Methods for immunosensing on lipid layers - Patents.com

Abstract

The present invention relates to diagnostic tests and techniques. In particular, the present invention relates to a method for determining an analyte suspected to be present in a sample, comprising contacting the sample with a sensor element comprising: i) an anchor layer present on a solid support; ii) a first binding agent attached to the anchor layer and capable of specifically binding to the analyte, comprising at least one detectable label; and iii) a second binding agent immobilized on the solid support, capable of specifically binding to the analyte when bound to the first binding agent, for a time and under conditions that allow specific binding of the analyte suspected to be present in the sample to the first binding agent and specific binding of the second binding agent to the analyte bound to the first binding agent; and detecting the formation of a complex of the first binding agent, the analyte and the second binding agent, thereby determining the analyte. Further, an apparatus for determining an analyte suspected to be present in a sample and its use for determining an analyte suspected to be present in a sample in the sample are provided. The present invention further contemplates a kit for determining, within said sample, an analyte suspected to be present in the sample.

Description

[Technical field]

[0001] The present invention relates to diagnostic tests and techniques. In particular, the present invention relates to a method for determining an analyte suspected to be present in a sample, comprising contacting the sample with a sensor element comprising: i) an anchor layer present on a solid support; ii) a first binding agent attached to the anchor layer and capable of specifically binding to the analyte, comprising at least one detectable label; and iii) a second binding agent immobilized on the solid support, capable of specifically binding to the analyte when bound to the first binding agent, for a time and under conditions that allow specific binding of the analyte suspected to be present in the sample to the first binding agent and specific binding of the second binding agent to the analyte bound to the first binding agent; and detecting the formation of a complex of the first binding agent, the analyte and the second binding agent, thereby determining the analyte. Further provided is an apparatus for determining an analyte suspected to be present in a sample, and its use for determining an analyte suspected to be present in a sample in the sample. Additionally, the present invention contemplates a kit for determining an analyte suspected to be present in a sample. [Background technology]

[0002] Immunoassays are widely used for various diagnostic purposes. Several setups for immunoassays have been developed. One of the most common immunoassays is the enzyme-linked adsorbent immunoassay (ELISA).

[0003] In an ELISA, a liquid sample containing or suspected to contain an analyte of interest is applied to a stationary solid phase with special binding properties due to the presence of antibodies or antibody-like molecules such as aptamers. After sample application, multiple reagents are added sequentially, incubated and washed to perform and stop the analytical detection reaction. After performing all these steps, a physical or chemical property in the setup, usually the liquid phase, changes and can be detected. Typically, an optical change occurs in the final liquid phase, such as the development of color due to the product of the enzymatic reaction. These changes correlate to the presence or abundance of the analyte of interest present in the investigated sample. The typically quantitative readout is usually based on the detection of the intensity of the transmitted light by spectrophotometry, which involves the quantification of the transmission of light of a specific wavelength through the liquid. The sensitivity of the detection depends on the amplification of the signal during the analytical reaction. Since the enzymatic reaction is a very well-known amplification process, the signal is generated by an enzyme linked to a detection reagent at a constant rate to allow accurate quantification.

[0004] For ELISA setup, the test substance binding agent, for example, an antibody, is immobilized on a solid support, such as a solid support structure. Usually, the antibody is coated and dried on the transparent bottom and sometimes also the sidewall of the well of an analytical multi-well plate or an analytical vial. However, nanoparticles or other beads can also be used as the solid phase of ELISA.

[0005] For research and diagnostic purposes, ELISA is often used in the so-called sandwich ELISA format. In the sandwich format, an immobilized capture antibody is used to capture, i.e. specifically bind, the test substance present in the sample applied to said immobilized antibody. After the capture antibody specifically binds to the test substance, the sample material is washed away. In a subsequent step, the test substance bound to the immobilized antibody is incubated with a detection antibody that specifically binds to the test substance or to the complex of the test substance and the capture antibody. The detection antibody typically includes a detectable linker or adapter molecule that allows it to attract such a detectable label from the solution.

[0006] However, in light of the fragile immune complexes required for signal generation and the various components used in such ELISAs in sandwich format, there are many possibilities for undesired binding of detection antibodies and thus the generation of false positive signals.Therefore, sandwich ELISAs used in research often require validation due to the risk of false positive results.In addition, due to the various shortcomings of such fragile multi-component assay formats, there are limitations in terms of sensitivity and specificity. Summary of the Invention

[0007] The technical problem underlying the present invention may be seen as the provision of means and methods for addressing the above-mentioned needs. This technical problem is solved by the embodiments characterized in the following claims and in the specification.

[0008] Accordingly, the present invention provides a method for determining a test substance suspected to be present in a sample, comprising the steps of: (a) placing the sample in a sensor element, (i) an anchor layer present on a solid support; (ii) a first binding agent that is affixed to the anchor layer and that can specifically bind to the test substance, the first binding agent comprising at least one detectable label; (iii) a second binding agent immobilized on the solid support, the second binding agent being capable of specifically binding to the test substance when bound to the first binding agent; for a time and under conditions that allow specific binding of the analyte suspected of being present in the sample to the first binding agent and specific binding of the second binding agent to the analyte bound to the first binding agent; (b) detecting the formation of a complex between the first binding agent, the test substance, and the second binding agent, thereby determining the test substance; and The present invention relates to a method comprising the steps of: DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] It should be understood that, in this specification and claims, "a" or "an" can mean one or more, depending on the context in which it is used. Thus, for example, a reference to "an" item can mean that at least one item can be utilized.

[0010] When used hereinafter, the terms "have", "comprise" or "include" are meant to have an open-ended or closed-ended meaning. Thus, these terms with a closed-ended meaning may refer to a situation in which no other features are present in the described embodiment apart from the features introduced by these terms, i.e., these terms have a closed-ended meaning in the sense of "consisting of" or "consisting essentially of". Terms with a closed-ended meaning refer to a situation in which, in addition to the features introduced by these terms, one or more other features are present in the described embodiment.

[0011] Furthermore, when used hereinafter, the terms "preferably", "more preferably", "most preferably", "particularly", "more particularly", "typically" and "more typically" or similar terms may be used in conjunction with additional or alternative features without limiting the possibilities for substitution.

[0012] Furthermore, as used herein, the term "at least one" will be understood to mean that one or more of the items followed by the term may be used in accordance with the present invention. For example, if the term indicates that at least one item should be used, this may be understood as one item or more than one item, i.e., two, three, four, five, or any other number. Depending on the item to which the term refers, a person skilled in the art will understand what upper limit, if any, the term may refer to.

[0013] The method according to the invention may consist of steps (a) and (b) as described above or may comprise further steps, such as pre-treating or isolating the sample before step (a) and / or after step (b), and may further comprise one or more steps of evaluating the determined test substance, for example by comparing it with a reference, in order to provide a diagnostic, prognostic, environmentally-related, agriculturally-related or analytically-related conclusion depending on the purpose of the determination of the test substance.

[0014] As used herein, the term "determining" encompasses any kind of qualitative or quantitative determination of a test substance. A qualitative determination aims to determine the presence or absence of a test substance in a sample, whereas a quantitative determination aims to determine the amount of the test substance. A quantitative determination, i.e., a determination of the amount, includes determining an absolute amount (e.g., the total amount by weight or number of molecules present in a sample) or a relative amount (e.g., the amount relative to the sample volume (concentration) or a classification such as a score (e.g., "high", "low", etc.). Typically, determining a test substance includes determining the presence, absence or amount of said test substance.

[0015] The term "test substance" as referred to herein relates to any type of molecule or agent suitable for determination by the method of the present invention. It will be understood that such molecule or agent may have a size and / or structure that allows the binding of the first and second binding agents referred to herein. Furthermore, there may be an upper size limit since the first and second binding agents and linking agents must be able to perform their functions as described in detail elsewhere herein. Typically, the test substance referred to herein is a small molecule such as a protein, peptide, nucleic acid, e.g. DNA or RNA, or lipid or metabolite, e.g. polyketides, including flavonoids and isoflavonoids, isoprenoids, including terpenes, sterols, steroids, carotenoids, xanthophylls, carbohydrates, phenylpropanoids, alkaloids, benzenoids, indoles, porphyrins, hormones, vitamins, cofactors, lignins, glucosinolates, purines, pyrimidines, nucleosides, nucleotides, alcohols, alkanes, alkenes, alkynes, aromatic compounds, ketones, aldehydes, carboxylic acids, esters, amines, imines, amides, cyanides, amino acids, thiols, thioesters, phosphate esters, sulfate esters, thioethers, sulfoxides or ethers. Small molecules can be, for example, toxins. However, the method of the present invention can also be used to determine viruses or even bacterial cells as test substances. Test substances determined by the present invention can also be molecules present in environmental samples and can be useful, for example, as indicators of environmental pollution, agriculture or other environmental conditions. Typically, the test agent is a protein, peptide, virus, bacterial cell or a small molecule, preferably a small molecule toxin.

[0016] As used herein, the term "sample" relates to any portion or aliquot of a composition that contains or is suspected to contain a test substance to be determined. Such a sample may typically be a biological sample isolated from an organism, such as a body fluid or a biopsy sample, or a composition that contains an organism, such as cultured cells. Typically, said biological sample is investigated by the method of the invention for medical purposes, such as the diagnosis or prediction of a disease or condition. The sample may also be an environmental sample or an artificial sample. An environmental sample may originate from any non-biological natural source, e.g. a solution present in the environment, such as water, or a composition, such as soil. An artificial sample may be a sample obtained from an artificial source, e.g. a product composition that may be manufactured or an intermediate composition that may occur during the manufacturing process of a product. Such an artificial sample may be investigated, for example, for quality control purposes or to determine the amount of a particular component. Typically, said sample according to the method of the invention is a biological sample, preferably a body fluid or a biopsy sample.

[0017] The term "sensor element" as used in accordance with the present invention includes a solid support having a surface covered by an anchor layer. Moreover, the sensor element further includes a molecular configuration as described above, i.e. a first binding agent capable of specifically binding to an analyte, which is fixed to the anchor layer and comprises at least one detectable label, and a second binding agent capable of specifically binding to an analyte when bound to the first binding agent, immobilized on the solid support.

[0018] The term "anchor layer" referred to herein includes any molecular layer that can associate molecules, particularly the first binding agent according to the present invention. The association of the first binding agent referred to according to the present invention shall allow molecular movement of the first binding agent or any anchor molecule attached thereto within or on the surface of the anchor layer. The anchor layer is typically a lipid layer or lipid bilayer. Those skilled in the art are well aware of how a solid support can be covered by such an anchor layer and which lipids can be used to create the lipid layer or lipid bilayer. A typical lipid layer or lipid bilayer suitable for use as an anchor layer according to the present invention may include, for example, a phospholipid layer or a phospholipid bilayer. Said phospholipid layer or phospholipid bilayer may in particular comprise one or more phosphatidylcholine(s) such as 1-oleoyl-2-palmitoyl-phosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine and / or 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl], or a mixture of one or more phosphatidylcholine(s) with cholesterol. Furthermore, supported lipid bilayers and tethered bilayer lipid membranes may find use as anchor layers according to the present invention. These are commonly used model lipid bilayers known in the art.

[0019] As used herein, the term "solid support" relates to a solid composition of matter that can serve as a basis for immobilizing molecules, in particular the second binding agent. The solid support may comprise inorganic or organic compounds or both. Typically, inorganic compounds suitable for solid supports may be selected from the group consisting of silica, porous glass, aluminosilicates, borosilicates, metal oxides (e.g., aluminum oxide, iron oxide, nickel oxide) and clays containing one or more of these. The solid support may also comprise a conductive compound such as a metal or graphite. Preferably, such a solid support may be or may comprise an electrode, a magnetic tunnel junction, a semiconductor, a plasmonic resonator, or any type of electrical circuitry. Alternatively, the solid support may comprise an organic compound such as a cross-linked polymer. Non-limiting examples of suitable cross-linked polymers may be selected from the group consisting of polyamides, polyethers, polystyrenes and mixtures thereof. The skilled person is well aware how to select a suitable solid support based on the type of sample to be investigated, the method envisaged for the detection of the analyte, the type of detectable label used to detect the analyte and / or the type of second binding agent used in the sensor element.

[0020] The sensor further comprises a first binding agent capable of specifically binding to the analyte, the first binding agent being anchored to the anchor layer, and at least one detectable label.

[0021] The term "first binding agent" referred to herein refers to a molecule that can specifically bind to a test substance, i.e., does not bind to and therefore does not cross-react with other molecules other than the test substance suspected to be present in a sample.Specific binding can in principle be tested by techniques well known in the art, including screening assays to identify agents that specifically bind to a test substance from a library containing different candidate agents.

[0022] Preferably, the molecule that can be used as the first binding agent that can specifically bind to the desired test substance can be an antibody. The antibody as a binding agent as meant in accordance with the present invention includes all types of antibodies that preferably specifically bind to the test substance. Preferably, the antibody of the present invention can be a monoclonal antibody, a polyclonal antibody, a single chain antibody, a chimeric antibody, or any fragment of such an antibody that can still specifically bind to the test substance. Such fragments included in the term antibody as used herein include bispecific antibodies, synthetic antibodies, Fab, F(ab)2Fv or scFv fragments, or chemically modified derivatives of any of these antibody fragments. Antibodies or fragments thereof that specifically bind to the desired test substance can generally be obtained by using the methods described, for example, in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. Monoclonal antibodies can be prepared by techniques including fusion of mouse myeloma cells with spleen cells from immunized mammals, preferably immunized mice (Koehler 1975, Nature 256, 495, and Galfre 1981, Meth. Enzymol. 73, 3). The skilled artisan is well aware of the methods by which specific binding can be tested by techniques well known in the art, such as immunoassays, cell sorting or immunological or biochemical techniques including Western blotting or plasmon surface resonance measurements.

[0023] Furthermore, the molecule that can be used as a first binding agent, preferably capable of specifically binding to a desired analyte, can be an aptamer. Aptamers as binding agents according to the present invention can be oligonucleotide acid or peptide molecules that bind to a specific target analyte (Ellington 1990, Nature 346(6287):818-22). Bock 1992, Nature 355(6360):564-6). Oligonucleotide acid aptamers are engineered by repeated selection or the so-called systematic evolution of ligands by exponential enrichment (SELEX technique). Peptide aptamers usually contain a variable peptide loop attached at both ends to a protein scaffold. This double structural constraint increases the binding affinity of peptide aptamers into the nanomolar range. The variable peptide loop length is preferably composed of 10-20 amino acids, and the scaffold can be any protein with improved solubility and compactness, for example thioredoxin-A. The selection of peptide aptamers can be carried out using different systems, including for example the yeast two-hybrid system (see for example Hoppe-Seyler 2000, J Mol Med. 78(8):426-30). Any fragment of said aptamer that is still capable of specifically binding to the test substance is also encompassed according to the present invention. Said fragments can be used in isolated form or can be part of a fusion molecule, i.e. a molecule that comprises said aptamer fragment as well as other moieties such as linker moieties or adapter molecules. The skilled person is well aware how specific binding can be tested by techniques well known in the art, such as plasmon surface resonance measurements.

[0024] Preferably, the molecule that can be used as the first binding agent capable of specifically binding to the desired test substance can be a receptor molecule. The receptor molecule as a binding agent referred to in accordance with the present invention is typically a protein that specifically binds to a ligand and is activated upon ligand binding to exert its biological function. Such receptor molecules or fragments thereof that can still specifically bind to a ligand can also be used as a binding agent according to the present invention for the ligand as a test substance, or for molecules derived from the ligand but still capable of being bound by the receptor molecule or fragments thereof, such as antagonistic or agonistic mutants of the ligand. Preferably, the receptor molecule envisaged as a binding agent according to the present invention can be a transmembrane receptor protein (G protein-coupled receptor, e.g. metabolic receptor, enzyme-linked receptor, e.g. receptor tyrosine kinase, e.g. growth factor receptor, immune receptor, e.g. viral receptor, cell surface antigen, T cell receptor, e.g. CD4, CD3 or CD8, etc.), or an MHC protein, a cell adhesion molecule (such as integrin, cadherin, selectin or syndecan), a neuronal receptor, or a pathogen receptor (such as Toll-like receptor). The receptor molecule may also be a nuclear receptor protein, such as a nuclear hormone receptor, for example a glucocorticoid receptor, a retinoic acid receptor or a thyroid hormone receptor. Those skilled in the art are well aware of receptor molecules or fragments thereof that specifically bind to the test substance or fragments thereof to be measured. Furthermore, specific binding can be tested by techniques well known in the art, such as plasmon surface resonance measurements.

[0025] More preferably, the molecule that can be used as the first binding agent that can specifically bind to the desired test substance can be a ligand molecule. The ligand molecule as a binding agent referred to according to the present invention is typically a protein or peptide that specifically binds to a receptor molecule and is activated when the receptor binds to said receptor molecule. Such a ligand molecule or a fragment thereof that can still specifically bind to a receptor molecule can also be used as a binding agent according to the present invention for the receptor as a test substance, or a molecule derived from the receptor molecule but still able to be bound by the ligand molecule or a fragment thereof, such as a soluble mutant of the receptor. Furthermore, the ligand can also be any antigen that can be used to determine a specific antibody in a biological sample. Preferably, the ligand molecules envisaged as binding agents according to the present invention may be peptide hormones, neurotransmitters, growth factors (such as angiopoietins, BMPs, neutrophil factors, EGF, epiphrin, EPO, FGF, GDNF, GDF, insulin or insulin-like growth factors), TGF, neutrophils, VEGF, cytokines (such as interleukins, interferons, lymphokines, monokines, colony-stimulating factors or chemokines), extracellular matrix proteins (such as fibronectin, vitronectin, collagen, ankyrin or laminin), etc. The skilled person is well aware of ligand molecules or fragments thereof that specifically bind to the test substance or fragments thereof to be measured. Furthermore, specific binding can be tested by techniques well known in the art, such as plasmon surface resonance measurements.

[0026] More preferably, the first binding agent can be a designed ankyrin repeat protein (DARPin). DARPins are engineered antibody-mimicking proteins that can be designed to achieve highly specific and high affinity target protein binding. They are derived from natural ankyrin repeat proteins. Typically, DARPins contain at least three repeat modules, of which the most N-terminal and the most C-terminal modules (also called "caps") protect the hydrophobic core of the protein (Binz 2003, Journal of Molecular Biology.332(2):489-503).

[0027] Typically, the first binding agent is selected from the group consisting of an antibody or fragment thereof, an aptamer, a receptor molecule or fragment thereof, and a ligand molecule or fragment thereof. More typically, the first binding agent is an antibody or fragment thereof or an aptamer.

[0028] Furthermore, the first binder according to the present invention is intended to be anchored to the anchor layer. The anchored first binder referred to according to the present invention is typically intended to be associated with or within the anchor layer. It is therefore typically limited in its movement in space to essentially two dimensions.

[0029] Preferably, the first binding agent is anchored to the anchor layer via an anchor molecule, more preferably a lipid. The lipid suitable for anchoring the first binding agent according to the present invention depends on the nature of the anchor layer and can be selected by the person skilled in the art without further ado, and the anchor molecule can typically be linked to at least one, preferably a plurality of first binding agents via a linker molecule. A suitable linker molecule allows many first binding agents and at least one anchor molecule to be attached. Preferably, a suitable linker molecule can be a circular DNA molecule. Thus, a plurality of first binding agents can be anchored to the anchor layer via the same anchor molecule.

[0030] As used herein, the term "detectable label" relates to a molecule that exhibits a physical or chemical property that can be detected in the context of the method of the invention. Preferably, said property is an optically detectable property, such as radiation, chemiluminescence, fluorescence or FRET emission, an electromagnetic property, such as interference with an electric or magnetic field, a detectable radioactivity, or a chemical property, such as the ability to perform or catalyze a specific chemical reaction. The detectable label may bind reversibly or permanently to the first binding agent. To this end, the detectable label may be a molecule that can bind reversibly to the first binding agent or may be a molecule or moiety that is already part of said first binding agent. Exemplary detectable labels include gold particles, latex beads, acridan esters, luminol, ruthenium, enzymatically active labels, magnetic labels, radioactive labels or fluorescent labels. Enzymatically active labels include horseradish peroxidase, alkaline phosphatase, beta-galactosidase or luciferase. Suitable substrates for the detection of such enzymatically detectable labels include di-amino-benzidine (DAB), 3,3'-5,5'-tetramethylbenzidine, NBT-BCIP (4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate). The appropriate enzyme-substrate combination may produce a colored reaction product, fluorescence or chemiluminescence, which can be measured by methods known in the art (e.g., using a light-sensitive film or a suitable camera system). Exemplary fluorescent labels include fluorescent proteins such as GFP, RFP, YFP, BFP or variants thereof, Cy3, Cy5, Texas Red, fluorescein, and Alexa dyes such as Alexa 568. The use of quantum dots as fluorescent labels is also contemplated. Exemplary radioactive labels include 35S, 125I, 32P, 33P, etc. Radioactive labels can be detected by any method known and appropriate, such as a light-sensitive film or a phosphor imager. Exemplary magnetic labels include iron-platinum nanoparticles, iron nanoparticles, nickel nanoparticles, or cobalt nanoparticles.Typically, the detectable label is selected from the group consisting of a fluorescent label, a chemiluminescent label, a radioactive label, a magnetic label, and an electrochemical label. Typically, the detectable label comprises a linker that can be covalently linked to a solid support by a linking agent.

[0031] The first binding agent may include at least one detectable label as part of the molecule. Alternatively, at least one detectable label may be linked to the first binding molecule by a linker. According to the present invention, permanent linkage as well as reversible linkage are envisioned. Reversible linkage can be achieved, for example, by using the biotin-streptavidin-based molecular adapter system that is well known in the art.

[0032] The sensor also includes a second binding agent capable of specifically binding to the analyte when bound to the first binding agent, the second binding agent being immobilized on a solid support.

[0033] The term "second binding agent" capable of specifically binding to a test substance relates to a molecule capable of specifically binding to a test substance or a test substance when bound to a first binding agent, i.e. a molecule that does not bind to and therefore does not cross-react with other molecules than the test substance suspected to be present in the sample or the complex of the test substance and the first binding agent. Typically, said second binding agent is selected from the group consisting of an antibody or a fragment thereof, an aptamer, a receptor molecule or a fragment thereof, and a ligand molecule or a fragment thereof. More typically, the second binding agent is an antibody or a fragment thereof or an aptamer. Typically, the second binding agent is from the same aforementioned molecular class as the first binding agent. The definitions of the antibody or a fragment thereof, the aptamer, the receptor molecule or a fragment thereof, and the ligand molecule or a fragment thereof made according to the first binding agent apply mutatis mutandis to the second binding agent.

[0034] The second binding agent shall be immobilized on a solid support. The immobilization of said second binding agent is preferably permanent immobilization. Suitable techniques for immobilizing a second binding agent on a solid support depend on the nature of the solid support and / or the nature of the second binding agent and are well known to those skilled in the art. It will be understood that when immobilized, the second binding agent cannot move its position within or outside the anchor layer. However, the second binding agent must be able to perform bending and other molecular movements or conformational changes necessary to bind to the test substance or the first binding agent bound to the test substance.

[0035] Furthermore, the interaction of the second binding agent with the test substance and the first binding agent should preferably be a reversible interaction. The association rate is typically greater than the dissociation rate, and it is assumed that the complex of the first binding agent, the test substance and the second binding agent remains stable over a period of time, allowing its determination by detecting the presence or abundance of at least one detectable label that is proximate to the location of the second binding agent during said time window.

[0036] Typically, the amount of the first binder exceeds the amount of the second binder by 10 to 100 times, 20 to 80 times, 30 to 70 times, or 40 to 60 times.

[0037] Furthermore, the sensor element preferably includes a detector for detecting the signal induced by the detectable label. Typically, the detector is located below the solid support, and the detector can selectively detect the signal in direct proximity to the second binding element of the gear. Depending on the detectable label, different detection techniques can be applied.

[0038] Optically detectable labels may be determined by measuring luminescence, fluorescence, FRET, polarization, refraction, etc. Exemplary optical detectors may be photomultipliers, phototubes, ionization detectors, active pixel sensors, phototransistors, photodiodes, quantum dot photoconductors or photodiodes, photovoltaic cells, semiconductor detectors, thermal detectors, photochemical detectors, and the like.

[0039] Electrochemically detectable labels can be determined by electrode configurations such as thin-layer electrodes suitable for voltammetric and typically amperometric measurements. The detector typically comprises at least two electrodes, at least one of which is the so-called working electrode. The electrodes can be made of all conventional electrode materials such as metals, noble metals, alloys or graphite, preferably noble metals such as gold or palladium or graphite. The various electrodes of the sensor can be made of the same or different materials. More typically, the electrodes are made of palladium.

[0040] It will be appreciated that the presence or absence or amount of the detected detectable label may then be transmitted to an evaluation unit, which may preferably include a data processing element such as a computer having an implemented algorithm for determining the presence or amount of an analyte in the sample based on the presence, absence or amount of the detected detectable label(s).

[0041] Such implementation algorithms may evaluate the measured signal induced from the detectable label(s) in the complex of the first binding agent, the test substance and the second binding agent for signal intensity, signal duration and other predefined parameters. Based on said evaluation, truly positive signals can be identified and verified, and noise signals can be identified and ignored for further evaluation. The skilled person is well aware which algorithms can be used and how they can be implemented in the device of the present invention. Preferably, the formation of the complex of the first binding agent, the test substance and the second binding agent is detected by measuring the intensity and / or duration of the signal induced by at least one detectable label. More preferably, said signal can be detected over a predefined characteristic period. The characteristics of the true signal are preferably the formation within a first time window according to one or more association constants of the complex comprising the first binding agent, the test substance and the second binding agent, the persistence of the predefined time window, and the dissociation within a second time window according to one or more dissociation constants of the complex comprising the first binding agent, the test substance and the second binding agent. Typically, the true signal can be detected for a significantly longer time than the signal induced by at least one detectable label contained by a freely moving first binding agent randomly passing through the anchor layer in proximity to an immobilized second binding agent, the latter of which will generate only a short noise signal.

[0042] Computer-implemented algorithms, in particular artificial intelligence algorithms, machine learning algorithms, etc., may be applied for accurate evaluation of the signal of the detectable label detected according to the method of the present invention and for determining the test substance based thereon.

[0043] It will be further understood that even single complex formation can be determined by the method of the present invention if the signal from the detectable label of the area of ​​the solid support containing a single or a predetermined amount of the second binding agent can be measured. This can be achieved by using wells, beads or other predetermined areas in which a single or a predetermined number of second binding agents can be immobilized and the signal from the detectable label can be determined separately. Such separate detection is also possible by using magnetically responsive detectors such as magnetic tunnel junctions or magnetic spin valves (see, for example, U.S. Pat. No. 5,981,297; Fernandes 2020, Nanomedicine: Nanotechnology, Biology, and Medicine 30, 102287 or Denmark 2019, Journal of Electronic Materials (48): 4749-4761), or nanopore-based detection techniques.

[0044] In step (a) of the method of the present invention, a sample containing or suspected of containing the analyte to be determined is contacted with a biosensor as specified in detail elsewhere in this specification.

[0045] As used herein, the term "contacting" refers to bringing the aforementioned components into physical proximity so that the first and / or second binding agent, if present in the sample, can bind to the test substance. It will be understood that the binding of the first binding agent to the test substance and the binding of the second binding agent to the test substance or to a complex of the first binding agent and the test substance, may require time and application of appropriate conditions. The skilled person is well aware of what time is required and what conditions need to be applied to achieve binding. For example, the sample and the binding agent may be dissolved or mixed with a buffer that adjusts the salt concentration and / or the pH value. It will be understood that suitable buffers and other auxiliary components that may be applied depend on the binding agent used and the chemical nature of the test substance to be determined.

[0046] In step (a) of the method of the invention, the first binding agent and the test substance are also contacted with a second binding agent immobilized on a solid support, which is capable of specifically binding to the test substance or the first binding agent when bound to the test substance. Typically, an arrangement on the sensor element comprising the first binding agent attached to an anchor layer and the immobilized second binding agent is provided prior to application of the sample, although the sample may be contacted with the first binding agent and applied to an anchor layer comprising the second binding agent immobilized on a solid support.

[0047] The first binding agent applied in the method of the present invention must be bound to a detectable label or may be linked to a linking agent capable of covalently or reversibly binding to at least one detectable label.

[0048] As a result of the above-mentioned activity occurring during step (a) of the method of the present invention, the test substance is bound by a first binding agent that includes a detectable label that is covalently attached or, optionally, reversibly attached via a linker molecule. The complex of the test substance and the first binding agent migrates through the anchor layer and is bound by a second binding agent immobilized on the solid support, generating a complex that includes the first binding agent, the test substance, and the second binding agent ("lock-on"). Due to molecular binding kinetics, this complex remains in close proximity to the fixed position of the second binding molecule on the solid support for a characteristic time window until it dissolves ("lock-off").

[0049] In step (b) of the method of the present invention, a complex of the above-mentioned first binding agent, the test substance and the second binding agent is detected, thereby determining the test substance.

[0050] As used herein, the term "detecting" relates to identifying the presence, absence and / or amount of a labeled molecule covalently bound to a solid support by measuring a physical, chemical, physicochemical and / or biological property of said labeled molecule. It will be understood that a detectable label typically generates a signal that can be measured. The signal intensity and / or duration typically indicates the amount of detectable labeled molecule present on the solid support. Depending on the chemical nature of the detectable label, the signal may be an active signal, such as the emission of light or other radiation, or a passive signal, i.e. a signal induced as a response to an external stimulus, such as applying an electromagnetic field, radiation, etc. The skilled person is well aware how the measurement of such a signal induced by a detectable labeled molecule can be performed, and it will be understood that the detection method used according to the method of the present invention depends on the detectable label used. Preferred measurement methods according to the present invention also include chemiluminescence measurements, fluorescence measurements, FRET measurements, electrochemiluminescence measurements, mass measurements including mass spectrometry, magnetic or electric field based measurements using e.g. ChemFET or other electrode arrangements, radioimmunoassay measurements, dissociation enhanced lanthanide fluoroimmunoassay (DELFIA) measurements, scintillation proximity assay measurements, quantum dot technology measurements, turbidimetric measurements or nephelometric measurements.

[0051] The detectable label may also be detected indirectly, i.e. by using an additional labeling molecule capable of specifically binding to the immobilized detectable label on the solid support. Such additional labeling molecule may be applied to the solid support after step (a) has been performed and before step (b). It will be understood that upon specific binding of one or more of said additional labeling molecules to the detectable label(s) on the solid support, said additional labeling molecule may be detected in step (b) by measuring a physical, chemical, physicochemical and / or biological property of said labeling molecule. Furthermore, additional molecules capable of specifically binding to the immobilized detectable label may also serve as adapters for other labeled molecules. Suitable molecules that may function as additional labeling molecules or adapter molecules may also be antibodies, aptamers or other molecules that allow specific binding of a target as described elsewhere herein. Suitable labels for such additional molecules are those described elsewhere herein for detectable labels.

[0052] In the method of the present invention, the first and second binding agents can be used to determine different analytes. In such a case, it will be understood that when complexes to such different analytes are formed, each complex must have a different detectable label. The different detectable labels can be preferably detected by using detectable physical, chemical, physicochemical and / or biological properties that are different between the detectable labels.

[0053] In a particular embodiment of the method of the present invention, the solid support is a well or a predetermined subdivided detection area. Preferably, each well or predetermined subdivided detection area has a predetermined amount of a second binding agent immobilized thereon. The method is particularly useful for determining the test substance and includes determining the amount of the test substance. Typically, the amount is determined by counting wells or predetermined subdivided detection areas in which complexes are formed. According to this particular embodiment, the presence or absence or amount of the test substance is detected for each well or predetermined subdivided area. The number of positive wells or predetermined subdivided areas is then determined. A positive well or predetermined subdivided area is one that exhibits a signal characteristic for suitable complex formation as described elsewhere herein with a predetermined intensity or duration, for example a measurable signal above a predetermined intensity threshold and / or over a predetermined time window. Based on the positive wells or predetermined subdivided areas and the predetermined amount of the second binding agent immobilized in each of the wells or predetermined subdivided areas, the amount of the test substance can be determined, for example by calculation. For example, if each well or predetermined subdivided area ideally contains one second binding agent, the presence of one molecule of test substance in the sample will generate one complex. Thus, when the presence of a complex in a well or on a predetermined subdivided area is determined, this presence reflects the presence of one test substance present in the sample. Thus, the use of a predetermined amount of second binding agent on an isolated article such as a well or any other predetermined subdivided area allows for quantitative or semi-quantitative determination of the test substance molecules present in the sample. This technology is also called "digital" detection.

[0054] Advantageously, according to the present invention, it has been found that the use of a first binding agent that is essentially restricted in its migration to the second dimension by association with an anchor layer, such as a lipid layer, in a sandwich assay format improves sensitivity and suppresses undesirable background noise. According to the present invention, an immune complex comprising a first binding agent, e.g. an antibody, an analyte to be determined, and a second binding agent, e.g. an antibody, forms with a specific kinetic, persists for a specific time window, and dissolves with a specific kinetic ("lock-on, lock-off" principle). Also, depending on the molecular configuration used, a signal of some intensity can be expected. Thus, in the method of the present invention, kinetic measurements of complex formation and complex dissolution are determined rather than single formation events. This allows dynamic real-time measurements and may even eliminate the need for washing steps and other treatments. The use of an anchor layer also reduces events that cause noise on the solid support. Depending on the nature of the detector, it is possible to essentially isolate the complex formation event. For example, when a magnetic response detector or a confocal detector is applied in conjunction with a single second binding agent that initiates complex formation in the presence of an analyte molecule, it will be understood that the complex formation is representative of said analyte molecule present in the sample. In such a digital format, the number of signals received by individual detectors may be used to count the analyte molecules present in the sample.

[0055] Thanks to the present invention, it is possible to determine molecular analytes in a sample with improved sensitivity and specificity. Moreover, even single molecule events can be determined.

[0056] The present invention also provides an apparatus for determining an analyte suspected to be present in a sample, comprising: (i) an anchor layer present on a solid support; (ii) a first binding agent that is affixed to the anchor layer and that can specifically bind to the test substance, the first binding agent comprising at least one detectable label; (iii) a second binding agent immobilized on the solid support, the second binding agent being capable of specifically binding to the test substance when bound to the first binding agent; The present invention relates to an apparatus comprising a sensor element comprising:

[0057] The term "device" as used herein relates to a system comprising the above-mentioned components operatively linked to each other to allow the determination of an analyte by the method of the present invention.

[0058] The sensor element according to the invention may be arranged in a reaction zone contained in the device. The reaction zone may directly allow sample application or may be connected to a loading zone where the sample is applied. In the latter case, the sample may be actively or passively transported to the reaction zone via a connection between the loading zone and the reaction zone. Furthermore, the reaction zone is also connected to a detector. Suitable detectors and detection techniques are described in detail elsewhere in this specification. The connection between the reaction zone and the detector must be such that the detector is capable of detecting the detectable label covalently bound to the solid support. The suitable connection depends on the technique used to measure the presence or amount of the detectable label(s). For example, for optical detection, light propagation may be required between the detector and the reaction zone, and for electrochemical determination, a fluid connection may be required, for example between the reaction zone and an electrode. The device according to the invention preferably further comprises a detector for detecting a signal induced by the detectable label, said detector being arranged below the solid support.

[0059] The present invention generally relates to the use of the above-mentioned device of the present invention for determining, within said sample, an analyte suspected to be present in said sample.

[0060] The present invention further provides a kit for determining an analyte suspected to be present in a sample, comprising a device of the present invention.

[0061] As used herein, the term "kit" refers to a collection of components necessary to carry out the method of the invention, including the device of the invention. Typically, the components of the kit are provided in separate containers or in a single container. The container also typically includes instructions for carrying out the method of the invention. These instructions may be in the form of a manual or may be provided by a computer program code that, when executed on a computer or data processing device, is capable of carrying out or supporting the determination of the test substance referred to in the method of the invention. The computer program code may be provided on a data storage medium or device, such as an optical storage medium (e.g., compact disc) or directly on the computer or data processing device, or may be provided in a download format, such as a link to an accessible server or cloud. Furthermore, the kit may typically include a test substance solution with a standardized amount or other reference amount for calibration or validation. The kit according to the invention may also include further components necessary to carry out the method of the invention, such as washing solutions, solvents and / or reagents necessary for the detection of the detectable label. Furthermore, it may partially or entirely include the device of the invention.

[0062] The following embodiments are specific preferred embodiments contemplated in accordance with the present invention, all definitions apply mutatis mutandis to the explanation of terms above.

[0063] Embodiment 1. A method for determining an analyte suspected to be present in a sample, comprising: (a) placing the sample in a sensor element, (i) an anchor layer present on a solid support; (ii) a first binding agent that is affixed to the anchor layer and that can specifically bind to the test substance, the first binding agent comprising at least one detectable label; (iii) a second binding agent immobilized on the solid support, the second binding agent being capable of specifically binding to the test substance when bound to the first binding agent; for a time and under conditions that allow specific binding of the analyte suspected of being present in the sample to the first binding agent and specific binding of the second binding agent to the analyte bound to the first binding agent; (b) detecting the formation of a complex between the first binding agent, the test substance, and the second binding agent, thereby determining the test substance; and A method comprising:

[0064] Embodiment 2. The method of embodiment 1, wherein the formation of the complex of the first binding agent, the test substance and the second binding agent is detected by measuring the intensity and / or duration of a signal induced by the at least one detectable label.

[0065] Embodiment 3. The method of embodiment 2, wherein the signal can be detected for a predetermined characteristic period of time.

[0066] Embodiment 4. The method of any one of embodiments 1 to 3, wherein the test substance is a protein, a peptide, a virus, a bacterial cell, or a small molecule.

[0067] Embodiment 5. The method of any one of embodiments 1 to 4, wherein the anchor layer is a lipid layer or a lipid bilayer.

[0068] Embodiment 6. The method of any one of embodiments 1 to 5, wherein the first binding agent is anchored to the anchor layer via an anchor molecule.

[0069] Embodiment 7 The method of embodiment 6, wherein the anchor molecule is a lipid.

[0070] Embodiment 8. The method of embodiment 6 or 7, wherein the anchor molecule is linked to at least one first binding agent via a linker molecule, preferably a circular DNA molecule.

[0071] Embodiment 9. The method of any one of embodiments 1 to 8, wherein the amount of the first binder exceeds the amount of the second binder by 10 to 100 times, 20 to 80 times, 30 to 70 times, or 40 to 60 times.

[0072] Embodiment 10. The method of any one of embodiments 1 to 9, wherein the first binding agent is selected from the group consisting of an antibody or fragment thereof, an aptamer, a receptor molecule or fragment thereof, and a ligand molecule or fragment thereof.

[0073] Embodiment 11. The method of any one of embodiments 1 to 10, wherein the second binding agent is selected from the group consisting of an antibody or fragment thereof, an aptamer, a receptor molecule or fragment thereof, and a ligand molecule or fragment thereof.

[0074] Embodiment 12. The method of any one of embodiments 1 or 11, wherein determining the test substance comprises determining the presence, absence, or amount of the test substance.

[0075] Embodiment 13. The method of any one of embodiments 1 to 12, wherein the detectable label is selected from the group consisting of a fluorescent label, a chemiluminescent label, a radioactive label, a magnetic label, and an electrochemical label.

[0076] Embodiment 14. The method of any one of embodiments 1 to 13, wherein the sensor element comprises a detector for detecting a signal induced by the detectable label, the detector being positioned below the solid support, and the detector being capable of selectively detecting a signal in direct proximity to the second binding element.

[0077] Embodiment 15. The method of any one of embodiments 1 to 15, wherein the sample is a biological sample, preferably a body fluid or a biopsy sample.

[0078] Embodiment 16. An apparatus for determining an analyte suspected to be present in a sample, comprising: (i) an anchor layer present on a solid support; (ii) a first binding agent that is affixed to the anchor layer and that can specifically bind to the test substance, the first binding agent comprising at least one detectable label; (iii) a second binding agent immobilized on the solid support, the second binding agent being capable of specifically binding to the test substance when bound to the first binding agent; An apparatus comprising a sensor element comprising:

[0079] Embodiment 17. The device of embodiment 16, wherein the device further comprises a detector for detecting a signal induced by the detectable label, the detector being positioned below the solid support.

[0080] Embodiment 18. Use of the device of embodiment 16 or 17 for determining, in said sample, an analyte suspected to be present in the sample.

[0081] Embodiment 19. A kit for determining an analyte suspected to be present in a sample, comprising a device according to embodiment 16 or 17.

[0082] All references cited herein are hereby incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification. [Brief description of the drawings]

[0083] [Figure 1] Schematic of the sensor device. On the left side, the lock state is shown, characterized by a complex of the first binder, the analyte and the second binder. The signal induced by the detectable label can be measured by dark-field excitation. Such a signal shall have a characteristic lock-on, lock-off signature. On the right side, the source of background noise is shown. The background noise arises from the first binder, which is suspended in the anchor layer and may or may not already be bound to the analyte molecule. The background signal shall typically not be very strong and not persist over a significant time window. [Diagram 2]A schematic diagram of a circular I-DNA molecule is shown, which is anchored to an anchor layer, e.g. a lipid layer, via three anchor molecules attached to it, and which comprises three antibody fragments as first binding agents according to the invention, with seven detectable labels, e.g. fluorescent labels, attached to the antibodies and to the circular I-DNA.

Claims

1. 1. A method for determining the presence, absence, or amount of a test substance suspected of being present in a sample, comprising: (a) subjecting the sample to a sensor element, (i) an anchor layer present on a solid support; (ii) a first binding agent capable of specifically binding to the test substance, anchored to the anchor layer and comprising at least one detectable label, wherein the first binding agent is associated with or within the anchor layer, thereby restricting movement of the first binding agent to essentially two dimensions in space; and (iii) a second binding agent immobilized on the solid support, which is capable of specifically binding to the test substance when bound to the first binding agent; and for a time and under conditions that allow specific binding of the analyte suspected of being present in the sample to the first binding agent, and specific binding of the second binding agent to the analyte bound to the first binding agent; (b) detecting the formation of a complex between the first binding agent, the test substance, and the second binding agent, thereby determining the test substance; and A method comprising:

2. 2. The method of claim 1, wherein the formation of the complex of the first binding agent, the test substance, and the second binding agent is detected by measuring the intensity and / or duration of a signal induced by the at least one detectable label.

3. The method of claim 2 , wherein the signal can be detected over a predetermined period of time.

4. The method according to any one of claims 1 to 3, wherein the anchor layer is a lipid layer or a lipid bilayer.

5. 5. The method of claim 1, wherein the first binding agent is anchored to the anchor layer via an anchor molecule, wherein the first binding agent is associated with or within the anchor layer, thereby restricting movement of the first binding agent to essentially two dimensions in space.

6. 6. The method of any one of claims 1 to 5, wherein the amount of the first binder exceeds the amount of the second binder by 10 to 100 times, 20 to 80 times, 30 to 70 times, or 40 to 60 times.

7. The method of any one of claims 1 to 6, wherein the first binding agent is selected from the group consisting of an antibody or fragment thereof, an aptamer, a receptor molecule or fragment thereof, and a ligand molecule or fragment thereof.

8. The method of any one of claims 1 to 7, wherein the second binding agent is selected from the group consisting of an antibody or fragment thereof, an aptamer, a receptor molecule or fragment thereof, and a ligand molecule or fragment thereof.

9. The method of any one of claims 1 to 8, wherein the detectable label is selected from the group consisting of a fluorescent label, a chemiluminescent label, a radioactive label, a magnetic label and an electrochemical label.

10. 10. The method of any one of claims 1 to 9, wherein the sensor element comprises a detector for detecting a signal induced by the detectable label, the detector being positioned below the solid support, and the detector being capable of selectively detecting a signal in proximity to the second binding agent.

11. 1. An apparatus for determining the presence, absence, or amount of a test substance suspected of being present in a sample, comprising: (i) an anchor layer present on a solid support; (ii) a first binding agent capable of specifically binding to the test substance, anchored to the anchor layer and comprising at least one detectable label, wherein the first binding agent is associated with or within the anchor layer, thereby restricting movement of the first binding agent to essentially two dimensions in space; and (iii) a second binding agent immobilized on the solid support, which is capable of specifically binding to the test substance when bound to the first binding agent; and 10. An apparatus comprising: a sensor element comprising:

12. 12. The device of claim 11, further comprising a detector for detecting a signal induced by the detectable label, the detector being positioned below the solid support.

13. 13. Use of the device of claim 11 or 12 to determine the presence, absence or amount of an analyte suspected to be present in a sample.

14. 13. A kit for determining the presence, absence or amount of an analyte suspected to be present in a sample, comprising the device of claim 11 or 12.

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