Method for immunosensing on a lipid layer

Abstract

To eliminate and improve the risk of false positive results in sandwich ELISA.SOLUTION: A method for determining an analyte suspected of being present in a sample, the method comprising: (i) an anchor layer present on a solid support; (ii) a first binding agent capable of specifically binding to the analyte, wherein the first binding agent is anchored to the anchor layer and comprises at least one detectable label; (iii) a second binding agent capable of specifically binding to the analyte when bound to the first binding agent, wherein the second binding agent is immobilized on a solid support; and contacting under conditions and for a time allowing 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, and detecting formation of a complex of the first binding agent, the analyte and the second binding agent, whereby the analyte is determined.SELECTED DRAWING: Figure 1

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, the method comprising contacting the sample with a sensor element comprising: i) an anchor layer present on a solid support; ii) a first binding agent anchored to the anchor layer and comprising at least one detectable label, capable of specifically binding to the analyte; 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 between the first binding agent, the analyte, and the second binding agent, thereby determining the analyte. Furthermore, 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 the sample in the sample are provided. 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 a variety of diagnostic purposes. Several setups for immunoassays have been developed. One of the most common immunoassays is the enzyme-linked adsorbent immunoassay (ELISA).

[0003] In ELISA, a liquid sample containing or suspected of containing an analyte of interest is applied to a stationary solid phase with specific binding properties due to the presence of antibodies or antibody-like molecules, such as aptamers. After sample application, multiple reagents are sequentially added, incubated, and washed to initiate and terminate the analytical detection reaction. After all these steps are performed, a physical or chemical property of the setup, usually the liquid phase, changes and can be detected. Optical changes, such as color development due to the product of an enzymatic reaction, typically occur in the final liquid phase. These changes correlate with the presence or abundance of the analyte of interest present in the investigated sample. Quantitative readouts are typically based on the detection of transmitted light intensity, usually by spectrophotometry, which involves quantifying the transmission of light of a specific wavelength through a liquid. The sensitivity of detection depends on signal amplification during the analytical reaction. Because enzymatic reactions are highly amplified processes, the signal is generated by an enzyme linked to a detection reagent at a constant rate, allowing for accurate quantification.

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

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

[0006] However, given the fragile immune complexes required for signal generation and the various components used in sandwich-format ELISAs, there is a high possibility of undesired binding of the detection antibody 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. Furthermore, due to various drawbacks of such fragile multi-component assay formats, they are limited in terms of sensitivity and specificity. Summary of the Invention

[0007] The technical problem underlying the present invention can 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 this specification.

[0008] Accordingly, the present invention provides a method for determining an analyte suspected to be present in a sample, comprising the steps of: (a) subjecting the sample to a sensor element, (i) an anchor layer present on a solid support; (ii) a first binding agent anchored to the anchor layer, the first binding agent comprising at least one detectable label, and capable of specifically binding to the test substance; (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 The present invention relates to a method, comprising: DETAILED DESCRIPTION OF THE INVENTION

[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 other than 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 an open-ended meaning refer to a situation in which one or more other features are present in the described embodiment in addition to the features introduced by these terms.

[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 mentioned following this term can be used in accordance with the present invention. For example, if this term indicates that at least one item should be used, this can 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 this term refers, those skilled in the art will understand what upper limit, if any, this term can 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. Qualitative determination aims to determine the presence or absence of a test substance in a sample, while quantitative determination aims to determine the amount of the test substance. Quantitative determination, i.e., determination of the amount, includes determining an absolute amount (e.g., total amount by weight or number of molecules present in a sample) or a relative amount (e.g., amount relative to sample volume (concentration)) or a classification such as a score (e.g., "high," "low," etc.). Typically, determining a test substance involves 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 molecules or agents may have a size and / or structure that allows for binding of the first and second binding agents referred to herein. Furthermore, there may be an upper size limit, as the first and second binding agents and linking agents must be able to perform their functions as described in detail elsewhere herein. Typically, the analyte referred to herein is a protein, peptide, nucleic acid, e.g., DNA or RNA, or a small molecule such as a lipid or metabolite, e.g., a polyketide, including flavonoids and isoflavonoids, an isoprenoid, e.g., a terpene, a sterol, a steroid, a carotenoid, a xanthophyll, a carbohydrate, a phenylpropanoid, an alkaloid, a benzenoid, an indole, a porphyrin, a hormone, a vitamin, a cofactor, a lignin, a glucosinolate, a purine, a pyrimidine, a nucleoside, a nucleotide, an alcohol, an alkane, an alkene, an alkyne, an aromatic compound, a ketone, an aldehyde, a carboxylic acid, an ester, an amine, an imine, an amide, a cyanide, an amino acid, a thiol, a thioester, a phosphate ester, a sulfate ester, a thioether, a sulfoxide, or an ether. Small molecules may be, for example, toxins. However, the methods of the present invention can also be used to determine viruses or even bacterial cells as analytes. The analyte determined by the present invention may also be a molecule present in an environmental sample and useful, for example, as an indicator of environmental pollution, agriculture, or other environmental conditions. Typically, the test agent is a protein, peptide, virus, bacterial cell or small molecule, preferably a small molecule toxin.

[0016] As used herein, the term "sample" refers to any portion or aliquot of a composition containing or suspected of containing 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 containing an organism, such as cultured cells. Typically, the biological sample is investigated by the method of the present invention for medical purposes, such as diagnosing or predicting a disease or condition. The sample may also be an environmental sample or an artificial sample. An environmental sample may be derived from any non-biological natural source, for example, 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, for example, 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, the sample according to the method of the present 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 with an anchor layer. Furthermore, the sensor element further includes a molecular configuration as described above, i.e., a first binding agent anchored to the anchor layer and comprising at least one detectable label, capable of specifically binding to the analyte, and a second binding agent immobilized on the solid support, capable of specifically binding to the analyte when bound to the first binding agent.

[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 in accordance with 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. Typical lipid layers or lipid bilayers suitable for use as anchor layers according to the present invention may include, for example, phospholipid layers or phospholipid bilayers. The 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" refers to a solid composition of matter that can serve as a basis for immobilizing molecules, particularly second binding agents. Solid supports can include inorganic or organic compounds, or both. Typically, inorganic compounds suitable for solid supports can 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. Solid supports can also include conductive compounds such as metals or graphite. Preferably, such solid supports can be or include electrodes, magnetic tunnel junctions, semiconductors, plasmonic resonators, or any type of electrical circuitry. Alternatively, solid supports can include organic compounds, such as cross-linked polymers. Non-limiting examples of suitable cross-linked polymers can be selected from the group consisting of polyamides, polyethers, polystyrenes, and mixtures thereof. Those skilled in the art are well aware of how to select an appropriate solid support based on the type of sample to be investigated, the method envisioned for detecting 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 test substance, the first binding agent being anchored to the anchor layer, and at least one detectable label.

[0021] The term "first binding agent" as referred to herein refers to a molecule that can specifically bind to a test substance, i.e., a molecule that 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 capable of specifically binding to the desired test substance can be an antibody. The antibody as a binding agent referred to in accordance with the present invention preferably includes all types of antibodies that 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 is still capable of specifically binding 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 the 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). Those skilled in the art are well aware of how 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, molecules that can be used as first binding agents capable of specifically binding to a desired analyte can be aptamers. Aptamers as binding agents according to the present invention can be oligonucleotide 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 aptamers are engineered by repeated selection or the so-called systematic evolution of ligands by exponential enrichment (SELEX) technique. Peptide aptamers typically contain a variable peptide loop attached at both ends to a protein scaffold. This dual structural constraint increases the binding affinity of peptide aptamers into the nanomolar range. The variable peptide loop length is preferably 10 to 20 amino acids, and the scaffold can be any protein with improved solubility and compactness, such as 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 able to specifically bind 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 comprising said aptamer fragment as well as other moieties such as a linker moiety or an adapter molecule. 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 referred to as a binding agent according to 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 binding agents according to the present invention for the ligand as a test substance, or molecules derived from the ligand but still capable of being bound by the receptor molecule or its fragment, such as antagonistic or agonistic mutants of the ligand. Preferably, the receptor molecule envisioned as a binding agent according to the present invention can be a transmembrane receptor protein (such as a G protein-coupled receptor, e.g., a metabolic receptor, an enzyme-linked receptor, e.g., a receptor tyrosine kinase, e.g., a growth factor receptor, an immune receptor, e.g., a viral receptor, a cell surface antigen, a T cell receptor, e.g., CD4, CD3, or CD8), an MHC protein, a cell adhesion molecule (such as an integrin, cadherin, selectin, or syndecan), a neuronal receptor, or a pathogen receptor (such as a Toll-like receptor). The receptor molecule may also be a nuclear receptor protein, such as a nuclear hormone receptor, e.g., 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 to be measured or a fragment thereof. Furthermore, specific binding can be tested by techniques well known in the art, such as plasmon surface resonance measurement.

[0025] More preferably, the molecule that can be used as the first binding agent capable of specifically binding to the desired test substance may be a ligand molecule. The ligand molecule referred to as a binding agent 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 the 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 the test substance, or a molecule derived from the receptor molecule but still capable of being 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 specific antibodies in a biological sample. Preferably, the ligand molecules envisaged as binders according to the present invention may be peptide hormones, neurotransmitters, growth factors (such as angiopoietin, BMP, neutrophil factor, EGF, epiphrin, EPO, FGF, GDNF, GDF, insulin or insulin-like growth factor), TGF, neutrophil, 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. Those skilled in the art are 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 may be a designed ankyrin repeat protein (DARPin). DARPins are genetically 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 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, and more typically, the first binding agent is an antibody or fragment thereof or an aptamer.

[0028] Furthermore, the first binding agent according to the present invention is anchored to an anchor layer. The anchored first binding agent referred to in accordance with the present invention is typically associated with or within the anchor layer. Therefore, it is typically limited in its movement to essentially two dimensions in space.

[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 of the present invention depends on the nature of the anchor layer and can be easily selected by those skilled in the art, and the anchor molecule can typically be linked to at least one, preferably multiple first binding agents via a linker molecule.A suitable linker molecule allows multiple first binding agents and at least one anchor molecule to be attached.Preferably, the suitable linker molecule can be a circular DNA molecule.Therefore, multiple first binding agents can be anchored to the anchor layer via the same anchor molecule.

[0030] As used herein, the term "detectable label" refers to a molecule that exhibits a physical or chemical property that can be detected in the context of the methods of the present invention. Preferably, the 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; detectable radioactivity; or a chemical property, such as the ability to perform or catalyze a specific chemical reaction. A detectable label may reversibly or permanently bind to a first binding agent. To this end, a detectable label may be a molecule that can reversibly bind to a first binding agent, or a molecule or moiety that is already part of the first binding agent. Typical 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 detecting such enzymatically detectable labels include diaminobenzidine (DAB), 3,3'-5,5'-tetramethylbenzidine, and NBT-BCIP (4-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate). Appropriate enzyme-substrate combinations may produce colored reaction products, fluorescence, or chemiluminescence, which can be measured by methods known in the art (e.g., using a light-sensitive film or an appropriate camera system). Typical 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. Typical radioactive labels include 35S, 125I, 32P, 33P, etc. Radioactive labels can be detected by any known and appropriate method, such as a light-sensitive film or a phosphor imager. Typical 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, both permanent and reversible linkages are envisioned. Reversible linkages can be achieved, for example, by using a biotin-streptavidin-based molecular adapter system, which is well known in the art.

[0032] The sensor also includes a second binding agent capable of specifically binding to the test substance 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 refers to the test substance or a molecule capable of specifically binding to the test substance when bound to the 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, the 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 antibodies or fragments thereof, aptamers, receptor molecules or fragments thereof, and ligand molecules or fragments thereof prepared according to the first binding agent apply mutatis mutandis to the second binding agent.

[0034] The second binding agent is to be immobilized on a solid support. The immobilization of the second binding agent is preferably permanent. 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 upon immobilization, the second binding agent cannot move its position within or outside the anchor layer. However, the second binding agent must be able to undergo 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 between the second binding agent and the test substance and first binding agent should preferably be a reversible interaction. The association rate is typically greater than the dissociation rate, and it is envisioned 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 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 disposed below the solid support, and the detector is directly adjacent to the second binding element of the gear so as to selectively detect the signal. Depending on the detectable label, different detection techniques can be applied.

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

[0039] Electrochemically detectable labels can be determined by electrode configurations such as thin-layer electrodes suitable for voltammetric and typically amperometric measurements. A detector typically includes at least two electrodes, at least one of which is a so-called working electrode. The electrodes can be made of any conventional electrode material, such as metals, noble metals, alloys, or graphite, and are preferably made of 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 implemented an 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 an implementation algorithm 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 predetermined parameters. Based on the evaluation, truly positive signals can be identified and verified, and noise signals can be identified and ignored for further evaluation. Those skilled in the art will be fully aware of which algorithms can be used and how they can be implemented in the device of the present invention. Preferably, the formation of a 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, the signal can be detected over a predetermined characteristic period. The characteristics of a true signal are preferably the formation of a complex comprising the first binding agent, the test substance, and the second binding agent within a first time window according to one or more association constants, the persistence of the predetermined time window, and the dissociation of the complex comprising the first binding agent, the test substance, and the second binding agent within a second time window according to one or more dissociation constants. 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, which will generate only a short noise signal.

[0042] Computer-implemented algorithms, particularly artificial intelligence algorithms, machine learning algorithms, etc., may be applied to accurately evaluate the signal of the detectable label detected according to the method of the present invention and to determine the test substance based thereon.

[0043] Furthermore, it will be appreciated that even single complex formation can be determined by the methods of the present invention, provided that the signal from the detectable label of a region of the solid support containing a single or predetermined amount of second binding agent can be measured. This can be achieved by using wells, beads, or other predetermined regions in which a single or predetermined number of second binding agents can be immobilized and the signals from the detectable labels can be separately determined. Such separate detection can also be achieved by using magnetically responsive detectors, such as magnetic tunnel junctions or magnetic spin valves (see, e.g., 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 technologies.

[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 herein.

[0045] As used herein, the term "contacting" refers to bringing the above 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 a complex of the first binding agent and the test substance may require time and the application of appropriate conditions. Those skilled in the art are well aware of what time is required and what conditions need to be applied to achieve binding. For example, the sample and binding agent may be dissolved or mixed with a buffer that adjusts the salt concentration and / or pH value. It will be understood that the appropriate buffer and other auxiliary components that may be applied depend on the chemical properties of the binding agent used and the test substance to be determined.

[0046] In step (a) of the method of the present invention, the first binding agent and the test substance are also contacted with a second binding agent immobilized on a solid support, which can specifically bind 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 anchored to an anchor layer and the immobilized second binding agent is provided before application of the sample, although the sample may also 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 that is capable of covalently or reversibly binding to at least one detectable label.

[0048] As a result of the above-described activity occurring during step (a) of the method of the present invention, the test substance is bound by a first binding agent containing a detectable label covalently attached or, optionally, reversibly attached via a linker molecule. The complex of the test substance and the first binding agent migrates within the anchor layer and is bound by a second binding agent immobilized on the solid support, producing a complex containing 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, the complex of the 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 the physical, chemical, physicochemical and / or biological properties 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 indicate the amount of detectably 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 the application of an electromagnetic field, radiation, etc. Those skilled in the art are well aware how the measurement of such a signal induced by a detectably labeled molecule can be performed, and it will be understood that the detection method used according to the method of the present invention will depend 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 configurations, radioimmunoassay measurements, dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) measurements, scintillation proximity assay measurements, quantum dot technology measurements, turbidimetric measurements or nephelometric measurements.

[0051] Detectable labels can also be detected indirectly, i.e., by using additional labeled molecules capable of specifically binding to the detectable labels immobilized on the solid support. Such additional labeled molecules can 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 the additional labeled molecules to the detectable label(s) on the solid support, the additional labeled molecules can be detected in step (b) by measuring the physical, chemical, physicochemical, and / or biological properties of the labeled molecules. Furthermore, additional molecules capable of specifically binding to the immobilized detectable labels can also serve as adapters for other labeled molecules. Suitable molecules that can function as additional labeled or adapter molecules can also be antibodies, aptamers, or other molecules that enable specific binding of targets, 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 may 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 preferably be detected by using detectable physical, chemical, physicochemical and / or biological properties that differ between the detectable labels.

[0053] In a specific 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 a 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 specific 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 of appropriate complex formation, as described elsewhere herein, having a predetermined intensity or duration, e.g., a measurable signal exceeding 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 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. Therefore, 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. Therefore, by using a predetermined amount of second binding agent on an isolated article such as a well or any other predetermined subdivided area, quantitative or semi-quantitative determination of the test substance molecules present in the sample is possible. This technology is also called "digital" detection.

[0054] Advantageously, the present invention has been found to improve sensitivity and suppress undesirable background noise by using a first binding agent whose migration in the second dimension is essentially restricted by association with an anchor layer, such as a lipid layer, in a sandwich assay format. 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 specific kinetics, persists over a specific time window, and dissolves with specific kinetics (the "lock-on, lock-off" principle). Furthermore, depending on the molecular configuration used, a certain degree of signal intensity can be expected. Therefore, in the method of the present invention, kinetic measurements of complex formation and complex dissolution are determined rather than single formation events. This allows for dynamic, real-time measurements and may even eliminate the need for washing steps and other processing. The use of an anchor layer also reduces noise-causing events on the solid support. Depending on the nature of the detector, it is possible to essentially isolate complex formation events. For example, when a magnetically responsive 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 complex formation represents the presence of said analyte molecule 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 analytes in a sample with improved sensitivity and specificity. Furthermore, 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 anchored to the anchor layer, the first binding agent comprising at least one detectable label, and capable of specifically binding to the test substance; (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 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 for the determination of an analyte by the method of the present invention.

[0058] The sensor element according to the present invention can be disposed in a reaction zone included in a 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 herein. The connection between the reaction zone and the detector must be such that the detector can detect the detectable label covalently bound to the solid support. The appropriate connection depends on the technique used to measure the presence or amount of the detectable label(s). For example, for optical detection, light transmission may be required between the detector and the reaction zone, while for electrochemical determination, a fluid connection may be required, for example, between the reaction zone and an electrode. The device according to the present invention preferably further comprises a detector for detecting a signal induced by the detectable label, said detector being disposed below the solid support.

[0059] The present invention generally relates to the use of the above-described 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 methods of the present invention, including the device of the present invention. Typically, the components of the kit are provided in separate containers or in a single container. The container also typically contains instructions for carrying out the methods of the present invention. These instructions may be in the form of a manual or may be provided by computer program code that, when executed on a computer or data processing device, can perform or support the determination of the test substance referred to in the methods of the present invention. The computer program code may be provided on a data storage medium or device, such as an optical storage medium (e.g., a compact disc) or directly on the computer or data processing device, or may be provided in a downloadable format, such as a link to an accessible server or cloud. Furthermore, the kit will typically include a test substance solution having a standardized amount or other reference amount for calibration or validation. The kit according to the present invention may also include additional components necessary to carry out the methods of the present invention, such as washing solutions, solvents, and / or reagents necessary for detecting the detectable label. Furthermore, it may partially or entirely include the device of the present 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) subjecting the sample to a sensor element, (i) an anchor layer present on a solid support; (ii) a first binding agent anchored to the anchor layer, the first binding agent comprising at least one detectable label, and capable of specifically binding to the test substance; (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:

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

[0065] Embodiment 3. The method of embodiment 2, wherein the signal can be detected over 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, peptide, virus, bacterial cell, or 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 anchored to the anchor layer, the first binding agent comprising at least one detectable label, and capable of specifically binding to the test substance; (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:

[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 the device of 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 herein. [Brief explanation of the drawings]

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

Claims

1. 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 anchored to the anchor layer, the first binding agent comprising at least one detectable label, and capable of specifically binding to the test substance; (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 characteristic 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. The method according to any one of claims 1 to 4, wherein the first binding agent is immobilized on the anchor layer via an anchor molecule.

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 determining the test substance comprises determining the presence, absence, or amount of the test substance.

10. The method of any one of claims 1 to 9, 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.

11. 11. The method of any one of claims 1 to 10, 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.

12. 1. 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 immobilized on the anchor layer, the first binding agent comprising at least one detectable label, and capable of specifically binding to the test substance; (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:

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

14. 14. Use of the device according to claim 12 or 13 for determining in said sample an analyte suspected to be present in the sample.

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

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