis, comprising an enzyme for eliminating peroxidase and an antioxidant in a layer different from the layer.
(2) The test piece for biochemical analysis according to (1), wherein the antioxidant is at least one antioxidant selected from the group consisting of ascorbic acid, an ascorbic acid derivative, glutathione, and a glutathione derivative.
(3) The test piece for biochemical analysis according to (2), wherein the antioxidant is ascorbic acid and / or an ascorbic acid derivative.
(4) The test piece for biochemical analysis according to any one of (1) to (3), wherein the enzyme that eliminates the antioxidant is ascorbate oxidase and / or γ-glutamyl transpeptidase.
(5) The test piece for biochemical analysis according to (4), wherein the enzyme that eliminates the antioxidant is ascorbate oxidase.
(6) The test piece for biochemical analysis according to any one of (1) to (5), wherein the concentration of the antioxidant is from 0.03 mg / cm 2 to 0.05 mg / cm 2 (7) The test piece for biochemical analysis according to any one of (1) to (6), wherein the concentration of the enzyme to be erased is 3 U / cm 2 or more and 100 U / cm 2 or less.
(8) The test for biochemical analysis according to any one of (1) to (7), wherein the leuco dye is 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine sodium (DA67) Piece.
(9) The test piece for biochemical analysis according to any one of (1) to (8), wherein the addition amount of the leuco dye is 0.03 mg / cm 2 or more and 0.10 mg / cm 2 or less.
(10) A group in which an enzyme that generates hydrogen peroxide by an oxidation reaction in the presence of a substrate and oxygen is composed of glucose oxidase, urate oxidase (uricase), cholesterol oxidase, creatinine amide hydrolase, lipase, ketoamine oxidase, and glycated amino acid oxidase The test piece for biochemical analysis according to any one of (1) to (9), which is one or more selected from the above.
(11) A biochemical analysis method comprising performing an enzymatic reaction using the biochemical analysis test piece according to any one of (1) to (10).
(12) A biochemical analyzer for performing biochemical analysis by performing an enzyme reaction using the biochemical analysis test piece according to any one of (1) to (10).

  According to the present invention, a leuco dye can be stabilized even in a test specimen for biochemical analysis in a dry analysis system, and a leuco dye having high sensitivity can be used. This contributes to more accurate measurement.

The present invention is described in detail below.
Examples of the “leuco dye” according to the present invention include triphenylmethane derivatives, phenothiazine derivatives, diphenylamine derivatives and the like. Specifically, 4,4′-benzylidenebis (N, N-dimethylaniline), 4,4′-bis [N-ethyl-N- (3-sulfopropylamino) -2,6-dimethylphenyl] methane 1- (ethylaminothiocarbonyl) -2- (3,5-dimethoxy-4-hydroxyphenyl) -4,5-bis (4-diethylaminophenyl) imidazole, 4,4′-bis (dimethylamino) diphenylamine, N- (carboxymethylaminocarbonyl) -4,4′-bis (dimethylamino) diphenylamine salt (DA64), 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine salt (DA67), etc. Can be mentioned.

Among these, a leuco dye is preferable because it has a high molar extinction coefficient, has a maximum absorption wavelength on the high wavelength side, can avoid the wavelength of coexisting substances in blood, and can easily detect the wavelength of the dye. 10- (carboxymethyl Aminocarbonyl) -3,7-bis (dimethylamino) phenothiazine salt (DA67) is more preferred.

  Examples of the “enzyme that generates hydrogen peroxide by oxidation reaction in the presence of substrate and oxygen” include glucose oxidase, peroxidase, cholesterol oxidase, ketoamine oxidase, glycated amino acid oxidase, and urate oxidase (uricase).

The “test strip for biochemical analysis” in the present invention is a reaction base material made of a polymer material for quantifying or qualifying a target measurement object by an enzyme reaction, and is sometimes called a chip, a slide or a strip.

The biochemical analysis method used for the biochemical analysis test piece of the present invention includes at least an enzyme and a dye in the test piece, and qualifies or quantifies the amount of enzyme reaction of the analyte sample to be measured by the color difference of the dye. There is a method that employs an enzyme colorimetric method. In the actual use of the test piece for biochemical analysis, a measuring instrument corresponding to the method adopted by the test piece for biochemical analysis is used. Although there are differences depending on the measuring instrument, a typical biochemical analysis method using the test piece for biochemical analysis is as follows: (1) A specimen sample is dropped onto the test piece for biochemical analysis, and then (2) the test piece. Is mounted on a measuring device, and (3) light emission is detected in the measuring device. Then, (4) the measured value is converted into concentration by calculation in the device, and (5) the measurement result is notified by screen or voice.

  The biochemical analysis in the present invention refers to analysis performed for measuring organic / inorganic compounds present in a specimen derived from a living body, and is also used to indicate analysis of measurement items used in clinical examinations. Examples of clinical tests in which biochemical analysis is used include blood tests and urine / fecal tests, and the present invention is suitably used for biochemical analysis belonging to blood tests. Examples of the analysis target items that can be used for the biochemical analysis in the blood test include blood glucose (glucose), hemoglobin A1c, glycoalbumin, insulin, total protein, albumin, cholinesterase, and lactic acid. Dehydrogenase (LD), alanine aminotransferase (ALT = GPT), aspartate aminotransferase (AST = GOT), γ-glutamine transpeptidase, alkaline phosphatase, bibilin, amylase, creatinine, creatine kinase, total cholesterol, neutral There are fats, HDL cholesterol, LDL cholesterol, uric acid.

  One of the embodiments in the field where the present invention is used is a test piece for biochemical analysis for analyzing blood glucose (glucose) using an enzyme colorimetric method. The test piece contains at least glucose oxidase, peroxidase and a dye. When a blood sample is dropped onto a test piece, glucose oxidase reacts with glucose in the blood plasma, and glucose is oxidized to generate gluconic acid and hydrogen peroxide. Next, peroxidase reacts in the presence of hydrogen peroxide and a dye to cause a color reaction. This color development reaction is measured by a measuring device different from the test piece, and the blood glucose level is calculated.

  Another embodiment in the field in which the present invention is used is a test strip for biochemical analysis for analyzing hemoglobin A1c (HbA1c) by an enzyme colorimetric method. The test piece contains at least a surfactant, protease, fructosyl amino acid oxidase and peroxidase, and a dye. When a blood sample is dropped on a test piece, hemoglobin is taken out from the blood cells by a surfactant, and then a protease cuts out the glycated peptide. Then, fructosyl amino acid oxidase reacts with glycated peptide HbA1c, and HbA1c is oxidized to generate hydrogen peroxide. Next, peroxidase reacts in the presence of hydrogen peroxide and a dye to cause a color reaction. HbA1c is calculated from the ratio of hemoglobin (Hb) and HbA1c measured separately by a measuring device different from the test piece.

  The test piece for biochemical analysis has a multilayer structure of two or more layers. In the test piece for biochemical analysis of the present invention, the antioxidant and the enzyme that erases the antioxidant are present in different layers, and the enzyme that generates hydrogen peroxide by the oxidation reaction in the presence of peroxidase, substrate, and oxygen. It is preferably present in a separate layer. For example, in the case of a two-layer structure, the lower layer preferably contains a leuco dye, one or more antioxidants, and an enzyme that generates hydrogen peroxide by an oxidation reaction in the presence of oxygen with a substrate. The upper layer preferably contains an enzyme that eliminates peroxidase and antioxidant. These two layers can contain a stabilizer, a pH adjusting agent, a surfactant, and other additives.

In the case of a three-layer structure, the lowermost layer preferably contains a leuco dye, one or more antioxidants, and an enzyme that generates hydrogen peroxide by oxidation reaction in the presence of oxygen with a substrate. Preferably contains an enzyme that eliminates the antioxidant. The top layer preferably contains peroxidase. Alternatively, the intermediate layer may contain peroxidase, and the uppermost layer may contain an enzyme that eliminates the antioxidant. Further, the three layers can contain a stabilizer, a pH adjusting agent, a surfactant, and other additives.

  The test piece for biochemical analysis is characterized in that an antioxidant and a leuco dye are present in the same layer, and an enzyme for eliminating the antioxidant is present in a separate layer. Leuco dyes have the problem of self-coloring due to oxidative degradation. Therefore, by allowing an antioxidant to coexist with the leuco dye, self-coloring of the dye can be suppressed. However, when the leuco dye and the antioxidant coexist, the oxidation reaction by the oxidase does not proceed. Therefore, an enzyme that erases the antioxidant impregnated in another layer dissolves in the liquid of the specimen and penetrates into the next layer, and first, the antioxidant is erased. The oxidation reaction, which is the main reaction thereafter, proceeds. These reactions make it possible to achieve both oxidation prevention (suppression of self-color development) of the leuco dye and oxidation reaction which is the main reaction.

  As the antioxidant contained in the test piece for biochemical analysis, ascorbic acid, glutathione, cysteine, ascorbic acid derivatives, and glutathione derivatives are preferably used. Only one of these antioxidants may be contained in the test piece, but two or more kinds of enzymes may be contained. Ascorbic acid and / or ascorbic acid derivatives are preferred.

  Examples of the enzyme that eliminates the antioxidant contained in the test piece for biochemical analysis include ascorbate oxidase, γ-glutamyl transpeptidase (γ-GT), and cysteine oxidase. The test piece may contain only one kind of enzymes for eliminating these antioxidants, but may contain two or more kinds of enzymes. These enzymes are selected depending on the type of antioxidant used. When ascorbic acid or a derivative of ascorbic acid is used as an antioxidant, the enzyme used as an enzyme that eliminates the antioxidant is preferably ascorbate oxidase.

  The reason why the combination of ascorbic acid and ascorbic acid oxidase is preferable as the enzyme that eliminates the antioxidant and the antioxidant contained in the test piece for biochemical analysis as described above is that the ascorbic acid according to the present invention is used to prevent oxidation of the dye. In other words, apart from the purpose of inhibiting self-color development, biochemical analysis has a problem that ascorbic acid contained in the blood is an interfering substance and needs countermeasures. For example, when measuring blood glucose by the enzyme method, since ascorbic acid has a reducing action, there is a problem that coloring of the dye is suppressed, measurement sensitivity is lowered, and an erroneous value is measured. As a countermeasure, it has been studied that ascorbate oxidase is used for the purpose of eliminating ascorbic acid in the reagent at the time of measurement. When ascorbic acid and ascorbate oxidase are used as dye antioxidants as in this case, there is a synergistic effect that ascorbate oxidase eliminates ascorbic acid in blood and improves measurement accuracy in addition to suppressing self-color development of dye. Because.

  The concentration of the antioxidant contained in the test piece for biochemical analysis is preferably 0.03 mg / cm 2 or more and 0.05 mg / cm 2. A concentration of less than 0.03 mg / cm 2 is not preferable because the self-color development of the leuco dye cannot be suppressed. On the other hand, the concentration of 0.05 mg / cm 2 or more is not preferable because the antioxidant inhibits the oxidation reaction which is the main reaction during biochemical analysis.

  The addition amount of the leuco dye contained in the test piece for biochemical analysis is preferably 0.03 or more and 0.10 mg / cm 2 or less. A concentration of less than 0.03 mg / cm 2 is not preferable because measurement sensitivity is insufficient. If it exceeds 0.10 mg / cm 2, self-coloring tends to occur and it is difficult to suppress with an antioxidant, and furthermore, the dye aggregates during preparation of the reagent, making it difficult to produce a test piece having a uniform concentration. More preferably, it is 0.03 or more and 0.05 mg / cm2 or less.

  The concentration of the enzyme that eliminates the antioxidant contained in the test piece for biochemical analysis is preferably 3 U / cm 2 or more and 100 U / cm 2 or less. When it is less than 3 U / cm 2, ascorbic acid cannot be eliminated in a short time reaction, and the oxidation reaction that is the main reaction does not proceed. If it exceeds 100 U / cm 2, the enzyme cannot be uniformly immobilized on the substrate at the time of preparing a test piece, which is not preferable.

  In the usage mode of the present invention, as a blood glucose sensor and a small biochemical analyzer for self-diagnosis by the patient himself / herself, the medical worker uses the biochemical analyzer for POC (Point of Care) for diagnostic purposes. Sometimes used.

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the examples. The physical properties in the specification were measured by the following methods.

(Test piece preparation method)
A test piece of 1 cm 2 was prepared from a fiber structure (woven fabric, plain weave, basis weight 80 g / m 2, thickness 110 μm), and used as a substrate for immobilizing enzymes and dyes.
The test piece of the example has a two-layer structure, and the upper layer contains an enzyme for eliminating the antioxidant and peroxidase, and the lower layer contains hydrogen peroxide by an oxidation reaction in the presence of a dye and an antioxidant, a substrate and oxygen. Contains enzymes that generate. After forming each layer, two layers were laminated to prepare an analytical test piece.
Comparative Example 1 to Comparative Example 7 differ from the reagent composition of Example 1 above, but the test piece preparation method was similarly prepared one layer at a time, and then two layers were stacked to prepare a test piece. Further, Comparative Examples 8 and 9 were evaluated with one layer.
Tables 1, 3, and 5 show detailed reagent amounts, for example, amounts of enzymes, dyes, and antioxidants.

(Absorbance evaluation)
The color development suppression state of the test piece prepared by the above method was measured with a reflected light measurement device (Chromatoscanner CS-9300PC, manufactured by Shimadzu Corporation), the measurement temperature was 20 ° C., the light source was 1 mm × 5 mm, and the dye λmax = 666 nm. Was measured from the lower surface of the second layer. The slides were stored at 200 lux at 20 ° C., and the absorbance after 3 days and 8 days was measured to evaluate the presence or absence of self-coloring.
Regarding the quantitative property, 10 μL of a fructosyl valyl histidine aqueous solution (F-VH) having three concentrations of 54.6 μmol / L, 500 μmol / L, and 1680 μmol / L was dropped on the test piece prepared by the above method, The reaction state was measured with a reflected light measurement device (Chromatoscanner CS-9300PC, manufactured by Shimadzu Corporation) at a measurement temperature of 20 ° C. and a light source of 1 mm × 5 mm, and the absorbance of the pigment at λmax = 666 nm was measured.
Further, the absorbance measured was obtained as a linearity of dilution from the relationship with the F-VH concentration, and the quantitativeness was evaluated from the correlation coefficient.

(Measurement of fructosyl amino acid oxidase (FAOD) activity)
The activity of fructosyl amino acid oxidase was calculated from the change in absorbance obtained by reacting hydrogen peroxide produced by the reaction of glycated valylhistidine and glycated amino acid oxidase in the presence of peroxidase with a redox coloring reagent. Here, at an optimum pH of fructosyl amino acid oxidase (pH = 6.5), the amount of enzyme that hydrolyzes fructosyl valyl histidine per minute at 37 ° C. to produce 1.0 μmol of hydrogen peroxide. Defined as 1U.

(Peroxidase (POD) activity measurement)
The activity of peroxidase is as follows: hydrogen peroxide and pyrogallol (P
pyrogallol (Purpurogall) produced by reaction
It was calculated from the change in absorbance derived from in). Here, the amount of enzyme that produces a color corresponding to 1.0 mg of purpurogallin at 20 ° C. for 20 seconds at the optimum pH of peroxidase (pH = 6.0) was defined as 1 U.

(Ascorbate oxidase (ASO) activity measurement)
The activity of ascorbate oxidase was defined as 1 U for the amount of enzyme that oxidizes 1.0 μmol of ascorbic acid per minute at 30 ° C. and pH 5.6 in the presence of ascorbate oxidase.

Example 1
About the preparation method of a test piece, as for the 1st layer, Toyobo Ascorbate oxidase (ASO) 62.6U and Toyobo Peroxidase (POD) 40U) were melt | dissolved in buffer 10 microliters (50 mM PIPES), and the reagent was adjusted. A fiber structure (woven fabric) was impregnated with this reagent and dried with hot air at 50 ° C. for 15 minutes to prepare a test piece for biochemical analysis.
The second layer consists of 10 U of enzyme (fructosyl amino acid oxidase (FAOD) manufactured by Toyobo) and 0.05 mg of dye (sodium 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine (DA67)). Antioxidant 0.05 mg of ascorbic acid was dissolved in 10 μL of buffer (50 mM PIPES) to prepare a reagent. A fiber structure (woven fabric) was impregnated with this reagent and dried with hot air at 50 ° C. for 15 minutes to prepare a test piece for biochemical analysis.
Table 1 shows the reagent composition of the test piece.

  The test piece prepared by the above method was subjected to absorbance evaluation and quantitative evaluation. The results are shown in Table 2.

In Table 2, Table 4, and Table 6, the meanings of the symbols in the self-coloring evaluation column are as follows.
A sufficient self-coloring inhibitory effect was observed at around 666 nm.
○ Self-coloring suppression effect was observed at around 666 nm.
Δ: Self-coloring inhibitory effect was observed at around 666 nm, but is insufficient.
× No self-coloring suppression effect was observed in the vicinity of 666 nm.

In addition, in the evaluation columns for quantitativeness in Tables 2, 4 and 6, the meanings of the symbols are as follows.
A high correlation between F-VH concentration and absorbance was observed.
○ Correlation between F-VH concentration and absorbance was observed.
Δ Correlation between F-VH concentration and absorbance was observed but insufficient.
X No correlation between F-VH concentration and absorbance was observed.

Further, in Tables 2, 4 and 6, the above self-coloring and quantitative properties were comprehensively evaluated, and those capable of achieving both self-coloring and quantitative properties were rated as ◎ or ○ otherwise.

As apparent from the results in Table 2, the reagent composition of Example 1 showed no self-coloring over time, and a sufficient self-coloring inhibitory effect was observed at around 666 nm. In the reagent composition of Example 1, the correlation coefficient between the substrate concentration and the absorbance was high, and quantitativeness was obtained. Moreover, it was possible to achieve both self-coloration inhibition and quantitativeness.

(Example 2)
The test method was the same as in Example 1. The reagent composition of the test piece is shown in Table 1.
About the preparation method of a test piece, the 1st layer dissolved the enzyme Ascorbic acid oxidase (ASO) 62.6U and Toyobo peroxidase (POD) 40U in the buffer 10 microliters (50 mM PIPES), and adjusted the reagent. A fiber structure (woven fabric) was impregnated with this reagent and dried with hot air at 50 ° C. for 15 minutes to prepare a test piece for biochemical analysis.
The second layer consists of 10 mg of enzyme (Fructosyl amino acid oxidase (FAOD) manufactured by Toyobo Co., Ltd.) and dye (10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine sodium (DA67)) and oxidized. A reagent was prepared by dissolving 0.03 mg of the inhibitor ascorbic acid in 10 μL of buffer (50 mM PIPES), impregnating the fiber structure (woven fabric) with this reagent, and drying for 15 minutes with hot air at 50 ° C. for biochemical analysis. Created a piece.

As is clear from the results in Table 2, the reagent composition of Example 2 showed no self-coloring over time, and a sufficient self-coloring inhibitory effect was observed at around 666 nm. In the reagent composition of Example 2, the correlation coefficient between the substrate concentration and the absorbance was very high, and quantitativeness was obtained. Moreover, it was possible to achieve both self-coloration inhibition and quantitativeness.

(Comparative Example 1)
The test method was the same as in Example 1. The reagent composition of the test piece is shown in Table 3.
About the preparation method of a test piece, as for the 1st layer, the enzyme Toyobo Ascorbate oxidase (ASO) 62.6U was melt | dissolved in buffer 10 microliters (50 mM PIPES), and the reagent was adjusted. A fiber structure (woven fabric) was impregnated with this reagent and dried with hot air at 50 ° C. for 15 minutes to prepare a test piece for biochemical analysis.
The second layer consists of enzymes (Toyobo's fructosyl amino acid oxidase (FAOD) 10U, Toyobo's peroxidase (POD) 40U and dye (10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine sodium). (DA67)) 0.05 mg and 0.2 mg of the antioxidant ascorbic acid were dissolved in 10 μL of buffer (50 mM PIPES) to prepare a reagent, which was impregnated into a fiber structure (woven fabric) and heated with hot air at 50 ° C. for 15 A test piece for biochemical analysis was prepared by drying for a minute.

As is clear from the results in Table 4, the reagent composition of Comparative Example 1 did not develop self-coloring over time, and exhibited a self-coloring inhibitory effect near 666 nm. In the reagent composition of Comparative Example 1, the correlation coefficient between the substrate concentration and the absorbance was low, and the quantitative property could not be obtained. Moreover, it was not possible to achieve both self-coloration suppression and quantitative properties.

(Comparative Example 2)
The test method was the same as in Example 1. The reagent composition of the test piece is shown in Table 3.
About the preparation method of a test piece, the 1st layer dissolved the enzyme Ascorbic acid oxidase (ASO) 62.6U and Toyobo peroxidase (POD) 40U in the buffer 10 microliters (50 mM PIPES), and adjusted the reagent. A fiber structure (woven fabric) was impregnated with this reagent and dried with hot air at 50 ° C. for 15 minutes to prepare a test piece for biochemical analysis.
The second layer consists of 10 mg of enzyme (Fructosyl amino acid oxidase (FAOD) manufactured by Toyobo Co., Ltd.) and dye (10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine sodium (DA67)) and oxidized. A reagent was prepared by dissolving 0.2 mg of the inhibitor ascorbic acid in 10 μL of buffer (50 mM PIPES), impregnating this reagent into a fiber structure (woven fabric), and drying it with hot air at 50 ° C. for 15 minutes to conduct a test for biochemical analysis. Created a piece.

As is clear from the results in Table 4, the reagent composition of Comparative Example 2 did not develop self-coloring over time and exhibited a self-coloring suppression effect in the vicinity of 666 nm. In the reagent composition of Comparative Example 2, the correlation coefficient between the substrate concentration and the absorbance was low, and the quantitative property could not be obtained. Moreover, it was not possible to achieve both self-coloration suppression and quantitative properties.

(Comparative Example 3)
The test method was the same as in Example 1. The reagent composition of the test piece is shown in Table 3.
About the preparation method of a test piece, the 1st layer dissolved the enzyme Ascorbic acid oxidase (ASO) 62.6U and Toyobo peroxidase (POD) 40U in the buffer 10 microliters (50 mM PIPES), and adjusted the reagent. A fiber structure (woven fabric) was impregnated with this reagent and dried with hot air at 50 ° C. for 15 minutes to prepare a test piece for biochemical analysis.
The second layer consists of 0.10 mg of enzyme (10 to U fructosyl amino acid oxidase (FAOD) and dye (sodium 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine (DA67)) and oxidized). A reagent was prepared by dissolving 0.2 mg of the inhibitor ascorbic acid in 10 μL of buffer (50 mM PIPES), impregnating this reagent into a fiber structure (woven fabric), and drying it with hot air at 50 ° C. for 15 minutes to conduct a test for biochemical analysis. Created a piece.

As is clear from the results in Table 4, the reagent composition of Comparative Example 3 exhibited self-coloring over time, and no self-coloring suppression effect was observed near 666 nm. In the reagent composition of Comparative Example 3, the correlation coefficient between the substrate concentration and the absorbance was very high, and a quantitative property was obtained. Moreover, it was not possible to achieve both self-coloration suppression and quantitative properties.

(Comparative Example 4)
The test method was the same as in Example 1. The reagent composition of the test piece is shown in Table 3.
About the preparation method of a test piece, the 1st layer dissolved the enzyme Ascorbic acid oxidase (ASO) 62.6U and Toyobo peroxidase (POD) 40U in the buffer 10 microliters (50 mM PIPES), and adjusted the reagent. A fiber structure (woven fabric) was impregnated with this reagent and dried with hot air at 50 ° C. for 15 minutes to prepare a test piece for biochemical analysis.
The second layer consists of 10 mg of enzyme (Fructosyl amino acid oxidase (FAOD) manufactured by Toyobo Co., Ltd.) and dye (10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine sodium (DA67)) and oxidized. A reagent was prepared by dissolving 0.1 mg of the inhibitor ascorbic acid in 10 μL of buffer (50 mM PIPES), impregnating this reagent into a fiber structure (woven fabric), and drying it with hot air at 50 ° C. for 15 minutes to conduct a test for biochemical analysis. Created a piece.

As is clear from the results in Table 4, the reagent composition of Comparative Example 5 did not develop self-coloring over time, and exhibited a self-coloring inhibitory effect near 666 nm. In the reagent composition of Comparative Example 5, the correlation coefficient between the substrate concentration and the absorbance was low, and the quantitative property could not be obtained. Moreover, it was not possible to achieve both self-coloration suppression and quantitative properties.

(Comparative Example 5)
The test method was the same as in Example 1. The reagent composition of the test piece is shown in Table 3.
About the preparation method of a test piece, the 1st layer dissolved the enzyme Ascorbic acid oxidase (ASO) 62.6U and Toyobo peroxidase (POD) 40U in the buffer 10 microliters (50 mM PIPES), and adjusted the reagent. A fiber structure (woven fabric) was impregnated with this reagent and dried with hot air at 50 ° C. for 15 minutes to prepare a test piece for biochemical analysis.
The second layer consists of 10 mg of enzyme (Fructosyl amino acid oxidase (FAOD) manufactured by Toyobo Co., Ltd.) and dye (10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine sodium (DA67)) and oxidized. A reagent was prepared by dissolving 0.01 mg of the inhibitor ascorbic acid in 10 μL (50 mM PIPES) of the buffer, impregnating the reagent into a fiber structure (woven fabric), and drying it with hot air at 50 ° C. for 15 minutes. It was created.

As is clear from the results in Table 4, the reagent composition of Comparative Example 6 exhibited self-coloring over time, and no self-coloring suppression effect was observed near 666 nm. In the reagent composition of Comparative Example 6, the correlation coefficient between the substrate concentration and the absorbance was very high, and quantitativeness was obtained. Moreover, it was not possible to achieve both self-coloration suppression and quantitative properties.

(Comparative Example 6)
The test method was the same as in Example 1. The reagent composition of the test piece is shown in Table 3.
About the preparation method of a test piece, the reagent of the 1st layer prepared the enzyme Toyobo peroxidase (POD) 40U in 10 microliters (50 mM PIPES) buffer. A fiber structure (woven fabric) was impregnated with this reagent and dried with hot air at 50 ° C. for 15 minutes to prepare a test piece for biochemical analysis.
The second layer is a buffer of 0.05 mg of enzyme (10 to U fructosyl amino acid oxidase (FAOD) and dye (sodium 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine (DA67))) A reagent was prepared by dissolving in 10 μL (50 mM PIPES) The fiber structure (woven fabric) was impregnated with this reagent and dried with hot air at 50 ° C. for 15 minutes to prepare a test piece for biochemical analysis.

As is clear from the results in Table 4, the reagent composition of Comparative Example 7 showed self-coloring over time, and no self-coloring suppression effect was observed near 666 nm. In the reagent composition of Comparative Example 7, the correlation coefficient between the substrate concentration and the absorbance was very high, and a quantitative property was obtained. Moreover, it was not possible to achieve both self-coloration suppression and quantitative properties.

(Comparative Example 7)
The test method was the same as in Example 1. The reagent composition of the test piece is shown in Table 5.
Regarding the preparation method of the test piece, there is no first layer, and the second layer is 62.6 U of enzyme Toyobo Ascorbate oxidase (ASO) and a dye (10- (carboxymethylaminocarbonyl) -3,7-bis (dimethyl). Amino) phenothiazine sodium (DA67)) 0.05 mg and the antioxidant ascorbic acid 0.2 mg were dissolved in 10 μL of buffer (50 mM PIPES) to prepare a reagent. A fiber structure (woven fabric) was impregnated with this reagent and dried with hot air at 50 ° C. for 15 minutes to prepare a test piece for biochemical analysis.

As is clear from the results in Table 6, the reagent composition of Comparative Example 8 did not develop self-coloring over time and exhibited a self-coloring inhibiting effect in the vicinity of 666 nm. In the reagent composition of Comparative Example 8, the correlation coefficient between the substrate concentration and the absorbance was low, and the quantitative property could not be obtained. Moreover, it was not possible to achieve both self-coloration suppression and quantitative properties.

(Comparative Example 8)
The test method was the same as in Example 1. The reagent composition of the test piece is shown in Table 5.
Regarding the preparation method of the test piece, there is no first layer, and the second layer is dye (10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine sodium (DA67) 0.05 mg) and antioxidant. Ascorbic acid 0.2 mg was dissolved in 10 μL of buffer (50 mM PIPES) to prepare a reagent. A fiber structure (woven fabric) was impregnated with this reagent and dried with hot air at 50 ° C. for 15 minutes to prepare a test piece for biochemical analysis.

As is apparent from the results in Table 6, the reagent composition of Comparative Example 9 did not develop self-coloring over time, and exhibited a self-coloring inhibitory effect near 666 nm. In the reagent composition of Comparative Example 9, the correlation coefficient between the substrate concentration and the absorbance was low, and the quantitative property could not be obtained. Moreover, it was not possible to achieve both self-coloration suppression and quantitative properties.

According to the present invention, it is possible to stably use a dye having a high sensitivity at the time of an enzymatic reaction of a simple dry-type test specimen for biochemical analysis, to provide a test piece that can obtain a stable enzyme reactivity and can be measured with high accuracy. Can do. Therefore, it is possible to improve the measurement accuracy when diagnosing diabetes and various diseases, and it is expected to greatly contribute to the industry such as disease prevention and diagnosis.

Claims (12)

A test piece for biochemical analysis comprising a leuco dye and a multilayer structure containing a substrate and an enzyme that generates hydrogen peroxide by oxidation reaction in the presence of oxygen,
(A) at least one layer containing a leuco dye and one or more antioxidants;
(B) A test piece for biochemical analysis, comprising an enzyme for eliminating peroxidase and an antioxidant in a layer different from the layer. The test piece for biochemical analysis according to claim 1, wherein the antioxidant is at least one antioxidant selected from the group consisting of ascorbic acid, an ascorbic acid derivative, glutathione, and a glutathione derivative. The test piece for biochemical analysis according to claim 2, wherein the antioxidant is ascorbic acid and / or an ascorbic acid derivative. The test piece for biochemical analysis according to any one of claims 1 to 3, wherein the enzyme that eliminates the antioxidant is ascorbate oxidase and / or γ-glutamyl transpeptidase. The test piece for biochemical analysis according to claim 4, wherein the enzyme that eliminates the antioxidant is ascorbate oxidase. The test piece for biochemical analysis according to any one of claims 1 to 5, wherein the concentration of the antioxidant is 0.03 mg / cm2 or more and 0.05 mg / cm2 or less. The test piece for biochemical analysis according to any one of claims 1 to 6, wherein the concentration of the enzyme that eliminates the antioxidant is 3 U / cm 2 or more and 100 U / cm 2 or less. The test piece for biochemical analysis according to any one of claims 1 to 7, wherein the leuco dye is 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine sodium (DA67). The test piece for biochemical analysis according to any one of claims 1 to 8, wherein the addition amount of the leuco dye is 0.03 to 0.10 mg / cm 2 or less. The enzyme that generates hydrogen peroxide by oxidation reaction in the presence of substrate and oxygen is selected from the group consisting of glucose oxidase, urate oxidase (uricase), cholesterol oxidase, creatinine amide hydrolase, lipase, ketoamine oxidase, and glycated amino acid oxidase The test piece for biochemical analysis according to any one of claims 1 to 9, which is one type or two or more types. A biochemical analysis method comprising performing an enzymatic reaction using the test piece for biochemical analysis according to claim 1. A biochemical analyzer for performing biochemical analysis by performing an enzyme reaction using the test piece for biochemical analysis according to any one of claims 1 to 10.