Rapid measurement of analyte contents

Abstract

The invention relates to a method for measuring the content, in particular the concentration of an analyte in a sample, and to the device for carrying out the method. The analyte is in particular a substrate or an enzyme inhibitor.

Description

[0001] Rapid determination of analyte levels

[0002] The invention relates to an enzyme-based method for determining the content, in particular the concentration, of an analyte in a sample, as well as a device for carrying out the method. The analyte is in particular a substrate or an inhibitor of an enzyme.

[0003] The invention is suitable for the analysis of samples, especially biological samples, especially human blood samples. It represents a novel principle for in vitro diagnostics, especially for rapid point-of-care tests. The method is characterized by the fact that the working enzyme is immobilized by binding to a specific ligand and displaced by the analyte. After washing out the matrix, the amount of analyte can be determined based on the activity of the remaining enzyme.

[0004] Technical background

[0005] One method used to purify enzymes is affinity chromatography. A protein or enzyme-binding molecule (a specific ligand) is covalently bound to a support material. This molecule can bind to the appropriate protein or enzyme from a sample and thereby immobilize it. After washing out non-binding substances, the enzyme can be detached from the support using an excess of soluble ligand. Dialysis or ultrafiltration removes the ligand, and the affinity-purified enzyme can be quantitatively determined and used further. Example: Dehydrogenases bind to NADH-Sepharose or Cibacron Blue-Sepharose and are released again by NADH (Mislovicovä, Danica; Gemeiner, Peter; Stratilovä, Eva; Horväthova, Marta (1990): Competitive elution of lactate dehydrogenase from Cibacron Blue — bead cellulose with Cibacron Blue — dextrans. In: Journal of Chromatography A 510, pp. 197-204). The technique is shown in Figure 1.

[0006] One method used to detect specific ligands is based on competitive immunoassays. A ligand-containing sample is mixed with an excess of a labeled antibody so that a portion of the antibody becomes saturated with ligand. If this mixture is brought into contact with the support-bound ligand, the remaining free antibodies bind to the bound ligand. After a washing step to remove non-immobilized antibody, the antibody remaining on the support can be detected due to its labeling. The more ligand present in the sample, the less label is present on the support. Figure 2 illustrates this technique. Plate-based assays—especially immunoassays—require time-consuming incubation and washing steps and are not suitable for use in the operating room.Calibrators must also be included in the same assay, so the effort is only justifiable for a large number of samples collected over a longer period of time.

[0007] For targeted treatment of bleeding complications, rapid determination of the levels, especially the concentration, of coagulation factors or anticoagulants is essential. This allows the cause of severe bleeding to be eliminated by administering factor concentrates or neutralizing the anticoagulant, preventing life-threatening blood loss and complications during wound healing. Fibrinogen (coagulation factor I) and direct oral anticoagulants (DOACs) are particularly relevant in this context.

[0008] Tests for many of the coagulation factors and anticoagulants are often performed in the clinical laboratory using enzyme-linked immunosorbent assays (ELISAs), which require time-consuming calibration and incubation steps.

[0009] The Clauss test, used for the coagulation factor fibrinogen, is also time-consuming. The Clauss turbidity test is standard for fibrinogen determinations. Faster tests, such as the viscoelastic method, require complex specialized equipment and only provide surrogate values ​​for fibrinogen concentration. Fibrinogen can also be determined using thromboelastometry, but the test is not accurate at low concentrations and is influenced by other blood components. Fibrinogen concentration also influences the flow distance in a simple paper assay (Saidykhan, Jerro; Selevic, Laura; Cinti, Stefano; May, Jennifer E.; Killard, Anthony J. (2021): Paper-Based Lateral Flow Device for the Sustainable Measurement of Human Plasma Fibrinogen in Low-Resource Settings. In: Analytical Chemistry 93 (41), pp. 14007–14013). Here, the viscosity of the sample plays a crucial role and influences from many other sample components must be taken into account.For further methods, see also Bialkower, Marek; Garnier, Gil (2022): Fibrinogen Diagnostics in Major Hemorrhage. In: Critical reviews in analytical chemistry 52 (1), pp. 194-209.

[0010] Thrombelastic methods require more sophisticated equipment and specially trained personnel. They provide surrogate values ​​that can be used as a guide, but often do not agree with the reference methods in the low fibrinogen concentration range.

[0011] Liquid chromatography combined with mass spectrometry (LC / MS) can be used to test for direct oral anticoagulants (DOACs). This method is the gold standard for direct concentration determination but is usually unavailable in hospitals. Recently, the ecarin clotting time can also be determined thromboelastometrically (ClotPro™). Meizothrombin, formed from prothrombin by ecarin, cleaves fibrinogen significantly more slowly in the presence of DOACs (Calatzis, A.; Wittwer, M.; Leyser, H.; Hipp, Q.; Spannagl, M. (2018): ClotPro — a new generation viscoelastic whole blood coagulation analyzer. In: Presented at the 62nd Annual Meeting of the Society of Thrombosis and Hemostasis Research (GTH), 2018). However, a normal coagulation test with physiologically cleaved thrombin is hardly affected by DOACs.Similar to the determination of fibrinogen concentration over the flow path in a paper LFT is the determination of DOACs in a microfluidic device (Frydman, Galit H.; Ellett, Felix; van Cott, Elizabeth M.; Hayden, Douglas; Majmudar, Maulik; Vanderburg, Charles R. et al. (2019): A New Test for the Detection of Direct Oral Anticoagulants (Rivaroxaban and Apixaban) in the Emergency Room Setting. In: Critical care explorations 1 (8)). Here, too, the sample runs and is stopped by clotting. The flow path is influenced by anticoagulants, so that the amount of DOACs can be determined by comparison with reference channels.

[0012] Point-of-care tests (PCTs) that provide accurate results within a few minutes with minimal effort, on the basis of which a therapeutic decision can be made, would be desirable here.

[0013] Summary of Revelation

[0014] The invention relates to an enzyme-based method for determining the content, in particular the concentration, of an analyte in a sample, as well as a device for carrying out the method. The invention particularly relates to a method for detecting soluble analytes that are enzyme inhibitors or substrates.

[0015] In some embodiments, the immobilized specific ligand can, for example, be one of two substrates of the enzyme, provided the enzyme requires two substrates. A single one of these substrates will then not be converted by the enzyme, but it can immobilize the enzyme if it is itself anchored to the surface. In such embodiments, the analyte therefore represents, in particular, the other of the two enzyme substrates. Only when the analyte and the specific ligand are present does the enzyme become mobilized. For enzymes that require a cosubstrate, the analyte can be either the cosubstrate or the substrate. The analyte does not necessarily have to be the other substrate.

[0016] The analyte can also be the sole substrate. In such embodiments, the specific ligand is, in particular, an inhibitor of the enzyme. This can prevent the enzyme from being released by reaction with the specific ligand. Both types of embodiments are therefore possible. If the analyte binds to the same site as the ligand, competition occurs, resulting in enzyme detachment. If the analyte is the second substrate, a reaction occurs, and the enzyme and products dissociate. In both pathways, the enzyme is mobilized.

[0017] Enzymes can be immobilized, in particular, by binding to specific ligands that bind to the active site or another specific substrate binding site. The inventors utilize this fact for the quantitative determination of analytes (particularly enzyme inhibitors or substrates). If a sample containing an analyte is added, the analyte binds to the active site and undergoes conversion, while the enzyme is displaced from the ligand binding site. When the analyte binds to another specific substrate binding site, the enzyme is also displaced from the ligand binding site. In both cases, the amount of displaced enzyme is inversely proportional to the amount of analyte in the sample. After washing out interfering components, for example, a signaling substrate is added to the immobilized enzyme. The signal intensity can be used to determine the amount of remaining enzyme and thus the amount of analyte.An embodiment of the method according to the invention is shown schematically in Figure 3.

[0018] The method differs from the state of the art in particular in that it involves a rapid competitive equilibrium (in contrast to immunoassays) and that this competition between support-bound specific ligand and analyte is exploited for the quantitative determination of the soluble substrate or inhibitor (instead of complete detachment from the support in affinity chromatography). In contrast to antibody binding with its high affinities with K values ​​in the low nanomolar range and below, the dissociation constants for enzyme-support-bound ligand are typically in the pM range; therefore, the dissociation rate constants are correspondingly higher. Thus, upon contact in the range of seconds to minutes, equilibrium can be established so that the support-bound enzyme is mobilized, i.e., transferred into the solution.

[0019] With antibody bindings that bind much more strongly and therefore dissociate more slowly, equilibration would require far too long a time, as documented by the long incubation times of immunoassays. Therefore, in a competitive immunoassay, the antibody must first be added to the sample, and only after the free ligand has bound is the mixture added to the carrier-bound ligand.

[0020] Another important difference is that in immunoassays, the antibody must be used in excess of the analyte. To determine higher concentrations of analytes, a pre-dilution step is required. This can be omitted in the method according to the invention because the equilibration of the carrier-bound enzyme / analyte-bound enzyme is controlled by the dissociation constants and not by the absolute amounts. Thus, it also works with a deficiency of enzyme.

[0021] The test principle described by the inventors can be carried out in a cuvette or microtiter plate format. However, it is also suitable for implementation in a lateral flow test (LFT) on a porous matrix. In both cases, immobilized specific ligand with bound enzyme is provided to the user on a suitable surface. The assay then proceeds in three steps: sample application, washing, and substrate addition. The LFT format is particularly advantageous here because the reagent solutions are transported through the reactive zone containing the immobilized enzyme by capillary forces of the porous matrix. Unlike with a cuvette or microtiter plate, the removal of this solution does not represent a separate step but simply occurs by applying the washing solution. Various porous matrices can be used to perform the assay in the LFT format.

[0022] When using paper and other cellulose-based materials, fusion proteins from known carbohydrate binding modules (CBMs) are suitable for immobilizing peptide-based enzyme ligands in the matrix of the reactive field. They can also be used to passivate the paper and prevent the nonspecific binding of proteins and other sample components. Oligosaccharides such as dextran or proteins that adsorb nonspecifically to surfaces, such as serum albumin, but render them inaccessible to other proteins, are suitable for passivating cuvettes or microtiter plates.

[0023] Brief description of the drawings

[0024] Figure 1 shows schematically the functional principle of affinity chromatography.

[0025] Figure 2 shows schematically the functional principle of the competitive immunoassay.

[0026] Figure 3 shows schematically the functional principle of an embodiment of the method according to the invention.

[0027] Figure 4 shows schematically the structure of a device for carrying out the method as a lateral flow test and reading out the generated signal.

[0028] Figure 5 shows the result of the NADH determination according to Example 1. Figure 6 shows the result of the fluorescence anisotropy measurement to determine the affinity of the peptide T-FX1 to FXa.

[0029] Figure 7 shows the determination of the IC50 of B-FX1 based on a plot of the initial velocity versus the peptide concentration.

[0030] Figure 8 shows the relationship between conversion of the substrate CS-11(22) and the amount of FXa used after FXa was previously immobilized via BFX1 peptide to an avidin-coated microtiter plate.

[0031] Figure 9 shows the effect of rivaroxaban on the activity of immobilized FXa.

[0032] Detailed description of the revelation

[0033] The invention particularly relates to a method for determining the content, in particular the concentration of an analyte in a sample, the method comprising the following steps: a) bringing the sample into contact with an enzyme immobilized by binding to an immobilized specific ligand, so that an equilibrium is established between binding of the enzyme to the specific ligand and binding of the enzyme to the analyte based on competition between the specific ligand and the analyte for binding to the enzyme, b) removing the sample and the portion of the enzyme mobilized by binding to the analyte from the non-mobilized portion of the enzyme, and c) determining the content, in particular the concentration of the analyte in the sample based on the enzymatic activity of the non-mobilized portion of the enzyme.

[0034] In particular, the steps are carried out in the specified order a) to c).

[0035] In some embodiments, the method may comprise, as a further step prior to step a), the step of providing the enzyme immobilized by binding to the immobilized ligand. As an even further prior step, the method may also comprise, in some embodiments, the step of providing the immobilized ligand.

[0036] "Specific ligands" are those that share the same binding site on the enzyme as the analyte. This ensures that only the analyte, and not other sample components, can displace the working enzyme.

[0037] "Enzymes" in the context of this invention are biocatalysts. They accelerate biochemical reactions by reducing the activation energy that must be overcome for a substance to be converted. Most enzymes are proteins.

[0038] A protein is a biological macromolecule composed of amino acids linked by peptide bonds. Macromolecules within the meaning of the invention are, in particular, molecules with a molar mass of at least 10 kDa.

[0039] In many embodiments of the invention, the enzyme is a protein. In some embodiments, the enzyme may be a ribozyme, particularly RNA- or DNA-based.

[0040] The method of the present invention is a method for determining the content, in particular the concentration, of an analyte in a sample. In some embodiments, the sample is a biological sample, in particular a blood sample, for example, a human blood sample. In other embodiments, the sample can also be a tissue sample, for example, a tissue extract.

[0041] The concentration of the analyte in the sample is preferably in a range determined by the K values ​​of the enzyme-support-bound ligand and enzyme-analyte bonds. It is advisable to choose a ligand that binds just well enough that only a small amount of enzyme is removed by washing. If the analyte binds significantly more strongly, then the method is particularly well suited for analyte concentrations in the range of 0.5 to 10 times the K value of the enzyme-ligand bond. If the ligand and analyte bind similarly well, then the preferred concentration shifts upwards, e.g., to the range of 10 to 100 times the K value of the enzyme-ligand bond. In some embodiments, the concentration of the analyte in the sample is, for example, in a range of 1 to 10 times the K value of the enzyme-ligand bond.For example, in some embodiments, the concentration of the analyte in the sample is in a range of 10 to 100 times the K value of the enzyme-ligand binding.

[0042] In some embodiments, the concentration of the analyte in the sample is, for example, in a range from 0 to 6 mg / ml, in particular from 0 to 4.5 mg / ml or from 0 to 4.0 mg / ml, for example in a range from 0.1 mg / ml to 3.0 mg / ml, from 0.2 mg / ml to 2.0 mg / ml or from 0.5 mg / ml to 1.5 mg / ml. In some embodiments, the concentration of the analyte in the sample is, for example, at most 6 mg / ml, in particular at most 4.5 mg / ml, at most 4.0 mg / ml, at most 3.0 mg / ml, at most 2.0 mg / ml or at most 1.5 mg / ml. In some embodiments, the concentration of the analyte in the sample is, for example, at least 0.1 mg / ml, at least 0.2 mg / ml or at least 0.5 mg / ml. The embodiments mentioned in this paragraph may in particular be embodiments in which the analyte is fibrinogen.

[0043] In some embodiments, the concentration of the analyte in the sample is, for example, in a range from 0 to 500 ng / ml, in particular from 10 to 400 ng / ml, from 15 to 300 ng / ml, from 20 to 250 ng / ml, from 30 to 200 ng / ml, or from 50 to 100 ng / ml. In some embodiments, the concentration of the analyte in the sample is, for example, at least 10 ng / ml, at least 15 ng / ml, at least 20 ng / ml, at least 30 ng / ml, or at least 50 ng / ml. The embodiments mentioned in this paragraph can in particular be embodiments in which the analyte is a DOAC.

[0044] The analyte can be a substrate or an inhibitor of the enzyme. A "substrate" is a substance that is converted in an enzymatically controlled reaction. An "inhibitor" is an inhibitor of an enzymatic reaction that reduces the rate of the catalyzed reaction.

[0045] In particular, the analyte is water-soluble. In particular, the analyte has a water solubility of greater than 10 pM, especially at a temperature of 20°C. In some embodiments, the analyte has a water solubility of at least 1 pM, especially at a temperature of 20°C.

[0046] According to step a) of the method according to the invention, the sample is brought into contact with an enzyme immobilized by binding to an immobilized ligand, so that an equilibrium is established between binding of the enzyme to the ligand and binding of the enzyme to the analyte based on competition between the ligand and the analyte for binding to the enzyme.

[0047] The binding of the enzyme to the ligand is particularly non-covalent. Due to the competition between the ligand and the analyte for binding to the enzyme, a portion of the enzyme is mobilized. This portion is transferred into the sample solution upon binding to the analyte.

[0048] The method according to the invention is primarily based on the competition between the ligand and the analyte for binding to the enzyme, in particular to the enzyme's active site or to exosites or allosteric binding sites of non-competitive inhibitors. For example, fibrinogen binds to two so-called "exosites" in addition to the active site of thrombin. The analyte can also be an allosteric inhibitor, which by definition binds outside the active site.

[0049] The analyte, the enzyme, and the immobilized specific ligand are thus coordinated with one another and thus form a unit to be described together. In some embodiments, enzymes can be immobilized, in particular, by binding to specific ligands that bind to the active site of the enzyme. In some embodiments, enzymes can be immobilized, in particular, by binding to specific ligands that bind to a specific substrate binding site other than the active site of the enzyme, for example, to exosites or to allosteric binding sites of non-competitive inhibitors.

[0050] To enable particularly short incubation times, it is advantageous if the dissociation constant KD of the enzyme-ligand bond is in the pM range.

[0051] The immobilized specific ligand binds, in particular, to the enzyme without being converted. In some embodiments of the invention, the ligand binds to the active site of the enzyme. The ligand can, in particular, be an inhibitor of the enzyme. In some embodiments, the ligand binds to an exosite or an allosteric binding site.

[0052] In some embodiments, particularly embodiments in which the analyte is a DOAC, the ligand is a derivative of the respective DOAC with reduced affinity for the respective working enzyme.

[0053] In some embodiments, particularly in embodiments where the enzyme is Factor Xa, the ligand is selected from the group consisting of Tyr-Chg-Arg-NH2(FX1), Ac-Tyr-Chg-Arg-Pip-NH2, Ac-Trp-Chg-Arg-Leu-Pro-NH2(SEQ ID NO:14) and Tyr-Ile-Arg-Leu-Pro-NH2(SEQ ID:15) (Ac: Acetyl, Arg: Arginine, Chg: Cyclohexylglycine, Glu: Glutamate, Gly: Glycine, Ile: Isoleucine, Leu: Leucine, Pip: Piperidine-2-carboxylic acid, Pro: Proline, Trp: Tryptophan, Tyr: Tyrosine).

[0054] In some embodiments, the enzyme is a hydrolase, particularly a protease, for example, an endoprotease. In some embodiments, the enzyme is a protease and the ligand is a non-convertible protease substrate, a small molecule inhibitor, or a peptidomimetic that binds to the active site or an exosite.

[0055] In some embodiments, the enzyme is Factor Xa (Stuart-Prower factor, thrombokinase) or a Factor X-like prothrombin activator from snake venom. In some embodiments, the enzyme is thrombin (Factor Ha) or a thrombin-like enzyme from snake venom (SVTLE), for example, batroxobin from Bothrops atrox or ancrod from Calloselasma rhodostoma.

[0056] In principle, the method of the present invention is suitable for determining the content, in particular the concentration, of any analyte that binds to an enzyme and competes with a ligand for this binding. Of particular clinical relevance is the rapid determination of the content, in particular the concentration, of analytes relevant to blood coagulation. Fibrinogen and direct oral anticoagulants (DOACs) are particularly relevant in this context.

[0057] In some embodiments, the analyte is a coagulation factor converted by thrombin, in particular fibrinogen, or a thrombin inhibitor, in particular dabigatran or dabigatran etelixate. In some embodiments, the analyte is a coagulation factor converted by Factor Xa or a Factor Xa inhibitor, in particular apixaban, edoxaban, or rivaroxaban. In some embodiments, the analyte is rivaroxaban.

[0058] In some embodiments of the invention, the analyte is fibrinogen or a direct oral anticoagulant. In some embodiments of the invention, the analyte is fibrinogen. In some embodiments of the invention, the analyte is a direct oral anticoagulant, in particular a thrombin inhibitor DOAC (e.g., dabigatran or dabigatran etelixate) or a Factor Xa inhibitor DOAC (e.g., apixaban, edoxaban, or rivaroxaban).

[0059] In some embodiments, the analyte is fibrinogen. In such embodiments, the immobilized ligand can be selected, in particular, from the group consisting of DOAC derivatives with poorer affinity, peptides from the fibrinogen sequence, or peptides from protein-based thrombin inhibitors, each of which binds to the active site and / or the exosites, as well as peptidomimetics derived therefrom. Fibrin-cleaving enzymes are suitable for fibrinogen detection, preferably enzymes with a low KM value (e.g., 0.1 to 5.0 pM), such as snake venom proteases, preferably batroxobin from Bothrops atrox (also known as reptilase) or ancrod from Calloselasma rhodostoma (commercially available as Viprinex). They are known to be insensitive to many thrombin inhibitors.In embodiments where the analyte is fibrinogen, the enzyme may be a snake venom enzyme, for example, a snake venom protease, in particular a snake venom thrombin-like enzyme (SVTLE). In embodiments where the analyte is fibrinogen, the enzyme may in particular be batroxobin from Bothrops atrox (also known as reptilase) or ancrod from Calloselasma rhodostoma (in particular, commercially available as Viprinex). In some embodiments where the analyte is fibrinogen, the enzyme is thrombin.

[0060] In some embodiments, the analyte is a direct oral anticoagulant (DOAC). Activated thrombin is suitable for the detection of DOACs that were developed as direct thrombin inhibitors. Activation by ecarin to meizothrombin is preferred because this enzyme form exhibits greater sensitivity to the DOACs (Steiner, T.; Böhm, M.; Dichgans, M.; Diener, HC; Ell, C.; Endres, M. et al. (2013): Recommendations for the emergency management of complications associated with the new direct oral anticoagulants (DOACs), apixaban, dabigatran and rivaroxaban. In: Clinical research in cardiology : official journal of the German Cardiac Society 102 (6), pp. 399-412). The active thrombin or meizothrombin is bound to the carrier-bound ligand. Activation of the body's own prothrombin should be avoided; It would bind the DOACs and thus falsify the test result. The inhibitor on the carrier and, if applicable,The DOAC in the sample ensures that blood clotting is not triggered by the active thrombin or meizothrombin in the reaction zone. As a further safety measure, relatively low activities of carrier-bound enzyme can be used so that only a small fraction of the substrate can be converted in the test time. This also applies to embodiments in which fibrinogen is the analyte. To prevent fibrin aggregation, the tetrapeptide GPRP (SEQ ID NO:1) can be added (Michelson, AD (1994): Platelet activation by thrombin can be directly measured in whole blood through the use of the peptide GPRP and flow cytometry: methods and clinical applications. In: Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis 5 (1), pp. 121-131).

[0061] In embodiments where the analyte is a thrombin inhibitor DOAC, the enzyme can be, for example, thrombin or meizothrombin. In such embodiments, the ligand is preferably selected from the group consisting of DOAC derivatives with reduced activity and peptides or peptidomimetics that specifically bind to the active site.

[0062] In some embodiments, the enzyme is thrombin, meizothrombin, batroxobin from Bothrops atrox, or ancrod from Calloselasma rhodostoma, and the analyte is fibrinogen, dabigatran, or dabigatran etelixate.

[0063] For the detection of DOACs that inhibit Factor Xa (thrombokinase, Stuart-Prower factor), in particular apixaban or rivaroxaban, activated Factor X or a Factor X homologous or similar prothrombin activator from snake venom, such as trocarin from Tropidechis carinatus, in carrier-bound form is suitable. In embodiments in which the analyte is a Factor Xa inhibitor DOAC, the enzyme can be, for example, activated Factor X or a Factor X homologous or similar prothrombin activator from snake venom, such as trocarin from Tropidechis carinatus. The terms "Factor Xa," ​​"FXa," and "activated Factor X" are used synonymously.

[0064] In some embodiments, the enzyme is Factor Xa or Trocarin from Tropidechis carinatus and the analyte is apixaban, edoxaban, or rivaroxaban.

[0065] If interfering interactions exist between different analytes, such as fibrinogen and DOACs, two tests (in particular meizothrombin for the detection of DOACs with a low-affinity ligand and batroxobin with a slightly higher affinity ligand for fibrinogen detection) can be performed in parallel and the results can be mutually corrected using an empirically determined algorithm.

[0066] There is no interfering interaction with fibrinogen when detecting FXa inhibitors.

[0067] According to step a) of the method according to the invention, the enzyme is immobilized by binding to an immobilized ligand. The ligand is thus immobilized. "Immobilized" means, in particular, that the ligand is not free in solution. The ligand is, in particular, bound to a support. The binding of the ligand to the support is, in particular, covalent. In some embodiments, the binding of the ligand to the support is a non-covalent binding of biotin-conjugated ligand to surface-bound streptavidin, avidin, or another high-affinity biotin-binding molecule.

[0068] Various materials and topologies can be used as supports. The most important factor is that the support enables the immobilization of the ligand. The immobilization of the ligand serves to enable separation between the mobilized portion of the enzyme and the non-mobilized portion of the enzyme, allowing the enzymatic activity of the non-mobilized portion of the enzyme to be determined. Based on this, the content, particularly the concentration, of the analyte in the sample can be determined. The support can be, for example, a cuvette or a microtiter plate or a porous matrix (e.g., a nonwoven fabric made of paper, polymer, or glass fibers).

[0069] According to step a) of the method according to the invention, an equilibrium is established between the binding of the enzyme to the ligand and the binding of the enzyme to the analyte based on the competition between the ligand and the analyte for binding to the enzyme. Such an equilibrium can also be referred to as a competitive equilibrium. At the beginning, i.e. at the moment the sample is added, the proportion of enzyme bound to the analyte is zero. This is followed by increasing mobilization of the enzyme through binding to the analyte. Equilibrium is reached when the rates of the forward and reverse reactions balance each other out, so that the mobilized proportion of the enzyme and the non-mobilized proportion of the enzyme are constant. How the two proportions are distributed in equilibrium depends on the concentration of the analyte in the sample.

[0070] It is a particular advantage of the present invention that equilibrium is established very quickly, in particular within a period of 1 second to 5 minutes, for example, from 1 second to 1 minute or from 10 seconds to 1 minute. Step a) of the method according to the invention can comprise an incubation step. The incubation time is in particular in a range from 1 second to 5 minutes, for example, from 1 second to 1 minute or from 10 seconds to 1 minute. The incubation time can, for example, be at least 1 second or at least 10 seconds. The incubation time can, for example, be at most 5 minutes or at most 1 minute.

[0071] After equilibrium is established, the two fractions (the mobilized and the non-mobilized fraction of the enzyme) must be separated from each other so that the enzymatic activity of the non-mobilized fraction can be determined. Therefore, the method according to the invention comprises step b) of removing the sample and the fraction of the enzyme mobilized by binding to the analyte from the non-mobilized fraction of the enzyme. Step b) comprises, in particular, one or more washing steps.

[0072] The number of washing steps is preferably in the range of 1 to 3. The details of sample removal and the portion of the enzyme mobilized by binding to the analyte depend essentially on the type of ligand immobilization.

[0073] If the immobilized ligand is present as a suspension, particularly in a reaction vessel, removal can be achieved, for example, by performing one or more washing steps, wherein each washing step comprises separating the suspension into solid and liquid components, removing the liquid components, and adding a washing solution, particularly a washing buffer. The separation of the suspension into solid and liquid components can be achieved, in particular, by centrifugation. Centrifugation for 0.5 to 2 minutes is preferred, with the required acceleration depending on the type of support.

[0074] In some embodiments, the ligand is immobilized in a microtiter plate or cuvette. In such embodiments, centrifugation is omitted. Instead, the solutions are actively removed.

[0075] If, however, the ligand is immobilized on a solid, such as the paper in a test strip, removal of the sample and the portion of the enzyme mobilized by binding to the analyte can be achieved, for example, by performing one or more washing steps, with each washing step comprising the addition of a washing solution to the solid, in particular to the test strip. The washing solution is preferably a washing buffer.

[0076] The washing solution can, for example, be applied to the solid at the same location where the ligand is immobilized. However, it is also possible to apply the washing solution to a location where the ligand is not immobilized. This may be a preferred option, especially if the solid, in particular the test strip, is designed in such a way that the washing solution can migrate through the solid, for example, through capillary forces, and thus be brought into contact with the immobilized ligand. This can ensure that components not bound to the ligand are removed.

[0077] The washing solution or washing buffer preferably has an approximately isotonic composition, e.g., 100 mM NaCl and 50 mM HEPES. The pH is preferably in a range from 6.0 to 9.0, more preferably from 7.0 to 8.0. The pH is preferably at least 6.0 or at least 7.0. The pH is preferably at most 9.0 or at most 8.0. The washing solution or washing buffer can, for example, also be PBS (phosphate-buffered saline), in particular 137 mM NaCl, 2.7 mM KCl, and 12 mM phosphate (HPO4 2- and H2PO4'), pH 7.4. The washing solution or the washing buffer may contain one or more biocides, for example NaNs (sodium azide), preferably in concentrations of 0.01 to 1.0 mM or of 0.05 to 0.5 mM, for example about 0.1 mM.

[0078] According to step c) of the method according to the invention, the content, in particular the concentration, of the analyte in the sample is determined based on the enzymatic activity of the non-mobilized portion of the enzyme. The determination is carried out in particular using a signal generated by the conversion of a detection substrate (catalyzed by the enzyme). The detection substrate can, for example, have a chromogenic or a fluorogenic group. In some embodiments of the invention, the signal is determined optically. For example, the signal can be determined using photometry. The signal can, in particular, be determined using fluorimetry. The details of the signal determination depend in particular on the fluorophore used.

[0079] A fluorimeter is suitable for detecting the generated signal, for example, one with LED illumination (e.g., 365 nm for substrates that release aminocoumarin) and a long-pass cutoff filter (e.g., 400 nm) in front of the detector. Alternatively, a reflectance photometer can be used to measure released p-nitrophenol at 405 nm. Suitable LEDs are available. In this case, the cutoff filter would be omitted. Figure 4 shows a schematic of the LFT and fluorimeter setup.

[0080] As an alternative to this optic, a cell phone camera or a device with a corresponding detector chip can be used. Blue and green pixels, with their sensitivities of 400-500 and 500-600 nm, are suitable. The ratio of blue to green intensities can also be evaluated. Blue pixels are suitable for measuring the reflectance of nitrophenol.

[0081] Thermostatic control is preferred because enzyme activity is temperature-dependent. Therefore, the measurement should be performed at the same temperature as the calibration curve. If the exact measurement temperature is known, a small deviation (e.g., 1-2 degrees) from the calibration temperature can also be compensated for mathematically.

[0082] Power supply, evaluation electronics, display instruments, interface for further processing of the results, etc. are known from the state of the art.

[0083] The optics can optionally measure fluorescence and / or absorbance during sample application and the subsequent steps to verify correct dosages and timing. A reflectance measurement to detect any incompletely flushed hemoglobin is also an option for result correction.

[0084] Since other sample components are washed out, nonspecific detection substrates are sufficient for the enzymes. At the same time, the detection substrates should have a KM value in the micromolar range or below to effectively compete with the binding of the enzyme to the immobilized ligand and mobilize the enzyme. Thus, the non-mobilized portion of the enzyme is mobilized to generate the signal. This mobilization only occurs after the sample and the portion of the enzyme mobilized by binding to the analyte have been removed.

[0085] The detection substrates can be, for example, tripeptides that carry a chromogenic group such as para-nitroaniline (pNA) or para-nitrophenol or a fluorogenic group such as aminomethylcoumarin (AMC) or aminotrifluoromethylcoumarin on the C-terminal side of the cleavage site. The commercially available substrates S-2222 (Bz-Ile-Glu(-OCH3)-Gly-Arg-pNA*HCl) (SEQ ID NO:16) and S2765 (ZD-Arg-Gly-Arg-pNA*HCl) are particularly suitable for factor Xa, while the substrate S2238 (HD-Phe-Pip-Arg-pNA*HCl) or Z-Gly-Pro-Arg-AMC are suitable for thrombin. Peptide derivatives derived from the respective cleavage site are suitable for the enzymes from snake venom. To achieve lower KM values, the peptide sequence can be extended towards the N-terminus.An extension to the C-terminus is also possible if both termini are equipped with a fluorophore that enables Förster resonance energy transfer (FRET), which is no longer possible after cleavage of the substrate, so that the fluorescence of the shorter-wavelength fluorophore is detected, or if one terminus carries a fluorophore and the other a suitable group to the fluorescence solution, so that the substrate itself does not fluoresce, but one of the cleavage products does.

[0086] In some embodiments, particularly in embodiments where the enzyme is Factor Xa, the detection substrate is selected from a peptide according to SEQ ID NO: 16, a peptide according to SEQ ID NO: 17, and mixtures thereof. In some embodiments, particularly in embodiments where the enzyme is Factor Xa, the detection substrate is selected from Bz-Ile-Glu(γOCH3)-Gly-Arg-pNA (SEQ ID NO: 16), Bz-Ile-Glu-Gly-Arg-pNA (SEQ ID NO: 17), and mixtures thereof.

[0087] The signal can, in particular, be proportional to the rate of the enzyme-catalyzed conversion of the detection substrate. The content, for example the concentration of the analyte in the sample, can be determined, in particular, via the temporal change of the signal, for example, via the initial slope of the signal, i.e., the slope of an initially linear increase. The content, in particular the concentration of the analyte in the sample, can be determined, for example, using a calibration curve. In a calibration curve, for example, the signal (or its temporal change) or the enzymatic activity determined from it can be plotted at known contents (in particular concentrations) of the analyte.If the signal (or its temporal change) or the enzymatic activity of the non-mobilized portion of the enzyme is determined using the method according to the invention, the corresponding content (in particular the corresponding concentration) of the analyte can be read from the calibration curve.

[0088] In some embodiments, the analyte concentration can be expressed directly in mg / ml using a calibration curve. However, such a competition test cannot differentiate between thrombin-inhibiting DOAC variants. The test result (the level) can therefore be expressed as thrombin inhibition (regardless of the drug). Eliminating this inhibition is the actual goal of treatment, and thus the extent of inhibition is the relevant value. The user can also select the specific medication on the associated device (e.g., dabigatran or argatroban) and obtain the appropriate concentration, e.g., in ng / ml. Conversion to ecarin coagulation times would also be possible using a corresponding comparison calibration. In the case of FXa inhibitors, the enzyme inhibition can also be expressed as a percentage of normal activity or, depending on the drug (rivaroxaban, edoxaban, or apixaban), as its concentration.

[0089] The invention relates to a method and a device for determining the content of an analyte in a sample. The content of the analyte in the sample is in particular the concentration of the analyte in the sample, for example in mg / ml or ng / ml. In some embodiments, the content of the analyte can also be the enzyme inhibition (for example in % or also as residual activity in % of the normal value), in particular in embodiments in which the analyte is an inhibitor of the enzyme, for example a DOAC. The content of the analyte can therefore be the extent of enzyme inhibition by the analyte. In some embodiments, the invention relates to a method and a device for determining the extent of enzyme inhibition by an analyte in a sample.

[0090] The invention particularly provides an enzyme bound to a carrier via a ligand. Enzymes are typically stabilized by such bonds. Nevertheless, it may occur that the enzyme activity decreases over the storage time of a test. This is either compensated for by a newly generated calibration curve or by using two fields with carrier-ligand-enzyme, with more or better binding ligand being used in one field than in the other (see also EP 3 538664 B1). Benzamidine inhibitors for thrombin and batroxobin with different inhibition constants are known from the literature (Stürzebecher, J.; Vieweg, H.; Wikström, P.; Turk, D.; Bode, W. (1992): Interactions of thrombin with benzamidine-based inhibitors. In: Biological chemistry Hoppe-Seyler 373 (7), pp. 491-496). Thrombin-inhibiting DOACs are related compounds; Here too, there are variants with different dissociation constants.Alternatively, peptide inhibitors can be used, which are adjusted by exchanging amino acids. In the field with stronger binding, the analyte will mobilize less enzyme, while in the other field, more. As described in EP 3 538664 B1, offsetting the activities of both fields can eliminate the unknown enzyme activity, allowing a calibration curve generated during test production to be used.

[0091] In a lateral flow test (LFT), three fields would expediently be arranged one behind the other: application field, reactive field with carrier-bound enzyme, and suction field. If two reactive fields are used, they could be arranged side by side. A serial arrangement would also be possible, whereby the field with the stronger enzyme bond should expediently be flowed through first, as this minimizes the influence of already mobilized enzyme on the subsequent field. The present invention also relates to a device for determining the content, in particular the concentration, of an analyte in a sample, in particular a test strip, for example in the form of a lateral flow test. The device is preferably a device for carrying out the method according to the invention.

[0092] The device comprises at least one reaction zone, preferably exactly one reaction zone.

[0093] The device preferably has three or more zones, in particular three zones. The device preferably comprises at least one application zone, at least one reaction zone, and at least one suction zone. The device may comprise a carrier (for example, made of a carrier material) to which the materials of the various zones are applied.

[0094] The reaction zone preferably contains an immobilized enzyme, in particular in an amount of 0.1 to 1000 pg, for example from 1.0 to 500 pg, from 2.0 to 200 pg, from 5.0 to 100 pg, or from 10 to 50 pg. In some embodiments, the amount can be, for example, at least 0.1 pg, at least 1.0 pg, at least 2.0 pg, at least 5.0 pg, or at least 10 pg. In some embodiments, the amount can be, for example, at most 1000 pg, at most 500 pg, at most 200 pg, at most 100 pg, or at most 50 pg. The amount of enzyme depends on its activity with the signaling substrate. The aim is an accurate determination of the residual enzyme activity in a few minutes, preferably in a maximum of one minute.

[0095] The immobilized enzyme is, in particular, a protein, for example, a protein with a molecular weight of 10 to 300 kDa, 10 to 100 kDa, or 12 to 70 kDa. The molecular weight of the enzyme can, for example, be at least 10 kDa or at least 12 kDa. The molecular weight of the enzyme can, for example, be at most 300 kDa, at most 100 kDa, or at most 70 kDa.

[0096] The enzyme is immobilized by binding to an immobilized ligand. The immobilization of the ligand preferably comprises one or more of the following measures:

[0097] Precipitation and adsorption

[0098] Ionic binding to DEAE- or CM-functionalized surfaces

[0099] Covalent bonding by formaldehyde, glutaraldehyde

[0100] Covalent bonding using EDC / NHS on amine- or COOH-functionalized support surface Covalent bonding using dimethyl suberimidate, dimethyl pimelimidate, dimethyl adipimidate on amine-functionalized support surface

[0101] Covalent bonding through carrier-bound epoxides, isocyanates

[0102] Covalent binding via amino groups to BrCN-activated polysaccharides, non-covalent binding via carbohydrate-binding modules (CBMs) to cellulose.

[0103] - Non-covalent binding of a ligand-biotin conjugate to streptavidin or avidin previously immobilized using one of the methods described above.

[0104] In some embodiments, the immobilization of the ligand comprises binding to cellulose via CBMs. This binding is particularly non-covalent. Carbohydrate Binding Modules (CBMs) are known (for example, CBM of cellobiohydrolase I (Cel7A, CBH1) from Trichoderma reesei or CBM3a from Clostridium thermocellum), which bind non-covalently but quite firmly (in particular, KD in a range of 0.1 to 10 pM, for example, from 0.5 to 10 pM or from 1.0 to 10 pM) to cellulose. They can be connected to other protein molecules via a linker. Such linkers serve as spacers between protein domains so that they do not interfere with each other during folding. In particular, the linkers are peptide linkers. Glycine, serine, or asparagine are frequently used. Examples of linker sequences: GAS: glycine-alanine-serine; Proline-threonine-rich linker (P / TL):

[0105] GSWPSTQPVTTPPATTKPPATTIPPS (SEQ ID NO:2); S3N10: SSSNNNNNNNNNN (SEQ ID NO:3), G10: GGGGGGGGGG (SEQ ID NO:4), GSn: (GSSGSS)n (n=1-9) (SEQ ID NOs:5-13). Die Sequenzen der SEQ ID NOs 5-13 lauten also wie folgt: GS1 : GSSGSS (SEQ ID NO:5), GS2: GSSGSS GSSGSS (SEQ ID NO:6), GS3: GSSGSS GSSGSS GSSGSS (SEQ ID NO:7), GS4: GSSGSS GSSGSS GSSGSS GSSGSS (SEQ ID NO:8), GS5: GSSGSS GSSGSS GSSGSS GSSGSS GSSGSS (SEQ ID NO:9), GS6: GSSGSS GSSGSS GSSGSS GSSGSS GSSGSS GSSGSS (SEQ ID NO: 10), GS7: GSSGSS GSSGSS GSSGSS GSSGSS GSSGSS GSSGSS GSSGSS (SEQ ID NO:11), GS8: GSSGSS GSSGSS GSSGSS GSSGSS GSSGSS GSSGSS GSSGSS GSSGSS (SEQ ID NO:12), GS9: GSSGSS GSSGSS GSSGSS GSSGSS GSSGSS GSSGSS GSSGSS GSSGSS GSSGSS (SEQ ID NO: 13).

[0106] A CBM construct consisting of CBM, linker, and peptide-based ligand can be produced using known methods. If paper is impregnated with a solution of the CBM construct, washed, and dried, the resulting paper contains specific enzyme ligands. Their concentration can be easily adjusted to meet specific requirements by varying the amount of CBM construct. In some embodiments, the immobilized enzyme is immobilized by binding to an immobilized peptide-based ligand. The ligand is linked to a linker via a peptide bond, and the linker is linked to a carbohydrate-binding module via a peptide bond. The linker is bound to a cellulose-containing support.

[0107] In some embodiments, the immobilized enzyme is immobilized by binding to an immobilized ligand, wherein the ligand is conjugated to biotin and linked via a streptavidin fusion protein to a carbohydrate-binding module bound to a support containing cellulose.

[0108] When non-covalently immobilizing enzymes by binding to the immobilized ligand, care must be taken to ensure that the enzymes do not bind non-specifically to the surface of the cuvette or microtiter plate or to the porous matrix. According to the inventors' findings, unstructured peptides and proteins with unstructured regions in particular bind very tightly to cellulose-based materials and are hardly washable under mild conditions. Therefore, when selecting materials, care must be taken to ensure that the surface is not prone to non-specific interactions with the enzyme, or that such interactions can be prevented by chemical modification, for example with oligoethylene glycol or dextran, or by coating with non-specifically binding proteins such as serum albumin or appropriately modified CBMs, e.g., CBP-PEG conjugates (ATssa, Kevin; Karaaslan, Muzaffer A.; Renneckar, Scott; Saddler, Jack N.(2019): Functionalizing Cellulose Nanocrystals with Click Modifiable Carbohydrate-Binding Modules. In: Biomacromolecules 20 (8), pp. 3087-3093).

[0109] When performing the procedure, interference with the measurement of enzymatic activity is not to be expected, since the vast majority of sample components are removed by the washing step.

[0110] The reaction zone is the zone of the device where the immobilized ligand is present. Thus, the immobilized enzyme is also present in the reaction zone. The mobilization of the enzyme, based on competition between the analyte and the ligand for binding to the enzyme, also occurs in the reaction zone.

[0111] The device preferably has exactly one reaction zone. Alternatively, two or more reaction zones may be provided.

[0112] The application zone, the reaction zone, and the suction zone can be made of the same materials or consist of the same materials. However, they can also be made of different materials or consist of different materials. Particularly suitable are all porous materials that allow capillary-driven transport of liquids and substances dissolved or suspended therein. Examples of such materials include nitrocellulose membranes, glass fiber fleeces, paper, filters made of paper or hydrophilized polymer fibers (e.g., polyester, polypropylene), as well as all composites made of the aforementioned materials. The materials can consist of woven or nonwoven fibers or threads, for example, or of open or closed foams.

[0113] Preferably, the device comprises or consists of the following materials: nonwoven webs made of paper, polymer or glass fibers

[0114] The device may have a housing, for example a housing made of plastic, preferably of a thermoplastic material.

[0115] In addition to the materials of the application zone, the reaction zone, and the suction zone, the device may comprise one or more other materials, such as a carrier material. The carrier material may consist, for example, of glass, Plexiglas, a polymer film, or even a coating that acts as a barrier to the liquid used in the device.

[0116] The application zone preferably comprises or consists of the following materials: nonwoven webs made of paper, polymer or glass fibers

[0117] The reaction zone preferably comprises or consists of the following materials: nonwoven webs made of paper, polymer or glass fibers

[0118] The absorption zone preferably comprises or consists of the following materials: paper or nonwoven fabric with an absorption capacity greater than that of the application and reaction zone. This can be achieved by increasing the thickness, length, or width of the fiber material in the absorption zone. Absorption capacity, not geometry, is crucial.

[0119] Preferably, the thickness or length of the suction zone is at least twice the thickness or length of the application zone or the thickness or length of the reaction zone. The application zone and reaction zone preferably have the same or substantially the same thickness. The thickness of the suction zone is preferably in a range from 2 times the thickness of the application zone or the reaction zone to 10 times the thickness of the application zone or the reaction zone.

[0120] Preferably, the absorption capacity of the suction zone is at least twice the absorption capacity of the application zone or the absorption capacity of the reaction zone. The application zone and reaction zone preferably have the same or substantially the same absorption capacity. The absorption capacity of the suction zone is preferably in a range from 2 times the absorption capacity of the application zone or the reaction zone to 10 times the absorption capacity of the application zone or the reaction zone.

[0121] The device can contain an application zone and a reaction zone in a combined application-reaction zone. Preferably, however, the application zone and reaction zone are located at spatially different positions within the device. This does not mean, however, that there is no connection between the application zone and the reaction zone. Quite the opposite: the device comprises at least one connection, preferably exactly one connection, between the application zone and the reaction zone.

[0122] The application zone is the zone of the device in which the sample and / or the washing solution and / or the detection solution are preferably applied. It is possible to provide different application zones, for example, at least one application zone for applying the sample, at least one application zone for applying the washing solution, and at least one application zone for applying the detection solution. However, the application zone of the device is preferably a combined application zone in which the sample, the washing solution, and the detection solution can be applied. The device preferably contains exactly one application zone.

[0123] The connection between the application zone and the reaction zone allows a sample and / or a wash solution or detection solution applied in the application zone to move from the application zone to the reaction zone. Transport from the application zone to the reaction zone can occur primarily through capillary forces.

[0124] The device preferably contains exactly one suction zone. Alternatively, two or more suction zones can be provided. The device preferably contains at least one connection, preferably exactly one connection, between the reaction zone and the suction zone. The suction zone serves in particular to remove the sample and the portion of the enzyme mobilized by binding to the analyte from the reaction zone. Excess washing solution or other solutions can also be removed from the reaction zone with the aid of the suction zone. Transport from the reaction zone to the suction zone can occur in particular by means of capillary forces.

[0125] The device preferably contains exactly two connections, namely a connection between the application zone and the reaction zone and a connection between the reaction zone and the suction zone. The connections are capillary active, thus ensuring uninterrupted liquid transport. If the zones are made of different materials, the connection between the zones can consist of or comprise the material of one of the two zones or of a mixture of the materials. It is also possible for the material of the connection to comprise or consist of another material that is not part of one of the two connected zones. If the zones are designed so that there is no change of material, the connections are preferably made of the same material. For example, the entire device can also be made of a nonwoven fabric, e.g. paper.

[0126] The device is in particular a test strip, for example a lateral flow test.

[0127] The present invention also relates to a system comprising a device of the invention and a washing solution and / or detection solution. The detection solution contains the detection substrate. For thrombin and thrombin-like snake venom enzymes such as batroxobin or ancrod, a single detection substrate and thus a single detection solution can be used, or different detection solutions with different detection substrates can be used. For Factor Xa and snake venom enzymes with Factor Xa-like activity, such as trocarin, a further detection substrate is required. Optionally, the system also includes control and / or calibration solutions, either separately for fibrinogen and DOACs, or mixtures for both parameters.

[0128] The invention also relates to the following aspects:

[0129] 1. A method for determining the content, in particular the concentration, of analytes (inhibitors or substrates) in biological samples, in which an enzyme is detached from a solid support by contact with the free analyte and, after washing, the enzyme remaining on the support is detected, characterized in that, a) in a first step, the sample solution is brought into contact with an adsorbed enzyme so that a competitive equilibrium of the enzyme is established between surface binding and binding to the analyte, whereby the enzyme present on the support is partially detached, b) in a washing step, mobilized enzyme and residual sample components are removed by means of a washing solution, c) the activity of the support-bound enzyme is detected with a suitable substrate from a detection solution, d) the content is determined using a calibration curve,in particular, the concentration of the ligand to be analyzed (the substrate or inhibitor) is determined.

[0130] 2. The method according to aspect 1, wherein the enzyme is bound to an immobilized specific ligand that binds to an exosite or allosteric binding site or to the active center of the enzyme without being converted, so that in step a, a competitive equilibrium of the enzyme is established between bound ligand and binding to the analyte, whereby the enzyme present on the support is partially detached

[0131] 3. The method of aspect 2, wherein the enzyme is a protease and the specific ligand is a non-convertible protease substrate, a small molecule inhibitor, or a peptidomimetic that binds to the active site or an exosite.

[0132] 4. The method according to aspect 3, wherein the enzyme is thrombin or a snake venom thrombin-like enzyme (SVTLE).

[0133] 5. The method according to aspect 4, wherein the analyte is a thrombin-converted

[0134] Coagulation factor, in particular fibrinogen, or a thrombin inhibitor, in particular dabigatran or dabigatran etelixate.

[0135] 6. A method according to aspect 3, wherein the enzyme Factor Xa (Stuart-Prower factor,

[0136] Thrombokinase) or a Factor X-like prothrombin activator from snake venom.

[0137] 7. The method according to aspect 6, wherein the analyte is a factor Xa-converted

[0138] coagulation factor or a factor Xa inhibitor, in particular apixaban, edoxaban or rivaroxaban.

[0139] 8. A method according to aspect 1, wherein the specific ligand is bound to the surface of a

[0140] reaction vessel into which the sample solution from step a) is added and removed again, the washing solution from step b) is added and removed again, and the detection solution c) from aspect 1 is added.

[0141] 9. A method according to aspect 8, wherein the specific ligand is present in the reaction vessel at a

[0142] Oligosaccharide matrix is ​​immobilized, which prevents non-specific interactions of the surface with the components of the sample solution.

[0143] 10. Method according to aspect 9, in which the ligand is bound to a porous matrix in a reactive field and the solutions from steps a), b) and c) from aspect 1 are applied to an application field and transported by capillary forces through the matrix via a reaction field into a suction field. Experienced according to aspect 10, in which the porous matrix is ​​a cellulose-based material on which the ligands are immobilized by means of cellulose-binding proteins or peptides. Experienced according to aspect 11, in which the cellulose-based matrix is ​​modified to avoid non-specific interactions with components of the sample. Experienced according to aspect 12, in which the cellulose-based matrix is ​​modified by binding further cellulose-binding proteins or cellulose-binding peptides to avoid non-specific interactions with components of the sample. Experienced according to at least one of the preceding aspects, wherein the enzyme substrate has a chromogenic group.Experienced according to at least one of the preceding aspects, wherein the enzyme substrate has a fluorogenic group. Experienced according to at least one of the preceding aspects, wherein the enzyme substrate has a fluorophore and a further functionality that absorbs the energy of the fluorophore, such that substrate cleavage leads to a significant change in fluorescence intensity. Method according to at least one of the preceding aspects, wherein the amount of reacted substrate is determined optically. Device, in particular for carrying out the method according to at least one of aspects 1 to 16, for the detection of fibrinogen or direct oral anticoagulants, wherein the device comprises a reaction zone and wherein the reaction zone comprises immobilized working enzyme. Device according to aspect 17, wherein the device has an application zone and a suction zone.Device according to at least one of aspects 17 to 18, wherein the device is configured such that the sample applied in an application zone can reach the reaction zone by capillary forces. Device according to at least one of aspects 17 to 18, with which the amount of reacted substrate is optically determined after addition of the reaction solution from aspect 1, step c).

[0144] Detailed description of the illustrations

[0145] Figure 1 schematically shows the functional principle of affinity chromatography (state of the art). A sample of complex composition is applied to a matrix 11 immobilized with ligand L (arrow 12). This can be an inhibitor, a specific binding partner that does not bind to the active site, or one of two reactable substrates, e.g., a coenzyme. The target protein 13 binds specifically to the ligand L, and the remaining sample components (not shown) are washed out. The target protein 13 is then eluted by adding ligand L (arrow 14). The ligand L is removed from the target protein 13 by dialysis or ultrafiltration (not shown).

[0146] Figure 2 schematically shows the functional principle of the competitive immunoassay (state of the art). A sample containing the analyte L is mixed with labeled antibodies 21 and incubated for a while. Part of the antibodies are occupied by the analyte molecules (22). The mixture is then added to a carrier-bound ligand (23) (arrow 24) and incubated again. The antibodies bound to free ligands are then removed by washing (arrow 25), and the labeling of the carrier-bound antibodies is measured (arrow 26). The label 26 is a radioisotope (radioimmunoassay, RIA), a fluorophore (fluorescence immunoassay, FIA), or an enzyme that converts a substrate into a detectable signal (chromophore, fluorophore, chemiluminescence) (ELISA).

[0147] Figure 3 schematically shows an embodiment of the method according to the invention. A ligand I is immobilized on a carrier 31. After addition (arrow 32) of enzyme 33, enzyme 33 is immobilized by binding to the immobilized ligand I. These steps are carried out during the preparation of the test strip. In the actual analysis, the sample is added. The analyte from the sample now competes with the immobilized ligand for the enzyme and detaches a portion of the bound enzyme 33 (low detachment at low analyte concentration (arrow 34a); high detachment at high analyte concentration (arrow 34b)), so that after washing (arrows 35a and 35b), a portion of enzyme 33 is removed and the activity in the detection step is reduced. This rapid equilibrium competition advantageously distinguishes the method according to the invention from the immunoassay.

[0148] Figure 4 schematically shows the structure of a device 41 for carrying out the method as a lateral flow test and reading the generated signal. Sample solution, washing solution, and detection solution are applied to the application zone 42 and transported by capillary forces through the reaction zone 43 to the suction zone 44. The reaction zone with the immobilized enzyme is excited by a light source 45 (e.g., a light-emitting diode (LED)), and the emitted fluorescent light is detected by a detector 46. When reacted with a fluorogenic substrate, the light passes through a filter 47, which transmits the emission light 49 but not the excitation light 48. When reacted with a chromogenic substrate, the detector measures the remission, and filter 47 is omitted.

[0149] Figure 5 shows the result of the NADH determination according to Example 1. The initial slope of the fluorescence decrease at 470 nm after excitation at 370 nm (Y-axis, arbitrary units, au) is plotted against the NADH concentration (X-axis, mM) in the competition step. The more NADH available to compete with the Cibacron Blue, the more LDH was detached and washed away. Accordingly, the higher the NADH concentration in the sample, the lower the activity in the assay.

[0150] Figure 6 shows the result of the fluorescence anisotropy measurement to determine the affinity of the peptide T-FX1 to FXa (n = 3, error bars represent ±SD (standard deviation). To investigate the binding of the FXa peptides to FXa, the dissociation constant for the TAMRA-labeled peptide was determined using fluorescence anisotropy measurement. The binding isotherm is shown in Figure 6. According to the evaluation by nonlinear regression using the Hill equation, the labeled peptide T-FX1 binds factor Xa with a dissociation constant of KD = 0.79 (R 2 = 0.984).

[0151] Figure 7 shows the determination of the IC50 of B-FX1 (also referred to as BFX1) based on a plot of the initial rate versus the peptide concentration (n=3, error bars represent ±SD). Figure 7 shows the plot of the initial slope of the absorbance of the chromophore released from the substrate versus the concentration of the added peptide B-FX1. For the biotinylated peptide B-FX1, an IC50 of 1.87 pM with a coefficient of determination of 0.944 was determined. According to the Cheng-Prusoff equation, this results in a KD of 1.23 pM with the KD of T-FX1.

[0152] Figure 8 shows the relationship between the conversion of the substrate CS-11(22) and the amount of FXa used, after FXa had been immobilized via BFX1 peptide to an avidin-coated microtiter plate (n=3, error bars represent ±SD). The detection limit is shown parallel to the x-axis. The coefficient of determination of the linear fit is 0.828.

[0153] Figure 9 shows the effect of rivaroxaban on the activity of immobilized FXa. (n=3, error bars represent ±SD) (R 2 =0.978). FXa was previously bound via BFX1 to an avidin-coated microtiter plate and is displaced from the plate upon incubation with rivaroxaban. The semi-logarithmic plot shows a sigmoidal decrease in the reaction rate with increasing rivaroxaban concentration.

[0154] Examples

[0155] Example 1

[0156] Cibacron Blue binds to the nucleotide binding site of lactate dehydrogenase (LDH), to which NADH normally binds as a cofactor. This is used for the affinity chromatographic purification of the enzyme LDH. It can also be used for the quantification of NADH using the method described here. For this purpose, Cibacron Blue is immobilized. Lactate dehydrogenase binds non-covalently to the dye and is competitively mobilized by NADH in a sample solution. After washing out free enzyme and analyte, the amount of remaining LDH can be quantified based on the enzymatic conversion of a substrate.

[0157] Methods

[0158] Production of Cibacron Blue Sepharose

[0159] Cibacron Blue (CB) Sepharose was prepared from Sepharose CL 6B and Cibacron Blue 3G-A in carbonate-alkaline solution at 60°C according to established procedures. The material was washed with 3 M NaCl and deionized water until the flowthrough was colorless. The final wash was performed with standard buffer (67 mM KH2PO4 / NaOH, pH 7.4). The material was stored as a 50% suspension. The standard buffer was used for all subsequent steps.

[0160] Loading of CB-Sepharose with lactate dehydrogenase

[0161] 7 ml of CB-Sepharose was mixed with 2.8 ml of rabbit muscle LDH (25 pg / ml in standard buffer) and incubated for 5 min at room temperature. The solution was then centrifuged for 3 min at 4000 rpm. The supernatant was discarded. Any remaining free enzyme was removed by twice making up to 50 ml with buffer and centrifuging.

[0162] Competition with the analyte NADH

[0163] 0.5 ml of LDH-CB-Sepharose was incubated in microcentrifuge tubes with NADH solutions at concentrations between 0 and 2 mM in a total volume of 1.0 ml for 5 min at room temperature. The solution was then centrifuged for 2 min at 8000 rpm, the supernatant discarded, and twice made up with 1 ml of buffer each time and centrifuged again.

[0164] Enzyme detachment

[0165] The LDH-CB-Sepharose was incubated with 0.6 mM NADH in 1 ml total volume for 5 min. After centrifugation, 300 μl of the supernatant was used for the enzyme assay.

[0166] Enzyme test

[0167] To test enzyme activity, a total volume of 3.0 ml of standard buffer was prepared with 0.3 ml of sample, NADH (0.3 mM), and pyruvate (1.0 mM). Fluorescence (excitation 370 nm, emission 470 nm) was monitored at 25°C. The decrease in fluorescence was plotted against time, and the initial slope was determined from the linear range.

[0168] Results

[0169] LDH immobilized on CB-Sepharose was incubated with different concentrations of NADH, washed, and the remaining LDH was eluted from the solid phase with NADH and reacted with NADH and pyruvate for quantification. The conversion of NADH to NAD + can be monitored spectroscopically by the fluorescence at 470 nm after excitation at 370 nm. The initial slope of the fluorescence decrease as a function of the NADH concentration in the sample is shown in Figure 5.

[0170] According to the competition principle, high analyte concentrations cause a large amount of enzyme to be detached and washed out, so that only a small amount of residual activity is detected in the enzyme test. At low analyte concentrations, the amount of remaining enzyme is correspondingly high. The inverse proportionality between the analyte and the signal is particularly favorable for sensitive determination of low concentrations, in this case from near zero to approximately 0.3 mM.

[0171] Example 2

[0172] Factor Xa-binding peptides are known from WO 95 / 29189 A1. Some of the sequences are listed below.

[0173] • Tyr-Chg-Arg-NH2(FX1) - WO 95 / 29189 A1, Table 3

[0174] • Ac-Tyr-Chg-Arg-Pip-NH2- WO 95 / 29189 A1, EXAMPLE XXXV, No. 17

[0175] Ac-Trp-Chg-Arg-Leu-Pro-NH2- WO 95 / 29189 A1, Table 3; here: SEQ ID NO:14 • Tyr-Ile-Arg-Leu-Pro-NH2- WO 95 / 29189 A1, Table 2; here: SEQ ID NO:15

[0176] (Ac: Acetyl, Arg: Arginin, Chg: Cyclohexylglycin, Glu: Glutamat, Gly: Glycin, Ile: Isoleucin, Leu: Leucin, Pip: Piperidin-2-carbonsäure, Pro: Prolin, Trp: Tryptophan, Tyr: Tyrosin)

[0177] According to our measurements, they bind the coagulation factor with KD values ​​between 0.70 and 1.99 pM and act as competitive inhibitors of Factor Xa with Ki values ​​between 0.29 and 1.56 pM. To quantify rivaroxaban as a model of an FXa inhibitor using the described method, the first peptide sequence – referred to as FX1 – is synthesized as a conjugate with N-terminal biotin (B-FX1) or tetramethylrhodamine (TAMRA, T-FX1). The latter variant serves to determine the affinity by measuring fluorescence anisotropy. The affinity of the biotinylated variant is determined in an activity assay in competition for the FXa substrate CS-11(22). Peptide B-FX1 is immobilized on avidin-coated microtiter plates via the biotin. FXa binds non-covalently to this peptide and is displaced by the example inhibitor rivaroxaban.After removal of the analyte solution, the amount of remaining FXa is determined by enzymatic conversion of the signaling substrate CS-11(22). This is a mixture of the peptides Bz-lle-Glu(yOCH3)-Gly-Arg-pNA (SEQ ID NO:16) and Bz-lle-Glu-Gly-Arg-pNA (SEQ ID NO:17) (Bz: benzyl).

[0178] Methods

[0179] Preparation of FX1 conjugates with biotin (B-FX1) and TAMRA (T-FX1)

[0180] Peptide FX1 is synthesized by standard Fmoc (fluorenylmethoxycarbonyl) / tBu (tert-butyl) solid-phase peptide synthesis. 100 mg of Rink amide resin is used. In each coupling step, 3 equivalents (eq.) of Fmoc-protected amino acid, 3 eq. of oxymethoxy, and 3 eq. of DIC (diisopropylcarbodiimide), dissolved in approximately 1 mL of DMF (dimethylformamide), are preactivated for 5 minutes with shaking. The first amino acid is coupled overnight, and the remaining amino acids are coupled in two consecutive coupling steps over one hour.

[0181] After synthesizing the peptide sequence, the batch is halved. To synthesize B-FXI, the peptide is biotinylated in three steps. For this purpose, a solution of biotin (1 eq.), HBTU ((2-(1 H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, 1 eq.), DIPEA (diisopropylethylamine, 1 eq.) in 1 mL DMSO (dimethyl sulfoxide) / DMF (1:1 v / v) is incubated on the shaker at 600 rpm and room temperature (RT) until the biotin is completely dissolved. The solution is added to the resin particles and incubated for 1 h at 600 rpm and RT. Fresh biotin coupling solution is then added (incubation for 1 h, 600 rpm). The particles are then washed three times with DMSO / DMF (1:1 v / v), followed by another coupling overnight. The resin particles are finally washed three times with DMSO, three times with DMSO / DMF (1:1 v / v) and twice with DMF.

[0182] In the second approach, 5(6)-carboxytetramethylrhodamine (TAMRA, 3 eq.) is ligated to the N-terminus of the peptide overnight using oxyma (3 eq.) and DIC (3 eq.), analogous to the first amino acid coupling.

[0183] The peptides are cleaved from the dried resin using a solution of 95% TFA (trifluoroacetic acid), 1.25% triisopropylsilane, 1.25% anisole, and 2.5% dH2O. This also removes the permanent protecting groups. The respective crude peptide is precipitated in ice-cold methyl tert-butyl ether, centrifuged, and the pellet is dissolved in 1 mL of eluent A (95% water, 5% acetonitrile, +0.1% TFA). After freezing in liquid nitrogen, the pellet is lyophilized. For purification, the prepared crude peptide is dissolved in eluent A at a concentration of 10 mg / mL.

[0184] For purification via reversed-phase HPLC, a YMC-Triart Prep C18-S column (250 x 10.0 mm) is used. The mobile phase consists of eluent A, with a continuous gradient of 0–100% eluent B (5% water, 95% acetonitrile, +0.1% TFA) used for elution. Peptides are detected by absorbance measurements at 220 nm (peptide bond), 280 nm (aromatic amino acids), and 555 nm (TAMRA labeling). The automatically collected fractions are analyzed by MALDI-ToF MS (matrix-assisted laser desorption ionization time-of-flight mass spectrometry). Successfully identified fractions are lyophilized and stored at -20°C. To characterize the purified peptide, a mass spectrum is recorded again and an analytical HPLC run is carried out on a YMC-Triart C18 column (150 x 4.6 mm) with 10 pL of the dissolved peptide.

[0185] The peptide B-FX1 has a calculated mass of 701.3 g / mol. MALDI-ToF-MS confirms a mass of 702.4 ([M+H] + ). The retention time in analytical HPLC is 2.18 min and the purity according to HPLC is 84.1%.

[0186] The peptide T-FX1 has a calculated mass of 887.4 g / mol. MALDI-ToF MS confirms a mass of 888.4 ([M+H] + ). The retention time in analytical HPLC is 12.38 min and the purity according to HPLC is 97.2%.

[0187] Binding of peptides to Factor Xa

[0188] For the determination of the dissociation constant of T-FX1 to FXa using

[0189] For fluorescence anisotropy measurement, a 1:2 FXa dilution series was prepared starting from a 26.67 pM stock solution in eight dilution steps in buffer e (35 mM NaCl, 40 mM sodium hydrogen phosphate, pH 7.4) and each diluted solution was spiked with T-FX1 peptide to a final concentration of 50 nM. After an incubation of 4 h, the anisotropy was measured on the Tecan Infinite M1000 (excitation: 530 nm, emission: 579 nm).

[0190] To determine the IC50 as a measure of the binding of the B-FX1 peptide to FXa, the peptide was mixed with 0.25 μl / ml FXa at different concentrations and incubated for 1 h at room temperature. After addition of 200 pM of the substrate CS-11(22), the absorbance was measured on a microtiter plate reader (Tecan Infinite M1000) at a wavelength of 405 nm to detect free p-nitroaniline. The control contained no peptide. The initial slope was determined by linear regression of the initial values, and this was plotted against the B-FX1 concentration. The IC50 value was determined by nonlinear regression using the [inhibitor] vs. response (variable slope) model. From this, the Kj value was determined using the Kj value via the Cheng-Prusoff equation.

[0191] Coating of microtiter plates

[0192] To coat the microtiter plates with avidin, following the protocol by Truett et al. (Truett, G., Walker, J., Wilson, J., Redmann, S. et al., Mammalian Genome 1998, 9, 629-632), a coating buffer is first prepared by dissolving 0.53 g of sodium carbonate in 90 mL of dH2O and adjusting the pH to 9.7. Then, 200 μL of avidin (1 mg / mL in dH2O) is added and the volume is made up to 100 mL. 100 μL of the coating buffer is pipetted into each of ten 96-well microtiter plates (High Binding, Transparent, flat bottom, Sarstedt). The plates are sealed with Parafilm and incubated overnight at 4°C. After washing four times with 200 pL of TBS-T (0.1 M Tris-HCl; 0.15 M NaCl; 0.05% Tween 20) each, 150 pL of blocking buffer (TBS-T + 2% BSA) is added and the plates are incubated for two hours. After removing the buffer, the plates are sealed with Parafilm and stored in a plastic wrap at -20°C until further use.Before use, the plates are brought to room temperature and rinsed twice with TBS-T.

[0193] To determine the loading capacity according to Dapron et al. (Dapron, J., Zobrist, J., Foster, K., Barbacci, L., Hassell, T., & Kappel, W., Life Science Quarterly 42003. See also https: / / www.sigmaaldrich.com / deepweb / assets / sigmaaldrich / product / documents / 189 / 244 / vol4-issuel-streptavidin.pdf?srsltid=AfmBOooTZiQWBU56xfCqAraAGDIzJg7AWUhmJHXUWncuVR W6i8KICgNZ), 25 pmol of biotin-fluorescein (in 200 pL TBS-T) are added to each of three wells. This corresponds to twice the theoretically calculated amount of approximately 12 pmol / well. After one hour of incubation, 100 pL of the supernatant is transferred to a black 96-well microtiter plate. Three 100 pL portions of the biotin-fluorescein loading solution are added. Fluorescence intensity is measured on a Tecan Infinite M1000 (excitation: 494 nm; emission: 518 nm). The total biotin-fluorescein bound to avidin per well is determined based on the ratio of the fluorescence units of the two samples.The loading capacity is 4.74 pmol / cavity.

[0194] Dependence of substrate turnover on the FXa concentration in the microtiter plate

[0195] The wells of an avidin plate used for the test are thoroughly washed twice with TBS-T. Each well is then filled with 100 pL of a 400 nM biotin-peptide solution in FXa buffer. After a one-hour incubation, the wells are washed twice with FXa buffer. Then, different concentrations of FXa between 0 and 500 nM are added in 100 pL portions. After another hour of incubation and another two-fold wash with FXa buffer, 100 pL of CS-11 (22) (200 pM) is added using a multichannel pipette. The change in absorbance at 405 nm is measured on the Tecam M1000.

[0196] Displacement of FXa by rivaroxaban

[0197] The wells of the avidin plate are washed twice with TBS-T and then incubated with 100 pL of biotin peptide (400 nM) for one hour. After two wash steps with FXa buffer, 100 pL of FXa (200 nM) is added to each well. After incubation for one hour and two wash steps, 100 pL of various concentrations (0–100 nM) of rivaroxaban are added. After a final incubation of 30 minutes and two further wash steps, 100 pL of CS-11(22) (200 pM) are added using a multichannel pipette. The absorbance is measured on the Tecan M1000 at a wavelength of 405 nm.

[0198] Results

[0199] FX1 was selected as the model peptide. It was prepared as a conjugate with N-terminal biotin (B-FX1) or tetramethylrhodamine (TAMRA, T-FX1) using the standard Fmoc / tBu method by solid-phase peptide synthesis, purified by RP-HPLC, and characterized by analytical HPLC and MALDI-ToF-MS. To investigate the binding of the FXa peptides to FXa, the dissociation constant for the TAMRA-labeled peptide was determined by fluorescence anisotropy measurement. The binding isotherm is shown in Figure 6. According to the evaluation by nonlinear regression using the Hill equation, the labeled peptide T-FX1 binds factor Xa with a dissociation constant of KD = 0.79 (R 2 = 0.984).

[0200] Binding of the peptides to the active site of FXa should result in inhibition. To verify this, FXa activity assays were performed in which the conversion of the substrate CS-11(22) by FXa from B-FX1 was inhibited. Figure 7 shows the initial slope of the absorbance of the chromophore released from the substrate plotted against the concentration of the added peptide B-FX1.

[0201] For the biotinylated peptide B-FX1, an IC50 of 1.87 pM with a coefficient of determination of 0.944 was determined. According to the Cheng-Prusoff equation, this results in a KD of 1.23 pM for T-FX1.

[0202] To test whether FXa can be specifically immobilized via the BFX peptides, the ELISA plate format was chosen. The wells of a 96-well high-binding microtiter plate were coated with avidin, and their loading capacity was determined using biotin-fluorescein to be 4.74 pmol / well. Subsequently, 100 pL of a BFX1 peptide solution (400 nM) was added to the wells. After one hour of incubation, the wells were washed, and a series of FXa concentrations (0–80 nM) were added. After further washing, the substrate CS-11(22) was added to quantify the bound FXa, and its conversion was measured. One well each without peptide and without enzyme loading served as controls. All measurements were performed in triplicate. The initial rates versus FXa concentration are plotted in Figure 8.

[0203] FXa was immobilized via the BFX peptides and was still present in sufficient quantities after two washing steps. In the presence of the substrate CS-11(22), FXa converted it. The initial rate increased with increasing FXa concentration. FXa concentrations below 10 nM were no longer detectable. Considering the loading capacity of 4.74 pmol / well, the maximum amount of FXa can be bound. When using 100 pL of 1 nM or 10 nM FXa solution, the amount of substance used corresponds to 0.1 or 1 pmol per well, respectively. The amount of FXa bound at equilibrium is apparently far below the determined KD value and is therefore insufficient for detection. For the competition experiments, 100 pL of 200 nM FXa was therefore chosen.

[0204] Since the peptides bind FXa via the active site, the substrate must displace them from this bond in order for it to be converted. The substrate conversion depends on the amount of enzyme used. A linear dependence of the reaction rate on the amount of FXa used was observed. This was to be expected in the case of an excess of free peptide binding sites compared to the amount of enzyme.

[0205] Competition with rivaroxaban

[0206] Rivaroxaban was added to microtiter plate wells loaded with BFX1 and FXa at concentrations ranging from 0 to 100 pM. After half an hour of incubation and repeated washing, substrate was added to quantify the bound FXa, and its conversion was measured. The change in absorbance at 405 nM was monitored on a Tecan Infinite M1000. As shown in Figure 9, the semi-logarithmic plot shows a sigmoidal decrease in the reaction rate with increasing rivaroxaban concentration.

[0207] This suggests that FXa was dissolved by BFX1 during incubation with rivaroxaban, depending on its concentration, and removed from the system during the washing steps. The remaining amount of FXa is inversely proportional to the rivaroxaban concentration.

Claims

Claims 1. A method for determining the content, in particular the concentration of an analyte in a sample, the method comprising the following steps: a) bringing the sample into contact with an enzyme immobilized by binding to an immobilized ligand, so that an equilibrium is established between binding of the enzyme to the ligand and binding of the enzyme to the analyte based on competition between the ligand and the analyte for binding to the enzyme, b) removing the sample and the portion of the enzyme mobilized by binding to the analyte from the non-mobilized portion of the enzyme, and c) determining the content, in particular the concentration of the analyte in the sample based on the enzymatic activity of the non-mobilized portion of the enzyme.

2. The method of claim 1, wherein the ligand is immobilized by binding to a support.

3. Method according to at least one of the preceding claims, wherein the sample is a blood sample.

4. The method according to at least one of the preceding claims, wherein step a) comprises an incubation step and wherein the incubation time is in a range of 1 second to 5 minutes.

5. The method according to at least one of the preceding claims, wherein step b) comprises one or more washing steps.

6. The method according to at least one of the preceding claims, wherein the analyte is a substrate or an inhibitor of the enzyme.

7. The method according to at least one of the preceding claims, wherein the ligand is an inhibitor of the enzyme.

8. The method according to at least one of the preceding claims, wherein the enzyme is thrombin, meizothrombin, batroxobin from Bothrops atrox or ancrod from Calloselasma rhodostoma and wherein the analyte is fibrinogen, dabigatran or dabigatran etelixate, or • wherein the enzyme is factor Xa or trocarin from Tropidechis carinatus and wherein the analyte is apixaban, edoxaban or rivaroxaban.

9. Device, in particular for carrying out the method according to at least one of claims 1 to 8, for determining the content, in particular the concentration, of an analyte in a sample, wherein the device comprises at least one reaction zone and wherein the reaction zone comprises immobilized enzyme.

10. Device according to claim 9, wherein the device comprises at least one application zone and wherein the device is configured such that sample applied in the application zone can reach the reaction zone by means of capillary forces.

11. Device according to at least one of claims 9 and 10, wherein the immobilized enzyme is immobilized by binding to an immobilized ligand and wherein the ligand is linked by means of a linker to a carbohydrate-binding module which is bound to a support containing cellulose.

12. System comprising a device according to at least one of claims 9 to 11 and washing solution and / or detection solution.

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