CN117529661A

CN117529661A - Solution phase single molecule capture and related techniques

Info

Publication number: CN117529661A
Application number: CN202280038229.7A
Authority: CN
Inventors: 阿什温·戈比纳特; 保罗·罗特蒙德; 里沙布·谢蒂; 肖恩·鲍文; 雷切尔·加利米迪; 袁大军
Original assignee: Private Placement Protein Body Operation Co ltd
Current assignee: Private Placement Protein Body Operation Co ltd
Priority date: 2021-05-27
Filing date: 2022-05-26
Publication date: 2024-02-06

Abstract

Provided 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 form a complex with a supramolecular structure in solution. The supramolecular structure of the complex may be detectable such that binding of the analyte molecule to binding sites of the array can be detected via one or more features of the supramolecular structure. The binding sites of the array include capture molecules that capture the bound complexes to facilitate detection.

Description

Solution phase single molecule capture and related techniques

Cross Reference to Related Applications

The present application claims priority from U.S. provisional application 63/194,005 filed on month 27 of 2021 and U.S. provisional application 63/249,367 filed on month 9 of 2021, the disclosures of which are hereby incorporated by reference in their entireties for all purposes.

Technical Field

Most of the current situation of personalized medical care is centered on genome, mainly focusing on quantifying genes existing in individuals. While this approach has proven to be extremely effective, it does not provide the clinician with an overall view of personal health. This is because the gene is the "blueprint" of the individual and it simply informs of the likelihood of getting ill. In an individual, these "blueprints" first need to be transcribed into RNA and then translated into various protein molecules (the "effectors" in the cell) to have an effect on the individual's health.

Protein concentration, interactions between proteins (protein-protein interactions or PPIs) and interactions between proteins and other molecules have a complex and intricate link to the health of different organs, self-regulating mechanisms and interactions of these systems with the external environment. Thus, quantitative information about proteins and protein interactions (such as PPIs) is essential to construct a full picture of the individual's health at a given point in time, as well as to predict any newly emerging health problems. The presence of these proteins and interactions between these proteins are also critical to drug development and are becoming increasingly a data set that is being pursued for capturing the individual proteomes and changes in the proteomes in response to environmental or other system events. The ability to detect and quantify proteins within a given sample and interactions of proteins with other molecules is an integral part of this health care development.

Disclosure of Invention

The present disclosure relates generally to systems, structures, and methods for detecting and quantifying analyte molecules in a sample.

In some embodiments, provided herein are solution-based techniques for detecting analyte molecules present in a sample. The techniques include a sample preparation step in which an analyte in solution is captured by or otherwise associated with a corresponding supramolecular structure. For example, in embodiments, individual analytes (e.g., protein molecules) are captured by an affinity binding agent of supramolecular structure. Once captured, the analyte-supramolecular structural complexes can be detected on a substrate that is part of a detection system, whereby individual binding sites of the substrate carry the affinity binding agent for the analyte of interest. The binding of the analyte-supramolecular structure complex at the binding site may be a sandwich arrangement, wherein the binding site affinity binding agent and the supramolecular structure affinity binding agent bind to different positions on the individual analyte molecules. Thus, the detected signal at a particular binding site may be correlated with the presence of a particular analyte of interest in the sample. The supramolecular structure of the complex may provide one or more of the following: 1) a tag or barcode (e.g., a nucleic acid having a unique barcode sequence) for identifying associated affinity binding agents of individual supramolecular structures, 2) a detectable initiator for providing a detectable signal that can be correlated with binding in a detection system, and 3) a physical scaffold coupled to an analyte that facilitates single molecule binding or low average of molecules at a binding site of a substrate, e.g., that spatially blocks or spatially blocks other molecules from binding at the same binding site.

The size exclusion type supramolecular structure complexes with analytes in solution, which creates greater flexibility for preparing substrates for detection of complex analytes. The disclosed technology works with more relaxed binding site preparations that include multiple immobilized affinity binders at each binding site, as compared to complex to manufacturing systems designed to associate a single affinity binder with each binding site. Thus, the supramolecular structure may be a single molecular binding entity, while the substrate may be arranged to permit multi-molecular binding at each binding site. The low number or nature of single molecule binding at each binding site (which contributes to spatially restricting the supermolecular structure of binding at each site) permits high throughput parallel characterization of multiple protein interactions on a single detection platform. Furthermore, the solution-based sample preparation is simplified relative to the substrate-based sample preparation.

As provided herein, a method for detecting an analyte molecule present in a sample may comprise: providing a sample comprising analyte molecules; and contacting the sample in solution with an aggregate of supramolecular structures to form an analyte molecule-supramolecular structure complex. The supramolecular structure may include a core structure comprising a plurality of core molecules and an affinity binding agent attached to the core structure. The individual analyte molecule-supramolecular structure complexes may comprise: a core structure comprising a plurality of core molecules; an affinity binding agent, said affinity binding agent being linked to said core structure; and analyte molecules of the analyte molecules that bind to the affinity binding agent, wherein different supramolecular structures of the collection of supramolecular structures comprise different affinity binding agents having different binding affinities for other analyte molecules of the analyte molecules. The method further comprises the steps of: contacting the analyte molecule-supramolecular structure complexes with an array, wherein the binding sites of the array comprise respective immobilized affinity binders having binding affinities for different analyte molecules; and detecting binding of the analyte molecule-supramolecular structure complex to the binding sites of the array.

As provided herein, a method for detecting analyte molecules present in a sample can include providing an array of supramolecular structures, wherein each individual supramolecular structure is immobilized on a respective binding site of a substrate. The individual supramolecular structures of the array comprise: a core structure comprising a plurality of core molecules; and a plurality of nucleic acid capture molecules attached to the core structure, wherein the nucleic acid capture molecules each comprise the same identification sequence distinguishable from the identification sequences of other supramolecular structures of the array. The method comprises contacting the array with an affinity binding agent collection linked to a single stranded nucleic acid tag having binding specificity for a different identification sequence such that different affinity binding agents bind to different binding sites of the array such that each binding site comprises a different subset of the affinity binding agent collection. The method further comprises the steps of: contacting the sample with the array such that the analyte molecules bind to individual affinity binders in the collection of affinity binders, wherein the analyte molecules are complexed with a supramolecular structure detection assembly; and detecting binding of the analyte molecule to the separate binding sites.

As provided herein, a method for detecting an analyte molecule present in a sample can include providing an array comprising single-stranded nucleic acids immobilized on the array such that individual binding sites of the array comprise a plurality of single-stranded nucleic acids comprising the same sequence distinguishable from sequences of other single-stranded nucleic acids immobilized on different binding sites of the array. The method comprises the following steps: contacting the array with an affinity binding agent aggregate such that immobilized single stranded oligonucleotides capture a subset of the affinity binding agents at binding sites of the array and such that the captured affinity binding agents form capture molecules immobilized at the respective binding sites; and contacting the sample with the array such that the analyte molecules bind to the individual capture molecules, wherein the analyte molecules are complexed with a supramolecular structure detection assembly. The method further comprises detecting binding of the analyte molecule to the separate binding sites.

As provided herein, a method for detecting an analyte molecule present in a sample may include forming nanospheres or functionalized nanostructures. The nanospheres or functionalized nanostructures comprise a plurality of individual oligonucleotides, each of the plurality of individual oligonucleotides being attached to a chemical moiety and each oligonucleotide being bound to a complementary nucleic acid of the nanospheres or functionalized nanostructures. The method comprises the following steps: providing a patterned array comprising a plurality of active sites; incubating the nanospheres or functionalized nanostructures with the patterned array to covalently attach active groups of individual active sites to chemical moieties of the plurality of individual oligonucleotides, thereby coupling individual nanospheres or functionalized nanostructures to the individual active sites; denaturing the plurality of individual oligonucleotides from the complementary nucleic acids of the nanospheres or functionalized nanostructures; washing the nanospheres or functionalized nanostructures from the array to leave the plurality of individual oligonucleotides immobilized on the individual binding sites, wherein the plurality of individual oligonucleotides are single stranded; and contacting the array with an affinity binding agent aggregate such that the immobilized plurality of individual oligonucleotides capture a subset of the affinity binding agents at the individual binding sites.

As provided herein, a method for detecting an analyte molecule present in a sample may comprise: providing a sample comprising analyte molecules; and contacting the sample in solution with an aggregate of supramolecular structures to form an analyte molecule-supramolecular structure complex. The individual analyte molecule-supramolecular structure complexes comprise: a core structure comprising a plurality of core molecules; an affinity binding agent, said affinity binding agent being linked to said core structure; sample-specific barcodes; an analyte molecule of the analyte molecules that binds to the affinity binding agent, wherein different supramolecular structures of the collection of supramolecular structures comprise different affinity binding agents having different binding affinities for other analyte molecules of the analyte molecules. The method comprises the following steps: combining the analyte molecule-supramolecular structure complex with other analyte molecule-supramolecular structure complexes associated with corresponding different sample-specific barcodes; contacting the combined analyte molecule-supramolecular structure complexes with an array, wherein the binding sites of the array comprise respective immobilized affinity binders having binding affinity for different analyte molecules; detecting binding of the analyte molecule-supramolecular structure complex to binding sites of the array; and correlating the detected binding with the sample-specific barcode.

As provided herein, a method for detecting an analyte molecule present in a sample is disclosed. The method includes providing a plurality of supramolecular structures. Each supramolecular structure comprises: a core structure comprising a plurality of core molecules; an antibody having binding affinity for an antigen and coupled to a nucleic acid capture chain; and a complementary strand of the nucleic acid capture strand, wherein the complementary strand is linked to the core structure and forms a duplex structure with the nucleic acid capture strand to couple the antibody to the core structure, wherein the plurality of supramolecular structures comprise antibodies that differ relative to each other with respective different binding affinities. The method comprises the following steps: contacting the plurality of supramolecular structures with a sample comprising the antigen such that the antigen binds to the antibody to form a complex; contacting the complex with beads carrying capture antibodies 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, wherein the released core structure is not coupled to the antibody; and detecting the core structure.

In some embodiments, any of the methods disclosed herein can comprise detecting the presence of an analyte molecule in the sample and/or quantifying the concentration of the analyte molecule. In some embodiments, any of the methods disclosed herein further comprise identifying the detected analyte molecule. In some embodiments, any of the methods disclosed herein further comprise detecting the analyte molecule based on a signal when the analyte molecule is present in the sample in a single molecule or higher count.

In some embodiments, for any of the methods disclosed herein, each core structure is a nanostructure. In some embodiments, for any of the methods disclosed herein, the plurality of core molecules of each core structure are arranged in a predefined shape and/or have a specified molecular weight. In some embodiments, the predefined shape is configured to limit or prevent cross-reactivity with another supramolecular structure. In some embodiments, for any of the methods disclosed herein, the plurality of core molecules of each core structure comprises 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 of the methods disclosed herein, each core structure independently comprises a scaffold deoxyribonucleic acid (DNA) fold, a scaffold ribonucleic acid (RNA) fold, a scaffold hybridization DNA: RNA fold, a single-stranded DNA tile structure, a multi-stranded DNA tile structure, a single-stranded RNA fold, a multi-stranded RNA tile structure, a DNA or RNA fold having a layered composition of multiple scaffolds, a peptide structure, or a combination thereof.

The affinity binding agent of the supramolecular structure may be linked to the core structure via a chemical bond. In some embodiments, the affinity binding agent based on the immobilization of the supramolecular structure of the solution and/or the binding site independently comprises a protein, peptide, antibody, aptamer (RNA and DNA), fluorophore, darpan (darpin), catalyst, polymerization initiator, polymer (such as PEG), or a combination thereof.

In some embodiments, for any of the methods disclosed herein, the detectable signal comprises a barcode indicative of a particular affinity binding agent, e.g., a binding site and/or a supramolecular structure. In some embodiments, each barcode provides a DNA signal or a trigger signal that corresponds to the affinity binding agent and provides information indicative of the specificity of the barcode for analyte molecules bound to the corresponding detector molecule. In some embodiments, the barcodes are analyzed using genotyping, qPCR, sequencing, or a combination thereof. In some embodiments, multiple analyte molecules in the sample are detected simultaneously by multiplexing. In some embodiments, for any of the methods disclosed herein, an affinity binding agent as provided herein is configured to bind to one or more specific types of analyte molecules.

In some embodiments, for any method comprising using a plurality of supramolecular structures disclosed herein, each core structure of the plurality of supramolecular structures is identical to each other. However, coupled affinity binders may vary for a plurality of supramolecular structures. Thus, in embodiments, the supramolecular structural assembly(s) are identical except for the coupled affinity binding agent and, in some embodiments, the identification moiety that identifies the coupled affinity binding agent (e.g., the affinity binding agent identifies a barcode nucleic acid sequence). For multiple samples detected on a single substrate or detection platform, the supramolecular structure associated with a particular sample can carry an identification moiety associated with the sample (e.g., sample barcode nucleic acid sequence) that identifies the sample.

In some embodiments, each supramolecular structure includes a specified shape, size, molecular weight, or combination thereof, which may reduce or eliminate cross-reactions between multiple supramolecular structures. In some embodiments, each supramolecular structure comprises only one affinity binding agent or multiple affinity binding agents. Where the supramolecular structure comprises multiple affinity binders on a single core structure, the multiple affinity binders may all have the same binding specificity, e.g., all specifically bind the same analyte. In some embodiments, each supramolecular structure comprises a defined stoichiometric amount of capture molecules and detector molecules in order to reduce or eliminate cross-reactions between multiple supramolecular structures.

In some embodiments, the substrate comprises a solid support, a solid substrate, a polymer matrix, or one or more beads. The substrate may include a plurality of binding sites patterned on the substrate and separated by a gap surface of the substrate, wherein each binding site includes a hole, and wherein at least one surface of the hole includes a first chemical group and the gap surface includes a second chemical group. Beads are loaded into each of the binding sites, wherein the first chemical group selectively binds to the beads and the second chemical group does not interact with or bind to the beads. Each bead comprises: a plurality of single stranded oligonucleotides immobilized on each bead such that individual binding sites of the array comprise a plurality of single stranded oligonucleotides comprising the same sequence distinguishable from sequences of other single stranded oligonucleotides immobilized on different binding sites of the array; and an affinity binding agent that is captured at a binding site of the array such that the captured affinity binding agent forms a capture molecule immobilized at the respective binding site. During detection, the analyte molecules bind to the individual capture molecules at the individual binding sites, wherein each analyte molecule is complexed with a supramolecular structure detection assembly. Detection of the detection assembly permits detection of an analyte bound to the substrate.

In some embodiments, a plurality of supramolecular structures are disposed on a substrate (such as a shaped or planar substrate), wherein the substrate comprises a plurality of binding sites, wherein each individual binding site is coupled to one or more affinity binders configured to bind to the same analyte molecule, e.g., such that an individual binding site is specific for an individual analyte molecule and different binding sites of the substrate are specific for different analyte molecules. The disclosed embodiments also include sample preparation reagents, substrates, and detection systems for performing the disclosed methods.

In some embodiments, for any of the methods disclosed herein, the sample comprises a complex biological sample. In some embodiments, for any of the methods disclosed herein, the one or more analyte molecules of the sample comprise a protein, peptide fragment, lipid, DNA, RNA, organic molecule, inorganic molecule, complex thereof, or any combination thereof. In some embodiments, for any of the methods disclosed herein, the sample comprises a biological particle or a biological molecule. In some embodiments, for any of the methods disclosed herein, the sample comprises an aqueous solution comprising a protein, peptide fragment, lipid, DNA, RNA, organic molecule, viral particle, exosome, organelle, or any complex thereof. In some embodiments, for any of the methods disclosed herein, the sample comprises a tissue biopsy, blood, plasma, urine, saliva, tears, cerebrospinal fluid, extracellular fluid, cell culture, culture medium, discard tissue, plant matter, synthetic proteins, prions, bacterial and/or viral samples, or fungal tissue, or a combination thereof. The sample may be an environmental sample, such as a wastewater or soil sample. The sample may also be a non-biological sample. In embodiments, the sample may be a sample from a chemical process step, a sample of a food or nutritional ingredient, or a sample of a packaging ingredient.

The sample may be treated to release an analyte from a cell or otherwise prepare the sample for analysis prior to contacting the sample with the supramolecular structures in solution as provided herein.

Drawings

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

Fig. 1 illustrates an example analyte-supramolecular structural complex after solution-based capture according to an embodiment of the present disclosure.

Fig. 2 illustrates an example workflow including solution-based single molecule capture sample preparation according to an embodiment of the present disclosure.

Fig. 3 illustrates an example of a substrate for analyte detection according to an embodiment of the present disclosure.

Fig. 4 shows an example of a binding site of a substrate with capture molecules and captured analyte-supramolecular structural complexes according to an embodiment of the present disclosure.

Fig. 5 shows an example of a substrate for analyte detection according to an embodiment of the present disclosure.

FIG. 6 illustrates a linked barcode nucleic acid structure for use with a substrate for analyte detection according to an embodiment of the present disclosure.

Fig. 7 shows an example of a substrate including the linked barcode nucleic acid structure of fig. 6 for analyte detection in accordance with an embodiment of the present disclosure.

Fig. 8 illustrates a nanoparticle structure for use with a substrate for analyte detection in accordance with an embodiment of the present disclosure.

Fig. 9 shows an example of a substrate including the nanoparticle structure of fig. 5 for analyte detection according to an embodiment of the present disclosure.

Fig. 10 shows an example of a substrate for analyte detection according to an embodiment of the present disclosure.

Fig. 11 shows an example of a bead for analyte detection according to an embodiment of the present disclosure.

Fig. 12 illustrates an example workflow for producing a substrate to accommodate beads for analyte detection according to an embodiment of the present disclosure.

Fig. 13 illustrates an example workflow including solution-based single molecule capture sample preparation for multiplex detection according to embodiments of the present disclosure.

Fig. 14A shows steps of an experimental workflow for antigen detection according to an embodiment of the present disclosure, including complexing an IgG affinity binding agent with a box-fold to form a supramolecular structure.

Fig. 14B shows additional steps of the experimental workflow of fig. 14A.

Fig. 15 shows different experimental subgroups evaluated using the experimental workflow of fig. 14A-14B.

Fig. 16 shows the results of solution-based optical detection in the case of using different antigen combinations of three antigens in the experimental workflow of fig. 14A to 14B.

Fig. 17 shows the solution-based optical detection results in the case of using TSH titration in the experimental workflow of fig. 14A to 14B.

Fig. 18 shows the results of a solution-based optical assay for antigen titration with or without the presence of another antigen.

Fig. 19 shows the results of solution-based optical detection of monomeric, dimeric and trimeric antigens.

Fig. 20 shows the results of solution-based optical detection of mixtures of unlabeled and labeled antigens.

Fig. 21 shows the results of solution-based optical detection of mixed or complex samples.

Fig. 22 shows the results of solution-based optical detection of titrated antigen samples.

FIG. 23 shows a block diagram of an example analyte detection system according to an embodiment of the present disclosure.

Detailed Description

Disclosed herein are structures and methods for detecting one or more analyte molecules present in a sample. In some embodiments, one or more analyte molecules are detected based on solution-based capture of one or more supramolecular structures. In some embodiments, one or more supramolecular structures comprise or are linked to an affinity binding agent that specifically binds to analytes present in the sample and solution for single molecule capture of the analytes. Binding of the analyte forms analyte molecule-supramolecular structure complexes in solution, and these complexes can be detected by a detection system as provided herein.

In embodiments, the complex analyte is captured by an immobilized affinity binding agent associated with a substrate or other detection platform of the detection system. The capture of the complex in turn immobilizes the complex on the substrate, and one or more characteristics of the immobilized substrate or its associated binding site can be characterized to characterize the analyte in the sample. In embodiments, the supramolecular structure comprises a detectable moiety, such as a unique identifier (e.g., a nucleic acid sequence, a peptide, a polysaccharide, phosphoramidite (acrydite)) and/or a molecule that includes other molecules that can be detected (e.g., optically, electrically, magnetically), interact with, or can be used to dock the other molecules. In some embodiments, the detectable moiety generates a DNA signal or other initiator signal such that detection and quantification of binding of the analyte molecule-supramolecular structure complex to the binding site can be detected by amplification of a unique identifier of the supramolecular structure and/or a unique identifier of the binding site. In some embodiments, the supramolecular structure is linked to an enzyme that converts a substrate into an optically detectable signal. In some embodiments, the supramolecular structure is coupled to a sensor on a substrate to generate an electrically or magnetically detectable signal. In embodiments, the supramolecular structure is a nucleic acid fold attached to or immobilized on a substrate. In embodiments, the supramolecular structure carries the affinity binding agent via a barcode bridge or linker that includes a unique identifier of the affinity binding agent and links the affinity binding agent to the scaffold of the supramolecular structure.

In some embodiments, the disclosed technology provides single molecule capture of analyte molecules in complex samples. The use of a supramolecular structure as a capture entity permits specific identification and (in embodiments) detection of associated affinity binders via interaction with the binding site of the substrate. Thus, as provided herein, individual affinity binders of a number of different affinity binder assemblies that an analyte binds to a substrate can be detected to produce an assay result, wherein the binding characteristics of the analyte assemblies for a plurality of different analytes are characterized. This in turn permits analysis of samples having uncharacterized analyte compositions for the presence and/or concentration of particular analytes of interest. For example, a human sample may be characterized to determine the presence and/or concentration of antibodies having binding specificity for a particular antigen in a set of antigen-affinity binders, such that the affinity binders represent a known set of infectious disease antigens. The assay results may show positive binding results associated with a particular antigen, which is indicative of the presence of antibodies in the subject providing the sample. In another embodiment, the identity of the analyte in the sample may be at least partially known, but its binding affinity may not be characterized for a particular affinity binding agent aggregate. For example, the affinity binding agent may be a set of candidate drugs and the analyte may be a molecule in human blood. The binding of the candidate drug to such a protein can be used to assess bioavailability or potential off-target binding. The assay results may display positive binding results associated with a particular drug candidate, which in turn may be mapped to a particular analyte, based on the identification of the particular analyte binding (e.g., identification of binding by barcode identification in a detector molecule assembly comprising antibodies specific for the analyte).

While conventional detection schemes for analytes such as proteins may include a detectable fluorescent signal generated by an enzyme linked to a detection antibody as an indicator of binding, the disclosed techniques may additionally or alternatively provide an amplified nucleic acid (or other initiator) signal from a unique identifier of the detector molecule assembly from which sequence information may be determined or from which optically detectable signals corresponding to the amplification are released (e.g., qPCR performed using primer/probe sets specific for the unique identifier). Thus, in certain embodiments, unique identity information of the captured complex at a particular binding site of the array permits specific identification of a particular affinity binding agent that has captured the analyte. The modular and customizable structure of the components of the supramolecular structure permits the provision of substantially identical bulk or common core structures and the barcoding of individual supramolecular structures with unique identifiers during the ligation of specific affinity binders. Thus, the identification sequence is associated with a unique one of the specific affinity binding agents. Other detection techniques may include optical, magnetic, and/or electrical detection techniques.

The disclosed embodiments relate to analyte detection, wherein the analyte is present in a sample, such as a biological sample. In some embodiments, the sample comprises an aqueous solution comprising a protein, peptide fragment, lipid, DNA, RNA, organic molecule, inorganic molecule, complex thereof, or any combination thereof. In some embodiments, the analyte molecules in the sample include proteins, peptides, peptide fragments, lipids, DNA, RNA, organic molecules, inorganic molecules, complexes thereof, or any combination thereof. In some embodiments, the analyte molecule comprises an intact protein, a denatured protein, a partially or fully degraded protein, a peptide fragment, a denatured nucleic acid, a degraded nucleic acid fragment, a complex thereof, or a combination thereof. In some embodiments, the sample is obtained from a tissue, a cell, an environment of a tissue and/or a cell, or a combination thereof. In some embodiments, the sample comprises a tissue biopsy, blood, plasma, urine, saliva, tears, cerebrospinal fluid, extracellular fluid, cell culture, culture medium, discard tissue, plant matter, synthetic protein, bacterial, viral sample, fungal tissue, or a combination thereof. In some embodiments, the sample is isolated from a primary source such as a cell, tissue, bodily fluid (e.g., blood), environmental sample, or a combination thereof, with or without purification. In some embodiments, the cells are lysed using a mechanical process or other cell lysis method (e.g., lysis buffer). In some embodiments, the sample is filtered using mechanical processes (e.g., centrifugation), microfiltration, chromatography columns, other filtration methods, or a combination thereof. In some embodiments, the sample is treated with one or more enzymes to remove one or more nucleic acids or one or more proteins. In some embodiments, the sample comprises an intact protein, a denatured protein, a partially or fully degraded protein, a peptide fragment, a denatured nucleic acid, or a degraded nucleic acid fragment. In some embodiments, the sample is collected from one or more individual individuals, one or more animals, one or more plants, or a combination thereof. In some embodiments, the sample is collected from an individual, animal, and/or plant having a disease or disorder, including infectious disease, immune disorder, cancer, genetic disease, degenerative disease, lifestyle disease, injury, rare disease, age-related disease, or a combination thereof.

The disclosed technology utilizes single molecule binding of an affinity molecule attached to a supramolecular structure. Single molecules of the analyte molecules combine to form a complex as shown in fig. 1, which can be detected by a detection system according to the disclosed techniques. Complex formation occurs in solution. Fig. 1 provides an exemplary embodiment of a supramolecular structure 10 comprising a core structure 13 and an affinity binding agent 2. In some embodiments, the supramolecular structure 10 comprises one or more affinity binding agents 2. In embodiments, the supramolecular structure 10 may refer to a complex comprising a core structure 13 and an affinity binding agent 2. In embodiments, the supramolecular structure 10 may refer to the core structure 13, and the supramolecular structure 10 may or may not comprise the affinity binding agent 2.

Accordingly, a supramolecular structure 10 is provided herein. In some embodiments, the supramolecular structure 10 is a programmable structure that can spatially organize molecules. In some embodiments, the supramolecular structure 10 comprises a plurality of molecules linked together. In some embodiments, at least some of the plurality of molecules of the supramolecular structure 10 interact with each other. In some embodiments, the supramolecular structure 10 includes a particular shape, such as a substantially planar shape having a longest dimension in the x-y plane. In some embodiments, the supramolecular structure 10 is a nanostructure. In some embodiments, the supramolecular structure 10 is a nanostructure comprising a specified molecular weight based on a plurality of molecules of the supramolecular structure 10. In some embodiments, the plurality of molecules are linked together by a bond, a chemical bond, a physical attachment, or a combination thereof. In some embodiments, the supramolecular structure 10 comprises macromolecular entities of a specific shape and molecular weight formed from a defined number of smaller molecules that interact specifically with each other. In some embodiments, the structural, chemical and physical properties of the supramolecular structure 10 are specifically designed. In some embodiments, the supramolecular structure 10 comprises a plurality of sub-components that are spaced apart by a prescribed distance. In some embodiments, at least a portion of the supramolecular structure 10 is rigid. In some embodiments, at least a portion of the supramolecular structure 10 is semi-rigid. In some embodiments, at least a portion of the supramolecular structure is flexible. In embodiments, the supramolecular structure 10 is at least 50-200nm in one dimension. In embodiments, the supramolecular structure 10 is at least 20nm in length in any dimension.

In some embodiments, 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, one or more core molecules include 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, or 500 unique molecules linked together. In some embodiments, the one or more core molecules comprise from about 2 unique molecules to about 1000 unique molecules. In some embodiments, one or more core molecules interact with each other and define a particular shape of the supramolecular structure. In some embodiments, the plurality of core molecules interact with each other through reversible non-covalent interactions.

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, all of the core structures 13 of the plurality of supramolecular structures 10 may have the same configuration, size and/or weight, but may differ in terms of their attached linker sequence and attached affinity binding agent 2. However, the plurality of supramolecular structures 10 may be otherwise identical, excluding different linkers 20 and affinity binders 2. In some embodiments, the 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 a combination thereof. In some embodiments, core structure 13 comprises a complete polynucleotide structure. In some embodiments, at least a portion of the core structure 13 is rigid. In some embodiments, at least a portion of the core structure 13 is semi-rigid. In some embodiments, at least a portion of the core structure 13 is flexible. In some embodiments, core structure 13 comprises a scaffold deoxyribonucleic acid (DNA) folder, a scaffold ribonucleic acid (RNA) folder, a scaffold hybrid DNA/RNA folder, a single-stranded DNA tile structure, a multi-stranded DNA tile structure, a single-stranded DNA folder, a single-stranded RNA tile structure, a multi-stranded RNA tile structure, a DNA and/or RNA folder having a layered composition of multiple scaffolds, a peptide structure, or a combination thereof. In some embodiments, the DNA fold is scaffold. In some embodiments, the RNA fold is scaffold. In some embodiments, the hybrid DNA/RNA fold is scaffold. In some embodiments, the core structure 13 comprises a DNA fold, an RNA fold, or a hybrid DNA/RNA fold comprising a defined two-dimensional (2D) or 3D shape.

In embodiments, the core structure 13 is a nucleic acid fold having at least one lateral dimension between about 50nm and about 1 μ. In embodiments, for example, the nucleic acid folds have at least one lateral dimension between about 50nm to about 200nm, about 50nm to about 400nm, about 50nm to about 600nm, about 50nm to about 800nm, about 100nm to about 200nm, about 100nm to about 300nm, about 100nm to about 400nm, about 100nm to about 500nm, about 200nm to about 400 nm. In embodiments, the nucleic acid folds have at least a first lateral dimension between about 50nm and about 1 μ and a second lateral dimension orthogonal to the first lateral dimension between about 50nm and about 1 μ. In embodiments, the nucleic acid folds have an area of about 200nm ² Up to about 1 mu ² Is provided.

As shown in fig. 1, in some embodiments, the core structure 13 is configured to be attached to the affinity binding agent 2. In some embodiments, the affinity binding agent 2 is immobilized relative to the core nanostructure 13 when attached thereto. However, the core structure 13 may be in solution and thus not fixed relative to the sample or reaction vessel. In some embodiments, as shown in fig. 1, the affinity binding agent 2 is attached to the core structure 13 by a linker 20. In some embodiments, the linker 20 comprises a polymer comprising a nucleic acid (double-stranded or single-stranded DNA or RNA) of a specific sequence associated with the linked affinity binding agent 2. Thus, the sequence of nucleotide linker 20 uniquely identifies affinity binding agent 2 among an aggregate of different affinity binding agents 2. The barcode may be at least 6 nucleotides, and may be 6-50 nucleotides. In fig. 1, a supramolecular structure 10 binds to an analyte molecule 14 having binding specificity for an affinity binding agent 2 to form an analyte molecule-supramolecular structure complex 40.

In some embodiments, any number of the one or more core molecules 13 comprises one or more linkers 20 configured to form a bond with the affinity binding agent 2. In some embodiments, the linker 20 is attached to one or more core molecules of the core structure 13 by chemical bonds. In some embodiments, the linker 20 may comprise a core reactive molecule. In some embodiments, each core reactive molecule independently comprises an amine, thiol, DBCO, NHS ester, maleimide, biotin, azide, phosphoramidite, 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 comprises a DNA sequence domain.

In some embodiments, the core structure 13 is attached to the affinity binding agent 2 at a defined location on the core structure 13.

In some embodiments, affinity binding agent 2 comprises a protein, peptide, antibody-derived reagent, aptamer (RNA and DNA), fluorophore, nanobody, darpan, catalyst, polymerization initiator, polymer (e.g., PEG), organic molecule, small molecule, pharmaceutical compound, candidate pharmaceutical compound, synthetic molecule, or a combination thereof. In some embodiments, a single affinity binding agent 2 is attached to the core structure 13. In some embodiments, a plurality of affinity binding agents 2 are attached 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 different analyte molecules that may bind to a multi-molecule complex (e.g., a protein-protein complex). In another example, multiple identical copies of the affinity binding agent 2 may be present on the core structure 13.

In some embodiments, each component of the supramolecular structure 10 may be independently modified or adjusted. In some embodiments, modifying one or more components of the supramolecular structure 10 may modify the 2D and 3D geometry of the supramolecular structure itself. In some embodiments, modifying one or more components of the supramolecular structure may modify the 2D and 3D geometry of the core structure 13. In some embodiments, this ability to independently modify the components of the supramolecular nanostructure enables precise control of the organization of one or more supramolecular structures.

As described herein, in some embodiments, one or more supramolecular structural complexes 40 enable detection of one or more analyte molecules in a sample. Fig. 2 is a schematic diagram of a workflow for forming analyte molecule-supramolecular structure complexes 40 in solution. The collection of supramolecular structures 10 having associated affinity binders 2 representing a series or set of affinities for respective different analytes is contacted with a sample 50 comprising a plurality of different analyte molecules 14. The analyte 14 in the sample 50 may be an uncharacterized or unknown analyte 14. In embodiments, the sample 50 may comprise one or more control analytes 14.

When the individual analyte molecules 14 and the individual affinity binding agent 2 pairs have binding specificity for each other, the analyte molecules 14 associate with the affinity binding agent 2 to form individual analyte molecule-supramolecular structure complexes 40. The reaction conditions permit binding of the analyte molecules 14 to the specific affinity binding agents 2. As provided herein, binding specificity may refer to interactions between the analyte molecules 14 and the affinity binding agent 2 that remain intact under the reaction conditions and after a washing or removal step for unbound reagents. Binding specificity may include formation of covalent or non-covalent bonds, ionic bonds, dipole interactions, hydrophilic or hydrophobic interactions, complementary nucleic acid binding, and the like. Specific binding may refer to binding of analyte molecules 14 that bind only to a particular affinity binding agent 2 and not to other affinity binding agents 2. Thus, certain affinity binders 2 of the collection of supramolecular structures 10 bind to certain analyte molecules 14 (e.g. the binding between a first analyte molecule 14a and a first affinity binder 2a or the binding between a second analyte molecule 14b and a first affinity binder 2 b). Some affinity binding agents 2 may not have a binding partner available in a given sample 50 and therefore will not specifically bind to any analyte molecules 14.

As provided herein, after formation of the complexes 40, any unbound analytes 14 may be removed prior to providing the complexes to the detection system. However, in other embodiments, no washing step is performed. Unbound analytes 14 in the solution may be permitted to interact with the detection system, but are unlikely to specifically bind at the binding sites and no detectable signal will be generated, as the unbound analytes do not carry the supramolecular structure 10.

The analytes 14 of the disclosed complexes 40 may be detected based on interactions with a patterned substrate 60 having an array of binding sites 66 distributed on or in the substrate 60, as generally shown in fig. 3-13. The substrate 60 may include a defined set of micropatterned binding sites 66 that are functionalized by having immobilized capture molecules 70.

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 pattern). 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 200nm. In some embodiments, the minimum distance between any two binding sites 66 is at least about 40nm to about 5000nm. In some embodiments, the geometry of the binding sites 66 includes a circular, square, triangular, or other polygonal shape. In some embodiments, the individual binding sites 66 are 20-200nm in diameter. The substrate 60 may be patterned as generally discussed in U.S. provisional application No. 63/119,316, filed 11/30, 2020, which is incorporated herein by reference.

The substrate 60 may comprise a glass or silicon wafer having one or more layers of silicon dioxide, silicon nitride, graphene, or silicon carbide. In embodiments, each binding site 66 houses a plurality of capture molecules 70 of the same type at each binding site 66, wherein different binding sites 66 have different capture molecule specificities or different chemistries. Patterned substrate 60 may be fabricated by a photolithographic process. Further, embodiments of the disclosed technology may include one or more regeneration steps that remove bound or "used" complexes 40 from the substrate 60 to permit binding of new complexes 40 in subsequent reactions.

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

Fig. 3 provides an exemplary illustration of forming a substrate 60 for detecting analyte molecules in a sample using a surface-based assay using capture molecules 70 (shown as antibodies, but which may be any suitable capture molecules that capture complexes 40, as described herein). In embodiments, the capture molecule 70 is an affinity binding agent as generally disclosed herein. In fig. 3, a patterned substrate 60 having a plurality of binding sites 66 functionalized with or attached to individual capture molecules 70 may be formed according to method 100. A substrate base layer 110 is provided. A passivation layer 112 is grown, assembled or deposited on the substrate layer 110, which may be selectively passivated. The one or more passivation layers 112 may include silicon nitride, graphene, quartz, metal, gold, silver, platinum, palladium, PDMS, a polymer film, or a combination thereof. The passivation layer 112 may be graphene, aluminum oxide, hfO ₂ 、Cr ₂ O ₃ (chromium oxide), titanium oxide, tantalum oxide, metal oxide, silicon dioxide (SiO) ₂ ) Or a combination thereof. Passivation layer 112 may be a self-assembling polymer such as polyacrylamide.

The passivation layer 112 is patterned, for example by removing portions of the top layer 112, to expose the locations 120 of the base layer 110 that will correspond to the bond sites 66 of the substrate 60. Patterning may be photolithography, electron beam lithography, nanoimprinting, spin coating of polymers, optical patterning, plasma activation, acid/base treatment, or other patterning means. The exposed sites 120 may be activated by chemical or plasma treatment to produce different reactive groups, depending on the individual chemistry of the layers.

The capture molecules 70 may be coupled to the activation sites 120 to create binding sites 66. In the example shown, capture molecules 70 may be printed on the binding sites 66. Each individual binding site 66 is printed with a preselected capture molecule 70. However, other attachment or coupling techniques for linking the capture molecules to the binding sites 66 are also contemplated. In embodiments, 1) each binding site includes a plurality (e.g., two or more) of capture molecules 70, and 2) all capture molecules 70 at an individual binding site have the same binding specificity for a particular analyte 14.

The capture molecules 70 may include one or more affinity binding agents, supramolecular structures, nucleic acids, as provided herein. Each binding site 66 of the substrate 60 comprises a plurality of capture molecules 70 that each have the same binding specificity for each individual binding site 66. In addition, adjacent or different binding sites 66 may have different binding specificities such that the first capture molecules 70a have different binding specificities than the second capture molecules 70 b.

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

Each binding site 66 may be coupled to a barcode or unique identification sequence that may be read via sequencing, amplification, or via hybridization assays as part of detection. The capture molecules 70 immobilized on the substrate 60 and the mapping of the spatial position on the substrate 60 may be obtained before performing the assay and performed as a quality control of the substrate 60. The plot may be stored in an analyte detection system as provided herein (see fig. 10) and used to generate a report of positive binding events to provide analyte information for the sample.

Once formed, the substrate 60 may be used in an analyte capture step following sample preparation (as shown in FIG. 2) to provide the complex 40. In some embodiments, a flow cell is used to bring the composite 40 into contact with a planar substrate. In some embodiments, the complex 40 is incubated on a substrate 60 having capture molecules 70 attached to the binding sites 66. In some embodiments, the incubation period may be from about 30 seconds to about 24 hours. In some embodiments, the incubation period may 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.

In some embodiments, the analyte molecules 14 of the complex 40 interact with capture molecules 70 on the binding sites 66. FIG. 4 shows an exemplary binding site 66 having capture molecules 70a that are specific for a particular analyte 14a of a complex 40 a. After interaction, binding can be detected via signal generation. Unbound complex 40b can be washed away prior to detection. The capture molecules 70a and corresponding analyte molecules 14a may also have binding specificity for each other. Because analyte molecules 14a form complexes 40a in solution prior to contact with substrate 60, capture molecules 70a, analyte 14a, and affinity binding agent 2a (attached to core structure 13 a) may form a sandwich-like binding arrangement, as shown in fig. 4. Thus, both capture molecule 70a and affinity binding agent 2a may be specific for the same analyte 14 a. However, capture molecules 70a and affinity binding agents 2a may bind to analyte 14a at different sites.

After these workflow steps, various bound complexes 40 remain on the array, each bound complex binds to a respective capture molecule 70 and can undergo various detection schemes to associate the analyte 14 with a specific supramolecular structural identity, which in turn is associated with a known affinity binding agent 2 and at a specific binding site 66, which in turn can be associated with a unique identifier (e.g., a barcode sequence). Thus, detection permits characterization of analyte-affinity binding agent binding.

In some embodiments, as shown in the workflow 150 of fig. 5, the binding sites 66 are functionalized via a single supramolecular structure 160 placed at the activation site 120 to form the binding sites 66. The binding sites 66 may be activated prior to placement of the supramolecular structure 160 as generally disclosed with respect to fig. 3. The supramolecular structure 160 may generally be arranged as a supramolecular structure 10 as provided herein, and may include a core structure 13 comprising DNA folds, wherein the supramolecular structure 10 is attached to each of the binding sites using DNA fold placement techniques that may include bonding via anchors. In some embodiments, DNA fold placement includes directed self-assembly techniques for organizing individual DNA folds (e.g., core structures) at binding sites 66. In some implementations, the planar substrate 60 may be stored in a clean environment for a substantial period of time after this step.

In the depicted embodiment, the supramolecular structure 160 is provided in an assembled form and has been attached to it a capture molecule 70. Each individual supramolecular structure with a corresponding different analyte specificity may be formed separately, for example in separate reaction tubes. A plurality of capture molecules 70 are attached to the core structure 13 of the supramolecular structure 160. The capture molecules 70 of the single supramolecular structure 160 at the single binding site 66 all have the same binding specificity. In addition, each supramolecular structure 160 includes a barcode or other identification sequence that uniquely identifies the type of associated capture molecule 70.

The placement of the preformed supramolecular structures 160 may be oriented such that the capture molecules 70 are located "right side up" on the binding sites 66. In other embodiments, the supramolecular structures 160 are all generally identical and are randomly placed on separate binding sites. After placement at different binding sites, the unique identification sequences and associated capture molecules 70 are linked.

Fig. 6-7 illustrate steps of forming a planar substrate 60 in which binding sites 66 are functionalized via a conjugate of linked nucleic acid products 200. FIG. 5 shows the steps of forming a linked nucleic acid product 200 from a circular template 210. The circular templates include barcode sequences 220 that uniquely distinguish the circular templates 210 from other circular templates 210. Rolling circle amplification with the strand displacement polymerase of extension primer 230 produces single stranded rolling circle amplification products comprising ligated nucleic acids having repeat units 252. Single-stranded probes 260 having reactive groups 262 hybridize to the complement of barcode sequence 220 and bind at a plurality of locations on the ligated nucleic acid product 200 where the barcode sequence 202 repeats as part of repeat unit 252. Thus, the end product of the rolling circle amplification used to create the binding sites is a linked nucleic acid product 200 having a plurality of bound probes 260 and associated reactive groups 262. In an embodiment, the formation of a substrate as provided herein includes producing a plurality of products 200 from respective different templates 210, whereby each product 200 has a distinguishable sequence based on the different templates 210. The corresponding different products 200 may be formed in different reaction vessels. However, in embodiments, different templates 210 may be combined to produce a combined product 200. Although each product 200 is typically associated with a binding site 66 at a ratio of 1:1, as provided in fig. 7, a pooled population of products 200 may be distributed throughout the substrate 60. However, independently producing the product 200 may prevent differences in amplification bias that may result in the production of unequal products 200 from certain templates 210, which in turn may result in the over-presentation of certain template sequences on the binding site 66.

Each template 210 includes a sequence that can associate or bond with a particular capture molecule 70 as a barcode or unique identifier for the capture molecule 70 as provided herein.

As shown in the workflow 270 of fig. 7, the active site 120 may be generated as generally disclosed with respect to fig. 3-5. Binding sites 66 are functionalized to form binding sites 66 via placement of a single linked nucleic acid product 200 having a plurality of bound probes 260 at activation sites 120. As shown, active site 120 includes active molecules 280 surrounded by passivation layer 112. The single stranded linked nucleic acid products 200 are nanospheres that are typically sized such that only one product 200 is contained on a single active site 120. Association of the product 200 with the active site during formation of the binding site 66 is achieved via the active group 262 of the bound (hybridized) probe 260. The interaction of the probe 262 with the active molecule may be accomplished via NHS-ester, thiol, DBCO, azide, maleimide interactions. For example, in one embodiment, the maleimide groups react specifically with thiol groups when the pH of the reaction mixture is between 6.5 and 7.5; the result is the formation of stable thioether linkages. Thus, in embodiments, reactive group 262 may be a maleimide reagent and reactive molecule 280 may be a thiol. Reactive group 262 reacts with reactive molecule 280 to form a stable conjugated thioether linkage. Thus, the probe 260 is immobilized on the active site 120. Nanosphere products can be removed by denaturation from probe 260 at a denaturation temperature and washing. This step immobilizes the probes 260 at the binding sites 66. The probe 260 has the same sequence along at least a portion of the oligonucleotide. In embodiments, probes 260 all have the same sequence relative to each other within binding sites 66, but have different sequences relative to probes 260 bound at some, most, or all of the other binding sites. In embodiments, 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 each other. Probes 260 between different binding sites 66 may have less than 50% sequence identity. Since the nanosphere product 200 is produced from a circular template 210 (see fig. 6), different templates 210 having different sequences correspondingly produce different nanosphere products 200. Thus, probes 260 complementary to repeat units 252 of different products 200 have different sequences based on the original template sequence.

Once immobilized, the probes 260 can be sequenced, amplified, or detected via labeled complementary oligonucleotides to map the binding sites 66 and their corresponding immobilized probes 260. The plot may be stored in a detection system (fig. 13).

Fig. 8-9 show alternative structural arrangements for placing capture molecules on the binding sites 66 of the substrate 60. In fig. 8, nanoparticles 300, which may be made of a polymer hydrogel, a cross-linked polymer, or an inorganic material, are incubated with single-stranded oligonucleotides 302 having chemical moieties 303 that may be covalently bonded to the nanoparticles 300. Subsequently, the nanoparticle 300, which has been functionalized with single-stranded oligonucleotide 302, is incubated with a second single-stranded oligonucleotide 306 having a second chemical moiety 305 to produce a functionalized nanoparticle 307 having immobilized double-stranded DNA and carrying the chemical moiety 305. Thus, the oligonucleotides 302, 306 interact based on complementarity to form double stranded DNA, wherein the chemical moiety is coupled to the nanoparticle 307 via hybridization. Examples of chemical moieties 303, 305 include, but are not limited to, thiols, amines, DBCO, maleimides, azides, NHS-esters.

The functionalized nanoparticles 307 in fig. 8 may be used to create monoclonal clusters of ssDNA on patterned substrate 60 by placement of nanoparticles 307, covalent bonding to the surface, and subsequent removal/denaturation. Steps 1 and 2 for patterning the substrate 60 may be performed as generally disclosed herein (see, e.g., fig. 3). In an embodiment, the workflow involves first passivating the surface and then lithographically patterning in accordance with the schemes as provided herein to produce a surface having two chemical properties, one within the binding sites 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 cause the nanoparticles 307 to organize within the binding sites 66. The driving force for this assembly is the interaction between the nanoparticle 307 and the binding site 66. The nanoparticles 307 are sized such that there is typically a single nanoparticle 307 organized or placed at a single binding site 66. Chemical moiety 305 coupled to nanoparticle 307 is covalently attached to surface 310 of binding site 66. Finally, nanoparticle 307 and complementary nucleic acid 302 on the nanoparticle are denatured to separate the oligonucleotide strands 302, 306, thereby creating a patterned surface with multiple copies of the same ssDNA306 per binding site. Single-stranded DNA (or RNA) 306 is covalently linked to surface 310 via chemical moiety 305.

FIG. 10 shows the coupling of capture molecules 70, shown as antibodies, to single stranded oligonucleotides 320 placed at separate binding sites 66. Single-stranded oligonucleotide 300 may be probe 260 coupled via reactive group 262 as shown in FIG. 7. Single-stranded oligonucleotide 300 may be an oligonucleotide 306 coupled via moiety 305 as shown in FIG. 9. The single stranded oligonucleotide 320 immobilized on the binding site 66 may be used directly as a capture molecule 70 (e.g., via hybridization). However, in embodiments, the unique sequence of oligonucleotide 320 may be used to design a complementary tag that is coupled to capture molecule 70 in a binding site-specific manner via hybridization. In embodiments, single stranded oligonucleotide 320 may be printed directly onto binding site 66 as shown in FIG. 3. Single stranded oligonucleotides 320 at individual binding sites 66 all have the same sequence or their sequences have at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity. Capture molecule 70 is coupled to oligonucleotide 300 by tag 290 comprising a complementary sequence. Each individual capture molecule 70 or each type of capture molecule 70 having binding specificity for an individual analyte is coupled to a tag 322 having a unique sequence that is distinguishable from other sequences coupled to other tags 322 of different capture molecules 70 having different specificities. Tag 322 is designed based on the known sequence of oligonucleotide 320. Thus, an assembly of different capture molecules 70 with corresponding unique tags 322 can be contacted with the binding site 66 comprising single stranded oligonucleotides 320.

As shown, each binding site 66 may be coupled to a plurality of capture molecules 70, which in turn may interact with one, two, three, or more complexes 40 via binding of an analyte to the capture molecules 70. The presence of the supramolecular structure 10 on the complex keeps the average number of molecules per binding site low. However, the presence of multiple capture molecules 70 with the same specificity at each binding site 66 promotes strong specific binding to the analyte.

Fig. 11 is an embodiment of capture molecules 70 coupled to beads 330. In the illustrated embodiment, the beads 330 are disposed within the wells 340 of the substrate 60, which are sized and shaped such that the individual binding sites 66 formed by the wells 340 accommodate a single bead 330. However, it should be understood that the beads 330 may be in solution and not associated with the substrate. Each bead is functionalized with a unique oligonucleotide 320 (all of the same or substantially the same sequence as provided herein), and capture molecules 70 with tags 322 hybridize to the oligonucleotide 320 only if they are complementary sequences.

Fig. 12 shows an example of binding site formation (e.g., for binding site 66 as in fig. 10-11). The binding sites 66 can be used to load beads 330 carrying a plurality of analytes 2 (e.g., DNA, RNA, or proteins/peptides as discussed herein) into lithographically defined nano/micro wells. The illustrated workflow produces a final product 350 (e.g., substrate 60) with an outer surface with nano/micro holes 360 that includes a first chemical group or functional group on the inner walls and bottom of the holes 390 and another different chemical group with different reactivity on the interstitial surfaces 362. Furthermore, the first chemical group is designed such that it will interact (electrostatically or covalently) with the bead 330, while the other chemical groups will not interact with the bead 300. In this case, when the beads 330 are incubated with the substrate 350, they will selectively bind to the interior of the wells 360, while none of the beads will bind to the gaps 360. In embodiments, the size or diameter of the beads 330 may be 20nm to 5 microns. In an embodiment, the holes may span 20nm to 5 microns (e.g., measured as the distance between adjacent gaps 362). The aperture 360 may be sized to accommodate only one bead 330. Thus, in certain embodiments, each well 360 may not be more than twice the diameter of the bead.

In the workflow of fig. 12, a silicon dioxide surface with a resin cover layer is provided and patterned via nanoimprinting. The patterned resin is treated with a plasma (e.g., O ₂ Plasma) and treated with a first silane. The treated surface is then provided with a coating, such as a polymethyl methacrylate (PMMA) coating. The oxygen etch exposes the top surface of the underlying patterned resin and removes the first silane from the gap 362 while retaining the first silane in the side and bottom surfaces of the hole 360. A second anti-fouling treatment is provided to the exposed gap and PMMA is removed to expose the pores 360. For example, the disclosed workflow is for producing a substrate 360. Substrate 360 may be used to load the beads 330 and used in conjunction with the disclosed techniques.

In some embodiments, as shown in fig. 13, multiple analyte molecules are detected simultaneously in a sample by multiplexing, wherein multiple different supramolecular structures 10 provide multiple signals (e.g., linker barcodes, sample barcodes) for sequencing and analyte identification, as well as sample de-multiplexing. In some embodiments, the methods described herein for detecting an analyte in a sample provide high throughput and high multiplexing capability through the use of multiple supramolecular structures 10. The plurality of different supramolecular structures 10a, 10b, 10c have associated different sets of affinity binders 2, which may be the same set or different sets between the plurality of sets or different sets. However, in embodiments, multiplex analyte detection may be used for different samples that are all in contact with the same set of affinity binding agents 2, all of which may be detected using the same functionalized substrate 60 having a specific set of capture molecules 70 compatible with the set of affinity binding agents 2.

The core structures 13 may generally be identical, whereby a plurality of single core structures 13 are coupled to different affinity binders 2. However, for a particular sample run, all core structures 13 may include or be coupled 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, the complexes 40a, 40b, 40c may be pooled together or run together on a sample substrate, but a detectable signal may be associated with a particular sample based on the detection of a particular sample-specific barcode 450.

Multiplexing may include a washing or separation step to separate complex 40 from unbound analyte 14. Such separations may include affinity separations via labels on the supramolecular structure 10, magnetic separations, size separations.

Embodiments of the present disclosure include multiplex kits, such as n-multiplex kits. The kit is provided with universal or common aptamers on the core structure 13. The aptamer served as a butt-joint for different n-fold barcodes. The number of barcodes may be selected based on the number of samples to be multiplexed, and each sample barcode may be added to a separate reaction vessel such that the sample-indexed core structures 13 are isolated from each other until labeling, complex formation, and separation from unbound analytes are performed.

Fig. 14A shows steps of an experimental workflow for antigen detection. The workflow includes functionalizing the supramolecular structure 10 to complex with specific affinity binders. For example, 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 signal transduction element, here shown as a fluorophore 500. However, the signal transduction element may be a nucleic acid signal generator, an optical signal generator, an electrical signal generator, a magnetic signal generator. The signal transduction element may be detected by a detection system as generally discussed with respect to fig. 23.

In the experiments discussed with respect to fig. 15-16, the affinity binding agent 2 was an IgG antibody 520 coupled to a single chain capture chain 520 that was complexed with a core structure 13 of a supramolecular structure according to an embodiment of the disclosure having a box-shaped folded structure 500 (e.g., a box-shaped structure formed from nucleic acid chains) with a plurality of associated fluorophores 510. However, it is to be understood that other antibody types or other affinity binding agents 2 are also contemplated in the disclosed embodiments. In the example shown, each different IgG represents a different affinity binding agent 2 with a corresponding different specificity for a different analyte (e.g., antigen 522). Thus, the workflow shown is shown to have triple detection capability, and each specific IgG antibody is coupled to a supramolecular structure with a specific fluorophore type. Thus, fluorophore 510a coupled to first IgG antibody 520a can be detected at a first fluorescence range, while fluorophore 510b coupled to second IgG antibody 520b can be detected at a distinguishable second fluorescence range, and fluorophore 510c coupled to third IgG antibody 520c can be detected at a distinguishable third fluorescence range. In an embodiment, each box-fold structure 500 is substantially identical except that it differs because different fluorescence detection wavelengths depend on different coupling fluorophores 510a, 510b, 510c. However, in embodiments, the aggregate of core structures 13 or box-fold structures 500 may be the same or different for a particular workflow.

At step 1, the single-stranded nucleic acid capture strand 522 conjugated to IgG is complexed with the single-stranded complementary strand 524 coupled to the box-fold structure 500 to form a duplex structure. Thus, igG antibodies complex with the core structure 13 via complementary binding under conditions that promote duplex structure formation. Each different antibody 520 and box-fold structure 500 may have a corresponding generic capture strand 522 and complementary strand 524 that are identical even for different affinity binding specificities to simplify and batch certain reagent preparation steps. However, in embodiments, each specific IgG antibody 520a, 520b, 520c, etc. may be coupled to a unique nucleic acid capture strand 522, which may include a barcode or other identifying information unique to the particular affinity binding agent 2.

While the experimental workflow is shown as a triple reaction with three different IgG antibodies 520a, 520b, 520c, permitting detection of three different antigens, single, double, or other multiplex arrangements are also contemplated. In embodiments, extended fluorophore signal discrimination can be achieved by controlling the number of fluorophores 510 coupled to each box-fold structure 500 to achieve a range of different signal intensities. Thus, the affinity binding agents 2 may be distinguished based on the detected fluorescence range and/or the detected intensity.

At step 2 of the workflow, the collection of supramolecular structures is contacted with a sample that may or may not contain the antigen 526 of interest. If antigen 526 to which an IgG antibody specifically binds is present, assembly 40 is formed, which is a complex 528 of supramolecular structure 10 that binds to antigen 526.

At step 3 of the workflow (which may be performed before, during, or after steps 1 and 2), the 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 coupled to streptavidin that binds to biotin-labeled antibodies 536. The capture antibodies 536a, 536b, 536c may have different binding specificities based on the specific binding of the IgG antibodies 520a, 520b, 520 c.

Fig. 14B shows additional steps of the experimental workflow of fig. 14A. At step 4, the supramolecular structure-antigen complex 528 is contacted with functionalized capture beads 540 to form a sandwich 550. For example, both IgG antibody 520 and capture antibody 536 bind to a particular antigen 526. In an embodiment, both IgG antibody 520a and capture antibody 536a are bound to separate antigens 526. Any box-fold 500 that does not bind antigen 526 will not couple to the magnetic beads 530 and thus can be removed from the sandwich 550 via magnetic capture.

At step 5, a displaced strand 560 is provided into the reaction mixture to break the duplex of the capture strand 520 and the complementary strand 522. In embodiments, the replacement is via foothold mediated replacement. Thus, in one example, the capture strand 522 includes a complementary region that binds to the complementary strand 522 and a foothold region that is non-complementary and remains unbound or unannealed prior to contact with the displacement strand 560. The displaced strand is complementary to both the complementary region and the footer region, thereby facilitating displacement from the complementary strand 522 via binding of the displaced strand 560 to the footer region to form a duplex of the displaced strand 560 and the capture strand 520. Thus, the substitution strand 560 shares sequence identity with the complementary strand 522 and also includes a foothold complementary region. Contact with the displacement strand causes the capture strand 520 and the displacement strand 560 to form a duplex that releases the box-fold 500 into solution, wherein the capture strand 520 and the displacement strand 560 become a duplex. In an embodiment, the released box-shaped fold 500 may be separated from the remainder 570 of the sandwich structure 550. For example, the remainder 570 retains the beads 530, which may be magnetically captured to leave the box-shaped folds in solution.

It should be appreciated that the substitutions may be reversed, wherein the complementary strand 522 may include a foothold region. The displaced strand 560 binds to the complementary strand 522 to break the duplex and release the box-fold into solution, wherein the capture strand 520 is single-stranded.

At step 6, the released box-fold 500 is imaged at a wavelength corresponding to the conjugated fluorophore 510, and the detectable signal is indicative of the presence of a particular antigen associated with a particular wavelength range. The signal may be evaluated for an expected or appropriate light intensity indicative of the presence of the antigen. For example, the light intensity may be. However, other detection means are also contemplated as discussed herein.

In embodiments, time interval cycling of different permutation chains 560 specific to different capture chains 520 can be used to increase the multiplicity of the workflow. For example, in a tri-fluorophore example, N number of tri-fluorophore sets can be coupled to a box-fold 500 having a set-specific unique capture strand 520 that is displaced by a unique set-specific displacement strand 56. For N number of sets, there may be N number of unique capture chain 520-permutation chain 560 pairs. Each unique substitution strand 560 of a known sequence may be added separately to release only some of the box-folds 500 carrying the corresponding capture strand 520. The released box-fold 500 is imaged to obtain antigen related data and the next substitution strand 560 may be added.

Fig. 15 shows different experimental subgroups evaluated using the experimental workflow of fig. 14A-14B. The antigens used were TSH, PSA and IL-6, and these antigens correspond to triple-differential antibody binding and detection. Different antigens are tested individually and in combination with each other to identify potential cross-binding or other interference. Antibodies were loaded in% antibody load as follows: 1.5% (Dynabeads). In the experiment, the incubation time was:

bead incubation for biotinylated-IgG was 30 min

For D-biotin, any free avidin sites were occupied for 10 minutes

For cassette-IgG and antigen 1 hour

For the cassette-IgG-antigen and IgG-beads 1 hour in 1x PBS pH 7.4+0.1% Tween+10 mM Mg. The displaced strand is a 1uM displaced strand in PBS Tween+10 mM Mg. The different fluorophores are as follows:

IL-6 readout was performed using the alexafluor 488 cassette

TSH read-out was performed using an alexafluor 647 cassette

PSA readout was performed using alexafluor 750 cartridges

Detection was performed via a Tecan microplate reader. It should be appreciated that the experimental workflow may be performed with other, more and/or fewer fluorophores.

Fig. 16 shows the experimental results of light intensity readout in the case of using different antigen combinations of three antigens in the experiment of fig. 15. Each individual antigen can be detected independently or in a mixture and without significant crosstalk or interference.

Fig. 17 shows the experimental results in the case where different antigen combinations of three antigens were used in the experiment of fig. 15 and TSH was titrated while PSA and IL-6 remained constant. The detected light output reflects the varying concentration of TSH in the sample.

Fig. 18 shows experiments in which TSH or PSA was titrated in a double reaction and shows the results of experiments in which varying antigen concentrations were detectable in the light output.

Figure 19 shows the results of a quadruple experiment quantifying four cytokines in a mixture.

TNFa: readout with alexafluor 488 cassette (128 imagers)

IL-10: readout with atto 565 cartridges (128 imagers)

INFy: readout with alexafluor 647 box (128 imagers)

IL-2: readout with alexafluor 750 cartridges (128 imagers)

The IgG input for the box-fold was 5nM

At 10mM MOPS+150mM NaCl+10mM MgCl ₂ Incubation with antigen and cassette-IgG in +0.1% Tween-20 for 2 hours

Incubation with cassette-IgG: antigen+IgG-beads for 1 hr

At 10mM MOPS+150mM NaCl+10mM MgCl ₂ Substitution of the cassette at 500nM in +0.1% Tween-20

Read on a Tecan, 15 uL/sample, gain 140 at 491, 550, 641nm excitation wavelength

Dilution of 100-fold dimer/trimer antigen with 2 out of 3 replicates had a greater readout.

Fig. 20 shows the results of experiments quantifying four cytokines in a mixture, where only one cytokine was detected per subgroup.

FIG. 21 shows experiments to detect IL-2, TNF alpha, IL-10 and IFN gamma in simulated serum mixtures, showing that optical detection can be performed for antigens in complex biological samples.

FIG. 22 shows a series of titrations of IL-8 and TNFα, where constant CRP concentrations show detectable antigen concentration differences in light output.

Experimental results demonstrate an effective solution-based detection of the proposed experimental workflow.

Embodiments of the present disclosure include one or more computer-implemented detection systems configured to perform certain methods of the disclosed embodiments. Fig. 23 shows an analyte detection system 1000 that includes 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, for example, to facilitate communications over the internet 1010 and/or over a wireless or wired network. The user interface 1006 facilitates user interaction with the characterized analyte detection results as provided herein.

Processor 1002 is programmed to receive analyte detection data and characterize the detected analyte. In one embodiment, after incubating the supramolecular structural complex 40 with an array of binding sites 66 and detecting characteristics of the binding sites 66, the complex 10, or both, the processor generates a report of the analytes detected in the sample. The report may include data for the optical signals generated at the respective binding sites corresponding to the detected analyte binding events. The report may include processed data such as a list of detected analytes or positive/negative binding results. The report may include a list of available affinity binders and/or available capture molecules 70 of the complex 10 indicating analyte detection capabilities.

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

The supramolecular structure 10 of the complex 40 that remains at the separate binding site 66 via interaction with the capture molecule 70 is detected to generate a detectable signal. For example, the barcode of the linker 20 serves as a binding site for a detectable signal transduction element in contact with the bound complex (e.g., via hybridization of complementary sequences). In some embodiments, the signal transduction element comprises a fluorescent molecule or microbead, a fluorescent polymer, a highly charged nanoparticle, or a polymer. In some embodiments, the barcode is amplified. For example, barcodes are used as polymerization initiators for the growth of highly fluorescent polymers in processes such as rolling circle amplification or hybridization chain reactions.

In some embodiments, the signal transduction element is optically active and may be measured using a microscope or integrated optical sensor within the substrate 60. In some embodiments, the signal transduction element is electroactive and may be measured using an integrated electrical sensor. In some embodiments, the signal transduction element is magnetically active and may be measured using an integrated magnetic sensor. In some embodiments, each signal event is associated with a 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 binding agent, whereby counting the number of locations where signal transduction elements are present gives a quantification of the analyte molecule in the sample.

In some embodiments, the supramolecular structure 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, wherein the affinity binding agent and the detector molecule are simultaneously attached to (e.g., bound to) the analyte molecule (e.g., sandwich formation).

In some embodiments, detecting the presence of an analyte molecule, as described herein, includes controllably releasing a single or multiple unique nucleic acid molecules into solution for identifying the analyte molecule from a sample and quantifying a property of the analyte molecule. In some embodiments, the unique nucleic acid molecules are provided by a capture barcode 20 of the corresponding supramolecular structure. In some embodiments, detecting the presence of an analyte molecule, as described herein, includes generating an optical or electrical signal related to a change in state, which can be counted to quantify the concentration of the analyte molecule in solution.

In some embodiments, each supramolecular structure 10 comprises a unique DNA barcode corresponding to an associated affinity binding agent. As provided herein, the linker barcode 20 can be used to uniquely identify an individual supramolecular structure 10. In turn, each supramolecular structure 10 is assembled such that the affinity binding agent 2 can associate with the capture barcode 20, for example, as stored in a look-up table of an analyte detection system. Thus, when the capture barcode 20 is authenticated, the identity of the affinity binding agent 2 is also available.

Analyte detection techniques may be used to characterize analyte binding. The bound complex 40 may be characterized based on: 1) The barcode 20 of the supramolecular structure 10, and (in embodiments) 2) the barcode of the capture molecule 70 co-located with the barcode 20. The barcode may be detected by a signal generated by a detector molecule coupled to one or more of the binding site 66, the complex 40, or the capture molecule 70. In embodiments, the location of capture molecules 70 may be determined prior to complex binding. That is, the array of binding sites 66 may be provided as a pre-mapped array, or the mapping may be a separate step. The mapping may include the step of detecting capture molecule barcodes, such as detecting unique optical, electrical, and/or magnetic patterns, as generally provided herein. In embodiments, detecting comprises sequencing the nucleotide sequence of the capture barcode. In embodiments, detection includes amplification and quantification of the amplified product, e.g., detection of a signal associated with the probe via qPCR.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. The following claims are intended to define the scope of the invention and the methods and structures within the scope of these claims and their equivalents are covered thereby.

Claims

1. A method for detecting an analyte molecule present in a sample, the method comprising:

providing a sample comprising analyte molecules;

contacting the sample in solution with an aggregate of supramolecular structures to form analyte molecule-supramolecular structure complexes, the individual analyte molecule-supramolecular structure complexes comprising:

a core structure comprising a plurality of core molecules;

an affinity binding agent, said affinity binding agent being linked to said core structure; and

analyte molecules of the analyte molecules bound to the affinity binding agent,

wherein different supramolecular structures of the collection of supramolecular structures comprise different affinity binders having different binding affinities for other ones of the analyte molecules;

contacting the analyte molecule-supramolecular structure complexes with an array, wherein the binding sites of the array comprise respective immobilized affinity binders having binding affinities for different analyte molecules; and

detecting binding of the analyte molecule-supramolecular structure complex to binding sites of the array.

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

3. The method of claim 2, wherein the identifying comprises: generating a detectable signal from the supramolecular structure and correlating the detectable signal with the 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 structure at the individual binding sites.

5. The method of claim 2, wherein the identifying comprises: amplifying the initiator of the supramolecular structure and detecting the amplification.

6. The method of claim 5, wherein the initiator is a nucleic acid.

7. The method of claim 2, wherein the identifying comprises: amplifying the initiators immobilized on the separate binding sites and detecting the amplification.

8. The method of claim 7, wherein the initiator is a nucleic acid.

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

10. The method of claim 1, wherein the binding of the analyte molecule-supramolecular structure to the immobilized affinity binding agent at the separate 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.

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

12. The method of claim 11, wherein each individual binding site comprises a plurality of nucleic acid capture molecules.

13. The method of claim 12, wherein the plurality of nucleic acid capture molecules at separate binding sites all have the same nucleic acid sequence.

14. The method of claim 12, wherein the plurality of nucleic acid capture molecules are coupled to an immobilized supramolecular structure.

15. The method of claim 1, wherein each individual binding site comprises a plurality of immobilized affinity binders, each having affinity for the same one of the analyte molecules.

16. The method of claim 1, wherein the diameter of each binding site is between 20 and 500 nanometers.

17. The method of claim 1, wherein each supramolecular structure of the aggregate is a nanostructure.

18. The method of claim 14, wherein each core structure is a nanostructure.

19. The method of claim 1, wherein each supramolecular structure of the aggregate is arranged in a predefined shape and/or has a defined molecular weight.

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

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

22. A method for detecting an analyte molecule present in a sample, the method comprising:

providing an array of supramolecular structures, wherein each individual supramolecular structure is immobilized on a respective binding site of a substrate, the individual supramolecular structures of the array comprising:

a core structure comprising a plurality of core molecules; and

a plurality of nucleic acid capture molecules linked to the core structure, wherein the nucleic acid capture molecules each comprise the same identification sequence that is distinguishable from the identification sequences of other supramolecular structures of the array;

contacting the array with an affinity binding agent assembly linked to a single-stranded nucleic acid tag having binding specificity for a different identification sequence, such that different affinity binding agents bind to different binding sites of the array, such that each binding site comprises a different subset of the affinity binding agent assembly;

Contacting the sample with the array such that the analyte molecules bind to individual affinity binders in the collection of affinity binders, wherein the analyte molecules are complexed with a supramolecular structure detection assembly; and

detecting binding of the analyte molecule to the separate binding sites.

23. The method of claim 20, wherein the supramolecular structure detection assembly is sized such that only one analyte molecule is capable of binding to each binding site.

24. The method of claim 20, wherein the nucleic acid capture molecule is single stranded.

25. The method of claim 20, wherein the supramolecular structure detection assembly is coupled to an affinity binding agent that binds the analyte molecule to form a complex.

26. The method of claim 20, wherein the supramolecular structure detection assembly comprises only a single affinity binding agent that binds the analyte molecule to form a complex.

27. A method for detecting an analyte molecule present in a sample, the method comprising:

providing an array comprising single stranded oligonucleotides immobilized on the array such that individual binding sites of the array comprise a plurality of single stranded oligonucleotides comprising the same sequence distinguishable from sequences of other single stranded oligonucleotides immobilized on different binding sites of the array;

Contacting the array with an affinity binding agent aggregate such that the immobilized single stranded oligonucleotides capture a subset of the affinity binding agents at binding sites of the array and such that the captured affinity binding agents form capture molecules immobilized at the respective binding sites;

contacting the sample with the array such that the analyte molecules bind to individual capture molecules at the individual binding sites, wherein the analyte molecules are complexed with a supramolecular structure detection assembly; and

detecting binding of the analyte molecule to the separate binding sites.

28. The method of claim 27, wherein the single stranded oligonucleotides are printed on the array.

29. The method of claim 27, wherein each binding site of the array comprises a well, and wherein single stranded nucleic acids are immobilized on beads located within the well.

30. The method of claim 27, wherein the subset of the affinity binding agents at each binding site comprises a plurality of affinity binding agents.

31. The method of claim 27, wherein immobilizing the single stranded oligonucleotide comprises: capturing rolling circle amplification products at the separate binding sites, the rolling circle amplification products comprising a plurality of complementary probes; and anchoring the plurality of complementary probes to the binding sites.

32. The method of claim 27, wherein immobilizing the single stranded oligonucleotide comprises: capturing functionalized nanoparticles at the individual binding sites, the functionalized nanoparticles comprising a plurality of double stranded nucleic acids; and anchoring one strand of the double-stranded nucleic acid to the separate binding site.

33. A method for detecting an analyte molecule present in a sample, the method comprising:

forming a nanosphere or functionalized nanostructure, the nanosphere or functionalized nanostructure comprising:

a plurality of individual oligonucleotides, each oligonucleotide of the plurality of individual oligonucleotides being linked to a chemical moiety and each oligonucleotide being bound to a complementary nucleic acid of the nanosphere or functionalized nanostructure;

providing a patterned array comprising a plurality of active sites;

incubating the nanospheres or functionalized nanostructures with the patterned array to covalently attach active groups of individual active sites to chemical moieties of the plurality of individual oligonucleotides, thereby coupling individual nanospheres or functionalized nanostructures to the individual active sites;

denaturing the plurality of individual oligonucleotides from the complementary nucleic acids of the nanospheres or functionalized nanostructures;

Washing the nanospheres or functionalized nanostructures from the array to leave the plurality of individual oligonucleotides immobilized on the individual binding sites, wherein the plurality of individual oligonucleotides are single stranded; and

contacting the array with an affinity binding agent aggregate such that the immobilized plurality of individual oligonucleotides capture a subset of the affinity binding agents at the individual binding sites.

34. The method of claim 33, wherein forming the nanospheres comprises producing rolling circle amplification products that hybridize to the plurality of individual oligonucleotides.

35. The method of claim 33, wherein forming a functionalized nanoparticle comprises covalently attaching a plurality of single stranded nucleic acids to a nanoparticle and hybridizing the plurality of individual oligonucleotides to the covalently attached single stranded nucleic acids.

36. A method for detecting an analyte molecule present in a sample, the method comprising:

providing a sample comprising analyte molecules;

A core structure comprising a plurality of core molecules;

an affinity binding agent, said affinity binding agent being linked to said core structure;

sample-specific barcodes; and

analyte molecules of the analyte molecules bound to the affinity binding agent,

combining the analyte molecule-supramolecular structure complex with other analyte molecule-supramolecular structure complexes associated with corresponding different sample-specific barcodes;

contacting the combined analyte molecule-supramolecular structure complexes with an array, wherein the binding sites of the array comprise respective immobilized affinity binders having binding affinity for different analyte molecules;

detecting binding of the analyte molecule-supramolecular structure complex to binding sites of the array; and

correlating the detected binding to the sample-specific barcode.

37. An array for analyte detection, the array comprising:

A plurality of binding sites patterned on a substrate and separated by a gap surface of the substrate, wherein each binding site comprises a pore, and wherein at least one surface of the pore comprises a first chemical group and the gap surface comprises a second chemical group;

a bead loaded into each of the binding sites, wherein the first chemical group selectively binds to the bead and the second chemical group does not interact with or bind to the bead, and wherein each bead comprises:

a plurality of single stranded oligonucleotides immobilized on each bead such that individual binding sites of the array comprise a plurality of single stranded oligonucleotides comprising the same sequence distinguishable from sequences of other single stranded oligonucleotides immobilized on different binding sites of the array;

an affinity binding agent that is captured at a binding site of the array such that the captured affinity binding agent forms a capture molecule immobilized at the respective binding site; and

an analyte molecule bound to an individual capture molecule at the individual binding sites, wherein each analyte molecule is complexed with a supramolecular structure detection assembly.

38. A method for detecting an analyte molecule present in a sample, the method comprising:

providing a plurality of supramolecular structures, wherein each supramolecular structure comprises

A core structure comprising a plurality of core molecules;

an antibody having binding affinity for an antigen and coupled to a nucleic acid capture chain; and

a complementary strand of the nucleic acid capture strand, wherein the complementary strand is linked to the core structure and forms a duplex structure with the nucleic acid capture strand to couple the antibody to the core structure, wherein the plurality of supramolecular structures comprises antibodies that differ relative to each other with respective different binding affinities;

contacting the plurality of supramolecular structures with a sample comprising the antigen such that the antigen binds to the antibody to form a complex;

contacting the complex with beads carrying capture antibodies 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, wherein the released core structure is not coupled to the antibody; and

and detecting the core structure.

39. The method of claim 38, wherein the core structure is a box-shaped fold formed from nucleic acids.

40. The method of claim 39, wherein the box-fold is coupled to a plurality of fluorophores that fluoresce at a particular wavelength, and wherein detecting the core structure comprises detecting fluorescence at the particular wavelength.

41. The method of claim 38, wherein the capture strand comprises a foothold region that does not bind to the complementary strand but does bind to the replacement strand.

42. The method of claim 38, wherein the complementary strand comprises a foothold region that does not bind to the capture strand but does bind to the displacement strand.

43. The method of claim 38, comprising separating the remainder of the sandwich structure from the released core structure prior to the detecting.

44. The method of claim 43, comprising isolating supramolecular structures of the plurality of supramolecular structures that are not bound to the antigen prior to the displacing.