JPS5819062B2 - Integrated analysis element - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION This invention relates to analytical chemistry, and more particularly to an improved multilayer analytical element used to analyze body fluids for specific components.

More specifically, this invention provides blood urea nitrogen (BU
N) Concerning elements useful for analyzing serum for blood content.

In recent years, a number of automated systems have been developed to perform chemical analysis of body fluid samples.

These systems have proven particularly advantageous in clinical laboratories, especially for the quantitative analysis of blood.

Continuous flow analysis, which consists of mixing the sample, diluent and test reagents together and then guiding the resulting mixture through an analytical device, is now very widely used. .

However, these analytical devices, for example, U.S. Patent No.
The analytical device described in No. 797149 is complex and expensive, requires technically sophisticated operation, requires a large amount of time and effort in repeated cleaning operations, and furthermore, This makes it impossible to use very small sample volumes, such as those used in microanalytical methods.

Efforts have been made to overcome these drawbacks, and various automated mechanical analysis devices have been proposed.

All of these devices utilize a continuous analytical tape on which the sample to be analyzed is deposited, and when using such devices, the components of the sample to be analyzed and the applied Quantitative analysis can be accomplished by spectroscopically measuring the color reaction between the tape and the test reagent carried by the tape.

Analyzers of this type are described in detail in a number of patents, including UK Patent No. 1,049,364 and US Pat. No. 3,036,893 and US Pat.

Analyzers utilizing continuous analytical tape are inherently much simpler than continuous flow analyzers.

However, the analytical tape analyzers described in patents such as those mentioned above have a number of notable drawbacks.
These drawbacks also hinder its industrial development.

Therefore, no test reagents are mixed within the tape itself and the tape is tested, for example in British Patent No. 10499
As described in certain embodiments of No. 64, when the sample and test reagents are used solely as a means of transporting the sample to be analyzed into the system, the sample and test reagents are each delivered at appropriate times and extreme pressures. It is absolutely necessary to apply it to the tape.

For example, US Patent No. 3,036,893 and British Patent No. 10
The use of separate tapes to accomplish various functions, such as sample filtration and reaction of the sample with Test 1 reagents, as described in certain other embodiments of No. 49364. is the cause of greatly increasing the complexity of the system.

Also, this makes it impossible to fully reproduce the simplicity characteristic of continuous tape analysis methods.

Furthermore, as described in U.S. Pat. No. 3,526,480, for example, the use of expensive analytical tapes that have complex properties and are difficult to manufacture is difficult to achieve in order to achieve an industrially practical system. This has become a serious obstacle.

In addition, other "dry" systems have also been developed to perform quantitative or semi-quantitative measurements of blood components.

These systems are primarily based on absorbent carrier materials such as paper or porous hydrophilic polymer layers (which may also include dialysis materials to remove unwanted blood components such as red blood cells by filtration). (which may or may not include the overcoat 1).

In particular, blood urea nitrogen (hereinafter abbreviated as BUN)
In this case, the urease is absorbed into the absorbent carrier on which the dialysis membrane is overcoated (US Pat. No. 3,145,086, US Pat. No. 3,249,513, US Pat. issue).

Also, according to what is taught in these patents, the ammonia released by the reaction of urea with oxygen in the presence of urease has the effect of initiating a color change in the absorbed pH indicator. .

However, the inherent utility of these systems is adversely affected by the presence of other basic materials, which have a spurious effect on the indicator.

The dialysis membrane, if used, is the only macrofilter located as the outermost shell layer.

Although it is disclosed that the barrier bands are selectable, these bands are used to longitudinally divide the test tube to obtain multiple regions with different sensitivities.

Furthermore, these barriers do not have a function that allows selective migration to the internal intermediate layer.

The disclosure of U.S. Pat.
It shows.

However, as disclosed in this patent, this barrier is a barrier to all substances and ammonia is released in a closed container by a gas discharge zone for atmospheric diffusion to the indicator zone. .

Other systems have also been developed based on urease reactions.

However, it is in dogs that these systems are involved in a variety of fluid reactions.

Representative patents demonstrating such an approach are, for example, US Pat. No. 3,409,508 and US Pat. No. 3,485,723.

Other patents that relate only to the general background of BUN analysis are, for example, U.S. Pat. No. 3,718,433, U.S. Pat.

US Pat. No. 3,723,064 proposes a multilayer test element for use in clinical analysis, in which two reagent layers are separated by an intermediate layer.

Some of these layers may contain varying concentrations of chemical traps that possess particular effects on the final product being tested.

The barrier may have an intermediate layer in the form of multiple vertically extending barrier compositions (eg, cellulose acetate) to control the concentration gradient of the fluid flowing through the intermediate layer.

These barriers flow from one reagent layer to another along the normal flow direction and therefore do not have the effect of suppressing interfering or undesired components.

Additionally, the concentrations of these barriers vary within the sample 1intercept area of the element.

US Pat. No. 3,901,657 describes a single layer of filter paper that acts as a physical separation layer between a reagent-containing layer and an indicator layer.

According to this specification, the liquid sample and all its components are forced to pass through this filter paper.

Also, regarding this paper layer, there is no suggestion in the specification that the paper layer should selectively allow only a portion of the sample used or a portion of the decomposition products of the analytical reaction to pass through. .

A recent approach to the problem of blood analysis is Belgian patent no.
No. 01742.

(This current approach) comprises a reagent layer, an isotropic porous diffusion layer, and additional layers that may include a dialysis layer, a filter layer, a reflective layer or any combination of these layers. The described elements are used to generate a detectable variable energy, such as a quantifiable shift in energy absorption or transfer, in response to the presence of an analyte in a test sample.

The reagent layer, like the additional layer, is in fluid contact with the diffusion layer, so that the component to be analyzed or its precursor or its reaction product will arrive at the reagent layer.

In this Belgian patent, one or more filter layers or dialysis layers proposed in the specification exclude the passage of filterable material present in the sample and at the same time sample components that are not trapped in the filter layer. It is stated that it has the function of passing through.

Such a filter layer or dialysis layer has also been considered to be directly or indirectly affected by substances (excluding potentially interfering substances) contained in the reagent layer located in front of the filter in the normal flow of fluid. It is not stated that there is a special relationship between these layers and the selective passage of decomposition products produced by the process.

Enzyme electrodes, which use semipermeable membranes that are selectively permeable only to selected substances, potentiometrically detect the enzyme layer and the degradation products produced by the enzyme, such as ammonia produced from urea in the presence of urease. It is used in conjunction with electrode means for recording.

Such a device is described, for example, in US Pat. No. 383'8033.
No. 3,896,008.

One object of this invention is to provide an improved dry method for complex fluid analysis of analytes that is protected from the intervention of undesired selectively reactive substances that have falsified readings. )'' is to provide analysis elements.

Another object of the invention is to provide such an element which allows quantitative analysis of blood components such as urea nitrogen.

In accordance with the present invention, an improved integrated analytical element for evaluating and analyzing analytes contained in a liquid sample is provided.

According to the invention, there is provided an integrated analytical element for the analysis of a liquid sample for a predetermined analyte, comprising the following reagents: (1) interacting with said analyte in the presence of said analyte; (2) a second reagent capable of reacting with the decomposition product or its final product to produce a detectable change. and the first and second reagents are separated by a barrier, and the barrier is substantially uniformly permeable to the vaporous decomposition products and free of liquid water and the interfering material. An integrated analytical element is provided which is characterized in that it is substantially impermeable.

Such elements are particularly useful in the analysis of BUN, and the enzyme urease contained in this element converts urea into NH
Converted to 3.

A problem with any analytical element that relies on the reaction of a reagent with an analyte is that the reagent has a certain tendency to induce undesired reactions. be.

Reagents can produce undesired and detectable changes by reacting with by-products produced by the reaction of the reagent with the analyte or by reacting with other components of the sample to be tested. obtain.

If such a situation actually occurred,
Errors will be introduced into the detection (results).

Hereinafter referred to as "interferences" are such reaction by-products or other liquid sample components that must be excluded from the reagents resulting in a detectable change in order to achieve quantitative results. Should.

According to the present invention, a novel integrated analytical element is provided which solves the above-mentioned problems by eliminating all interfering substances contained therein from the detection reagent.

The analytical element of the invention is simple in construction, easy to manufacture at reasonable cost, and made so that the analysis can be carried out according to a simple and effective technique.

The analytical element may be available in the form of a continuous strip or tape, or may be available in other forms, such as sheets or short strips, or pieces or chips.

The analytical element may or may not be mounted on a perforated card or other holding member.

All such forms of elements of the invention are considered equivalent and within the scope of the invention.

Furthermore, although a particularly preferred form of such elements is a multilayer element, the invention is not limited thereto.

Rather, the invention is directed to the separation of the two reagents (more on this below) by a barrier having the desired osmotic properties within the test period required for any given test chosen. (explained).

Additionally, as used herein, the terms ``substantially penetrable'' or ``permeable'' mean that substantially all of the decomposition products applied to the barrier material are removed from the barrier within the test period. It means actually passing through the inside of the object.

Quantitative analysis of specific components in a sample, for example the analysis of urea nitrogen in blood, can be done using a conventional spectrophotometer or by other techniques, i.e. by sensing the radiant energy formed in the analytical reaction. This can be easily achieved by using other techniques that can quickly and accurately measure the amount of product that can be produced.

As used herein, the term "product or seed material capable of detecting radiant energy" (5pe
The term J refers to a product or seed material that alters the radiant energy transmission or reflection properties of the element, either by formation or loss of fluorescence or other characteristics.

Furthermore, when used in this specification, the term ``decomposition'' includes treatments such as dissociation, dehydration, and hydrolysis, but the meaning of ``decomposition'' is not limited to these treatments.

The term "final product" means a substance formed by the reaction of the product of a decomposition reaction with one or more other substances (formulated in the component, e.g. reagents). There is.

As used herein, the term "reagent" means a substance that interacts with an analyte, a precursor of the analyte, a degradation product, or a final product.

Such interaction may be associated with chemical activity, catalytic activity in the formation of enzyme-substance complexes, or some other form of chemical or physical reaction.

For example, in the case of the chemical or physical reactions mentioned above, the ultimate result is that a change occurs within the element that can be detected by appropriate measurements of radiant energy, usually in the form of light energy. can.

The disclosure below is directed to particularly effective conjugates (i.e., the reagents included in the conjugate are an enzyme that forms a degradation product and an indicator that records a detectable change in response to the degradation product). However, other reagents, ie, other reagents, can also be used within the scope of the above definitions.

The element is preferably laminated in construction, so that when the element is viewed from the side in cross-section, a number of superimposed layers can be seen.

Such a structure allows the droplet-shaped sample to absorb gravity, capillary effects,
It allows diffusion from the outermost shell layer to one or more inner layers under the influence of wicking or the like.

Each layer of the element has a sample section area.

As used herein in reference to a single layer, the term "sample section zone" refers to the area in which any decomposition products of the applied sample or one of the components comprising the sample would normally move within the element. If an arbitrarily given element cross section (
refers to the total surface area of the layer that is made to be wetted by the sample or decomposition products (transverse to the direction of fluid flow).

In order to achieve a uniform effect within the layer, each component is preferably uniformly distributed within each sample section area of the respective layer.

Such structural uniformity is important in order to obtain quantitative measurements when measuring the results formed in the element by means of a radiometer, such as a reflectometer, for example.

As used herein, the term "uniform" is used when it is used to describe the structure of the layers making up the element; This means that it would contain substantially the same concentration of substance as some other similar volume.

Preferably, in forming the layer in question, it has a uniform thickness and a uniform amount of the reagent or substance (the barrier is composed of this reagent or substance throughout its thickness). By containing the above-mentioned uniformity, it is possible to achieve the above-mentioned uniformity.

In order to ensure that undesirable substances are excluded from the sample section area of the indicator layer during the test period, the sample section area of the barrier material in contact with the reagent layer previously brought into contact with the fluid is Advantageously, it is at least substantially coextensive with that of the contacting reagent layer.

Of course, it is also possible that the sample section area of the barrier is not of the same extent as before, but has a considerably larger extent.

In one preferred embodiment, the element is therefore provided with a support, overcoated on one side of the support, or integrally formed on one side of the support, and at least one indicator, barrier agent, etc. , a plurality of layers having an enzyme-containing layer, and a dispersion layer superposed above these layers for dispersing and metering the sample applied to the element.

Depending on the type of evaluation analysis to be carried out, one or more enzymes can be incorporated into the enzyme layer.

The accompanying drawings serve to explain the invention in more detail.

Each layer depicted in these figures represents the entire sample section area.

Therefore, the section area of each layer has the same size as that of the layer adjacent to it.

It can be seen from these figures that the respective layers can also be extended further beyond the illustrated sample section area, and that such extensions contain liquid sample, analyte and/or one or more It will be appreciated that the presence of reagents or barriers is not required as long as the decomposition products do not contact or penetrate into such extensions.

FIG. 1 shows one embodiment of the elements of the invention, in which element 10 includes a support 12, an indicator layer 14, a barrier 16, an enzyme layer 18, and an element to which a sample is applied. It is configured to include a scattering layer 20 defining a surface.

Although layer 20 is also described in detail below,
Other desirable functions may also be exhibited, such as filtration and/or reflection.

This layer 20 is separate and distinct from layer 18.

FIGS. 2 through 5 illustrate alternative embodiments of the elements of the invention, each of which has various rearrangements of its layers.

In FIG. 1, the same parts as already described have been given the same reference numerals, but the suffixes ua11 to d11 have been added to these numbers for further distinction.

Therefore, in FIG. 2, element 10. As described above, the layer a includes the support 12, the spray layer 20a, and the enzyme layer 18a.

However, rather than being placed as a fourth layer between the two layers containing the enzyme and indicator as in the previous embodiment, the barrier is incorporated into one bonding layer 30 with the indicator mixed therein. is formed.

In FIG. 3, element 10b includes a support 12b, an indicator layer 14, and a barrier 16b as in FIG. 1.

However, the enzyme layer 18a is mixed into the dispersed layer to form the binding layer 40.

In FIG. 4, element 10c is constructed with support 12c and only two further layers 30c and 40c, each of which corresponds to the barrier material of FIG.
It includes an indicator layer, and the scattering-enzyme layer of FIG.

In FIG. 5, element 10d includes a support 12d and a spreading layer 20d as in FIG. 1, and further includes an intermediate layer 50. In FIG.

Although the intermediate layer 50 includes the barrier composition throughout, both surface portions of the intermediate layer also include additional portions.

That is, there are two parts: a part 14d adjacent to the layer 12d containing an indicator for detecting decomposition products, and a part 18d adjacent to the sprayed layer 20d containing an enzyme.

The presence of central portion 52 (which does not contain both enzyme and indicator) is optional provided that portions 14d and 18d do not overlap each other.

In the embodiment shown in this drawing. Barriers also serve as binding agents for enzymes and indicators.

It will of course be readily appreciated that this embodiment is only suitable for certain tests, ie those in which the analyte as well as the degradation products formed therefrom are able to penetrate the barrier.

Also, if the analyte does not penetrate the barrier,
A portion of the enzyme must be included in layer 20d, as indicated by section 54 in FIG.

As is clear from the above description, no matter which aspect is used, certain functionality must necessarily be provided.

Namely: 1. The sample applied to the element is preferably uniformly distributed over the element by means of a spreading layer.

2. The selected analytes are preferably degraded by enzymes.

3. Decomposition products selectively permeate the indicator through the barrier.

4. An indication of the amount of degradation products is made as an indication of the amount of analyte present in the applied sample.

Preferably, the indicator is in the form of a dye precursor or a bleachable dye.

Each of these functions, as well as the preferred means thereof, will be discussed in detail in the following description, each time the function is explained.

In order to achieve a uniform concentration at the surface of the spreading layer which is in fluid contact with the adjacent layer(s), the sample applied to the element is first preferably coated with a coating material that performs this function. uniformly distributed by one or more layers.

This sample distribution layer included in a multilayer analytical element is generally the layer onto which the liquid sample to be analyzed, for example blood in general or serum, is deposited.

As used herein, "fluid contact"
What is meant by the term is such contact as to permit the passage or transport of a liquid or one or more of its components (liquid or gas) between the layers.

A sample in which one or more substances containing at least one component of a liquid sample applied to the element can be distributed or metered in a layer, as disclosed in the aforementioned Belgian Patent No. 801,742. A scattering layer can be provided.

Therefore, at any given time, it is facing the enzyme layer.

In other words, it is possible to achieve a homogeneous concentration of such substances in the surface area of the sprayed layer which is located close to it.

In this case it is unnecessary to limit the applied samples.

As will be appreciated, such concentrations, while immediately uniform, can be varied over a period of time without deleterious effects.

A concentration gradient may exist within the layer from top to bottom.

In the process of discussing a number of preferred embodiments, reference was made to ``isotropic porosity,'' which attests to the fact that there is substantial porosity in all directions within the dispersed layer. .

It will also be appreciated that the degree of porosity can be varied as needed or desired, for example with respect to pore size, porosity, etc.

The term ``isotropically porous'' (or isotropically porous) is used herein to describe a tear film that has continuous air bubbles between its surfaces. It will be understood that the term "imporous" or "ionotropic" is not to be confused with the terms "imporous" or "ionotropic," which are often used in reference to the lachrymal film of the human eye.

Similarly, the term ``isotropic porosity'' should not be confused with the term ``isotropic,'' which is used in contrast to the term ``anisotropic.'' The term refers to a membrane that has a thin "skin layer" along at least one side of the membrane).

For details regarding such terms, see, for example, Membra
-ne 5science and Technology
gy, James Fl inn ed, Plenum
Press, New York (1970).

As is clear, the degree of sparging depends in part on the volume of liquid to be sparged.

but. It can be emphasized that the uniform consistency achieved by sparging is substantially independent of the volume of the liquid sample and will generally occur as a function of varying the extent of sparging.

As a result, elements of the invention do not require precise sample application techniques,6 although it may be desirable to use specific liquid sample volumes to achieve preferred application times, etc.

The elements of the invention are capable of achieving quantitative results using very small sample volumes such that the entire volume can be absorbed within a scattering layer area having an advantageous size, e.g. 1 square centimeter. Therefore, it is unnecessary to remove excess moisture from the element after applying the liquid sample.

Furthermore, while the sparging takes place in the sparge layer and the sparged material is transported to the enzyme layer, an apparent substantial latent hydrostatic pressure is also unnecessary in this case, so that known analytical elements can be used. “Ringing” is often observed when using soluble reagents.
The problem is non-existence.

It is only necessary that the dusting layer forms a uniform concentration of dusting substance per unit area in its surface area facing the enzyme layer (with which it is in fluid contact). be.

Convenient methods for determining whether a particular layer may be suitable for application purposes include densitometry or other analytical methods.

Such methods include, for example, scanning an appropriate surface or reagent layer or other layer associated therewith to determine the apparent concentration of the sprayed material or any reaction products based on the concentration of this sprayed material. This can be achieved by

The test method described below is for illustrative purposes only.

Therefore, the selection of materials or test parameters described herein does not express or imply that other materials or test parameters cannot be considered suitable for similar purposes.

In conducting such tests, approximately 2 layers of clear gelatin are applied onto a clear photographic film support material, such as poly(ethylenetetraphthalate) with a subbing coating.
A gelatin coverage of 0.00 m9/dm2 can be applied.

Although gelatin may have different hardnesses, taking into account the purpose of the test, gelatin should harden to increase its layer thickness by about 300% when immersed in water at 22°C for 5 minutes. A layer of gelatin is suitable.

When dry, the gelatin layer will have a thickness of about 30 microns.

The layer to be evaluated for spreading purposes can be applied onto the gelatin layer by coating in the form of a solution or dispersion.

For the purposes of this test, a dry thickness of about 100 to 200 microns for the layer to be evaluated is suitable.

After drying these layers, a sample of the test solution or dispersion can be applied to the surface of the sprinkled layer to be evaluated.

The application amount of this sample is preferably small in order to prevent the entire layer from being wetted by the applied sample, but the wetting area can include an annular portion with a diameter of about 8-10 mm. in a desirable amount sufficient to form .

Choosing the test solution or dispersion is a matter of voluntary choice and depends, in part, on the sample or analyte (to which the layer will be exposed under actual conditions of use). It will depend on the type.

For example, in the case of low molecular weight materials, aqueous solutions of dyes can be used.

Taking 5olati-ne Pink■ as an example, a 0.0005% by weight solution thereof can be tolerated.

For relatively high molecular weight materials such as proteins, an aqueous dispersion of bovine albumin colored 5olatine Pink"'?C can be used.

After applying the liquid sample to the layer to be evaluated and allowing the sample to disappear from the surface of the layer and absorb into the interior of the layer, the test element can be turned over and The bottom surface of the proposed scattering layer can be observed through the transparent support material and gelatin layer.

Substantial evaporation of the solvent or dispersion medium is performed to ensure that if a previously colored spot is scanned by a densitometer having an annular aperture with a diameter of approximately 2-3 mm, the spot exhibits a substantially uniform color density. If so, continue spraying.

This makes it possible to achieve a uniform apparent concentration at the bottom of the test layer and/or at the gelatin layer.

This test layer is useful as a dispersion layer in analytical elements of the type described herein.

Although the term ``substantially uniform density'' as used herein refers to the density of the spot as a whole, the periphery of this spot may vary by ±10 to 15% from the average density.
It has maximum and minimum values within %.

Edge effects may cause featureless concentration gradients in the periphery of the spot, but it is necessary that these gradients do not affect the significance of the analytical results.

Although the periphery can vary between spots,
The kneading will normally not exceed about 20% of the total spot.

The periphery may be less than about 20 squares.

Such a scattering layer can be prepared using a variety of ingredients.

In one preferred embodiment, this layer is non-fibrous.

One perspective is the use of particulate materials to form such layers, i.e., layers in which isotropic porosity is provided by open-cell voids between the particles. I can do it.

Various types of such particulate materials are useful, and all are desirable as long as they exhibit chemical inertness to the sample components under analysis.

Pigments such as titanium dioxide, barium sulfate, zinc oxide,
Preferably lead oxide.

Other desirable particles are diatoms and fine quality colloidal materials derived from natural or synthetic polymers.

Such fine-quality materials can be found, for example, in the Journ.
alof Applied Polymer 5cie
nce Vol It.

0 published on pages 481-498 (1967)
,A.

Battista et al.’s paper” Co11oidal
Macromolecule-ular Phenomen
a, Part II, Novel Microcrys
tals of Polymers”.

Fine quality cellulose is an example of such a colloidal material and is sufficient for use in this invention.

It is also possible to use spherical particles with uniform dimensions, e.g. resin beads or glass beads, and where uniform air bubbles are advantageous, e.g. stiff beads for selective filtration purposes. is particularly preferred.

If the particulate material selected is not sticky, as is the case with glass peas or similar materials, the material may be treated to produce particles that can stick to each other at points of contact. Obtainable.

Moreover, this can further promote the formation of an isotropic porous layer.

An example of a suitable treatment is to apply a thin adhesive layer to the surface of non-adhesive particles, for example a solution of gelatin or a hydrocolloid such as polyvinyl alcohol, and to connect this non-adhesive surface with the coating. can be brought into contact with each other within the layer.

When the colloidal coating dries, the integrity of the layer is maintained and open cells remain between its component particles.

As an option, or in addition to such particulate materials, uniformly porous polymers can be used to prepare the scattering layer.

Such polymers can be prepared using techniques effective for forming side-bud "brushing" polymers.

The "plunging" polymer layer can also be formed by dissolving the polymer in a mixture of two liquids and applying the resulting solution onto the substrate.

Here, one of the two liquids is a good low-boiling solvent for the polymer, and the other is of the high-boiling type and is a non-solvent or at least a poor solvent for the polymer.

Subsequently, such polymers are applied onto a substrate and dried under controlled conditions.

Since lower boiling point solvents evaporate more quickly, the formed coating can remain concentrated in a liquid (poor solvent or non-solvent).

Under appropriate conditions, the polymer forms an isotropically porous layer during evaporation.

Isotropically porous ゛′ for use in this invention
A number of different polymers can be used alone or in combination in preparing the brushing polymer scattering layer.

Common such polymers include, for example, polycarbonates, polyamides, polyurethanes and cellulose esters, such as cellulose acetate.

Additionally, one or more reflective layers (which may optionally be used for detection) within the element to facilitate sensing according to reflective radiometry, e.g. reflectance photometry/methods or similar techniques. It is preferable to include some radiation (which may absorb radiation).

Such reflection can be effected, for example, by a layer which also acts as a diffusion layer, or by an additional layer which may otherwise have no additional function inside the element.

Pigments such as titanium dioxide and barium sulfate have reflective properties and can therefore be used advantageously in reflective layers.

Also, brushing polymers may constitute suitable reflective materials.

As can be seen, pigment-spread layers are very effective for this purpose.

In one preferred embodiment, pigments can also be incorporated into the brushing polymer layer to enhance the dispersion and/or reflection capabilities.

The amount of pigment that can be incorporated into the layer together with the brushing polymer can vary, e.g.
An amount of about 1 to about 10 parts by weight per part of brushing polymer is preferred, and an amount of about 3 to about 6 parts by weight per part of brushing polymer is most preferred.

Problems with adhesion of interlayers can be overcome without deleterious effects by applying surface treatment methods, including very thin applications of priming materials, such as those used in photographic film. .

Preferably, the sample is distributed to the appropriate enzymes by a sparge layer as described above to degrade the analyte and yield a product useful for detection.

The enzyme can be applied in the form of a separate enzyme layer or can be incorporated into a dispersed layer as described above.

The details of the enzyme, such as what kind of enzyme should be used, depend on the method of evaluation analysis.Urease is a particularly effective enzyme when analyzing BUN to generate ammonia, and the enzyme layer is preferably: About 3.0 to about 30.0? /m'
a coating amount of binder, an appropriate amount of enzyme, and a buffer system to maintain the pH value.

Enzymes generally only operate effectively within a relatively narrow pH range.

For this purpose, taking a normal case as an example, the effective pH of the enzyme is
It is necessary to buffer the enzyme-containing layer at certain pH values within a range.

However, such treatments are complex and must be balanced against the pH requirements of the desired decomposition products in equilibrium.

Therefore, although it is possible to detect some enzyme activities outside the ranges mentioned above, buffering the enzyme layer containing urease to bring its pH value to about 5-9.5, preferably about It is desirable to set it as 7.5-9.0.

Means for achieving this type of buffering effect are well known in the art and include, for example, dissolving or dispersing buffering agents in the dope prior to coating.

A suitable buffer for buffering to the above pH values is found in Good's 'Bioche-mistry'.
5,467 (1967).

Particularly effective buffers include phosphates, such as potassium phosphate, so-called 'Tris and Hepes' buffer A.
Contains IJ and dimethylglutarate.

The enzyme layer can also contain other additional compounds, such as dithiothreitol, an enzyme activator, at a coverage of about 0.5-5.0/m', as well as more detailed below. Coating aids such as those mentioned may be included.

The activators used herein have also been empirically shown to enhance the catalytic activity of enzymes according to some mechanisms, although not fully understood.

Furthermore, the enzyme layer can include compounds that together form complexes with heavy metal ions (which would otherwise suppress the activity of the enzyme).

The effective concentration of such complexing agents is 150~/m''~5
The range is from 00 to /m'.

In a particularly effective UN phase splitting embodiment, the enzyme layer is therefore coated with a binder, such as gelatin, agarose, polyvinyl alcohol, etc. from about 4000 U/m2 to about 500 Qo
U/m”, preferably about 10000 U/m2 to about 3
0000 U/m'' of urea, EDTAj Basin 2 as a metal ion complexing agent, and an orthophosphate activator.

An embodiment of this invention also exists after the formation of decomposition products by the catalytic effect of the enzymes described above.

That is, isolating the indicator from the interferent during the test period by preventing the interferent from accessing the indicator.

This objective can be achieved by a selectively permeable barrier that separates the enzyme reagent from the indicator reagent.

As used herein, the term ``barrier separating reagents'' refers to a barrier that physically separates the enzyme reagent from the indicator reagent.

Preferably, this barrier is in the form of a single layer between or spaced apart from separate adjacent layers containing reagents in the sample section area, for example as described above. Alternatively, the enzyme is dispersed or distributed in the layer or layer containing the enzyme and/or in the layer containing the indicator.

Such a barrier has the function of isolating the indicator, which forms a substance capable of detecting radiant energy (described in more detail below), from undesired obstructions.

This barrier is also commonly used in filter layers (all of these layers are limited in their choice of materials).

should not be confused with (not effective for excluding).

The barrier of this invention has uniform impermeability to obstructions.

It should be noted that, as used herein, the term ``uniform impermeable'' is present in or formed from a sample.

To the extent that some interference is excluded from the indicator while carrying out the analysis in question, i.e.
Impermeable or substantially impermeable to the same extent as across the sample section area.

Also, say here. However, the period of analysis changes each time depending on the analyte and the reagent used for evaluation analysis.

Such a barrier material provided according to one preferred embodiment of the element of the invention is such that the barrier material itself is homogeneous and coexists with at least the sectioned area of the enzyme layer.

The sample is in the form of a single layer with a section area.

Therefore, this barrier can be limited to a separate layer in contact with or intermediate the enzyme-containing layer and the indicator-containing layer, or else it can The barrier material may be dispersed in all or part of one of the two.

In some cases, enzymes and indicators may be incorporated into the surface portion of the layer formed by the barrier.

In this manner, any central portion containing a barrier substantially free of either indicator or enzyme can be formed.

The barrier chosen and its amount will of course depend on the decomposition products that are allowed to pass therethrough.

When carrying out an analysis of BUN, the barrier composition must be impervious to the liquid base present in blood or serum and permeable to ammonia vapor under atmospheric pressure.

In those cases where urease is an enzyme, the barrier composition is ideally impervious to liquids (water) and therefore impermeable to aqueous bases, while being permeable to ammonia vapor.

For this reason, at least a portion of the urease must be present outside the layer formed by the barrier.

In such cases, other bases that may react with the indicator and falsify the urea indication are also excluded.

The barrier also includes a layer of any thickness that allows the passage of ammonia in at least one direction while simultaneously inhibiting the passage of the aqueous base for the time allotted to completion of the test. Good too.

Note that the typical time required for the test is about 5 to about 20 minutes, especially in the case of BUN analysis.

Materials belonging to this type that are particularly effective for forming barriers having ammonia permeability and water impermeability include, for example, cellulose acetate butyrate (with a butyryl content of 10 to 60 parts by weight and 5 to 30 parts by weight). cellulose propionate valerate (with an acetyl content of 20 to 5
poly(methyl methacrylate) with a valeryl content of 0 parts by weight), and 19 parts or more, preferably about 40 parts by weight.
Included are polymers such as cellulose acetate, which has been acetylated to have a corresponding acetyl content.

Such a barrier allows for the desired reaction of the ammonia vapor with the indicator during a step in which only the ammonia vapor is allowed to pass during the BUN test.

This barrier can thus inhibit any interfering reactions that may occur at the same time as causing a color change.

Barriers used for BUN analysis generally have a concentration of 0.10 to 3.5, regardless of whether the barrier is used as a separate layer from the indicator layer or integrated with the indicator layer. @/m2, preferably 0.
It can have a coverage of 20 to 1.097 m''.

It is also possible to slightly hydrolyze the surface of the barrier, for example formed from cellulose acetate, in order to increase the adhesion of the layer above it.

The purpose of the indicator is to cause at least a portion of the test material to interact with the components of the analyte sample or with the degradation products of those components.

It is this indicator that produces a detectable change in response to the degradation product (or end product thereof) formed and selectively screened as described above.

Coatings containing one or more of the above-mentioned interreactive indicators in a hydrophilic colloid acting as a binder or vehicle, such as deionized gelatin, polyvinyl alcohol, agarose, etc., are suitable.

Also suitable are hydrophobic binders, such as cellulose acetate, cellulose acetate butyrate, and ethylcellulose, which are permeable to the desired component or to the decomposition products of that component.

In some cases, the barrier can also act as a binder, as already explained in connection with FIG.

In this case, it becomes unnecessary to prepare a separate binder for the indicator.

What kind of indicator is added to this layer?
The details of the indicator will of course depend on the liquid component to be analyzed and the analysis mechanism used.

In accordance with one embodiment of this invention, i.e., using an element to detect urea via the urease-catalytic decomposition product ammonia, the liquid component urea and the analytical mechanism, NH3 formation, include: It is necessary that the indicator undergoes a change in its absorption frequency in the presence of NH3.

Particularly effective indicators here are bleachable dyes or dye precursors.

The term "dye breaker" used here is
Dyes can be formed by structural changes in the molecular structure, for example by the dropping of one or more atoms, as in deprotonation, or by the combination of modified molecules with each other (autocoupling) or with color couplers. It refers to such compositions in general.

For example, the former includes base-activatable chromogens and the latter includes diazonium salts.

It will be appreciated that the particular dye precursor chosen herein will depend on the degradation products to be formed by the enzyme, as described below.

In BUN detection, the decomposition products are, for example, bases such as ammonia.

Among the particularly useful chromogens that can be activated with base are protonated or leuco dyes, such as leucocyanine dyes, nitro-substituted leuco dyes, and leukofthalein dyes (which, in the presence of a base, deprotonated to form a dye).

Leucocyanine dyes appear to form dyes in the presence of ammonia according to the following pathway.

(1) In the above formula, n represents a positive integer of 1 to 10, X○ represents an acid anion, R1 and R2 may be the same or different from each other, and each is a hydrogen atom or an alkyl group, preferably is a lower alkyl group having 1 to 4 carbon atoms (wherein the term "alkyl group" includes a substituted alkyl group, such as a substituted lower alkyl group having 1 to 4 carbon atoms, such as a human substituted xyalkyl group, alkoxyalkyl group, carboxyalkyl group, sulfoalkyl group,
sulfatoalkyl, acyloxyalkyl, alkoxycarbonylalkyl, aralkyl, alkenyl) or aryl having 6 to 20 carbon atoms (where "aryl" includes substituted aryl, e.g. phenyl, tolyl, naphthyl, methoxyphenyl, chlorophenyl, etc.)
And Zl and Z2 may be the same or different from each other, each containing the number necessary to complete the type of heterocyclic nucleus used in cyanine dyes having 5 to 6 atomic umbrellas in the heterocycle. Represents a group of nonmetallic atoms.

Moreover, the type referred to herein includes a type that can be protonated at a pH value of about 6 to about 9.

Particularly useful Zl and Z2 include, for example, benzimidazoles and 3,3-dialkyl-3H-indole nuclei.

The above-mentioned cyanine dyes are conventional and their synthesis can be carried out using known techniques, such as the theory of the Pho-tog, co-authored by Mees and James.
rapic Process 1 3rd edition, 206-20
It can be carried out according to the method described on page 7.

Other dyes useful in this invention are protonated and nitro-substituted dyes.

These dyes are deprotonated in a basic environment from compounds of the formula:

(2) In the above formula, (a) k is O or 1, (b) m is O or 1, (c) n is 1 or 2, and (d) L is each substituted. Methyl groups including methyl groups (e.g. -CH2, -C(CH3)2, etc.)
(e) A is, for example, oxygen←rumor-), sulfur (-8-)
or represents an electron-donating group such as -N-, --1-/-'-/-"""-norlT u
(g) p is 0 or 1, (h) R3 is the following (c) 1), (2), or (3):(1
) an alkyl group having 1 (18) carbon atoms, preferably a lower alkyl group having 1 to 4 carbon atoms, a sulfoalkyl group, preferably a sulfo-lower alkyl group having 1 to 4 carbon atoms in the alkyl component; A carboxyalkyl group, preferably a carboxy-lower alkyl group having 1 to 4 carbon atoms in the alkyl component, a sulfatoalkyl group, preferably a carboxy-lower alkyl group having 1 to 4 carbon atoms in the alkyl component; , an acyloxyalkyl group, preferably an acyloxy-lower alkyl group having 1 to 4 carbon atoms in the alkyl component, an alkoxycarbonylalkyl group, preferably a lower having 1 to 4 carbon atoms in both the alkoxy component and the alkyl component alkoxy-carbonyl-lower alkyl group, dialkylaminoalkylene group,
Preferably.

is a di-lower alkylamino-lower alkylene group having 1 to 4 carbon atoms in both the alkylene component and the alkyl component, a cycloaminoalkylene group, preferably a cycloaminoalkylene group having 4 to 6 atoms in the cycloamino component and alkylene Cycloamino-lower alkylene having 1 to 4 atoms in the component; (2) alkenyl group (including substituted alkenyl group)
, preferably a lower alkenyl group having 2 to 4 carbon atoms; (3) an aryl group (including substituted aryl groups), such as a phenyl group, a naphthyl group, a tolyl group, a xylyl group;
Halophenyl group, e.g. p-chlorophenyl group, p-7
- alkoxyphenyl groups, such as the romophenyl group, such as the methoxyphenyl group, the 2,4-dichlorophenyl group, and aralkyl groups, preferably aryl-lower alkyl groups having 1 to 4 carbon atoms in the alkyl component, such as the benzyl group, (i) Xe represents an acid anion; (J) Z3 is ortho- and ( or) an aryl ring which is para-nitro-substituted and can also be ortho- or para-substituted by another electron-withdrawing group, to which another substituent is attached and another carbocyclic ring is attached. good)
, preferably a phenyl ring or a naphthyl ring, and (k) Z' represents a heterocycle having an electron-donating atom (this ring contains a second heteroatom, e.g. oxygen, represents the group of non-metallic atoms necessary to complete a heterocyclic nucleus of the type used in cyanine dyes having 5 or 6 atoms in the cyanine dyes (which may contain nitrogen, selenium or sulfur).

In addition, the heterocyclic nucleus mentioned here preferably includes the following: thiazole nucleus including substituted and unsubstituted penzothiazole nucleus and naphthothiazole nucleus, substituted and unsubstituted benzoxazole nucleus and naphthoxazole nucleus, etc. oxazole nucleus, substituted and unsubstituted benzoselenazole nucleus, selenazole nucleus including naphthoselenazole nucleus, thiazoline nucleus, 2-pyridine nucleus, 4-pyridine nucleus,
3,3-dialkylindolenine nucleus, imidazole nucleus,
quinoline nucleus, imidazo(4,5-bl:l quinoxaline nucleus (Brooker and VanLare U.S. Pat. No. 3)
431111), 3H-pyrroloC2,3-
b) Pyridine nucleus, thiazolo[4゜5-b]quinoline nucleus,
selected from the group consisting of pyrylium nuclei (including benzopyrylium, thiapyrylium and benzothiapyryllium), and dithiolinium nuclei.

Although some of these dyes are not stable to light, such light-year stability is not a significant drawback in the case of this invention.

This is because reading in this case can be done virtually instantaneously, so there is little chance that the dye-bleach will be interfered with by ambient light.

Nitro-substituted dyes can be prepared according to conventional known techniques in the same manner as the cyanine dyes mentioned above.

Other effective indicators are phthalein dyes represented by the following general formula: (In the above formula,
-- R4, R5 and R6 may be the same or different from each other and are each selected from the group consisting of halogen, hydrogen, an alkyl group and an alkoxy group having 1 to 5 carbon atoms, and Z4 is natutalide and (in the above formula, R4 and Z4 are as defined above) Base-bleachable dyes useful in this invention include styryl - type dyes and pH-sensitive pyrylium dyes.

Such styryl-type dyes can be converted into colorless substances by breaking their conjugated chains in an alkaline medium (performed as in Section F).

(3) In the above formula, Z5 represents a group of nonmetallic atoms necessary to complete the 3,3-disubstituted 3H-indole nucleus or its fused homolog, and the 3゜3-substituent is lower alkyl radicals having 1 to 5 carbon atoms or 6 to 12 carbon atoms, when each radical is taken alone;
represents an aryl group having , and when these groups are considered as one, represents the atomic group necessary to complete the alicyclic ring, n is 0, 1 or 2, and R7 is 2 to 2 carbon atoms such that R7-OH can be a hydroxyethyl group or a hydroxypropyl group, such as a β-hydroxyethyl group, a δ-hydroxylpropyl group, etc.
Represents an alkylene group having three atoms.

The formation of an indicator based on chlorine bleaching of IJium salts can be achieved by eliminating the charged oxygen atoms contained in the dye to form either spiropyran or pyridine. can.

In the case of spiropyran, the formation reaction can be described as follows: in the above formula R8 and R9 are each an alkyl group having 1 to 10 carbon atoms or to complete the aromatic nucleus. The necessary atomic groups Rlo and R11 are each an atomic group necessary for forming an alicyclic ring, a hydrogen atom, or an alkyl group having 1 to 5 carbon atoms, Ar' is an aryl group having 6 to 10 carbon atoms and to which a hydroxyl group is bonded at the ortho position.

In the case of pyridine formation, the formation reaction can generally be described as follows.

(5) In the above formula, R8, R9El and R12 are each an alkyl group or an aryl group having 1 to 10 carbon atoms.

In the case of diazonium salts, the presence of a color coupler is necessary to achieve dye formation under a basic environment, such as provided by NH3.

It is well known to those skilled in the art that many diazonium salts are effective dye-forming agents.

Common examples of these salts include benzenediazonium salts represented by the following general formula.

In the above formula, M is 1. a hydrogen atom, 2. a halogen atom, 3. an aryl group, 4. an amine group including 7 substituted amino groups, and these groups are amine nitrogen atoms and other heteroatoms such as oxygen, It can be a cyclic group including sulfur, nitrogen, and the like.

5, a mercapto group, or 6, an alkyl or arylthioether group, and Xe is an acid anion.

These compounds also contain one of the carbons present in the benzene nucleus.
It can be substituted at one or more positions, for example by at least one halogen atom, aliphatic alkyl group, alkoxy group, acyl group, carbamyl group, carboxyl group or nitro group.

Additionally, the term "aliphatic alkyl" as used herein includes straight or branched alkyl groups having 1 to 8 carbon atoms, such as methyl, ethyl, isopropyl, tert, -butyl group, n-phenyl group,
Contains octyl groups.

Particularly effective diazonium salts include those represented by the general formula as described above, in which M represents an amine group, including a substituted amine group, or a thioether group, for example as described above; , the benzene nucleus constituting this diazonium salt is unsubstituted or substituted at least one of the 2-position and the 5-1 position with either an aliphatic alkyl group or an alkoxy group. Includes p-aminobenzenediazonium salts.

Effective diazonium salts belonging to this class can be represented by the following general formula.

In the above formula, 1, D represents a sulfur atom, or the formula: NQ4
2. When Q3 is considered alone, when D is NQ4, it represents a hydrogen atom and D is a sulfur atom.

or NQ4, lower aliphatic alkyl group, lower alkoxy group, formula N-C-T (T in the formula
is either an aryl group or an alkyl group as explained elsewhere.

3゜Q4 represents either a hydrogen atom, a lower alkyl group or a lower alkoxy group when considered alone, and 4, Q3 and Q4 are bonded together. When it is considered that they are integrated, a divalent group represented by the following general formula -CH2→1-- b-OCH2-(a in the formula is an integer of O or 1, and a and C is a positive integer, and the sum of a, b and C is 5), 5, Ql and Q2 are each a hydrogen atom, a lower aliphatic alkyl group (preferably a methyl group or an ethyl group) Alternatively, it represents either a lower alkoxy group (preferably a methoxy group or an ethoxy group), and 6 and X10 represent an acid anion.

Particularly useful p-aminobenzenediazonium salts include substituted aminobenzenediazonium salts represented by the general formula:

In the above formula, 1, Q5 and Q6? Each, when considered alone, represents a lower alkyl group, and 2, Q5 and Q6, when considered combined and integrated, represent the predetermined number of carbons and petroleum oxygen necessary to complete the morpholino group. 3, Q7 and Q8 each represent a hydrogen atom, a lower alkyl group or a lower alkoxy group, and 4 X20 represents an anion of chlorozincate, an anion of fluoroborate, or a sulfate Represents an anion, either a phosphate anion or a chlorostannate anion.

The most desirable benzenediazonium salts have the formula: 1, Q
When 5 and Q6 complete a morpholino group, Q7 and Q8 represent an alkoxy group, and when 2 Q5 and Q6 each represent a lower alkyl group, Q7 and Q8
each represents a hydrogen atom.

Ab dye couplers that can react with diazonium salts to form ab dyes include a variety of chemicals, such as Kosar, "Light-8en-sit".
lve Systems,”John Wileylln
c-1 New York (1,965), 220-2
The items listed on page 40 are in stock.

Particularly effective are phenolic couplers, and a more general description of this class is as follows.

1. 2-hydroxy-3-naphthanilide having the following formula as a blue coupler: (In the above formula, Q9 represents a phenyl group, preferably one of a lower alkyl group or a lower alkoxy group or a halogen atom. represents a phenyl group substituted with at least one phenyl group).

2. Ortho-naphthalene diol as a blue coupler.

3. 1-Hydroxy-2-naphthamide having the following formula as a blue coupler: (In the above formula, a, Q10 and Qll each represent a hydrogen atom when taken alone or a substituted compound such as the one described above) represents aliphatic alkyl groups, including aliphatic alkyl groups, cycloalkyl groups, aryl groups, and similar hydrocarbon groups or substituted hydrocarbon groups; (When a 6-membered heteropiperidino group or a morpholino group is considered as a 6-membered ring, it represents the carbon and oxygen atoms necessary to complete the 6-membered ring.)

4. As a yellow coupler, 2-acylamido-5 monosubstituted phenol having the following formula: H (In the above formula, Q12 represents either an alkyl group or an alkoxy group, and Q13 represents an aliphatic alkyl group, (represents an aryl group, an aralkyl group or an aralkoxy group).

The indicator layer therefore consists of a binder and optionally a desired indicator, the amount of binder present in this layer being 1
．． 0-20. Off/m”, preferably 5.0-15
．． OS'/m2.

It should be noted that the actual amount of binder present will of course depend on the desired thickness of the test element.

The concentration of the indicator must be adequate to indicate the concentration of the test component over the expected range.

In the case of BUN analysis, the expected BUN range is 0 to 1507 jllp for 1 dt of sample.

The indicator must respond within this range and therefore requires an amount within the range 3.0X10"mol/m2 to 1X10-2 mole/m".

Particularly advantageous indicator quantities are 5X10-' to 9.0X10'
mol/m".

For all of the materials mentioned above, supports for these materials can be provided optionally if none of the layers exhibiting other functions are self-supporting.

The support, if used, can be constructed from a material that is transparent to radiation (preferably light) but not permeable to liquids.

A large number of polymeric materials are suitable for this purpose, among which are particularly suitable, for example cellulose acetate,
Mention may be made of poly(ethylene terephthalate), polycarbonate or polystyrene.

The support may be of any suitable thickness, generally from 0.05 to 0.05 mm.
It can have a thickness of 25 mm.

As will be explained in detail below, the support may be exposed to at least electromagnetic radiation in a specific wavelength band (200-900 nm).
) is desirable.

In accordance with some particularly preferred embodiments, it is possible to use a support that is selectively transparent only for a relatively narrow range of specific wavelengths and is opaque to all other wavelengths. desirable.

In cases where the quantifiable substance is fluorescent, the support used should not exhibit an unacceptable level of fluorescence.

Layers such as those described above can be applied directly onto the support or, to aid in bonding the indicator layer to the support, an undercoat having energy transmitting properties similar to those of the support may be used. layers can be used.

In some cases, the support can be omitted, especially if at least one of the other layers is self-supporting.

In preparing the integrated analytical element of this invention, the layers can first be formed separately in advance and then the layers can be stacked into one integrated element.

Layers prepared according to such techniques are generally applied in the form of a solution or dispersion to a surface from which the dried layer can be physically peeled off.

By the way, an advantageous method that can avoid the need for multiple peel and lamination steps is to apply the first layer on the release surface or, if desired, on the support, and then
Thereafter, other layers are directly applied one after another on these previously applied coating films.

This kind of coating is applied with a knife. This can be done by hand using equipment or by machine using techniques such as dip coating or bead coating.

If mechanical coating methods are used, it is also possible to apply adjacent layers simultaneously using hopper coating methods, which are well known in the preparation of photographic light-sensitive films or papers. .

Problems with interlayer adhesion can also be addressed through the use of various surface treatment methods, including the use of subbing materials (applied very thinly), such as those used in photographic film, which can have detrimental effects. It can be resolved without any

If the layers described herein are formed by coating solutions or dispersions such as those described in the aforementioned Belgian Patent No. 801,742, uniform coating properties for the various layers may be obtained. It is often necessary to include the applied coating aid in the solution or dispersion.

Even if coating aids are used for this purpose, it is very important that these coating aids do not inhibit either the enzyme of the enzyme layer or the indicator present in the indicator.

Particularly effective coating aids for this purpose include nonionic surface active agents such as octylphenoxypolyethoxyethanol (p-nonylphenoxy) glycerol and polyethylene glycol.

According to the invention, there is no contact at the initial stage and then the ink + IJ-F as described in U.S. Pat. It is suitable to prepare elements with layers that can be separated by using elastic absorbent or deformable materials as described in No. 53 and No. 3,933,594.

As will be appreciated, if an element initially has non-adjacent layers, it may be necessary to apply a compressive force to the element, or in other cases,
It is necessary to provide means for bringing the layers of the element into fluid contact at the time the element is used to obtain analytical results.

When using an analytical tape or element as described above, the blood cells can first be separated from the serum by means such as centrifugation, and then the serum can be applied to the tape or element.

A particularly important advantage of the analytical element proposed here is the ability it possesses, i.e. it can be used to analyze either serum or whole blood. This means that it can be done.

Of course, when energy transmission methods are used, the scattering layer and any other layers must be uniformly transparent to the sensing radiation, and undesirable residues in whole blood must be avoided. It is recommended that serum obtained in some way be used to carry out the analysis unless some method is proposed to remove the preferable.

A typical automated analysis system utilizing the multilayered analytical element of the present invention involves placing the element below a disposer where a drop of the analytical sample, e.g. whole blood or serum, is collected.
to the surface of the element and then provide a means for sensing the element through one or more processing bands.

Such analytical measurements are generally carried out by guiding the element into a zone within which is suitable equipment for performing reflection, transmission or emission spectrophotometry. will be possible.

Next, the above aspects will be particularly explained in the following examples.

It should be understood that these examples are merely examples and do not limit the invention.

Examples 1-3 Utilizing different chromogens in the indicator layer, a series of three test elements for BUN were prepared having the configuration as illustrated in attached FIG. 1.

The preparation method was as follows.

Ammonia-permeable and relatively water-permeable containing three different chromogens (numbered 1-3) and 8.55 Vm'' of cellulose acetate butyrate as listed in Table 1 below. An impermeable layer was coated onto a poly(ethylene terephthalate) film support from an organic solvent mixture consisting essentially of dichloroethane and acetone.

Additionally, on top of each of these coatings, 30
A layer of cellulose propionate valerate (ammonia-permeable and relatively water-impermeable) having a valeryl content by weight was overcoated (this overcoat was applied from t-butyl alcohol). ).

The resulting element was then treated with 5.4Vm2(7) gelatin, 4300U/m2 of urease, 0.05@/m2
Dipotassium phosphate buffer, 0.067/m2 bisvinylsulfonyl methyl ether as curing agent, 0.067/m2. ．． 32
It was overcoated with a buffered gelatin layer (pH-7.5) containing 7/m2 of polyethylene glycol oleate as surfactant and 0.40 Vm'' of ethylenediaminetetraacetic acid tetrasodium salt.

To complete the element, 6.6? /city' cellulose acetate, 46.0 @/m2 titanium dioxide and 2.69
6. The reflection-scattering layer containing Vm2 of octylphenoxypolyethoxyethanol was coated with a mixture of acetone and dichloroethane (1:1) (double-fused with xylene);
4:1).

The completed analytical element was tested in the following manner.

An aqueous solution of reagent grade urea (contaminated with <10 mol NH3 in a 1 molar solution) was prepared with urea nitrogen (UN) in the range 1-150 ~/d,l (ref. For convenience, the normal range in serum is 10-20 BUN/a, g).

These solutions were spotted (10
μt spot).

A spectrophotometer was used to measure the reflection density occurring at specific wavelengths.

All of the measured values shown in Table 1 were determined after heating at a temperature of 50° C. for 10 minutes.

Table 1 shows preferred embodiments of the analytical element of this invention.

Here, since the extinction coefficient of the chromogens of Examples 1 and 2 is dog (approximately 104), in excess of 501n9AL, BUN
(7) It may be mentioned in particular that when determining the concentration it is advantageous to carry out the determination using kinetic analysis, the use of relatively short reaction times or off-peak readings of the dye.

Example 4 Analytical elements were prepared as follows.

Chromogen Tear 3.78Vm2 (7) 1-Ef Roux 4
The indicator layer containing (26-sinitrophenyl)methylquinolinium ethyl sulfonate and 6.43 @/m2 cellulose acetate was mixed with methanol and acetone (1
:2) was coated onto a polyethylene terephthalate film support.

An ammonia-permeable barrier layer of cellulose acetate butyrate with a butyryl content of 27 and an acetyl content of 21 was applied over the previous indicator layer at a coverage of 0.8 @/m2 (from dichloroethane).

Then copoly(methyl methacrylate; methacryloyloxytrimethylammonium methyl sulfate;
2-hydroxypropyl acrylate; 2-acetoacetoxyethyl methacrylate) (ratio = 40:20:
20:20) undercoat layer of 0.141? / m2 coverage from acetone.

Then, to the obtained elements, 21.6 f/m2 of deionized gelatin Q, 55@/m2 of polyethylene glycol oleyl ether Q, 4 f 7 m2 of tetrasodium ethylenediaminetetraacetic acid, 5.4? /m2 of N
, N-bis(2-hydroxyethyl)glycine, 0.2
8 @/m2 disodium orthophosphate, o, 9my/
A gelatin layer buffered at pH=8.0 containing m2 nodithiothreitol, 27000 U/m2 urease and 0.13 P/m2 bisvinylsulfonyl methyl ether was overcoated.

A subbing layer of poly(N-isopropylacrylamide) was applied from acetone at 0.32@/m2.

Finally, a scattering-reflection layer was overcoated as described in Example 1 above.

The completed analytical elements were evaluated according to the following method.

7% albumin and 0-150 groups (urea nitrogen)/d
An aqueous solution containing reagent grade urea at a fatty concentration was prepared.

These solutions were then applied to the test elements in 10 μt aliquots.

5 at an incubation temperature of 40°C
A spectrophotometer with a 30 nm band (centered at 463 nm) covering an interference filter, a Lanten 2B filter and a 0.4 ND neutral density filter was used to measure the reflection density after minutes.

The following results were obtained.

Example 5 An ammonia-permeable, relatively water-impermeable layer containing a diazo coupling system as an indicator was prepared as follows.

A sample of gelatin-subbed 0.18 mm poly(ethylene terephthalate) film support was coated with (1) 3.19 Vm'' of cellulose acetate butyrate, having a butyryl content of 27% and an acetyl content of 21% by weight; 2) 0
．． 635Vm" 0) 2-2) o -4-Hee IJ
Dinobenzenediazonium hexafluorophosphate, (3) 0.84 g, 3-(2-methoxyphenylcarbamoyl)-2-naphthol and (4) of /rn2
0.097'Vm2 polyethylene glycol 1540
A layer containing oleic acid ether of 30% was applied.

The test was then carried out by spotting an ammonium hydroxide solution having a concentration equal to 10% urea and 40% urea onto the coated light cream element.

For each element, after the solution was added, light green and dark green colors were observed.

This suggests that diazonium salts are suitable as dye precursors.

Example 6 - An ammonia-permeable, relatively water-impermeable layer containing an ammonia-bleachable dye as indicator was prepared as follows.

0.18m with basecoat as in Example 4 above
m of poly(ethylene terephthalate) sample, (1)
3,22 Vm2 of acetic butyric acid-t! with a butyryl content of 27% by weight and an acetyl content of 21%! :#0-
and (2) 0.322Vm2 (7) 2 (β-
A layer containing (2-hydroxy-1-naphthyl)-α-methylvinyl]-1-benzopyrylium verchlorate was applied.

Then each coated element (dark blue in color) has 10,771! The element was tested by spotting p% urea and ammonium hydroxide solution having a concentration equal to 20-% urea.

In areas where a low concentration of ammonium hydroxide was spotted, the dark blue elements were partially bleached;
On the other hand, in areas where a relatively high concentration of ammonium hydroxide was spotted, the element was completely bleached.

Equivalents of urea tested in this way
1ency) covers the normal BUN range, so
This Example 6 demonstrates the validity of bleachable dyes as indicators.
is considered to be explained.

In Examples 7-1, elements similar to those of Example 1 above were prepared.

However, in this case, in addition to placing an independent barrier formed by cellulose acetate butyrate (butyryl content 27% and 7 cetyl content 21%) between the indicator layer and the enzyme layer, 2.26 @/m2 of 4 (2,6-sinitro-4-chlorobenzyl)-1-probylquinolinium ethanesulfone) (cellulose acetate with a degree of acetylation of 40%) to formulate the barrier composition. The indicator layer was prepared by coating (middle).

The enzyme layer is about 8. Buffered to OpH value.

The analytical elements were evaluated according to the following method.

Aqueous solutions containing albumin and reagent grade urea at four concentrations ranging from about 15 to about 115 m9TJNc urea nitrogen) were prepared.

These solutions were then applied to the test elements in 10 μt aliquots.

Reflection density at 670 nm was measured after 5 minutes.

The results are shown in Table 2 below.

Although this invention has been described in detail with particular reference to preferred embodiments thereof, it will be understood that various changes and modifications may be made within the spirit and scope of the invention.

[Brief explanation of drawings]

1, 2, 3, 4, and 5 are cross-sectional views of analytical elements showing preferred embodiments of the present invention, in which 10 is the entire analytical element, 12 is a support, 14 is an indicator layer, 16 is a barrier composition, 18 is an enzyme layer, 20 is a scattering layer,
40 is a dispersion-enzyme layer, and 50 is an intermediate layer.

Claims (1)

[Scope of Claims] 1. An integrated analytical element for the analysis of a liquid sample with respect to a predetermined analyte, comprising the following reagents: a first which can form vaporous decomposition products;
(2) a second reagent capable of reacting with the decomposition product or its final product to produce a detectable change, and the first and second reagents are in a barrier. separated by a barrier material, and the barrier material is substantially uniformly permeable to the vaporous decomposition products and substantially impermeable to liquid water and obstructions. Integrated analysis element. 2. An integrated analytical element according to claim 1, wherein the first reagent is urease and the second reagent is a substance capable of reacting with ammonia to form a detectable product. 3. The integrated type according to claim 2, wherein the barrier material includes at least one polymer selected from the group consisting of cellulose propionate valerate, cellulose acetate butyrate, cellulose acetate, and poly(methyl methacrylate). analysis elements. 4. The integrated analytical element according to any one of claims 1 to 3, wherein one of the first and second reagents is distributed in the barrier material. 5. An integrated analytical element according to any one of claims 1 to 4, wherein the second reagent is a dye precursor capable of forming a dye in the presence of ammonia. 6. The integrated analytical element according to claim 5, wherein the dye precursor is a leuco compound that sheds protons in the presence of ammonia. 7. An integrated analytical element according to any one of claims 1 to 6, wherein the second reagent is an ammonia-bleachable dye.