JP2008148656A - Dry analytical element - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a dry analytical element designed to rapidly measure the concentration of an objective component of measurement such as glucose in blood with high accuracy. <P>SOLUTION: The dry analytical element is designed to measure the objective component of measurement in a liquid sample. The element is composed of an anisotropic membrane having a larger size of pores on the surface on the side for spotting the sample than that of the surface on the side for detecting coloring. The anisotropic membrane is impregnated with a reagent and an imidazole derivative leuco dye for detecting the objective component of measurement. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a dry analytical element for colorimetric determination for measuring the amount of a specific component in a specimen, such as measurement of glucose contained in a liquid such as blood.

  Dry analytical elements including enzyme-based compositions are widely used in clinics, clinics, hospitals and homes to test glucose levels in the blood. In fact, dry analytical elements have become a daily necessity for many diabetics. Diabetic patients have to measure blood glucose concentration several times a day, and according to the results, for example, dietary restriction and / or insulin injection must be performed, and it is very important to speed up measurement and improve measurement accuracy It is a difficult issue. To solve this problem, for example, as described in Japanese Patent No. 2545250 and Japanese Patent No. 2589053, an anisotropic porous membrane containing a reagent that causes a color change depending on the glucose concentration is used. The dry analysis element for shortening the time required for the development and diffusion of () is shown, but it is difficult to say that both are fast enough. Furthermore, WO 2004/086037 discloses a combination of 4-aminoantipyrine and its coupler reagent as an anisotropic porous membrane and a color-developing reagent, and measurement of the product in which the invention is used. Although the time is as fast as about 10 seconds, the measurement is performed in a time when the reaction is not sufficiently completed, so the quantitative property in the high concentration range cannot be ensured in particular, causing a decrease in measurement accuracy. Yes.

  There is also a problem that accurate blood glucose level measurement is difficult because sufficient sensitivity cannot be obtained in a low concentration range.

  Therefore, development of a dry analytical element for measuring glucose concentration in blood quickly and with high accuracy is required.

Japanese Patent No. 2545250 Japanese Patent No. 2589053 WO2004 / 086037

  The problem to be solved by the present invention is to provide a dry analytical element for measuring the concentration of a measurement target component such as glucose in blood quickly and with high accuracy.

  As a result of intensive studies to solve the above problems, the present inventor used an anisotropic membrane having a sample side having a relatively large pore size and a test side having a relatively small pore size, and was able to detect excess from glucose and oxygen. By containing a reagent that generates hydrogen oxide and causes a color change in the anisotropic film, and using an imidazole derivative leuco dye as a color-forming reagent that causes a color change, the concentration of the measurement target component can be adjusted quickly and with high accuracy. The inventors have found that it can be measured and have completed the present invention.

  That is, according to the present invention, a dry analysis element for measuring a component to be measured in a liquid sample, wherein the size of the pores on the surface on which the sample is spotted is the surface on the color detection side A dry analytical element comprising an anisotropic membrane larger than the pore size of the material, wherein the anisotropic membrane is impregnated with a reagent for detecting a component to be measured and an imidazole derivative leuco dye. Provided.

Preferably, the ratio of the average pore diameter between the surface on which the sample is spotted and the surface on the color detection side is 10 or less.
Preferably, the anisotropic membrane is a polysulfone membrane or a polyethersulfone membrane.

Preferably, the imidazole derivative leuco dye is a compound represented by the following formula (A).

(Wherein R1 and R2 are each independently a lower alkyl group, R3 and R4 are each independently a lower alkoxy group or a halogen atom, and R5 is a lower alkyl group, an aralkyl group, or C ₆ H ₅ —O. A lower alkyl group substituted with-.

Preferably, the imidazole derivative leuco dye is any one of the following compounds (1) to (5).

(In the formula, Me represents a methyl group, Et represents an ethyl group, Bz represents a benzyl group, and Ph represents a phenyl group.)

  The dry analytical element according to the present invention can improve the measurement speed as compared with the conventional anisotropic membrane glucose analytical element. Moreover, according to the dry analytical element of this invention, a measurement range can be improved so that it may become clear from the measurement example in an Example. Further, as is clear from the comparative example, the dry analytical element of the present invention enables highly sensitive measurement in a low concentration range using whole blood or the like as a sample. Furthermore, as is clear from the comparative example, the dry analytical element of the present invention enables high-precision measurement using whole blood or the like as a sample. As described above, the dry analytical material according to the present invention is extremely useful particularly in the analysis of components in a liquid using whole blood as a sample.

Hereinafter, embodiments of the present invention will be described in detail.
The present invention relates to a dry analytical element for measuring a component to be measured in a liquid sample. In particular, the size of the pore on the surface on which the sample is spotted is the surface on the surface on which color development is detected. The present invention relates to a dry analytical element comprising an anisotropic film larger than the size of the pores, wherein the anisotropic film is impregnated with a reagent for detecting a measurement target component and an imidazole derivative leuco dye.

  The anisotropic membrane used in the present invention is preferably a non-fibrous porous carrier. The anisotropic film used in the present invention has a plane (also referred to as a dense surface) provided with an opening portion having a relatively small hole diameter and a plane (hereinafter also referred to as a rough surface) provided with an opening portion having a relatively large hole diameter. An asymmetric shape is provided, and both apertures are in communication. In the present invention, a plane having an opening portion having a relatively large hole diameter is used as a surface on which a sample is spotted, and a plane having an opening portion having a relatively small hole diameter is used as a color detection side. Use as a surface. That is, in the anisotropic film used in the present invention, the pore size on the surface on which the sample is spotted is larger than the pore size on the surface on the color detection side. The ratio of the average pore diameter of the surface on which the sample is spotted and the surface on the color detection side is preferably 10 or less, and more preferably 5 or less. The ratio of the average pore diameter of the surface on which the sample is spotted and the surface on the color detection side can be, for example, 1.1 to 10, more preferably 1.1 to 5, Preferably it can be 1.1 to 3.5.

  The pore diameter of the rough surface of the anisotropic film used in the present invention (the surface on which the sample is spotted) has an upper limit of about 5.0 μm for securing a blood development area for photometry and for complete capture of blood cells, The lower limit is preferably about 0.5 μm. When measuring the glucose concentration by the color development method, a correct result cannot be obtained unless a sufficient color development area is secured, particularly when the amount of the sample liquid is very small. If the pore diameter of the rough surface of the non-fibrous porous carrier is larger than this, the blood development area becomes small, and the blood development area for photometry cannot be secured. Further, the pore diameter of the dense surface (that is, the surface on the color detection side) of the anisotropic film used in the present invention is preferably about 3.0 μm at the upper limit and about 0.2 μm at the lower limit.

  The material of the anisotropic film used in the present invention is preferably polysulfone or polyether tersulfone. Specifically, the polysulfone membrane manufactured by Fuji Photo Film Co., Ltd. is a membrane having an asymmetry such that the average pore size on the dense surface is 0.45 to 3.0 μm and the average pore size on the rough surface is 1.0 to 5.0 μm. And is a suitable material for the dry analytical element according to the present invention.

  Specific examples of the reagent component used in the present invention include glucose oxidase (GOD), peroxidase (POD), and imidazole derivative leuco dye (coloring reagent) for glucose measurement.

  The imidazole derivative leuco dye is a color indicator that is colored when a substance having an oxidizing action such as peroxidase acts as a catalyst in the presence of hydrogen peroxide. As the imidazole derivative leuco dye, for example, an imidazole derivative leuco dye described in the specification of JP-A-59-193352 can be used. The imidazole derivative leuco dye described in this publication has an absorption peak in the wavelength range of 600 or more, preferably 600 to 700 nm, and develops a blue color.

As the imidazole derivative leuco dye, a compound represented by the following formula (A) can be used.

(Wherein R1 and R2 are each independently a lower alkyl group, R3 and R4 are each independently a lower alkoxy group or a halogen atom, and R5 is a lower alkyl group, an aralkyl group, or C ₆ H ₅ —O. A lower alkyl group substituted with-.

  As used herein, the lower alkyl group means a linear, branched or cyclic alkyl group having 1 to 6 carbon atoms, such as a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group or a hexyl group. And so on.

  As used herein, the lower alkoxy group means a linear, branched or cyclic alkoxy group having 1 to 6 carbon atoms, such as methoxy group, ethoxy group, propoxy group, butoxy group, pentyloxy group or hexyl. An oxy group etc. can be mentioned.

  As used herein, a halogen atom means fluorine, chlorine, bromine or iodine.

  Examples of the aralkyl group in the present specification include those having 7 to 12 carbon atoms, preferably 7 to 9 carbon atoms, and specific examples include a benzyl group, a phenylethyl group, and a phenylpropyl group.

Specific examples of the imidazole derivative leuco dye include the following compounds (1) to (5).

(In the formula, Me represents a methyl group, Et represents an ethyl group, Bz represents a benzyl group, and Ph represents a phenyl group.)

  The compounds (2) and (4) can be synthesized by the method described in JP-A-59-193352, and the compounds (1), (3) and (5) are described in US Pat. No. 3,297,710. It can be synthesized by referring to the method.

  Among them, 2- (3,5-dimethoxy-4-hydroxyphenyl) -4- [4- (dimethylamino) phenyl] -5-phenethylimidazole or 2- (3,5-dimethoxy-4-hydroxyphenyl) It is particularly preferable to use -4- [4- (dimethylamino) phenyl] -5-benzylimidazole.

  Further, the dry analytical element of the present invention is not limited to the measurement of glucose in a liquid, but can also be used for the measurement of other components in a liquid. For example, by using an oxidase selected from the group consisting of cholesterol oxidase, lactate oxidase, and alcohol oxidase instead of glucose oxidase (GOD) contained in the glucose measuring reagent, cholesterol, lactic acid, and alcohol in a liquid are obtained. It can also be measured.

  As glucose oxidase, those derived from Aspergillus niger, Penicillium notatum and the like are preferably used.

  As the cholesterol oxidase, those obtained from Nocardia erythroporis, Brevibacterium, Pseudomonas, Mycobacterium, Suehirotake and the like are preferably used.

  As lactate oxidase, those obtained from Aerococcus viridans, Pediococcus sp and the like are preferably used.

  Alcohol oxidases include Basidiomycete, H. polymorpha and Kloeckera sp. It is preferable to use those obtained from the above.

  As peroxidase, peroxidase of plant origin or animal origin, or peroxidase of microbial origin can be used. It is preferable to use non-specific peroxidases of plant origin or microbial origin. It is more preferable to use a peroxidase extracted from horseradish or radish, or a peroxider extracted from a microorganism belonging to the genus Cochliobolus or Curvularia.

  The dry analytical element of the present invention can be obtained, for example, as follows. That is, the dry analytical element of the present invention can be prepared by impregnating or applying a non-fibrous porous carrier in which a reagent component is dissolved or dispersed in an appropriate solvent and then drying. At this time, in order to solve the solubility problem of the imidazole derivative leuco dye, which is a color development indicator, the application process is made in two or more stages, or the reagent components are emulsified and applied as a single solution in a one-stage application process. It is also possible to do. As the solvent to be used, a solvent that does not deform or collapse the structures of the polysulfone membrane and the polyethersulfone membrane is selected. For example, methanol, ethanol, acetone or the like may be used.

  Furthermore, the dry analytical element of the present invention may be one in which an anisotropic film is provided on the surface of a light-transmitting / water-impermeable support. Examples of light-transmitting / water-impermeable substrates include polymers such as polyethylene terephthalate, bisphenol A polycarbonate, polystyrene, cellulose esters (eg, cellulose diacetate, cellulose triacetate, cellulose acetate propionate, etc.) A film or sheet-like transparent support having a thickness of about 50 μm to about 1 mm, preferably about 80 μm to about 300 μm.

  The type of liquid sample applied to the dry analytical element of the present invention is not particularly limited, and any liquid sample (for example, body fluid such as whole blood, plasma, serum, lymph, urine, saliva, spinal fluid, vaginal fluid; The components to be measured in drinking water, alcoholic beverages, river water, factory wastewater, etc.) can be analyzed.

  The analysis using the dry analytical element according to the present invention is performed, for example, as follows. In other words, after processing the dry analytical element to a certain size and incorporating it into the device, a small amount of blood is spotted from the rough surface (sample side) of the dry analytical element, and at the same time, the color on the opposite side (test side) of the sample spotting surface This is done by observing the change in the reflection photometry.

  For example, the dry analytical element of the present invention is cut into small pieces, such as a square having a side of about 10 mm to about 30 mm or a circle of approximately the same size, and the Japanese Patent Publication No. 57-28331 (corresponding US Pat. No. 4,169,751). -142454 (corresponding U.S. Pat. No. 4,387,990), JP-A-57-63452, JP-A-58-32350, JP-T-58-501144 (corresponding international publication WO83 / 00391), etc. Use as a chemical analysis slide is preferable from the viewpoint of manufacturing, packaging, transportation, storage, measurement operation, and the like. Depending on the purpose of use, the tape can be used in a long tape form in a cassette or magazine, or a small piece can be attached to or stored in a card with an opening, or a cut piece can be used as it is. Thus, a container containing one or more pieces can have information such as item names, lot numbers, and calibrations. For example, IC, magnetism, or barcode can be used for these pieces of information, which can be easily read on hardware and useful for automation.

  In the dry analytical element of the present invention, for example, a liquid sample in the range of about 0.5 μL to about 30 μL, preferably 1 μL to 10 μL is spotted on the reagent layer. The spotted dry analytical element is incubated at a constant temperature in the range of about 20 ° C to about 45 ° C, preferably at a constant temperature in the range of about 30 ° C to about 40 ° C for 1 to 10 minutes. The color development or discoloration in the dry analytical element is reflected and photometrically measured from the support side, and the amount of the measurement target component in the liquid sample can be obtained by the principle of the colorimetric measurement method using a calibration curve prepared in advance.

  The measuring operation is disclosed in JP-A-60-125543, JP-A-60-220862, JP-A-61-294367, JP-A-58-161867 (corresponding to US Pat. No. 4,424,191). With the described chemical analyzer, highly accurate quantitative analysis can be performed with extremely easy operation. Depending on the purpose and required accuracy, semi-quantitative measurement may be performed by visually judging the degree of color development.

  The following examples further illustrate the present invention, but the present invention is not limited to the examples.

Examples 1 to 5: Production of dry analytical elements 1 to 5 for measuring glucose First, impregnating solutions (1) and (2) described in Tables 1 and 2 below were mixed and shown in Table 3 below. Using a non-fibrous porous membrane as a carrier, the mixed impregnation solution was impregnated for 30 seconds and then dried at 45 ° C. for 10 minutes. The obtained reagent-supporting non-fibrous porous carrier was cut into a 12 × 13 mm rectangle to obtain a glucose analysis material.

Comparative Example 1:
A mede-safe mini (Termo Co., Ltd. product), which is a self blood glucose measurement chip, was used. In this chip, a polyethersulfone membrane is used as a non-fibrous porous membrane, but an imidazole derivative leuco dye is not used as a coloring reagent, and 4-aminoantipyrine and its coupler reagent are used. Yes.

Measurement example:
Using the above glucose analysis material, the glucose concentration in whole blood was actually measured. Using human whole blood containing 10, 23, 42, 93, 329, 507 and 1047 mg / dl glucose as a liquid sample, 3 μl of whole blood was spotted on the rough side of the analysis material, and the elapsed time after spotting The color density on the dense surface side of the analytical material corresponding to 1 was measured at 650 nm with an instantaneous optical reflection measuring device MCPD3000 (manufactured by Otsuka Electronics Co., Ltd.). The glucose concentration of each sample was measured with a glucose analyzer (Hitachi, 7170).

  FIG. 1 shows the change of the reflection optical density at 650 nm with respect to the measurement time when the whole blood glucose concentration is 507 mg / dl for the analysis material of each type of membrane. In Examples 1 and 5, the color development reaction is faster than in Comparative Example 1, and the reflection optical density (OD) becomes saturated in about 10 seconds. Moreover, it turned out that Examples 2-4 have a high reflective optical density (OD) at the same time compared with a comparative example.

  FIG. 2 is a calibration curve prepared by plotting the reflection optical density at 650 nm with a reaction time of 10 seconds after spotting against the glucose concentration for each type of membrane analysis material. Compared with Comparative Example 1, it was found that Examples 1 to 4 had the same response to glucose over the entire concentration range, and Example 5 with a wide measurement range.

  Furthermore, in the low glucose concentration region (80 mg / dL or less), the sensitivities of the glucose analysis materials of Examples 1 to 5 and Comparative Example 1 were compared. The results are shown in Table 4. The sensitivity (dODr (80 mg / dL-10 mg / dL)) here is the reflection optical density OD (80 mg / dL) at 650 nm and the glucose concentration of 10 mg / dL when the glucose concentration is 80 mg / dL. Is defined as a difference in reflection optical density OD (10 mg / dL).

  From the results of sensitivity comparison in Table 4, it can be seen that the glucose analysis materials of Examples 1 to 5 have higher sensitivity in the low glucose concentration region than the glucose analysis material of Comparative Example 1. From this result, it is clear that the accuracy of glucose measurement in the low concentration region is high.

FIG. 1 shows changes in reflection optical densities (650 nm) of Examples 1 to 5 and Comparative Example 1 according to reaction elapsed time at each glucose concentration. FIG. 2 shows the analysis results of whole blood of various glucose concentrations obtained using the glucose analysis element according to the present invention. The vertical axis represents the reflection optical density at 650 nm when the reaction time is 10 seconds, and the horizontal axis represents the blood glucose concentration.

Claims (5)

A dry analytical element for measuring a component to be measured in a liquid sample, where the pore size on the surface on which the sample is spotted is larger than the pore size on the surface on the color detection side A dry analytical element comprising an anisotropic film, wherein the anisotropic film is impregnated with a reagent for detecting a component to be measured and an imidazole derivative leuco dye. The dry analytical element according to claim 1, wherein the ratio of the average pore diameter of the surface on which the sample is spotted and the surface on the color detection side is 10 or less. The dry analytical element according to claim 1 or 2, wherein the anisotropic membrane is a polysulfone membrane or a polyethersulfone membrane. The dry analytical element according to any one of claims 1 to 3, wherein the imidazole derivative leuco dye is a compound represented by the following formula (A).

(Wherein R1 and R2 are each independently a lower alkyl group, R3 and R4 are each independently a lower alkoxy group or a halogen atom, and R5 is a lower alkyl group, an aralkyl group, or C ₆ H ₅ —O. A lower alkyl group substituted with-. The dry analytical element according to any one of claims 1 to 4, wherein the imidazole derivative leuco dye is any one of the following compounds (1) to (5).

(In the formula, Me represents a methyl group, Et represents an ethyl group, Bz represents a benzyl group, and Ph represents a phenyl group.)

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