Solution-phase single molecule capture and related techniques

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 incorporated by reference in their entireties herein for all purposes. [Background technology]

[0002] The current state of personalized medicine is overwhelmingly genomic-centric, focusing primarily on quantifying the genes present within an individual. While such approaches have proven very powerful, they do not provide clinicians with a complete picture of an individual's health, as genes are the "blueprint" of an individual and only inform the likelihood of developing a disease. Within an individual, these "blueprints" first need to be transcribed into RNA and then translated into various protein molecular "actors" within the cell in order to affect the health of the individual.

[0003] The concentrations of proteins, interactions between proteins (protein-protein interactions or PPIs), as well as 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. Thus, quantitative information on proteins and protein interactions such as PPIs is essential to create a complete picture of an individual's health at a given time point, as well as to predict any emerging health problems. The presence and interactions between these proteins are also essential for drug development and are becoming highly sought-after data sets to capture individual proteomes and changes to the proteome in response to environmental or other systemic events. The ability to detect and quantify proteins and protein interactions with other molecules within a given sample is an essential component of such healthcare development. Summary of the Invention

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

[0005] In some embodiments, provided herein is a solution-based technique for detecting analyte molecules present in a sample, the technique being characterized in that the analytes in solution are ligated to their respective supramolecular structures. body or otherwise captured by the respective supramolecular structure body For example, in one embodiment, individual analytes (e.g., protein molecules) are separated into supramolecular structures. body Once captured, the analyte-supramolecular structure body The complexes can be detected on a substrate as part of a detection system whereby individual binding sites of the substrate carry affinity binders for the analytes of interest. body Binding at the binding site of the complex is determined by the binding site affinity binder and the supramolecular structure. body It can be a sandwich type arrangement in which affinity binding agents bind to different locations on individual analyte molecules. Thus, the signal detected at a particular binding site can be correlated with the presence of a particular 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) to identify the associated affinity binder; 2) a detectable initiator to provide a detectable signal that can be correlated with binding in a detection system; and 3) a physical scaffold attached to the analyte that promotes single molecule binding or low averaging of the molecule at a binding site on the substrate, e.g., that sterically hinders or sterically occludes binding of other molecules at the same binding site.

[0006] Size-exclusion supramolecular structure bodyprovides greater flexibility in preparing substrates for complexing with and detecting analytes in solution. In contrast to systems designed to have a single affinity binder associated with each binding site, which is complex to manufacture, the disclosed technology works with more permissive binding site preparations that include multiple immobilized affinity binders at each binding site. Thus, supramolecular structures body Although the substrate may be a single molecular binding entity, the substrate may be arranged to allow multi-molecular binding at each binding site. Supramolecular structures in which space limits binding at each site body The nature of few or single molecule binding at each binding site, facilitated by ELISA, allows for 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 an analyte molecule present in a sample includes providing a sample containing the analyte molecule, and dissolving the sample in a supramolecular structure in solution. body The analyte molecule-supermolecular structure is contacted with a pool of body and forming a complex. body can include a core structure that includes a plurality of core molecules and an affinity binding agent bound to the core structure. body The complex may comprise a core structure comprising a plurality of core molecules; an affinity binding agent bound to the core structure; and an analyte molecule bound to the affinity binding agent, the supramolecular structure comprising: body Pool of different supramolecular structures body The method also includes the step of: body contacting the complex with an array, the binding sites of the array comprising immobilized affinity binders each having a binding affinity for a different analyte molecule, and body detecting binding of the complex to the binding sites of the array.

[0008] As provided herein, a method for detecting an analyte molecule present in a sample comprises the steps of: body wherein each individual supramolecular structure body are immobilized on respective binding sites of the substrate. body a core structure including a plurality of core molecules; and a plurality of nucleic acid capture molecules linked to the core structure, each of the nucleic acid capture molecules being linked to another supramolecular structure of the array. body The method includes contacting the array with a pool of affinity binding agents linked to single-stranded nucleic acid tags having binding specificity for the different identification sequences, and binding different affinity binding agents to different binding sites of the array such that each binding site comprises a different subset of the pool of affinity binding agents. The method also includes contacting a sample with the array such that analyte molecules bind to individual affinity binding agents of the pool of affinity binding agents, wherein the analyte molecules form supramolecular structures. body This involves complexing, contacting with a detection assembly, and detecting binding of analyte molecules to the individual binding sites.

[0009] As provided herein, a method for detecting an analyte molecule present in a sample may include providing an array including single-stranded nucleic acids immobilized thereon, such that each individual binding site of the array includes a plurality of single-stranded nucleic acids including the same sequence that is distinguishable from the sequence of other single-stranded nucleic acids immobilized at different binding sites of the array. The method includes contacting the array with a pool of affinity binding agents, such that the immobilized single-stranded oligonucleotides capture a subset of the affinity binding agents at the binding sites of the array, such that the captured affinity binding agents form capture molecules immobilized at the respective binding sites, and contacting a sample with the array, such that the analyte molecules bind to the individual capture molecules, such that the analyte molecules form supramolecular structures. body The method also includes detecting binding of an analyte molecule to each of the binding sites.

[0010] As provided herein, a method for detecting an analyte molecule present in a sample can include forming a nanoball or functionalized nanostructure. The nanoball or functionalized nanostructure includes a plurality of individual oligonucleotides, each of the plurality of oligonucleotides being bound to a chemical moiety, each oligonucleotide being bound to a complementary nucleic acid of the nanoball or functionalized nanostructure. The method includes providing a patterned array including a plurality of active sites; incubating the nanoball or functionalized nanostructure with the patterned array to covalently bind an active group of the individual active sites to the chemical moiety of the plurality of individual oligonucleotides to bind the individual nanoball or functionalized nanostructure to the individual active sites; denaturing the plurality of individual oligonucleotides from the complementary nucleic acid of the nanoball or functionalized nanostructure; washing the nanoball or functionalized nanostructure from the array to leave a plurality of individual oligonucleotides immobilized at the individual binding sites, the plurality of individual oligonucleotides being single stranded; and contacting the array with a pool of affinity binding agents to cause the immobilized plurality of individual oligonucleotides to capture a subset of the affinity binding agents at the individual binding sites.

[0011] As provided herein, a method for detecting an analyte molecule present in a sample includes providing a sample containing the analyte molecule, and dissolving the sample in a supramolecular structure in solution. body The analyte molecule-supermolecular structure is contacted with a pool of body and forming a complex with the individual analyte molecule-supermolecular structure. body The complex comprises a core structure comprising a plurality of core molecules; affinity binders bound to the core structure; and an analyte molecule sample-specific barcode of the analyte molecule bound to the affinity binder, said supramolecular structure body Pool of different supramolecular structures body comprises different affinity binders having different binding affinities for said analyte molecule to other analyte molecules. body The complex is then transferred to another analyte molecule - the supramolecular structure bodyPooling the complex with other analyte molecules-supermolecular structures body The complexes are each associated with a different sample-specific barcode; pooling the pooled analyte molecules-supermolecular structures. body contacting the complex with an array, the binding sites of the array comprising immobilized affinity binders each having a binding affinity for a different analyte molecule; contacting the analyte molecule-supramolecular structure with the binding sites of the array; body detecting binding of the complex; and associating the detected binding with a sample-specific barcode.

[0012] As provided herein, a method for detecting an analyte molecule present in a sample is disclosed. The method comprises: body Each supramolecular structure body The present invention relates to a supramolecular structure comprising a core structure including a plurality of core molecules; an antibody having a binding affinity for an antigen and bound to a nucleic acid capture strand; and a complementary strand to the nucleic acid capture strand, the complementary strand being linked to the core structure and forming a duplex structure with the nucleic acid capture strand to bind the antibody to the core structure, and a plurality of supramolecular structures. body The method comprises the steps of: forming a complex comprising a plurality of supramolecular structures, each of which has a different binding affinity to the other antibodies; body contacting the complex with a sample containing an antigen; contacting the complex with a bead carrying a capture antibody having binding specificity for the antigen to form a sandwich structure; displacing the nucleic acid capture strand from the complementary strand using a displacement strand to release the core structure into solution, where the released core structure is not bound to an 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 an analyte molecule in a sample. In some embodiments, any method disclosed herein further includes identifying the detected analyte molecule. In some embodiments, any method disclosed herein further includes detecting the analyte molecule based on the signal if the analyte molecule is present in the sample at a count of single molecules or greater.

[0014] In some embodiments, for any method disclosed herein, each core structure is a nanostructure. In some embodiments, for any method disclosed herein, the multiple 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 a supramolecular structure. body In some embodiments, for any method disclosed herein, the multiple core molecules for each core structure comprise 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 comprises 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 organized DNA or RNA origami with multiple backbones, a peptide structure, or a combination thereof.

[0015] supramolecular structure body The affinity binding agent can be attached to the core structure via a chemical bond. In some embodiments, the solution-based supramolecular structure body The immobilized affinity binders of the affinity binders and / or binding sites include, independently, proteins, peptides, antibodies, aptamers (RNA and DNA), fluorophores, DARPins, catalysts, polymerization initiators, polymers such as PEG, or combinations thereof.

[0016] In some embodiments, for any of the methods disclosed herein, the detectable signal may be, for example, a binding site and / or a supramolecular structure. body In some embodiments, each barcode corresponds to an affinity binder and provides a DNA signal or an initiator signal that provides information indicating its specificity for the analyte molecule bound to the respective detector molecule. In some embodiments, the barcodes are analyzed using genotyping, qPCR, sequencing, or a combination thereof. In some embodiments, multiple analyte molecules in a sample are detected simultaneously by multiplexing. In some embodiments, for any method disclosed herein, the affinity binders provided herein are configured to bind to one or more specific types of analyte molecules.

[0017] In some embodiments, a plurality of the supramolecular structures disclosed herein body For any method comprising using a plurality of supramolecular structures body Each core structure of is identical to the others. However, the attached affinity binding agent may vary for the plurality. Thus, in one embodiment, the (e.g., plurality) of supramolecular structures body The pools are identical except for the bound affinity binders and, in some embodiments, an identifying moiety (e.g., an affinity binder identification barcode nucleic acid sequence) that identifies the bound affinity binder. In the case of multiplexed samples detected on a single substrate or detection platform, the supramolecular structures associated with a particular sample are body can have a sample-associated identifying portion (e.g., a sample barcode nucleic acid sequence) that identifies the sample.

[0018] In some embodiments, each supramolecular structure body Multiple supramolecular structures body In some embodiments, each supramolecular structure comprises a predetermined shape, size, molecular weight, or combination thereof that can reduce or eliminate cross-reactivity between the supramolecular structures. body The supramolecular structure may comprise a single affinity binding agent or multiple affinity binding agents. bodyWhen the supramolecular structure comprises multiple affinity binders on a single core structure, the multiple affinity binders can all have the same binding specificity, e.g., all specifically bind to the same analyte. In some embodiments, each supramolecular structure body Multiple supramolecular structures body The capture and detection molecules comprise a predetermined stoichiometric ratio to reduce or eliminate cross-reactivity between the capture and detection molecules.

[0019] In some embodiments, the substrate comprises a solid support, a solid substrate, a polymer matrix, or one or more beads. The substrate can comprise 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 surface comprising a second chemical group. A bead is loaded at each binding site, the first chemical group selectively binds to the bead, and the second chemical group does not interact with or bind to the bead. Each bead comprises a plurality of single-stranded oligonucleotides immobilized on each bead, such that each binding site of the array comprises a plurality of single-stranded oligonucleotides comprising the same sequence that is distinguishable from the sequence of other single-stranded oligonucleotides immobilized on different binding sites of the array, and the captured affinity binding agent forms a capture molecule immobilized on each binding site, and each analyte molecule is arranged in a supramolecular structure. During detection, an analyte molecule binds to an individual capture molecule at each binding site, and each analyte molecule is arranged in a supramolecular structure. body A complex is formed with the detection assembly, the detection of which allows for the detection of an analyte bound to the substrate.

[0020] In some embodiments, the plurality of supramolecular structures body is disposed on a substrate, such as a molded or planar substrate, the substrate comprising a plurality of binding sites, each individual binding site being bound to one or more affinity binding agents configured to bind the same analyte molecule, e.g., such that each individual binding site is specific for an individual analyte molecule, and different binding sites of the substrate have specificity for different analyte molecules. The disclosed embodiments also include sample preparation reagents, substrates, and detection systems for carrying out the disclosed methods.

[0021] In some embodiments, for any method disclosed herein, the sample comprises a complex biological sample. In some embodiments, for any method disclosed herein, the one or more analyte molecules of the sample comprise a protein, a peptide, a peptide fragment, a lipid, DNA, RNA, an organic molecule, an inorganic molecule, a complex thereof, or any combination thereof. In some embodiments, for any method disclosed herein, the sample comprises a biological particle or a biomolecule. In some embodiments, for any method disclosed herein, the sample comprises an aqueous solution comprising a protein, a peptide, a peptide fragment, a lipid, DNA, RNA, an organic molecule, a viral particle, an exosome, an organelle, or any complex thereof. In some embodiments, for any method disclosed herein, the sample comprises a tissue biopsy, blood, plasma, urine, saliva, tears, cerebrospinal fluid, extracellular fluid, cultured cells, culture medium, discarded tissue, plant material, synthetic proteins, prions, bacterial and / or viral samples, or fungal tissue, or a combination thereof. The sample may be an environmental sample, such as a 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 food or nutritional ingredient sample, or a packaging component.

[0022] The sample may be a supramolecular structure in solution as provided herein. body Prior to contacting with, the cells may be treated to release the analyte from the cell or to otherwise prepare the sample for analysis.

[0023] Specific embodiments of the disclosed devices, delivery systems, or methods will now be described with reference to the drawings. Nothing in this detailed description is intended to imply that any particular component, feature, or step is essential to the invention. [Brief description of the drawings]

[0024] [Figure 1] 1 shows an exemplary analyte-supramolecular structure complex after solution-based capture according to an embodiment of the present disclosure. [Diagram 2] FIG. 1 illustrates an exemplary workflow involving solution-based single molecule capture sample preparation according to embodiments of the present disclosure. [Diagram 3] 1 illustrates an example of a substrate for analyte detection according to an embodiment of the present disclosure. [Figure 4] 1 shows an example of a binding site of a substrate with a capture molecule and a captured analyte-supramolecular structure complex according to an embodiment of the present disclosure. [Diagram 5] 1 illustrates an example of a substrate for analyte detection according to an embodiment of the present disclosure. [Figure 6] 1 shows a linked barcode nucleic acid structure for use with a substrate for analyte detection according to an embodiment of the present disclosure. [Figure 7] 7 shows an example of an analyte detection substrate including the linked barcode nucleic acid structures of FIG. 6 according to an embodiment of the present disclosure. [Figure 8] 1 illustrates a nanoparticle structure for use with a substrate for analyte detection according to an embodiment of the present disclosure. [Figure 9] 6 illustrates an example of a substrate for analyte detection including the nanoparticle structure of FIG. 5 according to an embodiment of the present disclosure. [Figure 10] 1 illustrates an example of a substrate for analyte detection according to an embodiment of the present disclosure. [Figure 11] 1 shows an example of an analyte detection bead according to an embodiment of the present disclosure. [Figure 12] 1 illustrates an exemplary workflow for generating a substrate for housing beads for analyte detection according to an embodiment of the present disclosure. [Figure 13] 1 illustrates an exemplary workflow involving solution-based single molecule capture sample preparation for multiplexed detection according to embodiments of the present disclosure. [Figure 14A] FIG. 1 shows steps of an experimental workflow for antigen detection involving an IgG affinity binder conjugated to a box origami to form a supramolecular structure according to an embodiment of the present disclosure. [Figure 14B] FIG. 14B shows an additional step in the experimental workflow of FIG. 14A. [Figure 15]The different experimental subgroups evaluated using the experimental workflow in Figures 14A-B are shown. [Figure 16] Figure 14 shows solution-based optical detection results with different antigen combinations of the three antigens used in the experimental workflow in Figure 14A-B. [Figure 17] 14A-B show the solution-based optical detection results from the titration of TSH used in the experimental workflow of FIG. [Figure 18] 1 shows solution-based optical detection results for antigen titration with and without the presence of another antigen. [Figure 19] 1 shows solution-based optical detection results for monomeric, dimeric and trimeric antigens. [Figure 20] 1 shows the results of solution-based optical detection of a mixture of unlabeled and labeled antigens. [Figure 21] 1 shows the results of solution-based optical detection of mixed or complex samples. [Figure 22] 1 shows the solution-based optical detection results of titrated antigen samples. [Figure 23] FIG. 1 shows a block diagram of an exemplary analyte detection system according to an embodiment of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Disclosed herein are structures and methods for detecting one or more analyte molecules present in a sample. In some embodiments, the one or more analyte molecules are incorporated into one or more supramolecular structures. body In some embodiments, the detection is based on solution-based capture by one or more supramolecular structures. body The analyte-binding protein comprises or is linked to an affinity binder that specifically binds to an analyte present in a sample and in solution, and is used for single molecule capture of the analyte. The binding of the analyte is achieved by forming an analyte molecule-supermolecular structure in solution. body Complexes are formed and these complexes can be detected by the detection systems provided herein.

[0026] In an embodiment, the complexed analyte is captured by an immobilized affinity binder associated with a substrate or other detection platform of the detection system. The capture of the complex then immobilizes the complex on the substrate, and one or more features of the immobilized substrate or its associated binding site can be characterized to characterize the analyte in the sample. In one embodiment, the supramolecular structure body comprises a detectable moiety, such as a unique identifier (e.g., nucleic acid sequence, peptide, polysaccharide, acrydite), and / or a molecule that can be used to contain, interact with, or dock with other molecules that are detectable (e.g., optically, electrically, magnetically). In some embodiments, the detectable moiety generates a DNA signal or other initiator signal, thereby binding the analyte molecule-supramolecular structure to the binding site. body Detection and quantification of the binding of the complex is performed using a supramolecular structure. body In some embodiments, the supramolecular structure can be detected by amplification of the unique identifier of the binding site and / or the unique identifier of the binding site. body is linked to an enzyme that converts the substrate into an optically detectable signal. body is coupled to a sensor on the substrate to generate an electrically or magnetically detectable signal. body is a nucleic acid origami linked or immobilized to a substrate. In one embodiment, the supramolecular structure body comprises a unique identifier for the affinity binding agent, and associates the affinity binding agent with a supramolecular structure. body The affinity binding agent is carried via a barcode bridge or linker that links to the scaffold.

[0027] In some embodiments, the disclosed techniques provide for single molecule capture of analyte molecules in complex samples. Supramolecular structures as capture entities bodyThe use of allows for specific identification and, in some embodiments, detection of associated affinity binders via 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 generate assay results in which the binding properties of the analyte pool of multiple different analytes are characterized. This allows samples with uncharacterized compositions of analytes to be analyzed for the presence and / or concentration of a particular analyte of interest. For example, a human sample can be characterized to determine the presence and / or concentration of an antibody having binding specificity for a particular antigen in a panel of antigen affinity binders, such that the affinity binders represent a panel of known infectious disease antigens. The assay results can show a positive binding result associated with the particular antigen, indicating the presence of the antibody in the subject providing the sample. In another embodiment, the identity of the analytes in the 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. Binding of drug candidates to such proteins can be used to assess bioavailability or potential off-target binding. The assay results may indicate positive binding results associated with a particular drug candidate that can be mapped to a particular analyte based on identification of a specific detection agent binding (e.g., identifying binding by barcode identification in a detection agent molecular assembly including an antibody specific for the analyte).

[0028] While 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 indication of binding, the disclosed technology can additionally or alternatively provide an amplified nucleic acid signal from the unique identifier (or other initiator) of the detector 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 for the unique identifier). Thus, the unique identification information of the complex captured at a particular binding site of the array allows for specific identification of the particular affinity binder that captured the analyte in certain embodiments. Supramolecular Structure body The modular and customizable structure of the building blocks of the supramolecular structures are generally identical, with individual supramolecular structures having unique identifiers during binding of specific affinity binding agents. body This allows for the provision of a bulk or common core structure that allows for barcoding of the identification sequence. Thus, the identification sequence is bound to only one specific affinity binder. Other detection techniques may include optical, magnetic, and / or electrical detection techniques.

[0029] Disclosed embodiments relate to analyte detection where the analyte is present in a sample, such as a biological sample. In some embodiments, the sample comprises an aqueous solution comprising 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 comprise proteins, peptides, peptide fragments, lipids, DNA, RNA, organic molecules, inorganic molecules, complexes thereof, or any combination thereof. In some embodiments, the analyte molecules comprise intact proteins, denatured proteins, partially or fully degraded proteins, peptide fragments, denatured nucleic acids, degraded nucleic acid fragments, complexes thereof, or combinations thereof. In some embodiments, the sample is obtained from tissues, cells, the environment of tissues and / or cells, or combinations thereof. In some embodiments, the sample comprises tissue biopsy, blood, plasma, urine, saliva, tears, cerebrospinal fluid, extracellular fluid, cultured cells, culture media, discarded tissue, plant material, synthetic proteins, bacteria, virus samples, fungal tissues, or combinations thereof. In some embodiments, the sample is isolated, with or without purification, from a primary source such as cells, tissues, bodily fluids (e.g., blood), environmental samples, or a combination thereof. In some embodiments, the cells are lysed using a mechanical process or other cell lysis methods (e.g., lysis buffer). In some embodiments, the sample is filtered using a mechanical process (e.g., centrifugation), micron filtration, 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 comprises intact proteins, denatured proteins, partially or fully 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 having a disease or disorder, including an infectious disease, an immune disorder, a cancer, a genetic disease, a degenerative disease, a lifestyle-related disease, an injury, a rare disease, an age-related disease, or a combination thereof.

[0030] The disclosed technology is a supramolecular structure body The present invention utilizes single molecule binding of an affinity molecule linked to a core structure 13. Single molecule binding of an analyte molecule forms a complex as shown in Figure 1, which can be detected by a detection system according to the disclosed technology. The complex formation occurs in solution. Figure 1 shows a supramolecular structure comprising a core structure 13 and an affinity binder 2. body Ten exemplary embodiments are provided. In some embodiments, the supramolecular structure body 10 comprises one or more affinity binding agents 2. In one embodiment, the supramolecular structure body 10 may refer to a complex comprising a core structure 13 and an affinity binding agent 2. In one embodiment, the supramolecular structure body 10 may refer to the core structure 13, the supramolecular structure body 10 may or may not include an affinity binding agent 2.

[0031] Therefore, the supramolecular structure body 10 is provided. In some embodiments, the supramolecular structure body 10 is a programmable structure that can spatially organize molecules. In some embodiments, the supramolecular structure body 10 comprises a plurality of molecules linked together. In some embodiments, the supramolecular structure body The plurality of molecules of 10 interact with at least some of each other. body 10 includes a particular shape, for example a substantially planar shape with its longest dimension in the xy plane. In some embodiments, the supramolecular structure body 10 is a nanostructure. In some embodiments, the supramolecular structure body 10, supramolecular structure body In some embodiments, the supramolecular structure comprises a plurality of molecules of a predetermined molecular weight based on 10. In some embodiments, the plurality of molecules are linked together via bonds, chemical bonds, physical bonds, or combinations thereof. body 10 comprises a large molecular entity of a particular shape and molecular weight formed from a well-defined number of smaller molecules that specifically interact with each other. In some embodiments, the supramolecular structure bodyThe structural, chemical, and physical properties of 10 are explicitly designed. In some embodiments, the supramolecular structure body 10 includes a plurality of subcomponents spaced apart according to a predetermined distance. In some embodiments, the supramolecular structure body At least a portion of 10 is rigid. body At least a portion of 10 is semi-rigid. body At least a portion of the supramolecular structure is flexible. body 10 is at least 50-200 nm in one dimension. body 10 is at least 20 nm long in 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 comprises one or more core molecules linked together. In some embodiments, the one or more core molecules comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, or 500 unique molecules linked together. In some embodiments, the one or more core molecules comprise from about 2 unique molecules to about 1000 unique molecules. In some embodiments, the one or more core molecules interact with each other to form a supramolecular structure. body In some embodiments, the core molecules interact with each other via reversible non-covalent interactions.

[0033] In some embodiments, the particular shape of the core structure 13 is a three-dimensional (3D) configuration. In some embodiments, one or more core molecules provide a particular molecular weight. For example, multiple supramolecular structures body All the core structures 13 of 10 may have the same composition, size, and / or weight, but their binding linker sequences and binding affinity binders 2 may be different. However, apart from the different linkers 20 and affinity binders 2, multiple supramolecular structures may be body10 may be otherwise identical. In some embodiments, core structure 13 is a nanostructure. In some cases, the one or more core molecules include one or more nucleic acid strands (e.g., DNA, RNA, non-natural nucleic acids), one or more branched nucleic acids, one or more peptides, one or more small molecules, or combinations thereof. In some embodiments, core structure 13 comprises an entirely polynucleotide structure. In some embodiments, at least a portion of core structure 13 is rigid. In some embodiments, at least a portion of core structure 13 is semi-rigid. In some embodiments, at least a portion of core structure 13 is flexible. In some embodiments, core structure 13 comprises 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 organized DNA and / or RNA origami with multiple backbones, a peptide structure, or a combination thereof. In some embodiments, a DNA origami is scaffolded. In some embodiments, an RNA origami is scaffolded. In some embodiments, a hybrid DNA / RNA origami is scaffolded. In some embodiments, the core structure 13 comprises a DNA origami, an RNA origami, or a hybrid DNA / RNA origami comprising a predetermined two-dimensional (2D) or 3D 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 μ. In one embodiment, the nucleic acid origami has at least one lateral dimension of, for example, 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, or about 200 nm to about 400 nm. In one embodiment, the nucleic acid origami has a first lateral dimension of at least about 50 nm to about 1 μ and a second lateral dimension of about 50 nm to about 1 μ that is orthogonal to the first lateral dimension. In one embodiment, the nucleic acid origami has at least one lateral dimension of about 200 nm. 2 ~about 1μ 2 The planar footprint has an area of

[0035] As shown in FIG. 1, in some embodiments, the core structure 13 is configured to be linked to an affinity binder 2. In some embodiments, the affinity binder 2 is immobilized relative to the core nanostructure 13 when linked thereto. However, the core structure 13 may be in solution and thus not immobilized relative to the sample or reaction vessel. As shown in FIG. 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 that includes a specific sequence of nucleic acid (double-stranded or single-stranded DNA or RNA) associated with the linked affinity binder 2. Thus, the sequence of the nucleotide linker 20 uniquely identifies the affinity binder 2 among a pool of different affinity binders 2. The barcode may be at least 6 nucleotides and may be 6-50 nucleotides. In FIG. 1, the supramolecular structure body 10 binds to an analyte molecule 14 that has binding specificity for the affinity binding agent 2, forming an analyte molecule-supermolecular structure body A complex 40 is formed.

[0036] In some embodiments, any number of one or more core molecules 13 include one or more linkers 20 configured to form bonds with affinity binding agents 2. In some embodiments, the linkers 20 are attached 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, a thiol, DBCO, an NHS ester, a maleimide, a biotin, an azide, an acrydite, a single-stranded nucleic acid of a specific sequence (e.g., RNA or DNA), or a 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 an affinity binding agent 2 at a predetermined location on the core structure 13 .

[0038] In some embodiments, the affinity binder 2 comprises a protein, a peptide, an antibody, an antibody-derived reagent, an aptamer (RNA and DNA), a fluorophore, a nanobody, a DARPin, a catalyst, a polymerization initiator, a polymer such as PEG, an organic molecule, a small molecule, a pharmaceutical compound, a candidate pharmaceutical compound, a synthetic molecule, or a combination 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 may bind to different analyte molecules of a multimolecular complex, 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, the supramolecular structure body Each of the components of 10 may be independently modified or adjusted. In some embodiments, the supramolecular structure body By modifying one or more of the ten components, the supramolecular structure bodyThe two-dimensional and three-dimensional shapes of the supramolecular structures can be modified. body By modifying one or more of the components of the core structure 13, the two-dimensional and three-dimensional shape of the core structure 13 can be modified. In some embodiments, such an ability to independently modify the components of the supramolecular nanostructure allows one or more supramolecular structures to be modified. body This allows for precise control over the organization of the

[0040] As described herein, in some embodiments, one or more supramolecular structures body The complex 40 allows for the detection of one or more analyte molecules in a sample. body Schematic of a workflow for forming a complex 40. A supramolecular structure with associated affinity binders 2, each representing a panel or set of affinities for a different analyte. body The pool of 10 is contacted with a sample 50 that includes a plurality of different analyte molecules 14. The analytes 14 in the sample 50 may be uncharacterized or unknown analytes 14. In embodiments, the sample 50 may include one or more control analytes 14.

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

[0042] As provided herein, after formation of the complex 40, unbound analyte 14 can be removed before the complex is provided to a detection system. However, in other embodiments, no washing step is performed. Unbound analyte 14 in solution can interact with the detection system, but is unlikely to bind specifically at a binding site and is likely to form a supramolecular structure. body 10 and therefore produces no detectable signal.

[0043] Analytes 14 of the disclosed complexes 40 may be detected based on their 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 3-13. The substrate 60 may include a defined set of micropatterned binding sites 66 that are functionalized by having immobilized capture molecules 70.

[0044] In some embodiments, the binding sites 66 are micropatterned on the planar substrate 60. In some embodiments, the binding sites 66 on the surface are in a periodic pattern. In some embodiments, the binding sites 66 on the surface are in a non-periodic pattern (e.g., random). In some embodiments, a minimum distance is specified between any two binding sites 66. In some embodiments, the minimum distance between any two binding sites 66 is at least about 200 nm. In some embodiments, the minimum distance between any two binding sites 66 is at least about 40 nm to about 5000 nm. In some embodiments, the geometric shape of the binding sites 66 includes a circle, a square, a triangle, or other polygon. In some embodiments, the individual binding sites 66 are 20 to 200 nm in diameter. 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 with one or more silicon dioxide, silicon nitride, graphene, or silicon carbide layers. In one embodiment, each binding site 66 accommodates multiple capture molecules 70 of the same type at each binding site 66, with different binding sites 66 having different capture molecule specificities or different chemistries. The patterned substrate 60 may be fabricated by a lithographic process. Additionally, embodiments of the disclosed technology may include one or more regeneration steps to remove bound or "used" complexes 40 from the substrate 60 to allow for binding of new complexes 40 in a subsequent reaction.

[0046] In some embodiments, substrate 60 may include fiducial markers (not shown) having geometric features defined on its surface that are used as reference features for other features on substrate 60. In some embodiments, planar substrate 60 includes structures that facilitate detection, such as optical or electrical devices, such as FETs, ring resonators, photonic crystals, or microelectrodes, that are defined prior to formation of binding sites 66.

[0047] FIG. 3 provides an exemplary diagram of forming a substrate 60 used to detect analyte molecules in a sample using a surface-based assay using capture molecules 70 (shown as antibodies, but can be any suitable capture molecules to pull down complexes 40) as described herein. In one embodiment, the capture molecules 70 are affinity binders as generally disclosed herein. In FIG. 3, a patterned substrate 60 can be formed having a plurality of binding sites 66 functionalized or linked with individual capture molecules 70 according to a 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(s) 112 can include silicon nitride, graphene, quartz, metal, gold, silver, platinum, palladium, PDMS, polymeric film, or combinations thereof. The passivation layer 112 can be graphene, aluminum oxide, HfO2, Cr2O3 (chromium oxide), titanium oxide, tantalum oxide, metal oxide, silicon dioxide (SiO2), or combinations thereof. The passivation layer 112 may be a self-assembling polymer such as polyacrylamide.

[0048] The passivation layer 112 is patterned, for example, by removing portions of the top layer 112 to expose locations 120 of the base layer 110 that will correspond to the binding sites 66 of the substrate 60. The patterning may be by photolithography, e-beam lithography, nanoimprinting, polymer spin coating, optical patterning, plasma activation, acid / base treatment, or other patterning modalities. The exposed locations 120 can be activated by chemical or plasma treatment to generate different reactive groups, depending on the individual chemistries of these layers.

[0049] Capture molecules 70 can be attached to the activated locations 120 to generate binding sites 66. In the illustrated example, the binding sites 66 can be printed with capture molecules 70. Each binding site 66 is printed with a preselected capture molecule 70. However, other attachment or binding techniques for linking the capture molecules to the binding sites 66 are contemplated. In an embodiment, 1) each binding site includes multiple (e.g., two or more) capture molecules 70, and 2) all of the capture molecules 70 at each binding site have the same binding specificity for a particular analyte 14.

[0050] The capture molecule 70 may be one or more affinity binding agents, supramolecular structures, or the like, as provided herein. body , nucleic acids. Each binding site 66 of the substrate 60 includes multiple capture molecules 70 having the same binding specificity for each binding site 66. Additionally, adjacent or different binding sites 66 can have different binding specificities, such as a first capture molecule 70a having a different binding specificity than a second capture molecule 70b.

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

[0052] Each binding site 66 may be attached to a barcode or unique identification sequence that can be read out either via sequencing, amplification, or a hybridization assay as part of detection. Prior to running an assay, a map of the capture molecules 70 immobilized on the substrate 60 and with their spatial locations on the substrate 60 can be obtained and run as a quality control of the substrate 60. The map can be stored in the analyte detection system provided herein (see FIG. 10) and can be used to generate a report of positive binding events to provide analyte information of the sample.

[0053] Once formed, the substrate 60 can be used in an analyte capture step after sample preparation (shown in FIG. 2) to provide the complex 40. In some embodiments, the complex 40 is contacted with a planar substrate using a flow cell. In some embodiments, the complex 40 is incubated on the substrate 60 having the capture molecules 70 bound to the binding sites 66. In some embodiments, the incubation period can be from about 30 seconds to about 24 hours. In some embodiments, the incubation period can be from about 30 seconds to about 1 minute, from about 1 minute to about 5 minutes, from about 5 minutes to about 30 minutes, from about 30 minutes to about 1 hour, from about 1 hour to about 5 hours, from about 5 hours to about 12 hours, from about 12 hours to about 24 hours, from 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. FIG. 4 shows an exemplary binding site 66 with a capture molecule 70a having specificity for a particular analyte 14a of the complex 40a. After the interaction, the binding can be detected by signal generation. Unbound complexes 40b can be washed away before detection. The capture molecule 70a and each analyte molecule 14a may have binding specificity for each other. Because the analyte molecule 14a forms the complex 40a in solution before contacting the substrate 60, the capture molecule 70a, the analyte 14a and the affinity binding agent 2a (linked to the core structure 13a) can form a sandwich-like binding arrangement as shown in FIG. 4. Therefore, both the capture molecule 70a and the affinity binding agent 2a can have specificity for the same analyte 14a. However, the capture molecule 70a and the affinity binding agent 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 of which binds to a respective capture molecule 70 and binds the analyte 14 to a specific supramolecular structure. bodyIt can be subjected to various detection protocols to associate with its identity, which can then be associated with a known affinity binding agent 2, which can then be associated with a unique identifier, e.g., a barcode sequence, at the specific binding site 66. Detection thus allows characterization of the analyte-affinity binding agent binding.

[0056] In some embodiments, the binding site 66 is a single supramolecular structure located at the activation site 120 to form the binding site 66, as shown in workflow 150 of FIG. body The binding site 66 is functionalized via supramolecular structure 160. body Prior to placement of 160, it may be activated as generally disclosed with respect to FIG. body 160 generally refers to the supramolecular structures provided herein. body 10 and may include a core structure 13 comprising a DNA origami, where the supramolecular structure body 10 are attached to each of the binding sites using DNA origami placement techniques, which may include linkage via anchor molecules. In some embodiments, the DNA origami placement includes directed self-assembly techniques to construct 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 significant period of time after this step.

[0057] In the illustrated embodiment, the supramolecular structure body 160 is provided with assembled and already linked capture molecules 70. Individual supramolecular structures, each with different analyte specificities. body The capture molecules 70 may be formed separately, for example in separate reaction tubes. body 160 core structure 13. A single supramolecular structure in a single binding site 66. body All 160 capture molecules 70 have the same binding specificity. body 160 includes a barcode or other identification sequence that uniquely identifies the type of capture molecule 70 associated therewith.

[0058] A preformed supramolecular structure is formed such that the capture molecule 70 is positioned "right-side up" on the binding site 66. body The arrangement of 160 can be directional. body 160 are all generally the same and are randomly placed in the individual binding sites. After placement in the different binding sites, the unique identification sequences and associated capture molecules 70 are ligated.

[0059] 6-7 show steps in the formation of a planar substrate 60 in which binding sites 66 are functionalized via a binding agent for a ligated nucleic acid product 200. FIG. 5 shows steps in forming a ligated nucleic acid product 200 from a circular template 210. The circular template includes a barcode sequence 220 that uniquely distinguishes the circular template 210 from other circular templates 210. Rolling circle amplification with a strand-displacing polymerase extending a primer 230 produces a single-stranded rolling circle amplification product that includes a nucleic acid linked to a repeat unit 252. A single-stranded probe 260 having an active group 262 hybridizes complementary to the barcode sequence 220 and binds to multiple locations on the ligated nucleic acid product 200 where the barcode sequence 202 repeats as part of the repeat unit 252. Thus, the end product of rolling circle amplification for binding site generation is a ligated nucleic acid product 200 with multiple binding probes 260 and associated active groups 262. In one embodiment, forming a substrate as provided herein includes creating multiple products 200 from respective different templates 210, each product 200 having a distinguishable sequence based on the different templates 210. Each different product 200 can be formed in a different reaction vessel. However, in one embodiment, the different templates 210 can be pooled to generate a pooled product 200. As shown in FIG. 7, each product 200 generally associates with a binding site 66 in a 1:1 ratio, so the pooled population of products 200 may nevertheless be distributed around the substrate 60. However, generating the products 200 independently can prevent differences in amplification bias that result in unequal production of products 200 from particular species of templates 210, which may result in over-representation of a particular template sequence on the binding site 66.

[0060] Each template 210 includes a sequence that can associate with or key to a particular 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 FIG. 7, the active site 120 can be created as generally disclosed with respect to FIGS. 3-5. The binding site 66 is functionalized by placement of a single linked nucleic acid product 200 with multiple binding probes 260 at the activation location 120 to form the binding site 66. The active site 120 includes an active molecule 280 surrounded by a passivation layer 112 as shown. The single stranded linked nucleic acid product 200 is typically a nanoball of a size such that only a single product 200 can be accommodated on a single active site 120. The association of the product 200 with the active site during the formation of the binding site 66 is via the active group 262 of the bound (hybridized) probe 260. The interaction of the probe 262 with the active molecule can be through NHS-ester, thiol, DBCO, azide, maleimide interactions. For example, in one embodiment, maleimide groups react specifically with sulfhydryl groups when the pH of the reaction mixture is between 6.5 and 7.5; as a result, a stable thioether bond is formed. Thus, in one embodiment, active group 262 can be a maleimide reagent and active molecule 280 can be a sulfhydryl. Active group 262 and active molecule 280 are reacted to form a stable conjugated thioether bond. This immobilizes probe 260 at active site 120. The nanoball product can be removed by denaturing and washing from probe 260 at a denaturing temperature. This process leaves probe 260 immobilized at binding site 66. All of probes 260 have the same sequence along at least a portion of the oligonucleotide. In one embodiment, all of probes 260 have the same sequence as each other within binding site 66, but have different sequences from probe 260 bound to some, most, or all of the other binding sites. In one embodiment, the probes 260 of the 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. Because the nanoball products 200 are generated from circular templates 210 (see FIG. 6), different templates 210 with different sequences will generate correspondingly different nanoball products 200.Thus, the probes 260 complementary to the repeat units 252 of the different products 200 have different sequences based on the original template sequence.

[0062] Once immobilized, the probes 260 can be sequenced, amplified or detected via tagged complementary oligonucleotides to map the binding sites 66 and their respective immobilized probes 260. The map can be stored in the detection system (FIG. 13).

[0063] 8-9 show another structural arrangement for positioning capture molecules on the binding sites 66 of the substrate 60. In FIG. 8, a nanoparticle 300, which may be made of a polymer hydrogel, a cross-linked polymer, or an inorganic material, is incubated with a single-stranded oligonucleotide 302 having a chemical moiety 303 that can be covalently attached to the nanoparticle 300. The nanoparticle 300 functionalized with the single-stranded oligonucleotide 302 is subsequently incubated with a second single-stranded oligonucleotide 306 having a second chemical moiety 305 to obtain a functionalized nanoparticle 307 having immobilized double-stranded DNA and having a chemical moiety 305. Thus, the oligonucleotides 302, 306 interact based on complementarity to form a double-stranded DNA, and the chemical moiety is attached to the nanoparticle 307 via hybridization. Examples of chemical moieties 303, 305 include, but are not limited to, thiols, amines, DBCO, maleamide, azide, NHS-ester.

[0064] The functionalized nanoparticles 307 of FIG. 8 can be used to create monoclonal clusters of ssDNA on the patterned substrate 60 by positioning the nanoparticles 307, covalently binding to the surface, and then removing / denaturing. Steps 1 and 2 for patterning the substrate 60 can be performed as generally disclosed herein (see, e.g., FIG. 3). In one embodiment, the workflow involves first passivating the surface and then lithographically patterning according to the protocols provided herein to result in a surface with two chemical features, one within the binding site 66 and the other forming the passivated background 112. In step 3, the functionalized nanoparticles 307 of FIG. 8 are incubated on the patterned substrate 60 to result in the assembly of the nanoparticles 307 within the binding site 66. The driving force for this assembly is the interaction between the nanoparticles 307 and the binding site 66. The nanoparticles 307 are generally sized such that there is a single nanoparticle 307 organized or positioned at a single binding site 66. The chemical moieties 305 attached to the nanoparticles 307 are covalently attached to the surface 310 at the binding sites 66. Finally, the nanoparticles 307 as well as the complementary nucleic acid 302 on the nanoparticles are denatured to separate the oligonucleotide strands 302, 306, resulting in a patterned surface with multiple copies of identical ssDNA 306 at each binding site. Single stranded DNA (or RNA) 306 is covalently attached to the surface 310 via the chemical moieties 305.

[0065] FIG. 10 shows the binding of capture molecules 70, shown as antibodies, to single-stranded oligonucleotides 320 located at individual binding sites 66. The single-stranded oligonucleotides 300 can be probes 260 attached via active groups 262 as shown in FIG. 7. The single-stranded oligonucleotides 300 can be oligonucleotides 306 attached via moieties 305 as shown in FIG. 9. The single-stranded oligonucleotides 320 immobilized at the binding sites 66 can directly function as capture molecules 70 (e.g., hybridization). However, in embodiments, the unique sequence of the oligonucleotides 320 can be used to design complementary tags that are attached to the capture molecules 70 via hybridization in a binding site-specific manner. The single-stranded oligonucleotides 320 can, in embodiments, be printed directly on the binding sites 66, as shown in FIG. 3. All of the single-stranded oligonucleotides 320 at individual binding sites 66 have the same sequence or have sequences with at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity. The capture molecules 70 are attached to the oligonucleotides 300 by tags 290 that contain complementary sequences. Each capture molecule 70, or type of capture molecule 70 with binding specificity for each analyte, is attached to a tag 322 that has a unique sequence that is distinguishable from other sequences of other tags 322 attached to different capture molecules 70 with different specificities. The tag 322 is designed based on the known sequence of the oligonucleotide 320. Thus, a pool of different capture molecules 70 with their respective unique tags 322 can be contacted with the binding site 66 that contains the single-stranded oligonucleotide 320.

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

[0067] 11 is an embodiment in which capture molecules 70 are bound to beads 330. In the illustrated embodiment, beads 330 are placed in wells 340 of a substrate 60 that are sized and shaped such that each binding site 66 formed by the wells 340 accommodates a single bead 330. However, it should be understood that beads 330 may be in solution and not associated with a substrate. Each bead is functionalized with a unique oligonucleotide 320, all having the same or nearly the same sequence as provided herein, and capture molecules 70 bearing tags 322 hybridize to oligonucleotides 320 only if they are complementary sequences.

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

[0069] In the workflow of FIG. 12, a silicon dioxide surface with a resin coating layer is provided and patterned by nanoimprinting. The patterned resin is activated with plasma (e.g., O2 plasma) and treated with a first silane. A coating, such as a polymethylmethacrylate (PMMA) coating, is applied to the treated surface. An oxygen etch exposes the top surface of the underlying patterned resin and removes the first silane from the gap 362 while the first silane remains on the sides and bottom of the well 360. A second antifouling treatment is applied to the exposed gap and the PMMA is removed to expose the well 360. The disclosed workflow is for generating a substrate 360 ​​as an example. The substrate 360 ​​can be used to fill the beads 330 and can be used with the disclosed techniques.

[0070] In some embodiments, multiplexing allows for the simultaneous detection of multiple analyte molecules in a sample and the identification of multiple different supramolecular structures, as shown in FIG. body 10 provides multiple signals (e.g., linker barcodes, sample barcodes) for sequencing and analyte identification and demultiplexing of samples. In some embodiments, the methods described herein for detecting analytes in a sample include multiple supramolecular structures. body The use of 10 provides high throughput and high multiplexing capabilities. body Each of 10a, 10b, 10c associates a different set of affinity binders 2 which may be the same set or different sets among the multiple or different sets. However, in one embodiment, multiplex analyte detection can be used with different samples all contacted with the same set of affinity binders 2 which can all be detected using the same functionalized substrate 60 having a specific set of capture molecules 70 that are compatible with the set of affinity binders 2.

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

[0072] Multiplexing may include a washing or separation step to separate the complexes 40 from the unbound analytes 14. Such separation may involve the formation of supramolecular structures. body These separation methods may include affinity separation via tags, magnetic separation, and size separation.

[0073] An embodiment of the present disclosure includes a multiplexing kit, such as an n-plex kit. The kit includes a universal or common adapter on the core structure 13. The adapter serves as a dock for the different n-plex 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 such that the sample-indexed core structures 13 are separated from each other until tagging, complexing, and separation from unbound analytes.

[0074] FIG. 14A shows the steps of the experimental workflow for antigen detection. The workflow includes the steps of a supramolecular structure body23. The core structure 13 may be implemented as a generally box-shaped core structure 500. However, it should be understood that other shapes and implementations of the core structure 13 are also contemplated. Each core structure 13 is coupled to a signaling element, 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, a magnetic signal generator, or the like. The signaling element may be detected by a detection system, as generally described with respect to FIG. 23.

[0075] In the experiments discussed with respect to Figures 15-16, the affinity binding agent 2 is a supramolecular structure of multiple associated fluorophores 510 according to embodiments of the present disclosure. body In the illustrated example, the IgG antibody 520 was bound to a single-stranded capture strand 520 complexed to a core structure 13 having a box origami structure 500 (e.g., a box structure formed from a nucleic acid strand) having a specific fluorophore type. However, it should be understood that other antibody types or other affinity binders 2 are encompassed by the disclosed embodiments. In the illustrated example, each different IgG represents a different affinity binder 2, each with a different specificity for a different analyte, e.g., antigen 522. Thus, the illustrated workflow is shown with triple detection capability, where each specific IgG antibody is coupled to a supramolecular structure having a specific fluorophore type. body Thus, the fluorophore 510a bound to the first IgG antibody 520a can be detected in a first fluorescence range, the fluorophore 510b bound to the second IgG antibody 520b can be detected in a distinguishable second fluorescence range, and the fluorophore 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, 510c. However, in an embodiment, the core structures 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 is complexed with the single-stranded complementary strand 524 bound to the box origami structure 500 to form a duplex structure. Thus, the IgG antibody is complexed to the core structure 13 via complementary binding under conditions that promote duplex structure formation. Each different antibody 520 and box origami structure 500 can have a respective universal capture strand 522 and complementary strand 524 that are the same even for different affinity binding specificities to simplify and batch specific reagent preparation steps. However, in an embodiment, each specific IgG antibody 520a, 520b, 520c, etc. can be bound to a unique nucleic acid capture strand 522 that may include a barcode or other identifying information unique to the particular affinity binder 2.

[0077] Although the experimental workflow is shown as a triplex reaction with three different IgG antibodies 520a, 520b, 520c, it is also contemplated to allow detection of three different antigens, single, dual or other multiplex configurations. In one embodiment, enhanced fluorophore signal resolution can be achieved by controlling the number of fluorophores 510 attached to each box origami structure 500 to achieve a range of different signal intensities. Thus, affinity binding agents 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 is contacted with a sample that may or may not contain the antigen of interest 526. If an antigen 526 that IgG antibodies specifically bind to is present, the supramolecular structure bound to the antigen 526 is body An assembly 40 of ten complexes 528 is formed.

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

[0080] FIG. 14B shows an additional step in the experimental workflow of FIG. 14A. In step 4, the supramolecular structure body The box origami 500 without bound antigen 526 is not bound to the magnetic beads 530 and can be removed from the sandwich structure 550 by magnetic pull-down.

[0081] In step 5, a displacement strand 560 is provided in the reaction mixture to disrupt the duplex of the capture strand 520 and the complementary strand 522. In one embodiment, the displacement is via toehold-mediated displacement. Thus, in one example, the capture strand 522 includes a complementary region that binds to the complementary strand 522 and a toehold region that is non-complementary and remains unbound or unannealed prior to contact with the displacement strand 560. The displacement strand is complementary to both the complementary region and the toehold region, thus facilitating the binder-mediated displacement of the displacement strand 560 from the complementary strand 522 into the toehold region to form a duplex of the displacement strand 560 and the capture strand 520. Thus, the displacement strand 560 shares sequence identity with the complementary strand 522 and also includes a toehold complementary region. Upon contact with the displacement strand, the capture strand 520 and the displacement strand 560 form a duplex that releases the box origami 500 into solution, with the capture strand 520 now being duplexed with the displacement strand 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 solution.

[0082] It should be understood that the displacement may be reversed, as complementary strand 522 may contain a toehold region. Displacement strand 560 binds to complementary strand 522, breaking the duplex and releasing the box origami into solution, and capture strand 520 is 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 particular antigen associated with a particular wavelength range. The signal can be evaluated for an expected or appropriate light intensity that indicates the presence of the antigen. For example, light intensity is possible, but other detection modalities are also contemplated as described herein.

[0084] In one embodiment, time-separated cycles of different displacement strands 560 with specificity for different capture strands 520 can be used to increase the complexity of the workflow. For example, in the three fluorophore example, a set of N three fluorophores can be attached to the box origami 500, with a set-specific capture strand 520 that is replaced by a unique set-specific displacement strand 56. For N sets, there can be N unique capture strand 520-displacement strand 560 pairs. Each unique displacement strand 560 of known sequence can be added separately to release only the portion of the box origami 500 that carries the corresponding capture strand 520. The released box origami 500 can be imaged to obtain antigen-related data, and the next displacement strand 560 can be added.

[0085] Figure 15 shows the different experimental subgroups that were evaluated using the experimental workflow of Figures 14A-B. The antigens used were TSH, PSA, and IL-6, which corresponded to three different antibody binding and detection. The different antigens were tested alone and in combination with each other to identify potential cross-binding or other interferences. Antibody loading %: Antibodies were loaded at 1.5% (Dynabeads). In the experiments, the incubation times were as follows: 30 min for biotinylated IgG with beads incubation D-biotin occupying any free avidin sites for 10 min Time in 1 box IgG and antigen Box-IgG antigen and IgG-beads 1 hour in 1x PBS pH 7.4 + 0.1% Tween + 10mM Mg. Displaced chain was 1 uM displaced chain in PBS Tween + 10mM Mg. The different fluorophores were: IL-6 readout using alexafluor 488 box TSH readout using the alexafluor 647 box PSA reading using the alexafluor 750 box Detection was via a Tecan microplate reader. It is understood that the experimental workflow may be performed using other, more and / or fewer fluorophores.

[0086] Figure 16 shows the results of an experiment using a different antigen combination of the three antigens used in the experiment of Figure 15, showing 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 results of an experiment using different antigen combinations of the three antigens used in the experiment of Figure 15, where TSH is titrated but PSA and IL-6 are held constant. Changes in the concentration of TSH in the sample are reflected in the detected light output.

[0088] FIG. 18 shows the experimental results of an experiment using titration of TSH or PSA in a dual reaction, demonstrating that changes in the concentration of antigen are detectable in the light output.

[0089] FIG. 19 shows the results of a quadruplicate experiment to quantify four cytokines from a mixture. TNFa: readout with alexafluor 488 box (128 imager) IL-10: readout with atto 565 box (128 imager) INFy: readout by alexafluor 647 box (128 imagers) IL-2: readout with alexafluor 750 box (128 imager) Box origami IgG was input at 5nM. Incubation with antigen and box-IgG for 2 hours in 10 mM MOPS + 150 mM NaCl + 10 mM MgCl2 + 0.1% tween-20 Box-IgG: 1 hour incubation with antigen + IgG-beads Displacement of box with 500 nM in 10 mM MOPS + 150 mM NaCl + 10 mM MgCl2 + 0.1% tween-20 Read on tecan, 15uL / sample, 140 gain at excitation wavelengths of 491, 550, 641nm Two of the three replicates were diluted with 100x dimer / trimer antigen to obtain a larger reading.

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

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

[0092] FIG. 22 shows a series of titrations of IL-8 and TNFα, with constant CRP concentration demonstrating that antigen concentration differences are detectable in light output.

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

[0094] Embodiments of the present disclosure include one or more computer-implemented detection systems configured to perform certain 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 that stores instructions configured to be executed by the processor 1002. The controller 1001 includes a user interface 1006 and communication circuitry 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 is programmed to receive the analyte detection data and to characterize the detected analytes. In one embodiment, the processor is programmed to synthesize a supramolecular structure with an array of binding sites 66. body After incubation of the complexes 40 and detection of the characteristics of the binding sites 66, the complexes 10, or both, a report of the detected analytes in the sample is generated. The report may include data of the optical signals generated at the various binding sites corresponding to the detected analyte binding events. The report may include processed data such as a list of the analytes detected or positive / negative binding results. The report may include a list of available affinity binding agents of the complexes 10 and / or available capture molecules 70, which indicate analyte detection capabilities.

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

[0097] Supramolecular structure of complex 40 held at individual binding sites 66 via interactions with capture molecules 70 body10 is detected to generate a detectable signal. For example, the barcode of the linker 20 is used as a binding site for a detectable signaling element (e.g., via hybridization of a complementary sequence) that contacts the bound complex. In some embodiments, the signaling element comprises a fluorescent molecule or microbead, a fluorescent polymer, a highly charged nanoparticle or polymer. In some embodiments, the barcode is amplified. For example, the barcode is used as a polymerization initiator for the growth of a highly fluorescent polymer in a process such as rolling circle amplification or hybridization chain reaction.

[0098] In some embodiments, the signaling elements are optically active and can be measured using a microscope or integrated optical sensors in the substrate 60. In some embodiments, the signaling elements are electrically active and can be measured using integrated electrical sensors. In some embodiments, the signaling elements are magnetically active and can be measured using integrated magnetic sensors. 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) as determined by the corresponding detector and affinity binder, thus allowing quantification of the analyte molecules in the sample by counting the number of locations where the signaling element is present.

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

[0100] In some embodiments, detecting the presence of an analyte molecule as described herein comprises controllably releasing a single or multiple unique nucleic acid molecules from a sample into a solution that is used to identify and quantify a characteristic of the analyte molecule. In some embodiments, the unique nucleic acid molecules are each associated with a respective supramolecular structure. body is provided by the capture barcode 20. In some embodiments, detecting the presence of an analyte molecule as described herein comprises generating an optical or electrical signal associated with a state change that can be counted to quantify the concentration of the analyte molecule in solution.

[0101] In some embodiments, each supramolecular structure body 10 includes a unique DNA barcode corresponding to the associated affinity binder. As provided herein, the linker barcode 20 is a unique DNA barcode corresponding to the individual supramolecular structures. body 10 can then be used to uniquely identify each supramolecular structure. body 10 is assembled such that the affinity binding agent 2 is associated with the capture barcode 20, e.g., stored in a look-up table of the analyte detection system. Thus, once the capture barcode 20 is identified, the identity of the affinity binding agent 2 is also accessible.

[0102] Analyte detection techniques can be used to characterize analyte binding. The binding complex 40 comprises: 1) a supramolecular structure; body10 barcodes 20, and in an embodiment, 2) a barcode of a capture molecule 70 co-located with the barcode 20. The barcodes can be detected by signals generated by detector molecules bound to one or more of the binding sites 66, the complexes 40, or the capture molecules 70. In one embodiment, the location of the capture molecules 70 can be determined prior to complex binding. That is, the array of binding sites 66 can be provided pre-mapped or mapping can be a separate step. Mapping can include detecting the capture molecule barcodes as generally provided herein, such as detecting unique optical, electrical, and / or magnetic patterns. In one embodiment, detecting includes sequencing the nucleotide sequence of the capture barcode. In one embodiment, detecting includes amplifying and quantifying the amplification product, e.g., detecting 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 apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the present invention. It is understood that various alternatives to the embodiments of the present invention described herein may be employed in the practice of the present invention. It is intended that the following claims define the scope of the present invention, and that methods and structures within the scope of these claims, and their equivalents, are covered thereby.

Claims

1. 1. A method for detecting an analyte molecule present in a sample, comprising: Providing a sample containing an analyte molecule; contacting said sample with a pool of supramolecular structures in solution to form analyte molecule-supramolecular structure complexes, wherein each analyte molecule-supramolecular structure complex is A core structure comprising multiple nucleic acid strands; an affinity binding agent attached to the core structure; and comprising the analyte molecule bound to the affinity binding agent; forming said pool of supramolecular structures, wherein different supramolecular structures of said pool of supramolecular structures comprise different affinity binders having different binding affinities for other analyte molecules of said analyte molecule; contacting the analyte molecule-supramolecular structure complex with an array, the binding sites of the array comprising immobilized affinity binders each having a binding affinity for a different analyte molecule; detecting binding of said analyte molecule-supramolecular structure complex to binding sites of said array; The method comprising:

2. 2. The method of claim 1, comprising identifying the individual analyte molecules of the individual analyte molecule-supramolecular structure complexes bound to the individual binding sites of the array.

3. The method of claim 2 , wherein said identifying comprises generating a detectable signal from said supramolecular structure and associating said detectable signal with said individual binding sites.

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

5. 3. The method of claim 2, wherein said identifying comprises amplifying an initiator of said supramolecular structure and detecting said amplification, said initiator being a nucleic acid.

6. 3. The method of claim 2, wherein said identifying comprises amplifying an initiator immobilized at said individual binding sites and detecting said amplification, said initiator being a nucleic acid.

7. The method of claim 1 , wherein the affinity binding agent and the immobilized affinity binding agent are antibody molecules or portions of antibody molecules.

8. 2. The method of claim 1, wherein the binding of the analyte molecule-supramolecular structure to an immobilized affinity binding agent at an individual binding site comprises binding of the affinity binding agent of the supramolecular structure to a first portion of the analyte molecule and binding of the immobilized affinity binding agent to a second portion of the analyte molecule.

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

10. The method of claim 9 , wherein each individual binding site comprises a plurality of nucleic acid capture molecules.

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

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

13. The method of claim 1 , wherein each individual binding site comprises a plurality of immobilized affinity binders that all have affinity for the same analyte molecule of the analyte molecule.

14. The method of claim 1, wherein each binding site has a diameter of between 20 and 500 nanometers.

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

16. The method of claim 12 , wherein each core structure is a nanostructure.

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

18. 10. The method of claim 1, further comprising removing any analyte molecule-supramolecular structures that are not bound to binding sites on the array after contacting the sample with the array.

19. The method of claim 1 , wherein the analyte molecule comprises a protein, a peptide, a peptide fragment, a lipid, DNA, RNA, an organic molecule, an inorganic molecule, a complex thereof, or any combination thereof.

20. 1. A method for detecting an analyte molecule present in a sample, comprising: Providing a sample containing an analyte molecule; contacting said sample with a pool of supramolecular structures in solution to form analyte molecule-supramolecular structure complexes, wherein each analyte molecule-supramolecular structure complex is A core structure comprising multiple nucleic acid strands; an affinity binding agent bound to said core structure; A unique barcode for the sample, and the analyte molecule bound to the affinity binding agent; forming said pool of supramolecular structures, wherein different supramolecular structures of said pool of supramolecular structures comprise different affinity binders having different binding affinities for other analyte molecules of said analyte molecule; pooling said analyte molecule-supramolecular structure complex with other analyte molecule-supramolecular structure complexes, each of said other analyte molecule-supramolecular structure complexes being associated with a different sample-specific barcode; contacting the pooled analyte molecule-supramolecular structure complexes with an array, the binding sites of the array comprising immobilized affinity binders each having a binding affinity for a different analyte molecule; detecting binding of said analyte molecule-supramolecular structure complex to binding sites of said array; associating the detected binding with the sample-specific barcode; The method comprising:

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