JPS5818628B2 - Ittsutai Kei Eki Taibun Sekiyouso - Google Patents

Description

BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to an integrated multilayer analytical element for use in analyzing fluids such as biochemical and biological fluids.

Chemical analysis of liquids such as water, foods such as milk, and biological fluids is often desired and necessary.

Various analytical elements are known that facilitate liquid analysis.

Such analytical elements, upon contact with a liquid sample containing the substance to be analyzed (hereinafter referred to simply as the test substance), produce colored substances or other detectable substances depending on the presence of the test substance. They usually contain reagents that cause changes.

Such analytical elements may include, for example, paper or other highly absorbent carriers that are chemically active or inert and that develop color or color in response to contact with liquids containing hydrogen ions or other test substances. There are pH test strips and similar indicators impregnated with color-changing substances.

Depending on the selection of such responsive substances, said changes are usually qualitative and at best semi-quantitative.

In various fields, analytical techniques that can quickly obtain quantitative analysis results are often required.

It has recently been discovered that the testing of biological fluids, including body fluids such as blood, serum, urine, etc., must be performed quickly, conveniently, and with high quantitative properties to provide useful elements for diagnostic chemical analysis. A lot of development research is being carried out.

Solution chemical analysis techniques have gained wide acceptance in clinical testing, especially in the field of automated analysis.

However, this technique requires analytical equipment capable of handling and transporting complex solutions.

For example, U.S. Patent Nos. 2,797,149 and 30,368.
Various "wet chemistry" analytical devices, such as those exemplified in U.S. Pat. No. 93 and U.S. Pat. Therefore, it requires skill to keep it extremely clean.

As an alternative to this solution chemical analysis technique, various multilayer integrated analytical elements for non-solution type, essentially dry chemical analysis have been proposed.

In this specification, "integrated type" is used for analytical elements.
is an analytical element comprising two or more preferably independent layers superimposed in substantially continuous intimate contact with adjacent layers in the analytical element, and which layers are means an analytical element that cannot be separated without damaging the element.

Although dry analysis offers storage, handling, and other conveniences compared to wet analysis, the success of this "dry" approach is limited and is primarily used in the field of qualitative or semi-quantitative testing. It has been used for.

DESCRIPTION OF THE PRIOR ART The basic form of an integrated analytical element is described in U.S. Pat.
It is described in the specification of No. 5.

These multilayer analytical elements typically include chromophores,
An absorbent fibrous carrier impregnated with one or more reagents and a semipermeable material coated thereon are used.

When these analytical elements come into contact with the test liquid, the test substance passes through the semipermeable membrane to the fibrous carrier, producing an amount of color that is related to the concentration of the test substance.

The semi-permeable membrane prevents the passage and absorption of interfering components, such as red blood cells, which would interfere with accurate reading of the color that provides the test result.

Absorbing liquid sample collection and distribution in applications such as clinical testing? Analytical elements based on paper or other fibrous media have so far been less popular compared to wet chemical analysis.

This is probably due to the inability to obtain highly accurate and quantitative results with said analytical elements.

According to the literature, it has been reported that diagnostic analysis elements using impregnated absorbent materials such as fibrous filter paper cannot provide uniform test results.

U.S. Pat. No. 3,050,373 discloses that precipitation occurs in the impregnating solution, whereby the absorbent carrier or
It is stated that the uniform distribution of the reaction reagents in J-lux is impaired.

Analytical elements that use fibrous, absorbent materials are also prone to non-uniformity of test results, referred to as banding.

"Banding" refers to a phenomenon in which test results are excessively distributed in a part of the area of the analytical element showing the test results, such as the outer periphery of the area penetrated by the applied sample.

This phenomenon is probably due to the chromatographic effect.
This may be the result of excessive non-uniform migration of sample components or reaction reagents within the absorbent material.

U.S. Patent Nos. 3,061,523 and 3,104;
No. 209 lists gelatin and gelatin-like materials as useful constituents of the impregnating solution.

This is probably because gelatin and the like can limit the high-speed migration, and as a result, the test results can be made uniform.

However, the gelatin and gelatin-like materials in the fibrous absorbent matrix containing the reactive reagents reduce the rate of sample absorption compared to a more absorbent matrix that does not contain gelatin.

This reduction in absorbency results in surface liquid remaining on the analytical element, which must be cleaned to remove this excess liquid before test measurements are taken.

As a result, an upper limit of the amount of gelatin that can be impregnated into the absorbent matrix is typically specified.

Such properties are also characteristic of analytical elements that employ layers solely of gelatin or gelatin analogs, as discussed in US Pat. No. 3,526,480.

Integrated analytical elements for automated test analysis are also described, for example, in US Pat. No. 3,368,872 and US Pat. No. 3,526,480.

These include incorporating the applied sample into the element by immobilizing or immobilizing the reactive reagent or by using a simple porous member on the reactive reagent-containing absorbent material, such as fibrous P-paper. By including means to reduce the tendency to exert a cleaning effect on the reaction reagents,
Measures are described to avoid chromatographic effects (often referred to as ringing, targeting, tornating or banding) in the analytical element.

However, these descriptions do not only absorb a liquid sample into the analytical element, but also distribute sample components, such as the test substance, in a uniform apparent concentration over substantially the entire surface of the reactive reagent layer that is in contact with the applied sample. This does not go so far as to suggest the use of measures to coerce the government.

Such concentration uniformity is very important to obtain test results suitable for reading by densitometric, colorimetric, fluorescent, or other automatic readout methods.

This means that even in the absence of global non-uniformity such as that caused by chromatographic effects,
This is very important.

In dry analysis, a technique that can be called sample confinement is used to provide a uniform concentration of the test substance in the reaction reagent region of the analytical element.

A barrier is typically included on the analytical element, as described in U.S. Pat. No. 3,368,872.
The applied sample is confined to a predetermined area of the analytical element surface, such that excess liquid is present on the analytical element after sample application.

However, this technique has the inconvenience of handling and cleaning of residual excess sample on the analytical element, as well as the major problem of having to apply a very precise volume of sample when applying the sample to the analytical element. There is.

U.S. Patent No. 2,761,813, U.S. Patent No. 2,672,4
No. 31, No. 2,672,432, No. 2,677,
No. 647, No. 2,923,669, No. 3,814
, 670 and 3,843,452, there is some recognition of the need to optionally promote or prevent the migration of substances between the layers of an integrated analytical element. It will be done.

However, these relate to analytical elements that measure the presence of microorganisms, and the analytical elements described for this purpose typically contain at least one layer of a fibrous matrix and A non-discrete layer (norrdiscrete 1
aye-rs).

Until very recently, the test substance, test product, or other substance must be uniformly distributed within the layer to receive the sample components and provide an apparently uniform concentration to the cooperating layer exhibiting the analytical reaction or similar activity. No analytical elements have been suggested in the art that include layers or other means for distributing the sample components within the layer to achieve an apparent concentration.

As a practical matter, the structural and chemical customization of the absorbent and other fibrous materials used in most well-known analytical elements (e.g., absorbent cellulose filter paper, glass fiber paper, water condensation paper, etc.) It is widely recognized that such uniform concentration distribution is susceptible to being compromised due to physical limitations, non-uniform penetration of sample components, or undesired chemical binding.

Moreover, the selection of fibrous materials precludes highly accurate analytical measurements due to their non-uniformity in the strict sense of properties such as structure and texture.

For example, in paper manufacturing, raw fibers are often used as tendril fibers, which increase the strength of the resulting paper.
) are processed to create smaller constituent fibers called fibers.

As used herein to refer to paper-like materials,
The term "fibrous or fibrous" refers to materials prepared using preformed fibers or strands present in the final material.

For example, fibers used to prepare fibrous materials are described in US Pat. No. 3,867,258.

Traditionally, non-uniformity in detectable colored responses or other test results obtained using integrated analytical elements incorporating fibrous materials has been recognized as a problem with the use of such analytical elements. Ta.

There is a desire to overcome the problem of non-uniformity in analytical elements using such substances in the absorption layer.
This problem has not yet been resolved.

For example, U.S. Patent No. 3,723,064 states:
Analytical elements are disclosed that include permeable regions of differing efficiencies for the test substance or reaction products of the test substance and that produce a plurality of spaced apart threshold color indications as an assay result.

Although this shows a favorable smooth continuous response, analytical elements prepared as described in U.S. Pat. No. 3,723,064 can only provide approximate analytical results. , the accuracy of the analysis results changes directly in relation to the increase in the interval between thresholds.

In an attempt to increase the accuracy of the response in the dynamic range of interest, the difference in permeability between regions is reduced to narrow the spacing between thresholds, according to U.S. Pat. No. 3,723,064. The complexity of prepared analytical elements increases surprisingly.

Although there is no suggestion as to how to improve the uniformity and precision of continuously varying test results, U.S. Patent No. 3,723,0
The optimized analytical element described in '64 produces distinctly non-uniform, discontinuous responses within each permeation zone due to the non-homogeneous notes associated with the use of paper and other fibrous materials.

US Pat. No. 3,791,933 discloses a multicomponent device for the analysis of enzyme substrates and metabolites such as those found in body fluids.

However, this device is not monolithic; it captures the test sample, filters or otherwise removes large amounts of sample components (e.g., proteins), and produces detectable condensation (e.g., color development). It consists of a clamped array suitable for performing such test reactions.

Although it has been stated that glass fiber paper aids in the distribution of the reaction mixture through the plastic viewing window, such materials apparently aid in the outward diffusion of the liquid sample within the glass fiber layer. This merely expands the area in which test results of analytical elements are displayed, thereby making the test results easier to see.

There is no suggestion of a means of creating a concentrated homogenization of the test substance within the diffusion region, which is of course very important for the production of analytical results of uniform nature and high precision of detection.

An improved multilayer integrated analytical element is described in French Patent Application No. 7,323,599 (French Patent No. 2191734), filed June 28, 1973.

Such elements capture the liquid sample and spread the sample within the spreading layer of the analytical element, distributing the test substance, other continuous sample component, or test product to the analytical element in a uniform apparent concentration. However, in the presence of the test substance, methods such as spectrophotometry, fluorescence, etc. can be used to provide analytical results that can be measured quantitatively by automated equipment because of their uniformity.

The analytical element disclosed in French Patent No. 2,191,734 comprises a spreading layer and a reactive or interactive property which, by virtue of its activity, promotes a change in the analytical element detectable by radiological analysis, such as a color change. and a reaction reagent layer containing a substance.

However, the production of color or other analytical results is
It often requires a series of reactions that are difficult to control and subject to chemical or other interference.

As an example, by-products of the fluid under analysis or analytical measurements may produce components in the analytical element that interfere with the detection of test results.

This makes it possible to detect the substances that characterize the test results without the interference of said components that give undesired colors, fluorescence, etc.

Expression analysis elements are desired.

Therefore, while the analytical element described in French Patent No. 2,191,734 represents a substantial advance in the field of essential liquid dry analyzers, analytical elements of different or more advanced performance are desired. ing.

SUMMARY OF THE INVENTION According to the present invention, an integrated liquid analytical element comprising a radiation transparent support carrying in fluid contact a series of layers including at least one reactive reagent layer, comprising: (a) said reaction reagent layer; the reagent layer is permeable to the test substance or its precursor and comprises a reactive substance that produces a diffusible detectable substance in the presence of the test substance; (b) said element is in contact with the reactive reagent layer; Between the indicator and the
a detection layer permeable to and capable of detecting the detectable substance; and (c) the element is permeable to the detectable substance and transfers the detectable substance to the detection layer. An integrated liquid analytical element is provided which is characterized in that it does not allow interfering substances to migrate to the detection layer and that it comprises a radiation shielding layer located between the detection layer and the reaction reagent layer.

- The present invention provides a novel integrated analytical element for the analysis of fluids such as biological fluids.

As used herein, the terms "integral element" and "integrated analytical element" refer to a composite element that includes an "integral" arrangement of at least two superimposed layers.

The analytical elements of the present invention are capable of performing internally a variety of sample handling and/or processing functions.

The analytical element of the invention does not require skill for its use and, in particular in its preferred embodiments, does not require specific spotting or methods such as sample regulation, washing or other removal of excess sample. Quantitative analysis results can be generated without the need for

Furthermore, since the results obtained by the analytical element of the invention are virtually constant and do not cause harmful internal changes, automatic means of measuring electromagnetic radiation (radioanalytical techniques) may be used, if necessary or desired. can be used to detect the result with minimal error.

More particularly, the present invention relates to an integrated analytical element consisting of multiple superimposed layers that produces a detectable change within the analytical element that corresponds to the amount of test substance present in a liquid applied to the analytical element. Provide analysis elements that can be quickly provided.

The analytical element of the invention can be used for diagnostic analysis of biological fluids such as blood, serum or urine and is in fluid contact (1) permeable to at least the test substance or its precursor; (2) a reactive reagent layer having a composition comprising a reactive substance, such as a dye, capable of diffusing into the analytical element to produce a detectable substance in the presence of a test substance; and includes a detection layer in which the substance in question can be measured, for example by radioanalytical techniques.

The detection layer herein is generally prepared without the inclusion of chemically active substances or other substances that would interfere with the detection.

The various layers of the analytical element of the invention can be supported on a radiolucent injection support.

As used herein, the term "radiation-transparent" refers to supports and other layers of an analytical element that permit effective passage of electromagnetic radiation used to detect analytical results produced in the analytical element. .

This transparency includes the transmission of electromagnetic radiation of wavelengths within the range of 200 nm to 900 nm, and detection, such as produced by radioactivity, also includes the transmission of radiation.

If desired, the radiation transparent layer and support can be transparent, which is useful for measurements at low levels of radiation.

If the analytical element includes a support, the detection layer is interposed between the support and the reactive reagent layer, and is usually adjacent to the support.

The analytical element of the invention can include a radiation shielding layer, which is usually inserted between the reaction reagent layer and the detection layer.

This radiation shielding layer is a layer containing an opacifying agent of - or more, and which transmits electromagnetic radiation within or through the layer such as said wavelength used for the excitation and/or detection of the sensing substance in the detection layer. This is a layer that prevents the passage of.

Furthermore, the analytical elements of the present invention can include a material spreading layer in fluid contact with other layers of the element, such as reaction reagent layers and detection layers.

This sample spreading layer, also referred to herein as a spreading layer or a metering layer, distributes or meters into the layer a substance, including a test substance or its precursor, in a liquid sample applied to an analytical element. The substance can be distributed in a uniform apparent concentration over a predetermined period of time onto the surface of the spreading layer facing (ie in close contact with) the reactive reagent layer.

There is no need to limit the applied sample to obtain such a uniform concentration; the concentration will eventually be uniform at any point, but can be varied by an amount of time without deleterious effects. .

In various preferred embodiments, the spreading layer has isotropic pores 1'
4E(1sotr-opically porous
), i.e. porous in all directions within the layer.

As used herein, the term "isotropically porous" refers to porosity in all directions within the spread layer.

It goes without saying that the degree of porosity can be varied, for example in terms of pore size, porosity, etc., if necessary or desired.

As used herein, the term "isotropic porosity" (or isotropic porosity) is often used for filtration membranes to mean that the membrane has pores that communicate between both surfaces of the membrane. 1
soporous ) or ionotropic ( 1ono
tropic).

Similarly, the term isotropic is used as a contrast to anisotropic, which refers to a tear film with a thin skin (5 kin) along at least one surface of the mud film. It is also different from the word 1'l (1sotropic).

For example, Membrane 5ci
ence and Technology), James
s Flinn ed-, PlenumPress,
See New York (1970).

The reactive reagent layer is substantially uniformly permeable to at least one spreading material within the spreading layer and a diffusible detectable substance generated in the reactive reagent layer by interaction (reaction) as described herein. It is preferable that G).

Preferably, the detection layer has substantially uniform permeability to said detectable substance.

Uniform permeability of a layer is defined as the uniform permeability of a layer when a homogeneous liquid is applied uniformly to the surface of the layer by identical measurements of the concentration of the liquid within the layer (but measurements made through different areas of the layer surface). , usually refers to a permeability that gives substantially equal results.

Uniform permeability makes it possible to avoid undesirable concentration gradients, for example within the reaction reagent layer.

There is no need for every possible measurement technique to produce such a result.

The desirability of a particular technique and a particular measurement parameter depends on the physical properties of the layer, eg, its tendency to transmit, absorb, or scatter radiation.

The selection of an appropriate measurement technique (e.g. colorimetry, densitometry, fluorescence) and appropriate measurement parameters (e.g. pore size and arrangement) in any given case is self-evident to those familiar with the analytical method. Probably.

As explained herein, uniform permeability is not considered a custom feature of fibrous materials such as F paper.

Factors such as variable wicking within a fibrous material, differences in fiber size or spacing, etc., can affect the apparent percolation within the fibrous material and in cooperating materials in fluid contact with it. It is said that it can have an effect that causes a change in concentration.

This, of course, results in an undesirable bias between test results made within regions of different apparent concentrations.

Uniform permeability of reactive reagent layers, detection layers, or other layers within the analytical element is preferred as a means of facilitating convenient detection of analytical results.

The analytical significance of results produced in analytical elements that do not have a uniform permeation layer is limited.

Furthermore, it goes without saying that if, for example, irregular concentration or other discontinuities occur in the analytical element, which are read by the detection means, the efficiency of detecting the analytical result in such an analytical element is impaired.

As used herein, the term "fluidic contact" between the deployment layer, the reagent layer and/or other layers of an integrated analytical element refers to the presence of fluid, i.e. liquid or gas, in the element between overlapping regions of said layers. It means that it can pass through.

In other words, the term "fluid contacting" means that fluid components can be transported between the fluid contacting layers.

In the case of analysis of nitrogen-containing compounds, ammonia or other nitrogen-containing gaseous substances constitute the fluid that passes between the layers.

The fluid contacting layers can be adjacent or separated by an intervening layer.

However, the layers that physically intervene in fluid contact with each other must also be in fluid contact with those layers and must not prevent the passage of fluid between them.

As used herein, the term "diffusion" refers to diffusion when a substance is supported in a liquid present in the analytical element, such as a liquid sample solvent or dispersion medium applied to the analytical element. minutes by diffusion.

It refers to the ability to move effectively within an analytical element.

Similarly, the term "permeable" refers to the ability of substances supported in a liquid, or otherwise distributed in the liquid, by solution or dispersion, to permeate the layer.

In an analytical operation, an exemplary analytical element of the invention:

A liquid sample is captured and, if a test substance is present, the sample initiates a chemical reaction or other interaction in the reactive reagent layer to produce a diffusible, preferably radiometrically detectable, substance. This material diffuses from the reaction reagent layer into the detection layer.

Or if necessary. If desired, a radiation shielding layer can be provided between the reactive reagent layer and the detection layer in the analytical element.

Furthermore, a metering layer can be provided such that the applied sample passes through the metering layer before reaching the reaction reagent layer, and the test substance or its precursor is distributed within the metering layer and facing the reaction reagent layer. The substance is distributed in an apparently uniform concentration on the surface of the metering layer.

It is possible to obtain such uniform appearance concentrations for a wide range of sample volumes applied to the analytical element.

Owing to the fluid contact between the metering layer and the reactive reagent layer, and also because of the favorable permeability of the reactive reagent layer to the substance spread within the spreading layer or to the products produced by the action of said substance, uniform metering is achieved. The components are supplied from the spreading layer to the reactive reagent layer and can be passed through the reactive reagent layer without causing any essentially significant change in the apparent concentration of said substance or its product.

Due to the presence of reactive (e.g. chemically reactive) substances and due to the uniform appearance and concentration of the substances delivered from the metering layer to the reaction reagent layer, a uniform quantitative detectability change is produced in the analytical element. You can force it.

Such changes, which may be the production or decomposition of color or fluorescence, are quantitatively determined by radiometric techniques and, if desired, by automatic radiometric sensing devices such as photometric or fluorometric devices. can be detected.

DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION The integrated analytical elements of the present invention include a reactive reagent layer in fluid contact with a preferably radiation-transparent detection layer.

The analytical element can support these layers on a preferably radiation-transparent support.

No support is necessary if these layers exhibit adequate durability and integrity.

In a first preferred embodiment, the integrated analytical element of the invention comprises: (1) a detectable substance that is permeable to at least the test substance or its precursor and diffusible in the presence of the test substance; (2) a radiation shielding layer that is permeable to the detectable substance; and (3) a radiation shielding layer that is permeable to the detectable substance. It consists of a radiation-transparent support having in fluid contact a non-fibrous layer containing a radiation-transparent detection layer in which the substance is detected.

If desired, the detection layer can include a mordant for the detectable substance.

The detection layer is preferably interposed between the support and the radiation-shielding layer, and the radiation-shielding layer is interposed between the detection layer and the reactive reagent layer.

Preferably, the reactive reagent layer has substantially uniform permeability to the test substance (and, if appropriate, to the precursor of the test substance) and the diffusible detectable substance.

The detection layer has substantially uniform permeability to the detectable substance.

Normally, it is unlikely that the apparent concentration of the detectable substance supplied from the reaction reagent layer to the radiation shielding layer will change, but the radiation shielding layer has uniform permeability to the detectable substance. is preferable.

A preferred radiation shielding layer comprises an opacifying agent such as a pigment, a suitable form of a polymer, such as an opacified polymer, or both.

According to another aspect of the invention, there is provided an integrated analytical element in which a support carries in fluid contact a reactive reagent layer, a detection layer and a radiation shielding layer as described above for the preferred embodiments. Ru.

However, the element of this preferred embodiment further includes a metering layer, preferably of isotropic porous injection, and the reactive reagent layer is positioned within the element such that it is interposed between the detection layer and the metering layer. .

In one aspect of the invention, these layers are preferably non-fibrous.

The reactive reagent layer in the analytical element of the invention preferably has uniform permeability and, if appropriate, a substance which can be spread in the metering or spreading layer and the reaction product or the substance. can be porous to products formed as a result of the reaction or interaction of

As used herein, the term "permeability" includes permeability due to porosity, ability to swell, or other properties.

The reactive reagent layer can include a Mal-IJ sock in which a reactive substance is distributed, ie, dissolved or dispersed.

Of course, the choice of matrix material is variable and will depend on the intended use of the element.

Preferred matrix materials are natural substances such as gelatin, gelatin derivatives and hydrophilic cellulose derivatives and polysaccharides such as dextran, gum arabic and agarose, and water-soluble polyvinyl alcohols such as poly(vinyl alcohol) and poly(vinylpyrrolidone). Hydrophilic materials can include both chemical compounds as well as synthetic materials such as acrylamide polymers and the like.

Organophilic substances such as cellulose esters can also be useful substances, and in any case the selection of substances is carried out according to the intended use of the particular analytical element.

In order to increase the permeability of a reactive reagent layer when it is not porous, it is often useful to use a matrix material that is swellable in the solvent or dispersion medium of the liquid under analysis.

During manufacture of the element, it may also be necessary to select materials that are compatible with the application of adjacent layers, such as by coating means.

As an example, if the formation of discrete or discontinuous layers is desired and the analysis of interest is the analysis of aqueous solutions, a substantially water-soluble matrix may be used for the reactive reagent layer and for the adjacent layer, e.g. the spreading layer. It would be appropriate to select substantially organic soluble or organic dispersible components.

In this way, mutual solvent interaction can be minimized and a clearly demarcated layer structure can be constructed.

In many cases, as described herein, within the deployment layer,
In order to facilitate uniformity of the apparent concentration as described above, it is preferable to provide a reactive reagent layer having lower permeability than the spreading layer.

Relative permeability can be measured using known techniques.

A reactive substance is distributed in the reactive reagent layer in the presence of a test substance.

In some cases, the reactive substance is a precursor of the test substance, as in analytical elements for measuring cholesterol present in esterified form in serum or triglycerides, which are often analyzed on the basis of their glycerol content. Alternatively, it can be reacted with the reaction product.

As used herein, the term "reactivity" refers to chemical reactivity, catalytic activity, such as in the formation of enzyme-substrate complexes, or radiation-measurable, i.e., optical or Any other chemical or physical reaction that can produce or promote the production of a measurable diffusive substance by appropriate measurement of other electromagnetic radiation.

Distribution of the reactive substance can be achieved by dissolving or dispersing the reactive substance in the matrix material.

Although uniform distribution is often preferred, if the reactive substance is, for example, an enzyme, uniform distribution may not be necessary.

Reactive reagents or other reactive substances soluble in the liquid under analysis are advantageously immobilized (or immobilized) in the reactive reagent layer, especially if the reactive reagent layer is porous.

The specific reactive substances distributed within the reactive reagent layer are determined depending on the chosen analysis.

In many assays, an enzyme, such as an oxidase substance such as glucose oxidase or cholesterol oxidase, is included as a reactive substance in the reactive reagent layer of the element intended for the analysis of the test substance that is the substrate for that enzyme. is preferable.

As an example, dyes or other substances may be oxidized in the presence of peroxidase or peroxidizing substances and hydrogen peroxide produced by the reaction of peroxidase (or other peroxidizing substances) with oxidizing enzymes and their substrates. An oxidizing enzyme can be incorporated into the reaction reagent layer along with a substance or composition that produces a detectable substance.

In the practice of the invention, the detectable substance is diffusible;
□ can migrate into the permeable detection layer.

This diffusivity can be imparted to the naturally non-diffusible detectable substance described above by means well known in the art of chemical synthesis, usually by the addition of chemical groups that confer the desired solubility.

When analyzing aqueous liquids, hydroxyl groups, carboxyl groups,
Solubilizing groups such as sulfonic acid groups and the like are useful for solubilization purposes.

Substances or compositions that include oxidizing substances and can produce detectable substances include dye-imparting compositions.

In some embodiments, the dye-imparting material comprises a material that, when oxidized, is capable of coupling with itself or its reduced form to yield a dye.

Such autocoupling compounds include orthoaminophenols, including polyhydroxyl compounds such as cresols, pyrogallol, guaiacol, orcinol, catechol, chlorcurcinol, P,P-dihydroxydiphenyl, gallic acid, pyrocatexy, salicylic acid, etc. , 4-alkoxynaphthol, 4-amino-5
- Various hydroxyl compounds such as pyrazolones are included.

Compounds of this type are well known, for example in the theory of photographic processes.
tographic Process), Mees and James Ed-+ (1966), especially Chapter 17,
It is described in literature such as.

In other embodiments, the detectable substance can be formed primarily by oxidation of a leuco dye to give the corresponding dye form.

Representative leuco dyes include compounds such as leucomalachite green and leucophenolphthalene.

Other leuco dyes, called oxychromic compounds, are described in U.S. Pat. No. 3,880,658;
Furthermore, it is described that such compounds are diffusive even when they contain appropriate substituents.

The unstabilized oxychromic compounds described in US Pat. No. 3,880,658 are preferred in the practice of this invention.

In yet another embodiment, the detectable substance can be produced by a dye-imparting composition that includes an oxidizing compound capable of undergoing oxidative condensation with the coupler, such as one containing a phenol group or an active methylene group.

Typical oxidizing compounds include benzidine and its congeners such as P-phenylenediamines, P-aminephenols, and 4-amino-antipyrine.

A wide variety of such couplers, including numerous self-coupling compounds, are described, for example, by Mees and James, supra.
and Kosar's book on light-sensitive systems (Lig.
ht-8ensistive System) (19
65) described in literature such as pages 215-249.

Preferred dye-imparting compositions include, for example, 4-methoxy-1-naphthol, an autocoupling chemical, and 4-amine antipyrine (H
Cl) and 1,7-hydroxynaphthalene as a coupler.

To facilitate the detection of changes occurring in the analytical element, such as changes in color, optical density or fluorescence, the analytical element of the invention is formed in the reactive reagent layer and whose presence or absence is determined by It includes a layer that captures reaction products or other substances that are relevant to the detection of analytical results.

Such a layer, referred to herein as a detection layer, is permeable to the detectable substance generated within the analytical silica and is in fluid contact with the reactive reagent layer.

This detection layer can be separated from the reaction reagent layer by a radiation-shielding layer, such as a reflective and/or opalescent layer, to facilitate detection of analytical results by various radiation measurement techniques.

The detection layer, which is also preferably swellable in the liquid under analysis, can include hydrophilic colloids useful in reactive reagent layers.

This detection layer also needs to be radiation transparent to facilitate the detection of analytical results.

Additionally, if the detectable substance formed in the analytical element is a dye, the detection layer may include dye mordants such as those described as useful image dye mordants in color photographic film and paper. can.

Examples of mordants include vinylpyridine compounds of the type disclosed in U.S. Pat. No. 2,484,430, U.S. Pat.
No. 09690, No. 3488706 and No. 3557
Examples include polymers containing a quaternary ammonium group as described in the specification of No. 006.

A typical example of a mordant is N,N-dimethyl-N-benzyl-3
- Maleimidopropylammonium chloride.

As mentioned above, the analytical element of the present invention can preferably have a radiation shielding layer inserted between the reaction reagent layer and the detection layer.

The radiation shielding layer is permeable to the detectable substance produced in the analytical element and serves, for example, to block the passage of electromagnetic radiation at the wavelength used for detection.

The use of such a layer can protect the detection layer from color or other potential interference with the detection of the analytical result.

This layer contains an opacifier which, when incorporated into the layer, produces a radiation blocking effect by its absorption, reflection, etc. action.

In another embodiment, the radiation shielding layer may include a matrix containing an opacifying agent such as a pigment such as carbon or other inorganic pigments such as metal salts such as titanium dioxide, zinc oxide, barium sulfate and the like.

Generally reflective plush polymers can serve as opacifiers, and a layer of this plush polymer useful in a spreading layer can also be used as a radiation shielding layer.

If a single layer of microporous plush polymer is used as a radiation shielding layer, this layer can also act as a filtration layer.

In a preferred embodiment, the plush polymer layer can also incorporate reflective inorganic pigments, such as the highly reflective pigments described herein, to enhance spreadability and/or reflectivity.

The amount of pigment that can be included in the layer with the plush polymer can vary over a wide range, with pigment concentrations of from about 5% to about 1000% by weight of the plush polymer being preferred, and from about 100% to about 600% by weight of the plush polymer. A pigment concentration of % by weight is most preferred.

- In embodiments, the integrated analytical element of the present invention can include an isotropically porous spread layer in fluid contact with the reactive reagent layer.

The spreading layer is an isotropically porous or other layer in which a liquid sample is applied directly to the spreading layer or supplied to the spreading layer from one or more layers in fluid contact with the spreading layer. and reaction of the solvent or dispersion medium and at least one solute, dispersoid (constituent of the dispersed phase or internal phase) or solute or dispersoid in the sample in the spreading layer.

A spread layer facing the reactive reagent layer of the element in which the product has a uniform apparent concentration of said substance, i.e. a solute, a dispersoid or a reaction product thereof (which can be a test substance or a precursor thereof). distributed so as to give it to the surface of the

It goes without saying that such an apparent concentration can be achieved with a concentration gradient existing through the layer thickness or otherwise in the spread layer.

This gradient presents no difficulty for obtaining quantitative test results and can be adapted using known assay techniques.

The unfolding (or spreading) mechanism is not completely understood, but spreading is caused by hydrostatic pressure of the liquid sample, capillarity within the spreading layer, surface tension of the sample, and wicking of layers in fluid contact with the spreading layer. It is assumed to be defined by the result of the combination of forces and that combination.

It goes without saying that the extent of spreading will depend in part on the volume of liquid being spread.

However, the apparent uniformity of concentration is substantially independent of the volume of the liquid sample and occurs with varying degrees of spread.

As a result, the analytical elements of the present invention do not require precise sample application techniques.

However, a particular liquid sample volume may be desirable, such as to provide a favorable spreading time, etc.

The analytical element of the present invention allows quantitative analytical results to be obtained using very small sample volumes that can be completely absorbed within a conveniently sized area (e.g., li) of the spreading layer. , there is no need to remove excess moisture from the analytical element after application of the liquid sample.

Furthermore, because the spreading occurs in the spreading layer, the spreading material is delivered to the reactant layer in fluid contact, and there is no substantial lateral hydrostatic pressure, the "ringing" often seen in conventional analytical elements is avoided. No problems occur.

This spreading layer is only required to equalize the apparent concentration per unit area of the spreading substance on the surface facing the reaction reagent layer with which it is in fluid contact, and the particular layer is not suitable for this spreading purpose. It is very convenient to determine whether there is.

Such apparent concentration homogenization is accomplished by densitometry or other analytical techniques, e.g., to determine the apparent concentration of a spreading substance or a reaction product based on the concentration of a spreading substance, a reaction reagent or other related It can be measured by scanning the appropriate surface of the layer.

It goes without saying that the following tests are merely examples, and the selection of substances or test parameters does not imply that other substances or parameters are not suitable for similar purposes.

When conducting such tests, a prime coat (5u
bbed) poly(ethylene terephthalate), approx. 200
It can be applied to transparent photographic film support materials such as transparent gelatin layers with a gelatin coating of 1n9/dm2.

Although the gelatin can vary in hardness, for this test the gelatin layer is hardened such that the layer thickness swells by approximately 300% when immersed in water at 22° C. for 5 minutes.

When dry, the gelatin layer will be approximately 30 microns thick.

Layers evaluated for spreading purposes can be applied onto the gelatin layer, for example by coating a solution or dispersion.

The spreading layer can be designed to have a dry thickness that varies over a wide range, with thicknesses of about 100 to about 200 microns being convenient for testing purposes.

After drying the layer, a sample of the test solution or dispersion is applied to the surface of the spreading layer under evaluation, preferably with a wetted area, such as a circular section about 8 to 10 mm in diameter, but not so much that the entire layer is wetted with the applied sample. Apply a small amount sufficient to cause

Selection of the test solution or dispersion is a matter of choice and depends in part on the type of sample or test substance that will be applied to the layer during actual use.

For low molecular weight substances, aqueous dye solutions can be used, e.g.
”) can be used.

For high molecular weight substances such as proteins, an aqueous dispersion of bovine albumin stained with Thoratin Pink can be used.

After the liquid sample has been applied to the layer under evaluation and the liquid sample has disappeared from the surface of the layer and been absorbed into the layer, the test element can be turned over and the bottom surface of the expanded layer can be turned over. can be observed through the transparent support material and gelatin layer.

Substantial evaporation of the solvent or dispersion medium. If the test element exhibits a well-defined colored spot of substantially uniform color density when scanned with a densitometer having an aperture of approximately 5 μ x 100 μ prior to A uniform apparent concentration is achieved on the bottom surface of the test layer and/or on the gelatin layer.

Really. The term generally uniform density means that the density of the spot has maximum and minimum values that are less than about 20% from the average value, except at its outer periphery.

Due to the edge effect, a non-characteristic concentration gradient will occur around the spot periphery, but will have no effect on the analytical results.

No.

The outer periphery varies from spot to spot, but is generally less than about 20% of the total spot, and in reality will probably be less.

As noted herein, useful spreading or metering layers can be isotropically porous layers.

Such layers can be prepared using various ingredients.

In a first embodiment, particulate material can be used to construct the layer, where isotropic porosity is created by the interconnected spacing between the particles.

Various types of particulate materials are useful, preferably those that are chemically inert to the sample components under analysis.

For example, pigments such as titanium dioxide, barium sulfate, zinc oxide, lead oxide, etc. are preferred.

Other preferred particles are microcrystalline colloidal materials derived from diatomaceous earth and natural or synthetic polymers.

Such colloidal substances are reported in the Journal of Ap
pl ied Po-Iymer 5science 2nd
Vol. 481-489 (1967), A-Battista et al.
Macromolecular Phenome
na, Part I [.

Novel Microcrystals of Po
It is described in a paper entitled Lymersj.

Avicel” from FMC Corporation
Microcrystalline cellulose, commercially available under the registered trade name .), is an example of such a colloidal material and can be advantageously used in the present invention.

Spherical particles of uniform size, such as resin or glass peas, may also be used, with uniform porosity being particularly preferred, such as for selective filtration purposes.

If the selected particulate material is not adhesive, as in the case of glass beads, the particulate material is treated to obtain particles that can adhere to each other at points of contact, thereby facilitating the formation of an isotropic porous layer. be able to.

As an example of a suitable treatment, the non-adhesive particles can be coated with a thin adhesion layer, such as gelatin or a hydrophilic colloid solution such as polyvinyl alcohol, and brought into contact with each other in the layer.

When this colloidal coating dries, the integrity of the layer is maintained and open spaces remain between its constituent particles.

As an alternative or addition to such particulate materials, the spreading layer can be prepared using isotropically porous polymers.

The polymers can be prepared using any technique useful for constructing plush polymers.

A plush polymer layer can be formed on a substrate by dissolving the polymer in a mixture of a low-boiling liquid that is a good solvent for the polymer and a high-boiling liquid that is a non-solvent or at least a poor solvent for the polymer. can.

Such a polymer solution is then applied onto a substrate and dried under controlled conditions.

The low boiling point solvent evaporates quickly and the coating is concentrated in a liquid that is a poor solvent or a non-solvent.

When evaporation proceeds under appropriate conditions, the polymer becomes an isotropically porous layer.

Isotropic porous plush polymer used in the present invention [
A number of different polymers can be used alone or in combination to prepare the spreading layer, typical examples being polycarbonates, polyamides, polyurethanes, and cellulose esters such as cellulose acetate.

When preparing an integrated analytical element of the present invention, a plurality of layers can be individually preformed and laminated to form the entire element.

Layers prepared in this manner are typically applied as a solution or dispersion onto a surface from which the dry layer can be physically peeled off.

However, a convenient method that avoids the problem of multiple stripping and lamination steps is to apply a first layer, if desired, on a stripping surface or support, and then apply the next layer directly on these previously applied coatings. It is to apply a layer of.

Such coatings are applied manually using a blade applicator.
or can be accomplished mechanically using techniques such as dipping or bead coating.

When using mechanical coating methods, it is often possible to coat adjacent layers simultaneously using hopper coating techniques well known in the preparation of light-sensitive photographic film or paper.

If adjacent layers are discontinuous and it is not sufficient to maintain layer separation by adjusting the specific gravity of the coating composition as is possible in the case of porous spreading layers, suitable solutions for each layer, including solvents or dispersion media, may be used. By selecting suitable components, migration and solvent effects of layer components relative to each other can be minimized or reduced.

The problem of adhesion of layers to each other can be overcome without deleterious effects by means of surface treatment, including very thin applications of priming materials such as those used in photographic film.

For the reactive reagent layer, a coating solution or dispersion containing the Mad IJx and a reactive substance is prepared, coated as described above, and dried to form a dimensionally stable layer. Can be done.

The thickness and degree of penetration of the reactive reagent layer can vary widely and depend on the actual use.

Dry thicknesses of about 10 microns to about 100 microns are convenient, although widely varying thicknesses may be preferred under certain circumstances.

When relatively large amounts of reactive substances, such as polymeric substances such as enzymes, are required, it is preferable to use somewhat thicker layers of reactive reagents.

The radiation shielding layer and the detection layer can be prepared using methods and thicknesses as used for the reactive reagent layer, but
The constituent components used are those appropriate for the specific layer.

In the case of the detection layer, in addition to its permeability and radiation transparency,
Preferably, there is substantially no customization that would introduce flame or other noise that would interfere with the detection of the analytical results produced by the integrated analytical element of the present invention.

For example, changes in color or texture within the detection layer may be disadvantageous due to non-uniform reflection or transmission of the detection energy, such as occurs when a fibrous material such as paper is used as the penetration medium. be.

Furthermore, although fibrous materials such as filter paper and other papers are generally permeable throughout, some of these materials can typically exhibit a wide range of permeability, e.g. Due to structural variations such as size and spacing, such materials do not exhibit uniform permeability and are therefore not preferred for the detection layer and other layers of the analytical element of the invention.

Spreading layers can also be prepared by coating from solutions or dispersions.

As previously mentioned, the deployment and cooperating layers of the analytical element are in a superimposed relationship such that the deployment layer is in fluid contact with the reactive reagent layer.

The range of useful materials that can be included in the spreading layer can vary over a wide range, as discussed above, and generally are resistant to the liquid under analysis, i.e., substantially insoluble in the liquid and do not swell upon contact therewith. Contains mainly substances.

Swellings of about 10-40% based on the dry thickness of the layer are common.

The thickness of the spreading layer is variable and is consistent with the given sample volume that the spreading layer is supposed to be able to absorb for convenience and cleanability, and the void volume of the layer which also affects the amount of sample that can be absorbed into the layer. Depends on the department.

A spreading layer of about 50 microns to about 300 microns is particularly useful.

However, wider variations in thickness are also possible, and larger layer thicknesses may be preferred for particular analytes.

When preparing a spread layer for isotropic porous injection, it is useful to have a void volume of at least about 25% of the total volume of the layer, with 50 to 95% void volume being preferred.

Varying the void volume of a porous spreading layer can be usefully used to modify the customization of factors such as the total permeability of the spreading layer or the time required for spreading the sample.

The void volume within the layer can be controlled, for example, by selecting an appropriately sized particulate material, or by varying the solvent or drying conditions when an isotropically porous plush polymer is used in the spreading layer. It goes without saying that it can be done.

The void volume of such a layer is given by Chalkley Jou
rnal of the Nation-nal Can
cer Institute, Vol. 4, p. 47, 0943) and by direct weighing of the ratio of the actual weight of the layer to the weight of the solid material constituting the layer of the same volume as the volume of the layer. By doing this, calculations can be made with a moderate degree of ellipticity.

In any case, it goes without saying that the pore size should be large enough to allow the spread of sample components or other substances supplied to the reaction reagent layer.

As previously described herein, the integrated analytical element can be self-supporting or coated on a support.

Useful support materials include various polymeric materials such as cellulose acetate, poly(ethylene terephthalate), polycarbonate, polyvinyl compounds (eg, polystyrene), and the like.

The choice of support for a particular analytical element will be consistent with the intended mode of detection of the result.

A preferred support is radioactive radiation and about 200
a radiation transparent support material that is transparent to electromagnetic radiation at wavelengths from nm to about 900 nm.

For fluorescent detection of analytical results through a support, it is possible to transmit a slightly wider band than is necessary for non-fluorescent measurements, or alternatively to transmit in the absorption and emission spectra of the fluorophore used for detection. Preferably, the support is

This can be achieved, for example, by impregnating or coating the support with a one-plate colorant having a suitable absorption ++tq.

If the analytical element includes a support, the reactive reagent layer, the radiation shielding layer (if present) and the detection layer are interposed in the analytical element between the support and the spreading layer (if present). , the deployment layer is often the outermost layer of analysis elements.

The specific layer components and selected layer structure (or arrangement) of the integrated analytical element of the present invention will depend on the intended use of the analytical element.

As mentioned above, the pore size of the spreading layer can be selected such that the layer can screen out undesirable sample components, such as those that would interfere with the detection of analytical reactions or test results occurring within the analytical element.

For whole blood analysis, porous layers with pore sizes of 1 to about 5 microns are particularly useful for sorting blood cells, which typically have a size of about 7 to about 30 microns.

If desired, the analytical element can include multiple spreading layers, each with different spreading and filtration capabilities.

If it is necessary to limit the transport of substances within the analytical element beyond that provided by the spreading layer, a filter or dialysis layer can be included at a suitable location within the analytical element.

As an example, in the analysis of blood glucose, a dialysis layer, such as a semipermeable cellulose membrane, can prevent the passage of potential interfering substances to the protein or reaction reagent layer.

The layers of the analytical element can advantageously incorporate a monolithic surfactant, such as an anionic or neutral surfactant.

For example, these activators can increase the coverage of the layer structure and increase the extent and rate of spreading of spreading layers that are not easily wetted by liquid samples without the aid of things like surfactants. can be increased.

If desired, reactive substances can also be present in the developing layer for a particular analysis.

By way of example, proteins or other high molecular weight substances can be conveniently made more suitable for analytical reactions, such as by incorporating the substances into a spreading layer, such as enzymes such as proteases or esterases. There is also.

It can also be divided into lower molecular weight components that are more easily distributed.

It is also preferred that the layers of the analytical element contain substances that can render, by chemical reaction or otherwise, potentially harmful substances to the selected analysis inactive.

As an example, ascorbate oxidase can be incorporated into the analytical element to remove ascorbate ions that interfere with glucose analysis.

In yet other embodiments, the selection analysis requires multiple reactions;
The reaction is best accomplished in elements having multiple layers of reactive reagents, each layer suitable for enhancing or efficaciously performing a particular reaction step.

As an example, a continuous reaction can be used in the measurement of an enzyme known as serum glutamine-oxalacetate transaminase.

This enzyme converts alpha-ketoglutarate and aspartate ions to the corresponding oxal acetate and glutamate at a pH of about 7.4.

Oxal acetate is Fastponcy L (Fast
It can be determined by coupling with a diazonium salt of the dye known as Ponceau L).

To facilitate the first stage equilibrium to be established before coupling, the first stage equilibrium is established by separating the reaction reagents and incorporating each of them in a separate layer. It is preferred to provide a suitable time interval for the achievement of first stage equilibrium without being disturbed by the premature onset of the second stage reaction.

In this way, Fast Ponceau can be incorporated into a first reactive reagent layer coated onto a second reactive reagent layer containing said salt of the dye.

The integrated analytical element of the present invention is applicable to performing a wide range of chemical analyzes such as in the field of clinical chemistry as well as chemical experiments and chemical process control experiments.

Since clinical testing of body fluids such as blood, serum and urine often involves a large number of replicate tests and it is often necessary to provide test results within a short time after sample collection, the analytical element of the invention is particularly suitable for said clinical applications. ing.

For example, in the field of blood analysis, the multilayer analytical element of the invention is suitable for carrying out quantitative analysis of many commonly measured blood components.

That is, for example, the present analytical element can be used by appropriately selecting appropriate test reagents or other reactive substances.

It is easily adapted for the analysis of blood components such as albumin, birubirin, urea nitrogen, serum glucmin-oxalacetate transaminase, chloride, glucose, uric acid and alkaline phosphacus, as well as many other components.

When analyzing blood using the analytical element of the present invention, blood cells are first separated from serum by centrifugation or the like, and the serum is applied to the analytical element.

However, such separation is not necessary, especially when using reflectance spectrophotometric techniques to quantify or perform other analysis of the reaction products formed in the analytical element, and whole blood is directly transferred to the analytical element. The blood cells are separated by the action of the lachrymal layer.

Such as spectrophotometric analysis is transmitted through the support and detection layer and reflected by a radiation shielding layer or other reflective layer such that the detection radiation does not block the blood cells.

The presence of these blood cells in the analytical element does not interfere with the spectrophotometric measurements when performed by reflection techniques (G).

A particularly important feature of the integrated analytical element of the present invention is its ability to be used for either serum or whole blood analysis.

It goes without saying that a variety of different analytical elements can be prepared according to the present invention depending on the purpose of the analysis.

The analytical element can be configured in a variety of shapes, including stretched tapes, sheets or small chips of the desired width.

A particular element may be one or more tests of a single type or different types.

It is suitable for various types of tests.

For various tests of different types, it is possible to coat a common support with - or more strips or channels, which can be of different compositions, constituting composite elements suitable for carrying out the various desired tests. preferable.

Preferred embodiments of the analytical element of the present invention are as illustrated in the accompanying drawings.

The analytical element shown in FIG.
, a detection layer 12 carried thereon, a radiation-shielding layer 14 which can serve as a filter as well as providing a white background for the detection of analytical results, such as by reflection spectrophotometry, and a reactive reagent layer 16. .

Detection can be accomplished through a support that is moderately transparent to the detection wavelength.

Detection layer 12 can be a hydrophilic colloid such as gelatin.

Although the reactive reagent layer 16 may consist of a solution or dispersion of one or more test reagents in a binder such as gelatin, the layer 14 may have an isotropic porosity and/or a desired filtration effect. It can be a plush polymer with a pore size.

These layers are in fluid contact.

In FIG. 2, which shows another embodiment of the analytical element of the present invention, the analytical element has a spreading layer 26 and a reactive reagent layer 24 which are free from overeffecting and also provide a suitable reflective background for reflection spectrophotometric detection through the support 20. A radiation transparent support 20 having a detection layer 22 in fluid contact with the radiation transparent support 20 .

Also, layer 26 can be such that it is not reflective and detection can be done in a transmission mode.

Layer 26 can be, for example, a single layer of isotropically perforated plush polymer coated or laminated onto layer 24.

FIG. 3 shows yet another embodiment of the analytical element of the invention, which comprises a support 30, a detection layer 32, and a pigment such as titanium dioxide in a hydrophilic colloid such as gelatin. a radiation shielding layer 34 which can be composed of dispersed radiation;
It consists of a reactive reagent layer 36 and a spreading layer 38, such as a single layer of isotropic porous plush polymer capable of spreading and filtering functions.

These various layers are brought into fluid contact.

In FIG. 4 showing still another embodiment of the analytical element of the present invention, the analytical element of the present invention includes a support 40, a detection layer 42, a radiation shielding/filtration layer 44, a reactive reagent layer A46, and a reactive reagent. It is composed of a layer B 48 and a developing/filtering layer 50.

Layer 44 can be constructed, for example, by dispersing titanium dioxide in plush cellulose acetate, and layer 5
0 can be constructed by dispersing diatomaceous earth in plush cellulose acetate, or by adhering glass beads to each other using a hydrophilic colloid such as gelatin.

The analytical element of the present invention is used by applying an analytical liquid sample to the analytical element.

Typically, the analytical element is configured such that the applied sample contacts the spreading layer before the non-spreading reactive reagent layer and first contacts the surface of the developing layer remote from the reactive reagent layer. Ru.

The analytical ellipticity of the present invention is applied to the volume of the sample, especially when a spreading layer is present in the element.

Manual or mechanical sample application is possible as it is virtually unaffected by fluctuations.

However, for convenience of detection of analytical results, a reasonable match in sample volumes may be preferred.

As mentioned above, when a soluble reactive substance is used in the reaction reagent layer, the spreading layer is also highly preferred for minimizing the occurrence of ring formation.

In a typical analysis method using an analytical element of the present invention, which can be manual or automatic, the analytical element is taken from a supply roll, chip packet, or other source, such as a suitable diode.

Positioned to receive free drop, contact spot, or other form of liquid sample from the dispenser.

After application of the sample, and preferably after absorption of the liquid sample by the spreading layer, if present, the analytical element is preferably used to speed up or facilitate removal of the test result. Exposure to arbitrary conditions, such as heating, temperature supply, and other conditions.

In the case of automated analysis, it is preferable that the spreading layer performs its function in a few seconds, as opposed to the almost instantaneous uncontrolled diffusion that is obtained with absorbent fibrous papers (although it is preferable to provide metering). (It is preferable to leave it for a sufficient period of time.)

This can be conveniently achieved by appropriate selection of various parameters such as layer thickness, void volume of the porous layer, etc.

After the analytical result has been obtained as a detectable change, it is usually measured by passing the analytical element through a zone equipped with suitable equipment for reflection, transmission or fluorescence spectrophotometry.

Such devices work by directing an energetic beam, such as light, through the support and the detection layer.

Next, light, for example. It is either reflected from the radiation shielding layer in the analysis element to the detection means or, in the case of transmission detection, transmitted through the analysis element to the detector.

In a preferred manner, the analysis result is detected entirely within the area in which the analysis result occurred.

The use of reflection spectrophotometry is advantageous in this case, since it effectively avoids the interference of residues, such as blood cells, remaining on or in the layers of the analytical element.

When the detectable substance is a fluorescent substance, a general method of fluorescence spectrophotometry can also be used.

Detection is accomplished by using energy to excite the fluorescent substance and a detector sensitive to its fluorescent emission.

Additionally, permeation techniques can be used when testing serum or when means are provided to remove undesirable whole blood debris.
For example, U, V, visible or 1. R. Used to detect and quantify indicated reaction products by applying a stream of radiant energy, such as radiation, to one surface of an analytical element and measuring the delivery of that energy at the reactive surface of the analytical element. I can do it.

Although any radiation that can penetrate the analytical element and quantify the products formed in the reaction reagent layer can be used, electromagnetic radiation in the range of about 200 to about 900 nm is generally useful for such measurements. I discovered that.

Various assay techniques can be used to control the analysis.

As an example, a sample of the standard test solution can be applied adjacent to where the sample droplet was placed to allow the use of differential measurements in the analysis.

Examples are presented below to further explain the invention.

Example 1 The following layers were applied in succession on a poly(ethylene terephthalate) support (180μ thick) with a gelatin undercoat.

(1) 2.15 El of gelatin per square meter;
2.15g mordant (styrene and N,N-dimethy/L/
2) a porous, reflective radiation-shielding layer containing 151 g of gelatin and 11.4 g of titanium dioxide per square meter; , (3) 17.5g gelatin per square meter, 1
,! l potassium salt of 1-naphthol-2-sulfonic acid, 0.73 g disodium phosphate buffer, 0.459
potassium phosphate buffer, 0.38 g 4-aminoantipyrine (HCl), 1.6 g glycerin (plasticizer)
, 0.01 peroxidase (14014 U/ml)
and 0.374.9 glucose oxidase (404
(4) a spreading layer containing 97 g of cellulose acetate-I and 65.5 g of titanium dioxide per square meter.

The analytical element prepared in this way was
It was used to analyze glucose solutions with varying concentrations in dl.

A drop of 10 μl of aqueous glucose solution was deposited onto the sample of the analytical element.

After 1 hour, the density of the color was measured using a Matthies densitometer (Ma
Measurements were made by reflection using a Ceeth Densitometer (Model TD-504).

When a glucose solution is applied to the surface of the analytical element, it spreads into layer 4 and is metered into layer 3, where glucose reacts with oxygen and water in the presence of glucose oxidase to form gluconic acid and hydrogen peroxide. occurs.

These compounds, in the presence of peroxidase, undergo 4-
reacts with aminoantipyrine, then oxidizes, and this 4
-The oxidation product of aminoantipyrine is 1-naphthol-
Coupling reaction with 2-sulfonic acid potassium salt to form a dye.

The dye diffuses from the reagent layer 3 through the radiation shielding layer 2 and into the detection layer 1 where it is detected using a Macys TD-504 Densitomake.

The results are shown in the table of Example 2 below.

Example 2 An analytical element was prepared with the same structure as that prepared in Example 1, except that detection layer 1 contained gelatin as the only component with a coating weight of 4.3 og/rrt.

This analytical element was used according to the method described in Example 1.

The results obtained are shown in the table below. Although the present invention has been described above in detail with particular reference to its preferred embodiments, it goes without saying that various modifications and alterations can be made within the spirit and scope of the present invention.

[Brief explanation of drawings]

1, 2, 3, and 4 are enlarged cross-sectional views showing preferred embodiments of the integrated analysis element of the present invention. 10.20, 30, 40...Support: 12゜2
2.32,42...Detection layer: 14,34,44
...Radiation shielding layer: 16, 24, 36, 46° 48 ... Reaction reagent layer: 26, 38, 50...
...Development layer.

Claims (1)

Claims: 1. An integrated liquid analytical element comprising a radiation transparent support carrying in fluid contact a series of layers including at least one reactive reagent layer, comprising: (a) said reactive reagent layer; (b) the element comprises a reactive substance that is permeable to the test substance or its precursor and that produces a diffusible detectable substance in the presence of the test substance; Between,
(c) the element is permeable to the detectable substance and includes a detection layer capable of detecting the detectable substance; and (c) the element is permeable to the detectable substance and transfers the detectable substance to the detection layer. What is claimed is: 1. An integrated liquid analysis element characterized by comprising a radiation shielding layer located between the detection layer and the reaction reagent layer, which prevents interfering substances from migrating to the detection layer.