Structure and method for detecting a sample

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 are detected using one or more supramolecular structures. In some embodiments, the supramolecular structures are configured to form linkages with specific capture barcodes that are configured to form linkages with specific capture molecules. In some embodiments, the capture molecules are configured to interact with specific analyte molecules. In some embodiments, the location of the supramolecular structures is mapped onto a substrate having multiple binding locations according to the capture barcodes and / or another barcodes linked to the supramolecular structures. In some embodiments, the linkages between the analyte molecules and the supramolecular structures allow for the generation of a signal. In some embodiments, the generated signal allows for the identification and quantification of the analyte molecules in the sample based on the mapped location of the supramolecular structures on the substrate.

Description

Technical Field

[0001] Cross-reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 153,258, filed Feb. 24, 2021, which is hereby incorporated by reference in its entirety.

Background Art

[0002] Background The current state of personalized medicine is overwhelmingly genome-centric and primarily focuses on quantifying the genes that exist within an individual. While such an approach has proven to be extremely powerful, it does not provide physicians with a holistic picture of an individual's health. This is because genes are merely the "blueprint" of an individual and only inform the likelihood of developing a disease. These "blueprints" need to be first transcribed into RNA within the individual and then translated into various protein molecules, i.e., the actual "actors" within the cell, in order to have any impact on the individual's health.

[0003] Protein concentrations, protein-protein interactions (protein-protein interactions or PPIs), and interactions between proteins and small molecules are intricately linked to the health of various organs, homeostatic regulatory mechanisms, and the interaction of these systems with the external environment. Thus, quantitative information on proteins and PPIs is essential for constructing a comprehensive picture of an individual's health at a given point in time and for predicting any emerging health problems. For example, the amount of stress on the myocardium (e.g., during a heart attack) can be estimated by measuring the concentrations of troponin I / II and myosin light chains in peripheral blood. Similar protein biomarkers have also been identified, validated, and developed for a wide range of organ dysfunctions (e.g., liver disease and thyroid disorders), specific cancers (e.g., colorectal or prostate cancer), and infectious diseases (e.g., HIV and Zika virus infection). These protein-protein interactions are also essential and increasingly sought-after datasets for new drug development. The ability to detect and quantify proteins and other molecules in a given sample of body fluids is an indispensable element of such medical development. [Overview of the project] [Problems that the invention aims to solve]

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

[0005] In some embodiments, the Specified Method provides for detecting sample molecules present in a sample, comprising: a) i) providing a supramolecular structure comprising a core structure comprising a plurality of core molecules; and ii) a capture barcode linked to the core structure at a first location and configured to form a link with a capture molecule; b) linking the supramolecular structure to the capture molecule via the capture barcode; c) bringing the supramolecular structure into contact with a sample, thereby causing the sample molecule to interact with and bind to the capture molecule, thereby transitioning the supramolecular structure from a ground state to an excited state; d) generating a signal via the excited supramolecular structure; and e) detecting the sample molecule based on the signal.

[0006] In some embodiments, this specification discloses a method for detecting one or more sample molecules present in a sample, comprising: a) providing a plurality of supramolecular structures, each comprising i) a core structure comprising a plurality of core molecules, and ii) a capture barcode linked to the core structure at a first location; b) linking each of the plurality of supramolecular structures to a capture molecule via the corresponding capture barcode; c) contacting the plurality of supramolecular structures with a sample, thereby causing one or more capture molecules of the plurality of supramolecular structures to interact with the corresponding sample molecule of one or more sample molecules, thereby transitioning the corresponding supramolecular structures from a ground state to an excited state; d) generating a signal for each supramolecular structure in the excited state; and e) detecting each sample molecule based on the generated corresponding signal. In some embodiments, providing a plurality of supramolecular structures includes providing supramolecular structures attached to one or more widgets, one or more solid supports, one or more polymer matrices, one or more solid substrates, one or more molecular condensates, or combinations thereof. In some embodiments, each of the one or more solid substrates includes a planar substrate. In some embodiments, each planar substrate includes a plurality of binding sites, each of which is configured to attach to one of a plurality of supramolecular structures. In some embodiments, each binding site attaches to the supramolecular structure via a corresponding stationary molecule linked to the supramolecular structure. In some embodiments, the method further includes mapping the locations of the plurality of supramolecular structures attached to the plurality of binding sites, via 1) a corresponding capture barcode, 2) a stationary barcode linked to the supramolecular structure, and / or 3) another barcode linked to the supramolecular structure. In some embodiments, the mapping is performed before providing the plurality of supramolecular structures and / or before contacting the plurality of molecules with a sample. In some embodiments, the mapping allows for the identification of the capture molecule and the corresponding sample molecule, which are configured to be linked to the corresponding supramolecular structure attached to the corresponding binding site.In some embodiments, two or more supramolecular structures of multiple supramolecular structures are configured to form links with the same sample molecules of multiple sample molecules via corresponding capture molecules.

[0007] In some embodiments, any method disclosed herein further includes identifying each detected sample molecule. In some embodiments, any method disclosed herein further includes quantifying the concentration of each detected sample molecule. In some embodiments, any method or system disclosed herein, each capture molecule includes a protein, peptide, antibody, aptamer (RNA and / or DNA), small DNA molecule, affinity binder, or a combination thereof. In some embodiments, any method or system disclosed herein, each aptamer includes a modified aptamer. In some embodiments, any method or system disclosed herein is configured to specifically interact with a particular type of sample molecule. In some embodiments, any method disclosed herein further includes detecting each sample molecule based on a generated signal if the sample molecule is present in the sample in a number of one or more molecules. In some embodiments, any system disclosed herein is configured to detect each sample molecule based on a generated signal if the sample molecule is present in the sample in a number of one or more molecules. In some embodiments, any method or system disclosed herein includes a sample comprising a complex biological sample, and the method provides single-molecule sensitivity, thereby increasing the dynamic range and quantitative capture of various molecular concentrations in the complex biological sample. In some embodiments, any method or system disclosed herein includes one or more sample molecules comprising proteins, peptides, peptide fragments, lipids, DNA, RNA, organic molecules, inorganic molecules, complexes thereof, or any combination thereof. In some embodiments, any method or system disclosed herein includes a fluorescent signal and / or a visual signal. In some embodiments, any method or system disclosed herein includes a light signal, an electrical signal, or both.In some embodiments, in any method or system disclosed herein, the optical signal includes microwave signals, ultraviolet light, visible light, near-infrared light, light scattering, or a combination thereof.

[0008] In some embodiments, any method disclosed herein involves generating a signal by a) binding each sample molecule linked to the corresponding supramolecular structure in an excited state to a precursor molecule; and b) tagging each precursor molecule bound to the sample molecule with a fluorophore and / or a fluorescently labeled molecule to generate a fluorescent signal. In some embodiments, any method disclosed herein includes a biotin molecule. In some embodiments, any method disclosed herein includes an NHS-biotin molecule. In some embodiments, any method disclosed herein includes an amine-reactive NHS-biotin molecule. In some embodiments, any method disclosed herein includes a fluorescently labeled molecule including fluorescently labeled streptavidin, fluorescently labeled avidin, or both. In some embodiments, any method disclosed herein involves generating a signal by tagging each sample molecule linked to the corresponding supramolecular structure in an excited state with a dye molecule to generate a fluorescent signal. In some embodiments, in any method disclosed herein, the dye molecule comprises an NHS-dye molecule. In some embodiments, in any method disclosed herein, detection of each sample molecule includes obtaining a fluorescence readout of the generated signal(s) and correlating each corresponding supramolecular structure with the capture molecule and the sample molecule configured to be linked thereto. In some embodiments, in any method disclosed herein, correlating each corresponding supramolecular structure is based on the mapping described herein. In some embodiments, in any method disclosed herein, detection includes obtaining a fluorescence readout using a fluorescence microscope.

[0009] In some embodiments, any method disclosed herein involves generating a signal by: a) linking each sample molecule, which is linked to a corresponding supramolecular structure in an excited state, to a precursor molecule; and b) linking each precursor molecule linked to the sample molecule to a light-scattering molecule or nanoparticle, thereby generating a visual signal. In some embodiments, any method disclosed herein includes a biotin molecule. In some embodiments, any method disclosed herein includes an NHS-biotin molecule. In some embodiments, any method disclosed herein includes an amine-reactive NHS-biotin molecule. In some embodiments, any method disclosed herein includes a light-scattering molecule or nanoparticle by: a streptavidin molecule, an avidin molecule, or both. In some embodiments, any method disclosed herein includes a streptavidin molecule, an avidin molecule, or both by: a Qdot or metal nanoparticles. In some embodiments, in any method disclosed herein, the visual signal includes visualization of large streptavidin and / or avidin molecules linked to the precursor molecule. In some embodiments, in any method disclosed herein, detecting each sample molecule includes visualizing the interaction between each precursor molecule and light-scattering molecules or nanoparticles, and correlating each corresponding supramolecular structure with the capture molecule and the sample molecule configured to be linked thereto. In some embodiments, in any method disclosed herein, correlating each corresponding supramolecular structure is based on the mapping described herein. In some embodiments, in any method disclosed herein, detection includes using an interference scattering microscope.

[0010] In some embodiments, any method disclosed herein involves generating a signal by linking each sample molecule, linked to a corresponding supramolecular structure in an excited state, to a second capture molecule, each corresponding second capture molecule being either 1) fluorescently labeled to generate a fluorescent signal, or 2) unlabeled to generate a visual signal via sandwich formation through a complex formed with the corresponding sample molecule. In some embodiments, any method disclosed herein involves detecting each sample molecule by obtaining a fluorescent readout of the generated signal(s) and correlating each corresponding supramolecular structure with the capture molecule and the sample molecule configured to be linked thereto. In some embodiments, any method disclosed herein involves correlating each corresponding supramolecular structure based on the mapping described herein. In some embodiments, any method disclosed herein involves detection by obtaining a fluorescent readout using a fluorescence microscope. In some embodiments, any method disclosed herein involves detecting each sample molecule by visualizing the interaction between each sample molecule and the second capture molecule and correlating each corresponding supramolecular structure with the capture molecule and the sample molecule configured to be linked thereto. In some embodiments, in any method disclosed herein, correlating each corresponding supramolecular structure is based on the mapping described herein. In some embodiments, in any method disclosed herein, detection includes using an interference scattering microscope.

[0011] In some embodiments, the Specified System for detecting one or more sample molecules in a sample is disclosed, comprising: a) a substrate having a plurality of binding sites; b) a plurality of supramolecular structures, wherein each binding site is configured to receive one supramolecular structure of the plurality of supramolecular structures, and each supramolecular structure comprises: i) a core structure having a plurality of core molecules; and ii) a capture barcode linked to the core structure at a first site; c) a plurality of capture molecules, each capture barcode configured to be linked to one of the plurality of capture molecules; d) a sample having one or more sample molecules, wherein when the sample is brought into contact with the substrate, one or more sample molecules interact with corresponding capture molecules of the plurality of capture molecules, thereby causing the corresponding supramolecular structures to transition from a ground state to an excited state; e) a signal generation system capable of generating a signal based on the excited state supramolecular structures; and f) a detection system configured to detect each sample molecule linked to the excited state supramolecular structures based on the generated signal. In some embodiments, the signal includes a fluorescence signal, a visual signal, or both. In some embodiments, the detection system includes a fluorescence microscope and / or iSCAT. In some embodiments, the locations of multiple supramolecular structures at multiple binding sites are configured to be mapped.

[0012] In some embodiments, in any method or system disclosed herein, each supramolecular structure is a nanostructure. In some embodiments, in any method or system disclosed herein, each core structure is a nanostructure. In some embodiments, in any method or system disclosed herein, the multiple core molecules of each core structure are arranged in a predefined shape and / or have a predetermined molecular weight. In some embodiments, in any method or system disclosed herein, the predefined shape is configured to limit or prevent cross-reactivity with other supramolecular structures. In some embodiments, in any method or system disclosed herein, the multiple core molecules of each core structure include one or more nucleic acid chains, one or more branched nucleic acids, one or more peptides, one or more small molecules, or combinations thereof. In some embodiments, in any method or system disclosed herein, each core structure independently includes scaffold deoxyribonucleic acid (DNA) origami, scaffold ribonucleic acid (RNA) origami, scaffold hybrid DNA:RNA origami, single-stranded DNA tile structure, multi-stranded DNA tile structure, single-stranded RNA origami, multi-stranded RNA tile structure, DNA or RNA origami in a hierarchical configuration having multiple scaffolds, peptide structures, or combinations thereof. In some embodiments, in any method or system disclosed herein, each sample molecule interacts with the corresponding capture molecule through chemical bonding. In some embodiments, in any method or system disclosed herein, with respect to each supramolecular structure, a capture molecule is linked to a core structure through a capture barcode, the capture barcode includes a first capture linker, a second capture linker, and a capture bridge positioned between the first and second capture linkers, the first capture linker being linked to a first core linker bonded to a first location on the core structure, and the capture molecule and the second capture linker are linked to each other through bonding with a third capture linker. In some embodiments, in any method or system disclosed herein, the capture bridge includes a polymer core.In some embodiments, in any method or system disclosed herein, the polymer core of the capture bridge comprises a nucleic acid (DNA or RNA) of a specific sequence or a polymer such as PEG. In some embodiments, in any method or system disclosed herein, the first core linker, second core linker, third core linker, first capture linker, second capture linker, and third capture linker independently comprise a reactive molecule or a DNA sequence domain. In some embodiments, in any method or system disclosed herein, each reactive molecule independently comprises one or more polymers such as amines, thiols, DBCO, maleimide, biotin, azide, acrylic, NHS esters, single-stranded nucleic acids (DNA or RNA) of a specific sequence, PEG, or polymerization initiators, or a combination thereof. In some embodiments, in any method or system disclosed herein, the linkage between the capture barcode and 1) the first core linker and / or 2) the third capture linker comprises a chemical bond. In some embodiments, in any method or system disclosed herein, the chemical bond comprises a covalent bond. In some embodiments, in any method or system disclosed herein, the capture molecule is bonded to a third capture linker through a chemical bond. In some embodiments, in any method or system disclosed herein, the capture molecule is covalently bonded to the third capture linker. In some embodiments, in any method or system disclosed herein, each supramolecular structure further includes a fixed molecule linked to a core structure. In some embodiments, in any method or system disclosed herein, the fixed molecule is linked to the core structure via a fixed barcode, the fixed barcode includes a first fixed linker, a second fixed linker, and a fixed bridge positioned between the first and second fixed linkers, the first fixed linker being bonded to a third core linker bonded to a second location on the core structure, and the fixed molecule being linked to the second fixed linker.In some embodiments, in any method or system disclosed herein, the immobilized molecule includes one or more polymers such as amines, thiols, DBCO, maleimide, biotin, azide, acrylic, NHS esters, single-stranded nucleic acids (DNA or RNA) of a specific sequence, PEG, or a combination thereof. In some embodiments, in any method or system disclosed herein, the immobilized bridge includes a polymer core. In some embodiments, in any method or system disclosed herein, the polymer core of the immobilized bridge includes nucleic acids (DNA or RNA) of a specific sequence or a polymer such as PEG. In some embodiments, in any method or system disclosed herein, the third core linker, the first immobilized linker, the second immobilized linker, and the immobilized molecule independently include an immobilized reactive molecule or a DNA sequence domain. In some embodiments, in any method or system disclosed herein, each immobilized reactive molecule independently comprises one or more polymers such as amines, thiols, DBCO, maleimide, biotin, azide, acrylic, NHS esters, single-stranded nucleic acids (DNA or RNA) of a specific sequence, PEG, or polymerization initiators, or a combination thereof. In some embodiments, in any method or system disclosed herein, the immobilized molecule is linked to a second immobilized linker through chemical bonding. In some embodiments, in any method or system disclosed herein, the immobilized molecule is covalently bonded to a second immobilized linker. In some embodiments, in any method or system disclosed herein, the first location is located on the first side of the core structure, and the second location is located on the second side of the core structure. In some embodiments, in any method or system disclosed herein, one or more sample molecules in a sample are detected simultaneously by multiplexing via one or more supramolecular structures that have transitioned to an excited state. In some embodiments, in any method or system disclosed herein, the core structures of the multiple supramolecular structures are identical to each other.In some embodiments, in any method or system disclosed herein, each supramolecular structure includes a predetermined shape, size, molecular weight, or combination thereof. In some embodiments, in any method or system disclosed herein, each supramolecular structure includes a plurality of capture molecules. In some embodiments, in any method or system disclosed herein, each supramolecular structure includes a predetermined stoichiometric amount of capture molecules. In some embodiments, in any method or system disclosed herein, at least one of the plurality of supramolecular structures is configured to detect a sample molecule different from the other supramolecular structures. In some embodiments, in any method or system disclosed herein, the sample includes biological particles or biomolecules. In some embodiments, in any method or system disclosed herein, the sample includes an aqueous solution containing proteins, peptides, peptide fragments, lipids, DNA, RNA, organic molecules, viral particles, exosomes, organelles, or any complex thereof. In some embodiments, any method or system disclosed herein, the sample includes tissue biopsy, blood, plasma, urine, saliva, tears, cerebrospinal fluid, extracellular fluid, cultured cells, culture media, waste tissue, plant matter, synthetic proteins, bacterial and / or viral samples or fungal tissue, or combinations thereof.

[0013] In some embodiments, the supramolecular structure includes a predetermined shape, size, molecular weight, or combination thereof to reduce or eliminate cross-reactivity with other supramolecular structures. In some embodiments, the supramolecular structure includes multiple capture molecules and detection molecules. In some embodiments, the supramolecular structure includes capture molecules and detection molecules with a predetermined stoichiometry to reduce or eliminate cross-reactivity with other supramolecular structures.

[0014] In some embodiments, the sample comprises biological particles or biomolecules. In some embodiments, the sample comprises an aqueous solution comprising proteins, peptides, peptide fragments, lipids, DNA, RNA, organic molecules, viral particles, exosomes, organelles, or any complex thereof. In some embodiments, the sample comprises tissue biopsies, blood, plasma, urine, saliva, tears, cerebrospinal fluid, extracellular fluid, cultured cells, culture media, waste tissue, plant matter, synthetic proteins, bacterial and / or viral samples or fungal tissue, or combinations thereof.

[0015] Specific embodiments of the disclosed apparatus, delivery system, or method are described herein with reference to the drawings. Nothing in this detailed description is intended to imply that any particular component, feature, or step is essential to the invention. [Brief explanation of the drawing]

[0016] [Figure 1A] This represents an illustrative description of supramolecular structures and their associated partial configurations. [Figure 1B] Figure 1A shows the supramolecular structure and the capture molecules linked to it. [Figure 2] This provides an exemplary description of a method for detecting and quantifying sample molecules using multiple supramolecular structures attached to a planar substrate. [Modes for carrying out the invention]

[0017] Detailed description Throughout this application, various embodiments of the present disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an immutable limitation to the scope of the invention. Therefore, a range description should be considered to specifically disclose any possible sub-ranges, as well as all individual numerical values within that range. For example, a range description such as 1 to 6 should be considered to specifically disclose sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, as well as the individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0018] The terms "about" and "approximately" mean within an acceptable error range for a particular value determined by one of ordinary skill in the art, which depends in part on the method by which the value is measured or determined, i.e., the limitations of the measuring system. For example, "about" can mean within one or more standard deviations in the practice of the art. Alternatively, the term can mean within a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, the term can mean within one order of magnitude of the value, preferably within five-fold, more preferably within two-fold.

[0019] As used herein, the terms "sample" and "sample molecule" are used interchangeably.

[0020] As used herein, the terms "bind," "bound," and "interact" are used interchangeably and generally refer to non-covalent interactions between macromolecules (e.g., between a protein and a nucleic acid). In a state of non-covalent interaction, macromolecules are said to be "associated" or "interacting" or "bound" (e.g., when it is said that molecule X interacts with molecule Y, it means that molecule X binds to molecule Y in a non-covalent manner).

[0021] As used herein, the terms “attached,” “linked,” “linked,” and “linking” are interchangeable and generally refer to the joining of one object to another. For example, oligomers and primers may be attached to the surface of a capture site. With regard to the mechanism of attachment, the methods to be considered include means of attachment such as ligating, non-covalent bonding, or binding of biotinylated primers, amplicons, and probes to streptavidin, etc. Capture molecules may be attached directly to a supramolecular structure (e.g., via covalent bonding, biotin-streptavidin linking, DNA oligonucleotide linkers, or polymer linkers) or indirectly (e.g., via linking to a stationary strand, e.g., by conjugation, or through a linker such as a capture strand).

[0022] Performing single-molecule analysis assays in a high-throughput / parallel manner multiplexed on microfluidic chips is crucial in many commercially available instruments for multi-omic characterization of biological samples. A diverse range of such assays exist in the literature on DNA sequencing and single-molecule quantification. Mass spectrometry and other affinity-based methods (such as antibody-based measurements) for protein identification and quantification have classically dominated the field of high-content proteomics, but are limited by technical issues ranging from throughput to cross-reactivity. Protein-binding affinity conjugates, such as modified aptamers, represent a highly multiplexed technology that enables the quantification of the human proteome to unprecedented levels and the development of biomarkers for improved diagnostics and therapeutics with high sensitivity and specificity. An example of a modified aptamer is SOMAmer®. The SomaScan® assay has been used to identify potential biomarkers for various diseases such as malignancies, cardiovascular dysfunction, and inflammatory conditions. This rapid, scalable, large-scale parallel multiplexing technology is a powerful tool that enables the development of personalized diagnostics and therapies.

[0023] Disclosed herein are systems and methods for detecting and quantifying one or more analyte molecules present in a sample. In some embodiments, one or more analyte molecules are detected using one or more supramolecular structures and one or more capture molecules linked to the supramolecular structures, each capture molecule being configured to bind a unique analyte molecule. In some embodiments, each of the capture molecules includes an affinity binder. In some embodiments, each affinity binder includes an aptamer. In some embodiments, each aptamer includes a modified aptamer. In some embodiments, the one or more supramolecular structures are specifically designed to minimize cross-reaction with each other. In some embodiments, an analyte molecule bound to a corresponding capture molecule is configured to be detected through a generated signal. In some embodiments, the signal includes a fluorescence signal or a visual signal. In some embodiments, the signal correlates to a labeled analyte molecule. In some embodiments, a plurality of supramolecular structures are provided on an array substrate, and the supramolecular structures are barcoded to map the location of each supramolecular structure on the array. In some embodiments, the supramolecular structures are barcoded via a capture barcode that provides linkage to a specific capture molecule, and / or the supramolecular structures are barcoded through other barcodes assigned thereto. In some embodiments, the analyte molecules are detected and / or quantified using the mapped locations of the supramolecular structures on the substrate array.

[0024] Sample In some embodiments, the sample comprises an aqueous solution containing proteins, peptides, peptide fragments, lipids, DNA, RNA, organic molecules, inorganic molecules, complexes thereof, or any combination thereof. In some embodiments, the sample 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 sample molecules include intact proteins, denatured proteins, partially or completely degraded proteins, peptide fragments, denatured nucleic acids, degraded nucleic acid fragments, complexes thereof, or combinations thereof. In some embodiments, the sample is obtained from tissues, cells, tissue and / or cellular environments, or combinations thereof. In some embodiments, the sample includes tissue biopsies, blood, plasma, urine, saliva, tears, cerebrospinal fluid, extracellular fluid, cultured cells, culture media, waste tissue, plant matter, synthetic proteins, bacterial or viral samples, fungal tissue, or combinations thereof. In some embodiments, the sample is isolated from a primary source such as purified or unpurified cells, tissues, bodily fluids (e.g., blood), environmental samples, or combinations thereof. In some embodiments, cells are lysed using mechanical processes or other cell lysis methods (e.g., lysis buffers). In some embodiments, the sample is filtered using mechanical processes (e.g., centrifugation), filtration with micron filters, chromatography columns, other filtration methods, or combinations 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 contains intact proteins, denatured proteins, partially or completely degraded proteins, peptide fragments, denatured nucleic acids, or degraded nucleic acid fragments. In some embodiments, the sample is collected from one or more individuals, one or more animals, one or more plants, or combinations thereof. In some embodiments, samples are collected from individual people, animals, and / or plants having diseases or disorders including infectious diseases, immunodeficiencies, cancer, genetic disorders, degenerative diseases, lifestyle-related diseases, injuries, rare diseases, age-related diseases, or combinations thereof.

[0025] supramolecular structure In some embodiments, a supramolecular structure is a programmable structure that can spatially organize molecules. In some embodiments, a supramolecular structure is a supramolecular DNA origami structure. In some embodiments, a supramolecular structure comprises multiple molecules linked together. In some embodiments, multiple molecules of a supramolecular structure interact with at least some of each other. In some embodiments, a supramolecular structure comprises a specific shape. In some embodiments, a supramolecular structure comprises a predetermined molecular weight based on multiple molecules of the supramolecular structure. In some embodiments, a supramolecular structure is a nanostructure. In some embodiments, multiple molecules are linked together through bonds, chemical bonds, physical adhesion, or a combination thereof. In some embodiments, a supramolecular structure comprises a large molecular entity of a specific shape and molecular weight, formed from a clearly defined number of smaller molecules that specifically interact with each other. In some embodiments, the structural, chemical, and physical properties of the supramolecular structure are explicitly designed. In some embodiments, a supramolecular structure comprises multiple partial configurations spatially separated according to a predetermined distance. In some embodiments, at least a portion of the supramolecular structure is rigid. In some embodiments, at least a portion of the supramolecular structure is semi-rigid. In some embodiments, at least a portion of the supramolecular structure is flexible.

[0026] Figure 1A provides an exemplary embodiment of a supramolecular structure 40 comprising a core structure 13, a capture barcode 20, and a fixed molecule 18. In some embodiments, the supramolecular structure includes a supramolecular DNA origami structure, and the core structure includes a DNA origami structure. In some embodiments, the supramolecular structure does not include a fixed molecule. In some embodiments, the supramolecular structure is a polynucleotide structure.

[0027] In some embodiments, the core structure 13 comprises one or more core molecules linked together. In some embodiments, the one or more core molecules comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, or 500 unique molecules linked together. In some embodiments, the one or more core molecules comprise about 2 to about 1000 unique molecules. In some embodiments, the one or more core molecules interact with each other to define a specific shape of the supramolecular structure. In some embodiments, the multiple core molecules interact with each other through reversible non-covalent interactions. In some embodiments, the specific shape of the core structure is a three-dimensional (3D) configuration. In some embodiments, the one or more core molecules provide a specific molecular weight. In some embodiments, the core structure 13 is a nanostructure. In some examples, one or more core molecules include one or more nucleic acid strands (e.g., DNA, RNA, non-natural nucleic acids), one or more branched nucleic acids, one or more peptides, one or more small molecules, or combinations thereof. In some embodiments, the core structure includes a polynucleotide structure. In some embodiments, at least a portion of the core structure is rigid. In some embodiments, at least a portion of the core structure is semi-rigid. In some embodiments, at least a portion of the core structure is flexible. In some embodiments, the core structure includes scaffold deoxyribonucleic acid (DNA) origami, scaffold ribonucleic acid (RNA) origami, scaffold hybrid DNA / RNA origami, single-stranded DNA tile structure, multi-stranded DNA tile structure, single-stranded DNA origami, single-stranded RNA origami, single-stranded RNA tile structure, multi-stranded RNA tile structure, DNA and / or RNA origami in a hierarchical configuration with multiple scaffolds, peptide structures, or combinations thereof. In some embodiments, the DNA origami is a scaffold structure. In some embodiments, RNA origami is a scaffold structure. In some embodiments, hybrid DNA / RNA origami is a scaffold structure.In some embodiments, the core structure, which includes DNA origami, RNA origami, or hybrid DNA / RNA origami, includes a predetermined two-dimensional (2D) or three-dimensional shape.

[0028] As shown in Figure 1B, in some embodiments, the supramolecular structure is further configured to be linked to the capture molecule 2 via a capture barcode 20, as described herein. In some embodiments, the capture molecule 2 and / or the fixed molecule 18 are fixed with respect to the core nanostructure 13 once linked. In some embodiments, any number of one or more core molecules include one or more core linkers 12, 14 configured to form links with the capture molecule 2 and / or the fixed molecule 18. In some embodiments, any number of one or more core molecules are configured to be linked to one or more core linkers 12, 14 configured to form links with the core molecule 2 and / or the fixed molecule 18.

[0029] In some embodiments, one or more core linkers 12, 14 are linked to one or more capture molecules through chemical bonds. In some embodiments, at least one of the one or more core linkers 12, 14 contains a core reactive molecule. In some embodiments, each core reactive molecule independently contains an amine, thiol, DBCO, NHS ester, maleimide, biotin, azide, acrylic, a single-stranded nucleic acid (DNA or RNA) of a specific sequence, 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 contains a DNA sequence domain.

[0030] Referring to Figure 1A, in some embodiments, the core structure 13 is linked to 1) a capture barcode 20 at a predetermined first location on the core structure, and optionally to 2) a fixed molecule 18 at a predetermined second location on the core structure. In some embodiments, a designated first core linker 12 is located at the first location on the core structure. In some embodiments, one or more core molecules at the first location are modified to form a link with the first core linker 12. In some embodiments, the first core linker 12 is an extension of the core structure 13.

[0031] In some embodiments, a designated third core linker 14 is located at a second location on the core structure 13. In some embodiments, one or more core molecules at the second location are modified to form a link with the third core linker 14. In some embodiments, the third core linker 12 is an extension of the core structure 13. In some embodiments, the first location is located on the first side of the core structure 13, and the optional second location is located on the second side of the core structure 13.

[0032] Referring to Figure 1B, in some embodiments, capture molecule 2 includes proteins, peptides, antibodies, aptamers (RNA and / or DNA), fluorophores, nanobodies, DARPin, catalysts, polymerization initiators, polymers such as PEG, organic molecules, or combinations thereof. In some embodiments, the capture molecule includes a modified aptamer. In some embodiments, the capture molecule includes SOMAmer®. In some embodiments, one or more capture molecules include combinations of aptamers and modified aptamers, including combinations of SOMAmer® aptamers and non-SOMAmer® aptamers. In some embodiments, the modified aptamer includes a class of nucleic acid-based protein-binding reagents that are chemically modified to provide a unique fingerprint as an affinity binder. In some embodiments, the modified aptamer assay can convert the protein concentration in a mixture into a DNA signature, which can then be quantified, for example, by utilizing a commercially available DNA microarray platform. In some embodiments, modified aptamers possess a dual nature: a) a protein-binding folding entity with a specific shape having chemically modified properties, and 2) a unique nucleic acid sequence designed to be recognized by a hybridization probe. In some embodiments, the dual nature of modified aptamers makes them a powerful tool for highly multiplexed (over 1000 plexities) protein quantification. In some embodiments, capture molecules possess a unique shape and chemical properties configured to recognize and bind to a specific sample molecule (e.g., a protein). In some embodiments, the binding between the capture molecule and the sample molecule forms a capture molecule-sample molecule complex.

[0033] In some embodiments, the immobilized molecule includes a reactive molecule. In some embodiments, the immobilized molecule 18 includes a reactive molecule. In some embodiments, the immobilized molecule 18 includes a DNA strand containing a reactive molecule. In some embodiments, the immobilized molecule 18 includes an amine, thiol, DBCO, NHS ester, maleimide, biotin, azide, acrylic, single-stranded nucleic acid (DNA or RNA) of a specific sequence, or a polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, the immobilized molecule 18 includes a protein, peptide, antibody, aptamer (RNA and DNA), fluorophore, nanobody, DARPin, catalyst, polymerization initiator, polymer such as PEG, organic molecule, or a combination thereof.

[0034] In some embodiments, each component of the supramolecular structure may be modified or adjusted independently. In some embodiments, modifying one or more components of the supramolecular structure may modify the 2D and 3D geometric structure of the supramolecular DNA origami structure itself. In some embodiments, modifying one or more components of the supramolecular structure may modify the 2D and 3D geometric structure of the core structure. In some embodiments, the ability to independently modify components of such supramolecular structures allows for the organization of one or more supramolecular structures on a solid surface (e.g., a planar surface or microparticles) and precise control of their 3D volume (e.g., within a hydrogel matrix).

[0035] Capture barcode As shown in Figures 1A-1B, in some embodiments, the capture molecule 2 is linked to the core structure 13 through the capture barcode 20. In some embodiments, the capture barcode 20 forms a link with the capture molecule 2, and the capture barcode 20 forms a link with the core structure 13. In some embodiments, the capture barcode 20 is configured to form a link with a specific capture molecule (e.g., an aptamer). In some embodiments, the capture barcode is configured to form a link with a specific capture molecule through chemical linkage. In some embodiments, the chemical linkage includes maleimide-thiols, DBCO-azides, and amine-NHS esters. In some embodiments, the capture barcode is configured to hybridize with the capture molecule. In some embodiments, the capture barcode further provides a barcode for supramolecular molecular structures, which can be used to map the locations of supramolecular structures, for example, when multiple supramolecular structures are arranged at multiple binding locations on a planar substrate.

[0036] In some embodiments, the capture barcode 20 includes a first capture linker 11, a second capture linker 6, and a capture bridge 7. In some embodiments, the first capture linker 11 includes a reactive molecule. In some embodiments, the first capture linker 11 includes a reactive molecule comprising an amine, thiol, DBCO, NHS ester, maleimide, azide, acrydite, a single-stranded nucleic acid (DNA or RNA) of a specific sequence, or a polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, the first capture linker 11 includes a DNA sequence domain. In some embodiments, the second capture linker 6 includes a reactive molecule. In some embodiments, the second capture linker 6 includes a reactive molecule comprising an amine, thiol, DBCO, NHS ester, biotin, maleimide, azide, acrydite, a single-stranded nucleic acid (DNA or RNA) of a specific sequence, or a polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, the second capture linker 6 includes a DNA sequence domain. In some embodiments, the capture bridge 7 includes a polymer. In some embodiments, the capture bridge 7 includes a unique barcode sequence that can be used to map the location of a supramolecular structure and / or is configured to form links with individual capture molecules. In some embodiments, the capture bridge 7 includes a polymer containing nucleic acid (e.g., DNA or RNA) of a specific sequence. In some embodiments, the capture bridge 7 includes a polymer such as PEG. In some embodiments, the first capture linker 11 is attached to the capture bridge 7 at its first end, and the second capture linker 6 is attached to the capture bridge 7 at its second end. In some embodiments, the first capture linker 11 is attached to the capture bridge 7 via a chemical bond. In some embodiments, the second capture linker 6 is attached to the capture bridge 7 via a chemical bond. In some embodiments, the first capture linker 11 is attached to the capture bridge 7 via physical attachment. In some embodiments, the second capture linker 6 is attached to the capture bridge 7 via physical attachment.

[0037] In some embodiments, the captured barcode 20 is linked to the core structure 13 through a linkage between a first capture linker 11 and a first core linker 12. In some embodiments, the first core linker 12 described herein is located at a first location on the core structure 13. In some embodiments, the first capture linker 11 and the first core linker 12 are linked to each other through a chemical bond. In some embodiments, the first capture linker 11 and the first core linker 12 are linked to each other through a covalent bond. In some embodiments, the linkage between the first capture linker 11 and the first core linker 12 becomes reversible when subjected to a trigger. In some embodiments, the trigger includes interaction with a deconstructor molecule ("capture deconstructor molecule") or exposure to a trigger signal. In some embodiments, the capture deconstructor molecule includes nucleic acids (DNA or RNA), peptides, small organic molecules, or combinations thereof. In some embodiments, the trigger signal includes a light signal. In some embodiments, the trigger signal includes an electrical signal, a microwave signal, ultraviolet light, visible light, or near-infrared light.

[0038] In some embodiments, the captured barcode 20 is linked to the captured molecule 2 through a linkage between the second captured linker 6 and the third captured linker 5, which is bonded to the captured molecule 2. In some embodiments, the third captured linker 5 contains a reactive molecule. In some embodiments, the third captured linker 5 contains a reactive molecule including an amine, thiol, DBCO, NHS ester, maleimide, biotin, azide, acrydite, a single-stranded nucleic acid (DNA or RNA) of a specific sequence, or a polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, the third captured linker 5 contains a DNA sequence domain. In some embodiments, the captured molecule 2 is bonded to the third captured linker 5 through a chemical bond. In some embodiments, the captured molecule 2 is bonded to the third captured linker 5 through a covalent bond. In some embodiments, the second captured linker 6 and the third captured linker 5 are linked to each other through a chemical bond. In some embodiments, the second captured linker 6 and the third captured linker 5 are linked to each other through a covalent bond. In some embodiments, the linkage between the second capture linker 6 and the third capture linker 5 becomes reversible when subjected to a trigger. In some embodiments, the trigger includes interaction with a deconstructor molecule ("capture barcode releasing molecule") or exposure to a trigger signal. In some embodiments, the capture barcode releasing molecule includes nucleic acids (DNA or RNA), peptides, small organic molecules, or combinations thereof. In some embodiments, the trigger signal includes a light signal. In some embodiments, the trigger signal includes an electrical signal, a microwave signal, ultraviolet light, visible light, or near-infrared light.

[0039] In some embodiments, the capture barcode 20 is hybridized to the capture molecule 2 by means of nucleic acid hybridization or the like. In some embodiments, the capture barcode 20 is linked to the capture molecule 2 via hybridization such as nucleic acid hybridization. In some embodiments, the capture barcode 20 is linked to the capture molecule 2 via a covalent linkage between molecules 5 and 6, where both molecules 5 and 6 may be molecular pairs that react specifically with each other, such as DBCO-azide, amine-NHS ester, or thiol-maleimide.

[0040] In some embodiments, the trigger is used to disrupt the linkage between the first capture linker 11 and the first core linker 12, thereby disrupting the linkage of the captured molecule to the core nanostructure 13 at the first location. In some embodiments, the capture barcode 20 is configured to provide a signal for detecting the sample molecule once separated from the core structure 13 and the captured molecule 2. In some embodiments, the signal provided by the capture barcode 20 is a DNA signal.

[0041] Fixed barcode As shown in Figure 1, in some embodiments, the fixed molecule 18 is linked to the core structure 13 through a fixed barcode. In some embodiments, the fixed barcode forms a link with the fixed molecule 18, and the fixed barcode forms a link with the core structure 13. In some embodiments, the fixed barcode provides a barcode for a supramolecular molecular structure, which can be used to map the locations of the supramolecular structures, for example, when multiple supramolecular structures are arranged at multiple bonding locations on a planar substrate.

[0042] In some embodiments, the fixed barcode includes a first fixed linker 15, a second fixed linker 17, and a fixed bridge 16. In some embodiments, the first fixed linker 15 includes a reactive molecule. In some embodiments, the first fixed linker 15 includes a reactive molecule comprising an amine, thiol, DBCO, NHS ester, maleimide, biotin, azide, acrydite, a single-stranded nucleic acid (DNA or RNA) of a specific sequence, or a polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, the first fixed linker 15 includes a DNA sequence domain. In some embodiments, the second fixed linker 17 includes a reactive molecule. In some embodiments, the second fixed linker 17 includes a reactive molecule comprising an amine, thiol, DBCO, NHS ester, maleimide, biotin, azide, acrydite, a single-stranded nucleic acid (DNA or RNA) of a specific sequence, or a polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, the second fixed linker 17 includes a DNA sequence domain. In some embodiments, the fixed bridge 16 includes a polymer. In some embodiments, the fixed bridge 16 includes a polymer containing nucleic acid (DNA or RNA) of a specific sequence. In some embodiments, the fixed bridge 16 includes a polymer such as PEG. In some embodiments, the first fixed linker 15 is attached to the fixed bridge 16 at its first end, and the second fixed linker 17 is attached to the fixed bridge 16 at its second end. In some embodiments, the first fixed linker 15 is attached to the fixed bridge 16 via a chemical bond. In some embodiments, the second fixed linker 17 is attached to the fixed bridge 16 via physical adhesion. In some embodiments, the first fixed linker 15 is attached to the fixed bridge 16 via a chemical bond. In some embodiments, the second fixed linker 17 is attached to the fixed bridge 16 via physical adhesion.

[0043] In some embodiments, the fixed barcode is linked to the core structure 13 through a linkage between a first fixed linker 15 and a third core linker 14. In some embodiments, the third core linker 14 described herein is located at a third location on the core structure 13. In some embodiments, the first fixed linker 15 and the third core linker 14 are linked to each other through a chemical bond. In some embodiments, the first fixed linker 15 and the third core linker 14 are linked to each other through a covalent bond. In some embodiments, the linkage between the first fixed linker 15 and the third core linker 14 becomes reversible when subjected to a trigger. In some embodiments, the trigger includes interaction with a deconstructor molecule ("fixed deconstructor molecule") or exposure to a trigger signal. In some embodiments, the fixed deconstructor molecule includes nucleic acids (DNA or RNA), peptides, small organic molecules, or combinations thereof. In some embodiments, the trigger signal includes a light signal. In some embodiments, the trigger signal includes an electrical signal, a microwave signal, ultraviolet light, visible light, or near-infrared light.

[0044] In some embodiments, the fixed barcode is linked to the fixed molecule 18 through a linkage between the second fixed linker 17 and the fixed molecule 18. As described herein, in some embodiments, the fixed molecule includes a reactive molecule, a DNA sequence domain, a DNA sequence domain containing a reactive molecule, or a combination thereof. In some embodiments, the fixed molecule 18 is linked to the second fixed linker 17 through a chemical bond. In some embodiments, the fixed molecule 18 is linked to the second fixed linker 17 through a covalent bond. In some embodiments, the linkage between the second fixed linker 17 and the fixed molecule 18 becomes reversible when subjected to a trigger. In some embodiments, the trigger includes interaction with a deconstructor molecule ("fixed barcode-releasing molecule") or exposure to a trigger signal. In some embodiments, the fixed barcode-releasing molecule includes nucleic acids (DNA or RNA), peptides, small organic molecules, or a combination thereof. In some embodiments, the trigger signal includes a light signal. In some embodiments, the trigger signal includes an electrical signal, a microwave signal, ultraviolet light, visible light, or near-infrared light.

[0045] In some embodiments, the trigger is applied to break only the link between the first fixed linker 15 and the third core linker 14, thereby breaking the link of the fixed molecule to the core structure 13 at the third location.

[0046] In some embodiments, the capture deconstructor molecule and the capture barcode release molecule contain molecules of the same type. In some embodiments, the capture deconstructor molecule and the capture barcode release molecule contain molecules of different types. In some embodiments, the capture deconstructor molecule, the capture barcode release molecule, the immobilization deconstructor molecule, and the immobilization barcode release molecule contain molecules of the same type. In some embodiments, the capture deconstructor molecule, the capture barcode release molecule, the immobilization deconstructor molecule, and the immobilization barcode release molecule contain molecules of different types. In some embodiments, any combination of the capture deconstructor molecule, the capture barcode release molecule, the immobilization deconstructor molecule, and the immobilization barcode release molecule contains molecules of the same type.

[0047] In some embodiments, the core structure includes scaffold DNA origami in which a circular ssDNA molecule called a "scaffold" strand folds into a predefined 2D or 3D shape by interacting with two or more short ssDNA strands called "staple" strands, which interact with specific sub-units of the ssDNA "scaffold" strand.

[0048] In some embodiments of the supramolecular DNA origami structure, the core structure comprises DNA origami. In some embodiments, the core structure 13 comprises a first core linker 12 comprising a DNA sequence domain. In some embodiments, the first core linker 12 is complementary to a first capture linker 11 on a capture barcode strand 20. In some embodiments, the capture barcode strand 20 comprises a DNA strand containing a first capture linker 11 and a second capture linker at either end of the capture barcode strand. In some embodiments, the first capture linker 11 comprises a DNA sequence domain. In some embodiments, the second capture linker 6 comprises a DNA sequence domain. In some embodiments, the capture barcode strand 20 further comprises a unique barcode sequence 7 between the first capture linker 11 and the second capture linker 6. In some embodiments, the unique capture barcode sequence 7 comprises nucleic acid (DNA or RNA) of a specific sequence. In some embodiments, the unique capture barcode sequence 7 comprises a polymer such as PEG. In some embodiments, the capture barcode chain 20 includes a short domain called a toehold ("TH"). In some embodiments, the capture barcode sequence 7 includes a toehold ("TH").

[0049] In some embodiments, the second capture linker 6 is complementary to the third capture linker 5. In some embodiments, the third capture linker 5 is a DNA sequence domain. In some embodiments, the capture molecule 2 is bound to the third capture linker 5. In some embodiments, the capture molecule 2 is covalently bound to the third capture linker 5. In some embodiments, the capture molecule 2 is directly bound to the capture barcode strand 20. In some embodiments, the capture molecule 2 is directly bound to the capture barcode sequence 7.

[0050] In some embodiments, the core structure includes a second core linker 14 containing a DNA sequence domain. In some embodiments, the second core linker 14 is complementary to a first fixed linker 15 on another barcode strand 22. In some embodiments, the fixed barcode strand 22 includes a DNA strand containing a first fixed linker 15 and a second fixed linker 17 at either end of the fixed barcode section 22. In some embodiments, the first fixed linker 15 contains a DNA sequence domain. In some embodiments, the second fixed linker 17 contains a DNA sequence domain. In some embodiments, the fixed barcode strand 22 further includes a unique fixed barcode sequence 16 between the first fixed linker 15 and the second fixed linker 17. In some embodiments, the fixed barcode strand 22 includes a short domain called a toehold ("TH"). In some embodiments, the fixed barcode sequence 16 includes a toehold ("TH"). In some embodiments, the unique detector barcode sequence 16 includes nucleic acid (DNA or RNA) of a specific sequence. In some embodiments, the unique detector barcode sequence 16 includes a polymer such as PEG.

[0051] In some embodiments, the second immobilized linker 17 is complementary to the immobilized molecule 18. In some embodiments, the immobilized molecule 18 includes a DNA sequence domain. In some embodiments, the immobilized molecule 18 is linked to a terminal modification. In some embodiments, the terminal modification includes a reactive molecule. In some embodiments, the terminal modification includes a reactive molecule comprising an amine, thiol, DBCO, NHS ester, maleimide, biotin, azide, acrydite, a single-stranded nucleic acid (DNA or RNA) of a specific sequence, or a polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators).

[0052] Method for detecting sample molecules As described herein, in some embodiments, one or more supramolecular structures enable the detection of one or more sample molecules in a sample. In some embodiments, each supramolecular structure comprises a supramolecular DNA origami structure. In some embodiments, the supramolecular structure transitions from a ground state to an excited state via linkage with a given sample molecule (via a corresponding capture molecule linked to the supramolecular structure). In some embodiments, the excited-state supramolecular structure is configured to convert information about the presence of the given sample molecule in the sample into a signal. In some embodiments, the signal comprises a fluorescently labeled signal, an unlabeled signal, or a combination thereof. In some embodiments, the identification and / or quantification of a given sample molecule in a sample using the signal corresponds to a capture barcode located on the supramolecular DNA origami structure, and the locations of multiple supramolecular structures are mapped according to each capture barcode. In some embodiments, each capture barcode is configured to enable linkage with a specific capture molecule. In some embodiments, the capture molecule comprises a modified aptamer.

[0053] In some embodiments, detecting the presence of one or more sample molecules as described herein involves optical and / or electronic readout of signals from multiple fluorescently labeled and / or unlabeled events corresponding to one or more sample molecules linked to the corresponding supramolecular structure. In some embodiments, one or more sample molecules linked to the corresponding supramolecular structure are immobilized on a solid support(s) or planar fixed substrate(s), and the corresponding supramolecular structure and capture molecules are immobilized in a predefined manner. The terms “capture molecule” and “recognition molecule” as used herein are interchangeable.

[0054] In some embodiments, multiple sample molecules are detected simultaneously in the sample through multiplexing, and multiple supramolecular structures enable the detection of multiple signals (e.g., optical or electrical) for sample molecule identification. In some embodiments, the methods described herein for detecting samples in a sample provide high throughput and high multiplexing capability by using multiple supramolecular structures (e.g., supramolecular DNA origami structures). In some embodiments, high throughput and high multiplexing capability provide high accuracy for the detection and quantification of sample molecules. In some embodiments, the methods described herein for detecting samples in a sample are configured to rapidly characterize and / or identify biopolymers, including protein molecules, with high sensitivity and reproducibility. In some embodiments, multiple supramolecular DNA origami structures are configured to limit errors associated with cross-reactions. In some embodiments, errors associated with such cross-reactions include capture molecules of a supramolecular DNA origami structure interacting with capture molecules of another supramolecular DNA origami structure (e.g., intermolecular interactions). In some embodiments, the core structures of each of the multiple supramolecular DNA origami structures are identical to one another. In some embodiments, the structural, chemical, and physical properties of each supramolecular DNA origami structure are clearly designed. In some embodiments, identical core structures have a predetermined shape, size, molecular weight, a predetermined number of capture molecules, a predetermined distance between corresponding capture molecules (as described herein), or a combination thereof, in order to limit cross-reactions between supramolecular DNA origami structures. In some embodiments, the molecular weight of each core structure is identical and precise depending on the purity of the multiple core molecules. In some embodiments, each core structure has at least one capture molecule.

[0055] In some embodiments, multiple supramolecular DNA origami structures are configured to form links with different sample molecules (via corresponding capture molecules). In some embodiments, a change of state (from non-excited to excited) is primarily driven by the linkage between the capture molecule (linked to the supramolecular structure) and a specific sample molecule. In some embodiments, multiple supramolecular structures may share structural similarities because certain partial configurations are identical, but the linkage between the sample molecule from the sample and the supramolecular structure is defined by the corresponding capture molecule. In some embodiments, each capture barcode on a supramolecular structure described herein is configured to form a linkage with the same specific capture molecule. In some embodiments, each capture molecule on a given supramolecular DNA origami structure can specifically interact with a particular sample molecule in the sample, causing a change of state of the supramolecular structure through interaction with that specific sample molecule. In some embodiments, each supramolecular structure includes a unique DNA barcode (e.g., a capture barcode) corresponding to its respective capture molecule. In some embodiments, the capture molecule on a given supramolecular DNA origami structure is designed to interact with only one type of sample molecule in the sample. In some embodiments, the capture molecule on a given supramolecular DNA origami structure is designed to interact with more than one type of sample molecule in the sample.

[0056] In some embodiments, each supramolecular DNA origami structure is configured to be single-molecule sensitive to ensure the most likely dynamic range required to quantitatively capture a wide range of molecular concentrations in a typical complex biological sample. In some embodiments, single-molecule sensitivity includes a given supramolecular DNA origami structure configured to transition from a ground state to an excited state through interactions between a corresponding capture molecule (linked to a given supramolecular structure) and a single sample molecule, as described herein. In some embodiments, multiple supramolecular DNA origami structures limit or eliminate nonspecific interactions and the sample handling required to reduce any user-induced errors.

[0057] In some embodiments, multiple supramolecular structures are provided in solution. In some embodiments, multiple supramolecular structures are attached to one or more substrates. In some embodiments, multiple supramolecular structures are attached to one or more widgets. In some embodiments, multiple supramolecular structures are attached to one or more solid substrates, one or more polymer matrices, one or more molecular condensates, or a combination thereof. In some embodiments, one or more polymer matrices include one or more hydrogel particles. In some embodiments, one or more polymer matrices include one or more hydrogel beads. In some embodiments, one or more solid substrates include one or more planar substrates. In some embodiments, one or more solid substrates include one or more microbeads. In some embodiments, one or more solid substrates include one or more fine particles.

[0058] In some embodiments, the sample and the supramolecular DNA origami structure are incubated in an incubator under predetermined environmental conditions. In some embodiments, the sample is incubated with the supramolecular DNA origami structure for a period of about 30 seconds to about 24 hours. In some embodiments, the sample is incubated with the supramolecular DNA origami structure for a period of about 30 seconds to about 1 minute, about 1 minute to about 5 minutes, about 5 minutes to about 30 minutes, about 30 minutes to about 1 hour, about 1 hour to about 5 hours, about 5 hours to about 12 hours, about 12 hours to about 24 hours, and about 24 hours to about 48 hours.

[0059] In some embodiments, a method for detecting a sample molecule includes cleaving a capture barcode from a corresponding capture molecule that has interacted with the sample molecule. In some embodiments, the capture barcode is cleaved from the corresponding capture molecule through nucleic acid (DNA / RNA) strand substitution, optical cleavage, chemical cleavage, or a combination thereof.

[0060] In some embodiments, the cleaved capture barcodes are isolated from a solution containing a supramolecular DNA origami structure. In some embodiments, the cleaved capture barcodes are isolated from the solution via polyethylene glycol (PEG) precipitation. In some embodiments, the cleaved capture barcodes provide a signal correlated to each sample molecule bound to each capture molecule. In some embodiments, the capture barcodes described herein include a DNA strand. In some embodiments, the capture barcodes provide a DNA signal correlated to a sample molecule. In some embodiments, the isolated capture barcodes are analyzed to identify and / or quantify the corresponding sample molecule in the sample. In some embodiments, the analysis of the isolated capture barcodes includes genotyping, qPCR, sequencing, or a combination thereof.

[0061] In some embodiments, aligning capture molecules (e.g., modified aptamers) on an array based on the arrangement of DNA origami through DNA hybridization or other adhesion techniques (as described herein) provides an alternative platform to DNA microarray techniques and the use of the resulting DNA signature embedded in the modified aptamer for the quantification of protein-binding events. In some embodiments, solution-based assays can then be converted to chip-based assays as an alternative to the use of bead pull-down and UV cleavage strategies.

[0062] Detection of sample molecules using surface assays Figure 2 provides an example of a method for detecting sample molecules in a sample using a surface-based assay that utilizes a supramolecular structure, as described herein, for single-molecule counting of sample molecules in the sample (i.e., detecting sample molecules in the sample at single-molecule resolution). In some embodiments, the supramolecular structure includes a core structure 13 containing a DNA origami core. In some embodiments, a planar substrate 400 is provided, which includes (a) alignment markers 402 that serve as reference coordinates for all features on the substrate; (b) a defined set 406 of micropatterned binding sites to which individual core structures (e.g., DNA origami) can be fixed; and / or (c) background passivation 404 that minimizes or prevents interactions between the surface of the substrate 400 and the supramolecular structure (e.g., capture molecules, core structure molecules). In some embodiments, the alignment markers 402 include a geometric shape defined on the surface that is used as a reference feature for other features on the substrate 400. In some embodiments, the alignment marker 402 is coated with a polymer or self-assembled monolayer that does not interact with other molecules of the core structure or supramolecular structure (e.g., DNA origami). In some embodiments, background passivation 404 minimizes or prevents interaction between the substrate surface and the sample molecules of the sample. In some embodiments, in addition to background passivation required for preferential supramolecular structure binding (e.g., preferential DNA origami binding) to the binding site 406 on the substrate 400, the substrate 400 may be chemically treated with various blocking reagents to promote specific interactions between capture molecules (e.g., aptamers), sample molecules (e.g., protein samples), and label entities (e.g., NHS-biotin and streptavidin) and the supramolecular structure (e.g., DNA origami) molecules and / or molecules linked thereto. In some embodiments, the planar substrate 400 involves differential chemistry at the binding site 406. In some embodiments, the planar substrate 400 is prepared through lithography processes known in the art.In some embodiments, the planar substrate includes optical or electrical devices such as FETs, ring resonators, photonic crystals, or microelectrodes, which may be placed on the substrate before the formation of the bonding sites 406. In some embodiments, the bonding sites 406 are micropatterned on the planar substrate 400. In some embodiments, the bonding sites 406 on the surface are periodic patterns. In some embodiments, the bonding sites on the surface are aperiodic patterns (e.g., random). In some embodiments, a minimum distance is specified between any two bonding sites. In some embodiments, the minimum distance between any two bonding sites is at least about 200 nm. In some embodiments, the minimum distance between any two bonding sites is at least about 40 nm to about 5000 nm. In some embodiments, the geometric shape of the bonding sites includes circular, square, triangular, or other 2D or 3D polygonal shapes. In some embodiments, the chemical groups used for passivation include charge-neutral molecules such as trimethylsilyl (TMS), uncharged polymers such as PEG, zwitterionic polymers, or combinations thereof. In some embodiments, the chemical groups used to define the binding site include silanol groups, carboxyl groups, thiols, other groups, or combinations thereof.

[0063] In some embodiments, a single supramolecular structure 40 is attached to each binding site 406 (Step 1). Thus, in some embodiments, multiple supramolecular structures 40 are each attached to corresponding binding sites 406 on a substrate 400. Reference numeral 416 provides a description of the components of the supramolecular structures individually and constructed and placed on a planar substrate. In some embodiments, the supramolecular structure includes the components and arrangements described in Figures 1A-B of this specification. In some embodiments, the supramolecular structure 40 includes a core structure comprising DNA origami (e.g., M13mp18 scaffold and staples), and the supramolecular structure is attached to each of the binding sites 406 using DNA origami placement techniques (Step 1). In some embodiments, the supramolecular structure 40 is assembled before being attached to each binding site 406. In some embodiments, the DNA origami includes a unique shape and dimensions to facilitate binding to the binding sites using DNA origami placement techniques. In some embodiments, DNA origami arrangement involves a directed self-assembly technique for organizing individual DNA origami (e.g., core structures) on a surface (e.g., a micropatterned surface). In some embodiments, instead of DNA origami arrangement, reactive groups of the supramolecular structure 40 are bound to DNA origami pre-organized on the binding site 406. In some embodiments, the reactive groups include immobilized molecules described herein (e.g., Figure 1). In some embodiments, any of these methods for binding the supramolecular structure 40 to the corresponding binding site 406 rely on the ability to organize one or more molecules at the micropatterned binding site using the DNA origami arrangement technique. In some embodiments, the planar substrate can be stored in a clean environment for a significant period after this step.

[0064] In some embodiments, the supramolecular structure 40 is positioned on the binding site 406 by highly efficient single-molecule bonding within the binding site 406.

[0065] Referring to reference no. 416, in some embodiments, the supramolecular structure includes one or more capture barcodes. In some embodiments, all of the capture barcodes on a given supramolecular structure are configured to form links with the same type of capture molecule, thereby configuring all of the capture barcodes on a given supramolecular structure to form links with the same type of sample molecule (via a particular type of capture molecule). In some embodiments, the supramolecular structure includes one or more capture barcodes and further includes one or more additional barcode chains. In some embodiments, the supramolecular structure includes one or more fixed barcodes. In some embodiments, the supramolecular structure on a substrate is mapped via the capture barcodes, fixed barcodes, and / or other barcodes linked to the supramolecular structure to create a catalog of the locations of each particular sample binding site on the substrate 400 (e.g., a micropatterned surface). Therefore, a map of the binding sites (or locations) of a specific capture molecule, i.e., a specific sample molecule, on the substrate 400 is created via a unique capture barcode and / or another barcode (e.g., a fixed barcode, an additional barcode) linked to the supramolecular structure 40. In some embodiments, a map of the spatial locations corresponding to the unique capture molecule binding sites 406 on the substrate 400 is created using a dye-based hybridization assay or sequencing of the barcode region. In some embodiments, the mapping of the capture molecule binding sites is performed at the manufacturing site of the substrate 400 or before performing the assay. In some embodiments, each substrate may have a unique ID that can be looked up for mapping information. Alternatively, the mapping may be performed after the capture molecules have been immobilized on the substrate 400. In some embodiments, each supramolecular structure 40 includes one or more capture sites relating to a specific capture molecule as described herein. In some embodiments, one or more supramolecular structures 40 include capture sites relating to a specific capture molecule.

[0066] In some embodiments, the capture molecule 2 (as described herein) is brought into contact with a planar substrate 400 (step 2). In some embodiments, as described herein, the capture molecule 2 comprises an aptamer including a modified aptamer, or other affinity binders. In some embodiments, the modified aptamer includes SOMAmer®. In some embodiments, the capture molecule 2 is brought into contact with the planar substrate using a flow cell. In some embodiments, the capture molecule is provided in a solution that flows over the substrate 40, i.e., over the supramolecular structure 40. In some embodiments, the capture molecule is hybridized onto the substrate (40), and in some examples, this is similar to the process when the capture molecule is brought into contact with a DNA microarray pattern. In some embodiments, the capture molecule is linked to the supramolecular structure through linkages described in Figures 1A-B herein. Different capture molecules are S1, S2...S as shown in Figure 2. n It is identified as such. In some embodiments, the capture molecule is incubated on a planar substrate 400 together with a supramolecular DNA origami structure 40 attached to the binding site 416. In some embodiments, the incubation period is about 30 seconds to about 24 hours. In some embodiments, the incubation period is about 30 seconds to about 1 minute, about 1 minute to about 5 minutes, about 5 minutes to about 30 minutes, about 30 minutes to about 1 hour, about 1 hour to about 5 hours, about 5 hours to about 12 hours, about 12 hours to about 24 hours, and about 24 hours to about 48 hours.

[0067] In some embodiments, a capture molecule is captured by a capture barcode by interacting with a corresponding capture molecule on a plurality of supramolecular DNA origami structures. In some embodiments, a capture molecule is captured by a capture barcode by forming a linkage with a corresponding capture molecule (see reference no. 418). Thus, in some embodiments, a capture molecule is immobilized on the substrate 400 via linkage with a supramolecular structure (via the corresponding capture barcode). In some embodiments, a capture molecule is captured by a capture barcode via hybridization. In some embodiments, a capture molecule is captured by a capture barcode via a third capture linker, as shown in Figures 1A-B of this specification. In some embodiments, each capture barcode is configured to interact with a specific capture molecule (e.g., an aptamer, affinity binder, etc.).

[0068] In some embodiments, interference scattering microscopy (iSCAT), a label-free mass spectrometry method, is used to visualize the interaction (e.g., binding process) between a captured barcode and the corresponding captured molecule in a label-free form.

[0069] Referring to Figure 2, in some embodiments, a sample (as described herein) containing sample molecules 44 is brought into contact with a planar substrate 400 (step 3). In some embodiments, the sample is brought into contact with the planar substrate 400 using a flow cell. In some embodiments, the sample is flowed onto the substrate 400 containing the captured captured molecules 2. In some embodiments, the sample molecules 44 contain proteins. In some embodiments, the proteins contain one or more types of proteins. As shown in Figure 2, different sample molecules are P1, P2...P nIt is identified as such. In some embodiments, the sample is incubated on a planar substrate 400 together with the supramolecular structure 40 (attached to the corresponding binding site 416) and the corresponding capture molecule 2. In some embodiments, the incubation period is about 30 seconds to about 24 hours. In some embodiments, the incubation period is about 30 seconds to about 1 minute, about 1 minute to about 5 minutes, about 5 minutes to about 30 minutes, about 30 minutes to about 1 hour, about 1 hour to about 5 hours, about 5 hours to about 12 hours, about 12 hours to about 24 hours, and about 24 hours to about 48 hours.

[0070] In some embodiments, a sample molecule 44 in the sample interacts with a corresponding capture molecule 2 located on a supramolecular DNA origami structure 40 on a planar surface 400. As described herein, in some embodiments, the sample molecule 44 includes a protein. In some embodiments, a single copy of a particular sample molecule 44 binds to a corresponding capture molecule 2 captured by a capture barcode 20 (see reference no. 420). As described herein, each capture molecule 2 is configured to bind to a particular sample molecule 44. In some embodiments, the unique shape and chemical properties of a given capture molecule 2 (e.g., a modified aptamer) recognize and bind to a corresponding sample molecule 44 (e.g., a protein), forming a capture molecule-sample molecule complex at a given binding site 416 on the substrate 400 (S n -P n (See reference number 420 related to the complex). Thus, in some embodiments, the sample molecule is immobilized on the substrate 400 via interaction with the capture molecule. In some embodiments, the capture molecule interacts with and binds to a specific sample molecule. In some embodiments, the capture molecule interacts with and binds to only a specific sample molecule. In some embodiments, the capture molecule interacts directly with a specific sample molecule.

[0071] In some embodiments, the supramolecular structure 40 is linked to a sample molecule 44 (via a corresponding capture molecule 2) as described herein, and then the supramolecular structure is brought into contact with one or more other identification molecules to identify the supramolecular structure linked to the sample molecule in the sample, thereby identifying the sample molecule found in the sample. In some embodiments, the sample molecule is identified via a mapped location of the supramolecular structure as described herein. In some embodiments, the sample molecule is further quantified in the sample based on the amount of the sample molecule identified across the binding site 416 of the substrate 400.

[0072] In some embodiments, one or more identified molecules include biotin molecules 46. In some embodiments, the substrate 400 is brought into contact with the biotin molecules, thereby subjecting one or more sample molecules 44 to biotination (step 4). See reference no. 422. In some embodiments, subjecting to biotination corresponds to the sample molecule 44 interacting with the biotin molecule 46. In some embodiments, the sample molecule forms a linkage with the biotin molecule. In some embodiments, a solution containing one or more biotin molecules is flowed over the substrate 400. In some embodiments, the sample molecule 44 is subject to amine biotination, sulfhydryl biotination, carboxyl biotination, glycoprotein biotination, oligonucleotide biotination, nonspecific biotination, or a combination thereof. In some embodiments, one or more biotin molecules include NHS-biotin molecules or any other type of biotin molecule. In some embodiments, one or more biotin molecules include amine-reactive NHS-biotin molecules. In some embodiments, one or more amine-reactive biotin molecules label an amine by forming a permanent amide bond.

[0073] In some embodiments, after the sample molecule 44 is subjected to biotinylation (e.g., step 4), the sample molecule 44 is subsequently fluorescently labeled (step 5). In some embodiments, the substrate 400 is brought into contact with one or more fluorescently labeled molecules 48. In some embodiments, a solution containing one or more fluorescently labeled molecules 48 is flowed over the substrate 400. In some embodiments, one or more fluorescently labeled molecules include fluorescently labeled streptavidin molecules, fluorescently labeled avidin molecules, or other types of chemistry known for labeling sample molecules (e.g., proteins) with biotin. In some embodiments, the fluorescently labeled molecules interact with biotin molecules that have interacted with the sample molecule (see reference no. 424).

[0074] In some embodiments, fluorescent labeling of a sample molecule bound to a biotin molecule provides a fluorescent signal. In some embodiments, the fluorescent signal generated by the fluorescently labeled molecule is read out using a fluorescence microscope or any other device known in the art to detect the fluorescent signal (step 6 shown in Figure 2). In some embodiments, the fluorescent signal detected from a specific binding site 406 on the substrate 400 identifies the capture of a specific sample molecule (e.g., a protein) based on the mapping location of the supramolecular structure 40 and the corresponding capture molecule (as described herein). In some embodiments, the captured sample molecule is quantified based on the cumulative count of the fluorescent signals detected at the corresponding binding site 406 on the substrate 400. For example, if locations X1Y1, X3Y3, and X20Y20 on substrate 400 correspond to a captured molecule S1, such that they are mapped at those locations through a unique capture barcode on the molecule of the supramolecular structure 40 (e.g., a supramolecular DNA origami structure), then the fluorescent signals from these three locations after the streptavidin labeling step result in a count of 3 with respect to the sample molecule P1 (e.g., protein P1).

[0075] In addition to, or instead of, the fluorescent labeling step described above, in some embodiments, after the captured sample molecules are subjected to biotinylation (i.e., after step 4), the substrate 400 is brought into contact with one or more light-scattering molecules or nanoparticles to enable label-free imaging of the sample molecules 44. In some embodiments, a solution containing one or more light-scattering molecules or nanoparticles is flowed over the substrate 400. In some embodiments, the one or more light-scattering molecules or nanoparticles include streptavidin molecules, avidin molecules, or other types of chemistry known to interact with biotin molecules. In some embodiments, the one or more light-scattering molecules or nanoparticles include streptavidin-coated nanoparticles, streptavidin clusters, avidin-coated nanoparticles, other molecules and nanoparticles that interact with biotin molecules, or combinations thereof. In some embodiments, the light-scattering molecules or nanoparticles are labeled with Qdots and / or metal nanoparticles to enable label-free imaging of the sample molecules. In some embodiments, interference scattering microscopy (iSCAT) or other types of apparatus known in the art is used to visualize the complex formed via binding between light-scattering molecules or nanoparticles and biotin molecules (e.g., biotin-streptavidin complexes) at the location of the corresponding sample molecule 44 (i.e., the sample molecule immobilized via interaction with the corresponding capture molecule linked to the supramolecular structure) immobilized on the substrate 400, and to generate a visual signal. In some embodiments, the visual signal includes an optical signal, an electrical signal, or both. In some embodiments, the optical signal includes a microwave signal, ultraviolet light, visible light, near-infrared light, light scattering, or a combination thereof. In some embodiments, the visual detection of such a complex from a specific location on the substrate identifies the capture of a specific sample molecule (e.g., a protein) based on the supramolecular structure and the mapped location of the corresponding capture molecule (as described herein), thereby identifying the sample molecule 44 (step 6).In some embodiments, the captured sample molecule is quantified based on the cumulative count of biotin complexes visually detected at the corresponding binding sites 406 on the substrate 400 (step 6). For example, if sites X1Y1, X3Y3, and X20Y20 on the substrate 400 correspond to captured molecule S1 mapped at those sites through a unique capture barcode on the molecule of the supramolecular structure 40 (e.g., supramolecular DNA origami structure), then the visual detection of biotin complexes from these three sites results in a count of 3 with respect to sample molecule P1 (e.g., protein P1).

[0076] In some embodiments, instead of contacting the substrate 400 with biotin molecules, after step 3, the substrate 400 is contacted with a solution containing a second set of capture molecules. In some embodiments, the second set of capture molecules includes fluorescently labeled, unlabeled, or a mixture of both. In some embodiments, the second set of capture molecules is configured to interact with a specific sample molecule (as described herein with respect to the capture molecules). In some embodiments, the second set of capture molecules interacts with a corresponding sample molecule immobilized on the substrate 400, thereby enabling the formation of another sample molecule-capture molecule complex (i.e., thereby forming a “sandwich” configuration with the sample molecule located between the two capture molecules). Thus, in some embodiments, a patterned surface (substrate 400) of a single molecule having a corresponding capture barcode can be used as a sandwich assay with two capture molecules (e.g., modified aptamers) chemically synthesized to recognize the same sample molecule. In some embodiments, the second set of capture molecules is incubated with the substrate 400 (as described herein). In some embodiments, fluorescently labeled capture molecules from a second set of capture molecules fluorescently label the corresponding sample molecules that interact with the capture molecules, generating a fluorescence signal. In some embodiments, fluorescence readout (step 6) is performed to identify and quantify the sample molecules detected on the substrate, as described herein. In some embodiments, unlabeled capture molecules from a second set of capture molecules that interact with the corresponding sample molecules on the substrate 400 generate a visual signal, and the substrate 400 is optically interrogated using iSCAT or a similar apparatus known in the art to identify and quantify the sample molecules detected on the substrate based on the visual signal, as described herein (step 6). In some embodiments, as described herein, the visual signal includes an optical signal, an electrical signal, or both. In some embodiments, the optical signal includes a microwave signal, ultraviolet light, visible light, near-infrared light, light scattering, or a combination thereof.

[0077] In some embodiments, in an alternative step of contacting the substrate with biotin molecules, after step 3, the substrate 400 is contacted with a solution containing one or more NHS-dye molecules or other dye molecules known in the art (such as NHS-labeled quantum dots). In some embodiments, the NHS-dye molecules (or other types of dye molecules) are configured to interact with sample molecules 44, thereby generating a corresponding fluorescence signal, which enables fluorescence readout of the sample molecules immobilized on the substrate 400 (step 6) (as described herein). In some embodiments, the interaction between the NHS-dye molecules (or other types of dye molecules) and the sample 44 is a specific interaction. In some embodiments, fluorescence readout is performed to identify and quantify the sample molecules detected on the substrate 400, as described herein.

[0078] In some embodiments, the introduction of signaling elements, e.g., fluorescence and / or vision as described herein, results in a surface of the substrate 400 where the capture events of individual sample molecules (i.e., corresponding capture barcodes, capture molecules, linkage between the sample molecules, and subsequent biotinylation or other signal-generating events as described herein) result in a signaling element present at the location of each sample molecule 44 (on the substrate 400). As described herein, in some embodiments, the signaling elements are optically active and can be measured using a microscope or an integrated photosensor in the planar substrate 400. In some embodiments, the signaling elements are electrically active and may be measured using an integrated electrosensor. In some embodiments, the signaling elements are magnetically active and may be measured using an integrated magnetic sensor. In some embodiments, each signaling element comprises a fluorescent molecule or microorganism, a fluorescent polymer, a highly charged nanoparticle, or a polymer. In some embodiments, each signal event (at the corresponding binding site 406) is related to the capture of a sample molecule of the same type (a single copy of a sample molecule of the same type) and is determined by the corresponding capture molecule. Therefore, in some embodiments, counting the number of such binding sites 406 where a signaling element exists, based on the mapped locations 406 of a given capture barcode 20 on the substrate 400, gives a quantitative determination of the sample molecule in the sample corresponding to the given capture barcode.

[0079] In some embodiments, during any of the steps described herein (for example, steps 1-6 shown in Figure 2), the substrate 400 is cleaned to remove any unbound and / or non-adhered contents from the solution that has come into contact with the substrate 400.

[0080] In some embodiments, the high-density arrangement of DNA origami molecules (supramolecular DNA origami structures) on an array (i.e., multiple binding sites 406 on a substrate 400) allows for large-scale parallel assays for the quantification of sample molecules 44 (e.g., proteins), with complexity limited only by the number of unique capture molecules 2 bound to the supramolecular structures (e.g., origami molecules).

[0081] In some embodiments, the method for detecting a sample as described in Figure 2 enables the detection of a single type of sample molecule. In some embodiments, the method for detecting a sample as described in Figure 2 enables the detection of multiple types of sample molecules (multiplexed sample molecule detection). In some embodiments, each supramolecular structure (e.g., supramolecular DNA origami structure) is barcoded to uniquely identify the respective associated capture molecule, thereby enabling the identification of each captured sample molecule. In some embodiments, each supramolecular DNA origami structure is barcoded using its respective capture barcode and / or immobilized molecule.

[0082] In some embodiments, a patterned surface of a single molecule having a supramolecular structure (e.g., a supramolecular DNA origami structure) which may be a DNA origami nanostructure, can be used as a large-scale, multiplexed, high-throughput ligand phylogenetic evolution by exponential enrichment ("SELEX") platform to discover novel capture molecules (e.g., aptamers) that recognize sample molecules (e.g., proteins) already immobilized on the surface using a capture molecule-detection molecule complex. In some embodiments, the capture molecule-detection molecule complex corresponds to the use of a supramolecular structure comprising a capture molecule and a detection molecule, as described in U.S. Provisional Patent Application No. 63 / 078,837 ("Application 837"), filed September 15, 2020, which is incorporated in whole herein. In some embodiments, the capture molecule described in Application 837 refers to a specific capture molecule (e.g., an aptamer) described herein that is configured to interact with a particular sample molecule. In some embodiments, the detection molecule described in Application 837 refers to a specific capture molecule (e.g., an aptamer) described herein that is configured to interact with a particular sample molecule. In some embodiments, the capture molecules and detection molecules described in application 837 refer to the same type of specific capture molecules (e.g., aptamers) described herein that are configured to interact with specific sample molecules. In some embodiments, this capture molecule-detection molecule complex may need to be irreversibly bound, and the sample-capture molecule complex may also need to be irreversibly bound. In some embodiments, the capture barcodes are configured to be separated, and one or more separated capture barcodes are analyzed using genotyping, qPCR, sequencing, or a combination thereof. In some embodiments, multiple sample molecules in a sample are detected simultaneously through multiplexing via one or more supramolecular DNA origami structures that have transitioned to an excited state. In some embodiments, the SELEX platform may require cycling (washing and simultaneous flow-through) of tens to thousands of affinity binders.

[0083] Exemplary Embodiment of a Method for Detecting Sample Molecules In some embodiments, the Specified provides a method for detecting sample molecules present in a sample, comprising providing a supramolecular DNA origami structure arranged in an array at a predetermined location on a surface, comprising: i) a core structure comprising one or more molecules; ii) a capture molecule linked to the core structure at a first location comprising a barcode for the purpose of mapping the binding of a specific sample recognition molecule; iii) a fixed molecule linked to the core structure at a second location which may include a barcode for the purpose of mapping the binding of a specific sample recognition molecule and / or a barcode for covalently or noncovalently linking a DNA origami structure to the surface; and iv) detecting the sample molecule based on a signal provided by the supramolecular DNA origami structure through a fluorophore-labeled or non-labeled optical detection technique.

[0084] In some embodiments, the foregoing describes a method for detecting one or more sample molecules present in a sample, comprising: a) a plurality of supramolecular DNA origami structures, each comprising: i) a core structure comprising one or more molecules for binding unique recognition elements; ii) a capture molecule linked to the core structure at a predetermined location; and iii) detecting the sample molecule based on a signal provided by the supramolecular DNA origami structure through fluorophore-labeled or unlabeled optical detection techniques; b) contacting the recognition elements, i.e., SOMAmer or other affinity-binding entities, with the supramolecular DNA origami structure for capture at one or more locations on the structure through nucleic acid hybridization or other chemical linkage; and c) a predetermined location on the surface. A method is provided that includes: d) mapping the location of each unique recognition element through a fluorescence-based hybridization assay using multiple supramolecular DMA origami structures positioned at specific locations, or sequencing the sample; e) contacting the sample with recognition elements bound to supramolecular DMA origami structures at predetermined locations on the surface; e) generating a readable signal in optical form through various steps: i) biotinylating the captured sample from the sample immobilized by the recognition elements at pre-mapped locations on the surface, and ii) labeling the biotinylated locations with a fluorescent streptavidin moiety; f) quantifying the sample concentration by registering the mapping locations together with signals from specific sample-binding locations on the surface.

[0085] In some embodiments, the Specified provides a method for detecting sample molecules present in a sample, comprising: providing a supramolecular structure arranged in an array at predetermined locations on a surface, comprising: i) a core structure comprising one or more molecules; ii) capture molecules linked to the core structure at a first location, wherein the linkage between the capture molecules and the core structure comprises a capture barcode configured to map the interaction of the capture molecules onto the supramolecular structure; and iii) fixed molecules linked to the core structure at a second location, which may include a barcode intended to map the interaction between a specific capture molecule and the capture barcode, and / or a barcode for covalently or noncovalently linking a DNA origami structure to the surface; iv) contacting the sample with the supramolecular structure so that the capture molecules interact with the sample molecules; v) generating a signal based on the interaction between the capture molecules and the sample molecules; and vi) detecting the sample molecules based on the signal provided by the supramolecular DNA origami structure through fluorophore-labeled or non-labeled optical detection techniques. In some embodiments, the supramolecular structure comprises a supramolecular DNA origami structure. In some embodiments, the capture molecule comprises an aptamer containing a modified aptamer. In some embodiments, the sample molecule comprises a protein.

[0086] In some embodiments, the Specified Methods for detecting one or more sample molecules present in a sample are provided, comprising: a) providing a plurality of supramolecular structures, each comprising i) a core structure comprising one or more molecules, and ii) a capture molecule linked to the core structure at a predetermined location and configured to form a linkage with a specific capture molecule; b) contacting the supramolecular structure with one or more capture molecules (e.g., aptamers, modified aptamers including SOMAmers) or other affinity-binding entities at one or more locations on the supramolecular structure through nucleic acid hybridization or other chemical linkage; c) mapping the location of each unique capture molecule through a fluorescence-based hybridization assay or sequencing the sample by a plurality of supramolecular DMA origami structures positioned at predetermined locations on the surface; e) contacting the sample with the capture molecule linked to the supramolecular structure at the predetermined locations on the surface; f) generating a signal through fluorophore-labeled or unlabeled optical detection techniques; and e) identifying and quantifying the concentration of the sample molecule by registering the mapping locations together with signals from specific sample-binding locations on the surface. In some embodiments, the detection of the sample molecule involves detecting the signal in optical form through various steps: i. biotinylating the captured sample from the sample immobilized by a recognition element at a pre-mapped location on the surface; and ii. labeling the biotinylated location with a fluorescent, streptavidin moiety. In some embodiments, the supramolecular structure includes a supramolecular DNA origami structure. In some embodiments, the capture molecule includes an aptamer containing a modified aptamer. In some embodiments, the sample molecule includes a protein.

[0087] In some embodiments, any method disclosed herein further includes quantifying the concentration of a sample molecule in a sample. In some embodiments, any method disclosed herein further includes identifying the detected sample molecule. In some embodiments, any method disclosed herein further includes detecting the sample molecule based on a signal when the sample molecule is present in the sample as a single molecule or in a larger number of molecules. In some embodiments, in any method disclosed herein, the sample includes a complex biological sample, and the method provides single-molecule sensitivity, thereby increasing the dynamic range and enabling quantitative capture of various molecular concentrations in the complex biological sample. In some embodiments, in any method disclosed herein, the sample molecule includes a protein, peptide, peptide fragment, a complex thereof, or any combination thereof. In some embodiments, in any method disclosed herein, each supramolecular DNA origami structure is a 2D or 3D nanostructure.

[0088] In some embodiments, in any method disclosed herein, each core structure is a nanostructure. In some embodiments, in any method disclosed herein, a plurality of core molecules for each nanostructure are arranged in a predefined shape and / or have a predetermined molecular weight. In some embodiments, the predefined shape is configured to limit or prevent cross-reaction with another supramolecular DNA origami structure. In some embodiments, in any method disclosed herein, the plurality of molecules for each core structure include one or more nucleic acid strands, one or more branched nucleic acids, one or more peptides, one or more small molecules, or a combination thereof. In some embodiments, in any method disclosed herein, each core structure independently includes scaffold deoxyribonucleic acid (DNA) origami, scaffold ribonucleic acid (RNA) origami, scaffold hybrid DNA:RNA origami, single-stranded DNA tile structure, multi-stranded DNA tile structure, single-stranded RNA origami, multi-stranded RNA tile structure, DNA or RNA origami in a hierarchical configuration with multiple scaffolds, peptide structures, or a combination thereof.

[0089] In some embodiments, the trigger / readout signal includes an optical signal, an electrical signal, or both. In some embodiments, the trigger optical signal includes a microwave signal, ultraviolet light, visible light, near-infrared light, light scattering, or a combination thereof.

[0090] In some embodiments, in any method disclosed herein, a) a reactive sample molecule is 1) bound to a capture molecule of each supramolecular DNA origami structure through a chemical bond. In some embodiments, in any method disclosed herein, the capture molecule for each supramolecular DNA origami structure includes proteins, peptides, antibodies, aptamers (RNA and / or DNA), fluorophores, DARPin, catalysts, polymerization initiators, polymers such as PEG, or combinations thereof. In some embodiments, the aptamer includes a modified aptamer. In some embodiments, in any method disclosed herein, with respect to each supramolecular DNA origami structure, a) a capture molecule is linked to a core structure through a capture barcode, the capture barcode includes a first capture linker, a second capture linker, and a capture bridge positioned between the first and second capture linkers, the first capture linker being linked to a first core linker bonded to a first location on the core structure, and the capture molecule and the second capture linker being linked together through a bond with a third capture linker. In some embodiments, the polymer core of the capture bridge independently comprises a nucleic acid (DNA or RNA) of a specific sequence or a polymer such as PEG. In some embodiments, the first core linker, second core linker, first capture linker, second capture linker, and third capture linker independently comprise a reactive molecule or a DNA sequence domain. In some embodiments, each reactive molecule independently comprises one or more polymers such as amines, thiols, DBCOs, maleimides, biotin, azides, acrylics, NHS esters, single-stranded nucleic acids (DNA or RNA) of a specific sequence, PEG, or polymerization initiators, or a combination thereof. In some embodiments, the linkage between the capture barcode and 1) the first core linker and / or 2) the third capture linker comprises a chemical bond. In some embodiments, the chemical bond comprises a covalent bond. In some embodiments, in any method disclosed herein, the capture molecule is bonded to the third capture linker through a chemical bond. In some embodiments, the capture molecule is covalently bonded to the third capture linker.

[0091] In some embodiments, in any method disclosed herein, each supramolecular DNA origami structure further includes a fixed molecule linked to a core structure. In some embodiments, the fixed molecule is linked to the core structure via a fixed barcode, which includes a first fixed linker, a second fixed linker, and a fixed bridge positioned between the first and second fixed linkers, the first fixed linker being linked to a third core linker bonded to a second location on the core structure, and the fixed molecule being linked to the second fixed linker. In some embodiments, the fixed molecule includes amines, thiols, DBCO, maleimide, biotin, azide, acrylic, NHS esters, single-stranded nucleic acids (DNA or RNA) of a specific sequence, one or more polymers such as PEG or polymerization initiators, or a combination thereof. In some embodiments, the fixed bridge includes a polymer core. In some embodiments, the polymer core of the fixed bridge includes a nucleic acid (DNA or RNA) of a specific sequence or a polymer such as PEG. In some embodiments, the second core linker, the first fixed linker, the second fixed linker, and the fixed molecule independently comprise a fixed reactive molecule or a DNA sequence domain. In some embodiments, each fixed reactive molecule independently comprises an amine, thiol, DBCO, maleimide, biotin, azide, acrylic, NHS ester, a single-stranded nucleic acid (DNA or RNA) of a specific sequence, one or more polymers such as PEG or polymerization initiators, or a combination thereof. In some embodiments, the fixed molecule is linked to the second fixed linker through a chemical bond. In some embodiments, the fixed molecule is covalently bonded to the second fixed linker.

[0092] In some embodiments, any method disclosed herein includes a capture barcode corresponding to an excited supramolecular DNA origami structure. In some embodiments, any method disclosed herein further includes separating each capture barcode from the corresponding capture molecule with respect to at least one excited supramolecular DNA origami structure, so that the corresponding signal includes each capture barcode, which may be a nucleic acid-based sequence for the detection of the sample molecule bound to each capture molecule. In some embodiments, at least one separated capture barcode is analyzed using genotyping, qPCR, sequencing, or a combination thereof. In some embodiments, multiple sample molecules in a sample are detected simultaneously by multiplexing across one or more excited supramolecular DNA origami structures. In some embodiments, any method disclosed herein includes a capture molecule with respect to each supramolecular DNA origami structure, configured to bind to one or more specific types of sample molecules.

[0093] In some embodiments, in any method involving the use of multiple DNA origami structures disclosed herein, the core structures of the multiple supramolecular DNA origami structures are identical to one another. In some embodiments, each supramolecular DNA origami structure includes a predetermined shape, size, molecular weight, or combination thereof to reduce or limit cross-reactivity between the multiple supramolecular DNA origami structures. In some embodiments, each supramolecular DNA origami structure includes multiple capture molecules. In some embodiments, each supramolecular DNA origami structure includes capture molecules of a predetermined stoichiometry to reduce or limit cross-reactivity between the multiple supramolecular DNA origami structures.

[0094] In some embodiments, multiple supramolecular DNA origami structures are attached to one or more solid supports, one or more solid substrates, or a combination thereof. In some embodiments, each of the one or more solid substrates includes a planar substrate. In some embodiments, multiple supramolecular DNA origami structures are arranged on a planar substrate, the planar substrate includes a plurality of binding sites, each binding site is configured to link to a corresponding supramolecular DNA origami structure. In some embodiments, multiple supramolecular DNA origami structures are configured to detect the same sample molecule. In some embodiments, any method including the use of a planar substrate further includes providing a plurality of signaling elements configured to link to a captured sample molecule of at least one supramolecular DNA origami structure that has transitioned to an excited state (as described herein). In some embodiments, each signaling element includes a fluorescent molecule or microorganism, a fluorescent polymer, a highly charged nanoparticle, or a polymer. In some embodiments, at least one of the multiple supramolecular DNA origami structures is configured to detect a different sample molecule than the other supramolecular DNA origami structures. In some embodiments, any method including the use of a planar substrate further includes assigning a barcode to each supramolecular DNA origami structure in order to identify the location of each supramolecular DNA origami structure on the planar substrate. In some embodiments, any method including the use of a planar substrate includes providing a plurality of signaling elements configured to link to a captured sample molecule of at least one supramolecular DNA origami structure that has transitioned to an excited state. In some embodiments, each signaling element includes a fluorescent molecule or microbead, a fluorescent polymer, a highly charged nanoparticle or polymer.

[0095] In some embodiments, in any method disclosed herein, the sample comprises biological particles or biomolecules. In some embodiments, in any method disclosed herein, the sample comprises aqueous solutions comprising proteins, peptides, peptide fragments, lipids, DNA, RNA, organic molecules, viral particles, exosomes, organelles, or any complex thereof. In some embodiments, in any method disclosed herein, the sample comprises tissue biopsies, blood, plasma, urine, saliva, tears, cerebrospinal fluid, extracellular fluid, cultured cells, culture media, waste tissue, plant matter, synthetic proteins, bacterial and / or viral samples or fungal tissues, or combinations thereof.

[0096] In some embodiments, the Specified Provisions provide a substrate for detecting one or more sample molecules in a sample, comprising a plurality of supramolecular DNA origami structures, wherein each supramolecular DNA origami structure comprises a) a core structure comprising a plurality of core molecules, and b) a capture molecule linked to the supramolecular core at a first location, and the recognition of the sample molecule causes the interaction to induce each supramolecular DNA origami structure to transition to an excited state, thereby providing a signal for detecting each sample molecule.

[0097] In some embodiments, the Specified Provisions provide a substrate for detecting one or more sample molecules in a sample, comprising a plurality of supramolecular structures, wherein each supramolecular structure comprises a) a core structure comprising a plurality of core molecules, and b) a capture molecule linked to the supramolecular core at a first location, and the capture molecule is configured to interact with a specific sample molecule, the interaction inducing each supramolecular structure to transition to an excited state, thereby enabling the generation of a signal for detecting each sample molecule. In some embodiments, the supramolecular structures include supramolecular DNA origami structures.

[0098] In some embodiments, each sample molecule is 1) bound to a capture molecule through a chemical bond. In some embodiments, the capture molecule for each supramolecular DNA origami structure independently includes proteins, peptides, antibodies, aptamers (RNA and DNA), fluorophores, DARPin, catalysts, polymerization initiators, polymers such as PEG, or combinations thereof.

[0099] In some embodiments, the interaction between each sample molecule and the capture molecule includes each sample molecule that forms a link with the capture molecule. In some embodiments, the link includes a chemical bond. In some embodiments, the capture molecule for each supramolecular DNA origami structure independently includes a protein, peptide, antibody, aptamer (RNA and / or DNA), fluorophore, DARPin, catalyst, polymerization initiator, polymer such as PEG, or a combination thereof. In some embodiments, the aptamer includes a modified aptamer.

[0100] In some embodiments, the sample comprises biological particles or biomolecules. In some embodiments, the sample comprises an aqueous solution comprising proteins, peptides, peptide fragments, lipids, DNA, RNA, organic molecules, viral particles, exosomes, organelles, or any complex thereof. In some embodiments, the sample comprises tissue biopsies, blood, plasma, urine, saliva, tears, cerebrospinal fluid, extracellular fluid, cultured cells, culture media, waste tissue, plant matter, synthetic proteins, bacterial and / or viral samples or fungal tissues, or combinations thereof.

[0101] In some embodiments, the sample comprises a complex biological sample, and the method provides single-molecule sensitivity, thereby increasing the dynamic range and enabling quantitative capture of various molecular concentrations in the complex biological sample. In some embodiments, the sample molecules include proteins, peptides, peptide fragments, lipids, DNA, RNA, organic molecules, inorganic molecules, complexes thereof, or any combination thereof. In some embodiments, the supramolecular DNA origami structure is a nanostructure. In some embodiments, the core structure is a nanostructure. In some embodiments, multiple core molecules for the core structure are arranged in a predefined shape and / or have a predetermined molecular weight. In some embodiments, the predefined shape is configured to limit or prevent cross-reaction with another supramolecular DNA origami structure. In some embodiments, the multiple core molecules for each core structure include one or more nucleic acid chains, one or more branched nucleic acids, one or more peptides, one or more small molecules, or combinations thereof. In some embodiments, the core structure independently includes scaffold deoxyribonucleic acid (DNA) origami, scaffold ribonucleic acid (RNA) origami, scaffold hybrid DNA:RNA origami, single-stranded DNA tile structure, multi-stranded DNA tile structure, single-stranded RNA origami, multi-stranded RNA tile structure, hierarchical DNA or RNA origami having multiple scaffolds, peptide structures, or combinations thereof.

[0102] Preferred embodiments of the present invention are illustrated and described herein, but it will be obvious to those skilled in the art that such embodiments are provided only as examples. Numerous modifications, alterations, and substitutions will be conceivable to those skilled in the art without departing from the present invention. It should be understood that various alternative methods to the embodiments of the present invention described herein may be used in practicing the present invention. The following claims are intended to define the scope of the present invention, the methods and structures within the scope of these claims, and their equivalents.

Claims

1. A system for detecting sample molecules, Multiple DNA origami structures, each comprising at least one capture molecule configured to bind to a sample molecule, and a barcode that can be used to map the location of each DNA origami structure on a substrate; Each of these substrates includes multiple binding sites, each configured to receive one of the aforementioned DNA origami structures; A signal generation system configured to generate signals for each sample molecule bound to a corresponding capture molecule; A detection system configured to detect the generated signal, Includes, A system configured such that the locations of the DNA origami structures received at the multiple binding sites are mapped using the barcode, thereby enabling the creation of a map of unique capture molecular locations on the substrate.

2. The system according to claim 1, wherein each barcode is configured to be linked to one of the capture molecules.

3. The system according to claim 1 or 2, wherein the capture molecule is configured to interact with different types of sample molecules.

4. The system according to claim 1 or 2, wherein the capture molecules are configured to interact with sample molecules of the same type.

5. The system according to any one of claims 1 to 4, wherein the capture molecule attaches to the DNA origami structure through DNA hybridization.

6. The system according to any one of claims 1 to 5, wherein the DNA origami structure is a scaffold DNA origami structure.

7. A system for detecting sample molecules, Each of the multiple DNA origami structures contains at least one capture molecule configured to bind to a sample molecule; Each of these substrates includes multiple binding sites, each configured to receive one of the aforementioned DNA origami structures; A signal generation system configured to generate signals for each sample molecule bound to a corresponding capture molecule; A detection system configured to detect the generated signal, Includes, A system in which barcodes are linked to each DNA origami structure, and the DNA origami structures on the substrate are mapped via the barcodes, thereby enabling the creation of a map of uniquely captured molecular locations on the substrate.

8. The system according to claim 7, wherein the capture molecule is configured to interact with different types of sample molecules.

9. The system according to claim 7, wherein each of the capture molecules is configured to interact with sample molecules of the same type.

10. The system according to any one of claims 7 to 9, wherein the capture molecule attaches to the DNA origami structure through DNA hybridization.

11. The system according to any one of claims 7 to 10, wherein the DNA origami structure is a scaffold DNA origami structure.

12. A method for detecting sample molecules in a sample, To provide a plurality of DNA origami structures bound to a substrate at binding sites, each DNA origami structure comprising at least one capture molecule configured to bind to a sample molecule, wherein the DNA origami structures are assigned barcodes to map the location of each DNA origami structure on the substrate; Mapping the DNA origami structure on the substrate, thereby creating a map of the locations of trapped molecules on the substrate; The DNA origami structure is brought into contact with the sample so that the sample molecules bind to each capture molecule; To generate signals for each sample molecule bound to the corresponding capture molecule; The signal is detected, thereby allowing the detection of sample molecules present in the sample. Methods that include...

13. The method according to claim 12, wherein mapping the DNA origami structure on the substrate includes mapping the location of the barcode.

14. The method according to claim 12 or claim 13, wherein each barcode is configured to be linked to one of the capture molecules.

15. The method according to any one of claims 12 to 14, wherein mapping the DNA origami structure on the substrate is performed before bringing the substrate into contact with the sample to be detected.

16. The method according to claim 15, wherein mapping the DNA origami structure on the substrate is performed when the substrate is manufactured.

17. The method according to any one of claims 12 to 16, further comprising immobilizing the capture molecule on the substrate, wherein mapping the DNA origami structure on the substrate is performed after the capture molecule has been immobilized on the substrate.

18. The system according to any one of claims 12 to 17, wherein the capture molecule is configured to interact with different types of sample molecules.

19. The method according to any one of claims 12 to 17, wherein the capture molecules are configured to interact with sample molecules of the same type.

20. The method according to any one of claims 12 to 19, further comprising quantifying the concentration of the sample molecule present in the sample by registering the mapped capture molecule locations together with the detected signal.

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