CH615510A5 - Chemical analysis process and product for implementing this process - Google Patents

Abstract

The process is used for chemical analysis, and more particularly for chemical assaying of the constituents of a bodily fluid. In order to implement this process, a product is used which comprises a layer (18) of a substance capable of reacting with a fluid constituent to give a decomposition product, a barrier layer (16) affording a selective permeability to the decomposition product of the substance, and a layer (14) which contains an indicator providing a coloration by reaction with the decomposition product of the substance. The product furthermore comprises a layer (20) ensuring good spreading of the fluid sample which is deposited thereon and a substrate (12). Application to the assaying of urea in the blood by enzymatic means. <IMAGE>

Description

The present invention relates to chemical analysis, and in particular, a method for the determination of the constituents of body fluids by chemical means, as well as a product for the implementation of this method. The method according to the invention is, in particular, useful for the determination of nitrogen originating from urea in blood serum.

In recent years, many automated systems have emerged for chemical analysis of fluid samples. These systems have proved to be particularly advantageous for clinical analyzes carried out in the laboratory and, in particular, for quantitative analyzes carried out on blood. Systems have been widely used which operate by continuously analyzing samples with which diluents and analytical reagents are mixed, the whole then being conveyed through an analyzer. However, these continuous analyzers, such as, for example, that described in US Patent 2,797,149, are complicated and expensive devices requiring the intervention of qualified operators who must spend a lot of time and time. '' effort in repetitive cleaning operations; in addition, such devices do not allow the analysis of small quantities of samples such as those that can be used in micro-analysis techniques. In order to eliminate these drawbacks, automatic chemical analyzers have been proposed operating with a continuous analysis ribbon, on which a sample to be analyzed is deposited and with which a quantitative analysis operation is then carried out, for example by carrying out a reaction. chromogen between components of the sample and analysis reagents incorporated in the ribbon, then by performing a colorimetry measurement spectrophotometrically. Analyzers of this type are described in detail, for example in English patent 1,049,364 and in United States patents 3,036,893 and 3,526,480.

The analyzers which operate with a continuous ribbon are of simpler construction than the continuous flow analyzers. However, ribbon analyzers, such as those described in the patents cited above, have many drawbacks which have prevented their commercial development. When the analytical reagents are not incorporated into the ribbon and the latter only serves as a conveyor for the sample to be analyzed (as is the case of the system described in English patent 1 049 364), means must be provided for separately applying the sample and reagents when needed and in the right amount. It is obviously possible to provide separate ribbons each performing various functions, for example, sample filtration, reaction with analytical reagents, as described in United States patent 3,036,893 and English patent 1,049,364. But such a practice complicates the system in such a way that one no longer takes full advantage of the inherent simplicity inherent in the technique of analysis with continuous tape. If the analysis tape has a complex structure, its manufacture is difficult and of a high cost price, and it is then difficult to produce a commercially exploitable system.

Dry systems have been described, which make it possible to carry out quantitative or semi-quantitative analyzes of the constituents of the blood. Thus, provision has been made to incorporate reagents in a permeable support such as paper or a hydrophilic and porous polymer layer; a product is obtained which may possibly include overcoats or dialysis means to eliminate, by filtering them, certain constituents of the blood such as red blood cells. In particular, for the determination of nitrogen originating from urea in the blood, it is possible to impregnate a permeable support with a urease and apply to the product obtained, an overlay constituted by a dialysis membrane (US Patents d 'America 3 145 086.3 249 513.3 395 082, and the English patent 1 287 785). In all these patents, the reaction of urea with oxygen is used which, in the presence of urease, results in the release of ammonia which induces the change in color of a pH indicator impregnating the product. However, the presence of other alkaline substances can distort the result provided by the indicator. The dialysis membrane, if one is used, may simply be a macrofilter which is then the outermost layer. Optionally, barrier strips can be inserted; but they are used to separate the analysis product lengthwise in areas of different sensitivity; these barriers therefore have no role with regard to selective migration within the layers.

In U.S. Patent 3,011,874, it is noted that other barriers have been used for the determination of nitrogen

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from urea in the blood. However, according to this patent, layers are used which form a barrier to all substances; a release of gaseous ammonia occurs in a strip and, after release, the ammonia diffuses into the atmosphere of a closed container up to an indicator strip.

Other techniques are based on a reaction in the presence of urease, but most often these reactions are carried out in a liquid medium, as described in US Patents 3,409,508 and 3,485,723.

As for the technique for the determination of nitrogen originating from urea in the blood, it has been generally described in a certain number of patents and, in particular, in the patents of the United States of America 3,718,433 , 3,655,416, 3,567,364, 3,511,611 and 3,531,254.

In US Patent 3,723,064, a product for chemical analysis is described, comprising two layers of reagent separated by intermediate layers. One of these layers may have zones of chemical reagents of different concentrations, the action of which is specific for the final substance which is finally analyzed. Barriers can be provided at these intermediate layers, made, for example, of cellulose acetate, so as to adjust the concentration gradients when the substances pass through these layers.

Such barriers do not serve to trap undesirable substances, or those liable to give parasitic reactions, when such substances pass from one layer of reagent to the other in the normal direction of diffusion. In addition, their concentration is variable in the area of the product where the sample is intercepted.

In the patent of the United States of America 3,901,657, a layer of filter paper is described, the role of which is to physically separate a layer containing a reagent, and a layer containing an indicator; the liquid sample, in its entirety with its constituents, passes through this paper filter. It is not indicated that the filter paper could selectively oppose certain constituents of the sample, or a decomposition product from the analytical reaction.

A blood analysis technique has recently been described in Belgian patent 801,742. This patent describes a product which comprises a layer of reagent, a layer with isotropic porosity serving to spread the sample and other layers among which, optionally, a dialysis layer, a filtering layer, a reflective layer, or a layer performing several of these functions at the same time; this product further comprises means for forming a detectable change, for example a measurable change in absorption or transmission, depending on the presence of a component in the sample subjected to analysis. The continuous layer of reagent and the layer used for spreading are in contact in the presence of a fluid, which also ensures the connection with the other layers, so that the substance which one seeks to assay (or its precursor, or a reaction product of this substance) can reach the reagent layer. It is also indicated that the filtration or dialysis layers can be used to prevent the passage of components of the sample, which can thus be stopped by filtration, while other components of the sample are not stopped by the layer. filtration and can pass through it. Regarding such filtration or dialysis layers, it is not indicated that it ensures the preferential passage of a decomposition product formed directly or not in the reagent layer placed upstream of the filtration layer relative to the normal diffusion of the fluid sample, thus allowing the elimination of substances liable to interfere with the reaction.

Enzyme electrodes operating by means of semi-permeable membranes can also be used in the following manner: a membrane selectively permeable to the substance chosen is associated with an enzyme layer and an electrode enabling the formation, by potentiometer, to be followed. decomposition products resulting from the result of the enzymatic reaction; this decomposition product is, for example, ammonia produced from urea in the presence of urease. Such devices are described, for example, in United States patents 3,838,033 and 3,896,008.

The subject of the present invention is a method of analysis by a “dry” route of complex fluid, protected from interference which, when occurring with the reagents and by-products of the reaction, could disturb the measurements; the present invention further relates to an analytical product with indivisible structure for the implementation of this method.

The method of this invention allows in particular the quantitative analysis of blood components such as, for example, the determination of nitrogen from urea. This product may in particular have an indivisible structure and thus be used for the analysis of liquid samples which are applied thereto.

According to the invention, the method for the analysis in the fluid sample of a constituent determined in advance, uses:; a) a first substance capable of giving a reaction in the presence of the constituent to be analyzed, with formation of 'a decomposition product;

b) a second substance which reacts with this decomposition product, or with a transformation product so as to give a detectable modification.

This process is characterized in that the two substances are incorporated into an analytical product in which they are separated from each other by a barrier; this barrier has an almost uniform permeability to the aforementioned product of decomposition, while it remains practically impermeable to substances which could interfere with the analysis.

As just described, the method is particularly useful for the determination of nitrogen from blood urea, when this determination is carried out with urease which allows the transformation of urea. in ammonia.

According to one embodiment, the method is implemented using a product with an indivisible structure which comprises in superposition:

1) a first layer which contains a first substance capable of giving a reaction in the presence of the sample to be analyzed, with the formation of a decomposition product; and

2) a second layer which contains a second substance capable of giving a reaction with this decomposition product, or with the transformation product of this product, which thus forms a detectable modification. This product is characterized by the presence of a barrier separating the reactants from the first and from the second layer, which has an almost uniform permeability to the above-mentioned decomposition product and which 5, i is impermeable to parasitic substances.

In a method whose operation is based on the reaction between a substance and a constituent to be analyzed, any tendency of the substance to form undesirable reactions is a problem. The substances may lead to an undesirable detectable modification by giving parasitic reactions with certain substances which may form during the reaction (or by-products) which takes place between the reagent and the constituent to be analyzed, or by giving a reaction with the other compounds of the sample to be analyzed. If this really happens, the analysis is wrong. It is these intermediate products of the reaction or the other constituents of the liquid sample which are designated below as "interfering products" and which must be separated from the reagent responsible for the detectable modification, so as to provide results. quantitative. The method according to the invention solves the problem of interfering products by separating them from the detection reagent. As for the product according to the invention, its structure is simple, and it is easy to manufacture; its cost price is acceptable and it is

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designed to make simple and effective analyzes. The product can be used in the form of a ribbon or a continuous strip or in other forms for example, in the form of sheets of small area or of short strips which may optionally be mounted in window cards or by any other similar means. .

All these presentations of the product are equivalent embodiments and form part of the invention. In addition, although the preferred structure of the product is a multilayer structure, the invention is not limited to this embodiment. In fact, it relates to any product in which two reagents are separated by a barrier exhibiting the desired selective permeability for the time necessary for a determined analysis. The terms “permeable” or “practically uniform permeability” mean that all of the decomposition product which reaches the barrier, effectively crosses it during the duration of the analysis.

Quantitative analysis of the particular constituents of the sample is easily carried out, for example the analysis of nitrogen from blood urea, by conventional spectrophotometry, or by any other technique capable of rapidly giving an accurate measurement of the content of detectable product obtained by the analytical reaction.

It has been indicated that the "product formed by the analytical reaction is detectable by measuring the radiant energy"; this means that the products or the species thus formed modify the reflection or transmission characteristics of the radiant energy of the product by coloring or discoloration, by fluorescence or modification of any other characteristic.

The term “decomposition” designates, without being limiting, reactions of dissociation, dehydration, hydrolysis etc. The term "transformation product" designates a substance formed by the reaction of the decomposition product with one or more other substances such as an analytical reagent incorporated into the product.

The term "substance" refers to a body which gives a reaction with a constituent, a precursor of a constituent of the sample analyzed, a decomposition product or a transformation product.

These interactions can involve chemical reactivity, catalytic activity as linked to the formation of a complex between substrate and an enzyme, or any other form of chemical or physical interaction which results in ultimately providing in the analytical product, a change detectable by an appropriate measurement of the radiant energy, usually by a measurement of the light energy. Although the description below relates to a particularly useful combination where an enzyme is used to form the decomposition product and where an indicator sensitive to the decomposition product provides the detectable change. Other substances or additional reagents may be used, provided that they meet the definition given.

Preferably, the product for implementing the method according to the invention has a multi-layer structure hereinafter referred to as a laminated product. Such a structure allows the diffusion of a drop of sample by gravity, capillarity, or wicking effect, from the outer layer, to one or more inner layers.

Each layer of the product has a sample interception zone. The term “sample interception zone” referring to a layer designates the complete surface zone impregnated by a sample, or by a decomposition product of a constituent of the sample, during its normal movement through the product, at any level of its section, transversely to the direction of diffusion of the fluid. In the interception zone of each of the layers, there must be a uniform distribution of the constituents, so as to obtain uniform results.

Such uniformity is important for obtaining quantitative results when the measurements are made with a device using radiation such as a reflectometer. The term "uniform" used to describe the composition of the layers means that throughout its volume, the composition of such a layer is almost constant. Preferably, such uniformity is obtained with a barrier layer of constant thickness and composition.

It is preferable that the interception zone of the sample m of the barrier in contact with the layer containing the first substance first encountered by the liquid, have at least the same extent as that of this first, to separate the substances unwanted from the indicator layer interception area for the duration of the analysis. Naturally, the interception zone of the barrier sample can be much larger than that of the layer containing the analytical substance.

In a preferred embodiment, the product comprises a support, with on one face of this support, one or more 2o layers so as to produce a laminate with indivisible structure; this laminate comprises at least one indicator, a barrier, an enzyme-containing layer and an overlayer which is used for spreading and calibrating the sample deposited on the laminate. The enzyme layer may contain one or more enzymes depending on the analyzes envisaged.

Figures illustrate the invention. Only the interception zones of each layer have been shown; the sample interception zone of each layer has the same extent as that of the adjacent layers. Of course, each layer 10 can extend beyond the interception zone of the sample and such extensions do not require the presence of reagents or of a barrier as long as the liquid sample, the constituents and / or the decomposition products are not in contact or do not penetrate into these extensions. FIG. 1 illustrates the product 10 which comprises a support 12, an indicator layer 14, a barrier layer 16, an enzyme layer 18, and a spreading layer 20 determining the surface of the product to which the sample is applied . The layer 20 optionally fulfills other functions such as manufacturing and / or reflection, described in more detail below; this layer is distinct from layer 18.

Figures 2 to 5 illustrate variants where the layers are associated differently. The layers identical to those previously described have the same reference numbers 45 followed by the letters "a" to "d" to distinguish them.

Thus, in FIG. 2, the product 10a comprises a support 12a, a spreading layer 20a, an enzyme layer 18a. The barrier and the indicator are here combined in a single layer 30, unlike the product of FIG. 1, where the barrier layer was located between the enzyme layer and the indicator layer.

In FIG. 3, the product 10b comprises a support 12b, an indicator layer 14b, a barrier layer 16b, as in FIG. 1. In this embodiment, the enzyme is incorporated into the spreading layer, to obtain a single layer 40.

In Figure 4 the product 10c comprises a support 12c, and two other layers 30c and 40c comprising the indicator layer and the barrier of Figure 2 and the enzyme and spreading layer of Figure 3, respectively.

6 In FIG. 5, the product 10d comprises a support 12d, a spreading layer 20d as in FIG. 1, and a middle layer 50. The middle layer comprises the composition constituting the barrier; and each of the surface areas of layer 50 contains specific ingredients; an area 14d, 65 adjacent to layer 12d contains the indicator, and an area 18d, adjacent to layer 20d contains the enzyme.

The central zone 52, which is free of enzyme and of indicator is optional, as long as the zones 14d and 18d do not

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not overlap. In this embodiment, the barrier layer acts as a binder for the enzyme and the indicator. This embodiment is suitable in the case where the constituent to be analyzed, as well as its decomposition product, is capable of crossing the barrier. If the constituent to be analyzed does not cross the barrier, a certain amount of enzyme must be put into layer 20d, which is illustrated in FIG. 5 by layer 54.

From the above discussion, it emerges that certain functions must be fulfilled, whatever the embodiment, that is to say:

- the sample deposited on the product must be spread uniformly there, preferably using a spreading layer;

- the constituent to be dosed must be broken down, preferably by an enzyme; is

- the decomposition product must penetrate selectively towards the indicator, thanks to the barrier;

- an indication of the quantity of decomposition product must be provided, and this indication must represent the quantity

of constituent present in the deposited sample. Preferably, 2o the indicator is a dye precursor, or a deco-lorable dye. Each of these functions and the corresponding means must be studied separately and in detail according to their order of intervention.

A sample deposited on the product is, first of all, distributed uniformly, preferably by one or more layers which perform this function, in order to provide a uniform content on the surface of the spreading layer in contact in the presence of a fluid. with the adjacent layer (s). The spreading layer of the laminated product is generally that in which the liquid sample to be analyzed, such as a blood or serum sample, is deposited, most often in the form of a drop. The term “in contact in the presence of a fluid” designates a contact which allows a liquid or one or more of its constituents, liquid or gaseous, to pass through the layers. 35

As described in Belgian patent 801 742, a spreading layer can be used to uniformly distribute one or more substances in the layer, including at least one constituent of the liquid sample deposited, so that at a given time, the content of this substance is uniform on the face of the spreading layer 40 adjacent to the enzyme layer. The applied sample does not need to be kept in a tight space. It should be noted that such a uniform concentration, although it is achieved almost instantaneously, can vary after a certain time without causing harmful modifications. Different contents may even exist in the thickness of the product. In discussing the various embodiments, the term "isotropic porosity" means that the spreading layer is porous in all directions. The degree of this porosity can vary if necessary or desirable (for example, thanks to the size of the pores, the percentage of empty volume, etc.). The meaning of the term "isotropic porosity" (or isotropically porous) used in the present description, should not be confused with the meaning of these same terms when they are used for filtration membranes which have uninterrupted pores in their thickness. Likewise, the term isotropic used in the present description should not be confused with the term isotropic when the latter is used in opposition to the term anisotropic, to qualify filtration membranes which have a thin "skin" on one of their surfaces. One can refer, for example, to the work of James Flinn entitled “Membrane Science and Technology”, published with the Editions Plenum Press, of New-York (1970).

The spread depends in part on the volume of liquid. However, it should be emphasized that the uniform content obtained by spreading is completely independent of the volume of the liquid sample and that this result is obtained with varying degrees of spreading. The consequence is that the products according to the invention do not require precise techniques for the application of the sample. However, it is desirable to apply a determined volume of liquid sample to obtain preferred spreading times etc. Since the products according to the invention are designed to provide quantitative results using very small sample volumes occupying a calibrated area of the spreading layer (for example 1 cm 2), it is not necessary to remove excess moisture from the product after applying the liquid sample. In addition, since the spreading takes place on the spreading layer and the spreading substance is intended for the enzyme layer without significant lateral hydrostatic pressure, there is no risk of halo formation as is often the case in assay products when soluble reagents are used.

The spreading layer only serves to standardize the concentration of the spreading substance on the face adjacent to the enzyme layer with which it is in contact in the presence of a fluid. By densitometry or another analytical technique, it can be determined whether a particular layer is suitable for serving as a spreading layer. With light beam scanning methods of the surface of the reagent layer, or any other associated layer, the content of the spread substance or of any reaction product calculated on the content of the spread substance can be determined. The following essay is given as an example; the choice of substances or parameters does not expressly or implicitly indicate that other materials or parameters cannot be suitable for identical embodiments.

For this test, a transparent layer of gelatin is applied to a transparent photographic film support, for example made of polyethylene terephthalate, at a rate of approximately 200 mg / dm2. The tanning of gelatin can be variable but, in the test, a layer of tanned gelatin is used so that it swells by 300% when it is immersed in water for 5 min at 22 ° C. in the dry state, the gelatin layer has a thickness of approximately 30 microns. The layer for which the spreadability is checked can be applied to the gelatin layer from a solution or a dispersion. The appropriate layer must have a thickness of 100 to 200 n in the dry state. After the layers have been dried, a sample of the test solution or dispersion is applied to the surface of the spreading layer. to evaluate. Preferably, the application is made in small quantities, so that the surface of the layer is not completely impregnated with the sample; but the application is sufficient to produce a circular impregnated zone whose diameter is between 8 and 10 mm.

The test solution or dispersion is chosen in part depending on the type of sample or the sample constituent to which the layer will be exposed under the same conditions of practical use. Aqueous dye solutions are used for low molecular weight substances; such as 0.005% Solatin Pink ® solution. For higher molecular weight substances, such as proteins, an aqueous dispersion of beef albumin colored with "Solatine Pink®" is used. After having applied the liquid sample to the layer to be evaluated and after having allowed it to penetrate into the layer, the product is returned, which makes it possible to examine the spreading layer through the transparent support and the gelatin layer. If, before the complete evaporation of the solvent, or of the dispersion medium, the colored spot has a uniform color density when it is scanned by an opening densitometer equal to 2-3 mm in diameter, it is considered that the concentration of the deposited substance is uniform on the surface of the test layer and / or in the gelatin layer and therefore the test layer can be used as a spreading layer. The density is considered to be uniform if in the spot, with the exception of the periphery, the maximum values and

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minimum do not deviate more than 10 to 15% from the average value. Because of side effects, non-significant density gradients can be observed, but remain without influence on the result of the analysis. The peripheral zone can be variable from one spot to another, but usually it does not represent more than 20% of the spot and sometimes even less.

These spreading layers can be prepared from various ingredients. Preferably the layer is of non-fibrous structure. One can, in particular use a substance in the form of particles to form such layers; layers with isotropic porosity are obtained by connecting the spaces between the particles. Various substances can be used in the form of particles, preferably inert with respect to the constituents of the sample which it is desired to analyze. Pigments such as titanium dioxide, barium sulfate, zinc oxide, lead oxide, or even diatomaceous earth, particles of microcrystalline colloids, made from polymers of natural or synthetic origin , can be used for this purpose. Such microcrystalline substances are described in "Colloidal Macromolecular Phenomena, Part II, Novel Microcriystals of Polymers" by O.A. Battista et al, published in "Journal of Applied Polymer Science," Vol. II, pages 481-498 (1967). Microcrystalline cellulose is an example of such colloidal substances which give good results. It is also possible to use spherical particles of uniform size, which is particularly advantageous when it is desired to obtain uniform pores with a view to carrying out selective filtration. If a particular substance has no adhesion, as in the case of glass beads, it can be treated so that the particles used can adhere to each other and thus facilitate the formation of a layer with isotropic porosity. An appropriate treatment can consist in incorporating particles devoid of adhesion in a thin adherent layer, for example made of a hydrophilic colloid such as gelatin or polyvinyl alcohol, then in bringing these particles into contact within a layer . When the colloid layer has dried, the integrity of the layer is maintained with spaces between the particles.

The spreading layer can also be prepared from “white” layers of polymers with isotropic porosity. It is possible, in particular, to dissolve a polymer in a mixture of two liquids, one of which is a liquid with a low boiling point, a good solvent for the polymer and the other is a liquid with a higher boiling point which is a poor solvent for the polymer. This polymer solution is applied to a support and dried; the solvent with low boiling point evaporates more quickly and the layer is enriched in that of liquids which is a bad solvent for the polymer. If the evaporation is carried out under the appropriate conditions, the polymer makes it possible to obtain a “white” layer with isotropic porosity.

Various polymers or mixtures of polymers can provide such layers with isotropic porosity, usable as spreading layers, in particular polycarbonates, polyamides, polyurethanes and cellulose esters such as cellulose acetate.

A product according to the invention can also comprise one or more reflective layers, possibly capable of absorbing the radiation used for detection, so as to facilitate this detection by reflectrometric measurements. The reflectance can be provided by a layer which also serves as a spreading layer, or by another layer which may or may not have another function in the analysis product. Reflective pigments such as titanium dioxide or barium sulfate can be used, as can the "white" layers of polymers described above.

The pigment-based spreading layers are of interest. According to one embodiment, the “white” layers of polymer can contain a pigment to increase its spreading power and / or its reflecting power. The amount of pigment incorporated into this layer can be very variable and, for example, represent between 1 or 10 parts and preferably, between 3 and 6 parts by mass relative to the mass of polymer.

To solve the problems of adhesion between the layers, surface treatments can be used, in particular, the application of extremely thin layers of substrate such as those used for photographic products. In order for the component to be assayed to be broken down into a product allowing a detection reaction, the sample, via a spreading layer, is brought into contact with an appropriate enzyme. The enzyme can be incorporated into a particular layer or into the spreading layer itself. , 5 The choice of enzyme depends on the dosage envisaged. For the determination of urea, with a view to forming ammonia, urease is used.

The enzyme layer preferably includes a binder in an amount of from about 3.0 g / m2 to about 30.0 g / m2, an amount of suitable enzyme and a buffer to maintain the pH. The enzyme is effective only in a narrow pH range. Therefore, it is usually necessary to buffer the enzyme-containing layer at a pH within the enzyme's efficacy range. However, this pH range must take into account the equilibrium pH of the desired decomposition product. Thus, although it is possible to detect an enzymatic activity outside this range, it is preferable to buffer the enzyme layer containing the urease at a pH of between approximately 5 and 9.5 and preferably between 7.5 and 9.0. The techniques for buffering the pH are well known to the specialist and consist in dissolving or dispersing a buffering agent in the composition before applying it.

The buffering agents used to obtain the above-mentioned pHs are described in detail by Good in "Biochemistry" 5,467 i5 (1967). Particularly useful buffers are phosphates such as potassium phosphate, so-called "Tris and Hepes" buffers and dimethyl glutarate.

The enzyme layer may also include additional compounds such as dithiothreitol, which is an enzyme activator, at a rate of from about 0.5 mg / m2 to 5.0 mg / m2 and the sleeping aids mentioned above -after. The activator increases the catalytic activity of the enzyme by a poorly understood mechanism. In addition, compounds which form complexes with heavy metal ions which could otherwise inhibit the activity of the enzyme can be introduced. The useful contents of such complexing agents are between 150 mg / m2 and 500 mg / m2.

In one embodiment of the determination of nitrogen from blood urea, the enzyme layer may comprise a 50 binder such as gelatin, agarose, polyvinyl alcohol, etc., an appropriate amount of urease of between 4,000 U / m2 and 50,000 U / m2 and, preferably, between 10,000 U / m2 and 30,000 U / m2, a salt of ethylene diamine tetracetic acid as complexing agent for metal ions; and an activator such as ortho-phosphate.

After the formation of the decomposition product by the catalytic effect of the enzyme, described above, the indicator of the interfering products is isolated, in accordance with one of the aims of the invention, during the duration of the analysis. preventing their 6 „access to the indicator. This is made possible by the selectively permeable barrier separating the first reagent (the enzyme), and the second reagent (the indicator). The term "barrier" means that the barrier physically isolates the enzyme reagent and the indicator. Preferably, the barrier is in the form fl5 of a separate layer disposed between two adjacent layers which it separates; the reagents are thus themselves separated in an interception zone, as defined above. The barrier can also be in the form of a dispersion, or a distribution

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in the layer or in part of the layer, comprising the enzyme and / or in this case, the barrier actually forms part of these layers. Such barriers serve to isolate harmful interfering compounds and the indicator described in detail below. The barrier should not be confused with conventional filtration layers which are only effective in filtering only a limited part of the substances, but not all of them. The barrier according to the invention is uniformly impermeable to interfering compounds. The term "uniformly impermeable" means that the impermeability is complete for the duration of the analysis, to the point of preventing any interfering agent obtained in the sample, or initially present in the sample from being in contact with the indicator. in the sample interception area. The duration of the analysis varies according to the constituent and the reagents used in the analysis. According to one embodiment, a uniform barrier layer is used corresponding, at the surface and in position to the interception zone of the enzyme layer. Alternatively, the enzyme and the indicator may be incorporated into the surface portions of a layer constituting the barrier; the central part constituting the barrier is free of indicator or enzyme. The choice of barrier and the content of the constituents depends, of course, on the decomposition product which must pass through it. In the case of the determination of the urea nitrogen in the blood, the composition must be impermeable, under normal atmospheric pressure, to the liquid bases of the blood or the serum and permeable to ammonia vapors. When the enzyme is urease, the composition of the barrier is ideally impermeable to water and aqueous bases, but permeable to ammonia vapors. For this reason, at least part of the urease must be outside the barrier. In this way,

bases which can react with the indicator to distort the result of the urea test are excluded. The barrier may be a layer of thickness such that it allows the passage of ammonia in at least one direction, while it simultaneously prevents the passage of the aqueous bases during the duration of the analysis. A normal duration, particularly for the analysis of nitrogen in blood urea, is approximately 5 to 20 minutes. Particularly useful substances of this class, for constituting impermeable barriers and permeable to ammonia, include polymers such as cellulose aceto-butyrate containing 10% to 60% of butyryl radicals and 5% to 30% acetyl radicals, cellulose valero-propionate containing from 20% to 50% of valeryl radicals, polymethyl methacrylate, acetylated cellulose acetate at 19% and preferably at around 40%.

By allowing only ammonia vapors to pass for the duration of the analysis of the nitrogen in the urea of the blood, such a barrier allows the desired reaction with the indicator, which forms the variation in color while it prevents any interference reaction. In the determination of nitrogen in blood urea, the barrier can be applied in the form of a layer separate from the indicator layer or in intimate mixture with the latter; it has a content varying from 0.10 g / m2 to 3.5 g / m2 and preferably between 0.20 g / m2 and 1.0 g / m2. The surface of the barrier layer can be slightly hydrolyzed to increase the adhesion of the overlays applied to a barrier layer formed for example of a cellulose ester.

The indicator is used to supply at least some of the substances which react with the constituents or the breakdown products of the constituents of the sample to be analyzed. It is the indicator which generates a detectable modification, in proportion to the decomposition product obtained (or its trance formation product) and selectively isolated as described above.

A suitable layer comprises one or more indicator substances in a hydrophilic colloid serving as a binder or vehicle, such as deionized gelatin, polyvinyl alcohol, agarose, etc. Binders can also be hydrophobic, such as cellulose acetate, cellulose acetate butyrate, ethylcellulose, etc., which are permeable to a desired component or to a decomposition product of a component. According to a variant described in Figure 2, the barrier can act as a binder for the indicator.

The particular indicators incorporated into the layer naturally depend on the liquid compound to be analyzed and the mechanism used. If the product is used to measure the urea by a decomposition catalyzed by the urease generating ammonia, the constituent constituent (i.e. urea) and the reaction mechanism (the production of NH3 ), make it necessary to vary the frequency of absorption of the indicator in the presence of NH3. Particularly useful indicators are bleaching dyes and dye precursors.

The term “dye precursors” designates any composition capable of forming a dye by a modification of the structure of the molecule, for example by eliminating one or more atoms such as by deprotonation, or by association of modified molecules together, or by self-coating. or by 2o coupling with a colored coupler.

For example, it is possible to use chromogenic compounds activated in the presence of a base, and diazonium salts.

The choice of the dye precursor depends, of course, on the decomposition product provided by the enzyme.

In the determination of nitrogen in blood urea, the decomposition product is a base such as ammonia. The chromogenic compounds activated by a base, may be protected compounds, or leuco dyes such as the leuco cyanine dyes, the leuco dyes substituted by a nitro radical, the 3o leuco phthalene dyes, which lose a proton to give the colored form, in the presence of a base.

Cyanine dyes seem to form or dye in the presence of ammonia by the following mechanism:

zl-N

'(?) C - CH0 <CH = CH) \ ^ A' 2 n

'N 2 C @Z

nN /

40 O i

XW R

1 © c - CH t CH - CH)

not

XO R1

R

/ X <

c z 'I

R2

where n represents a number from 1 to 10, X® represents an acid anion; R1 and R2 are the same or different and each of them represents a hydrogen atom or an alkyl radical (preferably a lower alkyl radical of 1 to 4 carbon atoms) where the term alkyl radical includes substituted alkyl radicals such as substituted lower alkyl radicals of 1 to 4 carbon atoms, in particular hydroxyalkyl, alkoxyalkyl, carboxyalkyl, sulfoalkyl, sulfatoalkyl, acyloxyalkyl, 6-alkoxy (| carbonylalkyl, aralkyl, alkenyl, or any other alkyl radical of 6 to 20 carbon atoms, where the aryl or substituted aryl radical can be, for example, phenyl, tolyl, naphthyl, methoxyphenyl, chlorophenyl, etc.; and Z1 and Z2 are identical or different and each represent the organic atoms ft5 necessary to complete a heterocyclic nucleus of the type appearing in cyanine dyes, that is to say containing a heterocycle with 5 or 6 atoms, capable of fixing a proton at a pH of approximately 6 to approximately 9. Examples of the nuclei particu

615 510

8

The most useful that Z1 and Z2 can represent are the benzimidazoles and the 3,3-di-alkyl-3H-indoles.

The cyanine dyes described above are conventional and can be synthesized according to well known methods such as those described in the work of Mees and James, entitled "Theory of the Photography Process" 3rd edition - pages 206 and 207.

The other useful dyes are those substituted by a nitro radical, and which can lose a proton in basic medium; the formula of these nitro dyes is as follows:

, -Z-

! + <i t

A "4 = L-L. ■■ .1 I I Ç-CH - tn z

CH = CH — X

or:

(a) k equal to 0 or 1;

(b) m is 0 or 1;

(c) m equal to 1 or 2;

(d) each L represents an optionally substituted methine radical (for example -CH =, -C (CH3) =, etc;

(e) A represents an electron donor radical, such as an oxygen atom (-0-), a sulfur atom (-S-), or —N3—;

I

R

(f) E represents an electron acceptor group chosen from the class consisting of the nitro, cyano, ethoxycarbonyl and halomethyl radicals;

(g) £ is 0 or 1;

(h) R3 represents (1) an alkyl radical of 1 to 18 carbon atoms and preferably a lower alkyl radical of 1 to 4 carbon atoms; a sulfoalkyl radical and preferably a lower sulfoalkyl radical containing 1 to 4 carbon atoms in the alkyl radical; a carboxyalkyl radical, preferably a lower carboxyalkyl radical of 1 to 4 carbon atoms in the alkyl radical; a sulfatoalkyl radical, preferably a lower sulfatoalkyl radical of 1 to 4 carbon atoms in the alkyl unit; an alkoxyalkyl radical preferably a lower alkoxy lower alkyl radical of 1 to 4 carbon atoms, both in the alkoxy radical and in the alkyl radical; an acyloxyalkyl radical, preferably a lower acyloxy radical of 1 to 4 carbon atoms in the alkyl radical; an alkoxy carbonylalkyl radical, preferably a lower alkoxy carb-oxylalkyl radical of 1 to 4 carbon atoms both in the alkoxy radical and in the alkyl radical; a dialkylami-noalkylene radical, preferably where each of the alkylamino radicals and the alkylene radical are lower radicals of 1 to 4 carbon atoms; a cycloaminoalkylene radical and preferably a lower cycloaminoalkyl radical containing 4 to 6 atoms in the cycloamino radical and from 1 to 4 atoms in the alkyl radical; and (2) an alkenyl radical (including a substituted alkenyl radical) preferably a lower alkenyl radical of 2 to 4 carbon atoms, or else (3) an aryl radical (including a substituted aryl radical), for example phenyl, naphthyl, tolyl, xylyl, halo-phenyl such as p-chlorophenyl, g-bromophenyl, etc., alkoxy-phenyl such as methoxyphenyl, 2,4-di-chlorophenyl, etc; and an aralkyl radical, preferably an aryl radical, lower alkyl of 1 to 4 carbon atoms in the alkyl unit, for example, benzyl, p-phenethyl, omega-phenylbutyl, etc. ;

(i) X © represents an acid anion;

(j) Z3 represents the atoms necessary to complete an aryl ring (preferably phenyl or naphthyl) which, due to the presence of one or more nitro substituents and of the substituent "E" is substituted by a nitro radical in the ortho position and / or in the para position and can also be substituted in the ortho or para position by another electron accepting radical; Z3 can carry other substituents and be attached to other carbocycles, and;

(E).

• (KVn

(k) Z4 represents the organic atoms necessary to complete a heterocyclic nucleus encountered in the formula of cyanine dyes and containing 5 to 6 atoms in the heterocyclic nucleus with an electron donor atom conforming to the formula; the heterocyclic ring can contain a second heteroatom such as oxygen, nitrogen, selenium or sulfur. Preferably, the heterocyclic ring is a thiazole, benzothiazole or naphthothiazole ring, substituted or not; a substituted or unsubstituted oxazole, benzoxazole or naphthoxazole ring, etc. ; a selenazole, benzosélénazole or naphtosélénazole nucleus, substituted or not, etc. ; thiazoline, 2-pyridine, 4-pyridine, 3,3-dialkylindolenine, imidazole, quinoline, imidazo [4,5-b] quinoxaline nuclei (as described in US Patent 3,431,111); a 3H-pyrrolo [2,3-b] Pyridine ring; or a thiazolo [4,5-b] quinoline ring; a pyrylium, benzopyrylium, thiapyrylium or benzothiapyrylium nucleus or a dithiolinium nucleus.

Although some of these dyes are not light stable, this is not a significant defect in this case, since the reading must be instantaneous, the subsequent discoloration of the dye which could interfere with ambient light thus having little chance to interfere with the measurement.

These nitro dyes can be made by well known conventional methods.

Other useful indicator compounds are the phta-40 leïne dyes of the following formula:

HO

s *

R

C = 0

where R4, R5 and R6 are identical or different and are chosen from the group formed by halogens, hydrogen, alkyl and alkoxy radicals of 1 to 5 carbon atoms, Z4 represents the atoms necessary to complete a radical allowing to form a ring chosen from a naphthalide and a phthalide or else from the compounds of formula:

9

615 510

OH

OH

'© X-CH = (CH-CH * CH

(R ') OH jNH3

V

N (CH3) 2

'C-CH = (CH-CH) = CH-v / \ n

N 0

V

(CH3) 2

where R4 and Z4 have the meanings given above.

The base decolorizable dyes which are useful according to the invention include styryl class dyes and pH sensitive pyrylium dyes. The styryl dyes become colorless by attack of the conjugated chain in an alkaline medium, according to the following mechanism:

where Z5 represents the atoms necessary to complete a 3H-indole 3.3-disubstituted nucleus or a similar condensed nucleus; the substituents at 3.3 of the nucleus can be identical or different radicals, lower alkyl containing from 1 to 5! carbon atoms, or aryl containing from 6 to 12 carbon atoms; these substituents can together represent the atoms necessary to form an alicyclic radical; n is 0.1, or 2; and R7 represents an alkylene radical containing from 2 to 3 carbon atoms, such that R7-OH is a hydroxy-i propyl group, for example, ß-hydroxyethyl, 5-hydroxylpropyl, etc.

The production of an indicator by discoloration of a pyrylium dye in the presence of a base is done by elimination of an oxygen atom charged with the dye, so as to form either a spiropyran or a pyridine.

i In the case of a spiropyran, we can write the reaction as follows:

where R8 and R9 represent alkyl radicals of 1 to 10 carbon atoms, or they represent the atoms necessary to complete an aromatic ring R10 and R12 represent the atoms necessary to form an alicyclic ring, or else they represent ur hydrogen atom or an alkyl radical from 1 to 5

carbon atoms and Ar 'represents an alkyl radical of 6 to 10 45 carbon atoms to which the hydroxyl radical is linked in the ortho position. In the case of the formation of a pyridine, the reaction can be written as follows:

NH „

r8 ^ O / \ R ©

12

R

R

12

where R8, R9 and R12 each represent an alkyl radical or an aryl radical of 1 to 10 carbon atoms.

The diazonium salts must be placed in the presence of a colored coupler in a basic medium to form a dye; one can for example operate in the presence of NH3. Many diazonium salts are known to specialists as dye formers, in particular the benzene diazonium salts of formula:

where M represents one of the following possibilities: 1. a hydrogen atom,

615 510

10

2. a halogen atom,

3. an aryl radical,

4. an amino radical, including the substituted amino radicals, which may be cyclic, including the nitrogen atom of an amino radical and the other heteroatoms such as oxygen, sulfur, nitrogen, etc. ,

5. a mercapto radical, or

6. an alkyl or aryl thioether radical and,

X "represents an acid anion.

These compounds, on one or more of the carbon atoms of the benzene ring, can carry, for example, at least one of the following substituents: a halogen atom, the alkoxy, acyl, carbamyl, carboxyl or nitro radicals, the chain alkyl radicals straight or branched from 1 to 8 carbon atoms, such as methyl, ethyl, isopropyl, t-butyl, n-pentyl, octyl, etc.

Particularly useful diazonium salts include the p-aminobenzenediazonium salts according to the formula described above, in which M is either an amino or substituted amino radical, or a thioether radical as described above, and in which the nucleus Benzene is optionally substituted in one of positions 2 or 5 with either an aliphatic alkyl radical or an alkoxy radical. This class of useful diazonium salts can be represented by the following formula:

Q "D

in which:

1. D represents a sulfur atom or a radical of formula NQ4,

2. Q3 considered alone represents a hydrogen atom when D represents NQ4, or a lower aliphatic alkyl radical, a lower alkoxy radical an acyl radical of formula -C-T in which T represents an aryl radical,

II

O

or an alkyl radical as described here, or else a phenyl radical when D represents a sulfur atom or an NQ4 radical,

3. Q4 taken separately, represents a hydrogen atom, a lower alkyl radical or a lower alkoxy radical, so

4. Q3 and Q4 taken together, complete a divalent radical of formula:

_ (_ CH2—) a — Ob - (- CH2—) c—, ss where b is 0 or 1, a and c each represents an integer such that the sum of a, b and c is equal to 5.

in which:

1. Q5 and Q6 each represent a lower alkyl radical when taken separately,

2. Q5 and Q6 taken together, represent the number

; carbon atoms and oxygen heteroatoms needed to complete a morpholino radical,

3. Q7 and Q8 represent a hydrogen atom, a lower alkyl radical, or a lower alkoxy radical, and

4. X2 © represents anions chlorozincate, fluoroborate,) sulfate, phosphate, or chlorostannate.

More particularly suitable benzene diazonium salts are the fluoroborates where:

1. Q7 and Q8 represent radicals when Q5 and Q6 complement a morpholino radical, and

2. Q7 and Q8 each represent a hydrogen atom when R5 and R6 each represent a lower alkyl radical.

Couplers can be reacted with the diazonium salt to form an azo dye. These couplers are various chemical compounds such as those described in Kosar's book i, entitled "Light-Sensitive Systems", published by Editions John Wiley and Sons, INC., New-York (1965), p. 220-240. Phenolic couplers are particularly useful, the most common classes are:

(1) 2-hydroxy-3-naphtanilides, as blues-forming couplers, having the formula:

CONH

where Q9 represents a phenyl radical and preferably a phenyl radical substituted by at least one lower alkyl radical or a lower alkoxy radical, or a halogen atom;

(2) ortho-naphthalenediols, as blue-forming couplers,

(3) 1-hydroxy-2-naphthamides as yellow-forming couplers, having the formula:

.10

5. Q1 and Q2 each represent a hydrogen atom, a lower aliphatic alkyl radical (preferably a methyl or ethyl radical) or a lower alkoxy radical (preferably methoxy or ethoxy) and,

6. X1 © represents an acid anion.

Particularly useful g-aminobenzenediazonium salts include the substituted aminobenzenediazonium salts of formula:

CON.

where (a) Q10 and Q11 taken separately, each represent a hydrogen atom, an optionally substituted alkyl radical, such as those already described, a cycloalkyl radical, an aryl radical, etc; substituted or unsubstituted hydrocarbon radicals;

(b) Q10 and Q11 taken together, represent the carbon and oxygen atoms necessary to complete a heterocyclic piperidino or morpholino radical with six atoms, and

11

615 510

(4) phenols substituted in position 5 by a 2-acylamino radical, as yellow-forming couplers and having the following formula:

HCOQ

13

where Q12 represents an alkyl radical or an alkoxy radical, Q13 represents an aliphatic alkyl radical, an aryl radical, an aralkyl radical or an aralkoxy radical.

The indicator layer comprises a suitable binder and indicators, the binder being present in an amount of 1.0 g / m2 to 20.0 g / m2 and preferably from 5.0 g / m2 to 15 g / m2. The content obviously depends on the thickness of the product. The content of indicator must be such as to make it possible to dose the constituent of the sample analyzed over a fairly wide interval. In the case of nitrogen analysis of blood urea, the urea content expressed in mg of nitrogen per dl of blood is between 0 and 150. The indicator must therefore operate within this interval and for this, its content should be between 3.0 X 10 "4 mole / m2 and 1 X 10" 2 mole / m2, the preferred content being between 5 X 10 "4 and 9 X 10" 4 mole / m2.

All the aforementioned substances can optionally be applied on a support, if the layers cannot themselves play the support role.

If a support is used, it can be made of a substance impermeable to liquids, and also capable of transmitting radiation (preferably light). Different polymeric substances are suitable for this purpose, such as, for example, cellulose acetate, polyethylene terephthalate, polycarbonates or polystyrene. The thickness of the support, usually is between 0.05 and 0.25 mm.

As described in more detail below, it is desirable that the medium is capable of transmitting at least certain wavelengths of the electromagnetic spectrum in the range 200-900 nm. According to a particularly preferred embodiment, it is desired to use a support which selectively transmits radiation of certain specific wavelengths in a relatively narrow interval, and which is opaque to all other wavelengths. In the case where the assay product is fluorescent, the support must not be so fluorescent as to interfere with the measurement.

The layers described above are applied directly to a support, or an interlayer having energy transmission characteristics similar to those of the support can be used to make the connection between the indicator layer and the support.

According to a variant, the product does not include a separate support, particularly in cases where at least one of the other layers fulfills this function itself.

To obtain products according to the invention, the layers can first be formed separately, then applied to the support to obtain a product with an indivisible structure. According to this method, the layers are usually applied from a solution, or from a dispersion on a surface from which they can be physically separated after drying. However, an appropriate method which can avoid the operations of separation and superposition, consists in applying an initial layer on a lamination surface or support, if it is present, then in applying successive layers directly on those applied previously. Such compositions can be applied

by hand, or by using a blade coating device, or with a machine, or by using techniques such as dipping or meniscus coating. If machine coating techniques are used, it is often possible to apply adjacent layers simultaneously, using well-known dies to prepare emulsions and photographic papers. The adhesion problems can be solved without harmful effects, by surface treatments, or by the application in thin layer of sub-Ki tage substances such as those used for photographic films.

If the layers already described are formed by applying solutions or dispersions as described in Belgian patent 801,742, it is often necessary to introduce coating aids to impart uniform coating properties to the different layers.

Whatever coating aids are used for this purpose, it is important that they are not harmful to the enzyme in the enzyme layer or to any indicator present in the indicator layer. Particularly useful sleeping aids include non-ionic surfactants such as octylphenoxypolyethoxyethanols, (g-nonylphen-oxy) -glycerol and polyethylene glycols.

A product according to the invention can comprise layers which are not initially contiguous and which, in addition, can be spaced apart by interlayers described, for example, in United States patent 3,511,608, or by deformable supports, or elastic absorbent substances as described in US Patents 3,917,453 and 3,933,594. If the layers of the product are not initially contiguous, it may be necessary to apply a pressing force, or any other suitable means, to bring the layers of this product into contact with a fluid, at the time of use.

When using the product, or the above analysis ribbon, one can first separate the red blood cells from the serum by suitable methods such as by centrifugation, then apply the serum to the product. A particular significant advantage of the product for analysis described in the invention lies in its ability to be used to analyze serum or 4o of blood. Naturally, when energy transmission techniques are used, the spreading layer and the other layers must be uniformly permeable to detection radiation, unless a mechanism is provided to remove the undesirable constituents from the blood (for example example in 45 removing the spreading layer before the measurement); but it is preferable to use serum, obtained by any method, for the analysis.

A conventional automated analysis system with which the product for analysis with several layers according to the invention can be used, comprises the means, for placing the product below a dispenser which applies to the surface of this product, a drop of 'a sample, for example of blood or serum, then, to direct the product to one or more treatment stations. The analytical measurement is usually made by passing the product through a station which includes an appropriate device such as a spectrophotometer for measuring the reflection, the transmission, or the fluorescence.

The examples below illustrate the invention.

60 Examples 1-3

3 products are prepared for the determination of nitrogen from urea present in the blood, for each product, a different chromogenic substance is used in the indicator layer, the structure is that of FIG. 1.

65 On a polyethylene terephthalate film support, an indicator layer containing the desired chromogenic substances is applied from a mixture of organic solvents comprising mainly dichloroethane and acetone.

615 510

12

gnées under numbers 1 to 3 in Table I and 8.55 g of cellulose acetate butyrate per m2. The indicator layer is permeable to ammonia and relatively impermeable to water. On each layer, apply from a solution in t-butyl alcohol, a layer permeable to ammonia and relatively impermeable to water, containing 0.84 g / m2 of cellulose valero propionate (rate in valeryl groups: 30%). Each product is provided with an overlay of tempered gelatin at pH = 7.5 (5.4 g / m2) which also contains 4300 U / m2 of urease, 0.05 g / m2 of dipotassium phosphate as buffer , 0.06 g / m2 of bisvinylmethylsulfonylether as a tanning agent, 0.32 g / m2 of polyethylene glycol oleic ether as surfactant and 0.40 g / m2 of tetrasodium salt of ethylenediamine tetracetic acid.

To complete products, a spreading layer is applied which is also reflective, comprising 6.6 g / m2 of cellulose acetate, 46.0 g / m2 of titanium dioxide and 2.69 g / m2 of oc -tylphenoxypolyethoxyethanol. This composition is applied in a layer from a solution in a 1/1 mixture of acetone and dichloroethane, combined with xylene (respective proportions of the mixture and of sylene: 6.4 / l).

These products are used in the following test: aqueous solutions of urea are prepared (containing less than 10 −5 mole of NH 3 per mole of urea) the content of which varies between 1 mg and 150 mg of nitrogen per dl. In the sera usually subjected to analysis for the determination of urea, the nitrogen content is between 10 mg and 20 mg per dl. The products of the samples are deposited at the appropriate wavelengths.

The results indicated in Table I are read after 10 minutes of incubation at 50 ° C.

Table I Ex. Chromogenic substance

Amount of chromogenic substance

Wavelength at which the measurement is made

Reflection density urea content mg / dl

1 L-methyl-2- (2,4-dinitrophenyl) -methylquinolei- perchlorate

mum

5 mg / dl 10 mg / dl 20 mg / dl 50 mg / dl 100 mg / dl 150 mg / dl

1.08 gm / m2 585 nm 0.62 1.06

2 6- (2,4-Di-nitro-phenyl) -6H-pyri-do [2, l-a] isoindo-lium perchlorate

1.08 gm / m2 610 nm 0.45 0.81 1.22 2.06

3 phenolsulfone-phthalene (sodium salt)

0.8 gm / m2 565 nm 0.13 0.20 0.44 0.71 0.90 0.96

Table I shows the usefulness of the product according to the invention. Given the high extinction coefficients of the chromogenic substances of Examples 1 and 2 (approximately 104), it is preferable to carry out a kinetic analysis, to use a shorter reaction time or to follow the offset of the absorption peak, for the determination of a nitrogen level (from urea in the blood) greater than 50 mg / dl.

Examples 4

A product is prepared for analysis as follows:

On a film support of polyethylene terephthalate, an indicator layer containing 3.78 g / m2 of ethyl-4-ethyl sulfonate is applied from a mixture of methanol and acetone (1/1) (2,6-dinitrophenylmethylquinoline, as chromogenic substance and 6.43 g / m2 of cellulose acetate. A barrier layer permeable to ammonia is then applied; this barrier layer comprises 0.8 g / m2 of an acetobutyrate of cellulose (rate in butyryl groups: 27% o rate in acetyl groups: 21%, applied from a solution in dichloroethane.

A substrate comprising 0.14 g / m2 of a copolymer of methyl methacrylate, of methyl methacryloyloxytrimethylammonium sulfate, of 2-hydroxypropyl acrylate and of 2-acetoacetoxyethyl methacrylate is then applied.

45 (40/20/20/20); this copolymer is applied from a solution in acetone.

An overcoat is applied comprising 21 g / m2 of deionized gelatin, buffered at pH = 8.0, 0.65 g / m2 of polyethylene glycol oleyl ether, 0.40 g / m2 of tetrasodium acid 50 EDTA salt, 5.4 g / m2 of N, N-bis- (2-hydroxyethyl) -glycine, 0.9 g / m2 of dithiothreitol, 0.28 g / m2 of disodium orthophosphate, 27 U / m2 of urease, and 0.13 g / m2 bis-vinylsulfonylether.

Then, apply a substrate consisting of 0.32 g / m2 of poly (N-isopropyl-acrylamide), from a solution in acetone and finally a reflective layer also ensuring the spreading function, as in Example 1.

This product is used as follows:

7/100 aqueous solutions of albumin 60 are prepared containing amounts of urea corresponding to nitrogen values of between 0 and 150 mg / dl. We apply samples of 10 (il of these solutions to the product. To read the density measurements by reflection, we use a spectrophoto-meter equipped with an interference filter covering a 30 nm ft5 band centered at 463 nm, a Kodak Wratten 2B filter, and a neutral filter.

The measurements are made at 40 ° C. after an incubation time of 5 minutes. The following results are obtained:

13

615 510

Amount of nitrogen from urea in mg / dl in a 7/100 albumin solution

0 10 25 50 100 150

Density by reflection (after incubation for 5 minutes at 40 ° C)

0.04 0.26 0.57 0.97 1.69 2.33

Example 5

An ammonia permeable and waterproof layer is prepared, which contains a diazo coupling system as an indicator.

On a 0.18 mm thick polyethylene terephthalate film support, provided with a gelatin substrate, a layer is applied which contains:

(1) 3.19 g / m2 of a cellulose acetate butyrate, 27% in butyryl groups and 21% in acetyl groups;

(2) 0.635 g / m2 of 2-nitro-4-piperi-dinobenzenediazonium hexafluorophosphate;

(3) 0.84 g / m2 of 3 (2-methoxyphenylcarbamoyl) -2-naphthol and

(4) 0.097 g / m2 of polyethylene glycol 1,540 oleyl ether.

This light cream product is used with ammonium hydroxide solutions corresponding to amounts of 10 and 40 mg percent in urea. The formation of light green and dark green colorings is obtained respectively.

The nitrogen contents of these ammonium hydroxide solutions are equivalent to those of a blood comprising the urea contents which are usually metered. This example therefore shows the possibility of using this indicator system for such dosages.

Examples 7-16

A product similar to that of Example 1 is prepared, except that for the indicator layer, a layer comprising 2.26 g / m2 of ethane sulfonate of 4 (2,6-dinitro-4-chlorobenzyl) 1 is used. propyl quinolinium in a 40% cellulose acetate in acetyl group; in this way, the indicator layer also fulfills a barrier function. In addition, a separate barrier layer formed by a cellulose acetate butyrate 27% in butyryl groups and 21% in acetyl groups is applied, between the indicator layer and the enzyme layer. The latter is buffered at pH = 8.0, approximately.

The product is used with aqueous solutions of albumin and urea whose 4 concentration values are staggered between 15 and 115 mg of nitrogen per dl. Samples of 10 µl of these solutions are deposited.

The densities are read by reflection at 670 nm, after 5 minutes. The results obtained are reported in Table II.

Table 11

Amount of urea (mg of nitrogen per dl)

Example 6

A layer permeable to ammonia and impermeable to water is prepared containing, as an indicator, a dye which is removable by ammonia.

On a film support of polyethylene terephthalate 0.18 mm thick, provided with a gelatin substrate, a layer is applied containing:

(1) cellulose aceto-butyrate at 27% in butyryl groups and 21% in acetyl groups (3.22 g / m2);

(2) 0.322 g / m2 of 2 [ß- (2-hydroxy-1-naph-tyl) -a-methyl-vinyl] -1-benzopyrylium perchlorate.

This dark blue product is used with ammonium hydroxide solutions, the nitrogen concentrations of which are equivalent to urea contents of 10 and 20 mg per cent.

In the areas where the less concentrated solution is deposited, the dark blue coloration is partially discolored, while this coloration disappears completely in the areas where the more concentrated solution has been deposited.

Example

15.2

30.0

74.3

114.5

7

0.4251

0.6567

1.3280

8

0.4156

0.6475

1.3433

1.8942

9

0.4147

0.6403

1.3090

1.8859

10

0.4151

0.6627

1.3152

1.9255

11

0.4234

0.6536

1.3566

1.9097

12

0.4113

0.6546

1.3171

1.8790

13

0.4099

0.6559

1.3106

1.8739

14

0.4121

0.6428

1.3230

1.9024

15

0.4157

0.6586

-

1.8650

16

0.4094

0.6549

1.3335

1.8800

M "1

0.4149

0.6528

1.3268

1.8906

s <2>

0.0054

0.0071

0.0163

0.0192

s. XI00

1.30

1.08

1.23

1.01

M

(1) arithmetic mean M (2) estimate of the standard deviation S

1 drawing sheet

Claims (11)

615 510 2 1. Method for the analysis of a constituent in a fluid sample, using (1) a first substance capable of giving, with the constituent, a reaction leading to the formation of a decomposition product and (2) of a second substance capable of giving, with this decomposition product, or with a transformation product of the latter, a reaction generating a detectable modification, characterized in that the first substance and the second substance are incorporated into a product with an indivisible structure in which they are separated by a barrier having a uniform and selective permeability for the decomposition product. 2. Method according to claim 1, characterized in that the first substance and the second substance are each incorporated in a separate layer between which a barrier has been placed. 3. Method according to claim 2, characterized in that the barrier is contiguous both to the layer containing the 2 »first substance and to the layer containing the second substance. 4. Method according to claim 2, characterized in that the first layer comprises an interception zone of the sample, in registration with an interception zone of the barrier. 5. Method according to claim 1, characterized in that one of the two substances is incorporated into the barrier. 6. Method according to claim 1, characterized in that urease is used as the first substance and, as 3 »second substance, a reagent capable of reacting with ammonia to give a detectable substance. 7. Method according to claim 6, characterized in that a barrier is used which contains a polymer chosen from the class consisting of cellulose valero-propionate, cellulose acetate butyrate, cellulose acetate and polymethyl methacrylate. 8. Method according to claim 1, characterized in that a dye precursor is used as the second substance, capable of providing a dye in the presence of ammonia. 40 9. Method according to claim 8, characterized in that a leuco derivative capable of undergoing deprotonation in the presence of ammonia is used as a dye precursor. 10. Process according to claim 1, characterized in that a bleachable dye 45 is used as the second substance in the presence of ammonia. 11. Product for implementing the method according to claim 1, comprising said first and second substances, characterized in that the first substance and the second substance are incorporated into a product with an indivisible structure in which they are separated by a barrier with uniform and selective permeability for the decomposition product. 55