JPH01107137A - Liquid analysis - Google Patents

Abstract

PURPOSE:To enable clinical chemical analysis with high accuracy by a method wherein a standard liquid of different concentration is drip-deposited on at least two units of the same dry analysis element, permitting the collection of a sample even from a patient at home, for example. CONSTITUTION:When a specific component in liquid is to be analyzed using a dry analysis element, a sample is drip-deposited on a unit of said element by which the component is detected by its optical variation. At the same time, the standard liquid is drip-deposited on other two or more units of the dry analysis element, and optical variation on the element is measured. Therefore, analysis which permits the identification of the concentration of a specific component in a sample enabled highly accurate clinical chemical analysis.

Description

[Detailed Description of the Invention] [Prior Art and Its Disadvantages] Dry analytical elements, like so-called test strips and clinical chemistry analysis slides, contain all the reagents necessary for the detection of specific components in a dry state, and When brought into contact with this, a reaction with the reagents occurs, and by measuring the resulting optical and electrical changes, the specific component can be detected quantitatively or semi-quantitatively.

Conventionally, when the purpose of quantitative analysis is to spot a specimen on an analytical element, incubate it under certain conditions (for example, 5 minutes at 37°C) to cause a reaction, and then perform optical measurements etc. It was common practice to calculate the concentration of specific components inside.

Recently, devices that automatically perform operations after incubation have become commercially available, providing satisfactory reproducibility and accuracy. However, many of these devices require (1) a power source;

(2) It has a certain size.

(3) Because it is complex, precise, and expensive, etc.
They cannot be installed everywhere, so the installation of such devices is limited to hospitals or large clinics.

For example, if a diabetic outpatient at a hospital were to measure their blood sugar levels several times in their daily lives and bring the results with them when they visit the hospital, the treating doctor would be able to more accurately understand the patient's condition. .

There are devices that can be used for semi-quantitative measurements at home, but their accuracy is low and they are not suitable for the above purpose.

Another method is to place a specimen collected by the patient in daily life on an analytical element (element) and bring the analytical element to the hospital for measurement, but the Due to fluctuations in external conditions, changes in specimens and analysis elements, the accuracy is significantly reduced and is not useful for basolateral diagnosis.

Therefore, samples are collected during the patient's daily life at home etc.
Moreover, it is extremely desirable to be able to perform clinical chemical analysis with analytical accuracy that is not much different from that obtained when testing is performed using an automatic analyzer in a hospital or the like.

[Technical Problems to be Solved] The present invention makes it possible to collect a patient's specimen outside of a hospital where an automatic analyzer is installed, for example at home, and to collect the analytical elements at the place where the automatic analyzer is installed. The technical challenge is to enable clinical chemistry analysis with the same analytical accuracy as spot testing.

[Means for solving technical problems] The above problem is solved by spotting a sample in one unit area of the dry analysis element that detects the specific component by optical change when analyzing a specific component in a liquid using a dry analysis element. At the same time, after spotting the standard solution on two or more unit areas of the dry analysis element and storing those dry analysis elements under the given conditions, The problem was solved by an analytical method that measures optical changes and determines the concentration of specific components in a sample.

[Specific Structure of the Invention] The object of the present invention can be more preferably achieved by applying reference solutions of different concentrations to at least two unit areas of a dry analytical element. The higher the Na degree level, the easier it is to improve the analytical accuracy. However, in terms of the cost and effort required for analysis, it is better to have a lower concentration level, so choose it appropriately.

Dry analytical elements have at least one liquid-permeable porous layer. This layer may contain all or part of the reagent that reacts with the component to be detected, or a part of the reagent may be contained in layers at different depths of one porous layer. , or may have two or more porous or non-porous liquid-permeable layers that may contain some or all of the above reagents in another non-porous liquid-permeable layer.

Dry analytical elements may have a light-transmissive or transparent support, which may be liquid-permeable, such as paper, or liquid-impermeable, such as plastic.

Dry analytical elements are disclosed in U.S. Pat. No. 3,992,158 and U.S. Pat.
．． 292,272, on which a liquid-permeable layer containing at least a portion of the reagent and a further porous layer are successively provided - Imaging multilayer analysis Preferably, it is an element.

- Imaging multilayer analytical elements are also available in U.S. Patent 3,983,005
, 4,042,335, 4,066.403, 4
, 144,306, 4,132,528, 4,25
8,001, 4,357,363, 4,381,4
It may have a configuration as described in No. 21 or the like.

It may have a plurality of porous layers as described in JP-A-61-4959, JP-A-62-138756, JP-A-62-138757, and JP-A-62-138758.

- Even if the imaging multilayer analytical element is used in a format that detects the optical change near the substantial end point of the reaction between a specific component in the sample and the reagent in the analytical element, the reaction rate is However, since a considerable amount of time elapses from sample collection to optical measurement, a method that uses the end point of the reaction is more suitable.

Analytical elements that can be applied to the present invention can be measured colorimetrically, regardless of detection component, type of reaction, composition, measurement wavelength, etc. However, in extreme cases where the color completely disappears during storage, this Unsuitable for invention.

The present invention can also be applied to analytical elements made for analysis by rate assay, as long as the optical density when applied and left to stand corresponds to the concentration of the analyte.

In the present invention, the storage of the analytical element is different from the case where the analytical element is incubated under certain conditions in a colorimetric measuring device after spotting the sample solution, and then optically measured after a certain period of time (or at certain time intervals). The time required for storage does not have to be strictly constant; the storage period varies considerably depending on the case, and ranges from several tens of minutes to several tens of days. Of course, a constant temperature chamber or the like may be used to maintain constant storage conditions during all or part of the storage period.

It is preferable that the analysis unit area on which the standard solution is applied is two or more unit areas.It is preferable to apply two or more types of reference liquids with different concentrations in these unit areas in order to improve analysis accuracy. A reference solution with a certain concentration may be spotted on two or more units of analysis area, thereby increasing the accuracy of analysis.

It is preferable that each analysis unit area is physically continuous, but it may be discontinuous6. For example, a small piece of chemical analysis film may be fixed to a frame (for example, a parallel member with grooves on the inside). 0 For example, Saito et al.'s patent application No. 62-22803
Although the chemical analysis tape described in US Pat. It may also be something you have.

The optical change used for detection is not limited to color development, but may also be color change (change in absorption wavelength), fluorescence, luminescence, etc. The reaction used may be a reaction involving an enzyme in the sample or analytical element. , or may be an immune reaction.

The component to be detected (analyte) may be a low-molecular substance, an ion, or a high-molecular substance, and may be either a hydrophilic substance or a hydrophobic substance.The component to be detected may be an enzyme, an antigen, or a hydrophobic substance. The antigen or antibody may be an antibody; the antigen or antibody may be labeled with an enzyme or a fluorescent substance; or it may be bound to another polymer; the antigen may be a hapten.

Examples of analytes: Low molecules: urea, uric acid, ammonia, creatinine, lactic acid, pyruvic acid, glucose, galactose, neutral fat, cholesterol, hemoglobin, bilirubin, Ions: calcium, potassium, sodium, chloride ions, High molecules: protein (albumin, globulin, etc.), nucleic acids, riboproteins, enzymes: amylase, phosphatase, lipase, lactate dehydrogenase, creatine phosphokinase, alanine aminotransferase, asparagine aminotransferase The present invention is useful for diagnosis, treatment progress determination, health management, etc. The analysis method of the present invention is useful for quantifying various analytes in body fluids such as blood (whole blood, etc.) and urine. .

The analytical element used in the present invention contains at least one type of interaction composition. The interaction composition is an analyte or a reaction product of an analyte or with decomposition products or with each other,
In the analytical elements used in the present invention, which contain one or more interacting active ingredients, dyes that can be detected spectrophotometrically, either directly or indirectly (via other reactions) by such interaction, 6 Dyes that are generated, decomposed, or inactivated by fluorescent substances, etc. may be generated by interactions between specific components in the blood and dye-forming substances, or may be generated from diffusible dyes from non-diffusible dyes. Interactions J include chemical activity, catalytic activity, such as in the formation of enzyme-substrate complexes, immunogenic activity, such as in antigen-antibody reactions, as well as direct effects on dye concentration. Also included are any chemical or physical interactions that can liberate, form, or produce detectable dyes or the like that indirectly indicate the presence or concentration of a particular analyte.

The reagent composition contained in the analytical element of the present invention is a composition capable of producing an optically detectable substance, such as a dye, in the presence of a test component. Compositions that produce dyes by oxidation of leuco dyes (see, for example, U.S. Pat. No. 4,089,74
7, JP-A-59-193352, etc.), diazonium salts, and compositions containing compounds that produce dyes by coupling with other compounds when oxidized (e.g., 4 -aminoantipyrines and phenols or naphthols),
A compound consisting of a compound capable of producing a pigment in the presence of a reduced coenzyme and an electron transfer agent can be used.

Examples of leuco dyes include U.S. Pat. No. 4,089,747.
Examples include triarylimidazole leuco dyes such as those described in JP-A-59-193352, known triarylmethane leuco dyes, and the like.

A dye may be formed by a dye-forming composition comprising a condensation product of an oxidizable compound and a color former; examples of oxidizable compounds include benzidine and its congeners, p-phenylene diaminos, p- Examples include amine phenols, aminoantipyrine, such as 4-aminoantipyrine.

In addition, in the case of analytical elements for measuring enzyme activity, for example, p
- Containing a self-developing substrate (e.g. paranitrophenyl phosphate, paranitrophenyl maltopentaose, γ-glutamyl paranitroanilide) capable of liberating a colored substance such as nitrophenol in the reagent layer or developing layer. Can be done.

The reagent composition may contain an enzyme, and the one described in JP-A-62-138756 by Nagadai et al., pages 18 to 20, can be used.

The reagent composition may be contained in whole or in part in a substantially uniform layer with a hydrophilic polymer as a binder.
Examples of aqueous polymers include gelatin and derivatives thereof (e.g. phthalated gelatin), cellulose derivatives (e.g. hydroxypropyl cellulose, carboxymethyl cellulose), agarose, acrylamide polymers, methacrylamide polymers, acrylamide or methacrylamide and various vinyl monomers. Copolymers of polyvinylpyrrolidone, polyvinylpyrrolidone, and copolymers of polyvinylpyrrolidone and various vinyl monomers can be used.

A portion or all of the reagent composition may be included in the porous layer. In order to have at least one of the porous layers contain a reagent composition that develops color in the presence of the test component, an appropriate amount of the reagent composition may be included. A method of adhering a porous spreading layer pre-impregnated or coated with a suitable solution or dispersion onto another water-permeable layer, such as a reagent layer, as described in JP-A-55-164356, can be used.

After adhering the porous layer onto other water-permeable layers (e.g., undercoat layer, adhesive layer, water absorption layer) by the method described in JP-A-55-164356, a solution or dispersion of the reagent composition is applied. The liquid may be applied to the porous layer.

Known methods can be used for impregnating or coating the porous layer. For example, dip coating, doctor coating, hopper coating, curtain coating, etc. are selected and used as appropriate.

The reagent composition may be contained entirely in one porous layer, for example, in the porous layer closest to the support among two or more porous layers, or it may be contained separately in two or more porous layers. good.

The element may have, for example, a light blocking layer or a vortex layer as described in US Pat. No. 4,042,335.

The liquid spreading layer is constructed from any suitable fibrous or non-fibrous material, or mixtures thereof, having suitable porosity and average pore size capable of absorbing whole blood. Preferably, the liquid spreading layer provides a uniform amount of analyte liquid per unit area on the surface facing the adjacent water-permeable layer with which it is in liquid contact; useful liquid spreading layers are described in U.S. Pat. No. 4,292.
, No. 272 and JP-A-60-22276.
It can be constructed from a fibrous material such as the knitted fabric described in No. 9. It can also be constructed using non-fibrous isotropic porous materials (eg, plush polymers) as described in US Pat. No. 3,992,158.

When whole blood is used as a sample in the present invention, it is preferable that the analytical element has a layer that filters and removes substantially all or at least some of the blood cells, and the liquid spreading layer has this function at the same time. Those made of the above-mentioned woven or knitted fabrics are useful.

In the developing layer, in order to adjust the developing area, developing speed, etc., there are
It may contain a hydrophilic polymer or a surfactant as described in No. 61-122876 and No. 81-143754.

When a light-transmitting support is used, the practically preferred structure of the dry analytical element of the present invention is (1) having a reagent layer on the support and a liquid spreading layer thereon.

(2) A device having a detection layer, a reagent layer, and a liquid development layer in this order on a support.

(3) A support having a reagent layer, a light reflection layer, and a liquid development layer in this order.

(4) A support having a detection layer, a reagent layer, a light reflection layer, and a liquid development layer in this order.

(5) A support having a detection layer, a light reflection layer, a reagent layer, and a liquid development layer in this order.

In (1) or (3) above, a water absorption layer may be provided between the support and the reagent layer. In (1) or 3) above, a blood cell filter may be provided between the reagent layer and the detection layer or the liquid development layer. A vortex layer or other vortex layer may be provided between the light reflection layer and the detection layer, the reagent layer or the liquid spreading layer, between the reagent layer and the detection layer in (3) to 5) above, or A blood cell vortex layer or other filtration vortex layer may be further provided between the reagent layer and the liquid spreading layer.

The analytical element of the invention may have a light reflective layer.

For example, a light reflecting layer can be provided between the reagent layer and the detection layer or between the reagent layer and the liquid developing layer. The light-reflecting layer is used to measure detectable changes (color change, color development, etc.) in the detection layer, reagent layer, etc. from a light-transmissive support. It blocks the color of the test liquid, especially the red color of hemoglobin when the sample is whole blood, the yellow color of bilirubin, etc., and functions as a light reflective layer or background layer. The light-reflecting layer is preferably a water-permeable layer in which light-reflecting fine particles such as titanium oxide or barium sulfate are dispersed using a hydrophilic polymer as a binder. The binder may include gelatin, gelatin derivatives, polyacrylamide, etc. preferable.

A hardening agent may be added to hardenable polymers such as gelatin. Analytical elements may include a spreading layer, reagent layer,
The detection layer or the like may contain light-reflective particles such as titanium oxide.

The analytical element of the present invention may have a layer that substantially filters out whole blood cells, separate from the liquid rM layer.

For example, JP-A-58-70163, JP-A-61-495
No. 9, Patent Application No. 60-256408, No. 60-2798
The porous layers described in No. 59 to No. 279,861 are suitable.

One or more layers of the elements of the invention may contain one or more other various optional ingredients, such as surfactants,
It may contain a solvent for a coloring agent, a wiping agent, a binder, a hardening agent, and the like. These components can be present in amounts known to those skilled in the art. Representative component components are described, for example, in U.S. Pat.
No. 2,158, No. 4,042゜335, No. 4,144
, No. 306, No. 4,132,528, No. 4゜05G,
No. 898, No. 4,258,011, No. 4,275.1
No. 52, No. 4,292,272, etc.

To obtain more accurate and precise test results, the element may be incubated (heated) at two constant temperatures and for a certain period of time prior to optical measurement.

For spectrophotometric measurements known suitable devices are used, for example U.S. Pat.
, No. 488,810, No. 4,584,275, Japanese Unexamined Patent Publication No. 1983
No. 1-294367, No. 61-294368, No. 62
-184335.

No. 62-184336, Japanese Patent Application No. 61-25582,
No. 61-25583, No. 61-40890, No. 61
-87708 to 87710, 61-109
No. 402, No. 81-144258, No. 61-1854
No. 71, No. 61-185472, No. 61-20704
The devices described in Nos. 4 to 207049, etc., "Fuji Drychem 1ooo.

, same r2000. , r5000, analyzer (all manufactured by Fuji Photo Film Co., Ltd.), rEkta
cbes+ 400J.

rEktachem 700J, rEktache
m DT-60” analyzer (manufactured by Eastman Kodak Company), etc. can be used.

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

[Implementation PA1] 1> Preparation of chemical analysis film Yuka one color Δ baby box A leuco dye solution having the following composition A was prepared.

A: 2-(4-hydroxy-3,5-dimethoxyphenyl)
-4-(4-(dimethylamino)phenylcou 5-phenethylimidazole (leuco dye) acetate 5.02 Methylene chloride
10 mlN, N-diethyl laurylamide 90 companies 1 A gelatin solution having the following composition was prepared.

B: Alkali-treated gelatin 230g water
1400g glucose oxidase 40000 U peroxidase 7
0000 U (U represents the international unit) Sodium di-2-ethylhexyl sulfosuccinate 5 g Bis[(vinylsulfonylmethylcarbonyl)aminocomethane 2.3 g] However, 1 @ Housetsu B solution was mixed with TK O) - Homo mixer (special machine Solution A was added while stirring at about 6000 rpm using an emulsifier manufactured by Kogyo Co., Ltd., and dispersed for about 30 minutes to prepare an emulsion.

1- The above emulsion was coated with gelatin to a thickness of 180 μm.
It was applied onto a transparent polyethylene terephthalate (PET) film (support) at a rate of 150 g/m 2 and dried.

Filling 1 A light reflecting layer (dry layer thickness: 7 μN) consisting of the following coating amount for each component was provided on the coloring reagent layer by coating and drying an aqueous dispersion.

Alkali-treated gelatin 2.9 g/x2
Rutile type diacid type titanium oxide fine particles 13 g/x" nonylphenoxy polyglyside (average content of 10 glycide units > 400 x g/x
(1) An adhesive layer was provided on the reflective layer with the following coating amount (dry thickness: 5 μl) by coating and drying an aqueous dispersion.

Alkali-processed gelatin 6.717 m2 Nonyl phenoxy boriglyside (contains 10 glycide units on average) 600 layers g/jI
Water is almost uniformly supplied to the surface of the above adhesive layer at a ratio of 30g7x2 to moisten the surface of the adhesive layer, and on top of that, PE equivalent to 50 denier is applied.
A porous spreading layer was prepared by laminating and adhering a 36-gauge T-spun yarn knitted fabric with a thickness of about 250 μl using a light press.

Next, a polymer-containing ethanol dispersion was applied over the developing layer to the following coverage amount and dried to complete an analytical film for glucose determination.

hydroxypropylcellulose (28-30% methoxy groups.

Contains 7-12% hydroxypropoxy groups.

Viscosity of 2% aqueous solution at 20°C: 50 cps) 5 g7
1 nonylphenoxybolyethoxyethanol (average 40 oxyethylene units) 500 g/x22)
Analysis immediately after spotting Whole blood 1 and whole blood 2 were prepared by adding a small amount of glucose (powder, Wako Tsumugi Co., Ltd., WA) to whole blood collected from a human (heparin was used as an anticoagulant). , 10μ! each at two locations 30mm apart on the chemical analysis FILNO fabricated as described above. I spotted it.

On the other hand, control liquid rGLU-W control liquid for whole blood glucose analysis manufactured by Fuji Photo Film Co., Ltd.
10μ each! , was spotted on adjacent parts of the above analysis film.

Immediately after spotting, the glucose concentration was measured using a reaction measuring device "Fuji Drychem 2000" manufactured by Fuji Photo Film Co., Ltd., an incubator part of the analyzer (reaction temperature 37° C., reaction time 6 minutes) and an optical measuring section. The results are shown in Table 1.

Table 1 The above value will be referred to as the "true value" hereinafter.

3) Analysis of 18 release Xt* Next, on another sheet of the analysis film, apply control solution ■ to one place, control solution ■ to another place, and whole blood 1 to five places, 10μ each! I spotted it. In the same manner, whole blood 2 was spotted instead of whole blood 1 on another sheet. After leaving each film as it was at room temperature for 24 hours, the glucose concentration values were determined in the same manner as above using the Fuji Drychem Zooo analyzer, part of the incubator, and the optical measurement section. ” is called.

Using the true values and uncorrected values of the control solutions ■ and ■ obtained above, constants a and b in the relational expression % formula % [1] between the true values and the uncorrected values were determined.

And the formula (correction value) created using these constants a and b =
The uncorrected values obtained for whole blood 1 and whole blood 2 were substituted into aX (uncorrected value) and b [2] to obtain the corrected value. The results are shown in Table 2. In all cases, the deviation from the true value is +1 g/dl.
, -2mg7dl, which was a good result. Species type deviation S
D and coefficient of variation C■ are also sufficiently small.

Table 2 [Comparative Example 1] The same operation as in Example 1 was carried out, except that the correction using the control solutions (■) and (■) was omitted, and the uncorrected values of whole blood 1 and whole blood 2 were used as they were. The results are shown in Table 3, SD and Cv
are not much different from those in Table 2, but the deviations from the true values are -6mg7dl and -18H/dl, respectively, which is 1 from Example 1.
It was huge.

Table 3 Applicant: Fuji Photo Film Co., Ltd.

Claims (3)

[Claims] (1) A method for analyzing a specific component in a liquid using a dry analytical element, wherein a sample is placed in one of the unit areas of the dry analytical element, which has a plurality of detection unit areas for detecting the specific component by optical change. and at the same time, spot the reference solution on two or more other unit areas of the dry analysis element, and after storing the dry analysis element under the given conditions, apply it to each unit area of the dry analysis element. An analysis method characterized by measuring the optical changes that occur and determining the concentration of a specific component in a sample. (2) The analysis method according to claim (1), characterized in that reference solutions having different concentrations are spotted on at least two unit areas of the dry analysis element. (3) The dry analytical element is stored under the same external conditions and kept at a constant temperature for a certain period of time, and then the optical change occurring in the dry analytical element is measured. Analysis method for range (1).