Solution-phase single molecule capture and related technologies

Abstract

Provided herein are structures and methods for detecting one or more analyte molecules present in a sample. In some embodiments, one or more analyte molecules form a complex having a supramolecular structure in solution. The supramolecular structure of the complex can be detectable such that binding of the analyte molecule to the binding site of the array is detectable via one or more features of the supramolecular structure. The binding site of the array includes a capture molecule for capturing the binding complex to facilitate detection.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Application No. 63 / 194,005, filed May 27, 2021, and U.S. Provisional Application No. 63 / 249,367, filed September 28, 2021, the disclosures of which are hereby incorporated by reference in their entirety for all purposes.

Background Art

[0002] The current state of personalized medicine is overwhelmingly genome - centered, mainly focusing on the quantification of genes present within an individual. While such an approach has proven to be very powerful, it does not provide clinicians with a complete picture of an individual's health. This is because genes are the "blueprints" of an individual and only inform about the potential to develop diseases. Within an individual, these "blueprints" need to be first transcribed into RNA and then translated into various protein molecule "actors" within the cell in order to affect the individual's health.

[0003] Protein concentration, protein - protein interactions (protein - protein interaction or PPI), and interactions between proteins and other molecules are intricately related to the health of different organs, homeostatic regulatory mechanisms, and the interactions of these systems with the external environment. Therefore, quantitative information regarding protein interactions such as proteins and PPI is essential for creating a complete picture of an individual's health at a given point in time and for predicting any emerging health problems. The presence and interactions between these proteins are also essential for drug development and are becoming a much - sought - after dataset for capturing changes in individual proteomes and proteomes in response to environmental or other systemic events. The ability to detect and quantify protein interactions between proteins and other molecules within a given sample is an essential component of such healthcare development.

Summary of the Invention

[0004] This disclosure generally relates to systems, structures, and methods for the detection and quantification of analyte molecules in a sample.

[0005] In some embodiments, solution-based techniques for detecting analyte molecules present in a sample are provided herein. This technique detects the analyte in solution and their respective supramolecular structures. body Captured by, or otherwise each supramolecular structure body The process includes a sample preparation step associated with the following: For example, in one embodiment, individual analytes (e.g., protein molecules) are supramolecular structures body It is captured by the affinity binder. Once captured, the analyte-supramolecular structure body The complex can be detected on the substrate as part of a detection system, thereby allowing individual binding sites on the substrate to carry affinity binders for the analyte of interest. Analyte-supramolecular structure body Binding at the complex's binding site depends on the binding site affinity of the binder and the supramolecular structure. body The affinity binder may be in a sandwich configuration, binding to different positions on the individual analyte molecules. Therefore, signals detected at specific binding sites can be associated with the presence of the specific analyte of interest in the sample. Supramolecular structure of the complex body 1) Individual supramolecular structures body 1) A tag or barcode (e.g., a nucleic acid having a unique barcode sequence) for identifying the relevant affinity binder; 2) A detectable initiator for providing a detectable signal that can be correlated with binding in a detection system; and 3) One or more of the following physical scaffolds bound to the analyte that promote single-molecule binding or low-averaging of the molecule at the binding site on the substrate, for example, sterically interfering with or sterically occluding the binding of other molecules at the same binding site.

[0006] Size-excluded supramolecular structure bodyThis provides greater flexibility in preparing substrates for complexing with analytes in solution and detecting the complexed analytes. In contrast to systems designed to have a single affinity binder associated with each binding site, which is complex to manufacture, the disclosed technique works with more tolerable binding site preparations that include multiple immobilized affinity binders for each binding site. Thus, supramolecular structure body While a single-molecule binding entity may exist, the substrate may be arranged to allow for multi-molecule binding at each binding site. A supramolecular structure where space restricts binding at each site. body The nature of the few-molecule or single-molecule binding at each binding site, facilitated by this process, enables high-throughput parallel characterization of multiple protein interactions on a single detection platform. Furthermore, solution-based sample preparation is more streamlined compared to substrate-based sample preparation.

[0007] As provided herein, a method for detecting analyte molecules present in a sample provides a sample containing analyte molecules and the supramolecular structure of the sample in solution. body Contact with the pool to create an analyte molecule-supramolecular structure body It may include forming a complex. Supramolecular structure body This may include a core structure comprising multiple core molecules and affinity binders bonded to the core structure. Individual analyte molecule-supramolecular structure body The complex may include a core structure comprising multiple core molecules; an affinity binder bound to the core structure; and analyte molecules bound to the affinity binder, and the supramolecular structure body Different supramolecular structures in the pool body This method includes different affinity binders having different binding affinities for the analyte molecule to other analyte molecules. body The process involves contacting the complex with an array, wherein the binding sites of the array each contain immobilized affinity binders having binding affinity to different analyte molecules, and the analyte molecule-supramolecular structure body This includes detecting the binding of the complex to the binding site of the array.

[0008] As provided herein, a method for detecting analyte molecules present in a sample is a supramolecular structure body This may include providing an array of each individual supramolecular structure body The individual supramolecular structures of the array are immobilized at each bonding site on the substrate. body This comprises a core structure containing multiple core molecules; and multiple nucleic acid capture molecules linked to the core structure, each of which is a supramolecular structure of the array. body The method includes the same identification sequence that is distinguishable from the identification sequence. The method involves contacting the array with a pool of affinity binders linked to single-stranded nucleic acid tags having binding specificity to different identification sequences, and binding different affinity binders to different binding sites on the array such that each binding site includes a different subset of the pool of affinity binders. The method also involves contacting the sample with the array so that the analyte molecule binds to an individual affinity binder in the pool of affinity binders, and the analyte molecule has a supramolecular structure body This includes contacting the detection assembly to form a complex with it, and detecting the binding of analyte molecules to individual binding sites.

[0009] As provided herein, a method for detecting analyte molecules present in a sample may include providing an array containing single-stranded nucleic acids immobilized on an array such that each binding site of the array contains multiple single-stranded nucleic acids having the same sequence that is distinguishable from the sequences of other single-stranded nucleic acids immobilized on different binding sites of the array. The method includes contacting the array with a pool of affinity binders to cause the immobilized single-stranded oligonucleotides to capture a subset of the affinity binders at the binding sites of the array, so that the captured affinity binders form immobilized capture molecules at their respective binding sites, and contacting the sample with the array so that the analyte molecules bind to individual capture molecules, the analyte molecules being supramolecular structures body It is complexed with a detection assembly. This method also involves detecting the binding of analyte molecules to individual binding sites.

[0010] As provided herein, a method for detecting analyte molecules present in a sample may include forming nanoballs or functionalized nanostructures. The nanoballs or functionalized nanostructures comprise a plurality of individual oligonucleotides, each of which is bound to a chemical moiety, and each oligonucleotide is bound to a complementary nucleic acid of the nanoball or functionalized nanostructure. The method includes providing a patterned array comprising a plurality of active sites; incubating the nanoballs or functionalized nanostructures with the patterned array to covalently bond the active groups of the individual active sites to the chemical moieties of the plurality of individual oligonucleotides, thereby binding the individual nanoballs or functionalized nanostructures to the individual active sites; denaturing the plurality of individual oligonucleotides from the complementary nucleic acids of the nanoballs or functionalized nanostructures; washing the nanoballs or functionalized nanostructures from the array so as to leave a plurality of individual oligonucleotides immobilized at individual binding sites, wherein the plurality of individual oligonucleotides are single-stranded; and contacting the array with a pool of affinity binders to cause the immobilized plurality of individual oligonucleotides to capture a subset of the affinity binders at their individual binding sites.

[0011] As provided herein, a method for detecting analyte molecules present in a sample provides a sample containing analyte molecules and the supramolecular structure of the sample in solution. body Contact with the pool to create an analyte molecule-supramolecular structure body This may include the formation of complexes between individual analytes and supramolecular structures. body The complex comprises a core structure containing multiple core molecules; an affinity binder bound to the core structure; and an analyte molecule sample-specific barcode of the analyte molecule bound to the affinity binder, and the supramolecular structure body Different supramolecular structures in the pool body This method includes different affinity binders having different binding affinities for the analyte molecule to other analyte molecules. body The complex is a molecular-supramolecular structure of other analytes. bodyIt involves pooling with other analytes, molecules, and supramolecular structures. body The complex is pooled, each associated with a different sample-specific barcode; pooled analyte molecule-supramolecular structure body Contacting a complex with an array, wherein the binding sites of the array contain immobilized affinity binders having different binding affinities for different analyte molecules; analyte molecule-supramolecular structure to the binding sites of the array body This includes detecting the binding of complexes and associating the detected bindings with sample-specific barcodes.

[0012] A method for detecting analyte molecules present in a sample is disclosed herein, as provided herein. This method involves multiple supramolecular structures body This includes providing each supramolecular structure body It comprises a core structure containing multiple core molecules; an antibody having binding affinity to the antigen and bound to a nucleic acid capture chain; and a complementary chain to the nucleic acid capture chain, the complementary chain being linked to the core structure and forming a double-chain structure with the nucleic acid capture chain to bind the antibody to the core structure, and multiple supramolecular structures body This method involves multiple supramolecular structures, each having a different binding affinity. body The method includes: contacting the complex with a sample containing an antigen; contacting the complex with beads carrying a capture antibody having binding specificity to the antigen to form a sandwich structure; displacing the nucleic acid capture chain from the complementary chain using a substitution chain to release the core structure into a solution, wherein the released core structure is not bound to the antibody; and detecting the core structure.

[0013] In some embodiments, any method disclosed herein may include detecting the presence of and / or quantifying the concentration of analyte molecules in a sample. In some embodiments, any method disclosed herein further includes identifying the detected analyte molecules. In some embodiments, any method disclosed herein further includes detecting analyte molecules based on a signal when the analyte molecules are present in the sample at a count of single molecule or more.

[0014] In some embodiments, for any method disclosed herein, each core structure is a nanostructure. In some embodiments, for any method disclosed herein, the plurality of core molecules for each core structure are arranged in a predetermined shape and / or have a predetermined molecular weight. In some embodiments, the predetermined shape is configured to limit or prevent cross-reactivity with another supramolecular structure body and. In some embodiments, for any method disclosed herein, the plurality of core molecules for each core structure include one or more nucleic acid strands, one or more branched nucleic acids, one or more peptides, one or more small molecules, or a combination thereof. In some embodiments, for any method disclosed herein, each core structure independently includes a backbone deoxyribonucleic acid (DNA) origami, a backbone ribonucleic acid (RNA) origami, a backbone hybrid DNA:RNA origami, a single-stranded DNA tile structure, a multi-stranded DNA tile structure, a single-stranded RNA origami, a multi-stranded RNA tile structure, a hierarchically structured DNA or RNA origami having multiple backbones, a peptide structure, or a combination thereof.

[0015] Supramolecular structure body The affinity binder of can bind to the core structure via a chemical bond. In some embodiments, the affinity binder of the solution-based supramolecular structure body and / or the immobilized affinity binder of the binding site independently include a protein, a peptide, an antibody, an aptamer (RNA and DNA), a fluorophore, a darpin, a catalyst, a polymerization initiator, a polymer such as PEG, or a combination thereof.

[0016] In some embodiments, for any method disclosed herein, the detectable signal is, for example, a binding site and / or supramolecular structure. body The barcode includes a barcode indicating a specific affinity binder. In some embodiments, each barcode provides a DNA signal or initiator signal corresponding to the affinity binder, providing information indicating its specificity to the analyte molecule bound to the respective detection agent molecule. In some embodiments, the barcode is analyzed using gene typing, qPCR, sequencing, or a combination thereof. In some embodiments, multiple analyte molecules in the sample are detected simultaneously by multiplexing. In some embodiments, for any method disclosed herein, the affinity binder provided herein is configured to bind to one or more specific types of analyte molecules.

[0017] In some embodiments, multiple supramolecular structures are disclosed herein. body Any method including using multiple supramolecular structures body Each core structure is identical to the others. However, the bonded affinity binder can vary for multiple structures. Therefore, in one embodiment, (for example, multiple) supramolecular structures body The pool is identical except for the bound affinity binder and, in some embodiments, the identification portion that identifies the bound affinity binder (e.g., affinity binder identification barcode nucleic acid sequence). For multiplexed samples detected on a single substrate or detection platform, the supramolecular structure associated with a particular sample. body It may have a sample-related identification portion (e.g., a sample barcode nucleic acid sequence) that identifies the sample.

[0018] In some embodiments, each supramolecular structure body This is a multiple supramolecular structure body Includes a predetermined shape, size, molecular weight, or combination thereof that can reduce or eliminate cross-reactions between them. In some embodiments, each supramolecular structure body This includes one affinity binder or multiple affinity binders. Supramolecular structure bodyIf the supramolecular structure contains multiple affinity binders on a single core structure, all of the affinity binders may have the same binding specificity, for example, all of them may bind specifically to the same analyte. In some embodiments, each supramolecular structure body This is a multiple supramolecular structure body The system includes predetermined stoichiometric ratios of the capture molecule and the detection molecule to reduce or eliminate cross-reactions between them.

[0019] In some embodiments, the substrate comprises a solid support, a solid substrate, a polymer matrix, or one or more beads. The substrate may include a plurality of binding sites patterned on the substrate and separated by interstitial surfaces of the substrate, each binding site comprising a well, at least one surface of the well comprising a first chemical group, and the interstitial surfaces comprising a second chemical group. Beads are loaded onto each binding site, the first chemical group selectively binding to the bead, and the second chemical group not interacting with or binding to the bead. Each bead comprises a plurality of single-stranded oligonucleotides immobilized on each bead such that each individual binding site of the array comprises a plurality of single-stranded oligonucleotides having the same sequence distinguishable from the sequence of other single-stranded oligonucleotides immobilized on different binding sites of the array, and a captured affinity binder forming a capture molecule immobilized on each binding site, and the capture affinity binder forming a capture affinity binder at the binding site of the array. During detection, the analyte molecule binds to the individual capture molecule at the individual binding site, and each analyte molecule forms a supramolecular structure body It forms a composite with the detection assembly. Detection of the detection assembly enables detection of the analyte bonded to the substrate.

[0020] In some embodiments, multiple supramolecular structures body However, the substrate is placed on a molded or planar substrate, and the substrate comprises multiple binding sites, each individual binding site being bound to one or more affinity binders configured to bind to the same analyte molecule, for example, each individual binding site being specific to an individual analyte molecule, and different binding sites on the substrate being specific to different analyte molecules. The disclosed embodiments also include sample preparation reagents, substrates, and detection systems for carrying out the disclosed method.

[0021] In some embodiments, for any method disclosed herein, the sample includes a complex biological sample. In some embodiments, for any method disclosed herein, one or more analyte molecules of the sample include proteins, peptides, peptide fragments, lipids, DNA, RNA, organic molecules, inorganic molecules, complexes thereof, or any combination thereof. In some embodiments, for any method disclosed herein, the sample includes biological particles or biomolecules. In some embodiments, for any method disclosed herein, the sample includes an aqueous solution containing proteins, peptides, peptide fragments, lipids, DNA, RNA, organic molecules, viral particles, exosomes, organelles, or any complex thereof. In some embodiments, for any method disclosed herein, the sample includes tissue biopsy material, blood, plasma, urine, saliva, tears, cerebrospinal fluid, extracellular fluid, cultured cells, culture media, waste tissue, plant matter, synthetic proteins, prions, bacterial and / or viral samples or fungal tissue, or a combination thereof. The sample may be an environmental sample such as wastewater or soil sample. The sample may also be a non-biological sample. In one embodiment, the sample may be a sample from a chemical process step, a sample of food or nutritional components, or a packaging component.

[0022] The sample is a supramolecular structure in the solution provided herein. body Before contact, the sample may be treated to release the analyte from the cells or to prepare the sample for analysis in other ways.

[0023] Herein, specific embodiments of the disclosed apparatus, delivery system, or method will be described with reference to the drawings. Nothing in this detailed description is intended to imply that any particular component, feature, or process is essential to the invention. [Brief explanation of the drawing]

[0024] [Figure 1] This disclosure shows an exemplary analyte-supramolecular structure complex after solution-based capture according to embodiments of this disclosure. [Figure 2] This figure shows an exemplary workflow including solution-based single-molecule capture sample preparation according to embodiments of the present disclosure. [Figure 3] An example of a substrate for analyte detection according to an embodiment of this disclosure is shown. [Figure 4] An example of a binding site for a substrate having a captured molecule and a captured analyte-supramolecular structure complex according to an embodiment of the present disclosure is shown. [Figure 5] An example of a substrate for analyte detection according to an embodiment of this disclosure is shown. [Figure 6] This embodiment of the disclosure shows a linked barcode nucleic acid structure for use with a substrate for analyte detection. [Figure 7] An example of an analyte detection substrate containing the linked barcode nucleic acid structure shown in Figure 6, according to an embodiment of the present disclosure, is shown. [Figure 8] This invention illustrates a nanoparticle structure for use with a substrate for analyte detection, according to an embodiment of this disclosure. [Figure 9] An example of a substrate for analyte detection containing the nanoparticle structure shown in Figure 5, according to an embodiment of the present disclosure, is shown. [Figure 10] An example of a substrate for analyte detection according to an embodiment of this disclosure is shown. [Figure 11] An example of analyte detection beads according to an embodiment of this disclosure is shown. [Figure 12] This embodiment of the disclosure illustrates an exemplary workflow for generating a substrate for housing beads for analyte detection. [Figure 13] This disclosure illustrates an exemplary workflow including solution-based single-molecule capture sample preparation for multiplex detection according to embodiments of this disclosure. [Figure 14A] The present disclosure illustrates a step in an experimental workflow for antigen detection, comprising an IgG affinity conjugate complexed with box origami to form a supramolecular structure. [Figure 14B] Figure 14A shows additional steps in the experimental workflow. [Figure 15]Figures 14A and 14B show different experimental subgroups evaluated using the experimental workflow. [Figure 16] The experimental workflow shown in Figures 14A and 14B presents solution-based optical detection results using different antigen combinations of the three antigens. [Figure 17] Figures 14A-B show the solution-based optical detection results obtained by titration of TSH used in the experimental workflow. [Figure 18] This shows solution-based optical detection results for antigen titration, regardless of the presence or absence of other antigens. [Figure 19] Solution-based optical detection results for monomer dimer and trimer antigens are shown. [Figure 20] The results of solution-based optical detection of a mixture of unlabeled and labeled antigens are shown. [Figure 21] This shows the solution-based optical detection results for mixed or composite samples. [Figure 22] The solution-based optical detection results of the titrated antigen sample are shown. [Figure 23] A block diagram of an exemplary analyte detection system according to an embodiment of the present disclosure is shown. [Modes for carrying out the invention]

[0025] This specification discloses structures and methods for detecting one or more analyte molecules present in a sample. In some embodiments, one or more analyte molecules are one or more supramolecular structures body Detection is based on solution-based capture by several embodiments. In some embodiments, one or more supramolecular structures body It contains, or is linked to, an affinity binder that specifically binds to the analyte present in the sample and solution, and is used for single-molecule capture of the analyte. The binding of the analyte occurs in solution, forming an analyte molecule-supramolecular structure. body They form complexes, which can be detected by the detection systems provided herein.

[0026] In one embodiment, the complexed analyte is captured by an immobilized affinity binder associated with the substrate of the detection system or other detection platform. Capture of the complex then immobilizes the complex onto the substrate, and one or more features of the immobilized substrate or their associated binding sites can be characterized to characterize the analyte in the sample. In one embodiment, a supramolecular structure body This includes a detectable portion such as a unique identifier (e.g., nucleic acid sequence, peptide, polysaccharide, acridite) and / or other molecules that are detectable (e.g., optically, electrically, magnetically), interact with it, or can be used to dock it. In some embodiments, the detectable portion generates a DNA signal or other initiator signal, thereby enabling the analyte molecule-supramolecular structure to bind to the binding site. body Detection and quantification of complex binding, supramolecular structure body This can be detected by amplification of the unique identifier of the and / or the unique identifier of the binding site. In some embodiments, the supramolecular structure body It is linked to an enzyme that converts the substrate into an optically detectable signal. In some embodiments, a supramolecular structure body It is coupled to a sensor on a substrate to generate an electrically or magnetically detectable signal. In one embodiment, a supramolecular structure body This is nucleic acid origami linked to or fixed to a substrate. In one embodiment, a supramolecular structure body This includes a unique identifier for the affinity binder, and the affinity binder is supramolecular structure body The affinity binder is supported via barcode crosslinks or linkers connected to the scaffolding.

[0027] In some embodiments, the disclosed technique provides single-molecule capture of analyte molecules in a composite sample. Supramolecular structure as capture entity bodyThe use of this method enables specific identification and, in some embodiments, detection of relevant affinity binders through interaction with binding sites on the substrate. Thus, as provided herein, detectable analytes bind to individual affinity binders in a pool of many different affinity binders on a substrate to produce assay results characterized by the binding properties of an analyte pool of multiple different analytes. This makes it possible to analyze a sample having an uncharacterized analyte composition for the presence and / or concentration of a specific analyte of interest. For example, a human sample can be characterized to determine the presence and / or concentration of an antibody having binding specificity to a particular antigen in a panel of antigen affinity binders, such that the affinity binders represent a known infectious disease antigen panel. The assay results may show positive binding results related to a specific antigen, indicating the presence of an antibody in the subject the sample provides. In another embodiment, the identity of analytes in a sample may be at least partially known, but their binding affinity may not be characterized for a particular pool of affinity binders. For example, the affinity binders may be a set of candidate drugs, and the analytes may be molecules in human blood. The binding of such candidate drugs to proteins can be used to evaluate bioavailability or potential off-target binding. The assay results may show positive binding results associated with specific drug candidates that can be mapped to a particular analyte, based on the identification of specific detection agent binding (e.g., identifying binding by barcode recognition in a detection agent molecular assembly containing an antibody specific to the analyte).

[0028] Conventional detection protocols for analytes such as proteins may include a detectable fluorescent signal generated by an enzyme linked to a detection antibody as an indicator of binding. However, the disclosed technique can additionally or alternatively provide a nucleic acid signal amplified from a unique identifier (or other initiator) of the detection agent molecular assembly, from which sequence information can be determined, or from which an optically detectable signal corresponding to the amplification is emitted (e.g., qPCR using a primer / probe set specific to the unique identifier). Thus, the unique identification information of the complex captured at a specific binding site in the array allows for the specific identification of a particular affinity binder that captured the analyte in a particular embodiment. Supramolecular structure body The modular and customizable structure of its components is generally the same, with each individual supramolecular structure having a unique identifier during the binding of a specific affinity binder. body This makes it possible to provide a bulk or common core structure for barcoding. Therefore, the identification sequence is bound to only one specific affinity binder. Other detection techniques may include optical, magnetic, and / or electrical detection techniques.

[0029] The disclosed embodiments relate to the detection of analytes present in a sample, such as a biological sample. In some embodiments, the sample comprises an aqueous solution containing proteins, peptides, peptide fragments, lipids, DNA, RNA, organic molecules, inorganic molecules, complexes thereof, or any combination thereof. In some embodiments, the analyte molecules in the sample include proteins, peptides, peptide fragments, lipids, DNA, RNA, organic molecules, inorganic molecules, complexes thereof, or any combination thereof. In some embodiments, the analyte molecules include intact proteins, denatured proteins, partially or completely degraded proteins, peptide fragments, denatured nucleic acids, degraded nucleic acid fragments, complexes thereof, or combinations thereof. In some embodiments, the sample is obtained from tissue, cells, tissue and / or cellular environments, or combinations thereof. In some embodiments, the sample includes tissue biopsies, blood, plasma, urine, saliva, tears, cerebrospinal fluid, extracellular fluids, cultured cells, culture media, waste tissue, plant matter, synthetic proteins, bacterial or viral samples, fungal tissue, or combinations thereof. In some embodiments, the sample is isolated from a primary source such as cells, tissues, body fluids (e.g., blood), environmental samples, or combinations thereof, with or without purification. In some embodiments, cells are lysed using mechanical processes or other cell lysis methods (e.g., lysis buffers). In some embodiments, the sample is filtered using mechanical processes (e.g., centrifugation), microfiltration, chromatography columns, other filtration methods, or a combination thereof. In some embodiments, the sample is treated with one or more enzymes to remove one or more nucleic acids or one or more proteins. In some embodiments, the sample includes intact proteins, denatured proteins, partially or completely degraded proteins, peptide fragments, denatured nucleic acids, or degraded nucleic acid fragments. In some embodiments, the sample is collected from one or more individuals, one or more animals, one or more plants, or a combination thereof. In some embodiments, the sample is collected from individuals, animals, and / or plants with diseases or disorders including infectious diseases, immunodeficiencies, cancer, genetic diseases, degenerative diseases, lifestyle-related diseases, injuries, rare diseases, age-related diseases, or a combination thereof.

[0030] The disclosed technology involves supramolecular structures. body The system utilizes single-molecule bonding of affinity molecules linked to the core structure 13. The single-molecule bonding of the analyte molecules forms a complex as shown in Figure 1, which is detectable by the detection system according to the disclosed technique. Complex formation occurs in solution. Figure 1 shows the supramolecular structure including the core structure 13 and affinity binder 2. body Ten exemplary embodiments are provided. In some embodiments, supramolecular structures body 10 comprises one or more affinity binders 2. In one embodiment, a supramolecular structure body 10 may refer to a complex comprising a core structure 13 and an affinity binder 2. In one embodiment, a supramolecular structure body 10 may refer to the core structure 13, and the supramolecular structure body 10 may or may not contain affinity binder 2.

[0031] Therefore, in this specification, supramolecular structure body 10 is provided. In some embodiments, a supramolecular structure body 10 is a programmable structure in which molecules can be spatially organized. In some embodiments, supramolecular structures body 10 includes multiple molecules linked together. In some embodiments, a supramolecular structure body Ten or more molecules interact with each other in at least some ways. In some embodiments, a supramolecular structure body 10 includes a specific shape, for example, a substantially planar shape having the longest dimension in the xy plane. In some embodiments, a supramolecular structure body 10 is a nanostructure. In some embodiments, it is a supramolecular structure. body 10 is a supramolecular structure body A nanostructure having a predetermined molecular weight based on 10 or more molecules. In some embodiments, the molecules are linked to each other via bonds, chemical bonds, physical bonds, or a combination thereof. In some embodiments, a supramolecular structure body 10 comprises a large molecular entity of a specific shape and molecular weight, formed from a clearly defined number of smaller molecules that interact specifically with each other. In some embodiments, a supramolecular structure bodyThe 10 structural, chemical, and physical properties are explicitly designed. In some embodiments, supramolecular structures body 10 includes a plurality of subcomponents spaced apart according to a predetermined distance. In some embodiments, a supramolecular structure body At least a portion of 10 is rigid. In some embodiments, supramolecular structure body At least a portion of 10 is semi-rigid. In some embodiments, supramolecular structure body At least a portion of it is flexible. In one embodiment, a supramolecular structure body 10 is at least 50-200 nm in one dimension. In one embodiment, a supramolecular structure body 10 is at least 20 nm in length, of any dimension.

[0032] In some embodiments, the core structure 13 is a polynucleotide structure, a protein structure, a polymer structure, or a combination thereof. In some embodiments, the core structure 13 includes one or more core molecules linked together. In some embodiments, one or more core molecules include 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, or 500 unique molecules linked together. In some embodiments, one or more core molecules include about 2 to about 1000 unique molecules. In some embodiments, one or more core molecules interact with each other to form a supramolecular structure. body This defines a specific shape. In some embodiments, multiple core molecules interact with each other via reversible non-covalent interactions.

[0033] In some embodiments, the specific shape of the core structure 13 is a three-dimensional (3D) configuration. In some embodiments, one or more core molecules provide a specific molecular weight. For example, multiple supramolecular structures body All 10 core structures 13 may have the same composition, size, and / or weight, but their linker sequences and affinity binders 2 may differ. However, apart from the different linkers 20 and affinity binders 2, multiple supramolecular structures body10 may be otherwise identical. In some embodiments, the core structure 13 is a nanostructure. In some cases, one or more core molecules include one or more nucleic acid chains (e.g., DNA, RNA, non-natural nucleic acids), one or more branched nucleic acids, one or more peptides, one or more small molecules, or a combination thereof. In some embodiments, the core structure 13 includes a completely polynucleotide structure. In some embodiments, at least a portion of the core structure 13 is rigid. In some embodiments, at least a portion of the core structure 13 is semi-rigid. In some embodiments, at least a portion of the core structure 13 is flexible. In some embodiments, the core structure 13 includes a backbone deoxyribonucleic acid (DNA) origami, a backbone ribonucleic acid (RNA) origami, a backbone hybrid DNA / RNA origami, a single-stranded DNA tile structure, a multi-stranded DNA tile structure, a single-stranded DNA origami, a single-stranded RNA origami, a single-stranded RNA tile structure, a multi-stranded RNA tile structure, a hierarchically constructed DNA and / or RNA origami having multiple backbones, a peptide structure, or a combination thereof. In some embodiments, DNA origami is scaffolded. In some embodiments, RNA origami is scaffolded. In some embodiments, hybrid DNA / RNA origami is scaffolded. In some embodiments, the core structure 13 includes DNA origami, RNA origami, or hybrid DNA / RNA origami having a predetermined two-dimensional (2D) or three-dimensional shape.

[0034] In one embodiment, the core structure 13 is a nucleic acid origami having at least one lateral dimension of about 50 nm to about 1 μm. In one embodiment, the nucleic acid origami has, for example, at least one lateral dimension of about 50 nm to about 200 nm, about 50 nm to about 400 nm, about 50 nm to about 600 nm, about 50 nm to about 800 nm, about 100 nm to about 200 nm, about 100 nm to about 300 nm, about 100 nm to about 400 nm, about 100 nm to about 500 nm, and about 200 nm to about 400 nm. In one embodiment, the nucleic acid origami has at least a first lateral dimension of about 50 nm to about 1 μm and a second lateral dimension of about 50 nm to about 1 μm that is orthogonal to the first lateral dimension. In one embodiment, the nucleic acid origami is about 200 nm 2 ~about 1μ 2 It has a planar footprint with an area of ​​.

[0035] As shown in Figure 1, in some embodiments, the core structure 13 is configured to be linked to the affinity binder 2. In some embodiments, the affinity binder 2 is immobilized to the core nanostructure 13 when linked to it. However, the core structure 13 may be in solution and therefore may not be immobilized to the sample or reaction vessel. As shown in Figure 1, in some embodiments, the affinity binder 2 is linked to the core structure 13 via a linker 20. In some embodiments, the linker 20 comprises a polymer containing nucleic acids (double-stranded or single-stranded DNA or RNA) of a specific sequence associated with the linked affinity binder 2. Thus, the sequence of the nucleotide linker 20 uniquely identifies the affinity binder 2 in a pool of different affinity binders 2. The barcode may be at least 6 nucleotides, and may be 6 to 50 nucleotides. In Figure 1, the supramolecular structure body 10 binds to analyte molecule 14, which has binding specificity to affinity binder 2, forming an analyte molecule-supramolecular structure. body It forms a complex 40.

[0036] In some embodiments, any number of one or more core molecules 13 include one or more linkers 20 configured to form a bond with affinity binders 2. In some embodiments, the linkers 20 are bonded to one or more core molecules of the core structure 13 via chemical bonds. In some embodiments, the linkers 20 may include core reactive molecules. In some embodiments, each core reactive molecule independently includes an amine, thiol, DBCO, NHS ester, maleimide, biotin, azide, acridite, single-stranded nucleic acid of a specific sequence (e.g., RNA or DNA), or polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, at least one of the one or more core linkers includes a DNA sequence domain.

[0037] In some embodiments, the core structure 13 is linked to the affinity binder 2 at predetermined positions on the core structure 13.

[0038] In some embodiments, affinity binder 2 includes proteins, peptides, antibodies, antibody-derived reagents, aptamers (RNA and DNA), fluorophores, nanobodies, dalpins, catalysts, polymerization initiators, polymers such as PEG, organic molecules, small molecules, pharmaceutical compounds, candidate pharmaceutical compounds, synthetic molecules, or combinations thereof. In some embodiments, a single affinity binder 2 is linked to the core structure 13. In some embodiments, multiple affinity binders 2 are linked to the core structure 13. For example, different affinity binders 2 on the same core structure 13 may represent different binding sites for the same analyte molecule, or they may bind to different analyte molecules of a multimolecular complex of, for example, a protein-protein complex. In another example, multiple identical affinity binders 2 may be present on the core structure 13.

[0039] In some embodiments, supramolecular structure body Each of the 10 components may be independently modified or adjusted. In some embodiments, a supramolecular structure body By modifying one or more of the 10 components, the supramolecular structure bodyThe two-dimensional and three-dimensional shapes of the material itself can be modified. In some embodiments, the supramolecular structure body The two-dimensional and three-dimensional shapes of the core structure 13 can be modified by modifying one or more of its components. In some embodiments, such ability to independently modify the components of the supramolecular nanostructure is possible with respect to one or more supramolecular structures body This enables precise control over the organization of [the organization].

[0040] As described herein, in some embodiments, one or more supramolecular structures body Complex 40 enables the detection of one or more analyte molecules in the sample. Figure 2 shows the analyte molecule-supramolecular structure in solution. body This is a schematic diagram of the workflow for forming complex 40. It is a supramolecular structure having associated affinity binders 2 representing a panel or set of affinities for each different analyte. body Ten pools are brought into contact with a sample 50 containing multiple different analyte molecules 14. The analytes 14 in the sample 50 may be uncharacterized or unknown analytes 14. In embodiments, the sample 50 may contain one or more control analytes 14.

[0041] When individual analyte molecules 14 and individual affinity binders 2 have binding specificity with each other, the analyte molecules 14 associate with the affinity binders 2 to form individual analyte molecule-supramolecular structures. body A complex 40 is formed. The reaction conditions allow the binding of the analyte molecule 14 to the specific affinity binder 2. Where provided herein, binding specificity may refer to the interaction between the analyte molecule 14, which remains intact after the washing or removal step of the unbound reagent, and the affinity binder 2 under the reaction conditions. Binding specificity may include the formation of covalent or non-covalent bonds, ionic bonds, dipole interactions, hydrophilic or hydrophobic interactions, complementary nucleic acid bonds, etc. Specific binding may refer to binding to the analyte molecule 14 that binds only to a specific affinity binder 2 and not to other affinity binders 2. Thus, supramolecular structure bodyA specific affinity binder 2 in the pool of 10 binds to a specific analyte molecule 14 (for example, binding between a first analyte molecule 14a and a first affinity binder 2a, or binding between a second analyte molecule 14b and a first affinity binder 2b). A specific affinity binder 2 may not have a binding partner available in a given sample 50 and therefore will not bind to any specific analyte molecule 14.

[0042] As provided herein, after the formation of the complex 40, the unbound analyte 14 can be removed before the complex is provided to the detection system. However, in other embodiments, the washing step is not performed. The unbound analyte 14 in solution can interact with the detection system, but it is unlikely to bind specifically at the binding site, and the supramolecular structure body Since it does not carry 10, it does not generate a detectable signal.

[0043] The analytes 14 of the disclosed complex 40 can be detected based on interaction with a patterned substrate 60 having an array of binding sites 66 distributed on or within the substrate 60, as generally shown in Figures 3 to 13. The substrate 60 may contain a defined set of micropatterned binding sites 66 functionalized by having immobilized capture molecules 70.

[0044] In some embodiments, the bonding sites 66 are micropatterned on a planar substrate 60. In some embodiments, the bonding sites 66 on the surface are periodic patterns. In some embodiments, the bonding sites 66 on the surface are aperiodic patterns (e.g., random). In some embodiments, the minimum distance is specified between any two bonding sites 66. In some embodiments, the minimum distance between any two bonding sites 66 is at least about 200 nm. In some embodiments, the minimum distance between any two bonding sites 66 is at least about 40 nm to about 5000 nm. In some embodiments, the geometric shape of the bonding sites 66 includes circles, squares, triangles, or other polygons. In some embodiments, the individual bonding sites 66 have a diameter of 20 to 200 nm. The substrate 60 may be patterned as generally discussed in U.S. Provisional Application No. 63 / 119,316, filed November 30, 2020, which is incorporated herein by reference.

[0045] The substrate 60 may include a glass or silicon wafer having one or more silicon dioxide, silicon nitride, graphene, or silicon carbide layers. In one embodiment, each binding site 66 accommodates a plurality of capture molecules 70 of the same type, and different binding sites 66 have different capture molecule specificities or different chemistry. The patterned substrate 60 can be manufactured by a lithography process. Furthermore, embodiments of the disclosed technology may include one or more regeneration steps to remove the bound or "used" complex 40 from the substrate 60 to enable the binding of new complex 40 in subsequent reactions.

[0046] In some embodiments, the substrate 60 may include a reference marker (not shown) having a defined geometric feature on its surface that is used as a reference feature for other features on the substrate 60. In some embodiments, the planar substrate 60 includes a structure that facilitates the detection of optical or electrical devices such as FETs, ring resonators, photonic crystals, or microelectrodes, which are defined before the formation of the bonding site 66.

[0047] Figure 3 provides an exemplary diagram of forming a substrate 60 used to detect analyte molecules in a sample using a surface-based assay with a capture molecule 70 (shown as an antibody, but which can be any suitable capture molecule for pulling down the complex 40) as described herein. In one embodiment, the capture molecule 70 is an affinity binder as commonly disclosed herein. In Figure 3, a patterned substrate 60 having multiple binding sites 66 functionalized or linked with individual capture molecules 70 can be formed according to method 100. A substrate base layer 110 is provided. A passivation layer 112 is grown, assembled, or deposited on the base layer 110 which can be selectively protected. The passivation layer 112 may include silicon nitride, graphene, quartz, metal, gold, silver, platinum, palladium, PDMS, polymer film, or a combination thereof. The passivation layer 112 may also be graphene, aluminum oxide, HfO2, Cr2O3 (chromium oxide), titanium oxide, tantalum oxide, metal oxide, silicon dioxide (SiO2), or a combination thereof. The passivation layer 112 may be a self-assembled polymer such as polyacrylamide.

[0048] The passivation layer 112 is patterned, for example, by removing a portion of the top layer 112, to expose positions 120 in the base layer 110 that correspond to the bonding sites 66 of the substrate 60. Patterning may be done by photolithography, electron beam lithography, nanoimprint, polymer spin coating, optical patterning, plasma activation, acid / base treatment, or other patterning modalities. The exposed positions 120 can be activated by chemical or plasma treatment, depending on the individual chemical properties of these layers, to generate different reactive groups.

[0049] The capture molecules 70 can be bound to the activation site 120 to generate a binding site 66. In the illustrated example, the capture molecules 70 can be printed onto the binding site 66. Each binding site 66 is printed with pre-selected capture molecules 70. However, other attachment or binding techniques for linking the capture molecules to the binding site 66 are also conceivable. In embodiments, 1) each binding site contains multiple (e.g., two or more) capture molecules 70, and 2) all capture molecules 70 at each binding site have the same binding specificity for a particular analyte 14.

[0050] The capture molecule 70 is one or more affinity binders, supramolecular structures, as provided herein. body , may include nucleic acids. Each binding site 66 of the substrate 60 contains multiple capture molecules 70 having the same binding specificity for each individual binding site 66. Furthermore, adjacent or different binding sites 66 may have different binding specificities, such that the first capture molecule 70a has a different binding specificity from the second capture molecule 70b.

[0051] The capture molecules 70 immobilized at each binding site 66 (and not present at non-binding sites on the substrate 60) may include proteins, peptides, antibodies, aptamers (RNA and DNA), fluorophores, nanobodies, dalpins, catalysts, polymerization initiators, polymers such as PEG, organic molecules, or combinations thereof.

[0052] Each binding site 66 can be bound to a barcode or unique identification sequence that can be read out via any of the following hybridization assays: sequencing, amplification, or detection. Before performing the assay, a map of the capture molecules 70 immobilized on the substrate 60 and having spatial locations on the substrate 60 can be obtained and performed as quality control of the substrate 60. The map can be stored in the analytic detection system provided herein (see Figure 10) and used to generate a report of positive binding events to provide analytic information of the sample.

[0053] Once formed, the substrate 60 can be used in the analyte capture step after sample preparation (shown in Figure 2) to provide the composite 40. In some embodiments, the composite 40 is brought into contact with a planar substrate using a flow cell. In some embodiments, the composite 40 is incubated on the substrate 60 having capture molecules 70 bound to binding sites 66. In some embodiments, the incubation period may be about 30 seconds to about 24 hours. In some embodiments, the incubation period may be about 30 seconds to about 1 minute, about 1 minute to about 5 minutes, about 5 minutes to about 30 minutes, about 30 minutes to about 1 hour, about 1 hour to about 5 hours, about 5 hours to about 12 hours, about 12 hours to about 24 hours, or about 24 hours to about 48 hours.

[0054] In some embodiments, the analyte molecule 14 of the complex 40 interacts with the capture molecule 70 on the binding site 66. Figure 4 shows an exemplary binding site 66 having a capture molecule 70a that has specificity for a particular analyte 14a of the complex 40a. After the interaction, binding can be detected by signal generation. Unbound complex 40b can be washed away before detection. The capture molecule 70a and each analyte molecule 14a may have binding specificity to each other. Since the analyte molecule 14a forms the complex 40a in solution before contact with the substrate 60, the capture molecule 70a, analyte 14a, and affinity binder 2a (linked to the core structure 13a) can form a sandwich-like binding arrangement, as shown in Figure 4. Therefore, both the capture molecule 70a and the affinity binder 2a can have specificity for the same analyte 14a. However, the capture molecule 70a and the affinity binder 2a may be bound at different sites on the analyte 14a.

[0055] After these workflow steps, various binding complexes 40 remain on the array, each binding to its respective capture molecule 70, and the analyte 14 to a specific supramolecular structure. bodyIt can be subjected to various detection protocols to associate identity, which can then be associated with a known affinity binder 2, and then, at a specific binding site 66, with a unique identifier, such as a barcode sequence. Thus, detection enables characterization of analyte-affinity binder binding.

[0056] In some embodiments, as shown in the workflow 150 of Figure 5, the binding site 66 is located at the activation site 120 to form a single supramolecular structure that forms the binding site 66. body Functionalization occurs via 160. The binding site 66 is a supramolecular structure. body Before 160 is positioned, it may be activated as generally disclosed with respect to Figure 3. Supramolecular structure body 160 generally refers to the supramolecular structures provided herein. body It may be positioned as 10, and may include a core structure 13 containing DNA origami, where the supramolecular structure body 10 is attached to each of the binding sites using DNA origami arrangement techniques, which may include linking via anchor molecules. In some embodiments, the DNA origami arrangement includes directed self-assembly techniques for constructing individual DNA origami (e.g., core structures) on the binding sites 66. In some embodiments, the planar substrate 60 can be stored in a clean environment for a considerable period of time after this step.

[0057] In the illustrated embodiment, a supramolecular structure body 160 is provided with assembled and already linked capture molecules 70. Each individual supramolecular structure has different analyte specificities. body These can be formed separately, for example, in separate reaction tubes. Multiple capture molecules 70 form a supramolecular structure. body It is linked to 160 core structures 13. A single supramolecular structure at a single binding site 66. body All 160 capture molecules (70 of them) have the same binding specificity. Furthermore, each supramolecular structure body 160 includes a barcode or other identification sequence that uniquely identifies 70 types of related capture molecules.

[0058] A pre-formed supramolecular structure such that the capture molecule 70 is positioned "upper right" on the binding site 66. body The arrangement of 160 can be directional. In other embodiments, supramolecular structures body All 160 molecules are generally the same and are randomly placed at individual binding sites. After being placed at different binding sites, a unique identification sequence and associated capture molecule 70 are linked to them.

[0059] Figures 6-7 illustrate the steps in forming a planar substrate 60, in which the binding site 66 is functionalized via a binder for the linked nucleic acid product 200. Figure 5 illustrates the steps in forming the linked nucleic acid product 200 from a ring template 210. The ring template includes a barcode sequence 220 that uniquely distinguishes the ring template 210 from other ring templates 210. Rolling circle amplification by a strand-displacing polymerase extending a primer 230 produces a single-stranded rolling circle amplification product containing a linked nucleic acid with repeating units 252. A single-stranded probe 260 having an active group 262 hybridizes complementaryly with the barcode sequence 220 and binds to multiple locations on the linked nucleic acid product 200 where the barcode sequence 202 is repeated as part of the repeating units 252. Thus, the final product of rolling circle amplification for binding site generation is a linked nucleic acid product 200 having multiple binding probes 260 and associated active groups 262. In one embodiment, the formation of the substrate provided herein involves creating a plurality of products 200 from each different template 210, each product 200 having a distinguishable arrangement based on the different template 210. Each different product 200 can be formed in a different reaction vessel. However, in one embodiment, different templates 210 can be pooled to produce a pooled product 200. As shown in Figure 7, each product 200 associates with the binding site 66 in a generally 1:1 ratio, so the pooled population of products 200 can nevertheless be distributed around the substrate 60. However, producing the products 200 independently can prevent differences in amplification bias that result in uneven production of products 200 from a particular type of template 210, which may result in over-presentation of a particular template arrangement on the binding site 66.

[0060] Each template 210 includes an array that can associate with or key to a specific capture molecule 70 as a barcode or unique identifier for the capture molecule 70, as provided herein.

[0061] As shown in the workflow 270 of Figure 7, the active site 120 can be prepared as generally disclosed with respect to Figures 3 to 5. The binding site 66 is formed by functionalizing a single linked nucleic acid product 200 having multiple binding probes 260 at the activation site 120. The active site 120 contains an active molecule 280 surrounded by a passivation layer 112, as shown. The single-stranded linked nucleic acid product 200 is generally a nanoball sized such that only a single product 200 is accommodated on a single active site 120. The association of the product 200 and the active site during the formation of the binding site 66 is via the active group 262 of the binding (hybridize) probe 260. The interaction between the probe 262 and the active molecule may be due to NHS-ester, thiol, DBCO, azide, or maleimide interactions. For example, in one embodiment, the maleimide group reacts specifically with the sulfhydryl group when the pH of the reaction mixture is 6.5 to 7.5; as a result, a stable thioether bond is formed. Thus, in one embodiment, the active group 262 may be a maleimide reagent and the active molecule 280 may be a sulfhydryl. The active group 262 and the active molecule 280 are reacted to form a stable conjugated thioether bond. This immobilizes the probe 260 on the active site 120. The nanoball product can be removed by denaturing the probe 260 at a denaturation temperature and washing. This step leaves the probe 260 immobilized on the binding site 66. All probes 260 have the same arrangement along at least a portion of the oligonucleotide. In one embodiment, all probes 260 have the same arrangement as one another within the binding site 66, but have different arrangements from probes 260 bound to some, almost, or all other binding sites. In one embodiment, the probes 260 of individual binding sites have at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to one another. The probes 260 between different binding sites 66 may have less than 50% sequence identity. Since the nanoball products 200 are generated from a circular template 210 (see Figure 6), different templates 210 with different sequences will correspondingly generate different nanoball products 200.Therefore, the probes 260 complementary to the repeating units 252 of the different products 200 have different sequences based on the original template sequence.

[0062] Once immobilized, the probe 260 can be sequenced, amplified, or detected via tagged complementary oligonucleotides to map the binding site 66 and its respective immobilized probe 260. The map can be stored in a detection system (Figure 13).

[0063] Figures 8 and 9 show alternative structural configurations for positioning the capture molecule on the binding site 66 of the substrate 60. In Figure 8, nanoparticles 300, which can be made from a polymer hydrogel, crosslinked polymer, or inorganic material, are incubated with a single-stranded oligonucleotide 302 having a chemical moiety 303 that can be covalently bonded to the nanoparticles 300. Subsequently, the nanoparticles 300 functionalized with the single-stranded oligonucleotide 302 are incubated with a second single-stranded oligonucleotide 306 having a second chemical moiety 305 to obtain functionalized nanoparticles 307 having immobilized double-stranded DNA and the chemical moiety 305. Thus, oligonucleotides 302 and 306 interact based on complementarity to form double-stranded DNA, and the chemical moiety is bonded to the nanoparticles 307 via hybridization. Examples of chemical moieties 303 and 305 include, but are not limited to, thiols, amines, DBCOs, maleamides, azides, and NHS esters.

[0064] Using the functionalized nanoparticles 307 shown in Figure 8, monoclonal clusters of ssDNA can be created on a patterned substrate 60 by arranging the nanoparticles 307, covalently binding them to a surface, and then removing / denaturing them. Steps 1 and 2 for patterning the substrate 60 can be performed as generally disclosed herein (see, for example, Figure 3). In one embodiment, the workflow includes first passivating the surface and then patterning it by lithography according to the protocol provided herein, resulting in a surface having two chemical properties, one of which is within the binding sites 66 and the other forming the passivation background 112. In step 3, the functionalized nanoparticles 307 shown in Figure 8 are incubated on the patterned substrate 60 to result in the organization of the nanoparticles 307 within the binding sites 66. The driving force for this assembly is the interaction between the nanoparticles 307 and the binding sites 66. The nanoparticles 307 are typically sized such that there are single nanoparticles 307 organized or arranged within a single binding site 66. The chemical portion 305 bound to the nanoparticle 307 is covalently bonded to the surface 310 of the binding site 66. Finally, the nanoparticle 307 and complementary nucleic acid 302 on the nanoparticle are denatured to separate the oligonucleotide strands 302 and 306, obtaining a patterned surface in which each binding site has multiple copies of the same ssDNA 306. The single-stranded DNA (or RNA) 306 is covalently bonded to the surface 310 via the chemical portion 305.

[0065] Figure 10 shows the binding of a capture molecule 70, indicated as an antibody, to single-stranded oligonucleotides 320 positioned at individual binding sites 66. The single-stranded oligonucleotide 300 may be a probe 260 bound via an active group 262, as shown in Figure 7. The single-stranded oligonucleotide 300 may be an oligonucleotide 306 bound via a portion 305, as shown in Figure 9. The single-stranded oligonucleotide 320 immobilized at the binding site 66 may function directly as the capture molecule 70 (e.g., hybridization). However, in embodiments, a complementary tag can be designed using the unique sequence of the oligonucleotide 320 to bind to the capture molecule 70 via hybridization in a binding site-specific manner. In embodiments, the single-stranded oligonucleotide 320 may be printed directly onto the binding site 66, as shown in Figure 3. All single-stranded oligonucleotides 320 at individual binding sites 66 have the same sequence, or sequences with at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity. The capture molecule 70 is bound to the oligonucleotide 300 by a tag 290 containing a complementary sequence. Each capture molecule 70, or each type of capture molecule 70 having binding specificity to an individual analyte, is bound to a tag 322 having a unique sequence that is distinguishable from other sequences of other tags 322 bound to different capture molecules 70 having different specificities. The tag 322 is designed based on a known sequence of oligonucleotide 320. Thus, a pool of different capture molecules 70, each having its own unique tag 322, can be brought into contact with the binding site 66 containing the single-stranded oligonucleotide 320.

[0066] As shown, each binding site 66 can bind to multiple capture molecules 70 and interact with one, two, three or more complexes 40 via the analytes bound to the capture molecules 70. Supramolecular structure on the complex body The presence of 10 keeps the average number of molecules per binding site low. However, the presence of multiple capture molecules 70 with the same specificity at each binding site 66 promotes strong specific binding of the analyte.

[0067] Figure 11 shows an embodiment in which the capture molecule 70 is bound to the bead 330. In the illustrated embodiment, the bead 330 is placed in the wells 340 of a substrate 60 such that the individual binding sites 66 formed by the wells 340 accommodate a single bead 330. However, it should be understood that the bead 330 is in solution and does not need to be associated with the substrate. Each bead is functionalized with a unique oligonucleotide 320 having the same or substantially the same sequence as those provided herein, and the capture molecule 70 having the tag 322 hybridizes to the oligonucleotide 320 only if they have complementary sequences.

[0068] Figure 12 shows an example of binding site formation (for example, for a binding site 66 as shown in Figures 10-11). The binding site 66 can be used to fill lithographically defined nano / microwells with beads 330 carrying multiple analytes 2 (e.g., DNA, RNA, or protein / peptides discussed herein). The illustrated workflow produces a final product 350 (e.g., substrate 60) having nano / microwells 360 having an outer surface containing a first chemical group or functional group on the inner wall and bottom of the well 390 and other different chemical groups with different reactivity on the gap surface 362. Furthermore, the first chemical group is designed to interact with the beads 330 (electrostatically or covalently), while the other chemical group does not interact with the beads 300. In this situation, when the beads 330 are incubated with the substrate 350, they selectively bind to the wells 360 but not to the gaps 360. The size or diameter of the bead 330 may be 20 nm to 5 microns in one embodiment. The well may be 20 nm to 5 microns across (measured, for example, as the distance between adjacent gaps 362) in one embodiment. The well 360 may be sized to accommodate just one bead 330. Thus, in a particular embodiment, the size of each well 360 may be no more than twice the diameter of the bead.

[0069] In the workflow shown in Figure 12, a silicon dioxide surface having a resin coating layer is provided and patterned by nanoimprint. The patterned resin is activated with plasma (e.g., O2 plasma) and treated with a first silane. The treated surface is coated with a coating such as a polymethyl methacrylate (PMMA) coating. Oxygen etching exposes the upper surface of the underlying patterned resin and removes the first silane from the gaps 362, while retaining the first silane on the sides and bottom of the wells 360. A second antifouling treatment is applied to the exposed gaps to remove the PMMA and expose the wells 360. The disclosed workflow is, as an example, for producing a substrate 360. The substrate 360 ​​can be used to fill beads 330 and can be used in conjunction with the disclosed technology.

[0070] In some embodiments, as shown in Figure 13, multiplexing allows for the simultaneous detection of multiple analyte molecules in the sample, and multiple different supramolecular structures. body 10 provides multiple signals (e.g., linker barcode, sample barcode) for sequencing and analyte identification and demultiplexing of the sample. In some embodiments, the methods described herein for detecting analytes in a sample include multiple supramolecular structures body By using 10, high throughput and high multiplexing capability are provided. Different multiple supramolecular structures body 10a, 10b, and 10c each relate to different sets of affinity binder 2, which may be the same set or different sets, among multiple or different sets. However, in one embodiment, multiple analytes can be used for different samples that come into contact with the same set of affinity binder 2, all of which can be detected using the same functionalized substrate 60 having a specific set of capture molecules 70 that are compatible with the set of affinity binder 2.

[0071] The core structures 13 may generally be the same, so that individual core structures 13 can be bound to different affinity binders 2. However, for a particular sample run, all core structures 13 may contain, or be bound to, one or more sample-specific oligonucleotide barcodes 450a, 450b, 450c that are distinguishable among the samples 50. That is, each core structure 13 can contain one or more barcodes 450. Thus, the complexes 40a, 40b, 40c may be pooled on the sample substrate or run together, but the detectable signal can be associated with a particular sample based on the detection of a specific sample-specific barcode 450.

[0072] Multiplexing may include a washing or separation step to separate the complex 40 from the unbound analyte 14. Such separation may involve supramolecular structure body This may include affinity separation, magnetic separation, and size separation via tags on the 10th.

[0073] Embodiments of the present disclosure include multiplexing kits such as nPlex kits. The kit comprises a general-purpose or common adapter on a core structure 13. The adapter functions as a dock for different nPlex barcodes. The number of barcodes can be selected based on the number of samples to be multiplexed, and each sample barcode can be added to a separate reaction vessel so that the sample indexed core structure 13 is separated from each other until tagging, complex formation, and separation from unbound analytes.

[0074] Figure 14A shows the steps of the experimental workflow for antigen detection. The workflow involves supramolecular structure bodyThis includes functionalizing 10 and complexing it with a specific affinity binder. For example, the core structure 13 may be implemented as a substantially box-shaped core structure 500. However, it should be understood that other shapes and implementations of the core structure 13 are also possible. Each core structure 13 is bound to a signaling element, which is shown here as a fluorophore 500. However, the signaling element may be a nucleic acid signal generator, an optical signal generator, an electrical signal generator, or a magnetic signal generator. The signaling element can be detected by a detection system, as generally described with respect to Figure 23.

[0075] In the experiments discussed with respect to Figures 15-16, affinity binder 2 was found to be the supramolecular structure of multiple associated fluorophores 510 according to the embodiments of this disclosure. body The IgG antibody 520 was bound to a single-stranded capture chain 520, which was complexed with a core structure 13 having a box origami structure 500 (for example, a box structure formed from nucleic acid chains). However, it should be understood that other antibody species or other affinity conjugates 2 are also included in the disclosed embodiments. In the illustrated example, each different IgG represents a different affinity conjugate 2 having different specificity for different analytes, e.g., antigen 522. Thus, the illustrated workflow is shown in triple detection capability, and each specific IgG antibody has a supramolecular structure having a specific fluorophore type. body Fluorophores 510a bound to the first IgG antibody 520a can be detected in a first fluorescence range, fluorophores 510b bound to the second IgG antibody 520b can be detected in a distinguishable second fluorescence range, and fluorophores 510c bound to the third IgG antibody 520c can be detected in a distinguishable third fluorescence range. In one embodiment, each box origami structure 500 is generally the same except that it differs by different fluorescence detection wavelengths depending on the different bound fluorophores 510a, 510b, and 510c. However, in embodiments, the core structure 13 or the pool of box origami structures 500 may be the same or different for a particular workflow.

[0076] In step 1, the single-stranded nucleic acid capture strand 522 conjugated to IgG forms a complex with a single-stranded complementary strand 524 bound to the box origami structure 500 to form a double-stranded structure. Thus, the IgG antibody is complexed with the core structure 13 via complementary binding under conditions that promote double-stranded structure formation. Each different antibody 520 and box origami structure 500 may have their own universal capture strand 522 and complementary strand 524, which are the same even for different affinity binding specificities, in order to simplify and batch specific reagent preparation steps. However, in embodiments, each specific IgG antibody 520a, 520b, and 520c, etc., may be bound to a unique nucleic acid capture strand 522 that may contain a barcode or other identifying information specific to a particular affinity binder 2.

[0077] The experimental workflow is presented as a triple reaction with three different IgG antibodies 520a, 520b, and 520c, but it is also intended to allow detection of three different antigens, single, dual, or other multiple configurations. In one embodiment, extended fluorophore signal resolution can be achieved by controlling the number of fluorophores 510 bound to each box origami structure 500 to achieve a range of different signal intensities. Thus, affinity conjugates 2 can be distinguished based on the detected fluorescence range and / or detected intensity.

[0078] In step 2 of the workflow, the pool of molecular structures described above is brought into contact with a sample that may or may not contain the target antigen 526. If antigen 526 to which the IgG antibody specifically binds is present, the supramolecular structure bound to antigen 526 is formed. body An assembly 40, which is a composite 528 of 10 parts, is formed.

[0079] In step 3 of a workflow that can be performed before, between, or after steps 1 and 2, beads 530, such as magnetic beads, are functionalized with a capture antibody 536 to form functionalized capture beads 540. In one example, beads 530 may be bound to streptavidin that binds to biotin-labeled antibody 536. Capture antibodies 536a, 536b, and 536c may have different binding specificities based on their specific binding to IgG antibodies 520a, 520b, and 520c.

[0080] Figure 14B shows additional steps to the experimental workflow in Figure 14A. In step 4, the supramolecular structure body - The antigen complex 528 is brought into contact with the functionalized capture beads 540 to form a sandwich structure 550. For example, both the IgG antibody 520 and the capture antibody 536 bind to a specific antigen 526. In one embodiment, both the IgG antibody 520a and the capture antibody 536a bind to individual antigens 526. Box origami 500 that does not have antigen 526 bound to it does not bind to the magnetic beads 530 and can therefore be removed from the sandwich structure 550 by magnetic pull-down.

[0081] In step 5, a substitution chain 560 is introduced into the reaction mixture to break the double helix of the capture chain 520 and the complementary chain 522. In one embodiment, the substitution is via a foothold-mediated substitution. Thus, in one example, the capture chain 522 includes a complementary region that binds to the complementary chain 522 and a foothold region that is non-complementary and remains unbound or unannealed before contacting the substitution chain 560. The substitution chain is complementary to both the complementary and foothold regions and thus facilitates the substitution of the substitution chain 560 from the complementary chain 522 via a binder to the foothold region, thereby forming a double helix of the substitution chain 560 and the capture chain 520. Thus, the substitution chain 560 shares sequence identity with the complementary chain 522 and also includes a foothold-complementary region. Upon contact with the substitution chain, the capture chain 520 and the substitution chain 560 form a double chain that releases the box origami 500 into the solution, with the capture chain 520 being double-chained with the substitution chain 560. In one embodiment, the released box origami 500 can be separated from the remaining portion 570 of the sandwich structure 550. For example, the remaining portion 570 holds beads 530 that can be magnetically pulled down to leave the box origami in the solution.

[0082] It should be understood that the substitution can be reversed, as the complementary chain 522 can contain a foothold region. The substituted chain 560 binds to the complementary chain 522, breaking the double helix and releasing the box origami into the solution, while the capture chain 520 remains single-stranded.

[0083] In step 6, the released box origami 500 is imaged at a wavelength corresponding to the bound fluorophore 510, and a detectable signal indicates the presence of a specific antigen associated with a particular wavelength range. The signal can be evaluated for the expected or appropriate light intensity indicating the presence of the antigen. For example, light intensity is possible, but other detection modes are also possible, as described herein.

[0084] In one embodiment, the complexity of the workflow can be increased by using temporally separated cycles of different substitution strands 560 having specificity for different capture strands 520. For example, in the case of three fluorophores, N sets of three fluorophores can be bound to a box origami 500, each having a set-specific capture strand 520 that is substituted by a unique set-specific substitution strand 560. For N sets, there may be N unique capture strand 520-substitution strand 560 pairs. Each unique substitution strand 560 of a known sequence can be added separately to release only a portion of the box origami 500 carrying the corresponding capture strand 520. The released box origami 500 can be imaged to obtain antigen-related data, and the next substitution strand 560 can be added.

[0085] Figure 15 shows different experimental subgroups evaluated using the experimental workflows of Figures 14A-B. The antigens used were TSH, PSA, and IL-6, which corresponded to three different antibody bindings and detections. Different antigens were tested individually and in combination with each other to identify potential cross-binding or other interferences. Antibody filling %: Antibody was filled at 1.5% (Dynabeads). The incubation times in the experiments were as follows: 30 minutes of biotinylated IgG with bead incubation D-biotin occupying any free avidin site for 10 minutes Time in 1 box of IgG and antigen Box-IgG antigen and IgG-beads were incubated in PBS pH 7.4 + 0.1% Tween + 10 mM Mg for 1 hour. The substituted strand was a 1 μM substituted strand in PBS Tween + 10 mM Mg. The different fluorophores were as follows: IL-6 readout using an AlexaFluor 488 box TSH readout using an AlexaFluor 647 box PSA readout using the alexafluor 750 box Detection was performed via a Tecan microplate reader. It should be understood that the experimental workflow may be carried out using other, more, and / or fewer fluorophores.

[0086] Figure 16 shows the experimental results using different antigen combinations of the three antigens used in the experiment in Figure 15, and displays the light intensity readout. Each of the individual antigens can be detected independently or in a mixture without significant crosstalk or interference.

[0087] Figure 17 shows the experimental results using three different antigen combinations, as used in the experiment in Figure 15, in which TSH is titrated while PSA and IL-6 remain constant. Changes in the concentration of TSH in the sample are reflected in the detected light output.

[0088] Figure 18 shows experimental results demonstrating that changes in antigen concentration can be detected by light output using titration of TSH or PSA in a double reaction.

[0089] Figure 19 shows the results of a quadruple experiment to quantify four cytokines from the mixture. TNFa: Readout using an Alexafluor 488 box (128 imager) IL-10: Readout using the atto 565 box (128 imager) INFy: Readout using an AlexaFluor 647 box (128 imager) IL-2: Readout using an Alexafluor 750 box (128 imagers) Enter all values ​​for Box Origami IgG at 5nM. 2-hour incubation of antigen and VOC-IgG in 10 mM MOPS + 150 mM NaCl + 10 mM MgCl2 + 0.1% tween-20. Box-IgG: 1-hour incubation with antigen + IgG beads. Box substitution with 500 nM in 10 mM MOPS + 150 mM NaCl + 10 mM MgCl2 + 0.1% tween-20 Tecan reading, 15 μL / sample, 140 gain at excitation wavelengths of 491, 550, and 641 nm. Two of the three replicated samples were diluted with 100× dimer / trimer antigen to obtain larger readings.

[0090] Figure 20 shows the results of an experiment in which four cytokines were quantified from a mixture in which only one cytokine per subgroup was detected.

[0091] Figure 21 shows an experiment in which IL-2, TNFα, IL-10, and IFNγ were detected in a pseudo-serum mixture, demonstrating that optical detection of antigens in complex biological samples is possible.

[0092] Figure 22 shows a series of titrations of IL-8 and TNFα, demonstrating that a constant CRP concentration indicates that the antigen concentration difference is detectable at optical output.

[0093] The experimental results demonstrated effective solution-based detection for the proposed experimental workflow.

[0094] Embodiments of this disclosure include one or more computer-implemented detection systems configured to perform specific methods of the disclosed embodiments. Figure 23 shows an analyte detection system 1000 including a controller 1001. The controller 1001 includes a processor 1002 and a memory 1004 for storing instructions configured to be executed by the processor 1002. The controller 1001 includes a user interface 1006 and a communication circuit 1008 to facilitate communication, for example, over the Internet 1010 and / or a wireless or wired network. The user interface 1006 facilitates user interaction with the characterized analyte detection results provided herein.

[0095] The processor 1002 receives analyte detection data and is programmed to characterize the detected analyte. In one embodiment, the processor characterizes the supramolecular structure with respect to the array of binding sites 66. body Following the incubation of complex 40 and the detection of features of the binding site 66, complex 10, or both, a report of the detected analytes in the sample is generated. The report may include data of the photosignals generated at various binding sites corresponding to the detected analyte binding events. The report may include processed data such as a list of detected analytes or positive / negative binding results. The report may include a list of available affinity binders for complex 10 and / or available capture molecules 70, which indicates the analyte detection capability.

[0096] System 1000 also has a supramolecular structure. body 10 (and / or any immobilized supramolecular structure of the binding site 66) body The system includes an analytic detector 1020 that operates to detect analytic binding through the detection of one or more components of 160). The analytic detector 1020 includes a detection system having one or more sensors 1022. The analytic detector 1020 may also include a reaction controller 1024 that controls the incubation of the sample and the proper release of the reaction reagents and detector molecular assembly at appropriate time points. The sensors 1022 may be one or more of optical sensors (e.g., fluorescence sensors, infrared sensors), image sensors, electrical sensors, or magnetic sensors. In one embodiment, sensor 102 is a metal oxide semiconductor image sensor device.

[0097] Supramolecular structure of complex 40 held at individual binding sites 66 via interaction with capture molecule 70 body10 is detected and generates a detectable signal. For example, the barcode of linker 20 is used as a binding site for a detectable signaling element (e.g., via hybridization of complementary sequences) that comes into contact with the bound complex. In some embodiments, the signaling element includes fluorescent molecules or microbeads, fluorescent polymers, highly charged nanoparticles or polymers. In some embodiments, the barcode is amplified. For example, the barcode is used as a polymerization initiator for the growth of highly fluorescent polymers in processes such as rolling circle amplification or hybridization chain reactions.

[0098] In some embodiments, the signaling elements are optically active and can be measured using a microscope or an integrated optical sensor within the substrate 60. In some embodiments, the signaling elements are electrically active and can be measured using an integrated electrical sensor. In some embodiments, the signaling elements are magnetically active and can be measured using an integrated magnetic sensor. In some embodiments, each signal event is associated with the capture of the same type of analyte molecule (a single copy of the same type of analyte molecule) determined by the corresponding detector and affinity binder, and thus the analyte molecules in the sample can be quantified by counting the number of locations where the signaling elements are present.

[0099] In some embodiments, supramolecular structure body This converts information about the presence of a given analyte molecule in the sample into a DNA signal. In some embodiments, the DNA signal corresponds to sequence data of a capture barcode and / or detector barcode, and affinity binders and detector molecules are simultaneously linked (e.g., bound) to the analyte molecule (e.g., sandwich formation).

[0100] In some embodiments, as described herein, detecting the presence of analyte molecules involves the controllable release of one or more unique nucleic acid molecules into a solution used to identify and quantify the properties of analyte molecules from a sample. In some embodiments, the unique nucleic acid molecules are their respective supramolecular structures body The capture barcode 20 is provided. In some embodiments, detecting the presence of the analyte molecules described herein involves generating an optical or electrical signal associated with a change of state that can be counted to quantify the concentration of the analyte molecules in the solution.

[0101] In some embodiments, each supramolecular structure body 10 contains a unique DNA barcode corresponding to the associated affinity binder. As provided herein, the linker barcode 20 is an individual supramolecular structure body It can be used to uniquely identify 10. Next, each supramolecular structure body 10 is assembled so that the affinity binder 2 is associated with the capture barcode 20, for example, and stored in the lookup table of the analyte detection system. Therefore, once the capture barcode 20 is identified, the identity of the affinity binder 2 is also accessible.

[0102] Analyte detection techniques can be used to characterize analyte binding. The binding complex 40 is 1) supramolecular structure body10 barcodes 20, and in embodiments, 2) barcodes of capture molecules 70 located at the same locations as barcodes 20, can be characterized. The barcodes can be detected by a signal generated by a detector molecule bound to one or more of the binding sites 66, complex 40, or capture molecules 70. In one embodiment, the location of the capture molecules 70 can be determined before complex binding. That is, the array of binding sites 66 may be provided pre-mapped, or mapping may be a separate step. Mapping may include the step of detecting the capture molecule barcodes as commonly provided herein, such as detecting unique optical, electrical, and / or magnetic patterns. In one embodiment, detection includes sequencing the nucleotide sequence of the capture barcodes. In one embodiment, detection includes amplification and quantification of the amplification product, for example, detection of a signal associated with the probe by qPCR.

[0103] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided only as examples. Herein, those skilled in the art will anticipate numerous variations, modifications, and substitutions without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed in the practice of the present invention. The following claims define the scope of the present invention, and the methods and structures contained within these claims, as well as their equivalents, are intended to be encompassed thereby.

Claims

1. A method for detecting analyte molecules present in a sample, This method involves contacting a sample containing analyte molecules with a pool of supramolecular structures in solution to form analyte molecule-supramolecular structure complexes, wherein each analyte molecule-supramolecular structure complex is Core structure including DNA origami, An affinity binder which is an antibody or a part of an antibody molecule and is bound to the core structure via a linker, wherein the antibody or a part of an antibody molecule can bind to the analyte molecule, and The analyte molecule bound to the affinity binder The formation includes, wherein different supramolecular structures in the pool of supramolecular structures contain different affinity binders having different binding affinities to different analyte molecules, The process involves contacting the analyte molecule-supramolecular structure complex with an array, wherein the binding sites of the array contain immobilization affinity binders having different binding affinities for different analyte molecules. To detect the binding of the analyte molecule-supramolecular structure complex to the binding site of the array, The method, including the method described above.

2. The method according to claim 1, comprising identifying the individual analytes of the individual analytes of the individual analytes of the supramolecular structure complex bound to the individual binding sites of the array.

3. The method according to claim 2, wherein the identification includes generating a detectable signal from the supramolecular structure and associating the detectable signal with the individual binding sites.

4. The method according to claim 3, wherein the detectable signal is an optical, magnetic, or electrical signal indicating the presence of the individual analyte molecule-supramolecular structure at the individual binding sites.

5. The method according to claim 2, wherein the identification comprises amplifying a signal from a unique identifier of the affinity binder in the supramolecular structure and detecting the amplified signal, wherein the unique identifier is a barcode nucleic acid sequence.

6. The method according to claim 2, wherein the identification comprises amplifying a signal from a unique identifier of the immobilized affinity binder immobilized on the individual binding sites, and detecting the amplified signal, wherein the unique identifier is a barcode nucleic acid sequence.

7. The method according to claim 1, wherein the immobilized affinity binder is an antibody molecule or a part of an antibody molecule, and the antibody molecule or a part of an antibody molecule can bind to the analyte molecule.

8. The method according to claim 1, wherein the binding of the analyte molecule-supramolecular structure to the immobilized affinity binder at individual binding sites includes the binding of the affinity binder of the supramolecular structure to a first portion of the analyte molecule and the binding of the immobilized affinity binder to a second portion of the analyte molecule.

9. The method according to claim 1, wherein the immobilized affinity binder is linked to each individual binding site via a nucleic acid capture molecule.

10. The method according to claim 9, wherein each individual binding site includes multiple nucleic acid capture molecules.

11. The method according to claim 10, wherein all of the plurality of nucleic acid capture molecules at each binding site have the same nucleic acid sequence.

12. The method according to claim 10, wherein the plurality of nucleic acid capture molecules are bound to a supramolecular structure immobilized at the binding site.

13. The method according to claim 1, comprising a plurality of immobilization affinity binders, each of which has an affinity for the same analyte molecule.

14. The method according to claim 1, wherein each bonding site has a diameter of 20 to 500 nanometers.

15. The method according to claim 1, wherein each supramolecular structure in the pool is a nanostructure.

16. The method according to claim 1, wherein each DNA origami has at least one lateral dimension of about 50 nm to about 1 micron.

17. The method according to claim 1, wherein each supramolecular structure in the pool is arranged in a predetermined shape and / or has a predetermined molecular weight.

18. The method according to claim 1, further comprising contacting the sample with the array and then removing analyte molecules-supramolecular structures that are not bound to the binding sites of the array.

19. The method according to claim 1, wherein the analyte molecule comprises a protein, a peptide, or a peptide fragment.

20. A method for detecting analyte molecules present in a sample, This method involves contacting a sample containing analyte molecules with a pool of supramolecular structures in solution to form analyte molecule-supramolecular structure complexes, wherein each analyte molecule-supramolecular structure complex is Core structure including DNA origami, An affinity binder which is an antibody or a part of an antibody molecule and is bound to the core structure via a linker, wherein the antibody or a part of an antibody molecule can bind to the analyte molecule, Sample-specific barcode, and The analyte molecule bound to the affinity binder The formation includes, wherein the analyte molecule comprises a protein, peptide, or peptide fragment, and the different supramolecular structures in the pool of supramolecular structures comprise different affinity binders having different binding affinities to different analyte molecules, The pooling of the aforementioned analyte molecule-supramolecular structure complex with other analyte molecule-supramolecular structure complexes, wherein each of the other analyte molecule-supramolecular structure complexes is associated with a different sample-specific barcode, The pooled analyte molecule-supramolecular structure complex is brought into contact with an array, wherein the binding sites of the array contain immobilization affinity binders having different binding affinities for different analyte molecules. To detect the binding of the analyte molecule-supramolecular structure complex to the binding site of the array, The detected binding is associated with the sample-specific barcode, The method, including the method described above.

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