Test element for determining an analyte in blood and method for its preparation.

The invention relates to a test carrier for the determination of an analyte from whole blood with the aid contained in the test carrier reagents with an erythrocyte having a blood application side and an evaluation side, wherein on the blood application side, whole blood is supplied and the evaluation side of a detection means facing, in due the reaction of the reagents with the analyte takes place an optically detectable change. The invention is further directed to a method for manufacturing such a test carrier.

The qualitative or quantitative analytical determination of components of blood recently increasingly called carrier-bound tests are used. In these reagents in appropriate layers of a solid test carrier are embedded, which is brought into contact with the sample. The reaction of the sample and reagents leads to an optically detectable change, in particular a color change, which visually or with the aid of a device can be usually by reflection photometry. Instead of a color change, the reaction can also lead to the creation or modification of another optically detectable signal lead, for example, a fluorescence or luminescence. Test carriers are frequently constructed as test strips which consist essentially of an elongate supporting layer made of plastic material and test fields mounted thereon. However, test carriers are also known which are designed as square or rectangular plates. Carrier-bound tests are particularly distinguished by the ease of handling. All the more regrettable that the blood can not be used directly as a so-called whole blood with most previously known carrier-bound tests. Rather, it is necessary to separate the red blood cells (erythrocytes) in order to obtain colorless plasma or serum. This is typically done by centrifugation, ie it is an additional handling step required. There is also a device for centrifuging not available everywhere, especially carrier-bound tests are increasingly offered for laymen. The centrifugation requires a relatively large amount of sample, on the other hand the pursuit then goes in clinical diagnosis to make do with a small drop of blood, such as may be obtained by a finger prick.

There has been no shortage of attempts to provide test carriers are available, which allow analytical determinations directly from blood.

Thus, DE-B-1598153, a test carrier with a film layer made of an aqueous dispersion of natural or synthetic polymers known in the are necessary for the detection reagents. This test carrier, certain analytical determinations, in particular the determination of glucose, are carried out directly from blood. In other analytical determinations, however, no good results are obtained with such a test carrier, which may be due to the fact that the constituents of the sample can not penetrate sufficiently in this movie.

From EP-A-16387 a likewise based on the use of a dispersion film former test carrier layer is known, which thereby avoids this problem in that it contains relatively large amounts (in particular inorganic) small particles. Thus, although a universal test layer is obtained, which, however, no provision of thoroughbred allowed because not only larger samples to be analyzed ingredients, but also erythrocytes penetrate unhindered into this layer.

From EP-A 45 476 it is known to use glass fibers for the extraction of serum or plasma on a test carrier. This solution is universally applicable, but it is necessary to accommodate the fiber layers appropriately on the test carrier. This results in a relatively complicated test support structure, and method of manufacture is complicated.

Even where previously test carrier of the type described have been proposed in which on one side of the blood can be given up without erythrocyte separation and the evaluation is carried out on the other side, where there is an erythrocyte separation between the blood application side and the evaluation side, these attempts have not been to satisfy.

In the US-A-36 63 374 and in US-A-42 56 693 a membrane filter is used to prevent the passage of erythrocytes from the blood application side on the processing side. Although membrane filter for filtering off red blood cells are suitable in principle. Their use in test carriers, however, has not proven. The same applies to the also mentioned in these US patents combination of the membrane filter with a preceding fiber layer, which is to prevent the clogging of the membrane filter with coarser particles. The preparation of such test carriers would be very complicated, without that a satisfactory function is achieved.

In US-A-4069817 and several other US patents of the same Applicant also addresses the possibility to provide a passage of erythrocytes preventing intermediate layer in a test carrier which also contains radiation-blocking components to ensure that the light beams of the evaluation unit can not penetrate into the erythrocyte-containing layer. In this document, but no details are included, such as the filtering of the erythrocytes could be achieved.

Object of the present invention is therefore to provide a test carrier available, with the implementation of medical diagnostic determinations of whole blood is immediately possible, the test carrier easy to handle and should be simple and inexpensive to manufacture.

The object is achieved in a test carrier of the type described in that the erythrocyte includes a laminate which includes a first zone containing a polymeric film former, kieselguhr and a pigment and a formed thereon to form a transition region by liquid coating, a polymeric film former containing having second zone, the first zone of the blood application side and the second zone facing the evaluation side of the erythrocyte.

The invention provides a test carrier for whole blood analysis is provided, whose erythrocyte can be manufactured inexpensively with a simple to carry out double coating. The passage of the plasma from the first zone to the analysis zone is carried out in a few seconds, so that a rapid evaluation. The handling is easy, especially the applied blood must not be wiped or washed, as was often required in prior art test strip.

The inventive method for production of such a test carrier requires two different coating materials, which are formed in separate steps, each coating to a thin layer. includes the first coating composition, in addition to a carrier liquid in the dispersed or dissolved polymeric film former, kieselguhr and a pigment known adjuvants, such as buffers, wetting agents, thickeners, defoamers and the like. The second coating liquid also contains a polymeric film former dispersed or dissolved.

To produce a first of a zone and a second zone existing layer assembly is first formed, the first coating composition on a substrate in a thin layer and dried. Thereafter, the second coating composition is formed into a thin layer and dried in this layer. Here components of the second coating liquid penetrate into the first shaped layer. The first zone and the second zone are therefore not separated by a sharp boundary, but it forms a transition area in which they blend beautifully together. It is advantageous to comply with certain viscosity ranges of the two coating materials in the application of the layers. The first coating composition should have sec⁻¹ when applied to the substrate has a viscosity of 300-3000 mPas (milli Pascal seconds) at a shear rate of 492nd For the second coating composition of the corresponding value should be 100-1000 mpas. Both viscosities are after the German Industrial Standard (DIN) to determine 53019th

Suitable polymeric film formers are preferably organic plastics such as polyvinyl alcohols, polyvinyl acetates, polyacrylic esters, polymethacrylic, Polycrylamide, polyamides, polystyrene. Besides homopolymers and copolymers particularly, for example, from butadiene and styrene or of maleic acid esters and vinyl acetate, and terpolymers are also suitable. However, also other film-forming natural or synthetic organic polymers and mixtures thereof are used. Gelatin is not suitable.

The film-forming agent can be dissolved in suitable organic solvents. It is often advantageous to use a dispersion of a suitable film-forming agent. The preferred carrier liquid is water in this case.

Dispersion film containing submicroscopic insoluble in the carrier liquid polymer particles which are dispersed in suspension in the carrier liquid. If, during the film formation, the liquid removed by evaporation or evaporation, so the particles approach and finally touch. By occurring large forces and an accompanying film formation gain in surface energy, the particles grow together into a substantially closed film layer. For further details see, for example, the article "latex film formation" of JW Vanderhoff in Polymer News 1977, page 194 - refer 203rd

Kieselguhr is also known as diatomaceous earth. It is formed from the silica skeletons of diatom deposits that are mined in various places. The diatomaceous earth is preferably used has an average particle diameter 5-15 .mu.m, these values ​​being determined with a laser granulometer type 715, which is marketed by the company Pabisch, Munich, Germany.

The pigment is preferably composed of particles having an average diameter of between about 0.2 and 0.7 microns. Especially suitable, for example, titanium dioxide. However, other pigments suitable, and their particle sizes are usually approximately in the specified range corresponding to the wavelength range of visible light around. This maximum light scattering and thus a highly opaque pigment is achieved.

The reaction time in the layer composite is thereby shortened, that it is preferably at most 0.6 mm, more preferably is not more than 0.2 mm thick.

As backing for forming the first coating composition can for example be selected a plate of glass or other material from which can be easily removed, the film layer. This makes it possible to remove the finished laminate and, for example, be mounted on a transparent carrier film, wherein the processing side of the carrier foil facing, so that the blood application side of the layer assembly is freely accessible.

It is much simpler to manufacture and therefore preferred, it is to fasten the composite layer on a porous support layer, wherein the blood application side of the backing layer facing. This can be preferably characterized accomplish that one directly used as backing for forming the first coating the porous support layer. In such a layered composite is a free access of air to the detection zone is possible, whereby the reaction time in this zone, in many cases, is considerably reduced. This end-point determinations are possible.

As the porous support layer is in principle any open, planar composite structure, ie any structure which is extensively expanded and sufficiently open so that it is penetrated sufficiently rapidly from the blood. Good example would be a mesh-like structure made of a plastic material with many closely spaced holes arranged. However, the porous support preferably consists of a textile material, particularly a woven or knitted fabric, which may be made for example of polyamide, polyester, or silk. Possibly can also be a nonwoven fabric or paper may be used. Suitable materials are described in EP-A-113 896th

The invention is explained below with reference to an embodiment schematically shown in the figures; show it:<ul><li>FIG. 1 shows a test carrier according to the invention in perspective,</li><li>Fig. 2 shows a section through a test carrier according to FIG. 1 along the line S / S</li><li>Fig. 3 is a section corresponding to FIG. 2 with an alternative layer structure</li><li>Fig. 4 is a perspective view of an alternative embodiment of a test carrier according to the invention.</li></ul>

The illustrated in Fig. 1 test carrier 1 consists 2a essentially of a manufactured example, plastic frame 2 and a decision taken in this context, multi-layer test field.

The multilayer test field 2a consists in the case shown in cross-section of a acting as erythrocyte layer composite and a porous support layer 4. The layer composite 3 comprises a first zone 5 and a second zone 6, and a position indicated by a dashed double line transition region 7 between the two zones. The porous support layer 4 is the blood application side 8 of the laminate 3 faces, while the second zone 6 of the evaluation side faces. 9

Of course, the multi-layer test pad 2a contain further layers. The illustrated example is particularly simple in structure, as the first zone 5 and the second zone 6 also contain reagents for the detection reaction. However, it may also be appropriate to separate the functions of "erythrocyte separation" and "detection reaction" and provide for additional layers latter mainly on the detection side, but possibly also to the blood application side.

To carry out an analytical determination an optionally pre-measured amount of blood is applied to the blood application side 8, wherein the test carrier is usually maintained with this side facing up. The blood passes through the porous support layer 4 into the base region. 5 Due to the inventive structure of the layer composite, the red blood cells remain at the further penetration, so that no red blood dye passes into the detection zone. 6 The optical evaluation is carried out of the processing side 9 forth. The pigment blocks the radiation to such an extent that they can not get into areas of the laminate, in which red blood pigment is. The analysis is therefore not distorted by this.

The laminate according to the invention attained in its entirety so that what is 817 called for example in the aforementioned US-A-4 069 without the appropriate resources are provided, namely, filter out the red blood cells from the invading blood sample and at the same time necessary for the optical analysis of light radiation so block out that it does not enter the area of ​​the laminate, in which red blood pigment is.

Whereby the filter effect is achieved in the individual, is not fully understood. On the one hand it can be stated that the composition of the first zone 5 having (for example, applied to a porous support layer) layer does not have sufficient erythrocyte separation property. On the other hand it can be established that a significant proportion of the second coating composition in molding on the underlying layer penetrates into these. It must therefore be assumed that the particles of the dispersed or dissolved polymer to penetrate from the second coating composition in the previously prepared layer being set up, a gradient of polymer in the transition region 7 between the second zone 6 and the first zone. 5 It is likely that by the relatively open structure of the first zone 5 is being closed so far that on the one hand, the erythrocytes are retained, but that on the other hand relatively large to be analyzed components of the sample can penetrate into the second zone. 6

Details of the chemical process of analytical determination are not essential to the invention. It is essential only in so far that the characteristic for the desired concentration of optically detectable change in the second zone 6, or in a further arranged on the same side of the transition portion 7 of the test carrier layer and does not take place in the first zone. 5 In many usual in clinical chemistry test procedure, a color forming substance (eg, a chromogenic substrate of an enzyme involved in the reaction) formed or implemented in a way that it changes color, for example, as a final stage. Such a reaction component can be described as "an optically detectable signal producing component", shortly also signal producing component (SPK), respectively.

Preferably, such a component is in the second zone 6 of the laminate 3. Preferably, it is included in the second coating composition. However, it is also possible for the second zone 6 subsequently to be impregnated with a SPK or spray. This is particularly useful if the second coating composition is prepared based on an organically dissolved film-forming agent.

However, the SPK need not necessarily be present in the second zone 6 from the beginning. Rather, test procedures are known in which such components are formed in a different layer of a multilayer test carrier or present and arrive in the course of the reaction process in the actual detection area. Also in such a method, the invention is applicable where the components may be contained in the first zone 5, the porous supporting layer 4 or a further upstream layer initially.

Specifically, the layer composite of the invention is advantageously made in the form that the first coating composition is first applied to a slowly transported web of a suitable porous support layer as 4 material over its full width. The coating composition was about honey-like viscosity, so that it remains predominantly on one side of the textile support layer material, but sinks into the spaces between the preferred multifilament yarns. In the finished product can be seen from the blood application side, the material of the first zone in the interstices of the textile structure, but it should not envelop the threads total.

The connection between the support layer and the first zone-forming coating is so strong that they can not be separated without destroying.

The larger the proportion of pigment in the first coating composition, the better the red blood cells are retained, but the more slowly penetrates also in the plasma to the evaluation zone. Preferably, the diatomaceous earth and the pigment are in the first coating composition in a weight ratio of 1: 0.5 to 1: 2, particularly preferably in a weight ratio of 1: 0.8 to 1: 1. An appropriate weight ratio arises then, of course, in the first zone 5, a. In this context it should be noted that all concentration data of the zones 5 and 6 relate to the parts lying outside of the transition area. 7

The diatomaceous earth and the polymeric film formers in the first coating composition and, consequently, in the first zone 5 are preferably in a weight ratio of 1: 0.9 to each other: 0.2 to. 1

After application of the first coating mass is formed into such a thin layer, expediently a so-called doctor blade, that is, arranged above the transporting path of the porous support layer Abstreiflineal is used to set the desired layer height.

Accordingly, in a second expediently formed because of the need relatively long drying times of the first completely separate operation to the composite of porous support layer and disposed thereon from the first coating composition layer, the second coating composition is applied in a corresponding manner. This second layer should be applied very thinly. The more polymer is applied, the stronger the erythrocytes are held back. At the same time a slower formation of the optical signal can be observed. Preferably, the polymeric film-forming agent in the second coating composition having a basis weight of not more than 200g / m², preferably applied at most 100g / m².

As mentioned, it has been shown in practice that the second coating composition penetrates to a considerable extent in the underlying layer. Thus a consumption was found in second coating composition, for example, at a set amount of coating gap of 10μ, which corresponds to a 50μ high layer.

As mentioned above, the composite layer 3 also be prepared for example on a glass plate from which it can easily be removed after manufacture. However, since it is not mechanically stable, it is convenient to him to be applied to a carrier material. Fig. 3 shows an alternative embodiment of a test carrier according to the invention when as carrier material, a transparent film 10 is applied, which is bonded to the second zone 6. Such an embodiment may be useful in cases where the porous support layer would interfere. 4

The laminate according to the invention can be used in test carriers of very different external structure.

It is only essential that the blood of the first zone 5 is supplied and that the evaluation is carried out on the side of the second zone. 6 Otherwise, however, can be used paying rich further constructive characteristics, layers or reagents.

Fig. 4 shows, for example, one similar to a conventional test strip shaped test carrier 11 with a narrow, elongate base film 12, which serves to handle.

On the base film 12, a test zone 13 can be seen, in which a liquid transport layer 14 adhered for example with a melt adhesive strip 15 to the base film 12th The liquid transport layer 14 is partially covered by the test box 16, which is constructed in cross-section corresponding to the test area 2a in FIG. 2. It is with the porous support layer 4 down also by means of the hot melt adhesive strip 15 secured on the base sheet 12, that it is in fluid contact with the liquid transport layer fourteenth

In the test carrier shown in Fig. 4, the blood sample is applied to the overlapped not of the layer composite 16 partial region 13a of the liquid transport layer and penetrates from there into the liquid transport layer (under the action of Kapilarkraft) in the area below the layer composite 16, so that the blood in the first zone of this layer composite can penetrate 16th

The structure shown in Fig. 4 has the advantage that the blood application and the evaluation of the same test carrier side carried forth.

The invention can be used particularly advantageously in connection with the test carrier construction described in German Patent Application P 36 43 516th In this patent application is incorporated by reference.

The following examples serve to illustrate the invention.

example 1

Preparation of a test field for a test carrier for the detection of glucose in blood

198 g of acrylic acid ester copolymer dispersion (Acronal 14D from BASF, Ludwigshafen, Federal Republic of Germany, 55% in water) 174 g swollen, highly viscous methylhydroxyethylcellulose (0.5% in water) 336 g of diatomaceous earth 336 g of titanium dioxide 0.95 g tetraethylammonium perfluorooctane sulfonate 40 g of 0.5 M phosphate buffer pH 5.5 23 g of methanol 46 g 1-hexanol 69 g acetone 65 g water be a homogeneous first coating works ver and with 0.18 mm gap height on a 0.20 mm thick polyester filter fabric (2 F 777, Swiss Seidengazefabrik Thal, Switzerland) and dried.

On the support thus obtained, coated a second coating mass consisting of 102 g of acrylic acid ester copolymer dispersion (Acronal 14D from BASF, 55% in water) 38 g swollen, highly viscous methylhydroxyethylcellulose (0.5% in water) 3 g of sodium 36 KU glucose oxidase 1050 KU peroxidase 1.48 g of 3,3 ', 5,5'-tetramethylbenzidine 0.53 g 1-phenylsemicarbazide 28 g 1-methoxy-2-propanol 40 g 1-hexanol 38 g water processed into a homogeneous mass, coated with 0.02 mm gap height and dried.

The composite layer so obtained is used in a test strip according to FIG 4 and results in good gradation in the concentration range of 20 -. 250 mg of glucose / dl with the use of whole blood.

example 2

Preparation of a test field for a test carrier for the detection of blood triglyceride

37 g polyvinyl-dispersion (50% in water) 29 g swollen, highly viscous methylhydroxyethylcellulose (0.5% in water) 56 g of diatomaceous earth 56 g of titanium dioxide 3.2 g of sodium dodecylbenzenesulfonate 40 g of phosphate buffer 0.5 M, pH 5.5 3.8g methanol 7.7g 1-hexanol 11.5 g of acetone 22 g water be processed to a homogeneous first coating composition and with 0.15 mm gap height on a 0.09 mm thick pure silk fabric (type 541 Spinnhütte silk technology, Celle, Germany) and dried.

On the support thus obtained is of a second coating composition comprising 20 g polyvinyl-dispersion (50% in water) 0.28 g sodium alginate 70 g 0.2M phosphate buffer pH 7.5 0.58 g of adenosine-5'-triphosphate, disodium salt 0.59 g of magnesium sulfate 7-hydrate 1.0g dioctyl 0.45 g of 3,3 ', 5,5'-tetramethylbenzidine 15 mg 1- (4-methylphenyl) -semicarbazid 16.4 g of 1-hexanol 30 g acetone 2.0 g Triton X-100 27 KU cholesterol 8.0 KU glycerol phosphate 27 KU glycerokinase 73 KU peroxidase 50 g water, which were processed to a homogeneous mass, whose pH was adjusted to 7.5, coated with 0.01 mm gap height and dried.

The test fields thus obtained result after spotting blood on the fabric side of a good gradation in the concentration range 100 - 300 mg Triglyceride / dl.

example 3

Preparation of a test field for a test carrier for glucose detection of blood

. To 46.3 g of a 20% strength by weight solution of polyvinyl acetate (Mowilith 70 from Hoechst AG.) In acetone / 1-hexanol / methanol (3: 2: 1, v / v), a solution of 1.30 g dioctyl added to 29.4 g of acetone. In this mixture, 28 g of diatomaceous earth and 28 g of titanium dioxide are dispersed. This homogeneous first coating a 0.20 mm thick polyester filter fabric (2 F 777, Swiss Seidengazefabrik, Thal, Switzerland) is coated with a gap height of 0.2 mm and dried.

On the thus obtained coated carrier is a second coating composition consisting of 64 g of a 20 wt .-% solution of polyvinyl acetate (Mowilith 70) in acetone / 1-hexanol / methanol (3: 2: 1, v / v) 1.3g dioctyl 36 g acetone 264 mg 1-phenylsemicarbazide 740 mg of 3,3 ', 5,5'-tetramethylbenzidine 13.8 g 1-methoxy-2-propanol, have been processed to form a clear viscous solution applied with a gap height of 0.04 mm and dried. On the thus obtained test field the reagents a) by a further coating, b) can be applied by spraying:<ul><li>a) The coating composition for the coating consists of 20 g swollen, highly viscous methylhydroxyethylcellulose (0.5% in water) 36 kU glucose 1050 kU peroxidase and is applied with a gap height of 0.02 mm and dried.</li><li>b) The spray solution is made 72 kU glucose 2.1 MU peroxidase 40 ml water and eye sprayed with a consumption of 20-30 ml / m² and dried.</li></ul> The test fields thus obtained result after spotting blood on the fabric side of a good gradation in the concentration range of 150-600 mg of glucose / dl.