Methods for detecting analytes using structure-switching binding agents - Patents.com

Abstract

The present invention relates to diagnostic tests and diagnostic 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 at least one sensor element, the sensor element comprising at least one binding agent capable of specifically binding to the analyte and comprising at least one magnetic label, and a magnetic tunnel junction in functional proximity to the at least one binding agent, generating a signal that changes when the analyte binds to the binding agent, for a time and under conditions that allow specific binding of the analyte suspected to be present in the sample to the at least one binding agent, measuring a post-change signal generated by the magnetic tunnel junction when the analyte binds to the at least one binding agent comprising at least one magnetic label, and determining the analyte based on the post-change signal generated by the magnetic tunnel junction. Furthermore, the present invention relates to an apparatus for determining an analyte suspected to be present in a sample, and the use of such an apparatus. Furthermore, the present invention further relates to an aptamer capable of specifically binding to the analyte and comprising at least one magnetic label, and a method for identifying such an aptamer. Finally, the present invention relates to a kit for determining an analyte suspected to be present in a sample.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to diagnostic tests and diagnostic 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 at least one sensor element, the sensor element comprising at least one binding agent capable of specifically binding to the analyte and comprising at least one magnetic label, and a magnetic tunnel junction in functional proximity to the at least one binding agent, generating a signal that changes when the analyte binds to the binding agent, for a time and under conditions that allow specific binding of the analyte suspected to be present in the sample to the at least one binding agent, measuring a post-change signal generated by the magnetic tunnel junction when the analyte binds to the at least one binding agent comprising at least one magnetic label, and determining the analyte based on the post-change signal generated by the magnetic tunnel junction. Furthermore, the present invention relates to an apparatus for determining an analyte suspected to be present in a sample and for using such an apparatus. Furthermore, the present invention further relates to an aptamer capable of specifically binding to the analyte and comprising at least one magnetic label, and a method for identifying such an aptamer. Finally, the present invention relates to a kit for determining an analyte suspected to be present in a sample. [Background technology]

[0002] 2. Background of the Invention Immunoassays are widely used for various diagnostic purposes. Several setups have been developed for immunoassays. One of the most common immunoassays is the enzyme-linked sorbent immunoassay (ELISA).

[0003] In ELISA, a liquid sample containing or suspected to contain the 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 the application of the sample, multiple reagents are added in sequence, incubated and washed away in order to perform and stop the analytical detection reaction. After all these steps are performed, a physical or chemical property of the setup, usually in the liquid phase, changes and can be detected. Typically, optical changes occur in the final liquid phase, such as color development due to the product of the enzymatic reaction. These changes correlate to the presence or amount of the analyte of interest present in the sample under investigation. The typically quantitative readout is based on the detection of the intensity of the transmitted light, usually by spectrophotometry, which involves the quantification of the transmission of light of some specific wavelength through the liquid. The sensitivity of the detection depends on the amplification of the signal during the analytical reaction. The signal is generated by the enzyme, which binds to the detection reagent at a constant rate so that an accurate quantification is possible, since the enzymatic reaction is a very well-known amplification process.

[0004] In the setup of ELISA, the binding agent of the test substance, for example an antibody, is fixed to a solid phase, 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, it is also possible to use nanoparticles or other beads as the solid phase for 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 analyte present in the 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 to the complex of the analyte and the capture antibody. The detection antibody typically includes a detectable linker or an adapter molecule that allows such a detectable label to be attracted from the solution.

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

[0007] The present invention relates to a method for determining an analyte suspected to be present in a sample, comprising the steps of: (a) subjecting the sample to at least one sensor element, (i) at least one binding agent capable of specifically binding to an analyte and comprising at least one magnetic label; (ii) a magnetic tunnel junction in functional proximity to at least one binding agent, the magnetic tunnel junction generating a signal that changes when an analyte binds to the binding agent; for a time and under conditions that allow specific binding of an analyte suspected of being present in the sample to the at least one binding agent; (b) measuring a post-change signal generated by the magnetic tunnel junction when the test substance binds to at least one binding agent that includes at least one magnetic label; (c) determining the test material based on the changed signal generated by the magnetic tunnel junction; The present invention relates to a method comprising the steps of:

[0008] The present invention further provides an apparatus for determining the presence of an analyte suspected to be present in a sample, comprising: (i) at least one binding agent capable of specifically binding to an analyte and comprising at least one magnetic label; (ii) a magnetic tunnel junction in functional proximity to at least one binding agent, the magnetic tunnel junction generating a signal that changes when an analyte binds to the binding agent; The present invention relates to an apparatus comprising at least one sensor element including:

[0009] Furthermore, the present invention relates to an aptamer that can specifically bind to a test substance, contains at least one magnetic label, and is capable of changing the structure of the aptamer itself upon binding to the test substance, such that the position of the at least one magnetic label changes.

[0010] The present invention also relates to a method for identifying an aptamer as defined above, comprising the steps of: a) providing an aptamer library; b) identifying, by an iterative selection-amplification process, a plurality of candidate aptamers capable of specifically binding to the analyte of interest; c) synthesizing the aptamer candidates in an array format such that each aptamer candidate is capable of modulating a signal induced by a sensor element, preferably a magnetic tunnel junction, to which the aptamer candidate is bound; d) attaching at least one magnetic label to the candidate aptamers on the array; e) contacting the array with an analyte of interest; f) determining a response curve of the sensor element based on the induced signals; g) identifying an aptamer as defined above based on the evaluation of the individual response curves; The method includes:

[0011] The present invention also relates to a kit for determining an analyte suspected to be present in a sample, the kit comprising a device or an aptamer of the present invention. [Brief description of the drawings]

[0012] [Figure 1] Schematic of the aptamer in the unbound stage (A) or after specific binding of the analyte to be detected (B). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Detailed Description The present invention relates to a method for determining an analyte suspected to be present in a sample, comprising the steps of: (a) subjecting the sample to at least one sensor element, (i) at least one binding agent capable of specifically binding to an analyte and comprising at least one magnetic label; (ii) a magnetic tunnel junction in functional proximity to at least one binding agent, the magnetic tunnel junction generating a signal that changes when the analyte binds to the binding agent; contacting the analyte suspected of being present in the sample with at least one sensor element comprising the at least one binding agent for a time and under conditions that permit specific binding of the analyte to the at least one binding agent; (b) measuring a post-change signal generated by the magnetic tunnel junction when the test substance binds to at least one binding agent that includes at least one magnetic label; (c) determining the test material based on the changed signal generated by the magnetic tunnel junction; The present invention relates to a method comprising the steps of:

[0014] 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" element can mean that at least one of that element is available.

[0015] When used hereinafter, the terms "have," "comprise," or "include" are intended to have an open-ended or open-ended meaning. Thus, when they have a open-ended meaning, these terms may refer to a situation in which the described embodiment has no other features than those introduced by these terms, i.e., these terms have a open-ended meaning to the effect of "consisting of" or "consisting essentially of." When they have a non-open-ended meaning, these terms refer to a situation in which the described embodiment has one or more other features in addition to those introduced by these terms.

[0016] 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 and do not limit the possibilities for alternatives.

[0017] Furthermore, as used herein, the term "at least one" is understood to mean that the element followed by this term may be used one or more times according to the present invention. For example, when this term indicates that at least one element should be used, this may be understood as one element or two or more elements, i.e., two, three, four, five, or any other number. Depending on the element to which this term refers, a person skilled in the art will understand which upper limit, if any, this term may refer to.

[0018] The method according to the invention may consist of steps (a) to (c) as described above, or may comprise further steps prior to step (a), such as sample pretreatment or isolation, and after step (c), one or more further steps of evaluating the determined test substance, for example by comparison 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.

[0019] As used herein, the term "determination" encompasses any kind of qualitative or quantitative determination of an analyte. A qualitative determination aims to determine the presence or absence of an analyte in a sample, while a quantitative determination aims to determine the amount of the analyte. A quantitative determination, i.e., the 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 (concentration) relative to the volume of a sample) or a classification such as a score (e.g., "major amount" and "minor amount"). Typically, the determination of an analyte includes the determination of the presence or absence or amount of this analyte.

[0020] As referred to herein, the term "analyte" refers to any type of molecule or substance suitable for determination by the method of the present invention. It will be understood that such molecule or substance may have a size and / or structure that allows the first and second binding agents referred to herein to bind. Furthermore, there may be an upper size limit because the first and second binding agents and linking agents must be able to perform the functions as described in detail elsewhere herein. Typically, the analyte referred to herein is a biomolecule, such as a protein, a peptide, a nucleic acid, such as DNA or RNA, or a small molecule, such as a lipid or a metabolite, such as a polyketide, including flavonoids and isoflavonoids, an isoprenoid, such as a terpene, a sterol, a steroid, a carotenoid, a xanthophylls, 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, etc. The small molecule may be, for example, a toxin. However, the method of the present invention may also be used to determine that a virus or even a bacterial cell is the analyte. Furthermore, the analyte determined by the present invention may be a molecule present in an environmental sample and which may be useful, for example, as an indicator of environmental pollution or agricultural or other environmental conditions. Typically, said analyte is a protein, peptide, virus, bacterial cell, or small molecule, preferably a small molecule toxin.

[0021] As used herein, the term "sample" relates to any portion or aliquot of a composition that contains or is suspected to contain an analyte 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 be derived from any non-living natural source, such as a solution occurring in the environment, such as water, or a composition, such as soil. An artificial sample may be a sample of artificial origin, such as a manufactured product composition or an intermediate composition that may occur in the manufacturing process of a product. Such an artificial sample may be investigated, for example, for quality control purposes or for determining 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.

[0022] As used according to the present invention, the term "sensor element" includes at least one binding agent capable of specifically binding to an analyte and containing at least one magnetic label. The at least one binding agent is located in the sensor element in functional proximity to the magnetic tunnel junction. The magnetic tunnel junction must be in functional proximity so that the signal can change when the position of the at least one magnetic label of the at least one binding agent relative to the magnetic tunnel junction changes. Typically, the at least one binding agent may be immobilized on a solid support. As referred to herein, a solid support is a solid composition of matter that can serve as a basis for immobilizing molecules, in particular at least one binding agent and the magnetic tunnel junction. The solid support may include 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. Alternatively, the solid support may include organic compounds such as cross-linked polymers. Non-limiting examples of suitable cross-linked polymers may be selected from the group consisting of polyamides, polyethers, polystyrenes, and mixtures thereof. A person skilled in the art is well aware how to select a suitable solid support based on the type of sample to be investigated, the envisaged method for detecting the analyte, the type of magnetic label used to detect the analyte, and / or the type of magnetic tunnel junction used in the sensor element.

[0023] The term "binding agent" as referred to herein relates to a molecule capable of specifically binding to an analyte, i.e., a molecule that does not bind and therefore does not cross-react with other molecules other than the analyte 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 substances that specifically bind to the analyte from a library containing various candidate substances.

[0024] Preferably, the molecule that can be used as a binder capable of specifically binding to the desired analyte can be an antibody. The antibody as a binder as meant according to the present invention includes all kinds of antibodies that preferably specifically bind to the analyte. 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 analyte. Such fragments included in the term antibody as used herein include bispecific antibodies, synthetic antibodies, Fab, F(ab)2 Fv, or scFv fragments, or chemically modified derivatives of any of these antibody fragments. Antibodies or fragments thereof that specifically bind to the desired analyte 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 produced by techniques including the fusion of mouse myeloma cells to 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 Western blotting, or immunological or biochemical techniques including plasmon surface resonance measurements.

[0025] Furthermore, preferably, the molecules that can be used as binding agents capable of specifically binding to the desired analyte can be aptamers. Aptamers as binding agents according to the 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 consist of 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. Selection of peptide aptamers can be carried out using various 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 fragment can be used in isolated form or can be part of a fusion molecule, i.e. a molecule that comprises said aptamer fragment and 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. Preferred methods for identifying aptamers useful in the aforementioned methods of the present invention are described elsewhere herein. The binding agents according to the present invention are more typically aptamers identified by such methods.

[0026] Preferably, the molecule that can be used as a binding agent capable of specifically binding to the desired analyte may 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. Furthermore, such receptor molecules, or fragments of such receptor molecules that can still specifically bind to the ligand, may be used as binding agents according to the present invention with respect to the ligand as the analyte, or a molecule derived from the ligand, such as an antagonistic or agonistic variant of the ligand, to which the receptor molecule or its fragment can still bind. Preferably, the receptor molecule envisaged as a binding agent according to the present invention may be a transmembrane receptor protein, such as a G protein-coupled receptor, such as a metabolic receptor, an enzyme-linked receptor, such as a receptor tyrosine kinase, such as a growth factor receptor, an immune receptor, such as a viral receptor, a cell surface antigen, a T cell receptor, such as CD4, CD3, or CD8, or an MHC protein, a cell adhesion molecule, such as an integrin, a cadherin, a selectin, or a syndecan, a neuronal receptor, or a pathogen receptor, such as a toll-like receptor. Furthermore, the receptor molecule may 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 determined. Furthermore, specific binding can be tested by techniques well known in the art, such as plasmon surface resonance measurement.

[0027] Moreover, the molecule that can be used as a binding agent that can specifically bind to the desired analyte may 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 of such a ligand molecule that can still specifically bind to a receptor molecule, may be used as a binding agent according to the present invention for the receptor as an analyte, or a molecule derived from the receptor molecule, such as a soluble variant of the receptor, to which the ligand molecule or its fragment can still bind. Furthermore, the ligand may be any antigen that can be used to determine a specific antibody in a biological sample. Preferably, the ligand molecule envisaged as a binding agent according to the present invention can be a peptide hormone, a neurotransmitter, a growth factor, such as angiopoietin, BMP, neurotrophic factor, EGF, epiphrine, EPO, FGF, GDNF, GDF, insulin or insulin-like growth factor, TGF, neutrophil, VEGF, a cytokine, such as interleukin, interferon, lymphokine, monokine, colony-stimulating factor or chemokine, an extracellular matrix protein, such as fibronectin, vitronectin, collagen, ankyrin or laminin, etc. The skilled person is well aware of the ligand molecule or a fragment thereof that specifically binds to the test substance or a fragment thereof to be determined. Furthermore, specific binding can be tested by techniques well known in the art, such as plasmon surface resonance measurement.

[0028] More preferably, the binding agent can be a designed ankyrin repeat protein (DARPin). DARPins are engineered antibody mimetic 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).

[0029] Thus, typically, the 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 binding agent is an antibody or fragment thereof, or an aptamer.

[0030] At least one binding agent according to the present invention induces a post-change signal at the magnetic tunnel junction when the analyte is specifically bound to it. This is typically achieved by conformational changes or other molecular motions resulting from said specific binding, such that the position of at least one magnetic label changes and induces a post-change signal at the magnetic tunnel junction. Thus, preferably, said at least one binding agent changes its structure when bound to the analyte, such that the position of at least one magnetic label moves closer (positive mode) or farther (negative mode) from the sensing field of the magnetic tunnel junction.

[0031] At least one binding molecule according to the present invention may preferably also comprise an adapter for immobilization. Typically, said adapter allows at least one binding agent to be immobilized on the solid support contained in the sensor element. Typical adapter molecules useful according to the present invention for immobilization of binding agent are well known in the art and depend on the nature of the binding agent and the nature of the solid support used for immobilization in the sensor. More typically, such adapter is LNA, L-DNA or L-LNA. In such a case, more typically, the binding agent must be an aptamer as specified elsewhere herein.

[0032] As used herein, the term "magnetic label" refers to a molecule that can be detected in functional proximity to a magnetic tunnel junction. Thus, a magnetic label is typically envisaged according to the present invention as a detectable label. Exemplary magnetic labels include iron-platinum nanoparticles, iron nanoparticles, nickel nanoparticles, or cobalt nanoparticles. A magnetic label may be reversibly or permanently bound to a binding agent. To this end, a magnetic label may be a molecule that can be reversibly bound to a binding agent, or may be a molecule or moiety that is already part of said binding agent. A binding agent may include at least one magnetic label as part of a molecule. Alternatively, at least one magnetic label may be bound to a first binding molecule by a linker. Permanent and reversible binding are envisaged according to the present invention. Reversible binding may be achieved, for example, by using biotin-streptavidin-based molecular adapter systems well known in the art.

[0033] As used herein, the term "magnetic tunnel junction" refers to a magnetically responsive detector such as a magnetic tunnel junction or a magnetic spin valve (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). The magnetic tunnel junction of the sensor element is typically placed on the opposite side of at least one binding agent on a solid support. Depending on the magnetic label, different detection techniques can be applied. Furthermore, some detection techniques may require the application of a magnetic field to the magnetic tunnel junction.

[0034] It will be appreciated that the presence or absence or amount of the detected magnetic labels can then be transmitted to an evaluation unit, which may preferably comprise a data processing element such as a computer having implemented an algorithm for determining the presence or amount of analyte in the sample based on the presence or absence or amount of the detected magnetic labels.

[0035] Such an implemented algorithm can evaluate the measured post-change signal induced from the magnetic label for signal strength, signal duration, and other predetermined parameters. Based on said evaluation, true positive signals can be identified and verified, and noise signals can be identified and ignored for further evaluation. A person skilled in the art is well aware of which algorithms can be used and how they can be implemented. Preferably, measuring the post-change signal generated by the magnetic tunnel junction when the test substance binds to at least one binding agent includes measuring the intensity and / or duration of the generated signal. Furthermore, said measuring preferably includes measuring the time change of the post-change signal and evaluating said change by a validation algorithm, preferably implemented by a computer. Such validation algorithms typically include artificial intelligence algorithms, machine learning algorithms, etc.

[0036] 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 sensor element as specified in detail elsewhere in this specification.

[0037] As used herein, the term "contact" refers to bringing the aforementioned components into physical proximity such that at least one binding agent can bind to the analyte if the analyte is present in the sample. It will be understood that the binding of the binding agent to the analyte may require time and 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 the 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 binding agent used and the chemical nature of the analyte to be determined.

[0038] As a result of specific binding of the analyte to the at least one binding agent, the at least one binding agent changes its structure, for example by a conformational change or other molecular motion, such that the position of the at least one magnetic label moves closer (positive mode) or further away (negative mode) from the sensitive field of the magnetic tunnel junction. The change in position of the at least one magnetic label gives rise to a post-change signal on the magnetic tunnel junction that can be detected by a detector.

[0039] In step (b) of the method of the present invention, a post-change signal generated by the magnetic tunnel junction upon binding of the analyte to at least one binding agent comprising at least one magnetic label is measured.

[0040] The measurement may further include the application of conditions such as an external trigger that may be required for the detection of the post-change signal induced by the magnetic tunnel junction. In some cases, for example, when a superparamagnetic magnetic label is used, an external magnetic field may be applied. In this way, the post-change signal is triggered through at least one magnetic label by the application of an external magnetic field as an external trigger.

[0041] In step (c) of the method of the present invention, the analyte is determined based on the post-change signal generated by the magnetic tunnel junction.

[0042] The determination referred to in this context encompasses the identification of the presence or absence and / or amount of magnetic label. It will be understood that when the analyte binds to the binding agent, the magnetic label typically generates a post-change signal that can be measured by the magnetic tunnel junction. The signal strength and / or duration typically indicate the amount of magnetic label molecules present in functional proximity to the magnetic tunnel junction, and therefore also the amount of the analyte specifically bound to at least one binding agent. Therefore, preferably, measuring the post-change signal generated by the magnetic tunnel junction upon binding of said analyte comprises measuring the strength and / or the duration of the generated post-change signal. More preferably, said post-change signal can be detected over a predetermined characteristic period of time.

[0043] In the method of the present invention, different binding agents can be used to determine different analytes.In such a case, it will be understood that when such different analytes are bound by respective binding agents that can specifically bind to said different analytes, each type of binding agent must have different magnetic labels that can generate a post-change signal on the magnetic tunnel junction that differs in at least one signal characteristic.

[0044] In a particular embodiment of the method of the present invention, the sensor element comprises a solid support that is a well or a predetermined subdivided detection area. Preferably, a predetermined amount of a binding agent is immobilized in each well or predetermined subdivided detection area. The method is particularly useful for determining the amount of the analyte. Typically, the amount is determined by counting wells or predetermined subdivided detection areas in which a complex is formed. According to this particular embodiment, the presence or amount of the analyte 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 a well or area that exhibits a characteristic post-change signal resulting from the analyte specifically bound by a binding agent as described elsewhere herein (positive mode or negative mode). Based on the positive wells or predetermined subdivided areas and the predetermined amount of binding agent immobilized in each of the wells or predetermined subdivided areas, the amount of the analyte can be determined, for example by calculation. For example, if each well or predetermined subdivided area ideally contains one binding agent, the presence of one molecule of the analyte in the sample will generate one post-change signal. Thus, if a post-change signal is determined to be present in a well or a predetermined subdivided area, this presence reflects the presence of the analyte present in the sample. This technique is also called "digital" detection.

[0045] In the method of the invention, particularly for the purpose of digital detection, the sample is contacted with at least 100 sensor elements, at least 300 sensor elements, at least 500 sensor elements, at least 1,000 sensor elements, at least 5,000 sensor elements, at least 10,000 sensor elements, at least 50,000 sensor elements or at least 100,000 sensor elements. More preferably, in such a case, the determining is determining the amount of the analyte, more preferably, determining the amount of the analyte comprises counting the individual measured post-change signals generated by the magnetic tunnel junctions and subjecting the counts to a statistical analysis.

[0046] Advantageously, according to the present invention, it has been found that the use of a binding agent that generates a post-change signal of the magnetic tunnel junction upon binding with a specific analyte can be used to determine the analyte present in a sample. Furthermore, binding agents such as aptamers, which may change the structure of the aptamer itself as a result of the binding of the analyte, thereby changing the position of the magnetic label relative to the magnetic tunnel junction, may be particularly well suited to generate a post-change signal. In the method of the present invention, kinetic measurements of specific binding and dissolution, rather than single binding events, can also be determined. This allows for dynamic real-time measurements, and may even eliminate the need for washing steps and other treatments. Depending on the nature of the detector, it is possible to essentially isolate the binding events. In such a digital format, the number of post-change signals received by each detector may be used to count the analyte molecules present in the sample.

[0047] Thanks to the present invention, it is possible to determine analytes in a sample with improved sensitivity and specificity. Moreover, even single molecule events can be determined.

[0048] The present invention relates to an apparatus for determining an analyte suspected to be present in a sample, the apparatus comprising: (i) at least one binding agent capable of specifically binding to an analyte and comprising at least one magnetic label; (ii) a magnetic tunnel junction in functional proximity to at least one binding agent, the magnetic tunnel junction generating a signal that changes when the analyte binds to the binding agent; The present invention relates to an apparatus comprising at least a sensor element including:

[0049] Typically, the apparatus comprises: (iii) a detection unit capable of measuring a changed signal generated by the magnetic tunnel junction of the sensor element when the analyte binds to the at least one binding agent; (iv) an evaluation unit capable of determining the analyte based on the signal generated by the magnetic tunnel junction; It further comprises:

[0050] As used herein, the term "device" relates to a system comprising the above-mentioned components in operative association with each other to allow the determination of an analyte by the method of the invention. The sensor element according to the invention may be arranged in a reaction zone contained in the device. The reaction zone may allow direct 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 the connection between the loading zone and the reaction zone. Furthermore, the reaction zone is also connected to a detection unit. 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 able to detect the post-change signal from the magnetic tunnel junction.

[0051] The present invention generally contemplates the use of a device of the present invention for determining, within said sample, an analyte suspected to be present in the sample.

[0052] The present invention relates to an aptamer that can specifically bind to a test substance, contains at least one magnetic label, and is capable of changing the structure of the aptamer itself upon binding to the test substance such that the position of the at least one magnetic label changes.

[0053] Typically, the aptamers referred to herein are nucleic acid aptamers as specified in more detail elsewhere herein. Nucleic acid aptamers typically consist of DNA or RNA. They are preferably between 20 and 75 bases in length and have a three-dimensional structure that allows specific binding of a target molecule, such as a test substance, as referred to herein. More typically, the aptamer consists of at least 25 consecutive bases in length, preferably between 25 and 75 consecutive bases in length, between 25 and 50 consecutive bases in length, or more preferably between 25 and 35 consecutive bases in length.

[0054] Aptamers with specific binding and / or other properties can be generated by various techniques known in the art, including the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technique or the X-aptamer selection technique (see, for example, Kaur 2019, Nanoscale Advances. 1(6): 2123-2138 or Lokesh 2017, in Bindewald E, Shapiro BA (eds.). RNA Nanostructures. Methods in Molecular Biology. 1632. Springer New York. pp. 151-174). The aforementioned techniques may be used to generate aptamer libraries that can be used to identify aptamer species with desired binding and other properties. In particular, the methods for identifying aptamers according to the invention described hereinafter or in the accompanying examples may be applied.

[0055] Additionally, the aptamer typically includes at least one magnetic label selected from the group consisting of iron-platinum nanoparticles, iron nanoparticles, nickel nanoparticles, and cobalt nanoparticles.

[0056] Furthermore, the aptamer may typically include an adaptor for immobilization, which is more typically LNA, L-DNA, or L-LNA.

[0057] Furthermore, the present invention relates to a method for identifying an aptamer as previously defined herein, comprising the steps: a) providing an aptamer library; b) identifying, by an iterative selection-amplification process, a plurality of candidate aptamers capable of specifically binding to the analyte of interest; c) synthesizing the aptamer candidates in an array format such that each aptamer candidate is capable of modulating a signal induced by a sensor element, preferably a magnetic tunnel junction, to which the aptamer candidate is bound; d) attaching at least one magnetic label to the candidate aptamers on the array; e) contacting the array with an analyte of interest; f) determining a response curve of the sensor element based on the induced signals; g) identifying an aptamer as defined herein above based on the evaluation of the individual response curves; The present invention relates to a method comprising the steps of:

[0058] Preferably, steps e) and f) are repeated iteratively at least once, with incubation times and conditions being varied for each iteration.

[0059] The aforementioned aptamers according to the invention are preferably obtained by the aforementioned methods for identifying aptamers.

[0060] The present invention also relates to a kit for determining an analyte suspected to be present in a sample, the kit comprising a device or an aptamer 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 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 typically further comprises instructions for carrying out the method of the present invention. These instructions may be in the form of a manual, or may be provided by computer program code that, when implemented on a computer or data processing device, can carry out or assist in the determination of the analyte referred to in the method 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., compact disc), directly on the computer or data processing device, or in a downloadable form, such as a link to an accessible server or cloud. In addition, the kit may typically include a solution of the analyte with a standardized amount or other reference amount for calibration or validation. In addition, the kit according to the present invention may include further components necessary for carrying out the method of the present invention, such as washing solutions, solvents, and / or reagents necessary for the detection of the detectable label. In addition, the kit may partially or entirely comprise the device of the present invention.

[0062] The following embodiments are particularly preferred embodiments envisaged in accordance with the present invention, all of the definitions and explanations of terms given above apply mutatis mutandis.

[0063] Embodiment 1. A method for determining an analyte suspected to be present in a sample, comprising: (a) subjecting the sample to at least one sensor element, (i) at least one binding agent capable of specifically binding to an analyte and comprising at least one magnetic label; (ii) a magnetic tunnel junction in functional proximity to at least one binding agent, the magnetic tunnel junction generating a signal that changes when the analyte binds to the binding agent; contacting the analyte suspected of being present in the sample with at least one sensor element comprising the at least one binding agent for a time and under conditions that permit specific binding of the analyte to the at least one binding agent; (b) measuring a post-change signal generated by the magnetic tunnel junction when the test substance binds to at least one binding agent that includes at least one magnetic label; (c) determining the test material based on the changed signal generated by the magnetic tunnel junction; A method comprising:

[0064] Embodiment 2. The method of embodiment 1, wherein measuring a post-change signal generated by the magnetic tunnel junction when the test substance binds to at least one binding agent comprises measuring an intensity and / or a duration of the generated signal.

[0065] Embodiment 3. The method of embodiment 2, wherein said measuring comprises measuring a time change in a post-altered signal and evaluating said change by a validation algorithm, preferably implemented by a computer.

[0066] Embodiment 4. The method according to any one of embodiments 1 to 3, wherein the post-change signal is triggered via at least one magnetic label by application of an external magnetic field as an external trigger.

[0067] Embodiment 5. The method of any one of embodiments 1 to 4, wherein the test substance is a protein, a peptide, a virus, a bacterial cell, or a small molecule.

[0068] Embodiment 6. The method of any one of embodiments 1-5, wherein determining the test substance comprises determining the presence or absence of the test substance.

[0069] Embodiment 7. The method of any one of embodiments 1 to 6, wherein the sample is contacted with at least 100 sensor elements, at least 300 sensor elements, at least 1,000 sensor elements, at least 5,000 sensor elements, at least 10,000 sensor elements, at least 50,000 sensor elements, or at least 100,000 sensor elements.

[0070] Embodiment 8. The method of embodiment 7, wherein determining the analyte comprises determining an amount of the analyte.

[0071] Embodiment 9. The method of embodiment 8, wherein determining the amount of the analyte comprises counting each measured post-change signal produced by the magnetic tunnel junction and subjecting the counts to statistical analysis.

[0072] Embodiment 10. The method according to any one of embodiments 1 to 9, wherein the at least one binding agent changes its structure when bound to the test substance so that the position of the at least one magnetic label moves closer (positive mode) or farther (negative mode) to the sensing field of the magnetic tunnel junction.

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

[0074] Embodiment 12. The method of any one of embodiments 1 to 11, wherein the at least one magnetic label is selected from the group consisting of iron-platinum nanoparticles, iron nanoparticles, nickel nanoparticles, and cobalt nanoparticles.

[0075] Embodiment 13. The method of any one of embodiments 1 to 12, wherein the sample is a biological sample, preferably a body fluid or a biopsy sample. Embodiment 14. An apparatus for determining an analyte suspected to be present in a sample, comprising: (i) at least one binding agent capable of specifically binding to an analyte and comprising at least one magnetic label; (ii) a magnetic tunnel junction in functional proximity to at least one binding agent, the magnetic tunnel junction generating a signal that changes when an analyte binds to the binding agent; An apparatus comprising at least a sensor element including:

[0076] Embodiment 15. The device comprises: (iii) a detection unit capable of measuring a changed signal generated by the magnetic tunnel junction of the sensor element when the analyte binds to the at least one binding agent; (iv) an evaluation unit capable of determining the analyte based on the signal generated by the magnetic tunnel junction; 15. The apparatus of embodiment 14, further comprising:

[0077] Embodiment 16. An apparatus as described in embodiment 14 or 15, wherein measuring the post-change signal generated by the magnetic tunnel junction of the sensor element when the test substance binds to at least one binding agent comprises measuring the intensity and / or duration of the generated signal.

[0078] Embodiment 17. A device described in any one of embodiments 14 to 16, wherein the test substance is a protein, a peptide, a virus, a bacterial cell, or a small molecule, preferably a small molecule toxin.

[0079] Embodiment 18. An apparatus as described in any one of embodiments 14 to 17, wherein determining the test substance includes determining the presence or absence of the test substance.

[0080] Embodiment 19. The apparatus of any one of embodiments 14 to 18, wherein the sample is contacted with at least 100 sensor elements, at least 300 sensor elements, at least 500 sensor elements, at least 1,000 sensor elements, at least 5,000 sensor elements, at least 10,000 sensor elements, at least 50,000 sensor elements, or at least 100,000 sensors.

[0081] Embodiment 20. The apparatus of embodiment 19, wherein determining the analyte comprises determining an amount of the analyte.

[0082] Embodiment 21. The apparatus of embodiment 20, wherein determining the amount of the test substance comprises counting individual measured post-change signals generated by the magnetic tunnel junction.

[0083] Embodiment 22. A device described in any one of embodiments 14 to 21, wherein the at least one binding agent changes its structure when bound to the test substance so that the position of at least one magnetic label moves closer (positive mode) or farther (negative mode) from the sensing field of the magnetic tunnel junction.

[0084] Embodiment 23. A device described in any one of embodiments 14 to 22, wherein the at least one binding agent is selected from the group consisting of an aptamer, an antibody or fragment thereof, a receptor molecule or fragment thereof, and a ligand molecule or fragment thereof.

[0085] Embodiment 24. The device of any one of embodiments 14 to 23, wherein the at least one magnetic label is selected from the group consisting of superparamagnetic or ferromagnetic nanoparticles, iron-platinum nanoparticles, iron nanoparticles, nickel nanoparticles, and cobalt nanoparticles.

[0086] Embodiment 25. A device according to any one of embodiments 14 to 24, wherein the sample is a biological sample, preferably a body fluid or a biopsy sample.

[0087] Embodiment 26. Use of a device according to any one of embodiments 14 to 25 for determining, in said sample, an analyte suspected to be present in the sample.

[0088] Embodiment 27. An aptamer capable of specifically binding to a test substance, comprising at least one magnetic label, and capable of changing the structure of the aptamer itself upon binding of the test substance such that the position of the at least one magnetic label changes.

[0089] Embodiment 28. The aptamer of embodiment 27, wherein the aptamer is at least 25 consecutive bases in length, preferably between 25 and 75 consecutive bases in length, between 25 and 50 consecutive bases in length, or more preferably between 25 and 35 consecutive bases in length.

[0090] Embodiment 29. An aptamer according to embodiment 27 or 28, wherein the at least one magnetic label is selected from the group consisting of iron-platinum nanoparticles, iron nanoparticles, nickel nanoparticles, and cobalt nanoparticles.

[0091] Embodiment 30. The aptamer of embodiment 27 or 28, wherein the aptamer comprises an adaptor for immobilization.

[0092] Embodiment 31. The aptamer of embodiment 30, wherein the adapter is LNA or L-DNA or L-LNA.

[0093] Embodiment 32. A method for identifying an aptamer according to any one of embodiments 27 to 31, comprising: a) providing an aptamer library; b) identifying, by an iterative selection-amplification process, a plurality of candidate aptamers capable of specifically binding to the analyte of interest; c) synthesizing the aptamer candidates in an array format such that each aptamer candidate is capable of modulating a signal induced by a sensor element, preferably a magnetic tunnel junction, to which the aptamer candidate is bound; d) attaching at least one magnetic label to the candidate aptamers on the array; e) contacting the array with an analyte of interest; f) determining a response curve of the sensor element based on the induced signals; g) identifying an aptamer according to any one of embodiments 27 to 31 based on the evaluation of the individual response curves; A method comprising:

[0094] Embodiment 33. The method of embodiment 32, wherein steps e) and f) are repeated iteratively at least once, and the incubation time and conditions are varied for each iteration.

[0095] Embodiment 34. An aptamer according to any one of embodiments 27 to 31, wherein the aptamer is obtained by the method described in embodiment 32 or 33.

[0096] Embodiment 35. A kit for determining a test substance suspected to be present in a sample, comprising an apparatus according to any one of embodiments 14 to 25 or an aptamer according to any one of embodiments 27 to 31.

[0097] All references mentioned in this specification are hereby incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification. EXAMPLES

[0098] The examples are merely illustrative of the present invention and should not be construed as limiting the scope of the present invention in any way.

[0099] Example: Identification of aptamers that specifically bind to a test substance and change the structure of the aptamer itself upon specific binding

[0100] Aptamers that specifically bind to a test substance and change their own structure upon specific binding ("flipmers") are identified by carrying out the following steps: 1. Providing a magnetic tunnel junction on the chip; 2. Providing selected flipmer pairs, each carrying one or more magnetic labels, such as FePt nanoparticles, and a linker with a specific and complementary L-LNA sequence; 3. Providing specific immobilized L-LNA strands carrying a linker moiety, e.g. an alkyne, so that they can be covalently attached to the chip surface via click chemistry under copper-induced AFM catalysis in such a way that only one or two or three flipmers are located in close proximity at a single tunnel junction; 4. Assemble the assay platform by coating the chip with L-LNA anchored strands and then adding selected flipmers for the assay or multiplexed assay; 5.Read / record blank signal level; 6. Incubate with serum sample (eventually pre-diluted) and record the signal; 7. After some time, a sufficient positive signal is generated to allow the algorithm to calculate the concentration of the target in the sample. 8. After the assay, the chip may be regenerated by extensive washing off of all components except the immobilized L-LNA.

Claims

1. 1. A method for determining an analyte suspected to be present in a sample, comprising: (a) passing the sample through at least one sensor element, (i) at least one binding agent capable of specifically binding to said analyte and comprising at least one magnetic label; (ii) a magnetic tunnel junction in operative proximity to the at least one binding agent, the magnetic tunnel junction generating a signal that changes when the analyte binds to the binding agent; for a time and under conditions that allow specific binding of the analyte suspected of being present in the sample to the at least one binding agent; (b) measuring a post-change signal generated by the magnetic tunnel junction when a test substance binds to the at least one binding agent comprising the at least one magnetic label; (c) determining the analyte based on the changed signal generated by the magnetic tunnel junction; Including, The at least one binding agent is an aptamer or a fragment thereof, and when the at least one binding agent binds to the test substance, it changes its structure so that the position of the at least one magnetic label moves closer to (positive mode) or farther away (negative mode) from the sensing field of the magnetic tunnel junction.

2. 2. The method of claim 1, wherein measuring the post-change signal generated by the magnetic tunnel junction when a test substance binds to the at least one binding agent comprises measuring the intensity and / or duration of the generated signal.

3. The method of claim 2 , wherein the measuring comprises measuring a change in time of the altered signal and evaluating the change.

4. The method according to any one of claims 1 to 3, wherein the post-change signal is triggered via the at least one magnetic label by application of an external magnetic field as an external trigger.

5. The method of any one of claims 1 to 3, wherein the test substance is a protein, a peptide, a virus, a bacterial cell, or a small molecule.

6. The method of any one of claims 1 to 3, wherein said determining the analyte comprises determining the presence or absence of the analyte.

7. 4. The method of claim 1, wherein the sample is contacted with at least 100 sensor elements, at least 300 sensor elements, at least 1,000 sensor elements, at least 5,000 sensor elements, at least 10,000 sensor elements, at least 50,000 sensor elements, or at least 100,000 sensor elements.

8. The method of claim 7 , wherein determining the analyte comprises determining an amount of the analyte.

9. 10. The method of claim 8, wherein the determining the amount of the analyte comprises counting individual measured post-change signals produced by the magnetic tunnel junction and subjecting the counts to a statistical analysis.

10. The method of any one of claims 1 to 3, wherein the at least one magnetic label is selected from the group consisting of iron-platinum nanoparticles, iron nanoparticles, nickel nanoparticles, and cobalt nanoparticles.

11. The method according to any one of claims 1 to 3, wherein the sample is a biological sample.

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