DE2332760B2 - MATERIAL FOR THE QUANTITATIVE SPECTROPHOTOMETRIC ANALYSIS OF A LIQUID - Google Patents

Description

The invention relates to a material for quantitative specrophotometric analysis of a liquid, best s consisting of several layers arranged on a layer support.

In the past few years a number have been automated Methods for performing quantitative chemical analyzes of liquids have become known, which have proven particularly beneficial for clinical laboratories, especially for Blood analysis.

For example, from US Pat. No. 2,797,149 and 3,565,582 continuously operating analyzers become known, which consist of a multitude of containers in which the samples to be examined, Diluents and test reagents stored, mixed together and analyzed spectrophotometrically will. The liquids to be tested, diluents and test reagents are thereby carried out the analyzer is passed through a complex system of tubes or tubes. Disadvantageous at these continuously operating analyzers are that their structure is extremely complicated, that they are expensive to manufacture and that skilled personnel are required to operate them. Another disadvantage is that the analyzes to be carried out are very time-consuming, and that the cleaning of the Devices is very cumbersome and that the devices do not allow the analysis of very small ones Allow amounts of material to be tested, such as those used, for example, to carry out microanalytical Test procedures are sufficient.

To the disadvantages of this known, continuously 4s to overcome working analyzers, automated analyzers have become known, using a material in the form of an endless analytical tape is, on which the material to be analyzed is applied, and which onet analysis of the Allow applied material in a spectrophotometric way, namely by coloring Reactions between components of the test item to be examined and test reagents that are on the tape ss applied or contained therein. Tape-shaped materials for analysis are, for example, from GB-PS 10 49 364 known. A material shown in Fig. 4 of the patent has cutouts or recesses in which measured quantities of the liquid to be analyzed and the test reagent be introduced. The material shown in Fig. 9 of the Palentcription consists of a plurality of Ribbons in which a filtration or reaction occurs. From US-PS 30 36 893 it is also known <> to carry out such automated analysis processes to use a plurality of separate tapes which have the task of to absorb the liquid under investigation, to spread it out and to filter it and use it as a reaction medium to serve. In the analysis method known from US Pat. No. 3,036,893, the material to be analyzed is brought to a tape and then transferred from this to another tape. From US-PS 35 26 480 is Furthermore, a so-called analytical tape is known which has sections showing the measured quantities a test reagent and to which the material to be analyzed is applied.

The known analysis methods which use such endless analysis tapes are natural much simpler than the known flow analyzers. However, the well-known analysis tapes also have analysis methods using the disadvantages which make their use and dissemination strong have hindered. This is the case, for example, with analysis tapes that do not have the test reagent itself and which are mainly used to transport the sample to be analyzed through the device, such as it is the case in certain embodiments of GB-PS 10 49 364, disadvantageous that the to be analyzed Sample and test reagent in separate operations onto the tape at the right time and must be applied in the correct quantities.

Furthermore, the use of multiple tapes with different functions, e.g. B. with the function the spread of the sample to be tested, with the function of filtering the sample, and with the function, carry out the implementation of the compound to be analyzed with the test reagent, for example from US-PS 30 36 893 and from GB-PS 10 49 364 is known to that the process is complicated. Also the use of analytical tapes of a complex nature which are difficult to manufacture and expensive, e.g. B. the Use of the tapes known from US-PS 35 26 480, the spread of these tapes has been strong with special needs.

The object of the invention is to provide a material for the quantitative spectrophotometric analysis of a liquid indicate that is suitable for performing automated spectrophotometric analyzes, a has a simple structure, is easy to manufacture and with which a multitude of different analyzes in one continuously operating analyzer can be carried out in a simple and effective manner can.

It has been found that the problem can be solved with a material that consists of several on one Layer support arranged layers consists and characterized by the combination of the following features is:

1.) a liquid-impermeable, translucent layer support points to one of its two Pages at least one reagent layer with a reagent that is produced by reaction with a Part of the liquid to be analyzed, a color change within the reagent layer causes;

2.) a porous layer or a porous layer structure or the (a) on the layer support applied side of the reagent layer are arranged, (b) for the color-forming reaction with the The constituents of the liquid to be analyzed that are included in the reagent are permeable and (c) the Applied liquid spreads laterally in such a way that a constant volume of liquid per

Area unit is recorded and arrives at the reagent layer.

According to a particularly advantageous embodiment of the invention, the material according to the invention has the In the form of an endless strip or ribbon. However, it can be in another form, for example in In the form of a sheet of paper or a section of strip or in the form of small sections that appear on cards are attached, e.g. B. on cards with openings.

An essential feature of a material according to the invention is thus a reagent layer, which the for Execution of an analysis contains necessary reagents, so that when using one material only ensure that the sample to be analyzed is correctly applied to the material. To apply the sample to be analyzed can be automatically operated distribution or application devices the precisely measured quantities of the liquid to be analyzed at the correct point in time and apply to the right place on the material. Such automatically operating devices are known. With the help of such a device and a material according to the invention, quantitative Carry out various types of analysis, e.g. B. the glucose content of the blood can be analyzed by the treated material is fed to a conventional spectropholometer.

The material according to the invention is a material that all of the necessary to carry out the analytical process has required layers, in contrast to the known automated. Prior art systems using analyzer tapes in which two or more tapes are temporarily are brought into contact with each other and which are later separated from each other again.

Since the material of the present invention has a structure in which the substrate and various Layers are connected to each other, and as it is only required when using the material is. to apply the sample to be analyzed to the upper layer of the material and the material through a Carrying a spectrophotometer is what carrying it out an automated analysis required device is not expensive.

"Color-forming reactions" are also to be understood here in those cases in which the color-forming reactions within the reagent layer consist of reactions in which fluorescent compounds are generated.

For the production of materials according to the invention, the most varied of translucent, for Liquids impermeable substrates can be used, e.g. B. from cellulose acetate polyethylene terephthalate polycarbonates and polystyrene The thickness of the Support can be very different. It is expediently between 0.0508 and 0.254 mm.

The reagent layer can be applied directly to the layer support or to a translucent adhesive or intermediate layer which improves the bonding of the reagent layer to the layer support. The reagent layer contains the test reagents which enter into a color-forming reaction with components of the sample to be analyzed. The reagent layer can, for example, consist of a dispersion of one or more test reagents in a hydrophilic colloid that serves as a binding agent, for example gelatin or polyvinyl alcohol.

The test reagents of the reagent layer used in the individual case naturally depend on the material to be analyzed in the individual case and the color-forming reaction to be brought about in the individual case, which is used to identify the substance to be analyzed.

s was chosen.

There is or are on the side of the reagent layer that is applied to the support several layers, which have the function of uniformly spreading the applied liquid laterally

ίο direction to spread out.

This layer or these layers can optionally also have the task of from the analyzing sample to filter off constituents, the occurrence or the measurement of the color-forming

would interfere with ι s reaction.

This layer or layers can also have the function for the purpose of Carrying out a reflection spectrophotometric analysis of the light that has passed through the support reflect.

A material according to the invention thus has at least two layers in addition to the layer support, one of which is a reagent layer and the other is a layer which has a uniform lateral distribution the applied sample and possibly also has the other other functions mentioned. However, it is also possible to arrange a special layer for each individual function, in which case a material according to the invention besides the layer substrate

jo ger has, for example, four layers. on the other hand One layer can also perform two or three functions and another layer can perform the third function. It is also possible to use more than one layer for a specific purpose, i.e. h_ it is example

3s possible, two or more adjacent ones Layers to be used as reagent layers.

The layer with the spreading function is the layer furthest away from the substrate and the layer on top which the sample to be analyzed, e.g. B. is applied to a blood sample. Typically, that becomes too analyzing sample on the layer with the spreading function by means of an automatically operating distribution device applied, which is constructed in such a way. that they put a small drop of liquid on the able to apply the desired location on the top layer of the analysis element.

The layer with the spreading function is intended to distribute the drop uniformly in the lateral direction. The importance of this layer is to prevent the tenders of a "ring" formation in the reagent layer of the material. The phenomenon of the "ring" formation is for example in US-PS 30 36 89! and 35 26 480. If, accordingly, drops of a sample to be analyzed are applied directly to a reagent layer, they form a "ring" through lateral formation, with a reduced concentration of test reagent in the center of the ring and an increased concentration in the periphery of the ring. This leads to an imbalance

shaped color development, which in turn prevents quantitative measurement by spectrophotometric means

In the case of a material according to the invention thus practically the entire lateral movement di

Sample in the layer with the spreading function, so there when the sample reaches the reagent layer it does not spread further laterally and is not unevenly shaped ge distribution of the test reagent takes place.

Affected in the case of a material according to the invention a change in the volume of sample that is applied to the material, the diameter of the generated color stain in the reagent layer. The volume of liquid per unit area that the reagent layer reached, but remains practically constant regardless of changes in the volume of the applied sample. As a result, the density of the hue produced in the reagent layer by the color-forming reaction is generated by changing the size of the material applied Droplets are not noticeably affected and the hue is only dependent on the concentration of the component that enters into the color-forming reaction. As a result, when using a according to the invention material not required. To apply drops of the same size to the material or to determine the size of the drops applied in order to be able to carry out a quantitative analysis.

If the porous layer or a layer of the layer structure or the layer structure has the function of a filter layer, this means that components are to be filtered off from the sample which would interfere with the color-forming reaction in the reagent layer or which would hinder the spectrophotometric measurements to be carried out later. For example, in the case of a material intended for the analysis of blood specimens, a filter layer can serve to remove red blood cells but allow the serum to pass into the layer below. Such a filter layer can be constructed from any material which has a sufficient degree of porosity for the sample to be analyzed, the optimum porosity depending on the particular application of the analytical material. If the material according to the invention, for. B. in the form of an endless tape, are used for the spectrophotrimetric analysis of blood, it has proven to be useful if a filter layer is present. which has a pore size of 1 to 5 microns and is able to freeze blood cells, which are typically about 7 to 30 microns in size and which allows the serum to pass through. A filter sheet with a pore size that is too small. may limit the usefulness of the material, for example because it prevents the passage of blood proteins in the case of such analytical tests where it is important. that these blood proteins reach the reagent layer.

Is a material according to the invention for spectrophotometric analysis using reflection techniques are applied, it may contain a layer which has a function of a reflective layer and a suitable background for spectrophotometric measurements through the support side of the Material forms

The reflective layer represents a porous layer, thus components of the sample to be analyzed can pass this layer and get into the reagent layer. The reflective layer should be white to provide an effective background for the reflectance measurements.

In the case of a material according to the invention for the Analysis of blood will block the passage of blood cells through the filter layer, which is why they are the do not interfere with spectrophotometric measurements, as these are carried out through the layer support reflective layer serves as a white background.

When using an unused material according to the invention that is intended for reflection measurements Type, the photometer only records about 1% of the energy hitting the material. This 1% incident radiation then serves as a comparison value (100%) for subsequent reflection measurements with the Material to which a sample has been applied.

When evaluating the same material in the case of continuity measurements, the photometer records approximately 1.5 to 2% of the energy incident on the multilayer material. If the thickness of the above The spreading layers described are reduced by about half, so the permeability increases about 4% and in the case of materials with microcrystalline cellulose spreading layers of a thickness of about 100 to 300 microns the transmission increases to about 20% of the incident radiation. Consequently better signal-to-noise values are obtained if the permeability method is used as the determination method is used, which is why it may be more advantageous in many cases to use this method of determination apply.

Apart from the described spectronhotometric Determination methods can advantageously also use other methods of spectrophotometry are used to carry out the analytical procedures. Mention should be made, for example, of the Fluorescence spectrophotometry, in which case the reaction product of the sample with a test reagent consists of a fluorescent compound and the determination of the fluorescent compound with a Radiation occurs, which causes the reaction product to fluoresce. Other forms of spectrophotometry. which can be applied are those to which radiation is applied that of visible light is different. z. B. ultraviolet radiation or infrared light.

An example of a layer which has the function of a spreading layer and a filter layer and which also serves as a reflective layer at the same time is a so-called "blush polymer layer". Such layers can (cf. HF Payne "Organic Coating Technology". Verlag John Wiley and Sons. New York. 1954, Volume 1. Page 414) be produced on a carrier by dissolving a polymer in a mixture of two liquids, the one liquid being a good solvent for the polymer. whereas the other liquid, which has a comparatively higher boiling point, is a non-solvent for the polymer or at least a bad solvent, whereupon the polymer solution is applied to the carrier and dries up ; Polymer to the extent in the liquid. which is a poor solvent or a non-solvent for the polymer in that evaporation proceeds, which leads to the polymer being precipitated in the form of fine particles and forming an adhering porous layer on the support. For the production of such polymer layers the most varied of polymers can be used, for. B. Polycarbonates, polyamides and cellulose esters.

Instead of a "blush polymer layer" that dii Functions of specimen spread and filtratioi as well as the necessary reflection background for reflection measurements can be a ή correspondingly effective layer dadurc!

ίο

produced by placing a thin layer of a microporous filter membrane on top of the reagent layer laminated on. Such filter membranes consist of so-called "blush polymers". z. B. from cellulose esters, and contain microscopic pores. A large number of materials are used to produce such layers various pore sizes available in stores.

If the according to the invention: material has a single layer. which has the function of the sample spreading layer, the function of a filter layer and the function of a reflective layer, the material can e.g. B. in the form of a tape, consist of only two layers and the substrate, since apart from the layer with the function of a spreading layer, a filter layer and a reflective layer, only the reagent layer is absolutely necessary

However, it can also be beneficial. To use materials that have a layer that acts as a filter layer and reflective layer, and a special layer that has the function of a sample spreading layer or that have three separate layers that have the functions of sample spreading, sample filtration or exercise the light reflection. In this F which the layers are so deposited on the substrate, that the sample-spreading layer most distant from the substrate layer is Foigt whereupon the filter layer and the reflective layer, and finally, the reagent ichicht

An example of a shaft, which is effective as well as a reflecting layer both as a filter layer. is a layer of tkandioxid or barium sulfate, dispersed in a binder, ζ. Β Cellulose acetate, polyvinyl alcohol or gelatin. Such a layer has been found to be particularly effective in a material intended for the analysis of blood because the material is able to effectively filter out the blood cells while the layer allows the serum to pass through, while at the same time providing an effective white background for spectrophotometric measurements through the support supplies

A particularly advantageous layer with the function of a spreading layer for the analysis of blood is a layer consisting of a dispersion of diatomaceous earth in a binder, e.g. B. cellulose acetate. Diatomaceous earth has proven to be extremely effective for the uniform distribution di: s proven blood in the lateral direction. Advantageous sample spreading layers can furthermore be produced, for example, from microcrystalline colloidal products which are derived either from naturally occurring or synthetic polymeric substances. Such microcrystalline substances are described, for example, by OA B a 111 sta in the article “Colloidal Macromolecular Phenomena. Part IL Novel Microcrystals of Polymers, published in the journal of Applied Polymer Science, Volume II, Seilten 481-498 (1967). Microcrystalline cellulose, which can be used to produce such layers, is also commercially available.

Advantageous results are furthermore obtained when a sample cutout layer is used which is composed of inert spherical particles of uniform size in a matrix of a binder which binds the particles to the underlying layer.Examples of such spherical particles are beads made of glass and polymer Synthetic resin beads. Gelatin and polyvinyl alcohol, for example, have proven to be advantageous binders for filling such spheres. The binder should be used in small quantities if possible, so that it is avoided that a substantial proportion of the pore volume that is generated by the spherical particles is filled.

A spreading layer made up of spherical particles of uniform size can have advantages irr] Compared to a porous polymer layer such as a so-called "blush polymer layer", for example point. Such a layer can, for example! a drop of blood becomes uniform extremely quickly] and reproducibly, and so quickly that the spreading or spreading is finished before | a diffusion of blood komno to be considered nents in adjacent layers of the invention, a ßen material can be done. As a result, a | very uniform concentration of the sample components is achieved in the reagent layer and a uniform concentration moderate discoloration which is the basis for analysis! forms. While blood cells occasionally cause clogging of porous polymer layers such clogging does not occur. if the Sample spreading layer consists of spherical particles in a matrix of a binder. By using The formation of spherical particles of uniform size thus gives particularly advantageous results since these particles create an adequate or sufficient volume of pores while they keep the gaps on a relam narrow area limit. This leads to a standstill in the expansion of the blood when the spaces between the area to which the sample has been applied are completely filled, with rapid drainage at the same time Plasmas into the underlying layer is enabled when the spreading process has been completed E It has been found that spherical particles of a size of about 80 to 120 microns are particularly advantageous are when the spreading layer is used for a blood analysis Propagation of blood droplets is used. A drop of blood applied to such a layer spreads in a few seconds to a uniform circular surface. which covered after spreading Area is directly proportional to the sample volume

Advantageously, the spreading layer can be a material according to the invention, for. B. in addition to the spherical particles, a small amount of a surface-active compound is added to further accelerate the spreading process

In addition to the layers described, a material according to the invention can also have further layers exhibit. For example, it is possible to go directly to to arrange a dialysis layer in addition to the reagent layer. Such a layer can be used, for example the analysis of the glucose content of the blood be of advantage, since such a layer allows the passage of High molecular weight proteins, which could interfere with the glucose analysis. For the separation of high molecular weight substances of low molecular weight substances For example, semipermeable ceflulose membranes are suitable. which are therefore suitable as dialysis layers smd. Such layers act according to a different mechanism than the porous polymer layers, such as. B. the "blush polymer layers" that mainly act like a sieve or grid, in which the pores allow the passage of solutions and however, allow small particles to pass through

block of particles of a larger diameter.

The materials according to the invention are suitable for carrying out a wide variety of chemical analyzes, not only in the field of clinical chemistry, but rather also in the field of chemical research in general and in chemical laboratories that monitor the course of chemical processes. The materials according to the invention are, however, particularly suitable for clinical examinations, specifically for the analysis of body fluids such as blood and urine, since a large number of analyzes often have to be carried out here, analyzes often have to be repeated and the analysis results often in a very short time Time after taking the sample must be available. In the case of a blood analysis, a material according to the invention, for example, in the form of a tape, can be designed in such a way that it can be used for the quantitative analysis of many blood components that have to be routinely determined. For example, a material according to the invention, for. B. in the form of a tape, easy for the analysis of such blood components as albumin, bilirubin, urea nitrogen, serum glutamine-oxaloacetic acid transaminase, chloride, glucose. Uric acid and alkaline phosphatase can be used, as well as resistors ^r e components of the blood by appropriate test reagents are selected.

In the blood analysis using a material according to the invention, for. B. first the Separation of the blood cells from the serum take place, for example by centrifugation, whereupon the Serum is applied to the tape. However, the separation of the blood cells is not necessary, the entire blood can be applied directly to the material, with the blood cells passing through the Filter layer are filtered off. The presence of these blood cells on the material disrupts the spectrophotometric Analysis not, for example, if it is carried out using the reflection method, since the Layer support and the reagent layer are permeable to light and the light from the porous reflective layer is reflected. There is a particular advantage of a material according to the invention thus that it can be used for the analysis of serum as well as for the analysis of blood.

A wide variety of test reagents can be used to produce the materials according to the invention, depending on which component or which components of a test sample to be analyzed are to be determined and depending on the color-forming reaction to be carried out.

In the blood analysis, glucose can be determined, for example, by the fact that d »e Reagent layer contains a ferricyanide or a ferricyanide compound and that the decrease in the yellow Color tone is determined, which takes place through conversion of the ferricyanide with glucose. Because high molecular weight Proteins in the blood can lead to a certain reduction in ferricyanide and thus interfere with the method for determining glucose Avoidance of occurring difficulties a dialysis layer of the structure already described: · η placed on the material.

When analyzing uric acid in the blood, the reagent layer can, for example, consist of a mixture of copper sulfate and neocupromium, ie 2S- dimethyl-1. 10-nhenanthroline of the formula:

cn,

H ₁ C

dispersed in a binder. the Uric acid causes a reduction of copper (II) to copper (I), which then merges with neocuproin colored complex forms, the color density being direct

ι s is proportional to the concentration of uric acid.

When analyzing the as serum glutamine oxaloacetic acid transaminase known enzyme, a sequence of reactions can be carried out, for example The aforementioned system catalyzes the conversion of glutamic acid to oxaloacetic acid at pH of 7.4 and the oxaloacetic acid can be through a coupling reaction with the diazonium salt of a Determine the dye known as "Fast Ponceau L" (Color Index No. C. I. 37 151). To the expiration of the first

to facilitate reaction equation is before the color-forming coupling reaction, it may be advantageous to use the reagents in two separate layers accommodate to ensure that sufficient for the sequence of the first reaction

ίο time is available before the second Reaction can take place. So the glutamic acid can be accommodated in a first reagent layer, the placed over a second reagent layer containing the Fast Ponceau L salt.

is a typical automatic analyzer, in which can be used a tape-shaped material according to the invention, typically has Device parts for = unwinding or unwinding the tape from a supply roll as well as device parts, which lead the tape past a sample distributor through which a drop of the to be analyzed Sample, e.g. B. a blood or serum drop, is applied to the surface of the tape, as well Device parts that the tape by one or

4s lead several development zones and finally device parts through which the tape is wound up by means of a take-up roller. In the development zones mentioned, the tape is subjected to conditions which facilitate the flow of a color-forming sample and a component of the reagent layer. This means that the tape is subjected to conditioning, for example heating or the action of water or another level of solution in order to accelerate or support the course of the desired reaction.

The analytical measurements are then carried out in that the tape is passed through a zone in which spectrophotometric measurements can be carried out, for example in the case of the use of reflection spectrophotometry a device by means of which a light beam is directed through the support, which is then returned to the indicating Part of the spec.rophotometer is reflected. When using reflection spectrophotometric

6s measurement methods will cause optical interference Residues, such as blood cells, which are on or remained in the layers of the tape, avoided.

Another spectrophotometric measurement method that can be used is fluorescence spectrophotometry. To generate calibration standards, drops of saturated solutions of the to to be applied adjacent to the area to which the drop is to be analyzed analyzing sample is applied, whereby differential measurements are made possible.

The materials of the invention can be made in various sizes. In case of Tapes, a typical tape has a width of 16 mm, for example, which is for example on a reel can be wound up and enclosed in a cassette.

The automated analytical systems can be designed to include only a single type of Band can be used or the device can be constructed so that the a wide variety of tapes can be used, either simultaneously or sequentially, with each tape contains the reagents necessary for the analysis of a specific component of the to be analyzed Sample are required. A single analytical tape can also be used to analyze two or more Components of a sample are used when there are test reagents for performing the analysis contains each of the two components, the test reagents being mixed with one another in a Reagent layers can be present or in two or more separate reagent layers, so long as only the reagents are chosen so that they do not cause the reaction of the other reagent disturb.

The reagent layer can optionally also be in the form of a plurality or multiplicity of parallel, adjacent Strips of various compositions are available, with each strip having one or more test reagents that are intended for performing an analysis of a specific component of the sample.

As already stated, a material according to the invention need not be in the form of an endless belt. Rather, for example, smaller sections can be attached to cards, in particular cards with openings, which can be passed through a sample application zone, one or more conditioning zones, e.g. B. Zones in which there is an action of heat or a solvent, and a zone in which spectrophotometric measurements are carried out. A large number of different materials according to the invention can be affixed to such a card, each material ¹ containing the reagent required for the analysis of a particular component of a sample, so that such a kanc can be used for carrying out a large number of tests.

Various embodiments are shown in the drawing materials according to the invention in section, enlarged, shown. In detail are shown in

F i g. 1 a material consisting of a layer support 10 with a reagent layer applied thereon 12, a reflective layer 14, which has a white background for reflection measurements through provides the support 10, a filter layer 16 and a sample spreading layer 18. The reagent / .layer 12 can, for example, consist of a dispersion of one or more test reagents in a binding agent, bcivr> ir> kwpi <; p Gelatin, insist while each of the Layers 14, 16 and 18 made of a so-called "blush polymer layer" can exist with a pore size which is adapted in each case to the function of the individual layer; F i g. 2 a material consisting of a layer support 20 with a reagent layer 22 and a layer 24, which has the function of spreading the sample to be applied and filtering it, and which further allows a suitable background for reflection measurements through the layer carrier 20. Layer 24

ίο can, for example, consist of a »blush polymer layer« which was applied to layer 22, or a layer of microporous filter membrane, which has been laminated to layer 22;

F i g. 3 a material composed of a layer support 30, a reagent layer 32, a dialysis layer 34, which consists of a semipermeable membrane layer was formed, and a layer 36, e.g. B. a "blush polymer layer" which has the function of spreading the sample to be analyzed and filtering the sample, and

jo which further provides a suitable background for Provides reflection measurements d.rch the substrate 30;

F i g. 4 a material consisting of the layer support 40, a first reagent layer 42. a second

: 5 reagent layer 44. a layer 46 serving as a filter layer and light reflective layer, and a sample spreading layer 48. Layer 46 may, for example consist of a dispersion of titanium dioxide in cellulose acetate and the layer 48 can, for example, consist of a dispersion of diatomaceous earth in cellulose acetate or of glass beads in gelatin.

The following examples are intended to illustrate the invention in more detail.

Be ι sρ ie I 1

A reagent layer consisting of a dispersion of copper sulfate and neocuproin in gelatin was applied to a 0.10 mm thick polyethylene terephthalate support in such a way that 0.84 mg of copper sulfate, 1.28 mg of neocuproin and 53.9 mg of gelatin were ^{applied to a support area of dm 2.}

A layer of microporous filter material 180 thickness was then placed on top of the reagent layer Microns and an average pore size of 1.2 microns. The filter material consisted of a commercially available microporous cellulose acetate nitrate membrane. The membrane was laminated on the reagent layer by making the reagent layer first exposed to steam for several seconds to soften it whereupon the filter layer was pressed onto the reagent layer. The two layers were then exposed to low pressure by means of a roller.

A strip-shaped material produced in this way is suitable, for example, for the determination of uric acid in the blood, the presence of uric acid being indicated by the appearance of a pinkish-red color Hue is displayed in the reagent layer.

do B e i s ρ i e 1 2

Another tape-like material for the determination of uric acid in blood was produced in the manner described in Example 1, with the exception (χ; however, that polyvinyl alcohol was used as a binding agent for the reagent layer instead of gelatin. The polyvinyl alcohol was used in a concentration of 43.1 mg / dm ² used.

Example 3

Another bandiormiges material for the determination of chloride in blood was prepared in the manner described in Example 1, with the exception, however, that the reagent layer consisted of a gelatin layer in which silver chromate was dispersed. 53.9 mg of gelatin and 18.0 mg of silver were allotted to a support area of 1 dm ^2. The presence of chloride in a sample to be analyzed is indicated in this case by a change in the color of the reagent layer from reddish-brown to yellow.

Example 4

According to the method described in Example 1, another tape-shaped material for the determination of albumin in blood was produced, this time a reagent layer was produced, which consists of 4.66 ng of hydroxyphenylazobenzoic acid, dispersed in 21.0 ng of a copolymer of acrylic acid and 2-acetoaceloxyethyl methacrylate, each based on an area of 1 dm ² existed. The presence of albumin in a sample to be analyzed results from the development of a pink-orange hue in the reagent layer.

Example 5

A reagent layer of 3.15 mg glucose oxidase, 3.95 mg peroxidase, 4.42 mg o-dianisidine hydrochloride and 215 mg gelatin was ^{applied to a 0.10 mm thick polyethylene terephthalate carrier, each per 1 dm 2 of} carrier area. Furthermore, a slurry was prepared by mixing 12.0 g of TiO ₂ and 50 ml of a 3% strength by weight solution of cellulose acetate in acetone and diluting by adding 80 ml of a mixture of equal parts by volume of acetone and xylene. The slurry was then applied to the reagent layer in a layer thickness corresponding to 300 mg TiO ₂ and 37.2 mg cellulose acetate, in each case per dm ² , whereby a layer was obtained which acted both as a filter layer and as a light-reflecting layer. A spreading layer of a slurry of diatomaceous earth, salicylic acid and cellulose acetate in a mixture of 1.2 parts by volume of dichloroethane and 1 part by volume of acetone was then applied to this layer.

There were three different tape-shaped materials using different concentrations made of diatomaceous earth in the spreading layer, with each produced band being based on its ability to draw blood spread has been studied. A 10 microliter sample of blood was applied to each tape. Determined became the time it took for the blood to completely diffuse into the ligament. Furthermore, the The diameter of the spot produced is determined through the substrate. It became the get the following results:

The tape-like materials described in this example are suitable for determining the Glucose content of the blood, the presence of glucose by the formation of a brown hue is displayed in the reagent layer.

Example 6

A reagent layer of the composition given in Example 1 was applied to a 0.10 mm thick polyethylene terephthalate support, whereupon a microporous filter layer of the type described in Example 1 was laminated onto the reagent layer. The microporous filter layer served as a filter layer and as a layer that provided a suitable background for making reflectance measurements. A spreading layer was then applied to the filter layer, including glass beads with a diameter of 0.08 to 0.012 mm in a ratio of 1 g of beads to 0.5 ml of a solution containing 2.5% by weight of gelatin and 0.01% by weight. % of a surface active compound, namely para-isononylphenoxypolyglycidol having 10 glycidol units, was mixed, and the resulting slurry was coated in the form of a thin layer on the microporous filter layer so that 1 g of beads was per an area of 27 cm ² .

10 microliters of blood was then applied to the material obtained, which was found to be 10 microliters of blood were spread in less than 1 second, to a circular area of about 1 cm in diameter.

Example! 7th

A reagent layer composed of 2.15 mg of glucose oxidase and 1.07 mg of peroxidase was first placed on a 0.18 mm thick polyethylene terephthalate support. 3.20 mg of o-dianisidine hydrochloride and 215 mg of gelatin, in each case per dm ^{2 of} layer support area, were applied. A microporous filter membrane, as described in Example 1, was then laminated onto the reagent layer.

The effect of the size of the sample to be analyzed on the surface and the reflection density were determined of the colored stain that was created in the reagent / M'hicht by the action of different sizes Samples of a glucose solution that are distilled per 100 ml Water contained 100 mg glucose.

The reflection densities were read 5 minutes after the samples were applied to the material. It the results compiled in the following table were obtained.

Sample amount
in microliters

Patch District
(mm ² )

30th
50
63
86
92

Reflection density

0.66
0.71
0.70
0.65
0.70

attempt
No.

Diatoms

C'ltc

Salicylic acid

Cellulose
acetate

time in seconds

(mg / dm ² ) (mg / dm?) (mg / dm ² )

1,300

2,375
T 450

3.00
3.75
4.50

37.2
37.2
37.2

Diffusion spots fto From the results obtained, it can be seen that the Reflection density values were practically independent of the size of the applied drop. The received Results show accordingly that a material according to the invention a quantitative analysis of a Component of a sample to be analyzed, without the need for a precisely defined amount of a sample is applied to the material.

26 41 31

(Spot) diameter in cm

1.05 1.00 1.00

Example 8
The following solutions were made:

(a) a solution of 3.23 g of agarose (polysaccharide) that solidifies at low temperature in 70 ml of a 0.3 molar 2-amino-2-hydroxymethyl-1,3-propanediol buffer solution;

(b) a solution made up of 108 international units from Glycerol dihydrogenase, 108 mg human serum albumin, 108 mg resazurin, and 54 mg Diaphorase;

(c) a solution of 54 mg nicotamide adenine dinucleotide in 10 m! a 0.3 molar 2-AmiriO-2-hydroxymethyl-1,3-propanediol buffer solution.

The solutions described were mixed with one another and then applied to a 0.254 mm thick cellulose triacetate layer support in such a way that an area of 1 m ^{2 was} covered.

The resulting reagent layer was then coated with a spreading layer of the type described in Example 1 coated.

Drops of an aqueous glycerol solution were placed on the surface of the material produced applied in various amounts from 50 to about 500 mg glycerine per deciliter. After drying the generated fluorescent areas were determined by means of a fluorescence spectrophotometer, wherein It was found that the fluorescence generated is proportional to the concentration of the sample solution applied glycerin was present. The fluorescent areas were excited with radiation a wavelength of 540 nm, with fluorescence occurring at 590 nm.

Example 9

Initially, the following solutions were made:

(a) a solution of 107.6 international units of glycerol dihydrogenase and 107.6 mg of human Serum albumin, dissolved in 20 ml of a 03 molar glycine buffer solution at a pH value of 9.6;

(b) a solution of 2,150 g of an agarose (polysaccharide) that solidifies at low ίο temperature in 70 ml of a 0.3 molar glycine buffer solution with a pH of 9.6;

(c) 504 mg of nicotamide adeninucleotide in 10.7 ml of a 0.3 molar glycine buffer solution of pH of 9.6.

Solutions (a), (b) and (c) were ^{mixed with one another at 40 °} C. and applied to a cellulose triacetate layer support in such a way that a layer area of 1 m ^{2 was} covered. This reagent layer was then coated with a spreading layer as described in Example \.

Various amounts of an aqueous solution were then applied to the surface of the material obtained of glycerine applied with 50 mg glycerine per deciliter of water. After the applied Samples within 40 minutes at room temperature became the reaction product generated in the reagent layer by irradiation with light of one wavelength excited by 350 nm, with a fluorescence at a

Wavelength of 445 nm occurred. The strength of the fluorescence generated at 445 nm that with a usual Fluorescence spectrophotometer was measured, proved proved to be proportional to the concentration of glycerin in the test sample.

1 sheet of drawings

Claims (19)

Claims. 1. Material for the quantitative spectrophotometric analysis of a liquid, consisting of several layers arranged on a layer support, characterized by the combination of the following features:
1.) a liquid-impermeable, translucent layer support (Fig. 1:10; Fig. 2:20; Fig. 3:30; Fig. 4:40) has at least one reagent layer on one of its two sides (Fig. 1: 12 ; Fig. 2:22; Fig. 3:32; Fig. 4:42) with a reagent which, by reacting with a constituent of the liquid to be analyzed, causes a color change within the reagent layer; 2.) a porous layer (Fig. 2:24) or a porous layer structure (Fig. 1: 14-18; Fig. 3: 34-36; F i g. 4: 44-48), the (a) on the side facing away from the substrate Reagent layer are arranged, (b) for the color-forming reaction with the reagent incoming Components of the liquid to be analyzed are permeable and (c) the applied Liquid spreads laterally in such a way that a constant volume of liquid per unit area is picked up and comes onto the reagent layer. 2. Material according to claim 1, characterized in that that the porous layer (Fig. 2:24) or at least one layer of the porous layer structure (Fig. 1: 4-18; Fig. 3: 34-36; Fig. 4: 44-48) • consists of a polymer layer. d, e by solving of a polymer in a mixture of two liquids, one of which is a good solvent for the polymer and the other a non-solvent or at least is a poor solvent for the polymer and has the higher boiling point. Apply the solution to the reagent layer or a layer above it and allow it to dry of the layer with evaporation of the solvents. 3. Material according to claims 1 and 2, characterized in that the porous layer (F i g. 2:24) or at least one layer of the porous layer structure (Fig. 1: 14-18; Fig. 3: 34-36; Fig. 4: 44-48) forms a reflective background for spectrophotometric measurements. 4. Material according to claims 1 to 3, characterized in that it is in the shape of an endless one Band owns. 5. Material according to claims 1 to 4, characterized in that the reagent layer (Fig. 1:12; Fig. 2:22; Fig. 3: 32; Fig. 4: 43) from a dispersion of at least one test reagent in a hydrophilic colloid. 6. Material according to claims 1 to 5, characterized in that the reagent layer (F i g. 1:12; Figures 2:22; Figures 3:32; Fig. 4: 42) from a <> o Dispersion of at least one test reagent in gelatin consists. 7. Material according to claims 1 to 5, characterized in that the Re.agenzschicht (F i g. 1:12; F i g. 2:22; F i g. 3:32; F i g. 4:42) from the dispersion < > s at least one test reagent in polyvinyl alcohol consists. 8. Material according to one of claims I to 7, characterized in that the layer support (Fig. 1:10; Fig. 2:20; Fig. 3:30; Fig. 4:40) from Cellulose acetate or polyethylene terephthalate is made. 9. Material according to claim 2, characterized in that there are a plurality of porous Has layers that were created by dissolving a polymer in a mixture of two liquids, one of which is a good remedy! a nonsolvent for the polymer and the other or at least a poorer solvent for the polymer and the higher boiling point owns. Applying the solution to the reagent layer (Fig. 1:22; Fig. 2:22; Fig. 3:32; Fig. 4:42] or an overlying layer and drying of the layer with evaporation of the solvent, where it applies that the individual porous layers of the porous layer structure produced one have different porosities from one another. Ό. Material according to Claims 1 to 9, characterized in that the porous layer (Fig. 2:24) a filter and reflective layer consisting of a dispersion of titanium dioxide in one Binder, or that in the case of a porous layer structure (Fig. 1: 14-18; Fig. 3: 34-36 Fig. 4: 44-48) this dressing has at least one such layer. 11. Material according to claims 1 to 9, characterized characterized in that the porous layer (Fig. 2: 24) consists of a filter and reflective layer, c from a dispersion of barium sulfate in a "binder," or that in the case of a porous Layered association (Π g 1. 14-18; Fi g. 3: 34-36 Fig. 4: 44-48) this has at least one such layer. 12. Material according to claims 1 to 9, characterized in that the porous layer (F i g. 2:24) a spreading layer consisting of diatomaceous earth dispersed in a binder is, or i that in the case of a porous layer structure: (Fig. 1: 14-18; Fig. 3: 34-36; Fig. 4: 44-48; this contains at least one such layer. 13. Material according to claims 1 to 9, characterized in that the porous layer (F i g. 2: 24 ^ is a spreading layer composed of a dispersion of inert spherical particles more uniformly Size in a binder or in the case of a porous layer structure (Fig. 1: 14-18 Figure 3: 34-36; Fig. 4: 44-48) at least one contains such a layer. 14. Material according to claims 1 to 9, characterized in that the porous layer (Fig. 2:24 is a spreading layer made up of a dispersion of glass beads of uniform size ße in a binder, or that in the case of a; porous layer structure (Fig. 1: 14-18 Figure 3: 34-36; Fig. 4: 44-48) of these at least; contains such a layer. 15. Material according to claims 1 to 14, characterized in that it is adjacent to the reagent layer has a dialysis layer. 16. Material according to claims] to 9, characterized in that the porous layer (F i g. 2:24 consists of a cellulose acetate layer or that in the case of a porous layer structure (Fig. 1 14-18; Figure 3: 34-36; Fig. 4: 44-48) which has at least one cellulose acetate layer. 17. Material according to claims 1 to 16, characterized marked that there is a plural number of each other Has adjacent reagent layers. 18. Material according to claims 1 to 17, characterized in that the reagent layer is glucose oxidase. Contains peroxidase and o-dianisidine hydrochloride. 19. Material according to claims 1 to 17, characterized in that the reagent layer copper sulfate and contains neocuproin.