DE2332760C3 - Material for the quantitative spectrophotometric analysis of a liquid - Google Patents

Description

The invention relates to a material for the quantitative spectrophotometric analysis of a liquid, consisting of made up of several layers arranged on a substrate.

In the past few years a number of automated methods of implementation have become more quantitative chemical analyzes of liquids have become known, which are particularly useful in clinical laboratories have proven beneficial, especially for blood analysis.

For example, from US Pat. No. 2,797,149 and US Pat. No. 3,565,582, continuously operating analyzers have become known which consist of a large number of containers in which samples to be examined, diluents and test reagents are stored, mixed with one another and analyzed spectrophotometrically. The liquids, diluents and test reagents to be tested are passed through the analysis device through a complex system of tubes or tubes. The disadvantage of these continuously operating analysis devices is 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 cleaning the devices is very laborious ^! This means that the devices do not allow the analysis of very small amounts of material to be tested, such as are sufficient, for example, to carry out microanalytical test procedures.

In order to overcome the disadvantages of these known, continuously operating analyzing devices, automated analyzing devices have become known in which a material in the form of an endless analytical tape is used on which there is. ' material to be analyzed applied .vird, and which allow an analysis of the applied material in a spectrophotometric way, namely by coloring reactions between components of the test specimen to be examined and test reagents that are applied to the tape or contained in it. Tape-shaped materials for analysis are known, for example, from GB-PS 10 49 364. A material shown in FIG. 4 of the patent has cutouts or recesses. into which measured amounts of the liquid to be analyzed and the test reagent • are placed. A material shown in FIG. 9 of the patent consists of a plurality of • changes ₍ in which a filtration or reaction takes place. From US Pat • to use other »which have the task of absorbing the liquid to be examined, spreading it out and filtering it and which serve as a reaction medium A so-called analytical tape is also known from US Pat

ίο analyzing material 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 analyzing methods using the disadvantages that their use and dissemination are 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 in certain embodiments of the GB-PS i0 49 364 the fail is disadvantageous that the? u analyzing Sample and test reagent in separate operations onto the tape at the right time and must be applied in the right 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 r.iit 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 make the procedure complicated.

Also the use of analytical tapes of a complex nature that are difficult to manufacture and expensive, e.g. B. the Use of the tapes known from US-PS 35 26 430, the spread of these tapes has been strong with special needs.

From US-PS 36 63 374 a spot test method for rough estimation of enzyme activity in rilut and other liquids is also known, to carry out spot test elements consisting of an upper and a lower Tctplatte each with several circular openings, each two discrete glass fiber disks over the openings of the upper test plate as well as a membrane filter disc and a cellulose acetate disc / wipe the openings of both plates can be used. The glass fiber disks and the membrane filter disk each have a double function. Firstly, they each contain at least one herauslöv from the disk bare component of the reagent system and / to other they o.er.en in blood analysis / separation for dnr white or red blood cells, so that the au Cclluloseacetatschfcibe the incident sample ^f on the one hand for the test reaction contains the necessary reagents, but on the other hand is free of interfering blood cells. The enzyme activity is estimated by comparison with Fn / ym standards. which are arranged on different cellulose acetate discs.
_t From US-PS 33 68 872 further eih automatic device is known for the chemical analysis of liquids, in which a drop of de-analyte F! is üs igkeil applied to a tape-shaped analytical material containing reagents with the Component of the liquid droplet that is supposed to have a color reaction

are able to go, and in which the tape-shaped material, which may have a stain of color is fed to a measuring station. The band-shaped material used in the known method consists it consists of a so-called "combined" tape, namely a "porous" tape and a test tape. The test tape contains the reagents required for the color reaction, while the "porous" Tape is intended to prevent "ring formation" in the test tape.

The "porous" tape can (a) consist of a separate tape made of cellophane, cellulose acetate or cellulose nitrate exist or (b) are generated "in situ" on the test tape, e.g. B. by spraying a polymer solution on the test tape.

Although it is permeable to liquids. However not porous in the usual sense. The well-known "porous" tapes are rather film-like tapes, which can also be referred to as so-called dense membranes or around perforated bands (cf. Encyklopedia of Polymers Science and Technology, Volume 8. Pages 620 to 621).

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 large number of the most varied analyzes in one continuously operating analyzer can be carried out in a simple and effective manner can.

The material should make it possible to achieve a shade that is independent of the volume applied of the liquid sample always has a constant density and, moreover, is uniform.

It has been found that the problem can be solved with a material that consists of several Layers arranged in an integrated manner in a layer support and by the combination of the following features is marked:

1) a liquid impermeable. more translucent Layer support has on one of its two sides at least one reagent layer with at least a reagent in a hydrophilic colloid. that by reaction with a component of the liquid to be analyzed causes a color change within the reagent layer:

2) a microporous membrane made of a non-fibrous, porous layer or a porous layer structure, the 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 em 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 Form of a sheet or a strip section or in the form of wine sections, which are attached to cards are, ζ. B. on cards having openings.

To apply the sample to be analyzed to the material, automatic distribution or application devices can be used precisely measured amounts of the liquid to be analyzed at the right time and at the right time Apply point of the material. Such automatically operating devices are known. With the help of a Such a device and a material according to the invention can be quantitative analyzes of various Perform type, e.g. B. lets see the glucose content of the Analyze blood by feeding the treated material into a standard spectrophotometer.

The material according to the invention is a material that all of the necessary to carry out the layers required for the analytical process, in contrast to known automated ones. Prior art systems using analytical tapes in which / wet or multiple tapes 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 top layer of the material and pass the material through a Conducting a spectrophotometer is the device required to perform automated analysis not expensive.

By "color-forming reactions," and those are here t all to understand where the color-forming reactions exist within the reagent in reactions in which fluorescent compounds are produced.

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 substrate can vary widely. Appropriate it is 0.0508 to 0.254 mm.

The reagent layer can be applied directly to the layer support or to a translucent one Adhesive or intermediate layer, which improves the bonding of the reagent layer to the support The reagent layer contains the test reagents, which have a color-forming reaction with components of the too to be analyzed. The reagent layer can, for example, consist of a dispersion of one or several test reagents in a hydrophilic colloid, which serves as a binder, e.g. B. gelatin or Polyvinyl alcohol.

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

On the side of the reagent layer that is attached to the substrate is applied, there is one or more layers that have the function that applied liquid spread uniformly in the lateral direction and the microporous membrane forms or forms.

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 the 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.

Thus, an inventive material has at least two layers out of the layer support, one of which is a reagent layer ISL and the other a Schichtj causes that a uniform lateral distribution of the introduced p _ro b _e and optionally also has the further different functions mentioned. However, it is also possible to arrange a special layer for each individual function, in some cases a material according to the invention has, for example, four layers in addition to the layer carrier. 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 just one layer for a specific purpose, ie it is possible, for example, to use two or more adjacent layers s! S Rc! «~ Ci ?? * chicht € ti to v ^A rwcndcii

The layer with the spreading function, i.e. the layer forming the microporous membrane, is that of the Layer support furthest layer and the layer on which the sample to be analyzed, e.g. B. a blood sample is applied. The sample to be analyzed is typically applied to the layer with the spreading function applied by means of an automatically operating distribution device which is constructed in this way is that they put a small drop of liquid in the desired location on the top layer of the Able to apply analysis element.

The layer with the spreading function is supposed to hold the drops Distribute evenly in the lateral direction. The importance of this layer is the tendency to prevent a "ring" formation in the reagent layer of the material. The phenomenon of the "ring" - Formation is described, for example, in US Pat. No. 3,036,893 and 35 26 480. As a result, drops of a sample to be analyzed are placed directly on a When the reagent layer is applied, they form a "ring" due to their lateral formation, with a / diminished Concentration of test reagent occurs in the center of the ring and an increased concentration in the Periphery of the ring. This leads to non-uniform color development, which in turn is quantitative Measurement prevented by spectrophotometric means.

In the case of a material according to the invention, practically the entire lateral movement of the Sample in the layer with the spreading function so that when the sample reaches the reagent layer, it will does not spread further laterally and the test reagent is no longer distributed unevenly.

The function of the porous layer is therefore also different from that of the membrane filter disc, which is in the frame of the spot test method known from US Pat. No. 3,663,374 is used, because the latter is primarily used as a filter to separate the red blood cells and as a reagent carrier.

In the case of a material according to the invention, a change in the sample volume affects the the material is applied, the diameter of the generated color patch in the reagent layer. That Volume of liquid per unit area that reaches the reagent layer, however, remains practically constant, regardless of changes in the volume of the sample applied. As a result, the density of the Color tone that is generated in the reagent layer by the color-forming reaction, by a The change in the size of the drop applied to the material is not noticeably affected The hue only depends on the concentration of the component that causes the color-forming reaction

$ is received. As a result, when using a Material according to the invention is 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.

ίο Has the porous layer or a layer of the layer structure «; or the shift unit aUcli the function a filter layer, this means that from the sample Components that interfere with the color-forming reaction in the reagent layer are to be filtered off or which would interfere with the spectrophotometric measurements to be carried out later. For example, in the case of a material intended for the analysis of blood components, a

remove but allow the serum to pass into the underlying layer. Such a filter layer can be made up of any material that has a sufficient degree of porosity for the to be analyzed Sample, the optimal porosity of the specific use of the analytical material depends. If the material according to the invention, for. B. in the form of an endless tape, to be used for the spectrophotometric analysis of blood, so it has proved to be useful when a filter layer is present. which have a pore size of 1 to 5 microns and is able to filter off the blood cells, which typically have a size of about 7 to 30 Microns and which allows the serum to pass through. A filter sheet with a pore size that is too small can limit the usability of the material, for example because it allows 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, the passage of blood cells blocked by the filter layer which is why they do not interfere with the spectrophotometric measurements because these take place through the layer support, the reflective layer serving 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 that on the multilayer material

striking energy. If the thickness of the spreading layers described above by about Reduced by half, the permeability increases to about 4% and in the case of materials with microcrystalline Cellulose broad layers of a thickness of about 100 to 300 microns increase the permeability about 20% of the incident radiation, as a result, better signal-to-noise values are obtained then if the method of determination is the permeability 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 spectrophotonic Determination methods can advantageously also use other methods of spectropholometry 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 ius 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 spectropholometry, those which can be applied are those to which radiation is applied which is caused by 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 I, 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 poor solvent, whereupon the polymer solution is applied to the carrier and dries up. Since the good solvent for the polymer evaporates faster due to its low boiling point, the polymer accumulates in the liquid that is a bad solvent or a nonsolvent for the polymer to the extent that evaporation proceeds, resulting in the polymer is excreted in the form of fine particles and forms an adhesive porous layer on the carrier. A wide variety of polymers can be used to produce such polymer layers, 7. B. Polycarbonates, polyamides and cellulose esters.

Instead of a "blush polymer layer" that the Performs the functions of specimen spreading and filtration as well as providing the necessary reflective background for reflection measurements, an in correspondingly effective layer can be produced by the fact that one is on the reagent layer Thin layer of a microporous filter membrane laminated on. Such feeding membranes consist of so-called "blush polymers", e.g. B. from cellulose esters, and contain microscopic pores. For the production A large number of materials of different pore sizes are commercially available for such layers.

The inventive material has a single material Layer on, soften the function of the sample spreading layer, has the function of a filter layer and the function of a reflective layer, so can the material z.!? ·> in the form of a tape, made of merely consist of two layers and the substrate, since apart from the layer with the function of spreading layer, a filter layer and a reflective layer, only the reagent layer is absolutely necessary is.

However, it can also be advantageous to use materials that have a layer that acts as Filtefschicht and reflective layer acts, and one special layer that has the function of a sample spreading layer or the three separate layers which have the functions of sample propagation, sample filtration and light reflection exercise. In this case, the layers are applied to the substrate in such a way that the sample Auv broad layer is the layer furthest from the substrate, followed by the filter layer and then the reflective layer and finally the reagent layer.

An example of a layer that works as both a filter layer and a reflective layer is effective is a layer of titanium dioxide or barium sulfate dispersed in a binder, e.g. B.

■ 25 cellulose acetate, polyvinyl alcohol or gelatin. One such a layer has proven to be particularly effective in one Material that is intended for the analysis of blood. as the material is the blood cells able to filter effectively, while the layer lets the serum through, while at the same time one provides an effective white background for spectrophotometric measurements through the support.

A particularly advantageous layer with the function of a spreading layer for the analysis of blood is one Layer consisting of a dispersion of diatomaceous earth in a binder, e.g. B. cellulose acetate. Diatomaceous earth has been found to be extraordinarily effective for the uniform distribution of blood in lateral Proven direction. Advantageous sample spreading

AO layers can also be produced from microcrystalline colloidal products, for example, which are derived either from naturally occurring or synthetic polymeric substances. Such microcrystalline substances are, for example, from O.

A. B a 11 is t a in the article “Colloidal Macromolecular Phenomena. Part II, Novel Microcrystals of Polymers, ”published in the Journal of Applied Polymer Science, Volume II, pp. 481-498 (1967). Microcrystalline cellulose used for manufacture such layers are usable is also commercially available.

Advantageous results are also obtained when a sample spreading layer is used that of inert spherical particles becomes more uniform Size in a matrix of a binder that binds the particles to the underlying layer, Examples of such spherical particles are beads made of glass and polymer Synthetic resin beads. As a beneficial binder for such beads have been found to be gelatin and polyvinyl alcohol, for example. The binder should be used in small amounts if possible, so that it is avoided that a substantial Part of the pore volume that is generated by the spherical particles is filled.

An overlay made up of spherical ones Particles of uniform size can have advantages over a porous polymer layer such as

for example a so-called "blush polymer layer". Such a layer can, for example, make drops of blood uniform extremely quickly and spreading reproducibly, and so rapidly that the spreading or spreading is finished. before a possible diffusion of blood components into adjacent layers of the invention Material can be done. As a result, the concentration of the sample constituents becomes more uniform reached in the reagent layer and a uniform Discoloration, which forms the basis for the analysis. While blood cells occasionally become one Clogging of porous polymer layers, such clogging does not occur if the Sample breakout layer consists of spherical particles in a fine matrix of a binder. By using spherical particles of uniform size thus obtain particularly advantageous results, since these particles create an adequate or sufficient volume of pores while they limit the spaces to a relatively narrow area. This brings the spread to a standstill of the blood when the spaces between the area to which the sample has been applied are complete are filled, with rapid drainage of the at the same time Plasma is allowed into the underlying layer when the spreading process has been completed. It It has been found that spherical particles of a size of about 80 to 120 microns are particularly advantageous when the spreading layer is used to spread blood droplets in a blood analysis. One on one Layer of applied blood drops spreads into a uniform circular shape in a few seconds Area, the area covered after spreading is directly proportional to the sample volume.

Advantageously, the spreading layer of a material according to the invention, for. B. in addition to the spherical particles, a small amount of a surface-active compound can be added to 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 add a dialysis layer directly above the reagent layer to arrange. Such a layer can be advantageous, for example, when analyzing the glucose content of the blood because such a layer prevents the passage of high molecular weight proteins, which the Could interfere with glucose analysis. For the separation of high molecular weight substances from low molecular weight substances are for example semipermeable cellulose membranes, which are therefore suitable as dialysis layers. Such layers act like a mechanism other than the porous polymer layers, such as the "blush polymer layers" that act mainly like a sieve or grid, in which jie pores allow the passage of solutions and allow small particles, but block the passage of particles of a larger diameter.

The materials according to the invention are suitable for carrying out a wide variety of chemical analyzes, and not only in the field of clinical chemistry, but rather also in the field of chemical research in general as well as in chemical laboratories that control the flow of chemical processes monitor. The materials according to the invention are suitable however, it is 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 one Period of time must be available after the sample has been taken. In the case of a blood analysis, this can be done for example a material according to the invention, e.g. B. in the form of a tape, so that it is for the quantitative analysis of many blood components that must be routinely determined is used can be. For example, a material according to the invention, for. B. in the form of a tape, easy to 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 for other components of the blood by selecting appropriate test reagents.

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 components of a test sample to be analyzed should be determined and depending on the color-forming reaction to be carried out.

In the blood analysis, glucose can be determined, for example, by UjB die The reagent bar is a ferricyanide or a ferricyanide compound contains and that the decrease in the yellow hue is determined, soft by implementing the Ferricyanides is made with glucose because high molecular weight proteins of the blood cause a certain reduction of the Ferricyanides and can thus interfere with the determination method for glucose, can lead to Avoidance of occurring difficulties a dialysis layer of the structure already described in placed on the material.

When analyzing uric acid in the blood, the Reagent layer, for example, from a mixture of copper sulfate and neocuproin, i.e. 2,9-dimethyl-1, 10-phenanthrolm of the formula:

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

When analyzing the serum glutamine oxaloacetic acid transaminase known enzyme, a sequence of reactions can be carried out, for example. The mentioned system catalyzes the conversion of glutamic acid into oxaloacetic acid at a pH value to 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 It can be advantageous to facilitate the reaction equation before the end of the color-forming coupling reaction be to place the reagents in two separate layers to ensure that for a sufficient period of time is available for the course of the first reaction before the second Reaction can take place. So the glutamic acid can be accommodated in a first reagent layer.!, The is arranged over a second reagent layer. which contains the salt of Fast Ponceau L.

A typical automatic analyzer, in which can be used a tape-shaped material according to the invention, typically has Vo. -lighting 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 sample to be analyzed, e.g. B. a blood or serum drop on the Surface of the tape is applied, as well as Vornchtungsteile. which the tape through an or lead several development zones and finally Vornchtung parts through which the tape by means of a Take-up roller is wound. In the development zones mentioned, the tape is subject to such conditions subjected to the course of the reaction between a color-forming sample and a component of the reagent layer. This means that the Band is subjected to conditioning, for example heating or the action of Water or another solvent for acceleration or support the process 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 application a reflection spectrophotometry a device, by means of which a light beam is directed through the substrate, which is then returned to the displaying Part of the spectrophotometer is reflected. When using reflection spectrophotometric Measurement methods are an optical interference from residues, such as blood cells, which on or remained in the layers of the tape, avoided.

Another spectrophotometric measurement method that can be used is fluorescence spectra trophctometry, for generating calibration standards drops of saturated solutions of the component to be analyzed can be adjacent to the district are applied to which the drop of the sample to be analyzed for 6s is applied, whereby Difference 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 provided, 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 markings can be made on cards, in particular cards with openings. ..: fc ^5f igt which by means of special devices for such cards through a sample application zone, one or more conditioning zones. z. B. Zones in which there is an action of heat or a solvent, and a zone in which spectrophotometric measurements are carried out. A variety of different materials in accordance with the invention may be affixed to such a card, each material containing the reagent required to analyze a particular component of a sample, so that such a card can be used to perform a variety of tests.

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

F 1 g. 1 a material consisting of a layer support 10 with a reagent layer 12 applied thereon, a reflective layer 14 provides a white background for reflection measurements through 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 or Several test reagents exist in a binder, such as gelatin, while each of the layers 14.16 and 18 from a suction. "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 the sample to be applied spread and filter them, and which further provide a suitable background for reflectance measurements made possible by the layer support 20. Layer 24

can, for example, consist of a »blush polymer layer« consist, which was applied to the layer 22, or a layer of a 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 semi-permeable! Membrane layer was formed, and a layer 36, e.g. B. a "blush polymer layer" soft performs the function of spreading the sample to be analyzed and filtering the sample, and which furthermore provides a suitable background for reflection measurements through the layer support 30 supplies;

F i g. 4 a material consisting of the layer support 40, a first reagent layer 42, a second Reagent layer 44, a layer 46 which serves as a filter layer and a light reflective layer, and a sample spreading layer 48. The layer 46 can, for example, consist of a dispersion of titanium dioxide in cellulose acetate and the layer 48 may, for example, consist of a dispersion of diatomaceous earth in cellulose acetate or made up of glass beads in gelatin.

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

example 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 layer so 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 on the Reagent layer laminated in that the reagent layer initially several for the purpose of softening Seconds to the action of steam, whereupon the filter layer on the reagent layer was pressed on. The two layers were then subjected 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.

Example 2

Another tape-shaped material for the determination of uric acid in blood was used in Example 1 described manner, with the exception, however, that as a binder for the reagent layer Polyvinyl alcohol was used instead of gelatin. The polyvinyl alcohol was daboi in one Concentration of 43.1 mg / dm '' used.

Example 3

Another tape-like material for the determination of chloride in blood was produced 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 dm ² 53.9 mg gelatine and 18.0 mg silver 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

Another tape-like material for the determination of albumin in blood was produced according to the method described in Example 1, this time producing a reagent layer consisting of 4.66 mg of hydroxyphenylazobenzoic acid dispersed in 21.0 mg of a copolymer of acrylic acid and 2-acetoacetoxyethyl 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 support, each per 1 dm 2 of support 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. 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 TiOj 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 slurry of diatomaceous earth spread over this layer. Salicylic acid and cellulose acetate are applied in a mixture of 1.2 parts by volume of dichloroethane and 1 part by volume of acetone.

There were three different tape-shaped materials using different concentrations made of diatomaceous earth in the spreading layer.

each manufactured tape based on its ability. 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:

Ver · Diatoms Salicylic Cellu Diffusion stains 5 ° search Earth acid loose time in (Spot) - No acetate Seconds By- knife (mg / dm ² ) (mg / dm ² ) (mg / dm ^J ) in cm ss 1 300 3.00 37.2 26th 1.05 2 375 3.75 37.2 41 1.00 3 450 4.50 37.2 31 1.00

The tape-shaped materials described in this example are suitable for the determination of the

Glucose content of the blood, with the presence

of glucose is indicated by the formation of a brown hue in the reagent layer.

_6s examples

On a 0.10 mm thick polyethylene terephthalate 'layer support was a reagent layer in Example! 1 specified composition applied, on what

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 slide, which provided a suitable background for performing reflection measurements. A spreading layer was then applied to the filter layer, including glass ogels 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 gelatin .-% of a surface-active compound, namely para-isononylphenoxypolyglycidol with 10 glycidol units, were mixed, and the resulting slurry was applied in the form of a thin layer on the microporous filter layer so that 1 g of beads per an area of 27 cm ² .

10 microliters of blood was then applied to the obtained material, it was found that these 10 microliters of blood were spread in less than 1 second, namely z's of a circular area of approximately 1 cm Durchi.-resser.

Example 7

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

Was determined the size of the dip effect analyzing sample on the area and the reflection density of the colored spot, dei in the xeagent layer was generated by the action of differently sized samples of a glucose solution which distilled per 100 i .1 Water contained 100 mg glucose.

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

Sample amount Patch District Reflective in microliters (mm ² ) density 5 30th 0.66 8th 50 0.71 10 63 0.70 12th 86 0.65 15th 92 0.70

From the results obtained, it can be seen that the reflection density values are practically independent of the Size of the applied drop. The results obtained accordingly show that a Material enables a quantitative analysis of a component of a sample to be analyzed, without the need to apply a precisely defined amount of a sample to the material is applied.

Example! 8th
The following solutions were made:

(a) a solution of 3.23 g one at lower Temperature setting agarose (Polysacchä

rid) in 70 ml of an O ^ molar 2-amino-2-hydroxymethyl-1,3-propanedioI buffer solution;

(b) a solution of 108 international units of glycerol dihydrogenase, 108 mg of human serum albumin, 108 mg resazurin and 54 mg diaphorase;

(c) a solution of 54 mg nicotamide adenine dinucleotide in 10 ml of a 0.3 molar 2-amino-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 an O ^ molar glycine buffer solution of a pH value of 9.6;

(b) a solution of 2.150 g of agarost (polysaccharide) that solidifies at low 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 I.

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, the reaction generated in the reagent layer product excited by irradiation with light with a wavelength of 350 nm, with a fluorescence at a Wavelength of 445 nm occurred. The strength of the fluorescence generated at 445 nm that <iinit was / proved to be measured with a conventional fluorescence spectrophotometer proved to be proportional to the concentration of glycerin in the test sample.

1 sheet of drawings

Claims (18)

Patent claims: 1. Material for quantitative spectrophotometric Analysis of a liquid, consisting of several arranged integrated on a layer support Layers characterized by the combination of the following features: 1) a liquid impermeable, translucent one Support (Fig. I: 10; Fig. 2: 20; Fig. 3:30; Fig. 4:40) points to one of his at least one reagent layer on both sides (Fig. 1:12; Fig. 2:22; Fig. 3:32; Fig. 4: 42,44) with at least one reagent as a dispersion in a hydrophilic colloid, which by Reaction with a component of the liquid to be analyzed causes a color change within the reagent layer effects; 2) a porous layer (Fig. 2:24) or a porous one Layered dressing (Fig. 1: 14-18: Fig. 3: 34-36; Fi g. 4: 46-48) who (a) on the ^ o Layer carriers are arranged facing away from the side of the reagent layer, (b) for the color-forming Reaction with the reagent incoming component of the liquid to be analyzed are permeable and (c) the applied liquid in the manner of a blush polymer layer 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 Jie porous layer (F i g. 2:24) or at least one layer of the buttocks bandage (Fig. 1: 14-18; Figure 3: 34-36; F i g. 4: 46-48) best from a polymer layer, which by dissolving of a polymer in a mixture of two liquids, one of which is a good solvent for the polymer and the other a nonsolvent or at least a bad solvent for is the polymer and has the higher boiling point, applying the solution to the reagent layer or an overlying layer and drying of the layer with evaporation of the solvents, was generated. 3. Material according to claims 1 and 2, characterized in that the porous layer (Fig. 2: 24) or at least one layer of the porous layer structure (Fig. 1: 14-18; Fig. 3: 34-36; Fig. 4: 46-48) a reflective background for spectrophotometric Making measurements. 5c 4. Material according to Claims 1 to 3, characterized in that it is in the form of an endless one Band owns. 5. Material according to claims I to 4, characterized in that the reagent layer (Fig. 1:12; F i g. 2: 22. Γ i g. 3: 32: F i ρ 4: 42, 44) from e ^; ner dispersion consists of at least one test reagent in gelatin. 6. Material according to claims I to 4, characterized in that that the reagent layer (Fig. I: 12: P i g. 2:22; F 1 g. 3:32; F 1 g. 4: 42, 44) from the dispersion at least one Tesu reagent consists of poly * vinyl alcohol. 7. Material according to one of claims I to 6, characterized in that the layer support (Fig. I: 10; Fig. 2:20; Fig. 3: 30; Fig. 4:40) Cellulose acetate or polyethylene terephthalate is made. 8. Material according to claim 2, characterized in that that it has a plurality of porous layers created by dissolution of a polymer in a mixture of two liquids, one of which is a good solvent a nonsolvent for the polymer and the other or at least a poorer solvent for the polymer and the higher boiling point possesses, applying the solution to the R =: agent layer (Fig. 1:12; Fig. 2:22; Fig. 3:32; Fig. 4:42, 44) or an overlying layer and drying of the layer with evaporation of the solvent, it is good that the individual porous layers of the produced porous layer structure have a different porosity from one another. 9. Material according to claims 1 to 8, characterized in that the porous layer (F i g. 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; F i g. 4: 46-48) this association has at least one such association Has layer. 10. Material according to claims 1 to 8, characterized in that the porous layer (F i g. 2:24) a filter and reflective layer, consisting of a dispersion of barium sulfate in one Binder, or that in the case of a porous layer structure (F 1 g. 1: 14-18; F i g. 3: 34-36: Fig. 4: 46-4S) of these at least one such Has layer. 11. Material according to claims I to 8, characterized in that the porous layer (Fig. 2: 24) is a spreading layer consisting of diatomaceous earth dispersed in a binder, or that in the case of a porous layer arrangement (Fig. 1: 14-18; Fig. 3-34-36: Fig. 4: 46-48) this contains at least one such layer. 12. Material according to claims I to 8 thereby characterized in that the porous layer (Fig. 2: 24) is a spreading layer, built up from one Dispersion of inert spherical particles of uniform size in a binder or in the case a porous layer structure (Fig. 1: 14-18; Fig. 3: 34-36; Fig. 4: 46-48) at least one contains such a layer. 13. Material according to claims I to 8, characterized characterized in that the porous layer (Fig. 2:24) is a spreading layer made up of one Dispersion of glass beads of uniform size in a binding agent or in the case of a porous layer structure (Fig. 1: 14-18; Fig. 3: 34-36; Fig. 4: 46-48) this at least contains such a layer. 14. Material according to claims I to 13 thereby characterized in that it is adjacent to the reagent layer has a dialysis layer. 15. Material according to claims 1 to 8 thereby characterized in that the porous layer (Fig. 2:24) consists of a cellulose acetate or that in the case of a porous layer structure (Fig. 1: 14-18; Fig. 3: 34-36; Fig. 4: 46-48) this has at least one cellulose acetate layer. 16. Material according to claims 1 to 15, characterized in that there are a plurality of one another Has adjacent reagent layers. 17. Material nadh claims 1 to 16, characterized characterized in that the reactant layer is glucose oxidase, Peroxidase and o-dianisidine hydrochloride contains. 18. Material according to claims 1 to 16, characterized in that the reagent layer is copper sulfate and contains neocuproin.