DE2332760A1 - MULTI-LAYER ANALYTICAL ELEMENT FOR QUANTITATIVE SPECTROPHOTOMETRIC ANALYSIS - Google Patents

Description

educational analytical element for quantitative

spectrophotometric analysis 9 ^ ^? 7 R Π

The invention relates to a multilayer analytical element for the quantitative spectrophotometric analysis of a liquid, in particular in the form of an analytical tape for the automated spectrophotometric analysis of fluids, especially body fluids, for example Blood.

In the past few years a number of automated systems for performing quantitative chemical analysis have been used by Liquids have become known which have proven particularly advantageous for clinical laboratories, in particular for blood analysis.

For example, US Pat. Nos. 2,797,149 and 3,565,582 have disclosed continuous flow analysis systems which consist of a large number of containers in which samples to be examined, diluents and test reagents are stored, mixed together and / or analyzed spectrophotometrically will. The liquids, diluents, and test reagents to be tested are grounded through the analyzer guided through a complex system of tubes or tubes. The disadvantage of these continuously operating analysis devices is that their structure is extremely complex and that they are expensive and require skilled personnel to operate. Another disadvantage is that the Analyzes are very time-consuming, that the cleaning of the devices is also very cumbersome and that the known devices do not allow the analysis of very small amounts of material to be tested, as for example sufficient to carry out microanalytical test procedures.

In order to overcome the disadvantages of the known, continuously operating flow analyzers, automated chemical analyzers become known at

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which an endless analytical tape is used to which the material to be analyzed is applied, and which one Allow analysis of the applied material by spectrophotometric means, through color-forming reactions between Components of the test item to be examined and test reagents that are applied to the tape or contain it are. A number of endless analysis tapes of various structures have already become known. Two analysis tapes various structures are known from GB-PS 1,049,364, for example. The one shown in Fig. 4 of the patent Tape has cutouts or recesses into which measured amounts of the liquid to be tested and the Test reagent can be introduced. The other band shown in Fig. 9 of the patent consists of a plurality of Ribbons in which a filtration or reaction occurs. It is also known from US Pat. No. 3,036,893 to carry out such automated analysis methods to use a plurality of mutually separate tapes that perform the task have to absorb and spread the liquid to be examined, filter it and serve as a reaction medium. In the analytical method known from US Pat. No. 3,036,893, the material to be analyzed is placed on a belt and then transferred from this to another tape. From US Pat. No. 3,526,480, an analytical tape is also known which Has cutouts containing measured amounts of test reagent and in which the material to be analyzed is applied.

The known analysis systems with endless analysis belts are naturally much simpler than the known flow analysis devices. However, the known analysis methods using analysis tapes also have disadvantages which prevent their use and have severely hampered diffusion. This is the case, for example, with analysis tapes that do not have the test reagent themselves

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included and mainly the transport of the to be analyzed Serve sample through the device, as is the case with certain embodiments of GB-PS 1 049 364, disadvantageous, that the sample to be analyzed and the test Pveagent in separate operations on the analysis tape at the right time and must be applied in the right quantities. i) It further carries out the 'use of multiple tapes with different Functions, e.g. B, with the function of spreading the sample to be tested, with the function of filtering the sample, and with the function of converting the compound to be analyzed to be carried out with the test reagent, as is known, for example, from US Pat. No. 5,036,893 and from British Pat. No. 1,049,364 is to make the procedure complicated. Also the use analytical tapes of a complex nature that are difficult to manufacture and expensive, e.g. B, the use of the US patent 3,526,480 known tapes, has severely hampered the spread of these tapes.

The object of the invention is to provide a multilayer analytical element for the quantitative spectrophotometric analysis of a Specify liquid that is suitable for performing automated spectrophotometric analyzes and which one has a simple structure, is easy to manufacture and inexpensive, and with which a large number of the most varied analyzes in a continuous analyzer · can be carried out in a simple and effective manner.

It has been found that the task at hand can be realized by a multilayered analytical element which is made up of a layer support, a reagent layer, which contains one or more test reagents for the reaction with contains components of the test item to be tested, and a porous medium consisting of one or more layers, which has the task of extracting the liquid to be tested

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and to filter and further serves to facilitate the spectrophotometric analysis through the support.

The subject of the invention is thus a multilayer analytical Element for the quantitative spectrophotometric analysis of a liquid, characterized by 1. one for liquids impermeable, translucent layer support, 2. at least one arranged on one side of the layer support Reagent layer with a reagent that changes color when it reacts with a component of the liquid to be analyzed within 'the reagent layer, and 3. one on the side of the reagent layer facing away from the support arranged porous layer or a porous layer structure, which or which for the color-forming reaction with the reagent incoming component of the liquid to be analyzed is permeable and the or the applied liquid in such a way spreads laterally so that a constant volume of liquid per unit area is obtained and onto the reagent layer got.

According to a particularly advantageous embodiment of the invention, the multilayer analytical element according to the invention has in the form of an endless strip or ribbon. However, it can also be in another form, for example in form a sheet of paper or a short strip or in the form of small sections attached to cards, e.g. B. Openings exhibiting cards.

Characteristic of the multilayer analytical according to the invention Elements is thus a reagent layer which contains the reagents required to carry out an analysis, see above that when using a multilayer analytical element according to the invention, the person performing the analysis only for a suitable application of the sample to be analyzed

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has to worry about the item. To apply the to be analyzed Sample can be used automatically operating distribution or application devices that accurately measured Apply quantities of a liquid to be tested at the right time and to the right place on the analytical element. Such automatically operating devices are known. With the help of such a device and a multilayer analytical Element according to the invention, for example, quantitative analyzes of certain constituents of the components to be tested Carry out liquid, ζ. B. the glucose content of the blood can be analyzed by adding the treated analytical element is fed to a conventional spectrophotometer.

The multilayer analytical element according to the invention is a so-called integral multilayer element Element that has all the layers required to carry out the analytical process, as opposed to the known prior art automated systems using analysis tapes in which two or more tapes are temporarily brought into contact with each other "and after the formation of a so-called composite structure again from each other be separated.

Since the multilayer analytical element of the present invention is a has integral structure in which the ski support and the different layers are connected to each other, and since it is only necessary when using the element, apply the sample to be analyzed to the top layer of the element and close the element through a spectrophotometer lead, the device required to carry out an automated analysis is not expensive, so that the endless analytical tapes can take full advantage of their own simplicity in an analysis system.

The terms “color change” and “color-forming reaction” here also include the cases in which the color-forming reaction exists within the reagent layer in a reaction producing a fluorescent compound.

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For the production of multilayer analytical elements according to the invention, the most varied of light-permeable, liquid-impermeable layer supports can be used. For example, a wide variety of polymers, e.g. b. Cellulose acetate, polyethylene terephthalate, polycarbonates and polystyrenes can be used. The thickness of the support can vary widely. The layer thickness is expediently about 0.0508 to 0.254 mm.

The reagent layer can be applied directly to the layer support or to a translucent adhesive or intermediate layer which supports the bonding of the reagent layer to the layer support. The reagent layer contains the test reagents, which enter into a color-producing 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 which serves as a binding agent, e.g. b. Gelatin or polyvinyl alcohol. .

The test reagents of the reagent layer used in the individual case depend of course on the material to be analyzed in the individual case and the color-forming to be produced in the individual case Reaction used to identify the one to be analyzed Substance has been selected.

On the side of the reagent layer facing away from the support is, is there one or more layers th, which have the function of spreading the applied liquid uniformly in the lateral direction.

This layer or these layers can optionally also have the task of extracting from the sample to be analyzed To filter off constituents ^ Sas occurrence or measurement would interfere with the color-forming reaction.

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This layer or layers can also have the puncture for the purpose of carrying out a reflection spectrophotometric Analysis to reflect the light that has passed through the layer support.

A multilayer analytical element according to the invention thus has at least two layers in addition to the support, of which one of these layers is a reagent layer and the other is a layer which causes a uniform lateral distribution of the applied sample and possibly the other other functions mentioned Has. However, it is also possible to arrange a special layer for each individual function, in which case a multilayer element according to the invention has, for example, four layers in addition to the support. 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 as reagent layers.

The layer with the spreading function is the layer furthest away from the layer support and the layer on which the one to be analyzed is applied Sample, e.g. B. blood is applied. Typically, the sample to be analyzed is, as already stated, on the layer with the spreading function applied by means of an automatically operating distribution device, which in such a way is designed to place a small drop of liquid at the desired location on the top layer of the analysis element able to muster.

The layer with the spreading function should make the drop uniform distribute in a lateral direction. The importance of this layer is to tend to form a "ring" in

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the reagent layer of the element. The phenomenon of "ring" formation is described, for example, in US Pat. No. 5,500 and 3,526,480. If, accordingly, drops of a sample to be analyzed are applied directly to a reagent layer, so they form a "ring" through lateral formation, with a reduced concentration of test reagent in the center of the ring occurs and an increased concentration in the periphery of the ring. This leads to uneven color development, which in turn is a quantitative measurement based on spectrophotometric Paths prevented.

In the case of a multilayer analytical element according to the invention, practically the entire lateral movement takes place of the sample in the layer with the spreading function, so that when the sample reaches the reagent layer, it no longer spreads laterally spreads, no more uneven distribution of the test reagent takes place.

In the case of a multilayer analytical element according to the invention, a change in the sample volume affects the is applied to the element, the diameter of the colored spot created in the reagent layer, but the volume of liquid per unit area that reaches the reagent layer, remains practically constant regardless of changes in the volume of the sample applied. As a result, the density of the The hue generated in the reagent layer by the color-forming reaction is not noticeable by a change the size of the drop applied to the element and the color tone is only dependent on the concentration of the Component that enters into the color-forming reaction. As a result, it is when using a multilayer analytical Element according to the invention not required to apply drops of uniform size on the element or the size of the to determine applied drops in order to carry out a quantitative analysis can.

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If the porous layer or a layer of the layer structure or the layer structure has the function of a filter layer, so this means that components are to be filtered off from the sample, which the color-forming reaction in the reagent layer would disturb or which would be carried out later hinder spectrophotometric measurements. For example, in the case of a multilayer analytical element for the analysis of constituents of the blood, a layer of film serve to remove red blood cells but allow the serum to pass into the underlying layer. One such filter layer can be constructed from any material which has a sufficient degree of porosity for the to be analyzed Sample having the optimal porosity depending on the specific use of the multilayer analytical Element depends. Should the analytical element, e.g. B. in the form of an endless belt, for reflection-spectrophotometric Analysis of blood are used, so it has proven to be useful if a filter layer is present, the one Has pore size from 1 to 5 microns. A filter layer with such a pore size is able to filter out the blood cells, which are typically about 7 to 30 microns in size, with the serum passing through the layer able. When using a filter layer with a pore size that is too small, the usability of the element or band, for example because it prevents the passage of blood proteins, namely in the case such analytical tests where it is important that these blood proteins reach the reagent layer.

As previously stated, the multilayer analytical element of the invention can be used in an analytical system be determined using reflectance techniques of spectrophotometric analysis of why the element contains a layer which has the function of a reflective layer and a suitable background for spectrophotometric Forms measurements through the layer support side of the element.

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

In the case of a multilayer analytical element, e.g. B. tape, for the analysis of blood is the passage of blood cells or blood cells blocked by the filter layer, so they do not interfere with the spectrophotometric measurements, since these take place through the layer support, with the reflective layer serving as a white background.

When reading an unused analytical element of the type intended for reflection-spectrophotometric measurements, the phctometer detector only records about 1 % of the energy incident on the element.This 1% of incident radiation then serves as a comparison value (10 %) for subsequent reflection measurements of the element on which a sample was applied. This low collection efficiency is the result of the diffuse reflectivity of the spreading layers of the type described and the low acceptance angle required to distinguish it from diffuse light.

When reading or evaluating the same element in the case of passage measurements, the detector records about 1.5 to 2 % of the energy incident on the multilayer element. If the thickness of the spreading layers described above is reduced by about half, the permeability increases to about 4 ° and in the case of elements with microcrystalline cellulose spreading layers of a thickness of about 100 to about 300 microns the permeability increases to about 20 % of the incident radiation. As a result-

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better signal-to-noise values of this are obtained if the permeability method is used as the method of determination which is why it may be more advantageous in many cases to use this method of determination.

Apart from the spectrophotometric determination methods described In an advantageous manner, other forms of spectrophotometry can also be used to carry out the analysis method can be applied. Mention should be made, for example, of fluorescence spectrophotometry, in which case the reaction product of the sample with a test reagent from a fluorescent Connection exists and the determination of the fluorescent Connection with radiation takes place, which causes the reaction product to fluoresce. Other forms of spectrophotometry, those that can be applied are those upon which radiation is applied that is from visible light is different, e.g. B. ultraviolet radiation or infrared

Light. The term “color-forming reaction” therefore also includes the cases in which a fluorescent compound is obtained in the reaction.
As already stated, the analytical element according to the invention can have 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.

An example of a layer that can perform all of these three functions is a so-called "bush polymer layer". Such layers can be known (see. H. F. Payne "Organic Coating Technology", Verlag John Wiley and Sons, New York, 1954, Vol. I, page 414) can be produced on a substrate by a polymer in a mixture dissolves from two liquids, one being a liquid is a good solvent for the polymer, whereas the other liquid, which has a comparatively higher boiling point, is or at least is a nonsolvent for the polymer

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a bad solvent, whereupon the polymer solution is applied to the substrate and dried. Because the good solvent For the polymer evaporates faster due to its lower boiling point, the polymer accumulates in the liquid at showing a bad solvent or a nonsolvent for the polymer is to the extent to which the evaporation progresses, which results in the polymer becoming finer in shape Particles is precipitated and forms an adhesive porous layer on the substrate. For the production of such "Llush polymer layers", which can be part of an analytical element according to the invention can be the most varied of polymers be used, e.g. B. polycarbonates, polyamides and cellulose esters.

Instead of a "Llush polymer layer" as a suitable layer, which performs the functions of sample propagation and filtration as well as the necessary reflection background for reflection spectrophotometric represents measurements, a correspondingly effective layer can be produced in that a thin layer of a microporous filter membrane is laminated onto the reagent layer. Such filter membranes are or consist of so-called "blushed polymers" z. B. from cellulose esters, and contain microscopic pores Size. A large number of materials of various pore sizes are commercially available for the production of such layers available, examples of fabrics of this type that can be used to produce corresponding layers and those commercially available are the filters and filter materials sold under the trade name Millipore by from the US company Millipore Corporation and, for example, in the company brochure "Millipore Catalog MC / 1 "of August 1972 by Millipore Corporation of Bedford, Hass., USA 01730, and also those Filters and filter materials under the trade name "Metricel from the US Gelnian Instrument Company are available.

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If the analytical element according to the invention has a single layer which functions as the sample spreading layer, has the function of a filter layer and the function of a reflective layer, the analytical element, z. B. in the form of a tape, consist of only two layers and the substrate, as except for the layer with the function of a spreading layer, a filter layer and a reflective layer only the reagent layer absolute is required. Of course, the element can also have other layers if so desired is, d. H. z. B. two or more adjacent reagent layers can be arranged to allow sequential reactions allow in each of the layers.

As already explained, however, it can also be advantageous To use elements that have a layer that acts as a filter layer and a reflective layer, and a special one Layer that has the function of a sample spreading layer or elements using the three separate layers have, which perform the functions of sample spreading, sample filtration and light reflection. In this In the case of course, the layers are applied to the substrate in such a way that the sample spreading layer corresponds to the from The backing is the furthest layer, followed by the filter layer and then the reflective layer and finally the reagent layer.

An example of a layer that functions as both a filter layer and a reflective layer is one Layer of titanium dioxide or barium sulfate dispersed in a binder, e.g. B. cellulose acetate, polyvinyl alcohol or gelatin. Such a layer has been found to be particularly effective in a multilayer element, e.g. B. Volume, proven that is intended for the analysis of blood, since that

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The element or band is able to effectively filter out the blood cells while the layer allows the serum to pass through it at the same time an effective white background for spectrophotometric Provides measurements through the substrate.

Line particularly advantageous layer with the function of a Sample spreading layer in a multilayer element the invention for the analysis of blood is a layer consisting of a dispersion of diatomaceous earth in a binder, z. B. cellulose acetate. Diatomaceous earth has been found to be extraordinarily effective in uniformly distributing the blood proven in the lateral direction. Advantageous sample spreading layers can also consist of microcrystalline Colloidal products are made that are either naturally occurring or synthetic derive polymeric substances. Such microcrystalline substances are, for example, by 0. A. Battista in the article "Colloidal Macromolecular Phenomena, Part II, Novel llicrocrystals of Polymers "published in the Journal of Applied Polymer Science, Volume II, pp. 481-498 (1967). Microcrystalline cellulose, which is used for the production of such Layers which can be used are commercially available, for example under the trade name Avicel, manufacturer FMC Corporation, USA (see Food Chemical Codex, 1st edition).

Advantageous results can furthermore be obtained when a sample spreading 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 polymeric Kunstharzkügelclien, ie so _ö e; uannie ^fi glass beads '' and "polymeric resin beads". As vortti "iliaf *, <ί IAn J, - ^ iitt el for such kitchens have?] / · I ε I sv, ei ί ο gelatine ' ^τ ' κΐ ^ olyvinyl-

"P. ¹ IP ä '"! · '*

, -> U ύ ο C j <" ι

ν- ¹ ^ *

Alcohol proven, uas binding agent should be used if possible be used in small amounts, so that it is avoided that a substantial proportion of the pore volume caused by the spherical Particle is generated, is filled.

nine spreading layer, made up of spherical particles
Uniform size can have advantages over a porous polymer layer, such as a so-called "blush polymer layer". Such a layer is able, for example, to spread a drop of blood extremely quickly, uniformly and reproducibly, so quickly that the spreading or distribution is completed before blood components can be drawn into adjacent layers of the multilayer element. As a result, a very uniform concentration of the sample components in the reagent layer is achieved and consequently a uniform discoloration, which is the basis for the
Analysis forms. While blood cells can occasionally clog porous polymer layers, such clogging does not occur when the sample spreading layer consists of spherical particles in a matrix of a binder. By using spherical particles
of uniform size, particularly advantageous results are thus obtained, since these particles have an adequate or
Create a sufficient volume of pores while keeping the
Limit spaces to a relatively narrow area. This leads to a halt in the spread of the blood when the spaces between the area on which the sample is applied
have been completely filled, with rapid drainage of the plasma into the underlying layer at the same time
is enabled when the spreading process has ended. Vs 3iat has been shown that spherical particles with a size of about. 80 to 120 lik of are particularly advantageous when the spreading layer is used in a folutanalysis for the spread of

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Blood droplet is used. One applied to such a layer Blood drop spreads to a uniform circular area in a few seconds, which after spreading area covered is directly proportional to the sample volume.

The spreading layer of an analytical Element according to the invention, for. B. in addition to the spherical particles, a small amount of a surface-active Compound can be added to further accelerate the spreading process.

In addition to the layers described, an analytical element according to the invention can also have further layers. So is it is possible, for example, to additionally arrange a dialysis layer directly over the reagent layer. Such a layer can For example, when analyzing the glucose content of the blood, this can be advantageous because such a layer prevents the passage of Allows glucose into the reagent layer, but inhibits the passage of high molecular weight proteins that cause the Could interfere with glucose analysis. For the separation of high molecular weight substances from nxedrxgmolecular substances, for example semipermeable cellulose membranes, which are therefore suitable as dialysis layers. Such layers work by a different mechanism than the porous polymer layers, such as. B. the "blush polymer layers", the act mainly like a sieve or grid, in which the pores allow the passage of solutions and small particles, however, block the passage of larger diameter particles.

The analytical elements according to the invention can be used to carry out The most diverse chemical analyzes are used, and not only in the field of clinical

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Chemistry, but rather also in the field of chemical research in general as well as in chemical laboratories that monitor the course of chemical processes. The inventive analytical However, elements are particularly suitable for clinical trials, specifically for the analysis of body fluids, such as blood and urine, since a large number of analyzes must often be carried out here, analyzes often have to be repeated and the analysis results often in a very short period of time after taking the sample must be available. In the case of a blood analysis, for example, an analytical element, e.g. B. in shape of a tape, designed so that it is used for the quantitative analysis of many blood components that are routinely determined must be. can be used. For example, an analytical element according to the invention, e.g. B. in the form of a Tape, easy to analyze 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 for other components of the blood, selected in ^ the appropriate test reagents will.

For example, in blood analysis with an analytical tape according to the invention, the blood cells can first be separated from the serum, for example by centrifugation, after which the serum is applied to the tape. On the other hand, however, it is not necessary to perform such a separation of the blood cells because all of the blood can be applied directly to the analytical tape, with the blood cells being filtered out by the filter layer. The presence of these blood cells on the tape does not interfere with the spectrophotometric analysis if this is carried out, for example, by the spectrophotometric reflection method, since the support and the reagent layer for

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Light is permeable and the light from the porous reflective Layer is reflected. A particular advantage of an analytical tape according to the invention is thus that it can be used to analyze both serum and blood.

A wide variety of test reagents can be used to produce the analytical elements 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, the determination of glucose can for example take place in that the reagent layer contains a ferricyanide or a ferricyanide compound and that the decrease in the yellow color of the ferricyanide is determined, which takes place by reacting the ferricyanide with glucose. Because high molecular weight Blood proteins can lead to a certain reduction in ferricyanide and thus the method for determining glucose can interfere, a dialysis layer of the structure already described can be used to avoid difficulties be placed in the element or band.

When analyzing uric acid in the blood can use the reagent layer for example from a mixture of copper sulphate and neocuproin, d. H. 2,9-dimethyl-l, 10-phenanthroline of the formula:

CH.

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dispersed in a binder. The uric acid causes a reduction of copper (II) to copper (I), which then forms a colored complex with the neocuproin, whereby the color density is directly 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 system mentioned catalyzes the conversion of glutamic acid into oxaloacetic acid at a pH value of 7.4 and the oxaloacetic acid can be by a Coupling reaction with the diazonium salt of a dye, known as "Fast Ponceau L" (Color Index No, CI. 37151). In order to facilitate the process of the first reaction equation before the color-forming coupling reaction takes place, can it may be advantageous to separate the reagents in two Lay layers to ensure that sufficient time is allowed for the initial reaction to proceed Becomes available before the second reaction can proceed. So the glutamic acid can be accommodated in a first reagent layer placed over a second layer of reagent containing the Fast Ponceau L salt.

A typical automatic analyzer in which an analytical Tape according to the invention can be used, typically has device parts for unwinding or unwinding of the tape from a supply roll and device parts that guide the tape past a sample distributor which one drop of the sample to be analyzed, e.g. B. a blood or serum drop applied to the surface of the tape as well as device parts that guide the belt through one or more development zones, and finally device parts, through which the tape is wound up by means of a take-up roller. In the mentioned development zones the tape

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subjected to such conditions that the course of a color-forming Facilitate reaction between a component of the sample to be analyzed 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 solvent to accelerate or assist the desired reaction to proceed.

The analytical measurements are then made by passing the tape through a zone in which spectrophotometric measurements are taken can be carried out, for example, in the case of the use of reflection spectrophotometry, a device by means of which a beam of light is directed through the substrate, which is then returned to the indicating parts of the spectrophotometer is reflected. When using reflection spectrophotometric measuring methods, an optical interference becomes by residues, for example blood cells, which have remained on or in the layers of the tape, avoided.

As already stated, however, other spectrophotometric Measurement methods are used, for example fluorescence spectrophotometry. For generating comparison standards drops of saturated solutions of the component to be analyzed can be applied adjacent to the area, on which the drop of the sample to be analyzed is applied, whereby differential measurements are made possible.

The analytical elements according to the invention can be in various Size to be made. In the case of tapes, for example, a typical tape has a width of 16 mm, the for example, it can be wound onto a spool and enclosed in a cassette.

The automated analytical systems can be so constructed be that in them there is only one type of analytic

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Tape can be used in the case of systems in which only a single analysis is to be carried out,. with a large number of samples to be analyzed, or the device can be constructed so that a wide variety of tapes can be used in it, namely either simultaneously or sequentially, each band containing the reagents necessary for the analysis of a particular component of the sample to be analyzed. A single analytical tape can also be used to analyze two or multiple components of a sample are used if there are test reagents for performing the analysis of each of the two Contains components, wherein the test reagents can be present in a mixture with one another in a reagent layer or in two or more separate reagent layers, as long as the reagents are selected so that they Do not interfere with the reaction of the other reagent.

The reagent layer can optionally also be in the form of a plurality or a plurality of parallel, adjacent strips of different compositions are present, each strip having one or contains multiple test reagents designed for performing an analysis of a specific component of the sample.

As stated earlier, the analytical element of the invention needs not in the form of an endless belt, but rather, for example, smaller sections on cards, in particular cards with openings, be attached, wel-

en
before using a special device for such cards 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 analytical elements according to the invention can be fastened in or on such a card, each element being used for analyzing a specific component of a sample

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contains necessary reagent so that such an aperture card can be used to conduct a variety of tests can be used.

In the drawing, different embodiments are multilayered analytical elements according to the invention in section, enlarged, shown. In detail are shown in:

1 shows an analytical element consisting of a layer support 10 with a reagent layer 12 applied thereon, a reflective layer 14 which has a white background provides for reflection-spectrometric measurements through the layer 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 binder, for example gelatin, while each of the layers 14, 16 and 18 consists 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.

2 shows an analytical element 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 filter them, and which further provide a suitable background for reflectance spectrophotometric measurements made possible by the layer carrier 20. The layer 24 can, for example consist of a "blush polymer layer" applied to the layer 2 2, or a layer of a microporous one Filter membrane which has been laminated onto layer 22.

! / ig. 3 an element made up of a layer support 30, a reagent layer 32, a dialysis layer 34, which consists of a semiperineablen Membrane layer was formed, and a layer 36, e.g. B.

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a "blush polymer layer" which has the function of spreading the sample to be analyzed and the filtration of the sample and which also provides a suitable background for reflection-spectrophotometric Provides measurements through the substrate.

4 shows an analytical element consisting of the layer support 40, a first reagent layer 42, and 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 can, for example, consist of a dispersion of diatomaceous earth in cellulose acetate or of glass beads in gelatin be constructed.

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

example 1

On a 0.10 mm thick polyethylene terephthalate film support a reagent layer consisting of a dispersion of copper sulfate and neocuproin in gelatin was applied in such a way that that on a support area of 1 dm 0.84 mg of copper sulfate, 1.28 mg of neocuproin and 53.9 mg of gelatin were allotted.

A layer of microporous Filter material 180 microns thick and an average Laminated pore size of 1.2 microns. The filter material consisted of a commercially available microporous one Cellulose acetate nitrate membrane commercially available under the trade name Millipore MF Filters, manufactured by Millipore Corporation, USA. The membrane was applied to the

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Reagent layer laminated by the reagent layer was initially exposed to the action of steam for several seconds for the purpose of softening, after which the filter layer was pressed onto the reagent layer, whereupon the two layers for the purpose of connection by means of a roller have been subjected to low pressure.

An analytical tape produced in this way is suitable, for example, for the determination of uric acid in blood, whereby the presence of uric acid is indicated by the creation of a pinkish-red hue in the reagent layer.

Example 2

Ls became an analytical tape for the determination of uric acid prepared in the blood in the manner described in Example 1, with the exception, however, that as a binder for the reagent layer polyvinyl alcohol was used instead of gelatin. The polyvinyl alcohol was in a concentration of 4 3.1 mg / dm used.

Example 3

An analytical tape for the determination of chloride in blood was produced in the manner described in Example 1, except, however, that the reagent layer consisted of a gelatin layer in which silver chromate is dispersed was. 53.9 mg of gelatine and 18.0 mg of silver were used on a support area of 1 dm. The presence of chloride in a sample to be analyzed is indicated in this FaTLe by changing the color of the reagent layer from reddish-brown after yellow.

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ORIGINAL INSPECTED

Example 4

An analytical tape for the determination of albumin in blood was produced according to the method described in Example 1, this time creating a reagent layer consisting of 4.66 mg of Ilydroxyphenyiazobenzoic acid dispersed in 21.0 mg of a copolymer !! from acrylic acid and 2-acetoacetoxy-

2 ethyl methacrylate, each based on an area of 1 dm. 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 film support, each per 1 dm support area. A slurry was then 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 50 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 ₇ and 37.2 mg cellulose acetate, each per dm, whereby a layer was obtained which acted both as a filter layer and as a light-reflecting layer. A spreading layer was then applied to this layer, namely 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.

Three different analytical tapes were made using various concentrations of diatomaceous earth in the spreading layer, v; above each tape produced

309803/1343

has been tested for its ability to spread blood, wherein a 10 microliter sample of blood was applied to each tape became. The time it took for the blood to flow was determined diffuse completely into the tape. Furthermore was the diameter of the spot produced is determined, through the layer support. Ls were the following results obtain:

Ver Diatomsen- Salicyl- Cellulose- Diffusion- Spots (Spot) -such earth ₇ acid ₇ acetate ₇ time in diameter Kr. (Mg / dm) (mg / dm) (mg / dm ") seconds in cm

1,300 3.00 37.2 26 1.05

2,375 3.75 37.2 41 1.00

3,450 4.50 37.2 31 - 1.00

The analytical tapes described in this example are suitable for determining the glucose content of the blood, whereby the presence of glucose is indicated by the formation of a brown hue in the reagent layer.

Example 6

At a 0.10 mm thick polyethylene terephthalate ilmschicntträger a reagent layer of the composition given in Example 1 was coated!, After which the reagent layer is a microporous filter layer of the type described in Example 1 is laminated i \ ^r urde. The microporous filter layer served as a filter layer and as a layer that provided a suitable background for making reflectance spectrophotometric 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. -Ό a surface-active compound, and

309883/1343

233: ^ 60

although para-isononylphenoxypolyglycidol with 10 clycidol units, Were blurred, whereupon the resulting slurry in shape a thin layer was applied to the microporous filter layer in such a way that 1 g of beads was applied to one surface of 27 cm "were omitted.

1N microliters were then applied to the analytical element obtained Blood which was found to spread that 10 microliters of blood into one in less than 1 second circular area about 1 cm in diameter.

Example 7

On a 0.18 mm thick polyethylene terephthalate film support First, a reagent layer was made of 2.15 mg of glucose oxidase, 1.07 mg of peroxidase, 3.20 mg of o-dianisidine hydrochloride and 215 mg of gelatin, in each case per dm layer support area, applied. A microporous i-'il termembrane, 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 of the colored sample was determined The stain produced in the reagent layer by the action of variously sized samples of a glucose solution which contained 100 mg of glucose per 100 ml of distilled water.

The reflection densities were read 5 minutes after application of the samples on the analytical tape. The results compiled in the following table were obtained.

.3 09883/1343

ORiGfNAL INSPECTED

2332760 Spots (spot) -
District (mm ² ) Reflection
density 30th 0.66 50 0.71 63 0.70 86 0.65 92 0.70

Sample amount
in microliters

10
12th
15th

From the results obtained, it is found that the reflection density values were practically independent of the size of the applied drop. The results obtained therefore show that that an analytical element according to the invention a quantitative analysis of a component of a to be analyzed Sample allows, without the need for a precisely defined amount of sample to be applied to the element will.

Example "8

The following solutions were made:

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

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

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

309883/1343

The solutions described were mixed together and then applied to a 0.254 mm thick cellulose triacetate film layer
de.

2 carriers applied in such a way that an area of 1 m was covered.

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

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

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 0.3 molar glycine buffer solution at a pH value of 9.6;

(b) a solution of 2.150 g of a low-temperature solidifying agarose (polysaccharide) in 70 ml of a 0.3 molar Glycine buffer solution, pH 9.6;

(c) 504 mg of nicotamide adenine dinucleotide in 10.7 ml of a 0.3 molar Glycine buffer solution with a pH of 9.6.

3 0 9883/1343

in 2 ⁷ 6o

Solutions (a), (b) and (c) were mixed together at 40 ° C and so on a cellulose triacetate support

applied so that a layer area of 1 m was covered. These Reagent layer was then coated with a spreading layer as described in Example 1.

Various amounts of an aqueous solution of glycerine containing 50 mg of glycerine per deciliter of water were then applied to the surface of the element obtained. After the applied samples had dried on within 40 minutes at room temperature, the reaction product generated in the reagent layer was excited by irradiation with light having a wavelength of 350 nm, fluorescence at a wavelength of 445 nm
occurred. The strength of the fluorescence generated at 445 nm that
measured with a conventional fluorescence spectrophotometer was found to be proportional to the concentration of glycerin in the test sample.

3 0 9883/1343

Claims (1)

PATENT CLAIMS ] 1 ·) Multi-layer analytical element for quantitative spectrophotometric analysis of a liquid, characterized by 1. a liquid impermeable, translucent Layer support, 2. at least one reagent layer arranged on one side of the layer support a reagent that reacts with a component of the liquid to be analyzed to cause a color change within the reagent layer, and 3. one arranged on the side of the reagent layer facing away from the layer carrier porous layer or a porous layer structure that or the constituent of the liquid to be analyzed which is responsible for the color-forming reaction with the reagent is permeable and the or the applied liquid spreads laterally such that a constant volume of liquid per Unit area is obtained and applied to the reagent layer. 2. Multi-layer analytical element according to claim 1, characterized in that the porous layer or at least one Layer of the porous layer structure consists of a polymer layer which was produced by dissolving a polymer in one Mixture of two liquids, one a good solvent for the polymer and the other a nonsolvent or at least is a poor solvent for the polymer and has the higher boiling point, applying the Solution on the reagent layer or a layer above it and drying of the layer with evaporation of the solvent. 309883/1343 3. Multi-layer analytical element according to claims 1 and 2, characterized in that the porous layer or at least a layer of the porous layer structure forms a reflective background for spectrophotometric measurements. 4. Multi-layer analytical element according to claims J. to 5, characterized in that it is in the form of an endless belt. 5. Multi-layer analytical element according to claims 1 to 4, characterized in that the reagent layer consists of a dispersion at least one test cage consists of a hydrophilic colloid. 6. Multi-layer analytical element according to claims 1 to 5, characterized in that the reagent layer consists of a Dispersion of at least one test reagent in gelatin consists. 7. Multi-layer analytical element according to claims jj to S, characterized in that the reagent layer consists of the dispersion of at least one test reagent in polyvinyl alcohol consists. 8. Multi-layer analytical element according to one of the claims 1 to 7, characterized in that the layer support consists of Cellulose acetate or polyethylene terephthalate is made. C. Multi-layer analytical element according to claim 2, characterized in that it has a plurality of porous layers which have been produced by dissolving a polymer in a mixture of two liquids, of which the. one is a good solvent for the polymer and the other is a solvent or at least a poor solvent for the polymer and has the higher boiling point Ί09883 / 1343 BATH ORIGINAL the solution onto the reagent layer or a layer above it and drying the layer with evaporation of the solvent, with the proviso that the individual porous. Layers of the produced porous layer structure one from the other have different porosities. 10. Multi-layer analytical element according to claims 1 to 9, characterized in that the porous layer is a filter and reflective layer consisting of the dispersion of Titanium dioxide in a binder, or that in the case of a porous layer structure, this structure is at least one such Has layer. 11. Multi-layer analytical element according to claims 1 to 9, characterized in that the porous layer has a filter and reflective layer consisting of a dispersion of barium sulfate in a binder, or that in the case a porous layer structure that has at least one such layer. 12. Multi-layer analytical element according to claims 1 to 9, characterized in that the porous layer is a spreading layer, consisting of diatomaceous earth dispersed in a binder, or that in the case of a porous layer structure this contains at least one such layer. 13. Multi-layer analytical element according to claims 1 to 9, characterized in that the porous layer is a spreading layer is composed of a dispersion of inert spherical particles of uniform size in a binder, or in the case of a porous layer structure contains at least one such layer. 309883/13 43 14. Multi-layer analytical element according to one of claims 1 to 9, characterized in that the porous layer is a Spreading layer is made up of a dispersion of crystal beads of uniform size in a binder, or in the case of a porous layer structure this at least contains such a layer. 15. Multi-layer analytical element according to one of claims 1 to 14, characterized in that it is adjacent to the reagent layer has a dialysis layer. 16. Multi-layer analytical element according to one of claims 1 to 9, characterized in that the porous layer consists of a cellulose acetate layer or, in the case of a porous layer structure, there is at least one cellulose acetate layer having. 17. Multi-layer analytical element according to claims 1 to 16, characterized by having a plurality of reagent layers adjacent to one another. 18. Multi-layer analytical element according to claims 1 to 17 ", characterized in that the reagent layer contains glucose oxidase, peroxidase and o-dianisidine hydrochloride. 19. Multi-layer analytical element according to claims 1 to 17, characterized in that the reagent layer contains copper sulfate and neocuproin. 309883/1343