Spreading layers, wetting agents for their preparation and their use in test strips

The present invention relates to spreading layers comprising a porous sheet impregnated with N-acyl-N-alkyl-glycinates, the production of this spreading material using the N-acyl-N-alkyl-glycinates and test strips which contain the spreading material according to the invention.

So-called carrier-bound tests are often used for the qualitative or quantitative analytical determination of constituents of body fluids, in particular blood. In these, reagents are present on or in corresponding layers of a solid test carrier which is brought into contact with the sample. The reaction of the liquid sample and reagents leads to a detectable signal, in particular a color change, which can be evaluated visually or with the aid of a device, usually by reflection photometry.

Test carriers are often designed as test strips, which essentially consist of an elongated base layer made of plastic material and detection layers attached thereon as test fields. However, test carriers are also known which are designed as square or rectangular plates. In the following description, the term “test strip” is also intended to include test carriers which do not have the strip shape.

Test carriers of the type described at the outset are known, for example, from German Patent 21 18 455. There, diagnostic test carriers for the detection of analytes in liquids are described which consist of a support layer and at least one detection layer containing the detection reagents, the surface of which does not lie on the support layer is provided with a cover layer. The cover layer can consist of a fine-mesh network in the form of a woven, knitted or non-woven fabric. Plastic meshes are given as preferred networks in order to achieve a quick wetting of the detection layer with sample liquid and to avoid disturbing chromatography effects. To detect an analyte in a liquid, such a diagnostic test carrier is immersed in a corresponding liquid, preferably urine. The detection layer thus comes into contact with a very large excess of liquid which cannot be absorbed by the test carrier. Depending on the duration of contact of the detection layer with the liquid to be examined, however, different color intensities can be observed.

As a rule, the longer the contact time, the more positive results are obtained. With a large sample excess, a correct quantitative analyte determination is therefore not possible in this way.

On the other hand, a sample volume that is too small for a test carrier construction is a common cause of incorrect measured values ​​in diabetes monitoring, that is, the regular monitoring of the blood of people with diabetes for the content of glucose.

Test carriers with the smallest possible volume requirements are therefore the target of a variety of current developments.

DE-A-3042857 discloses test strips which have a sample distribution layer (spreading layer) on their multilayer analysis elements, which has the task of distributing sample liquid applied in a punctiform manner uniformly over the entire test element. This spreading layer consists of a cloth or a foam layer which is hydrophilized by impregnation with a wetting agent and either pressed onto the still moist top gelatin layer of the analysis element or fixed thereon by means of an additional adhesive layer.

In the exemplary embodiments of this publication, a cotton fabric or a filter cloth is used as the spreading layer; the nonionic wetting agent polyoxyethylene nonylphenoxy ether is used for the hydrophilization.

Diagnostic test carriers in the form of test strips which offer significant progress in terms of reproducibility of the test results even with application of different sample volumes and in terms of hygienic handling are known from EP-A-0 821 233.

They contain a base layer with a detection layer arranged thereon for the determination of analyte in a liquid sample and a layer covering the detection layer from a network, which is larger than the detection layer and on both sides of the detection layer on the base layer, preferably via a spacer, is attached, on the other hand, without attachment directly, ie touching the entire surface without a gap. The network used as a support should be hydrophilic but not capillary-active on its own. Here too, the network for hydrophilization is impregnated with a wetting agent, namely sodium dioctyl sulfosuccinate.

In this construction, the network covering the detection layer preferably consists of a coarse-titer, relatively coarse-mesh monofilament fabric with a sufficiently large mesh size for liquid to pass through the network onto the detection layer (page 3, line 12). In the example of this application, a monofilament fabric with a mesh size of 280 µm is used. It has an essential function: it quickly transfers sample liquid applied to its surface to the detection layer underneath. If the detection layer is saturated, any excess sample is drained off into the edge areas of the network that go beyond the detection layer. In this way, small amounts of sample are made completely available to the detection layer, but the longer exposure to excess sample, which can lead to false-positive results, is avoided.

This publication also describes an embodiment which has two or more detection layers arranged next to one another, which are intended to measure the same or different analytes.

The use of test carriers in this embodiment naturally offers tempting advantages in terms of labor and cost savings, since it should be possible to carry out two or even more measurements with a single sample application. In practice, however, difficulties arise when trying to use this embodiment, which can be attributed to the fact that the spreading action of the relatively coarse-meshed network in multi-field test strip constructions is not sufficient to distribute the analyte samples over both detection fields. Only with the most careful application of an analyte sample exactly above the limit of both test fields - which is rarely possible in practice - can both test fields be wetted, however a minimum sample volume of over 15 µl is then required to completely wet the test fields.

Even the attempt to wet both test fields by placing the fields directly next to each other without a gap does not lead to success. It can be seen that even in this case the tiny distance of 5 to 10 µm between the test fields still prevents the blood from "jumping" from one field to the other.

The prior art thus shows approaches for further improving and economizing the analytical methods using test strips, but there are still considerable obstacles to their use in practice.

There is no technical teaching to distribute analyte samples quickly and evenly over a test field arrangement, especially if small sample volumes are available and / or even several test fields are arranged next to one another. There is a need, among other things, for the quantitative detection of analytes, for example glucose, in capillary blood in a single-field test about 3 µl blood in less than 2 seconds over the relatively large area of ​​30 mm² to distribute evenly. In a doubt field test, approximately 5 µl of blood should be taken over two test fields of approximately 30 mm each² be distributed.

The time factor is also of great importance in such methods: it is not just about accelerating the test procedure, but it is important to distribute the analyte evenly over the entire test layer with the least possible delay, because, as already mentioned above, it can a time offset is reflected in a gradient of the analysis result over the test field, so that the determination is burdened with a considerable uncertainty factor.

To the knowledge of the patent applicant, the tasks resulting from these claims have not yet been satisfactorily solved in a simple manner.

It has now surprisingly been found that the sample volume required for the measurement can be reduced to approximately 4 μl and that the sample can be distributed very evenly over several detection fields arranged next to one another in a time of less than 2 seconds if the detection fields are used covers the spreading material according to the invention described below.

The spreading material according to the invention comprises a porous sheet impregnated with a wetting agent, which is characterized in that the wetting agent is an N-acyl-N-alkyl-glycinate of the formula I.         R-CO-N (R¹) -CH₂-COOMe (I) is in which R is an aliphatic radical with 9 to 23 C atoms, in particular with 11 to 17 C atoms, which is saturated or has one to three double bonds,<ul id="ul0001" list-style="none" compact="compact"><li>R¹ Hydrogen or lower alkyl and</li><li>Me is a hydrogen or metal atom.</li></ul>

In particular, R is the aliphatic chain of lauric acid, myristic acid, palmitic acid, stearic acid, palmitoleic acid, oleic acid (oleic acid), linoleic acid, linolenic acid and their isomers.

It is also very advantageous to use an N-acyl-N-alkylglycinate mixture in which the radicals R correspond in terms of their structure and their proportion in the mixture of the structure and the proportion of their occurrence to the alkyl radicals of natural fatty acids, for example tallow fatty acid or coconut fatty acid . In technical parlance, such alkyl mixtures are referred to as "tallow fatty alkyl" or "coconut fatty alkyl".

For R¹ standing lower alkyl groups are preferably linear and have 1 to 4, in particular 1 or 2, carbon atoms. Particularly preferred for R¹ is the methyl group.

The metal atom Me is expediently chosen so that the glycinate of the formula I is water-soluble. Particularly suitable are the alkali metals, preferably sodium and potassium. A spreading material according to the invention which is impregnated with sodium N-oleoyl sarcosinate is particularly preferred. Sodium N-oleoyl sarcosinate can easily be prepared by reacting N-oleoyl sarcosinate (eg commercial product Crodasinic® O from Croda, Nettetal) with an equivalent amount of sodium hydroxide solution.

In connection with the material description of the spreading material according to the invention, the feature “impregnated” means that the material bears a coating of the impregnating agent on its surface that is accessible to liquids, ie that pores and intervening filament spaces are also “lined” with this agent. The application of the compound of formula I brings about a particularly advantageous hydrophilization of the porous fabrics, which justifies their good suitability as spreading material.

Naturally, the local surface concentration of the impregnation agent depends on the accessibility of the surface element in question. However, this is of minor importance for the hydrophilizing effect of the superficial layer.

To achieve a hydrophilically effective coating on spreading materials according to the invention, the amount is usually from 0.01 to 2.0% by weight, preferably from 0.03 to 0.5% by weight, of N-acyl-N-alkyl Glycinates of the formula I, based on the weight of the material before the impregnation, are sufficient.

The superior spreading action of the spreading material according to the invention is closely linked to the presence of a wetting agent of the formula I in the material. The superiority of the wetting agent of formula I used according to the invention over that used in the closest comparable prior art, EP-A-0 821 233, can be demonstrated by a simple experiment:

A nonwoven fabric (Viledon® FO 2451/121 from Freudenberg) is mixed in a 0.5% by weight aqueous solution of sodium dioctyl sulfosuccinate (the wetting agent used in EP-A-0 821 233) Saturation impregnated and air dried. A blood sample of 5 to 10 µl is placed on an approximately 3x3 cm large, horizontally clamped piece of the impregnated nonwoven. The blood remains hemispherical on the top of the nonwoven, the bottom does not get wet.

If the same amount of blood is applied to a piece of the nonwoven fabric impregnated with sodium dioctyl sulfosuccinate, which lies on a reagent film, the drop of blood is also on the top of the nonwoven without wetting the film and thus causing a reaction. Only when the blood is mechanically pressed through at one point, for example by touching the fleece with the pipette tip, does blood reach the underside of the fleece and spreading and reaction take place.

If the experiment described is repeated in exactly the same way with the only difference that instead of the sodium dioctyl sulfosuccinate solution, an aqueous 0.1% by weight solution of sodium N-oleoyl sarcosinate is used to impregnate the nonwoven fabric , the applied blood penetrates the fleece and hangs hemispherically on the underside. If the impregnated fleece lies on a reagent film when the blood is applied, the blood will spontaneously reach the bottom and spread and react.

The demonstrated superiority of the compounds of formula I when used as spreading agents is very surprising. Wetting agents are known in large numbers and from various groups of chemical compounds.

What they have in common is that they have a hydrophilic and a hydrophobic section of the molecule.

In "Ullmanns Encyclopedia of Technical Chemistry", Vol.5, wetting agents with non-ionic, anionic, cationic and amphoteric character are described. On page 778, from the group of anionic wetting agents, succinic acid derivatives, in particular sulfosuccinic acid dialkyl esters, are highlighted as wetting agents with outstanding effectiveness, which have found an extraordinarily broad technical application.

On page 751 of this standard work, derivatives of aminocarboxylic acids, including aminoacetic acid - which is also a structural element of the compounds of the formula I to be used according to the invention - are mentioned as wetting agents and are classified in the amphoteric wetting agent group. On pages 795/796 it is stated that these have gained only little technical importance, in particular because of the strong dependence of their properties on the pH.

It was therefore not foreseeable that compounds of the formula I, when used as spreading agents, would be considerably superior to the sulfosuccinic acid esters known as excellent wetting agents.

The porous fabric on which the spreading material according to the invention is based is a textile fabric made of monofilaments or corresponding multifilament yarns, which itself is not capillary-active but liquid-permeable, and whose construction and material and / or hydrophilization equipment is selected such that it rests on a base, 10 ul water an area of ​​more than 300 mm² spreads.

Textile fabrics that meet this requirement can be found under commercially available materials. The rough selection of suitable textile materials can basically be made according to the motto "as thin as possible with the smallest possible basis weight". The following spreading test can be used to select suitable materials:

10th mm wide and at least 100 mm long strips of the textile material to be tested, if necessary hydrophilized, are placed on the matt side of a polycarbonate film. Then 10 µl of water is applied in a dot shape in the middle of the strip and its spread is observed. With a suitable material, the spreading process should be completed within 5 to 10 seconds and the wetted area should be at least 30x10 mm.

With this test, for example, the serious differences between the polyester fabric PE 280 HC to be used as spreading material according to EP-A-0 821 233 and textile materials to be used according to the invention, for example polyester fabric PE 38 HC or Viledon nonwoven fabric FO 2451/121, can be demonstrated as follows:

10th mm-wide strips of the textile materials to be tested are impregnated and dried with the same overlay (approx. 0.25% by weight, based on the weight of the material before impregnation) of sodium N-oleoyl sarcosinate. Then the impregnated textile strips are placed on the matt side of a polycarbonate film. If you now apply 10 µl of water in a punctiform manner, the water spreads over the areas specified in the following table in 5 to 10 seconds and then comes to a standstill:<tables id="tabl0001" num="0001"><table frame="all"><tgroup cols="3" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="52.50mm" /><colspec colnum="2" colname="col2" colwidth="52.50mm" /><colspec colnum="3" colname="col3" colwidth="52.50mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" align="left">According to</entry><entry namest="col2" nameend="col2" align="left">material</entry><entry namest="col3" nameend="col3" align="left">surface</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">EP-A-0821233</entry><entry namest="col2" nameend="col2" align="left">PE 280 HC</entry><entry namest="col3" nameend="col3" align="left">8th x 10 mm</entry></row><row><entry namest="col1" nameend="col1" align="left">invention</entry><entry namest="col2" nameend="col2" align="left">PE 38 HC</entry><entry namest="col3" nameend="col3" align="left">40 x 10 mm</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">invention</entry><entry namest="col2" nameend="col2" align="left">Viledon FO 2451/121</entry><entry namest="col3" nameend="col3" align="left">50 x 10 mm</entry></row></tbody></tgroup></table></tables>

Textile materials that are particularly suitable for spreading small amounts of blood are woven, knitted or nonwoven fabrics with a thickness of 20 to 200 μm, preferably 30 to 100 μm and / or a pore volume of 30 to 85, preferably 40 to 75% . proven. The basis weight of these materials, which are particularly suitable for spreading small sample volumes, is 10 to 200, preferably 10 to 50 g / m².

In this context, materials with a thickness of less than 100 μm, in particular less than 50 μm and basis weights of less than 50, preferably less than 25 g / m, are particularly preferred².

Preferred fabrics with the dimensions mentioned are woven and nonwovens. Of the fabrics, those are preferred which have a mesh size below 200 μm, preferably from 20 to 150 μm, in particular from 20 to 60 μm, are resistant to sliding and made from monofilaments with titers in the range from 2 to 20 dtex, preferably from 4 to 15 dtex, are made in plain weave. The fabrics may have anti-slip equipment applied after weaving. However, the resistance to displacement of the fabrics is preferably achieved in that a wavy deformation is impressed on the monofilaments in the course of fabric manufacture and / or that warp and weft filaments are easily fused together at the crossing points, thereby securing them against displacement.

Nonwovens used according to the invention are random webs, preferably bonded spunbonded webs. They consist of endless monofilaments whose titer is in the range from 0.5 to 2.5 dtex, preferably from 0.8 to 2.0 dtex. The bonding can be done by treating the nonwovens with bonding agents; however, "autogenous" bonding is preferred, in which the filaments are easily fused together at their crossing points. The pores of such nonwovens naturally have considerable differences in size. Surprisingly, nonwovens with the features specified above are eminently suitable for use in accordance with the invention, despite the wide range of pore sizes.

The low or nonexistent capillary activity of the fabric is shown by the fact that it has a rising height of water at 25 ° C. of less than 2 mm in 10 seconds.

Suitable fiber materials for the textile materials to be used according to the invention are natural fibers such as cellulose or protein fibers, semisynthetic fibers such as acetate or viscose silk or fully synthetic fibers such as polyester, polyamide, polyurethane, polyacrylonitrile, polyethylene or polypropylene fibers or mixtures of the fiber types mentioned .

Preferred cellulose fibers are cotton fibers, preferred protein fibers are wool and natural silk. Suitable acetate fibers are 2.5 or triacetate fibers depending on the desired degree of hydrophilicity. Preferred synthetic fibers consist of polyethylene terephthalate, polyamide-6, polyamide-6,6 or modified polyacrylonitrile.

Because of the high stability against environmental influences and the possibility to modify mechanical and chemical properties during production, synthetic, in particular thermoplastic, fibers are preferred over the natural ones.

Commercial textile fabrics that can be used according to the invention, in particular after hydrophilization, are, for example, the monofilament polyester fabric type PE nn HC or the monofilament nylon fabric type NY nn HC or HD from ZBF Mesh + Technology, Rüschlikon, CH, where nn of 20 varies up to 150, especially polyester fabric type PE 38 HC or the monofilament nylon fabric type NY 41 HC. The "Viledon® nonwovens FO 2451, in particular the type FO 2451/121, from Freudenberg, Weinheim, Germany, are also very well suited for use in accordance with the invention.

The use of the spreading material according to the invention is of course not limited to the use in the construction of test strips, although they are of particular importance in this context, in particular in the construction of diagnostic test strips. Rather, the spreading material according to the invention can be used in technology wherever the rapid and uniform distribution of liquids over relatively large areas is important, for example in the development of extremely large-format silver halide images with a limited amount of developer and in certain sound separation processes in artistic photography.

The present invention also relates to the use of compounds of the formula I in which the radicals R, R¹ and Me have the meanings given above, for the production of a spreading material.

The compounds of the formula I can be used for the preparation of the spreading material in pure form or in the form of preparations, in particular in the form of solutions or liquid preparations.

A preparation can consist of a solution or a finely divided dispersion of one or more compounds of the formula I in water, an inert organic solvent or a solvent / water mixture. It can also be an emulsion of an aqueous solution of the compounds of the formula I in a water-immiscible solvent.

In addition to the compounds of the formula I, the preparations can also contain further additives and / or auxiliaries which either interact synergistically with them or which act, for example, as impregnation auxiliaries, as stabilizers or as protective colloids. In addition to the compounds of the formula I, the preparations can also contain substances which impart additional functions or other advantageous properties to the spreading material. For example, the preparations can also contain finely dispersed inorganic or organic filter materials or fillers. Dyes which give the spreading material a marking color or which are suitable for improving the recognizability of color reactions of the detection layers or the complete wetting of the spreading material or which absorb UV radiation are examples of additives which are used in the preparations of the compounds of the invention to be used Formula I can be included.

The invention also relates to a method for producing a spreading material by impregnating a porous fabric with a wetting agent or a wetting agent preparation, if necessary adjusting the impregnated sheet to a predetermined wetting agent absorption and optionally drying the material, which is characterized in that at least one compound of the formula I in which the symbols R, R¹ and Me have the meanings given above, is used.

Wetting agent preparations to be used in the method according to the invention have already been described above.

All measures which are suitable for a statistically uniform distribution of the wetting agent of the formula I in the porous fabric are suitable for the impregnation. As a rule, the wetting agent is applied in the form of a solution or a liquid preparation.

The impregnating agents used advantageously contain 0.01 to 2% by weight, preferably 0.03 to 1% by weight, in particular 0.07 to 0.3% by weight, of wetting agents of the formula I.

The application can take place in any customary manner, for example by dipping, spraying or by brush application. It is also possible to powder the porous materials with fine dust of the compounds of formula I or solid preparations thereof.

The compounds of the formula I are preferably applied in the form of aqueous solutions.

The applied amount of the compounds of the formula I or the corresponding preparations is measured or an applied excess is removed to such an extent that an overlay of 0.01 to 2.0% by weight, preferably 0.03 to 0, is applied to the porous material , 5 wt .-% of the N-acyl-N-alkyl-glycinate of formula I, based on the weight of the material before impregnation, remains.

Since the wetting agent of the formula I is generally applied in the form of solutions or liquid preparations, drying of the impregnated material is necessary after the impregnation. This can be done in any way suitable for textile materials or open-cell foams. The material is usually dried at temperatures between 10 ° C. and the boiling point of the liquid phase contained in the application medium, preferably at 20 to 80 ° C. The drying process can be supported by vacuum and / or air circulation. The heat can be supplied by convection with a heat transfer medium, by contact with heating elements or by radiation. The fabrics are expediently dried in the expanded state.

In addition to the spreading material according to the invention, a test strip is also the subject of the present invention, consisting of a flexible sheet-like carrier on which one or more test fields are arranged next to one another, each carrying one or more detection layers lying one above the other, which is characterized in that the test fields are characterized by an above-described Spreading material according to the invention are covered.

The test strip should preferably be used for diagnostic purposes.

Particularly advantageous test strips have two immediately adjacent ones - although in practice there is also a microscopic gap of approx. 5-10 µm - or single or multilayer test fields for the same or different analytes separated by a gap. It is also possible to place more than two test fields on one test strip.

The detection layers on the test fields contain reagents for the detection of a diagnostically usable analyte. The detection of the same analyte in two separate test fields can be of interest if the detection layers are to enable a qualitative, semi-quantitative or quantitative assessment of very different concentrations of the analyte or if an assessment of the reproducibility of the measurement result is simply desired. Of particular interest is, of course, the case where the two detection layers permit the simultaneous qualitative detection or a semi-quantitative or quantitative determination of two different, in particular diagnostically interesting, analytes.

The spreading material is not fixed on the detection field or fields, but only lies loosely. In contrast to the teaching for the production of test strips known from DE-A-30 42 857 (page 10), they are not pressed into the detection layers or glued to them. Such a fixed connection makes the spreading function described impossible.

The spreading material is attached to the test strip only on both sides of the test field or test fields, preferably on spacers provided with adhesive layers. With this type of fastening, the spreading material can, in unfavorable cases, form a fold over the test area if the generally flexible test strips are subjected to a bend when used. Wrinkling affects the distribution of the reagent sample across the test area. This wrinkling can be avoided if the support of the spreading material according to the invention consists of one or more sheet-like support elements which are fastened on the flexible test strip in such a way that at least a part of their area is opposite to the area of ​​the test strip covered by this part in the direction of the bending the curvature generated is freely displaceable. This preferred type of attachment of a support element of the support according to the invention to the flexible test strip is carried out by means of at least one, preferably at least two, attachment points which lie in a coherent surface area (attachment area) of the support element. which extends between the edges of the support element lying in the direction of curvature and whose delimitation from the freely displaceable part of the support element extends essentially in a straight line and transverse to the direction of curvature.

In the event that the test strip has two test fields, their covering by a spreading pad according to the invention of the type described above is particularly practical, in which the test fields are covered by two support elements which are fixed on the test strip in such a way that their displaceable areas are directed towards one another and overlap.

In this case, an optimal distribution of the sample is achieved when the overlap of the two support elements lies above the dividing line between the two test fields and preferably symmetrically to it.

Especially in the case of test strips, the proper function of which is of particular importance, which must also be ensured under various conditions of use and when used by more or less experienced laymen, the type of fastening according to the invention, by means of which wrinkles are avoided when the test strip is bent, is very valuable Contribution to application security.

The test fields are preferably mounted one behind the other on the test carrier in the direction of its longitudinal axis, and the spreading support elements, seen in the same direction, are fixed on the flexible carrier in a surface area in front of and behind the test fields.

It is further preferred that the spreading support elements are fixed on spacers which have approximately the thickness of the detection layers.

Finally, it has proven to be expedient if the arrangement of detection layers and overlays on the test strip is covered with an inert sheet-like material in such a way that only in the area of ​​the overlap of the overlay elements in the direction of the longitudinal axis of the test strip is there sufficient distance left for sample application , which is usually 2 to 5 mm.

The carrier of the test strip consists of a transparent material and / or has openings of the same or different shape in the area of ​​the test fields through which the underside of the detection layers can be inspected.

It is advantageous, in particular for automation of the test evaluation, if the carrier of the test strip has alignment markings in the form of additional bores, punched-out areas or notches.

FIGS. 1 to 6 serve to illustrate the above and the following explanations.

FIG. 1 shows a perspective top view, FIG. 2 shows a section along the section line A-A ', and FIG. 3 shows a top view of the underside of an embodiment of a test strip according to the invention.

FIG. 4 shows a perspective top view, FIG. 5 shows a section along the section line A-A ', FIG. 6 shows a top view of the underside of an embodiment of a test strip according to the invention in which the spreading pad according to the invention consists of two support elements, which preferably lie on the Test strips are attached.

The reference symbols used in the figures have the following meaning:<dl id="dl0001" compact="compact"><dt>1:</dt><dd>Test strips</dd><dt>2:</dt><dd>Flexible carrier</dd><dt>3rd and 3a:</dt><dd>Detection layers</dd><dt>4th and 4a:</dt><dd>Spacers</dd><dt>5 and 5a:</dt><dd>Adhesive layers</dd><dt>6:</dt><dd>Edition according to the invention</dd><dt>6 and 6a:</dt><dd>Support elements according to the invention</dd><dt>7 and 7a:</dt><dd>Fastening area</dd><dt>8th and 8a:</dt><dd>Movable area</dd><dt>9 and 9a:</dt><dd>Protective cover</dd><dt>10:</dt><dd>Order area</dd><dt>11 and 11a:</dt><dd>Marking the limits of the detection fields</dd><dt>12:</dt><dd>Marking the direction of insertion</dd><dt>13:</dt><dd>Positioning hole</dd><dt>14 and 15:</dt><dd>Observation and measurement openings</dd></dl>

FIG. 1 shows a perspective top view, FIG. 2 shows a section along the section line A-A ′ in FIG. 1, and FIG. 3 shows a top view of the underside of an embodiment of a test strip according to the invention with a detection area and a spreading pad according to the invention. This representation is made without a scale to make the structure clearly visible. A concrete dimensioning of this embodiment can be found in embodiment 1.

The diagnostic test strip (1) according to the invention shown in perspective in FIG. 1, in section in FIG. 2 and from below in FIG. 3 has a detection layer (3) on a base layer (2), which is covered by the spreading pad (6) . In addition to the detection layer (3), the spreading pad (6) is attached to the base layer (2) by means of spacers (4,4a) and adhesive layers (5,5a). In practice, these spacers can also be hot-melt adhesive surfaces or double-sided adhesive tapes that fix the spreading pad (6) on the base layer (2). Ideally, the spacers with their adhesive surfaces have approximately the same thickness as the detection layer (3). The structure shown here also has covers (9, 9a) which are attached to the base layer (2) and the spreading pad (6). They are arranged in such a way that they cover the areas protruding beyond the detection layer (3) and part of the surface of the spreading pad (6) lying above the detection layer. However, they leave an area above the middle of the detection layer that represents the sample application site (10). The sample liquid to be examined is then applied. The left cover (9) contains a printed arrow (12), which shows the user with which end the test carrier (1) should be placed or pushed into a measuring device. The positioning hole (13) serves to hold the test strip at a precisely predetermined point on the apparatus in the case of an apparatus measurement, for example a reflection photometric measurement. This can be done by, for example, a pin protruding into the positioning hole (13) and thus holding the test carrier (1) at a predetermined location.

FIG. 3 shows the underside of the test strip according to the invention with the positioning bore (13) made in the carrier (2) and the round observation opening (14) through which the detection layer can be inspected and measured.

FIG. 4 shows a perspective top view, FIG. 5 shows a section along the section line AA 'in FIG. 4, FIG. 6 shows a top view of the underside of an embodiment of a test strip according to the invention with two immediately adjacent detection surfaces and a spreading pad according to the invention, which is shown in the above described particularly preferred type of one-sided attachment is fixed above the detection layers. This representation is also done without a scale to make the structure clearly visible. A concrete dimensioning of this embodiment can be found in embodiment 2.

The diagnostic test strip (1) according to the invention shown in perspective in FIG. 4, in section in FIG. 5 and from below in FIG. 6 has two detection layers (3, 3a) on a support layer (2), immediately adjacent to one another the spreading pad elements (6, 6a) are covered. In addition to the detection layers (3,3a), the support elements (6,6a) are attached to the base layer (2) by means of spacers (4,4a) and adhesive layers (5,5a). In practice, these spacers can also be hot-melt adhesive surfaces or double-sided adhesive tapes that fix the spreading support elements (6, 6 a) on the base layer (2). Ideally, the spacers with their adhesive surfaces have approximately the same thickness as the detection layers (3,3a). The structure shown here also has covers (9, 9a) which are attached to the base layer (2) and the spreading pads (6, 6a). They are arranged in such a way that they cover the areas protruding beyond the detection layers (3,3a) and part of the surface of the supports (6,6a) lying above the detection layers. However, they leave the overlap area of ​​the support elements (6,6a) above the limit of the detection fields free. This area represents the sample application point (10). The sample liquid to be examined is then applied. If the covers are transparent, markings (11, 11a) can be placed on them above the outer boundaries of the detection fields, so that the user can see whether the conditions have completely saturated the detection fields with sample liquid. If this is the case, the amount of sample has been sufficient, otherwise there is a suspicion that the amount of sample was too small and an incorrect measurement may be taking place. The left cover (9) contains printed arrows (12) which show the user with which end the test carrier (1) is to be placed or pushed into a measuring device. The positioning hole (13) serves to hold the test strip at a precisely predetermined point on the apparatus in the case of an apparatus measurement, for example a reflection photometric measurement. This can be done by, for example, a pin protruding into the positioning hole (13) and thus holding the test carrier (1) at a predetermined location.

FIG. 6 shows the underside of the test strip according to the invention with the positioning bore (13) made in the carrier (2) and the differently shaped observation openings (14 and 15) through which the detection layers can be inspected and measured.

In a diagnostic test carrier according to the invention, those materials that do not absorb the liquids to be examined are particularly suitable for the base layer. These are so-called non-absorbent materials, with plastic films made of polystyrene, polyvinyl chloride, polyester, polycarbonate or polyamide being particularly preferred. However, it is also possible to impregnate absorbent materials, such as, for example, wood, paper or cardboard, with water-repellent agents or to coat them with a water-resistant film, silicones or hard fats being used as hydrophobizing agents and nitrocellulose or cellulose acetate, for example, as film-forming agents. Metal foils or glass are suitable as further carrier materials.

In contrast, for a detection layer it is necessary to use materials which are capable of absorbing the liquid to be examined with the ingredients contained therein. These are so-called absorbent materials, such as nonwovens, woven fabrics, knitted fabrics, membranes or other porous plastic materials or swellable materials, such as gelatin or dispersion films, which can be used as layer materials. The materials that are suitable for the detection layer must of course also be able to carry reagents that are required for the detection of the analyte to be determined. In the simplest case, all the reagents required for the detection of the analyte are on or in one layer. However, cases are also conceivable for which it is more advantageous to distribute the reagents over a plurality of absorbent or swellable material layers, which are then arranged one above the other, touching the entire surface. The term “detection layer” used in the following is intended to encompass both cases in which the reagents are located either only in or on one layer or in two or more layers arranged as described above.

Preferred materials for the detection layer are papers or porous plastic materials, such as membranes. Of these, particular preference is given to asymmetrically porous membranes which are advantageously arranged such that the sample liquid to be examined is applied to the large-pored side of the membrane and the analyte is determined from the fine-pored side of the membrane. Polyamide, polyvinylidene difluoride, polyether sulfone or polysulfone membranes are very particularly preferred as porous membrane materials. Polyamide 66 membranes and hydrophilized asymmetric polysulfone membranes are particularly suitable. The reagents for determining the analyte to be detected have generally been introduced into the above-mentioned materials by impregnation or applied on one side by coating. When coating asymmetrical membranes, the fine-pored side is advantageously coated.

However, so-called open films are also suitable for the detection layer, as described, for example, in EP-B-0 016 387. For this purpose, solids are added as fine, insoluble, organic or inorganic particles to an aqueous dispersion of film-forming organic plastics and the reagents required for the detection reaction are additionally added. Suitable film formers are preferably organic plastics, such as polyvinyl esters, polyvinyl acetates, polyacrylic esters, polymethacrylic acid, polyacrylamides, polyamides, polystyrene, copolymers, for example of butadiene and styrene or of maleic acid ester and vinyl acetate or other film-forming, natural and synthetic organic polymers and mixtures thereof in the form of aqueous dispersions. The dispersions can be spread on a base to form a uniform layer, which results in a waterproof film after drying. The dry films have a thickness of 10 µm to 500 µm, preferably 30 to 200 µm. The film can be used together with the support as a support or can be applied to another support for the detection reaction. Although the reagents required for the detection reaction are normally added to the dispersion used to prepare the open films, it may also be advantageous if the film formed is impregnated with the reagents after it has been produced. Pre-impregnation of the fillers with the reagents is also possible. The person skilled in the art knows which reagents can be used to determine a specific analyte. This need not be explained in more detail here.

Another example of a detection layer preferred according to the invention is a film layer as described in WO-A-92 15 879. This layer is produced from a dispersion or emulsion of a polymeric film former, which additionally contains a pigment, a swelling agent and the detection reagent in a homogeneous distribution. Particularly suitable polymeric film formers are polyvinyl esters, polyvinyl acetates, polyacrylic esters, polymethacrylic acid, polyvinylamides, polyamides and polystyrene. In addition to homopolymers, copolymers, for example of butadiene, styrene or maleic acid esters, are also suitable. Titanium dioxide is a particularly suitable pigment for the film. The swelling agent used should have particularly good swelling properties, with methyl vinyl ether maleic acid copolymer being particularly recommended. It is up to the person skilled in the art to determine which reagents are used to determine a particular analyte.

It is very particularly preferred to use a test field as a detection layer in a diagnostic test carrier according to the invention, which is constructed from two layers. This test field comprises a transparent film, on which a first and a second film layer are applied one above the other in this order. It is essential that the first layer on the transparent film is significantly less light-scattering when wet than the second layer above it. The non-coated side of the transparent film is referred to as the detection side and the side of the second layer that lies opposite the side with which the second layer rests on the first is referred to as the sample application side.

The film layers are made from dispersions or emulsions of polymeric film formers. Dispersion film formers contain microscopic polymer particles which are insoluble in the carrier liquid (usually water) and which are dispersed in the finest distribution in the carrier liquid. If the liquid is removed by evaporation during film formation, the particles approach and finally touch. Due to the large forces that occur and a gain in surface energy associated with film formation, the particles grow together to form a largely closed film layer. Alternatively, an emulsion of the film former can be used, in which the latter is dissolved in a solvent. The dissolved polymer is emulsified in a carrier liquid that is immiscible with the solvent.

Particularly suitable polymers for such film formers are polyvinyl esters, polyvinyl acetates, polyacrylic esters, polymethacrylic acid, polyvinylamides, polyamides and polystyrene. In addition to homopolymers, copolymers, e.g. B. of butadiene, styrene or maleic acid suitable.

The two film layers mentioned are located on a transparent film in the test field. Plastic foils which are impermeable to liquids are particularly suitable for this purpose. Polycarbonate film has proven to be particularly preferred.

The two film layers can be produced from coating compositions which contain the same polymeric film-forming agent or they can be produced from coating compositions which contain different polymeric film-forming agents.

If there are special test tasks and / or test conditions, such as when determining glucose in whole blood, it is advisable to design the layers so that, in addition to good erythrocyte separation, they also have optical features that facilitate observation of the detection reaction and the accuracy of the assessment and improve the metrological recording.

For this purpose, the first layer expediently contains a swelling agent and optionally a weakly light-scattering filler, the second layer contains a swelling agent and at least one strongly light-scattering pigment. In addition, the second layer can also contain non-porous fillers and porous fillers, such as diatomaceous earth, in small amounts without becoming permeable to erythrocytes.

By adding a swelling agent that swells well (i.e. a substance that increases its volume when water is taken up), not only layers are obtained that are penetrated relatively quickly by sample liquid, but also good erythrocyte and also blood pigment separation properties despite this opening effect of the swelling agent have. The swelling properties should be so good that for a test in which the speed of color formation - such as a glucose detection reaction - depends primarily on the penetration of the sample liquid through the layer, the optically detectable reaction can be measured after a maximum of one minute. Xanthan gum and methyl vinyl ether maleic acid copolymer have proven to be particularly suitable swelling agents.

Diatomaceous earth is also known as diatomaceous earth. These are deposits formed from the silica skeletons of the diatom species, which are broken down at various locations. The preferred diatomaceous earth used has an average particle diameter of 5-15 μm, these values ​​being determined using a type 715 laser granulometer, which is marketed by Pabisch, Munich, Federal Republic of Germany.

The strongly light-scattering pigment fraction of the second layer is at least 25% by weight, based on the dried and ready-to-use double layer of the test field. Since the weakly light-scattering fillers and the strongly light-scattering pigments are essentially responsible for the optical properties of the film layers, the first and second film layers have different fillers and pigments.

The first film layer should either contain no fillers or fillers whose refractive index is close to the refractive index of water. Precipitated silicas, silicon dioxide, silicates and aluminum silicates have proven to be particularly suitable for this. A sodium aluminum silicate with the trade name Transpafill® is particularly preferred.

The second layer should be as light-scattering as possible. Ideally, the refractive index of the pigments in the second film layer is at least 2.5. Titanium dioxide is therefore preferably used. Particles with an average diameter of approximately 0.2 to 0.8 μm have proven to be particularly advantageous. Easily processable types of titanium dioxide in the anatase modification are very particularly preferred.

Reagent systems for detecting certain analytes by color formation are known to the person skilled in the art. It is possible that all components of the reagent system are in one film layer. However, it is also possible for the components of the reagent system to be distributed over both film layers. The color-forming reagent system is advantageously located at least in part in the first film layer.

In the context of the present invention, color formation is understood not only to mean the transition from white to color, but also any color change, of course those color changes are particularly preferred which are associated with the greatest possible shift in the maximum absorption waveline (λ max).

For the optimization of the test field in the diagnostic test carrier according to the invention, it has proven to be particularly advantageous if both film layers contain a non-hemolyzing wetting agent. Neutral, that is, non-charged wetting agents are particularly suitable for this. N-Octanoyl-N-methyl-glucamide is very particularly preferred.

In addition, further wetting agents which promote the homogeneity of the coatings, for example sodium-N-methyl-N-oleoyl taurate, can be contained in the film layers.

To produce a test field of a diagnostic test carrier according to the invention, the respective film layers are produced one after the other from a homogeneous dispersion of the constituents mentioned. For this purpose, the transparent film is used as a base for shaping the coating composition for the first film layer. After the coating composition for the first film layer has been applied in a specific layer thickness, the layer is dried. The coating composition for the second layer is then also applied to this layer in a thin layer thickness and dried. After drying, the thickness of the first and second film layers should total a maximum of 0.2 mm, preferably a maximum of 0.12 mm, particularly preferably a maximum of 0.08 mm.

The attachment can be carried out according to methods known to those skilled in the art from test carrier technology. For example, it can be attached using hot melt adhesive or hardening cold adhesive. A punctiform or grid bond is advantageous because the capillary-active liquid transport is particularly possible in this case. Double-sided adhesive strips have also proven to be advantageous. In all cases, however, it is important that the support is attached to the base layer in such a way that capillary-active liquid transport from the detection layer into the part of the base that is attached to the base layer is possible. This capillary-active liquid transport must be possible in particular if the detection layer is saturated with liquid. Adhesive tapes with natural or synthetic rubber have proven particularly suitable for processing. It is very particularly advantageous if the means used to attach the support to the base layer has approximately the same thickness as the detection layer (s). It then serves, as it were, as a spacer in order to keep the support according to the invention even on a continuous surface outside the area of ​​the detection layer (s).

To determine the analyte to be detected in the sample liquid, the detection layer is visible in the diagnostic test carrier according to the invention, but at least the reaction areas, that is, reagent-bearing areas of the detection layer (s) that can be observed and measured for signal formation, are visible through the support layer. As already stated above, this can be achieved in that the base layer is transparent. However, it is also possible for the base layer to have a perforation which is covered by the detection layer or layers. The detection layer or the detection layers, or at least the reaction areas of the detection layers, are then visible through the perforation. In a preferred embodiment of the diagnostic test carrier according to the invention, there is a hole in the base layer below a detection layer through which the detection layer or a reaction area can be observed. The hole has a somewhat smaller diameter than the smallest length dimension of the detection layer, so that the detection layer lies on the base layer outside the hole and can be fastened there. Advantageously, the detection layer is adequately fixed by double-sided adhesive tapes arranged on both sides and the overlay according to the invention lying above the detection layer and its attachment to the base layer. However, the detection layer itself is preferably also attached to the base layer by means of thin adhesive tape.

However, several reaction areas of a detection layer can also be visible through a hole.

The perforation of a diagnostic test carrier according to the invention can also consist of two or more holes which can be used to determine analyte (one or more analytes). Different detection layers can be arranged above the holes or just one detection layer with several reaction areas, so that one detection layer or one reaction area can be observed through each hole. It is also possible that several reaction zones can be observed through one hole.

An inert cover made of sample-impermeable, generally water-impermeable and non-absorbent material can be expediently arranged over the spreading pad of the diagnostic test carrier according to the invention so that the area of ​​the pad is covered outside the detection layer. Ideally, the cover also protrudes into the area of ​​the detection layer, but in any case leaves a middle part of the overlay according to the invention, which covers the detection layer, free. This free part of the edition is called the sample application point.

Plastic films have proven to be particularly advantageous as a cover. If the cover and the overlay according to the invention have different colors, for example white and yellow or white and red, the location to which the sample liquid to be examined is to be applied can be identified very well.

The cover can also be used, for example, to indicate with one or more arrows in which direction, that is, the end with which a diagnostic test carrier according to the invention is to be placed or pushed into a measuring device.

A sample application point can be achieved particularly easily by a cover by means of two band-shaped plastic films which leave a band-like area of ​​the support according to the invention covering the detection layer free. If 2 or more sample application points are provided, 3 or more band-shaped plastic films must be used. The foils used for covering are attached to the support according to the invention and, if necessary, to the base layer. Suitable for such attachment are hot-melt adhesives, which are preferably applied at points or in a grid pattern on the base layer or the underside of the cover, or adhesive tapes if the films are not themselves adhesive. The sample application point is preferably located above the perforation in the base layer, through which a signal formation in the detection layer can be observed.

To carry out a method for determining analyte in a liquid sample with the aid of a diagnostic test carrier according to the invention, sample liquid is applied to the side of the overlay facing away from the detection layer, ideally so much that the liquid passing through the overlay according to the invention completely saturates the detection layer. Body fluids such as blood, plasma, serum, urine, saliva etc. are particularly suitable as sample fluids. Blood or liquids derived from blood such as plasma or serum and urine are particularly preferred sample liquids. A signal can then be detected in the detection layer in the presence of the analyte to be determined. Such a signal is advantageously a color change, which means both color formation, color loss and color change. The intensity of the color change is a measure of the amount of analyte in the examined liquid sample. It can be evaluated visually or with the aid of a device, usually by quantitative reflection photometry, and calibration curves created in preliminary tests can be used. Alternatively, the analyte content can also be displayed directly via the device software.

A great advantage of the diagnostic test carrier according to the invention is that no predetermined volume of a sample liquid has to be applied to the test carrier.

It has been shown that when using a test strip that was constructed using the preferred materials mentioned above, an excess sample is not absorbed by the strip but remains above the application point. Another significant advantage of the constructions according to the invention is that the test strip is "self-dosing". If you bring your application site into contact with a drop of blood on the fingertip or attached to it, the strip only takes the amount required to soak the detection layer (s), the rest remains on the finger.

In this way, the signal intensity which arises in the presence of an analyte is independent of the amount and the duration of contact of the sample liquid with the detection layer. The color, which usually appears within a few seconds to a few minutes after the detection reaction has ended, remains unchanged for the measurement. It is only determined by the stability of the coloring system. False positive results are also avoided and quantitative analyte determination is made possible.

By covering parts of the support according to the invention and thus marking the sample application site, care is taken to ensure that liquid can only reach the detection layer at the optimal location. In combination with a detection layer, which absorbs only a little liquid and still ensures intensive signal formation, it is ensured that reliable analyte determinations are possible even with very small sample volumes. The fact that the test carrier according to the invention consists of only a few components that can be put together easily and quickly makes it very inexpensive to manufacture.

The following exemplary embodiments illustrate the production of spreading pads and test tires according to the invention.

example 1

<ul id="ul0002" list-style="none"><li>A.) Production of a spreading pad according to the invention.</li><li>A.1) 55.0 g of N-oleoyl-sarcosine ("®Crodasinic O" from Croda, Nettetal) are added to 55 kg of distilled water and, with stirring, 11.0 g of 32% strength by weight sodium hydroxide solution are added adjusted to pH 6.0. A wetting agent solution to be used according to the invention with a content of approximately 0.106% by weight of active substance is obtained.</li><li>A.2) With this solution a 1 m wide, 700 m long ®Viledon fleece, type FO 2451/121, from Freudenberg, Weinheim, (thickness 50 µm, basis weight 18 g / m²) pulled at a speed of 5 m / min. The soaked fleece is then in a horizontal dryer of 30 m in length at 80 ° C and an air flow of 50 m³/ min dried. The fleece absorption of the fleece was 45 ml / m², so that the content of active substance on the impregnated fleece is approximately 0.26% by weight.</li></ul><ul id="ul0003" list-style="none"><li>B.) Production of test strips according to the invention.</li><li>B.1) A 5 mm wide double-sided adhesive tape (polyester carrier and synthetic rubber adhesive) is parallel to a tape-shaped, 50 mm wide titanium dioxide-containing polyester base layer at a distance of 18.6 mm (measured from the left edge of the adhesive tape) from its left edge ) applied. Two holes, a positioning hole and an inspection and measuring hole, are punched out of this composite at a distance of 6 mm, the center points of which lie on a straight line perpendicular to the longitudinal axis of the carrier strip. The first hole, the positioning hole, is circular, 2.6 mm in diameter and its center is 4 mm from the left edge of the carrier strip. The second hole is also round with a diameter of 4 mm. The center point distance of the second hole from the left edge of the carrier strip is 21 mm. The protective paper on the double-sided adhesive tape is then removed. The procedure for producing a detection layer which is composed of 2 film layers is as follows:</li><li>B.2) The following components are combined in a beaker as pure substances or in the form of stock solutions in the following composition and mixed by stirring:<ul id="ul0004" list-style="none" compact="compact"><li>Water: 820.0 g</li><li>Citric acid 1 hydrate: 2.5 g</li><li>Calcium chloride 2 hydrate: 0.5 g</li><li>Sodium hydroxide: 1.4 g</li><li>Xanthan gum: 3.4 g</li><li>Tetraethylammonium chloride: 2.0 g</li><li>Sodium N-methyl-N-oleoyl taurate: 0.29 g</li><li>N-octanoyl-N-methyl-glucamide: 2.1 g</li><li>Polyvinylpyrrolidone (MW 25000): 3.5 g</li><li>Transpafill ((sodium aluminum silicate): 62.1 g</li><li>Polyvinyl propionate dispersion (50% by weight in water): 60.8 g</li><li>Bis- (2-hydroxyethyl) - (4-hydroximinocyclohexa-2,5-dienylidine) ammonium chloride: 1.2 g</li><li>2,18-phosphoromolybdic acid hexasodium salt: 16.1 g</li><li>Pyrroloquinoline quinone: 32 mg</li><li>Glucose dehydrogenase rec. out</li><li>Acinetobacter calcoaceticus, 1.7 MU EC 1.1.99.17: (2.4 g)</li><li>1-hexanol: 1.6 g</li><li>1-methoxy-2-propanol: 20.4 g</li></ul> The total mass is adjusted to a pH of about 6 with NaOH and then with a basis weight of 89 g / m² applied to a 125 µm thick polycarbonate film and dried.</li><li>B.3) The following components are combined in a beaker as pure substances or in the form of stock solutions in the following composition and mixed by stirring:<ul id="ul0005" list-style="none" compact="compact"><li>Water: 579.7 g</li><li>Sodium hydroxide: 3.4 g</li><li>Gantrez ((methyl vinyl ether-maleic acid copolymer): 13.8 g</li><li>Sodium N-methyl-N-oleoyl taurate: 0.25 g</li><li>N-octanoyl-N-methyl-glucamide: 3.6 g</li><li>Tetraethylammonium chloride: 9.7 g</li><li>Polyvinylpyrrolidone (MW 25000): 20.2 g</li><li>Titanium dioxide: 177.1 g</li><li>Diatomaceous earth: 55.3 g</li><li>Polyvinyl propionate dispersion (50% by weight in water): 70.6 g</li><li>2,18-phosphoromolybdic acid hexasodium salt: 44.3 g</li><li>Potassium hexacyanoferrate (III): 0.3 g</li><li>1-hexanol: 1.6 g</li><li>1-methoxy-2-propanol: 20.4 g</li></ul> The total mass is adjusted to a pH of about 6 with NaOH and then with a weight per unit area of ​​104 g / m² applied to the coated polycarbonate film as described above under A. and dried.</li><li>B.4) A 5 mm wide strip of the detection layer produced in this way is glued with the foil side onto the punched double-sided adhesive tape on the base layer. Adjacent to the detection layer, double-sided adhesive tapes (PVC backing and natural rubber adhesive) are placed on both sides as spacers</li></ul>

Carrier film glued on. In the present example, one spacer is 6 mm wide and the other is 9 mm wide. The protective film of the two double-sided adhesive tapes is then removed.

A 20 mm wide strip of the spreading fleece produced in section A is placed on this composite and glued by pressing.

Two one-sided adhesive tapes (PVC backing and natural rubber adhesive) are glued on the spreading fleece as covers so that the spacers are completely covered and at least a slight overlap with the reactive area takes place. The tape goods are now complete.

The tape goods are cut into 6 mm wide test carriers so that the measuring hole is in the center of the test carrier.

Example 2

Production of a diagnostic test carrier according to the invention with two detection fields for the determination of glucose in low and in high concentration.

A test carrier according to FIGS. 4, 5 and 6 is produced in the following working steps:

A 10 mm wide double-sided adhesive tape (polyester backing and synthetic rubber adhesive) is applied in parallel to a tape-shaped, 50 mm wide titanium dioxide-containing polyester base layer at a distance of 18.6 mm (measured from the left edge of the adhesive tape) to its left edge. Three holes, one positioning hole and two inspection and measuring holes, are punched out of this composite at a distance of 6 mm, the center points of which lie on a straight line perpendicular to the longitudinal axis of the carrier strip. The first hole, the positioning hole, is circular, 2.6 mm in diameter and its center is 4 mm from the left edge of the carrier strip. The second hole is also round with a diameter of 4 mm, the third hole is rectangular with an edge length of 3 mm in the longitudinal direction of the strip and 4 mm in the transverse direction. The second and third holes are both 5.1mm apart on the tape. the center distance of the second hole from the left edge of the carrier strip is 21 mm.

The protective paper on the double-sided adhesive tape is then removed.

The procedure for producing the first detection layer, which is composed of 2 film layers, is as follows:<ul id="ul0006" list-style="none"><li>A. In a beaker, the following components are combined as pure substances or in the form of stock solutions in the following composition and mixed by stirring:<ul id="ul0007" list-style="none" compact="compact"><li>Water: 820.0 g</li><li>Citric acid 1 hydrate: 2.5 g</li><li>Calcium chloride 2 hydrate: 0.5 g</li><li>Sodium hydroxide: 1.4 g</li><li>Xanthan gum: 3.4 g</li><li>Tetraethylammonium chloride: 2.0 g</li><li>N-octanoyl-N-methyl-glucamide: 2.1 g</li><li>Polyvinylpyrrolidone (MW 25000): 3.5 g</li><li>Transpafill ((sodium aluminum silicate): 62.1 g</li><li>Polyvinyl propionate dispersion (50% by weight in water): 60.8 g</li><li>Bis- (2-hydroxyethyl) - (4-hydroximinocyclohexa-2,5-dienylidine) ammonium chloride: 1.2 g</li><li>2,18-phosphoromolybdic acid hexasodium salt: 16.1 g</li><li>Pyrroloquinoline quinone: 32 mg</li><li>Glucose dehydrogenase rec. from Acinetobacter calcoaceticus, 1.7 MU EC 1.1.99.17: (2.4 g)</li><li>1-hexanol: 1.6 g</li><li>1-methoxy-2-propanol: 20.4 g</li></ul> The total mass is adjusted to a pH of about 6 with NaOH and then with a basis weight of 89 g / m² applied to a 125 µ thick polycarbonate film and dried.</li><li>B. The following components are combined in a beaker as pure substances or in the form of stock solutions in the following composition and mixed by stirring:<ul id="ul0008" list-style="none" compact="compact"><li>Water: 579.7 g</li><li>Sodium hydroxide: 3.4 g</li><li>Gantrez ((methyl vinyl ether-maleic acid copolymer): 13.8 g</li><li>N-octanoyl-N-methyl-glucamide: 3.6 g</li><li>Tetraethylammonium chloride: 9.7 g</li><li>Polyvinylpyrrolidone (MW 25000): 20.2 g</li><li>Titanium dioxide: 177.1 g</li><li>Diatomaceous earth: 55.3 g</li><li>Polyvinyl propionate dispersion (50% by weight in water): 70.6 g</li><li>2,18-phosphoromolybdic acid hexasodium salt: 44.3 g</li><li>Potassium hexacyanoferrate (III): 0.3 g</li><li>1-hexanol: 1.6 g</li><li>1-methoxy-2-propanol: 20.4 g</li></ul></li></ul>

The total mass is adjusted to a pH of about 6 with NaOH and then with a weight per unit area of ​​104 g / m² applied to the coated polycarbonate film as described above under A. and dried. The layer thickness is 60 µm after drying.

The procedure for producing the second detection layer, which is also composed of 2 film layers, is as follows:<ul id="ul0009" list-style="none"><li>A. In a beaker, the following components are mixed as pure substances or in the form of stock solutions in the specified amounts with stirring:<ul id="ul0010" list-style="none" compact="compact"><li>Water: 820.0 g</li><li>Citric acid 1 hydrate: 2.5 g</li><li>Calcium chloride 2 hydrate: 0.5 g</li><li>Sodium hydroxide: 1.4 g</li><li>Xanthan gum: 3.4 g</li><li>Tetraethylammonium chloride: 4.22 g</li><li>N-octanoyl-N-methyl-glucamide: 2.1 g</li><li>Sodium N-methyl-N-oleoyl taurate 0.29 g</li><li>Polyvinylpyrrolidone (MW 25000): 3.5 g</li><li>Transpafill ((sodium aluminum silicate): 62.1 g</li><li>Polyvinyl propionate dispersion (50% by weight in water): 60.8 g</li><li>N- (4-nitrosophenyl) -N'-carboxymethyl-piperazine: 1.0 g</li><li>2,18-phosphoromolybdic acid hexasodium salt: 20.9 g</li><li>Pyrroloquinoline quinone: 32 mg</li><li>Glucose dehydrogenase rec. out</li><li>Acinetobacter calcoaceticus, 1.7 MU (EC 1.1.99.17): (2.4 g)</li><li>1-hexanol: 1.6 g</li><li>1-methoxy-2-propanol: 20.4 g</li></ul> The total mass is adjusted to a pH of approx. 6.0 with sodium hydroxide solution and then with a basis weight of 89 g / m² applied to a 125 µm thick polycarbonate film and dried.</li><li>B. The following components are mixed in a beaker as pure substances or in the form of stock solutions in the specified amounts with stirring:<ul id="ul0011" list-style="none" compact="compact"><li>Water: 579.7 g</li><li>Sodium hydroxide: 3.4 g</li><li>Gantrez ((methyl vinyl ether-maleic acid copolymer): 13.8 g</li><li>Tetraethylammonium chloride: 6.71 g</li><li>N-octanoyl-N-methyl-glucamide: 2.74 g</li><li>Sodium N-methyl-N-oleoyl taurate 0.25 g</li><li>Polyvinylpyrrolidone (MW 25000): 15.6 g</li><li>Titanium dioxide: 136.7 g</li><li>Polyvinyl propionate dispersion (50% by weight in water): 54.6 g</li><li>N- (4-nitrosophenyl) -N'-carboxymethyl-piperazine: 1.51 g</li><li>2,18-Phosphomolybdic acid hexasodium salt: 33.13 g</li><li>Potassium hexacyanoferrate (III): 0.28 g</li><li>1-hexanol: 1.6 g</li><li>1-methoxy-2-propanol: 20.4 g</li></ul></li></ul>

The total mass is adjusted to a pH of about 6.0 with sodium hydroxide solution and then with a basis weight of 102 g / m² applied to the coated polycarbonate film as described above under A and dried. The layer thickness after drying is 55 µm.

Each 5 mm wide strip of the detection layers produced in this way is glued with the foil side onto the punched double-sided adhesive tape on the base layer in such a way that the strips run side by side directly adjacent to one another.

Directly adjacent to the detection layers, double-sided adhesive tapes in the thickness of the detection strips (PVC backing and natural rubber adhesive) are stuck onto the backing film as spacers. In the present example, one spacer is 6 mm wide and the other 9 mm wide. The protective film of the two double-sided adhesive tapes is then removed.

Then a 10 mm wide strip of the spreading fleece produced in accordance with Section A of Example 1 is placed on the 9 mm wide spacer in such a way that the cut edge of the fabric strip extends 0.5 to 0.6 mm above the boundary line between the detection strips and is fixed by pressing. A 10 mm wide strip of the same spreading fleece according to the invention is then placed on the 6 mm wide spacer in such a way that it overlaps the cut edge of the first fabric strip by 1 to 1.2 mm and is fixed by pressing.

Then two single-sided adhesive tapes (PVC backing and natural rubber adhesive) are glued on both sides of the structure as covers so that a gap of 2 to 2.5 mm remains uncovered symmetrically to the boundary line of the detection strips. The tape goods are now complete.

The tape goods are cut into 6 mm wide test carriers so that the measuring and inspection holes and the positioning hole are in the center of the test carrier.

Example 3

According to Example 2, test strips are produced which have the same detection layers in both detection fields. A PE 38 HC polyester fabric impregnated with 0.25% by weight sodium N-ol eoyl sarcosinate is used as the spreading layer. The strips are inserted into a GLUCOTREND device set up for simultaneous measurement of both test fields, whereby they are subjected to a slight bend to fix them in the measurement position.

The strips are spotted with increasing volumes of EDTA venous blood with 102 mg / dl glucose. 5 series of 10 test strips were measured per volume (n = 5, N = 50). From this, 5 CV values ​​were calculated for each volume. (The VK value is defined as the relative standard deviation<maths id="math0001" num=""><math display="block"><mrow><mtext>VK = standard deviation / mean</mtext></mrow></math><img file="EP0995992A2_D0001.tif" /></maths> and is given in%.)

The following table shows the medians of the measurement results and the medians of the VK values ​​of the 5 series per volume: <tables id="tabl0002" num="0002"><table frame="all"><tgroup cols="5" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="31.50mm" /><colspec colnum="2" colname="col2" colwidth="31.50mm" /><colspec colnum="3" colname="col3" colwidth="31.50mm" /><colspec colnum="4" colname="col4" colwidth="31.50mm" /><colspec colnum="5" colname="col5" colwidth="31.50mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col3" align="center">Field 1</entry><entry namest="col4" nameend="col5" align="center">Field 2</entry></row><row><entry namest="col1" nameend="col1" align="center">volume</entry><entry namest="col2" nameend="col2" align="center">Median of the measured value [mg / dl]</entry><entry namest="col3" nameend="col3" align="center">Median of sales value [%]</entry><entry namest="col4" nameend="col4" align="center">Median of the measured value [mg / dl]</entry><entry namest="col5" nameend="col5" align="center">Median of sales value [%]</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="center">3rd µl</entry><entry namest="col2" nameend="col2" align="center">Error*</entry><entry namest="col3" nameend="col3" align="char" char=",">-</entry><entry namest="col4" nameend="col4" align="char" char=",">63,4**</entry><entry namest="col5" nameend="col5" align="char" char=",">9,3</entry></row><row><entry namest="col1" nameend="col1" align="center">4th µl</entry><entry namest="col2" nameend="col2" align="center">100</entry><entry namest="col3" nameend="col3" align="char" char=",">2,5</entry><entry namest="col4" nameend="col4" align="char" char=",">102</entry><entry namest="col5" nameend="col5" align="char" char=",">2,7</entry></row><row><entry namest="col1" nameend="col1" align="center">5 µl</entry><entry namest="col2" nameend="col2" align="center">102</entry><entry namest="col3" nameend="col3" align="char" char=",">2,3</entry><entry namest="col4" nameend="col4" align="char" char=",">103</entry><entry namest="col5" nameend="col5" align="char" char=",">2,6</entry></row><row><entry namest="col1" nameend="col1" align="center">10th µl</entry><entry namest="col2" nameend="col2" align="center">101</entry><entry namest="col3" nameend="col3" align="char" char=",">2,2</entry><entry namest="col4" nameend="col4" align="char" char=",">102</entry><entry namest="col5" nameend="col5" align="char" char=",">2,7</entry></row><row><entry namest="col1" nameend="col1" align="center">15 µl</entry><entry namest="col2" nameend="col2" align="center">103</entry><entry namest="col3" nameend="col3" align="char" char=",">2,4</entry><entry namest="col4" nameend="col4" align="char" char=",">102</entry><entry namest="col5" nameend="col5" align="char" char=",">2,7</entry></row><row rowsep="1"><entry namest="col1" nameend="col5" align="justify">Remarks: </entry></row></tbody></tgroup><tgroup cols="5" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="31.50mm" /><colspec colnum="2" colname="col2" colwidth="31.50mm" /><colspec colnum="3" colname="col3" colwidth="31.50mm" /><colspec colnum="4" colname="col4" colwidth="31.50mm" /><colspec colnum="5" colname="col5" colwidth="31.50mm" /><tbody valign="top"><row><entry namest="col1" nameend="col5" align="justify">*: An uneven reaction color is recognized in field 1 by the 2-LED optics of the device (described in EP-A-819 943). The device therefore only outputs an error message, but no measured value.</entry></row><row><entry namest="col1" nameend="col5" align="justify">**: Field 2 is only illuminated with an LED, so that an uneven coloring of the test field is not recognized. As a result, the VK value is greatly increased. However, by comparing both test fields, the device can give the error message "reaction depth of different depths of both fields". This function was switched off during the measurement carried out here.</entry></row></tbody></tgroup></table></tables>

The experiments show that from a sample volume of 4 µl both test fields indicate the same color, ie the same glucose concentration. From this one can see the excellent spreading effect of the support according to the invention over both measuring fields. With higher sample volumes, the value does not change because the excess sample material remains above the application gap. With smaller volumes, the wetting and thus also the coloring of the two reaction fields is incomplete, which can be detected by the 2-LED optics of field 1. Suitable software measures can be used to prevent such measurements from displaying (false-negative) values. Instead, an error message is displayed to inform the user that the sample volume is too low.