HK1008565B - Volume-independent diagnostic test carrier and methods in which it is used to determine an analyte - Google Patents

Description

Volume-independent diagnostic test carrier and method for determining an analyte using the same

Technical Field

The present invention relates to a diagnostic test carrier comprising a support layer, one or more detection layers disposed thereon containing reagents required to determine an analyte in a liquid sample, and a web larger than the detection layer and adhered to the support layer overlying the detection layer. In addition, the invention relates to the use of the diagnostic test carrier for the determination of analytes in liquids and to a method for the determination of analytes in liquid samples with the aid of the diagnostic test carrier according to the invention.

Background

The so-called bound carrier test is usually used for qualitative or quantitative analytical determination of body fluid components, in particular blood components. In these tests, the reagents are present on or in an appropriate layer of the solid test support which is in contact with the sample. The reaction of the liquid sample with the reagent produces a detectable signal, in particular a color change which can be analyzed with the naked eye or with the aid of an instrument, usually with a reflectance photometer.

The test carrier is usually in the form of a test strip, which essentially consists of an elongated carrier layer made of plastic material and a detection layer arranged thereon as test field. However, test carriers are also known which are small plates of quadrilateral or rectangular shape.

A test carrier of the above-mentioned type is known from German patent document 2118455. This document describes a diagnostic support for detecting an analyte in a liquid, which support consists of a support layer and at least one detection layer containing a detection reagent, which detection layer has a cover layer on its surface which is not located on the support layer. The cover layer may be composed of a fine mesh in the form of a fabric, a braid or wool. Plastic fabrics are recommended meshes in order to quickly wet the detection layer with the sample liquid and avoid disturbing the chromatographic effect. For the determination of analytes in liquids, such diagnostic test carriers are immersed in a corresponding liquid, preferably urine. The detection layer is thus in contact with a large excess of fluid that the test carrier cannot absorb. However, depending on the contact time of the detection layer with the liquid to be measured, different color intensities are observed. Longer contact times generally lead to stronger positive results. Therefore, it is impossible to quantitatively determine the analyte correctly in this manner.

One common reason for erroneous measurements in the monitoring of diabetes (i.e. the regular control of the glucose content in the blood of a diabetic patient) is on the one hand that the sample volume is not suitable. The need for the smallest possible volume of test carrier is one of the goals of current development. However, such test carriers must not only be able to obtain correct measurements with very small sample volumes (about 3. mu.l), but also reliably perform measurements with relatively large sample volumes (about 15-20. mu.l) and must also retain the sample fluid. If the liquid leaks out of the test carrier, hygiene problems may arise, for example if potentially infectious, foreign blood is to be measured, or if the test carrier is to be instrumented, there is a risk of contamination of the instrumentation. To the best of the applicant's knowledge, this aim has not been achieved in a simple and satisfactory manner so far.

Disclosure of Invention

It is therefore an object of the present invention to provide a diagnostic test carrier for the quantitative determination of analytes in liquids, on which carrier a variable amount of sample liquid can be applied. A sample volume of about 3. mu.l should be suitable. However, an excess of sample liquid does not lead to time-dependent false positive results. Furthermore, an excess of sample liquid should not cause hygiene problems, and the test carrier should also be as simple to produce as possible.

The present invention achieves this object, and features of the present invention are reflected in the following embodiments.

1. A diagnostic test support 1 comprising a support layer 2, a detection layer 3 containing reagents required to determine an analyte in a liquid sample on the support layer, and a web 4 covering the detection layer 3, the web 4 being larger than the detection layer 3 and being adhered to the support layer 2, wherein the web 4 is hydrophilic but not capillary active per se, and a cover 5 made of a material impermeable to the sample is placed over a region of the web 6 extending beyond the detection layer such that spots 7 on the region of the web 4 covering the detection layer remain free, the web 4 creating capillary active voids between the cover 5 and the detection layer 3 and between the cover 5 and the support layer 2 or between the cover 5 and a spacer 10 on the support layer 2.

2. The diagnostic test carrier according to 1, wherein several detection layers are placed one after the other on the support layer.

3. The diagnostic test carrier according to 1 or 2, wherein the support layer is perforated and the detection layer is placed on the perforations.

4. The diagnostic test carrier according to 3, wherein the spotting points are located on the perforations of the support layer.

5. The diagnostic test carrier according to 3, wherein the spotting points are not located on the perforations of the support layer.

6. The diagnostic test carrier according to 3, wherein the support layer comprises a plurality of holes as perforations, on which one or more detection layers are placed.

7. The diagnostic test carrier according to 2, wherein the support layer comprises several wells as perforations, on each of which a different detection layer is placed.

8. The diagnostic test carrier according to 1, wherein the support layer comprises a well, and a detection layer comprising several adjacent reaction zones is disposed on the well.

9. A diagnostic test carrier according to one of claims 7 or 8, wherein the spots are located on several or all detection layers or reaction zones in each case.

10. The diagnostic test carrier according to one of claims 7 or 8, wherein the spotting is located only on one of the detection layers or one of the reaction zones.

11. The diagnostic test carrier according to 1, wherein the mesh is a monofilament fabric.

12. The diagnostic test support according to 1, wherein the web is adhered to the support layer by means of an adhesive tape preferably comprising natural or synthetic rubber.

13. The diagnostic test carrier of one of items 1-12 is used in the determination of an analyte in a liquid.

14. Method for determining an analyte in a liquid sample with the aid of a test carrier according to one of claims 1 to 12, wherein the sample liquid is applied to a sample application point, excess liquid not absorbed by the detection layer and the areas of the network (4) lying thereon is conducted to the areas of the network extending beyond the detection layer, and the signal generated at the detection layer is observed and used to determine the presence or quantity of the analyte in the liquid sample to be determined.

The gist of the invention is a diagnostic test carrier with a support layer and a detection layer (which is arranged on the support layer and contains the reagents required for the determination of an analyte in a liquid sample). The detection layer is covered with a mesh that is larger than the detection layer and is secured to the support layer outside the detection layer. The network of diagnostic test carriers of the present invention is hydrophilic but not capillary active when used alone. An inert cover layer made of a material impermeable to the sample liquid is placed over the area of the mesh extending beyond the detection layer, so that the area of the mesh located above the detection layer for the sample application remains free.

Furthermore, the invention relates to the use of this diagnostic test carrier for the determination of analytes in liquids. Therefore, a method for determining an analyte in a liquid sample by means of this diagnostic test carrier, in which a sample liquid is added to a sampling point, is also the gist of the present invention. The web directs excess liquid from the detection layer into the region of the web extending beyond the detection layer, and the generation of a signal from the detection layer can then be observed. The signal is generated to determine the presence or amount of the analyte in the sample to be tested.

The web of diagnostic test carrier of the invention should not have capillary activity or absorbency in itself, so that the sample liquid can be utilized as completely as possible by the detection layer. Those nets where the water rises less than 2mm up the net when vertically immersed in water have proved suitable. Preferably, a hydrophilic coarse mesh monofilament fabric is used as the mesh. For this purpose, the textile material itself can be hydrophilic or made hydrophilic by treatment with, for example, a wetting agent. It is particularly preferred to use polyester as the web material, in which case the web made of this material is then used after treatment with a wetting agent.

The thickness of the web must be such that the cover placed on it and the thin layer under the web are at a distance from each other that the liquid will be absorbed from the impregnated detection layer, retain the liquid and then wick into the web filling the network in the area under the cover by capillary forces, leading the liquid away from the sampling points. For this reason, the thickness of the net is usually preferably 50 to 400. mu.m.

The mesh of the mesh must have a width large enough to allow liquid to pass through the mesh onto the detection layer. The nature of the mesh is such that liquid does not spread horizontally across the mesh on the surface of the mesh, but flows vertically through the mesh onto the detection layer.

Materials which are particularly considered as support layer materials in the diagnostic test carrier according to the invention are those which do not absorb the liquid to be tested. These materials are so-called non-absorbent materials, plastic foils made of, for example, polystyrene, polyvinyl chloride, polyester, polycarbonate or, particularly preferably, polyamide. However, it is also possible to impregnate absorbent materials, such as wood, paper or cardboard, etc., with water repellents or to coat water repellent films, in which case silicon or stearin may be used as the water repellent, while, for example, cellulose nitrate or cellulose acetate may be used as the film former. In addition, metal foils and glass are also suitable as carrier materials.

In contrast, the detection layer requires the use of a material capable of absorbing the liquid to be measured together with the components contained therein. These materials are so-called absorbent materials (such as wool, textiles, knits, membranes or other porous plastic materials, etc.) or swelling materials (such as gelatin or dispersion membranes which can be used as membrane materials, etc.). Of course, it is contemplated that the material used for the detection layer must also be capable of carrying the reagents required to detect the analyte to be detected. In the simplest case, all reagents required for the analyte test are on or in one layer. However, it is also conceivable to dispense the reagent in several absorbent or swelling material layers placed one above the other. "detection layer" as used hereinafter includes those instances where the reagent is either on or in only one layer, or in two or even more thin layers as described above.

Furthermore, the detection layer may also comprise a thin layer capable of separating plasma or serum from whole blood, such as a fleece of glass fibres, as known for example from EP-B-0045476. One or several such separation layers may be located on one or several thin layers carrying detection reagents. This structure is also included in the "detection layer".

Preferred materials for the detection layer are paper or porous plastic materials such as membranes. Of these, an asymmetric porous membrane is particularly preferred, and it is preferable to arrange the porous membrane so that the sample liquid to be measured is applied to the large pore side of the membrane and the analyte is detected from the fine pore side of the membrane. Polyamide, polyvinylidene fluoride, polyethersulfone or polysulfone membranes are most preferred as porous membrane materials. Polyamide 66 membranes and hydrophilic asymmetric polysulfone membranes are particularly suitable. The reagents for detecting the analyte to be detected are usually introduced by immersion in the above-mentioned materials or by coating one side of the membrane. When an asymmetric membrane is applied, the pore side is preferably applied.

However, the detection layer may also be considered as a so-called open film (open film) as described in, for example, EP-B-0016387. For this purpose, an aqueous dispersion of a film-forming organic plastic solid of insoluble organic or inorganic fine particles may be added, with the reagents required for the detection reaction being additionally added. Suitable film formers are preferably organic plastics (such as polyvinyl esters, polyvinyl acetates, polyacrylates, polymethacrylic acids, polyacrylamides, polyamides, polystyrenes, etc.), mixed polymers (such as mixed polymers of butadiene and styrene or mixed polymers of maleates and vinyl acetates, etc.) or other natural or synthetic film-forming organic polymers and mixtures thereof in the form of aqueous dispersions. The dispersion can be applied to a substrate to form a thin uniform layer that dries to form a water-resistant film. The dry film thickness is 10-500 μm, preferably 30-200 μm. The membrane may be used as a carrier with the substrate or may be placed on another detection reaction carrier. Although the reagents required for the detection reaction are typically added to the dispersion used to create the apertured film, it may also be advantageous to impregnate the formed film with the reagents after production. The filler may also be pre-impregnated with the agent. Reagents that can be used to detect an analyte are known to those skilled in the art. This need not be elaborated upon in more detail here.

Another preferred example of A detection layer according to the invention is the film layer described in WO-A-9215879. The layer is produced from an emulsion or dispersion of a polymeric film former that also contains a pigment, a swelling agent, and a detection agent in a homogeneous dispersion. Polyvinyl esters, polyvinyl acetates, polyacrylates, polymethacrylic acids, polyvinyl amides, polyamides and polystyrenes are particularly suitable as polymeric film formers. In addition to homopolymers, mixed polymers such as butadiene, styrene or maleic esters are also suitable. Titanium dioxide is a particularly suitable film pigment. The swelling agent used should have particularly good swelling properties, with methyl vinyl ether maleic anhydride copolymers being particularly preferred. The question of which reagent is suitable for use in detecting a particular analyte is left to the person skilled in the art.

In the diagnostic test carrier according to the invention, it is particularly recommended to use a test field consisting of two layers as detection layer. The test zone comprises a transparent sheet and a first film layer disposed thereon and a second film layer disposed over the first film layer. It is important that the first layer on the transparent sheet scatters light in the wet state substantially less than the second layer on the first layer. The uncoated side of the transparent sheet is called the detection side, while the side of the second layer opposite the side of the first layer is called the spotting side.

The film layer is produced from an emulsion or dispersion of a polymeric film former. Dispersion film formers comprise polymer particles that are microscopically visible and insoluble in the carrier liquid (usually water) and are well dispersed in the carrier liquid. If the liquid is removed by evaporation during film formation, the particles approach each other and contact each other well. The large forces generated in the process and gained in the surface energy associated with film formation cause the particles to actually form a tight thin film layer. Alternatively, an emulsion of the film former in a solvent may be used. The dissolved polymer is emulsified in the carrier liquid and is immiscible with the solvent.

Polyvinyl esters, polyvinyl acetates, polyacrylates, polymethacrylic acids, polyvinyl amides, polyamides and polystyrenes are particularly suitable as polymers for such film formers. In addition to homopolymers, mixed polymers such as butadiene, styrene or maleic esters are also suitable.

Two so-called membrane layers are located on the transparent sheet in the test zone. For this purpose, plastic foils which can be impregnated with liquids are conceivable. Polycarbonate sheets have proven to be particularly suitable.

Two film layers can be produced by coating with a compound containing the same polymeric film former or by coating with a compound containing different polymeric film formers. The first film layer contains a swelling agent and optionally a filler which scatters light weakly, while the second film layer requires a swelling agent and in any case at least one pigment which scatters light strongly. In addition, the second film layer also contains a non-porous filler and a small amount of a porous filler such as diatomaceous earth, without permeability to red blood cells.

By adding a swelling agent with good swelling properties (i.e. a substance that increases in volume when absorbing water), despite this opening effect, we do not only obtain film layers that penetrate the sample fluid rather quickly, but also these film layers have very good pigment separation properties for red blood cells as well as for blood. The swelling properties of the swelling agent should be so good that the colour production rate of the test (such as glucose test reactions etc.) is mainly dependent on the penetration of the sample liquid through the membrane layer, optionally after up to 1 minute a detectable reaction is determined. Particularly suitable swelling agents have proven to be methyl vinyl ether maleic anhydride copolymers, synthetic biopolymers and methyl vinyl ether maleic anhydride copolymers.

Diatomaceous earth (Kieselghur) is also known as diatomic earth (diatomous earth). These diatomaceous earths are deposits formed by skeletons of diatomaceous silica mined in different places. The diatomaceous earth preferably used has an average particle diameter of from 5 to 15 μm, as determined by means of a laser particle size analyzer of the type 715, sold by the Pasisch company, Munich, Germany.

The amount of strongly scattering pigment in the second layer is at least 25% by weight of the dried bilayer as determined in the test area. Since the weakly scattering optical filler and the strongly scattering optical pigment are necessary for the optical properties of the thin film layer, the first and second thin film layers contain different fillers and pigments.

The second film layer should either be free of fillers or contain those having a refractive indexA near water refractive index filler. Silicon dioxide, silicates and aluminum silicates have proven particularly suitable for this purpose. The trade name of the specially recommended commodity is Traspafill^Sodium aluminum silicate.

According to the invention, the second layer should scatter light very strongly. The refractive index of the pigment in the second film layer should ideally be at least 2.5. Therefore, silica is preferably used. Particles having an average diameter of 0.2 to 0.8 μm have proven particularly advantageous. Titanium dioxide of the type which is easily processable in the anatase modification is particularly preferred.

Reagent systems for detecting specific analytes by color generation are known to those skilled in the art. It is possible that all components of the reagent system are located in one film layer. However, it is also possible for the components of the reagent system to be distributed in two film layers. Preferably, the color-producing reagent system is at least partially disposed on the first film layer.

Color generation within the scope of the invention is to be understood not only as a change from white to colored, but also as any change in color, of course particularly preferably with the wavelength of maximum light absorption (. lamda.)_max) Such color changes are related to changes in (b).

In order to optimize the test fields of the diagnostic test carrier according to the invention, the respective film layers are produced continuously with a homogeneous dispersion of the components in each case. For this purpose, a transparent sheet is used as a substrate to produce a coating compound for the first thin film layer. After the first thin film layer coating compound is applied to have a specific thin film thickness, the thin film layer is dried. Then, a second thin-film layer coating compound is applied to this layer so as to also have a thin-film thickness, and then dried. After drying, the total thickness of the first film layer and the second film layer should not exceed 0.2mm, preferably not exceed 0.12mm, particularly preferably not exceed 0.08 mm. The dried second film layer is preferably 2-5 times thicker than the first film layer.

The test carrier according to the invention can have a detection layer. However, it is also possible to have several detection layers placed one after the other. In the case of several detection layers, these detection layers may be identical or different, so that one and the same analyte can be detected in parallel in several detection layers, or different analytes can be detected in one detection layer each. However, it is also possible that several spatially separated reaction zones are located one after the other on the detection layer, so that, in this case, several times of the same analyte can be detected in parallel in the same detection layer, or different analytes can be detected. In the latter case, the material of the film layer is the same material that does not contain the analyte detection reagent. Different reagents are located in different reaction zones. The different reaction zones may be present side by side and in contact with each other or separated by an intervening zone where no signal is generated by the same analyte.

In the diagnostic test carrier of the present invention, the mesh covering the detection layer is larger than the detection layer beneath it. The portion of the web extending beyond the detection layer (i.e., the portion of the web not in contact with the detection layer) is secured directly or indirectly via a spacer to the support layer beyond the detection layer. Immobilization may be carried out by methods known to those skilled in the art of test carrier technology. For example, the fixing may be performed with a thermosetting adhesive or by hardening a cold-setting adhesive. Since capillary-active liquid transfer is particularly well performed in this case, point bonding or pattern bonding is preferably used in this case. Double-sided adhesive tapes have also proven to be advantageous. In all cases, however, it is important to adhere the web to the support layer so that it is possible to transfer liquid from the detection layer to the portion of the web adhered to the support layer by capillary activity. This capillary-active liquid transfer is particularly likely to occur when the detection layer is saturated with liquid. Adhesive tapes made of natural or synthetic rubber have proven particularly suitable for this process. It is particularly advantageous for the adhesive used to adhere the web to the support layer to be the same thickness as the detection layer. It is used more or less as a spacer to fix the mesh all in a continuous plane also outside the detection layer area.

If the diagnostic test carrier of the invention contains several detection layers one after the other, the mesh may cover all detection layers, or several meshes may be used.

For detecting the analyte to be detected in the sample liquid, the detection layer and at least the reaction zone (i.e.the area of the reagent-carrying detection zone which can be observed and which determines the generation of a signal) can be seen through the support layer of the diagnostic test carrier according to the invention. This can be achieved with a transparent support layer. However, the support layer may also have perforations which are covered with a detection layer or layers. The detection layer or layers and at least the reaction zone of the detection layer can be seen through the aperture. In a preferred embodiment of the diagnostic test support of the present invention, the support layer below the detection layer has holes through which the detection layer or the reaction zone can be observed. The diameter of the hole is slightly less than the smallest linear length of the detection layer so that the detection layer outside the hole is located on and can be adhered to the support layer. Double-sided adhesive strips located near both sides of the detection layer are preferably secured to the web above the detection layer and suitably adhered to the support layer. However, the detection layer itself is also preferably adhered to the support layer by a thin adhesive tape.

However, it is also possible to see several reaction zones of the detection layer through one hole.

The wells of the diagnostic test carrier of the present invention may be comprised of two or more wells for detecting an analyte(s). Different detection layers or only one detection layer with several reaction zones can be placed on the well, so that in each case one detection layer or one reaction zone can be observed through one well. Several reaction zones can also be observed through one hole.

An inert cover made of a non-absorbent material that is impermeable to the sample (typically water) is placed over the mesh of the diagnostic test support of the present invention so that the area of the mesh outside the detection layer can be covered. It is desirable that the cover also extends slightly beyond the area of the detection layer. In any case, however, a substantial portion of the mesh covering the detection layer remains free. The free portion of the mesh represents the spot.

Plastic foils have proven particularly suitable as coverings. If the cover and the mesh have different colors, for example white and yellow or white and red, this may very well mark the site where the sample liquid to be tested should be added.

If, for example, one or several arrows are printed on the cover, it is also possible to clearly see the direction (i.e. one end) in which the diagnostic test carrier according to the invention should be placed or inserted into the meter.

The spot can be obtained particularly simply by two strip-shaped plastic foils which leave free the strip-shaped area of the web covering the detection layer. If two or several spots are provided, three or more strip-shaped plastic foils must be used. The plastic sheet used for covering is adhered to the web and optionally also to the support layer. If the plastic foil itself is not an adhesive, hot-melt adhesives, for example in the form of dots or a grid, for the bottom side of the support layer or the cover, are suitable for such attachments or adhesive tapes. In any case, however, care must be taken that the capillary voids created by the mesh remain under the cover where excess sample liquid can be absorbed from the liquid-saturated detection layer. The spots are preferably located on perforations in the support layer through which the generation of the signal in the detection layer can be observed.

To carry out the method for determining an analyte in a liquid sample with the aid of the diagnostic test carrier according to the invention, the sample liquid is applied to the side of the web which is not facing the detection layer, ideally such that the liquid completely penetrates the detection layer through the web. Body fluids such as blood, plasma, serum, urine, saliva, etc. are particularly considered as sample fluids. Blood or liquids produced from blood (such as plasma or serum) and urine are particularly preferred sample liquids. Excess liquid is directed out of the detection layer through the mesh and into the area of the mesh that extends beyond the detection layer. A signal can then be detected in the detection layer when the analyte to be detected is present. This signal is preferably a change in color, which can be understood as the generation of color, the loss of color, and a change in color. The intensity of the color change can determine the amount of analyte in the liquid sample to be tested. The evaluation can be carried out visually or quantitatively with the aid of instruments, usually with the aid of a reflectance photometer.

If too little liquid reaches the detection layer, i.e. less liquid than is necessary to saturate the detection layer, the area of the detection layer can be seen to remain dry from above or from below, since the liquid can only reach the detection layer vertically through the web, without spreading horizontally over the surface of the web. Since the signal is only generated in the fully wetted areas of the detection layer if the analyte is present, the generation of a non-uniform signal can be seen or instrumentally detected through the web and through the support layer. This clearly indicates to the test person that too little sample liquid is used and the test result may be erroneous. Even if no analyte is present in the sample, a visual or reflectometric determination of several partial areas of the detection layer can for example confirm that only a part of the detection layer is moist and therefore that too little sample liquid is used.

In addition to marking the spots, the cover also maintains capillary forces that draw excess liquid away from the detection layer. Furthermore, the cover also prevents excess liquid that is conducted out of the detection layer from coming into contact with the outside and this liquid from easily dripping off the test carrier.

The main advantage of the diagnostic test carrier of the present invention is that it is not necessary to add a predetermined volume of sample liquid to the test carrier. Excess liquid is conducted out of the detection layer through the web extending beyond the detection layer as described above. Hygiene issues are also considered due to excess liquid being conducted away from the detection layer. Dripping of liquid from the test carrier or contact of liquid with instrument parts on which the test carrier is placed, for example, is reliably avoided for instrument detection. This is very important in the detection of blood or samples produced from blood, such as plasma or serum.

The size of the area of the web extending beyond the detection layer (the portion of the web extending beyond the detection layer) depends on the maximum sample volume actually expected, so that a real excess of liquid can also be conducted out of the detection layer. In this way, the intensity of the signal generated in the presence of the analyte is independent of the amount of sample liquid in contact with the detection layer and the contact time. The color developed after completion of the detection reaction (usually within a few seconds to a few minutes) remains unchanged when the measurement is performed. It is determined solely by the stability of the color-generating system and not by, for example, the analyte with which excess liquid diffuses back into the detection layer. False positive results can also be avoided and quantitative determination of the analyte is possible.

Covering the web portion and thus marking the spots ensures that the liquid can only be placed at the optimal location on the detection layer. This, together with the detection layer which only absorbs small amounts of liquid, but nevertheless ensures that an enhanced signal is generated, ensures that reliable detection of the analyte is possible even with very small sample volumes. Since the test carrier according to the invention consists of only a few components which can be assembled simply and quickly, it can be produced very inexpensively.

Drawings

Preferred embodiments of the diagnostic test carrier of the present invention are shown in FIGS. 1-23.

FIG. 1 shows a perspective view of a diagnostic test carrier of the present invention having spots.

FIG. 2 shows a top view of the bottom surface of the test carrier of FIG. 1 of the present invention with circular holes under the detection layer.

FIG. 3 shows a cross-sectional view of the diagnostic test carrier of the present invention of FIG. 1 taken along section A-A.

Fig. 4 shows an enlarged view of a portion of the cross-sectional view of fig. 3.

FIG. 5 shows a perspective view of a diagnostic test carrier of the present invention having a spot.

FIG. 6 shows a top view of the bottom surface of the diagnostic test carrier of FIG. 5 of the present invention having perforations (including circular and rectangular holes) under two separate detection layers.

FIG. 7 shows a cross-sectional view of the diagnostic test carrier of FIG. 5 taken along section A-A in accordance with the present invention.

FIG. 8 shows a perspective view of a diagnostic test carrier of the present invention having a very large spot size.

FIG. 9 shows a top view of the bottom surface of the diagnostic test carrier of FIG. 8 of the present invention with perforations (including circular and rectangular holes) under a very large detection layer.

FIG. 10 shows a cross-sectional view of the diagnostic test carrier of FIG. 8 taken along section A-A according to the present invention.

FIG. 11 shows a perspective view of a diagnostic test carrier of the invention having spots on one of the two detection layers.

FIG. 12 shows a top view of the bottom surface of the diagnostic test carrier of FIG. 11 of the present invention having perforations (including circular and rectangular holes) under two separate detection layers.

FIG. 13 shows a cross-sectional view of the diagnostic test carrier of FIG. 11 taken along section A-A in accordance with the present invention.

FIG. 14 shows a perspective view of a diagnostic test carrier of the present invention having a very large spot size.

FIG. 15 shows a top view of the bottom surface of the diagnostic test carrier of FIG. 14 of the present invention having an aperture (comprising a very large rectangular hole) under the detection layer having two adjacent reaction zones.

FIG. 16 shows a cross-sectional view of the diagnostic test carrier of FIG. 14 taken along section A-A in accordance with the present invention.

FIG. 17 shows a perspective view of a diagnostic test carrier of the present invention having a spot on one of two reaction zones.

FIG. 18 shows a top view of the bottom surface of the diagnostic test carrier of FIG. 17 of the present invention having an aperture (comprising a very large rectangular hole) under the detection layer having two adjacent reaction zones.

FIG. 19 shows a cross-sectional view of the diagnostic test carrier of FIG. 17 taken along section A-A in accordance with the present invention.

FIGS. 20-23 show calibration curves 1-4 generated according to the method described in example 2.

The reference numerals used in the figures have the following meanings.

1 diagnostic test Carrier

2 supporting layer

3 detection layer

4 mesh

5 covering

6 network region extending beyond the detection layer

7 sampling point

8 holes

9 reaction zone

10 shim

11 capillary active gap

12 sample liquid

13 positioning hole

14 adhesive tape attachment for detection layer

The diagnostic test carrier 1 according to the invention, which is shown in a perspective view in FIG. 1 and in a sectional view in FIG. 3, is in the form of a test strip. On the support layer 2 is placed a detection layer covered by a larger mesh 4. The web 4 is attached to the support layer in the vicinity of the detection layer 3 by means of a spacer 10. These spacers, which may be hot-melt adhesive zones or double-sided adhesive tapes, secure the web 4 to the support layer 2. The thickness of the spacer is ideally the same as the thickness of the detection layer 3. A thin layer serving as a cover is attached to the support layer 2 and the mesh 4. The meshes are positioned such that they cover the area of the mesh 4 that extends beyond the detection layer 3. The cover 5 also extends slightly beyond the detection layer 3. However, they leave most of the portion of the mesh 4 covering the detection layer 3 free. This area represents the sampling point 7. A sample liquid 12 to be tested is added to this area. The alignment holes 13 allow the test strip to maintain an accurate predetermined position on the instrument (such as a reflectance photometer) when measured with the instrument such as a reflectance photometer. This can be done with a needle inserted into the positioning hole 13, thus holding the test carrier 1 in a predetermined position. The cover 5 on the left contains a printed arrow indicating to the user which end of the test carrier 1 should be placed or inserted into the meter.

FIG. 4 shows an enlarged cross-sectional view through the diagnostic test carrier of the present invention shown in FIGS. 1 and 3. The figure is intended to illustrate how the method of detecting an analyte in a liquid sample is performed. For this test, a sample liquid is applied to the spot 7 of the web 4. The liquid penetrates vertically through the web 4 into the detection layer 3, which is adhered to the support layer 2 with double-sided adhesive tape 14. The adhesive tape attachment 14 contains a hole corresponding to the eyelet 8 of the support layer 2 and also located exactly above the eyelet 8. If sufficient sample liquid is added, the liquid is dispersed throughout the reaction zone 9 in the detection layer 3. If the liquid volume is very small, the detection layer may even suck up the web 4 above, since the web 4 itself is not capillary active. In the case of a large liquid volume medium, the liquid first fills the empty spaces of the web 4 above the detection layer 3 and subsequently fills the capillary spaces under the cover 5. In order for these capillary voids to function properly, the cover 5 must at least slightly overlap the area of the detection layer 3 under the cover 5. The reaction zone 9 of the detection layer 3 can be observed through the aperture 8. In this regard, FIG. 2 shows a top view of the bottom surface of the diagnostic test carrier of FIGS. 1 and 3. If the analyte is present in the added sample liquid, the reaction zone 9 will change. A signal is generated, e.g. a color change, and the color intensity is used to determine the amount of analyte in the sample liquid.

The diagnostic test carrier of the invention shown in fig. 5-7 has two detection layers 3 allowing the sample liquid 12 to enter through two spots 7 located on the detection layers. The spots 7 are formed by three strip-like covers 5 covering the area of the web extending beyond the detection layer 3. In the example shown, a continuous web 4 is used. However, it is also possible to use two separate webs 4 with a liquid barrier interposed between them (such as for example adhesive tape or a strip of hot-melt adhesive, etc.). The well 8 in the support layer 2 of the test carrier 1 comprises two holes, each of which allows the reaction zone 9 of one of the two detection layers 3 to be observed. This test carrier 1 is suitable, for example, for the simultaneous detection of two different analytes. In this case, it is preferable to use a spatially separated detection layer 3 if the reagents or reaction products can interfere with each other.

The diagnostic test carrier 1 according to the invention of fig. 8 to 10 has very large spots 7 on the detection layer 3, which can be observed through the aperture 8 comprising two holes. Different reaction zones 9 containing reagents for different analytes may for example be placed on two wells. Thus, two analytes of one sample can be detected. However, the two reaction zones may also be used to detect different sensitivities of the same sample.

The two detection layers 3 of the diagnostic test carrier 1 according to the invention shown in FIGS. 11 to 13 are located on the aperture 8 comprising two holes. One detection layer 3 is placed on each of the holes 8. In this case, the spots 7 are located on only one of the two detection layers 3. Thus, the sample liquid 12 first enters the detection layer 3 below the spotting point 7, and then the excess liquid enters the detection layer 3 on the right by capillary forces in the area of the mesh 4 below the cover 5 on the right, which can be observed through the rectangular hole in the support sheet 2. This test carrier is suitable, for example, for detecting analytes with two detection layers of different sensitivity. The less sensitive general area is preferably located directly below the spot and the other, more sensitive area is located near the general area. The test carrier enables the measurement to be carried out with a universal zone in the case of small sample volumes and with two zones in the case of large sample volumes.

The test carrier 1 of FIGS. 14-16 has a very large spot 7 on the detection layer 3 carrying two adjacent reaction zones 9. The two reaction zones are visible from the bottom surface of the support layer 2 through the perforations 8 (which in this case consist only of once single rectangular holes). The sample liquid 12 added to the center of the spotting point 7 permeates into the detection layer 3 through the web 4 and reaches the two reaction zones 9 simultaneously. This test carrier can be used, for example, to detect two different analytes of a sample.

The test carrier 1 shown in FIGS. 17 to 19 corresponds essentially to the test carrier of FIGS. 14 to 16. However, the spotting point 7 is located on only one of the two reaction zones 9. The cover 5 on the right prevents the sample liquid 12 from being added directly to the reaction zone 9 on the right. The sample liquid 12 can reach this reaction zone only in the area of the web 4 under the right cover by capillary forces.

Detailed Description

The invention is illustrated in more detail by the following examples.

Example 1

Production of diagnostic test vectors of the invention for the detection of glucose

The test vehicle of fig. 1 was produced by the following production steps:

a 5mm wide double-sided adhesive tape (polyester support and elastomer adhesive) was placed on a polyester support layer containing titanium dioxide. The composite was drilled at a 6mm hole-to-hole distance to create an assay hole. The protective paper of the double-sided adhesive tape was then removed.

A detection layer consisting of 2 thin film layers was produced as follows:

A. the following ingredients, either pure or in stock form, were added together in a beaker with stirring to mix:

water: 820.0g

Citric acid monohydrate: 2.5g

Calcium chloride dihydrate: 0.5g

Sodium hydroxide: 1.4g

Synthesizing a biopolymer adhesive: 3.4g

Tetraethylammonium chloride: 2.0g

N-octanoyl-N-methyl-glucamide: 2.1g

Polyvinylpyrrolidone (MW 25000): 3.5g

Transpafill^(sodium aluminum silicate): 62.1g

Polyvinyl propionate dispersion (50% by weight in water): 60.8g

Bis- (2-hydroxyethyl) - (4-hydroxyimino-cyclohex-2, 5-dienyl) -ammonium chloride: 1.2g

2, 18-hexasodium phosphomolybdate: 16.1g

Pyrroloquinoline-quinone: 32mg of

Glucose dehydrogenase from Acinetobacter calcoaceticus, 1.7MU

EC1.1.99.17： (2.4g)

1-hexanol: 1.6g

1-methoxy-2-propanol: 20.4g

The total composition was adjusted to about pH6 with NaOH and then applied at an areal weight of 89g/qm onto polycarbonate sheets having a thickness of 125 μm and then dried.

B. The following ingredients, either pure or in stock form, were added together in a beaker with stirring to mix:

water: 579.7g

Sodium hydroxide: 3.4g

Grantrez^(methyl vinyl ether maleic acid copolymer): 13.8g

N-octanoyl-N-methyl-glucamide: 3.6g

Tetraethylammonium chloride: 9.7g

Polyvinylpyrrolidone (MW 25000): 20.2g

Titanium dioxide: 177.1g

Diatomite: 55.3g

Polyvinyl propionate dispersion (50% by weight in water): 70.6g

2, 18-hexasodium phosphomolybdate: 44.3g

Potassium hexacyanoferrate (III): 0.3g

1-hexanol: 1.6g

1-methoxy-2-propanol: 20.4g

The total composition was adjusted to about pH6 with NaOH and then applied to polycarbonate sheets by the method described in a. at an areal weight of 104g/qm, and then dried.

The 5mm wide test layer strip produced in this way was accurately fixed and bonded to a support layer, the sheet side of which was placed on a perforated double-sided adhesive tape.

Double-sided adhesive tape as a spacer (PVC support and natural rubber adhesive) was adhered to the support sheet on both sides of the support layer and directly attached to the detection layer. In an embodiment of the invention, one shim is 6mm wide and the other shim is 9mm wide. The two protective sheets of double-sided adhesive tape were subsequently removed.

A yellow monofilament coarse polyester fabric, Scrynel PE 280 HC ("Zurcher Beuteltuchfabrik, rusclikon, Switzerland) impregnated with wetting agent was placed on the compound structure and bonded by pressing.

Two double-sided adhesive tapes (PVC support and natural rubber adhesive) were adhered to the yellow mesh as a cover so as to completely cover the gasket and overlap the reaction zone at least a little more. Thus, the band-shaped material is completed.

The strip material was cut into test carriers of 6mm width so that the measurement wells were in the middle of the test carriers.

Example 2

Volume independence of the diagnostic test Carrier of the invention

The test carrier of example 1 was measured with a reflectance photometer. Reflectance values for determining color intensity can be converted to glucose concentration when a calibration curve is obtained. If "relative reflectance" is used, this refers to the reflectance of the dried test support.

A. A large number of venous blood samples with different glucose concentrations were assayed and a calibration curve was established. The reflectance values and glucose concentrations of these venous blood samples determined by the reference method can be used to establish a calibration curve.

In calibration variant 1, 10. mu.l of venous blood are applied to the test carrier from example 1 and the reflectance is determined after 21 seconds. Calibration curve 1 (fig. 20) was determined by regression calculation of the mean reflectance of 10 test carriers and the reference value of the blood sample.

In calibration variant 2, 10. mu.l of venous blood were also applied to the test carrier of example 1 and the reflectance was determined after 30 seconds. The calibration curve 2 (fig. 21) was determined by regression calculation of the mean reflectance of 10 test carriers and the reference value of the blood sample.

In calibration variant 3, 10. mu.l of venous blood were also applied to the test carrier of example 1, and the reflectance was measured every 3 seconds. The measurement was terminated as long as the difference in reflectance was less than 0.3 in two consecutive times, and the reflectance value was then used for evaluation. The calibration curve 3 (fig. 22) was determined by regression calculation using the mean reflectance of 10 test carriers and the reference value of the blood sample.

In calibration variant 4, 10. mu.l of venous blood were also applied to the test carrier of example 1, and the reflectance was measured every 3 seconds. The measurement was terminated as long as the difference in reflectance was less than 0.9 in two consecutive times, and the reflectance value was then used for evaluation. The calibration curve 4 (fig. 23) was determined by regression calculation of the mean reflectance of 10 test carriers and the reference value of the blood sample.

B. In assay variant 1, different volumes of venous blood were applied to the test carrier of example 1 and the reflectance was determined after 21 seconds. The respective reflectance was converted to glucose concentration using the corresponding calibration curve of fig. 20. The deviation in accuracy, which is shown in table 1, is determined from the mean concentration of 10 test carriers and the reference value of the blood sample.

In assay variant 2, different volumes of venous blood were also applied to the test carrier of example 1 and the reflectance was determined after 30 seconds. The respective reflectance was converted to glucose concentration using the corresponding calibration curve of fig. 21. The deviation in accuracy, which is shown in table 2, is determined from the mean concentration of 10 test carriers and the reference value of the blood sample.

In assay variant 3, different volumes of venous blood were also applied to the test carrier of example 1, and reflectance was measured every 3 seconds. The measurement was terminated as long as the difference in reflectance was less than 0.3 in two consecutive times, and the reflectance value was then used for evaluation. The respective reflectance was converted to glucose concentration using the corresponding calibration curve of fig. 22. The deviation in accuracy, which is shown in table 3, is determined from the mean concentration of 10 test carriers and the reference value of the blood sample.

In assay variant 4, different volumes of venous blood were also applied to the test carrier of example 1, and reflectance was measured every 3 seconds. The measurement was terminated as long as the difference in reflectance was less than 0.9 in two consecutive times, and the reflectance value was then used for evaluation. The respective reflectance was converted to glucose concentration using the corresponding calibration curve of fig. 23. The deviation in accuracy, which is shown in table 4, is determined from the mean concentration of 10 test carriers and the reference value of the blood sample.

Table 1:determination of the volume tolerance of the test strips in variant 1

Sample volume	Measured relative reflectance [% ]]	Concentration calculated according to calibration Curve 1	Deviation of reflectance value [% ]]
				3μl	42.8	117.5	-0.5
5μl	42.9	117.1	-0.8

8μl	42.6	118.5	0.4
				10μl	41.8	122.1	3.4
20μl	41.9	121.6	3.0

Table 2:determination of the volume tolerance of the test strips in variant 2

Sample volume	Measured relative reflectance [% ]]	Concentration calculated according to calibration Curve 2	Deviation of reflectance value [% ]]
				3μl	37.4	117.5	-0.5
5μl	37.6	117.0	-0.9
				8μl	37.4	117.7	-0.3
10μl	37.2	118.6	0.4
				20μl	37.0	119.4	1.1

Table 3:determination of the volume tolerance of the test strips in variant 3

Sample volume	Measured relative reflectance [% ]]	Concentration calculated according to calibration Curve 3	Deviation of reflectance value [% ]]
				3μl	33.4	120.2	1.5
5μl	34.0	117.7	-0.6
				8μl	33.9	118.0	-0.3
10μl	34.1	117.0	-1.2
				20μl	33.8	118.5	0.1

Table 4:determination of the volume tolerance of the test strips in variant 4

Sample volume	Measured relative reflectance [% ]]	Concentration calculated according to calibration Curve 4	Deviation of reflectance value [% ]]
				3μl	35.2	119.2	0.8
5μl	35.3	118.7	0.3
				8μl	35.6	117.3	-0.8
10μl	35.6	117.4	-0.8
				20μl	35.4	118.0	-0.3

C. As can be seen in the table, the test carriers of the present invention are largely volume independent.

Claims (14)

1. A diagnostic test carrier (1) comprising a support layer (2), a detection layer (3) containing reagents required for the determination of an analyte in a liquid sample on the support layer, and a web (4) covering the detection layer (3), the web (4) being larger than the detection layer (3) and being adhered to the support layer (2), wherein the network (4) is hydrophilic but not capillary active per se, and a cover (5) made of a material impermeable to the sample is placed over the area (6) of the network extending beyond the detection layer, such that the spots (7) on the area of the web (4) overlying the detection layer remain free, the web (4) creating capillary-active voids between the cover (5) and the detection layer (3) and between the cover (5) and the support layer (2) or between the cover (5) and the pad (10) on the support layer (2).

2. The diagnostic test carrier according to claim 1, wherein several detection layers are placed one after the other on the support layer.

3. A diagnostic test carrier according to claim 1 or 2, wherein the support layer is perforated and the detection layer is placed on the perforations.

4. A diagnostic test carrier according to claim 3, wherein the spots are located on perforations in the support layer.

5. A diagnostic test carrier according to claim 3, wherein the spots are not located on the perforations in the support layer.

6. A diagnostic test carrier according to claim 3, wherein the support layer comprises a plurality of apertures as perforations over which one or more detection layers are placed.

7. A diagnostic test carrier according to claim 2, wherein the support layer comprises several wells as perforations, on each of which a different detection layer is placed.

8. A diagnostic test carrier according to claim 1, wherein the support layer comprises a well over which the detection layer comprising several adjacent reaction zones is placed.

9. The diagnostic test carrier according to one of claims 7 or 8, wherein the spots are located on several or all detection layers or reaction zones in each case.

10. The diagnostic test carrier according to one of claims 7 or 8, wherein the spotting is located only on one of the detection layers or one of the reaction zones.

11. The diagnostic test carrier according to claim 1, wherein the mesh is a monofilament fabric.

12. A diagnostic test support according to claim 1, wherein the web is attached to the support layer by adhesive tape.

13. The diagnostic test carrier according to claim 12, wherein the adhesive tape comprises natural or synthetic rubber.

14. Use of a diagnostic test carrier according to claim 1 in the determination of an analyte in a liquid sample, wherein sample liquid is applied to a sample application site, excess liquid not absorbed by the detection layer and the areas of the network (4) located thereon is directed to the areas of the network extending beyond the detection layer, and the signal generated at the detection layer is observed and used to determine the presence or amount of analyte in the liquid sample to be determined.