Structures and methods for detecting a sample specimen

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 facilitate the binding of a single detector molecule. In some embodiments, the steady state supramolecular structures are configured to provide a signal for analyte molecule detection and quantification. In some embodiments, the signal is correlated with a DNA signal such that detection and quantification of the analyte molecule comprises converting the presence of the analyte molecule into a DNA signal.

Description

Technical Field

[0001] The present invention relates to a structure and method for detecting a sample analyte. 〔Cross - Reference to Related Applications〕 This application claims priority and the benefit thereof to U.S. Provisional Patent Application No. 63 / 152,607, entitled "STRUCTURE AND METHODS FOR DETECTION OF SAMPLE ANALYTES", filed on February 23, 2021, the disclosure of which is hereby incorporated by reference in its entirety.

Background Art

[0002] The current state of personalized healthcare is predominantly genomics - centered, focusing mainly on quantifying genes that exist within an individual. While such techniques have been found to be very powerful, they do not provide a complete picture of an individual's health to a clinician. This is because genes are an individual's "blueprint", which only conveys the potential to develop diseases. For these "blueprints" to have any effect on an individual's health, they first need to be transcribed into RNA and then translated into various protein molecules, which are the actual "actors" within the cell.

[0003] The concentration of proteins, the interactions between proteins (protein-protein interactions or PPIs), and the interactions between proteins and other molecules are intricately linked to the health of various organs, homeostatic mechanisms, and the interaction of these systems with the external environment. Therefore, quantitative information regarding protein interactions such as proteins and PPIs is highly important for generating an overall 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 inferred by measuring the concentrations of troponin I / II and myosin light chain present in peripheral blood. Similar protein biomarkers have also been identified, validated, and rolled out for a wide range of organ dysfunctions (e.g., liver diseases and thyroid abnormalities), certain cancers (e.g., colorectal cancer or prostate cancer), and infectious diseases (e.g., HIV and Zika virus). The interactions between these proteins are also indispensable for drug development and have become highly sought-after datasets. The ability to detect and quantify the protein interactions between proteins and other molecules within a given body fluid sample is an essential component of such healthcare development.

Summary of the Invention

Problems to be Solved by the Invention

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

Means for Solving the Problems

[0005] Provided herein, in some embodiments, is a method for detecting an analyte molecule present in a sample, the method comprising: a) providing a supramolecular structure comprising a core structure having a plurality of core molecules and a capture molecule linked to the core structure at a first location; b) contacting the sample with the supramolecular structure; c) providing a detector molecule assembly; and d) detecting the analyte molecule based on a signal provided by the detector molecule assembly and a related signal provided by the supramolecular structure.

[0006] In some embodiments, any method disclosed herein further comprises quantifying the concentration of analyte molecules in a sample. In some embodiments, any method disclosed herein further comprises identifying the detected analyte molecules. In some embodiments, any method disclosed herein further comprises detecting the analyte molecules based on a signal when the analyte molecules are present in the sample at a single molecule count value or more. In some embodiments, for any method disclosed herein, the sample comprises a complex biological sample, and the method provides single molecule sensitivity, thereby increasing the dynamic range and quantitative capture of various molecular concentrations within the complex biological sample. In some embodiments, for any method disclosed herein, the analyte molecules comprise proteins, peptides, peptide fragments, lipids, DNA, RNA, organic molecules, inorganic molecules, complexes thereof, or any combination thereof. In some embodiments, for any method disclosed herein, each supramolecular structure is a nanostructure.

[0007] In some embodiments, for any of the methods disclosed herein, each core structure is a nanostructure. In some embodiments, for any of the methods disclosed herein, the plurality of core molecules for each core structure are arranged in a predetermined shape and / or have a defined molecular weight. In some embodiments, the predetermined shape is configured to limit or prevent cross-reactivity with another supramolecular structure. In some embodiments, for any of the methods disclosed herein, the plurality of core molecules for each core structure comprise one or more nucleic acid strands, one or more branched nucleic acids, one or more peptides, one or more small molecules, or combinations thereof. In some embodiments, for any of the methods disclosed herein, each core structure independently comprises a backbone deoxyribonucleic acid (DNA) origami, a backbone ribonucleic acid (RNA) origami, a backbone hybrid DNA:RNA origami, a single-stranded DNA tile structure, a multi-stranded DNA tile structure, a single-stranded RNA origami, a multi-stranded RNA tile structure, a hierarchical DNA or RNA origami having multiple backbones, a peptide structure, or combinations thereof.

[0008] In some embodiments, for any of the methods disclosed herein, each analyte molecule is bound to a capture molecule of each supramolecular structure through a chemical bond and / or bound to a detector molecule of a detector molecule assembly through a chemical bond. In some embodiments, for any of the methods disclosed herein, the capture molecule and the detector molecule independently comprise a protein, a peptide, an antibody, an aptamer (RNA and DNA), a fluorophore, a darpin, a catalyst, a polymerization initiator, a polymer such as PEG, or a combination thereof. In some embodiments, for any of the methods disclosed herein, for each supramolecular structure, a) the capture molecule is linked to the core structure through a capture barcode, the capture barcode comprising a first capture linker, a second capture linker, and a capture bridge disposed between the first capture linker and the second capture linker, the first capture linker being bound to a first core linker bound to a first location on the core structure, the capture molecule and the second capture linker being linked to each other through a bond to a third capture linker; b) the detector molecule assembly comprises a detector barcode, the detector barcode comprising one or more linkers. In some embodiments, the capture bridge and the detector molecule assembly independently comprise a polymer core. In some embodiments, the polymer core of the capture bridge and the polymer core of the detector molecule assembly independently comprise a nucleic acid (DNA or RNA) of a specific sequence or a polymer such as PEG.

[0009] In some embodiments, for any of the methods disclosed herein, each supramolecular structure further comprises an anchor molecule linked to a core structure. In some embodiments, the anchor molecule is linked to the core structure through an anchor barcode, and the anchor barcode comprises a first anchor linker, a second anchor linker, and an anchor bridge disposed between the first anchor linker and the second anchor linker, the first anchor linker being coupled to a third core linker coupled to a third location on the core structure, and the anchor molecule being linked to the second anchor linker. In some embodiments, the anchor molecule comprises an amine, a thiol, a DBCO, a maleimide, a biotin, an azide, an acridite, an NHS ester, a single-stranded nucleic acid (RNA or DNA) of a specific sequence, one or more polymers such as PEG, a polymerization initiator, or a combination thereof. In some embodiments, the anchor bridge comprises a polymer core. In some embodiments, the polymer core of the anchor bridge comprises a nucleic acid (DNA or RNA) of a specific sequence or a polymer such as PEG. In some embodiments, the third core linker, the first anchor linker, the second anchor linker, and the anchor molecule each independently comprise an anchor reactive molecule or a DNA sequence domain. In some embodiments, each anchor reactive molecule independently comprises an amine, a thiol, a DBCO, a maleimide, a biotin, an azide, an acridite, an NHS ester, a single-stranded nucleic acid (RNA or DNA) of a specific sequence, one or more polymers such as PEG, a polymerization initiator, or a combination thereof. In some embodiments, the anchor molecule is linked to the second anchor linker through a chemical bond. In some embodiments, the anchor molecule is covalently linked to the second anchor linker.

[0010] In some embodiments, for any of the methods disclosed herein, the signal comprises a detector barcode, a capture barcode, or a combination thereof, corresponding to a supramolecular structure bound to an analyte bound to a detector molecule assembly. In some embodiments, each detector barcode provides a DNA signal corresponding to a detector molecule and provides information indicative of the specificity of the detector molecule for an analyte molecule bound to each detector molecule. In some embodiments, the detector barcode is analyzed using genotyping, qPCR, sequence analysis, or a combination thereof. In some embodiments, multiple analyte molecules in a sample are detected simultaneously by multiplexing. In some embodiments, for any of the methods disclosed herein, the capture molecules and detector molecules for each supramolecular structure are configured to bind to one or more specific types of analyte molecules.

[0011] In some embodiments, for any method comprising using a plurality of supramolecular structures disclosed herein, each core structure of the plurality of supramolecular structures is identical to one another. In some embodiments, each supramolecular structure has a defined shape, size, molecular weight, or a combination thereof that reduces or eliminates cross-reactivity between the plurality of supramolecular structures. In some embodiments, each supramolecular structure comprises a plurality of capture molecules. In some embodiments, each supramolecular structure has a defined stoichiometry of capture molecules and detector molecules that reduces or eliminates cross-reactivity between the plurality of supramolecular structures.

[0012] In some embodiments, one or more supramolecular structures are attached to a hydrogel porous matrix. In some embodiments, each supramolecular structure is copolymerized with the hydrogel through corresponding anchor molecules linked to respective core structures of the corresponding supramolecular structures. In some embodiments, one or more supramolecular structures are embedded within the hydrogel. In some embodiments, a plurality of supramolecular structures are disposed on a substrate such as a shaped substrate or a planar substrate, the substrate comprising a plurality of binding sites, each binding site configured to link with a corresponding supramolecular structure. In some embodiments, a plurality of supramolecular structures are configured to detect the same analyte molecule. In some embodiments, for any method comprising the step of using a substrate, the method further comprises the step of providing a plurality of signal transmitting elements linked to detector molecules. In some embodiments, each signal transmitting element comprises a fluorescent molecule or microbeads, a fluorescent polymer, highly charged nanoparticles or polymers. In some embodiments, at least one of the plurality of supramolecular structures is configured to detect an analyte molecule different from other supramolecular structures.

[0013] In some embodiments, for any method comprising the step of using a planar substrate, the method further comprises the step of barcoding each supramolecular structure to identify the location of each supramolecular structure on the planar substrate. In some embodiments, for any method comprising the step of using a planar substrate, the method comprises the step of providing a plurality of signal transmitting elements provided herein configured to link with detector molecules.

[0014] In some embodiments, for any of the methods disclosed herein, the sample comprises a biological particle or a biomolecule. In some embodiments, for any of the methods disclosed herein, the sample comprises an aqueous solution comprising a protein, a peptide, a peptide fragment, a lipid, DNA, RNA, an organic molecule, a viral particle, an exosome, an organelle, or a complex of any of these. In some embodiments, for any of the methods disclosed herein, the sample comprises a tissue biopsy material, blood, plasma, urine, saliva, tear fluid, cerebrospinal fluid, extracellular fluid, cultured cells, a culture medium, discarded tissue, a plant, a synthetic protein, a prion, a sample of bacteria and / or virus, or a fungal tissue, or a combination thereof. The sample can be processed to release the analyte from the cells or otherwise prepare the sample for analysis prior to contacting it with the supramolecular structures provided herein. The sample can be an environmental sample such as wastewater or a soil sample. The sample can be a non-biological sample. In some embodiments, the sample can be a sample from a chemical treatment step, a food sample or a nutritional component, or a packaging component.

[0015] Provided herein, in some embodiments, is a substrate for detecting one or more analyte molecules in a sample, the substrate comprising a plurality of supramolecular structures each comprising a) a core structure comprising a plurality of core molecules and b) a capture molecule linked to the supramolecular core.

[0016] In some embodiments, each core structure of the plurality of supramolecular structures is identical to each other. In some embodiments, the substrate comprises a solid support, a solid substrate, a polymer matrix, or a molecular condensate. In some embodiments, the sample comprises a complex biological sample, and the method provides single molecule sensitivity, thereby increasing the dynamic range and quantitative capture of various molecular concentrations within the complex biological sample. In some embodiments, the one or more analyte molecules comprise proteins, peptides, peptide fragments, lipids, DNA, RNA, organic molecules, inorganic molecules, complexes thereof, or any combination thereof. In some embodiments, each supramolecular structure is a nanostructure. In some embodiments, each core structure is a nanostructure. In some embodiments, the plurality of core molecules for each core structure are arranged in a predetermined shape and / or have a defined molecular weight. In some embodiments, the predetermined shape is configured to limit or prevent cross-reactivity with another supramolecular structure. In some embodiments, for any of the methods disclosed herein, the plurality of core molecules for each core structure comprise one or more nucleic acid strands, one or more branched nucleic acids, one or more peptides, one or more small molecules, or a combination thereof. In some embodiments, each core structure independently comprises a backbone deoxyribonucleic acid (DNA) origami, a backbone ribonucleic acid (RNA) origami, a backbone hybrid DNA:RNA origami, a single-stranded DNA tile structure, a multi-stranded DNA tile structure, a single-stranded RNA origami, a multi-stranded RNA tile structure, a hierarchical structured DNA or RNA origami having multiple backbones, a peptide structure, or a combination thereof. In some embodiments, each analyte molecule is 1) bound to a capture molecule through a chemical bond and / or 2) bound to a detector molecule through a chemical bond. In some embodiments, the capture molecule and the detector molecule independently comprise proteins, peptides, antibodies, aptamers (RNA and DNA), fluorophores, darpins, catalysts, polymerization initiators, polymers such as PEG, or a combination thereof.

[0017] In some embodiments, for each supramolecular structure of the substrate, a) the capture molecule is linked to the core structure through a capture barcode, the capture barcode comprising a first capture linker, a second core linker, and a capture bridge disposed between the first capture linker and the second core linker, the first capture linker being bound to a first core linker bound to a first location on the core structure, and the capture molecule and the second core linker being linked to each other through a bond to a third capture linker. The capture molecule binds to an analyte that binds to a detector molecule of the detector molecule assembly. In some embodiments, the detector molecule assembly comprises discrete supramolecular structures that are not linked to the capture molecules and are not immobilized on the substrate.

[0018] In some embodiments, the supramolecular structure comprising the capture molecule interacts directly with the substrate material to immobilize the supramolecular structure on the substrate. In some embodiments, the supramolecular structure comprising the capture molecule further comprises an anchor molecule provided herein linked to the core structure, the anchor molecule being linked to the substrate to immobilize the supramolecular structure on the substrate.

[0019] In some embodiments, the signal read or detected from the substrate comprises a detector barcode, a capture barcode, or a combination thereof that can be analyzed using an optical sensor, a magnetic sensor, and / or an electrical sensor. The detection techniques include electrochemical sensing, genotyping, qPCR, sequence analysis, or a combination thereof. In some embodiments, one or more supramolecular structures are configured to multiplex samples such that multiple analyte molecules within the sample are detected simultaneously. In some embodiments, the capture molecule for each supramolecular structure is configured to bind to one or more specific types of analyte molecules.

[0020] In some embodiments, the sample comprises a complex biological sample, and the method provides single molecule sensitivity, thereby increasing the dynamic range and quantitative capture of various molecular concentrations within the complex biological sample. In some embodiments, the analyte molecules comprise proteins, peptides, peptide fragments, lipids, DNA, RNA, organic molecules, inorganic molecules, complexes thereof, or any combination thereof. In some embodiments, the supramolecular structure is a nanostructure. In some embodiments, the core structure is a nanostructure. In some embodiments, the supramolecular structure has a defined shape, size, molecular weight, or combination thereof that reduces or eliminates cross-reactivity with another supramolecular structure. In some embodiments, the supramolecular structure comprises a plurality of capture molecules.

[0021] In some embodiments, the plurality of core molecules for the core structure are arranged in a predetermined shape and / or have a defined molecular weight. In some embodiments, the predetermined shape is configured to limit or prevent cross-reactivity with another supramolecular structure. In some embodiments, for any of the methods disclosed herein, the plurality of core molecules for each core structure comprise one or more nucleic acid strands, one or more branched nucleic acids, one or more peptides, one or more small molecules, or any combination thereof. In some embodiments, the core structure independently comprises a backbone deoxyribonucleic acid (DNA) origami, a backbone ribonucleic acid (RNA) origami, a backbone hybrid DNA:RNA origami, a single-stranded DNA tile structure, a multi-stranded DNA tile structure, a single-stranded RNA origami, a multi-stranded RNA tile structure, a hierarchical DNA or RNA origami having multiple backbones, a peptide structure, or any combination thereof.

[0022] Reference is now made to the drawings to describe certain embodiments of the devices, delivery systems, or methods of the present disclosure below. None of the detailed descriptions are intended to suggest that any of the specific components, features, or steps are essential to the present invention.

Brief Description of the Drawings

[0023]

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Mode for Carrying Out the Invention

[0024] Disclosed herein are structures and methods for detecting one or more analyte molecules present in a sample. In some embodiments, the one or more analyte molecules are detected based on capture by one or more supramolecular structures. In some embodiments, the one or more supramolecular structures comprise or are linked to capture molecules that specifically bind to analytes present in the sample. The bound analyte then interacts with or binds to a detector molecule of a detector molecule assembly having a detectable moiety such as a unique identifier (e.g., nucleic acid sequence, peptide, polysaccharide, acridite) and / or other molecules that can be detected (e.g., optically, electrically, magnetically). In some embodiments, the detector molecule assembly generates a DNA signal, in which case detection and quantification of the binding of the analyte molecule to the capture molecule comprises the step of converting the presence of the analyte molecule into a DNA signal by amplification of the unique identifier of the supramolecular structure. In some embodiments, the detector molecule assembly is linked to an enzyme that converts the substrate into an optically detectable signal. In some embodiments, the supramolecular structure is a nucleic acid origami linked to or immobilized on a substrate. In some embodiments, the supramolecular structure holds the capture molecule through a barcode that contains a unique identifier for the capture molecule and links the capture molecule to the backbone of the supramolecular structure.

[0025] In some embodiments, the disclosed technology of the present invention enables a single molecule enzyme-linked immunosorbent assay (ELISA) where a supramolecular structure and associated capture molecules act as capture entities and a detector molecule assembly acts as a detection entity used to generate a detection signal (e.g., when reacting with an appropriate detection reagent sensed using a sensor of a detection system). The use of the supramolecular structure as a capture entity allows for specific discrimination and, in multiple embodiments, enables mapping the location of each individual capture molecule immobilized on a substrate. Further, the supramolecular structure is configured to be organized on or within a porous material on a substrate to allow for single molecule binding.

[0026] That is, as provided herein, to generate assay results characterizing the binding properties of a sample pool composed of a plurality of different samples, the binding of a detectable sample can be associated with individual capture molecules within many different capture molecules on a substrate. This association enables analyzing a sample having an unknown sample composition with respect to the presence and / or concentration of a particular target sample. For example, a human sample can be characterized to determine the presence and / or concentration of an antibody having binding specificity to a certain antigen within a panel of antigen capture molecules corresponding to a known panel of infectious disease antigens. The assay results can indicate a positive binding result for a particular antigen, and this result indicates the presence of the antibody in the subject who provided the sample. In another embodiment, the identity of the sample within the sample may be at least partially known, but the binding affinity for a particular pool of capture molecules may not be characterized. For example, the capture molecules can be a set of drug candidates, and the sample can be molecules in human blood. The binding of the drug candidate to such proteins can be used to evaluate bioavailability or potential off-target binding. The assay results may indicate a positive binding result for a particular drug candidate, and then this result can be mapped based on the identification of the binding of a particular detector to a particular sample (e.g., binding identification by identifying a barcode within a detector molecule assembly containing a specific antibody for the sample).

[0027] Conventional ELISA procedures may include a detectable fluorescence signal generated by an enzyme linked to a detection antibody as an indicator of binding, but the techniques disclosed in the present invention can, in addition to or instead of this, generate an amplified nucleic acid signal from a unique identifier of a detector molecule assembly, sequence information can be determined from this signal, or an optically detectable signal corresponding to amplification (e.g., qPCR using a specific primer / probe set for the unique identifier) is emitted. That is, in certain embodiments, the unique identification information regarding the detector molecule assembly enables the specific identification of a particular detector molecule linked to a capture molecule through the bound analyte. However, in embodiments, the detector molecule assembly may not carry a unique identifier. Other detection techniques can include optical, magnetic, and / or electrical detection techniques.

[0028] Sample Embodiments of the disclosure of the present invention relate to analyte detection when the analyte is present in a sample such as a biological sample. In some embodiments, the sample comprises an aqueous solution comprising proteins, peptides, peptide fragments, lipids, DNA, RNA, organic molecules, inorganic molecules, complexes thereof, or any combination thereof. In some embodiments, the analyte molecules in the sample comprise proteins, peptides, peptide fragments, lipids, DNA, RNA, organic molecules, inorganic molecules, complexes thereof, or any combination thereof. In some embodiments, the analyte molecules comprise intact proteins, denatured proteins, partially or completely degraded proteins, peptide fragments, denatured nucleic acids, degraded nucleic acid fragments, complexes thereof, or combinations thereof. In some embodiments, the sample is obtained from tissue, cells, the environment of tissue and / or cells, or any combination thereof. In some embodiments, the sample comprises tissue biopsy material, blood, plasma, urine, saliva, tears, cerebrospinal fluid, extracellular fluid, cultured cells, culture medium, discarded tissue, plant bodies, synthetic proteins, bacterial samples, viral samples, fungal tissue, or any combination thereof. In some embodiments, the sample is isolated from a primary source such as cells, tissue, body fluid (e.g., blood), environmental sample, or any combination thereof, either by purification or without purification. In some embodiments, the cells are lysed using mechanical treatment or other cell lysis methods (e.g., lysis buffer). In some embodiments, the sample is filtered using mechanical treatment (e.g., centrifugation), micron filtration, chromatography columns, other filtration methods, or any combination thereof. In some embodiments, the sample is treated with one or more enzymes to extract one or more nucleic acids or one or more proteins. In some embodiments, the sample comprises 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 taken from one or more individuals, one or more animals, one or more plants, or any combination thereof. In some embodiments, the sample is taken from an individual, animal, and / or plant having a disease or disorder comprising an infectious disease, immune disorder, cancer, genetic disease, degenerative disease, lifestyle disease, injury, rare disease, age-related disease, or any combination thereof.

[0029] Supramolecular structure In some embodiments, the supramolecular structure is a programmable structure capable of spatially organizing molecules. In some embodiments, the supramolecular structure comprises a plurality of molecules linked to each other. In some embodiments, the plurality of molecules of the supramolecular structure interact with at least a portion of each other. In some embodiments, the supramolecular structure has a specific shape. In some embodiments, the supramolecular nanostructure has a defined molecular weight based on its plurality of molecules. In some embodiments, the supramolecular structure is a nanostructure. In some embodiments, the plurality of molecules are linked to each other by bonds, chemical bonds, physical attachment, or combinations thereof. In some embodiments, the supramolecular structure comprises a large molecular entity having a specific shape and molecular weight formed from a 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 specifically designed. In some embodiments, the supramolecular structure comprises a plurality of subcomponents spaced apart according to a defined 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 flexible.

[0030] Figure 1 provides an exemplary embodiment of a supramolecular structure 40 comprising a core structure 13, a capture molecule 2, and an anchor molecule 18. In some embodiments, the supramolecular structure comprises one or more capture molecules 2 and optionally one or more anchor molecules 18. In some embodiments, the supramolecular structure does not comprise an anchor molecule. In some embodiments, the supramolecular structure is a polynucleotide structure.

[0031] In some embodiments, the core structure 13 comprises one or more core molecules linked to each other. In some embodiments, the one or more core molecules comprise two, three, four, five, six, seven, eight, nine, ten, twenty, fifty, one hundred, two hundred, or five hundred unique molecules linked to each other. In some embodiments, the one or more core molecules comprise from about two to about one thousand 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 plurality of core molecules interact with each other by reversible non-covalent interactions.

[0032] In some embodiments, the specific shape of the core structure is a three-dimensional (3D) configuration. In some embodiments, one or more core molecules confer a specific molecular weight. In some embodiments, the core structure 13 is a nanostructure. In some cases, one or more core molecules comprise 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 comprises 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 comprises a backbone deoxyribonucleic acid (DNA) origami, a backbone ribonucleic acid (RNA) origami, a backbone hybrid DNA / RNA origami, a single-strand DNA tile structure, a multi-strand DNA tile structure, a single-strand DNA origami, a single-strand RNA origami, a single-strand RNA tile structure, a multi-strand RNA tile structure, a hierarchical DNA or RNA origami having multiple backbones, a peptide structure, or combinations thereof. In some embodiments, the DNA origami is backbone-modified. In some embodiments, the RNA origami is backbone-modified. In some embodiments, the hybrid DNA:RNA origami is backbone-modified. In some embodiments, a core structure comprising a DNA origami, an RNA origami, or a hybrid DNA:RNA origami has a defined two-dimensional (2D) or 3D shape.

[0033] In an embodiment, the nucleic acid origami has a lateral dimension between about 50 nm and about 1 μm. In some embodiments, the nucleic acid origami has, by way of example, at least one lateral dimension between about 50 nm and about 200 nm, between about 50 nm and about 400 nm, between about 50 nm and about 600 nm, between about 50 nm and about 800 nm, between about 100 nm and about 200 nm, between about 100 nm and about 300 nm, between about 100 nm and about 400 nm, between about 100 nm and about 500 nm, between about 200 nm and about 400 nm. In some embodiments, the nucleic acid origami has a first lateral dimension between about 50 nm and about 1 μm and a second lateral dimension between about 50 nm and about 1 μm that is orthogonal to the first dimension. In some embodiments, the nucleic acid origami is about 200 nm 2 to about 1 μm 2 and has a planar footprint with an area between.

[0034] As shown in FIG. 1, in some embodiments, the core structure 13 is configured to be linked to a capture molecule 2, an anchor molecule 18, or a combination thereof. In some embodiments, the capture molecule 2 and / or the anchor molecule 18 are immobilized relative to the core nanostructure 13 when linked thereto. In some embodiments, any number of one or more core molecules comprise one or more core linkers 12, 14 configured to form a link with the capture molecule 2 and / or the anchor molecule 18. In some embodiments, any number of one or more core molecules are configured to be linked to one or more linkers 12, 14 configured to form a link with the capture molecule 2 and / or the anchor molecule 18. In some embodiments, one or more linkers are linked to one or more core molecules through a chemical bond. In some embodiments, at least one of the one or more core linkers comprises a core reactive molecule. In some embodiments, each core reactive molecule independently comprises an amine, a thiol, a DBCO, an NHS ester, a maleimide, a biotin, an azide, an acridite, a single-stranded nucleic acid of a specific sequence (e.g., RNA or DNA), or a polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, at least one of the one or more core linkers comprises a DNA sequence domain.

[0035] In some embodiments, the core structure 13 is 1) linked to the capture molecule 2 at a defined location on the core structure and optionally 2) linked to the anchor molecule 18 at a different defined location on the core structure.

[0036] In some embodiments, a first core linker 12 is disposed at a 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.

[0037] In some embodiments, a defined third core linker 14 is disposed at a third location on the core structure 13. In some embodiments, one or more core molecules at the third location are modified to form a link with the third core linker 14. In some embodiments, the third core linker 14 is an extension of the core structure 13. In some embodiments, the first and second locations are disposed on a first side of the core structure 13, and an optional third location is disposed on a second side of the core structure 13.

[0038] In some embodiments, the capture molecule 2 comprises a protein, peptide, antibody, aptamer (RNA and DNA), fluorophore, nano - body, darpin, catalyst, polymerization initiator, polymer such as PEG, organic molecule, or a combination thereof. In some embodiments, the anchor molecule comprises a reactive molecule. In some embodiments, the anchor molecule 18 comprises a reactive molecule. In some embodiments, the anchor molecule 18 comprises a DNA strand comprising a reactive molecule. In some embodiments, the anchor molecule 18 comprises an amine, thiol, DBCO, NHS ester, maleimide, biotin, azide, acridite, single - stranded nucleic acid of a specific sequence (e.g., RNA or DNA), or a polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, the anchor molecule 18 comprises a protein, peptide, antibody, aptamer (RNA and DNA), fluorophore, nano - body, darpin, catalyst, polymerization initiator, polymer such as PEG, organic molecule, or a combination thereof. In some embodiments, a single capture molecule 2 is linked to the core structure 13. In some embodiments, a plurality of capture molecules 2 are linked to the core structure 13. In some embodiments, multiple pairs of capture molecules 2 are spaced apart from each other to minimize crosstalk. For example, different capture molecules 2 on the same core structure 13 can correspond to different binding sites for the same analyte molecule or can bind to different analyte molecules. In another example, multiple identical capture molecules 2 can be present on the core structure 13.

[0039] In some embodiments, each component of the supramolecular structure can be modified or adjusted independently. In some embodiments, by modifying one or more of the components of the supramolecular structure, the 2D and 3D geometric shapes of the supramolecular structure itself can be modified. In some embodiments, by modifying one or more of the components of the supramolecular structure, the 2D and 3D geometric shapes of the core structure can be modified. In some embodiments, such a function for independently modifying the components of the supramolecular nanostructure enables precise control over the organization of one or more supramolecular structures on a solid surface (e.g., a flat surface or microparticle) and in a 3D volume (e.g., within a hydrogel matrix).

[0040] Capture barcode As shown in FIG. 1, 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 a link with the core structure 13. In some embodiments, the capture barcode 20 comprises a first capture linker 11, a second capture linker 6, and a capture bridge 7. In some embodiments, the first capture linker 11 comprises a reactive molecule. In some embodiments, the first capture linker 11 comprises a reactive molecule comprising an amine, a thiol, a DBCO, an NHS ester, a maleimide, an azide, an acridite, a single-stranded nucleic acid of a specific sequence (e.g., RNA or DNA), or a polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, the first capture linker 11 comprises a DNA sequence domain. In some embodiments, the second capture linker 6 comprises a reactive molecule. In some embodiments, the second capture linker 6 comprises a reactive molecule comprising an amine, a thiol, a DBCO, an NHS ester, a biotin, a maleimide, an azide, an acridite, a single-stranded nucleic acid of a specific sequence (e.g., RNA or DNA), or a polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, the second capture linker comprises a DNA sequence domain. In some embodiments, the capture bridge 7 comprises a polymer. In some embodiments, the capture bridge 7 comprises a polymer comprising a nucleic acid of a specific sequence (e.g., DNA or RNA). In some embodiments, the capture bridge 7 comprises 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 through a chemical bond. In some embodiments, the second capture linker 6 is attached to the capture bridge 7 through a chemical bond.In some embodiments, the first capture linker 11 is attached to the capture bridge 7 by physical attachment. In some embodiments, the second capture linker 6 is attached to the capture bridge 7 by physical attachment.

[0041] In some embodiments, the capture barcode 20 is linked to the core structure 13 by a linkage between the first capture linker 11 and the first core linker 12. In some embodiments, as described herein, the first core linker 12 is disposed 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.

[0042] In some embodiments, the capture barcode 20 is linked to the capture molecule 2 by a linkage between the second capture linker 6 and a third capture linker 5 attached to the capture molecule 2. In some embodiments, the third capture linker 5 comprises a reactive molecule. In some embodiments, the third capture linker 5 comprises a reactive molecule comprising an amine, thiol, DBCO, NHS ester, maleimide, biotin, azide, acridite, single-stranded nucleic acid of a specific sequence (e.g., RNA or DNA), or a polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, the third capture linker 5 comprises a DNA sequence domain. In some embodiments, the capture molecule 2 is linked to the third capture linker 5 through a chemical bond. In some embodiments, the capture molecule 2 is linked to the third capture linker 5 through a covalent bond. In some embodiments, the second capture linker 6 and the third capture linker 5 are linked to each other through a chemical bond. In some embodiments, the second linker 6 and the third capture linker 5 are linked to each other through a covalent bond.

[0043] Anchor barcode As shown in FIG. 1, in some embodiments, the anchor molecule 18 is linked to the core structure 13 through an anchor barcode. In some embodiments, the anchor barcode forms a link with the anchor molecule 18 and the anchor barcode forms a link with the core structure 13. In some embodiments, the anchor barcode comprises a first anchor linker 15, a second anchor linker 17, and an anchor bridge 16. In some embodiments, the first anchor linker 15 comprises a reactive molecule. In some embodiments, the first anchor linker 15 comprises a reactive molecule comprising an amine, a thiol, a DBCO, an NHS ester, a maleimide, biotin, an azide, an acridite, a single-stranded nucleic acid of a specific sequence (e.g., RNA or DNA), or a polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, the first anchor linker 15 comprises a DNA sequence domain. In some embodiments, the second anchor linker 17 comprises a reactive molecule. In some embodiments, the second anchor linker 17 comprises a reactive molecule comprising an amine, a thiol, a DBCO, an NHS ester, a maleimide, biotin, an azide, an acridite, a single-stranded nucleic acid of a specific sequence (e.g., RNA or DNA), or a polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, the second anchor linker 17 comprises a DNA sequence domain. In some embodiments, the anchor bridge 16 comprises a polymer. In some embodiments, the anchor bridging portion 16 comprises a polymer comprising a nucleic acid (DNA or RNA) of a specific sequence. In some embodiments, the anchor bridge 16 comprises a polymer such as PEG. In some embodiments, the first anchor linker 15 is attached to the anchor bridge 16 at its first end and the second anchor linker 17 is attached to the anchor bridge 16 at its second end. In some embodiments, the first anchor linker 15 is attached to the anchor bridge 16 through a chemical bond. In some embodiments, the second anchor linker 17 is attached to the anchor bridge 16 by physical attachment. In some embodiments, the first anchor linker 15 is attached to the anchor bridge 16 through a chemical bond.In some embodiments, the second anchor linker 17 is attached to the anchor bridge 16 by physical attachment.

[0044] In some embodiments, the anchor barcode is linked to the core structure 13 by a linkage between the first anchor linker 15 and the third core linker 14. In some embodiments, as described herein, the third core linker 14 is disposed at a third location on the core structure 13. In some embodiments, the first anchor linker 15 and the third core linker 14 are linked to each other through a chemical bond. In some embodiments, the first anchor linker 15 and the third core linker 14 are linked to each other through a covalent bond.

[0045] In some embodiments, the anchor barcode is linked to the anchor molecule 18 by a linkage between the second anchor linker 17 and the anchor molecule 18. As disclosed herein, in some embodiments, the anchor molecule comprises a reactive molecule, a reactive molecule, a DNA sequence domain, a DNA sequence domain comprising a reactive molecule, or a combination thereof. In some embodiments, the anchor molecule 18 is attached to the second anchor linker 17 through a chemical bond. In some embodiments, the anchor molecule 18 is attached to the second anchor linker 17 through a covalent bond.

[0046] Figure 2 is a schematic diagram of a supramolecular structure 40 bound to a corresponding analyte molecule 44 having binding specificity for the capture molecule 2. The supramolecular structure 40 has the function of binding to one or more analyte molecules 44 as a function of a specific capture molecule 2 associated therewith. Further, the analyte molecule 44 has the function of binding to a detector molecule assembly 46. The detector molecule assembly 46 comprises a detector molecule 1 that binds using the specificity of the analyte molecule 44. Figure 2 illustrates a sandwich-type binding arrangement in which the capture molecule 2 and the detector molecule 1 bind to different sites on the analyte molecule 44. In some embodiments, the detector molecule 1 comprises a protein, peptide, antibody, aptamer (RNA and DNA), fluorophore, nano-body, darpin, catalyst, polymerization initiator, polymer such as PEG, organic molecule, or a combination thereof. As shown in Figure 2, in some embodiments, the detector molecule 1 is linked to a detector barcode 21. In some embodiments, the detector barcode 21 can form a link with the detector molecule 1 and comprise one or more intervening components. In other embodiments, the detector molecule 1 is linked to a detectable tail or linker but does not retain unique barcode information.

[0047] Figure 3 illustrates an exemplary arrangement of detector barcodes 21. In some embodiments, the detector barcode comprises one or more detector linkers including a first detector linker 4. The detector barcode 21 can comprise a dock 8 that functions as an attachment site or an extension / amplification site to facilitate detection. In some embodiments, the linker 4 comprises a reactive molecule. In some embodiments, the linker 4 comprises a reactive molecule comprising an amine, a thiol, a DBCO, an NHS ester, a maleimide, biotin, an azide, an acridite, a single-stranded nucleic acid of a specific sequence (e.g., RNA or DNA), or a polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, the linker 4 comprises a DNA sequence domain. In some embodiments, the dock 8 comprises a polymer. In some embodiments, the dock 8 comprises a polymer comprising a nucleic acid (DNA or RNA) of a specific sequence, e.g., a single-stranded or double-stranded nucleic acid. In some embodiments, the dock 8 comprises a polymer such as PEG. In some embodiments, the linker 4 is attached to the dock 8 at its end, and another detector linker 4 is attached to the dock 8 at its second end. The attachment can be by a chemical bond or physical attachment.

[0048] In some embodiments, the detector barcode 21 is linked to the detector molecule 1 by a linkage between a plurality of linkers shown as detector linker 4 and a second detector linker 3 bound to the detector molecule 1 in this figure. In some embodiments, the second detector linker 3 comprises a reactive molecule. In some embodiments, the second detector linker 3 comprises a reactive molecule comprising an amine, a thiol, a DBCO, an NHS ester, a maleimide, a biotin, an azide, an acridite, a single-stranded nucleic acid of a specific sequence (e.g., RNA or DNA), or a polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, the second detector linker 3 comprises a DNA sequence domain. In some embodiments, the detector molecule 1 is bound to the second detector linker 3 through a chemical bond. In some embodiments, the detector molecule 1 is bound to the second detector linker 3 through a covalent bond. In some embodiments, the second detector linker 4 and the second detector linker 3 are bound to each other through a chemical bond. In some embodiments, the second detector linker 4 and the second detector linker 3 are bound to each other through a covalent bond.

[0049] As provided herein, the capture molecule assembly comprises a supramolecular structure 40 having a capture molecule 1 and a core structure 13. In certain embodiments, the detector molecule assembly 46 is linked or bound to the core structure 13 and thus this detector molecule assembly is also a supramolecular structure provided herein. In this case, as shown in FIG. 4, the detector molecule that binds to the capture analyte molecule 44 associated with the supramolecular structure 40 of the capture molecule assembly generates a supramolecular structure sandwich. Generally, the supramolecular structure 40 of the capture molecule assembly or the detector molecule assembly 46 is immobilized to enable the flow of the capture entity or the detection entity. In some embodiments, the capture molecule assembly is immobilized on a surface or within a porous material.

[0050] The supramolecular structure 40 and / or the detector molecule assembly 46 can comprise a DNA origami. In some embodiments, a subcomponent of the core structure 13 of the supramolecular structure 40 and / or the detector molecule assembly 46 comprises a DNA origami and one or more extended nucleic acid strands. In some embodiments, the core structure 13 of the supramolecular structure 40 and / or the detector molecule assembly 46 comprises a backbone DNA origami folded into a predefined 2D or 3D shape by interaction of a circular ssDNA molecule, called the “backbone” strand, with two or more short ssDNAs, called “staple” strands, that interact with specific subsections of the ssDNA “backbone” strand.

[0051] As described herein, in some embodiments, one or more supramolecular structures enable the detection of one or more analyte molecules in a sample. As shown in the schematic of FIG. 5, the supramolecular structure 40 is exposed to the analyte molecule 44 and the detector molecule assembly 46 along with the relevant capture molecule. When an individual analyte molecule 44 and an individual capture molecule 2 have binding specificity for each other, the analyte molecule 44 is associated with the capture molecule 2. Next, a detector molecule assembly 46 having binding specificity for the analyte molecule 44 is associated with the analyte molecule 44 to produce a bound detection structure 50.

[0052] As shown in FIG. 6, an analyte molecule 44 that does not have binding specificity for the capture molecule 2 is not associated with the supramolecular structure 40. Further, the detector molecule assembly 46 is also not associated with the supramolecular structure 40, and the bound detection structure 50 is not achieved. In a substrate comprising an immobilized array of supramolecular structures 40, when the sample comprises a suitable analyte molecule 44 having binding specificity for the capture molecule 2, each individual site can have a binding reaction that produces a bound detection structure 50.

[0053] The depicted embodiment shows a detector molecule assembly 46 and an analyte molecule 44 brought into contact with the supramolecular structure 40 in one step, but as provided herein, it should be understood that the analyte molecule 44 and the detector molecule assembly 46 can be added in separate steps such that any unbound analyte molecule 44 is removed prior to the addition of the detector molecule assembly 46. FIG. 7 illustrates an exemplary method workflow for adding a pool of analyte molecules 44 to a group or array of capture molecule assemblies implemented as the supramolecular structure 40. The analyte molecules 44 can represent different analytes present in a sample such that different analyte molecules 44 within the pool have different degrees of binding specificity to an array of capture molecules 2 available.

[0054] These reaction conditions enable the binding of the analyte molecule 44 to a specific capture molecule 2. As provided herein, binding specificity can mean the interaction between the analyte molecule 44 and the capture molecule 2 that remains intact under these reaction conditions and after a washing or removal step with respect to unbound reagents. Binding specificity can comprise information such as covalent or non-covalent bonds, ionic bonds, dipole interactions, hydrophilic or hydrophobic interactions, complementary nucleic acid binding. Specific binding can mean the binding to an analyte molecule 44 that binds only to a specific capture molecule 2 and not to other capture molecules 2. Thus, within a given sample, a certain capture molecule 2 of the array (e.g., capture molecule 2a) binds to the analyte molecule 44, while another capture molecule (e.g., capture molecule 2b) has no corresponding binding partner and thus does not bind to any analyte molecule 44 by specificity. Any unbound analyte can be removed from the immobilized capture molecule assembly as provided herein.

[0055] The detector molecule 1 and the analyte molecule 44 can likewise have binding specificity for each other. As disclosed in various embodiments, the array substantially contacts a detector molecule assembly 46 that can all be the same or different. Any unbound detector molecule assembly 46 is removed, for example, by washing. After these workflow steps, various bound detector structures 50, each bound to a respective analyte and detector molecule assembly 46, remain on the array and can undergo various detection procedures to associate the analyte with the identity of a specific supramolecular structure related to the known capture molecule 2. That is, detection enables characterization of analyte-capture molecule binding.

[0056] FIG. 8 illustrates an exemplary detection step of evaluating various unique capture barcodes (20a, 20b, 20c, and 20d) of the bound detector structure 50 to associate a specific capture barcode with a binding event. In some embodiments, the supramolecular structure converts information about the presence of a given analyte molecule in a sample into a DNA signal. In some embodiments, the DNA signal corresponds to sequence data regarding a capture barcode and / or a detector barcode when a capture molecule and a detector molecule are simultaneously linked (e.g., bound) to the analyte molecule.

[0057] In some embodiments, the step of detecting the presence of an analyte molecule as described herein comprises controllably releasing one or more unique nucleic acid molecules into a solution that will be used to identify the nature of the analyte molecule from the sample. In some embodiments, these unique nucleic acid molecules are provided by the capture barcode 20 of each supramolecular structure. In some embodiments, the step of detecting the presence of an analyte molecule as described herein comprises generating an optical or electrical signal that can be counted to quantify the concentration of the analyte molecule in the solution in connection with a change in state.

[0058] In some embodiments, multiple analyte molecules in a sample are detected simultaneously by multiplexing, and the multiple supramolecular structures provide multiple signals (e.g., detector barcodes, capture barcodes) for sequence analysis and analyte identification. In some embodiments, the methods for detecting analytes in a sample described herein provide high-yield and high-multiplex capabilities by using multiple supramolecular structures. In some embodiments, the high-yield and high-multiplex capabilities provide high accuracy for the detection and quantification of analyte molecules. In some embodiments, the methods for detecting analytes in a sample described herein are configured to rapidly, highly sensitively, and highly reproducibly characterize and / or identify biopolymers comprising protein molecules. In some embodiments, the multiple supramolecular structures are configured to limit errors in cross-reactivity relationships. In some embodiments, such errors in cross-reactivity relationships involve the interaction (e.g., intermolecular interaction) of the capture molecule and / or detector molecule of one supramolecular structure with the capture molecule and / or detector molecule of another supramolecular structure. In some embodiments, each core structure of the multiple supramolecular structures is identical to each other. In some embodiments, the structural, chemical, and physical properties of each type of supramolecular structure are clearly designed. In some embodiments, the same core structure has a defined shape, size, molecular weight, defined number of capture molecules and detector molecules, a predetermined distance between the corresponding capture molecule and detector molecule (as described herein), a defined stoichiometry between the corresponding capture molecule and detector molecule, or a combination thereof that limits cross-reactivity between supramolecular structures. In some embodiments, the molecular weights of all core structures are the same and are accurate up to the purity of the core molecule. In some embodiments, each core structure has at least one capture molecule.

[0059] In some embodiments, although the plurality of supramolecular structures may be thought to share structural similarities due to the same among certain sub-components, the interaction between the analyte molecules and the supramolecular structures from the sample is predetermined by the corresponding capture molecules and detector molecules. In some embodiments, each detector molecule and capture molecule that bind on a given bound detection structure 50 can specifically interact with a particular analyte molecule in the sample. In some embodiments, each supramolecular structure comprises a unique DNA barcode corresponding to the associated capture molecule. In some embodiments, the capture molecules are designed to interact with more than one analyte molecule in the sample.

[0060] As provided herein, the individual supramolecular structures 40 can be uniquely identified using the capture barcode 20. Next, the supramolecular structures 40 are collected such that the capture molecules 2 can be associated, for example, with the capture barcode 20 stored in a lookup table of an analyte detection system (see FIG. 16). Thus, when the capture barcode 20 is identified, the identity of the capture molecule 2 is also accessible.

[0061] In some embodiments, each supramolecular structure is configured for single molecule sensitivity to ensure the largest possible dynamic range required to quantitatively capture a wide range of molecular concentrations in a typical complex biological sample. In some embodiments, the plurality of supramolecular structures limit or eliminate non-specific interactions, as well as sample manipulations required to reduce any user-induced errors.

[0062] FIG. 9 illustrates an exemplary analyte detection technique that can characterize the binding of an analyte using various locations of a supramolecular structure 40 having different respective capture molecules 2, along with unique capture barcode information. A sample comprising a pool of different analyte molecules 44 is contacted with an immobilized supramolecular structure 40 having different respective capture molecules 2. The analyte molecules 44 and the capture molecules 2, which have binding specificity for each other, are contacted under conditions that allow the interaction to occur. A detector molecule assembly 46 is bound to the analyte molecules 44 associated with the supramolecular structure. The bound detector structure can be characterized based on 1) a capture barcode 20 and 2) a signal generated by the bound detector molecule assembly that corresponds to a location map of a particular capture barcode 20. In some embodiments, the locations of the supramolecular structure 40 can be determined along with capture barcode information prior to the binding of the analyte. That is, pre-mapping can be provided to the array, or the mapping can be an individual step. The mapping can comprise a step of detecting a capture barcode as generally provided herein, such as a step of detecting an optical, electrical, and / or magnetic unique pattern. In some embodiments, the detection comprises a step of sequencing the nucleotide sequence of the capture barcode. In some embodiments, the detection comprises amplification and quantification of an amplification product, e.g., detection by qPCR of a signal for a probe.

[0063] Serological tests search for antibodies in a patient's blood to identify past infections by pathogens. In one example, a COVID-19 serological assay detects the presence of IgG or IgM antibodies against the spike protein or nucleocapsid. The capture molecule 2 is in the patient sample and retains the antibody analyte 44 to which the detector molecule assembly 46 is also bound. In some embodiments, the detector molecule assemblies 46 can all be of the same type and / or all have the same detector molecule 1. The detector molecule 1 can be an anti-human antibody that binds regardless of antibody-antigen specificity to any human antibody. Thus, the detector molecule 1 has the function of binding a variety of different analytes 44 associated with each antigen capture molecule 2. An analyte 44 indicating a positive binding event by a detectable signal from the detector molecule assembly 46 can be linked to a specific supramolecular structure 40 based on a specific barcode 20 for identifying a positive antibody result. The disclosed technology of the present invention can be used to generate assays for one or more infectious diseases such as COVID-19, influenza, RSV, and pneumonia. In some embodiments, the capture molecule 2 comprises a pool of various antigens of different infectious diseases, each associated with a different supramolecular structure 40. In addition to or instead of this, the assay can comprise multiple isotypes and multiple potential antigens of infectious diseases, and antigens from other respiratory pathogens including but not limited to influenza, RSV, and pneumonia. In embodiments, the assay can enable a distinction between innate and vaccine-acquired immunity through the specific addition of vaccine protein targets. The assay can also include a distinction between IgG specificity, IgM specificity, and IgA specificity. This assay can be updated or modified to appropriately examine the current pathogen situation when new infectious diseases occur. The improvements made to the current workflow by this assay will provide a higher insight into the patient's humoral immune system and help provide information for vaccine development. For example, the antibody response or circulating antibody population of a patient can be evaluated with respect to binding to various antigen candidates.

[0064] The detector molecule assemblies 46 can all be of the same type or can be detected using the same detection method. The detection signal may not carry any unique barcode information. In one example, the detector tail can include reaction molecules that generate a signal. In some embodiments, the detector molecule assembly 46 is detected based on the enzymatic conversion of a substrate into an optically detectable product. The optical detection relates to spots on the array of the supramolecular structures 40. In some embodiments, each detector molecule 1 of the detector molecule assembly 46 can all be of the same type and / or can have the same binding specificity. In one example, all the analyte molecules 44 to be detected are human antibodies, and the detector molecules are anti-human antibodies having a general binding specificity for a wide range of human antibodies regardless of antigen specificity. Other embodiments are also contemplated. For example, the analyte molecule 44 can undergo a processing step of adding a tag or a tag (e.g., biotin) that enables binding to the streptavidin detector molecule 1. In the depicted embodiments, the pool of detector molecule assemblies 46 is not diverse, and the detector molecules 1 and the associated tails or linkers can all be of the same type, so the step of providing the detector molecule assembly 46 can be made less complex. Further, since no sequence information or barcode information can be obtained from the detector side, the detection step can also be made less complex. Thus, in an embodiment, the unique identification information is the capture barcode 20 used together with the location information regarding each capture barcode and the location of the signal generated by the detector. The binding of the analyte is characterized using the correlation between the detector signal and the location of a specific barcode. It should be understood that the analyte detection method of FIG. 9 can be carried out using a detector molecule assembly having a unique barcode and reaction molecules that generate a detector signal.

[0065] FIG. 10 illustrates an embodiment of a specimen detection method similar to that of FIG. 9, but with a diverse pool of detector molecule assemblies 46. The diverse pool of detector molecule assemblies 46 holds different respective detector molecules 1 and detector barcodes 21 (shown as detector barcodes 21a, 21b, 21c, and 21d). The bound detector structure 50 comprises both a unique capture barcode 20 associated with a specific capture molecule 2 to a specific specimen molecule 44 and a specific unique detector barcode 21 to the specimen molecule 44. One or both of the unique capture barcode 20 or the unique detector barcode 21 can be evaluated to characterize the binding of the specimen. Detection of the unique detector barcode 21 can be performed using the techniques discussed with reference to the unique capture barcode 20 that comprises optical, electrical, and / or magnetic detection. Detection can comprise generating sequence data or amplified data of the unique detector barcode 21.

[0066] In some embodiments, a sample comprising one or more specimens contacts one or more supramolecular structures 40. In some embodiments, as described herein, a plurality of supramolecular structures attached to one or more solid substrates are provided. FIGS. 11-14 provide examples of supramolecular structures attached to patterned solid substrates. FIG. 15 illustrates an example of a supramolecular structure incorporated within a porous hydrogel matrix. The disclosed techniques of the present invention can be performed with respect to a patterned substrate comprising binding sites dispersed on or within the binding sites. In some embodiments, each binding site accepts a single supramolecular structure 40 having differential chemical action. The patterned substrate can be processed by lithographic processes. Further, embodiments of the disclosed techniques of the present invention can comprise one or more regeneration steps of removing the bound detector structure 50 or the “used” bound detector structure 50 from the substrate to incorporate a new supramolecular structure 40.

[0067] FIG. 11 provides an exemplary diagram of a method for detecting analyte molecules in a sample using a surface-based assay that uses supramolecular structure 40 as described herein towards single molecule counting of analytes in a sample (i.e., the step of detecting analyte molecules in a sample with single molecule resolution). In some embodiments, the supramolecular structure comprises a core structure comprising a DNA origami core. In some embodiments, a planar substrate 60 is provided that comprises: (a) a fiducial marker 62 that acts as a reference coordinate for all features on the substrate 60; (b) a predefined set of binding sites 66 in a micropattern arrangement that can immobilize individual core structures (e.g., DNA origamis); and (c) a background passivation portion 64 that minimizes or prevents interaction between the surface of the substrate 60 and the supramolecular structure (comprising capture molecules, core structure molecules). In some embodiments, the fiducial marker comprises geometric features that are predefined on the surface and are to be used as reference features for other features on the substrate. In some embodiments, the fiducial marker 62 is coated with a polymer or self-assembled monolayer that does not interact with the core structure of the supramolecular structure (e.g., DNA origami) or other molecules. In some embodiments, the background passivation portion 64 minimizes or prevents interaction between the surface of the substrate 60 and the analyte molecules of the sample. In some embodiments, the planar substrate 60 comprises an optical device or an electrical device such as a FET, a ring resonator, a photonic crystal, or a microelectrode that is predefined prior to the formation of the binding sites 66. In some embodiments, the binding sites 66 are micropatterned on the planar substrate 60. In some embodiments, the binding sites 66 on the surface are in a periodic pattern. In some embodiments, the binding sites 66 on the surface are in an aperiodic (e.g., random) pattern. In some embodiments, a minimum distance is defined between any two binding sites 66. In some embodiments, the minimum distance between any two binding sites 66 is at least about 200 nm. In some embodiments, the minimum distance between any two binding sites 66 is from at least about 40 nm to about 5000 nm. In some embodiments, the shape of the binding sites 66 includes circular, square, triangular, or other polygonal shapes.In some embodiments, the chemical groups used for the passivation part 64 comprise neutral charged 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 66 comprise silanol groups, carboxyl groups, thiols, other groups, or combinations thereof.

[0068] In some embodiments, a single supramolecular structure 40 is attached to each binding site 66 (step 1). The reference numeral 70 presents a diagram of the components of the supramolecular structure 40 individually and assembled and arranged on a planar substrate (the components are described herein, for example, in FIGS. 1-4). In some embodiments, the supramolecular structure 40 comprises a core structure 13 comprising a DNA origami and is attached onto each of the binding sites using DNA origami placement techniques (step 1). In some embodiments, the supramolecular structure 40 is assembled before being attached to each binding site 66. In some embodiments, the DNA origami has specific shapes and dimensions that facilitate binding to the binding sites using DNA origami placement techniques. In some embodiments, DNA origami placement includes a directed self-assembly technique for organizing individual DNA origamis (e.g., core structures) on a surface (e.g., a surface with micro-patterns). In some embodiments, instead of DNA origami placement, the reactive groups of the supramolecular nanostructure 40 are bound to pre-organized DNA origamis on the binding sites. In some embodiments, both of these methods for binding the supramolecular nanostructure to the corresponding binding sites rely on the function of organizing one or more molecules using DNA origami placement techniques on the binding sites of the micro-pattern placement. In some embodiments, the planar substrate can be considered to be stored in a clean environment for a significant period of time after this step.

[0069] Continuing with reference to FIG. 11, in some embodiments, in the sample capture step, a sample (described herein) comprising analyte molecules contacts a planar substrate (step 2). In some embodiments, the sample contacts the planar substrate using a flow cell. In some embodiments, the sample is cultured on a planar substrate to which a supramolecular structure is attached at the binding site 66. In some embodiments, the culture period can be from about 30 seconds to about 24 hours. In some embodiments, the culture period can be from about 30 seconds to about 1 minute, from about 1 minute to about 5 minutes, from about 5 minutes to about 30 minutes, from about 30 minutes to about 1 hour, from about 1 hour to about 5 hours, from about 5 hours to about 12 hours, from about 12 hours to about 24 hours, from about 24 hours to about 48 hours.

[0070] In some embodiments, analyte molecules 44 in the sample interact with supramolecular structures 40 on the flat surface 60. In some embodiments, a single replica of a particular analyte molecule 44 binds to a capture molecule. Continuing with reference to FIG. 11, in step 3, a detector molecule assembly 46 contacts the captured analyte. In step 4, the detector molecule assembly is detected and a detectable signal is generated. For example, the detector barcode 21 is used as a binding site for a signal transmitting element 76 that contacts the bound detector molecule assembly 46. In some embodiments, the signal transmitting element 76 comprises a fluorescent molecule or microbeads, fluorescent polymers, highly charged nanoparticles or polymers. In some embodiments, one or more signal transmitting elements 76 are left to interact with the supramolecular structures on the planar structure. In some embodiments, the signal transmitting element 76 is introduced into a flow cell comprising a planar substrate. In some embodiments, as shown in the depicted embodiment, the detector barcode is amplified. For example, the detector barcode is used as a polymerization initiator for the growth of highly fluorescent polymers by a process such as rolling circle amplification or hybridization chain reaction. In some embodiments, the detectable signal described in step 4 results in a surface where the signal transmitting element 76 is present at the location of each analyte (linked to the capture molecule and the detector molecule), as a result of all individual analyte capture events.

[0071] In some embodiments, the signal transmitting element 76 has optical activity and can be measured using a microscope or an integrated optical sensor within the planar substrate 60. In some embodiments, the signal transmitting element has electrical activity and can be measured using an integrated electrical sensor. In some embodiments, the signal transmitting element 76 has magnetic activity and can be measured using an integrated magnetic sensor. In some embodiments, each signal event relates to the capture of the same analyte molecule (a single replica of the same analyte molecule) and is determined by the corresponding detector molecule and capture molecule, and thus, the step of counting the number of locations where the signal transmitting element 76 is present provides quantification of the analyte molecules in the sample.

[0072] FIG. 12 illustrates an arrangement having a planar substrate 60 with coupling sites 66 similar to those of FIG. 11. The planar substrate 60 has assembled supramolecular structures 40 immobilized (step 1) at respective coupling sites 66. After analyte capture (step 2), the detector molecule assembly contacts the captured analyte molecule 44. In this case, each detector molecule assembly 46 comprises a coupled core structure (e.g., core structure 13, see FIG. 1) such as a DNA origami that acts as an integral signal transmitting element 76. FIG. 13 illustrates an arrangement having a planar substrate 60 (which can be formed as described above with respect to FIG. 11) having assembled supramolecular structures 40 immobilized (step 1) at respective coupling sites 66. Individual processing associates analyte 44 with respective detector molecule assemblies 46. In some embodiments, each detector molecule assembly 46 holds a signal element 76 such as a supramolecular core structure. The associated analyte and detector molecule assembly 46 are cultured with the planar substrate 60 and the immobilized supramolecular structures 40. The associated analyte and detector molecule assembly 46 bind to a supramolecular structure having a capture molecule with binding specificity for the analyte, and the bound analyte can be characterized, for example, by detecting a signal generated from a signal transmitting element (which can be part of the detector molecule assembly 46 or added after the association of the analyte and detector molecule assembly 46 with the capture molecule).

[0073] In FIG. 14, a patterned substrate 60 having a plurality of binding sites 66 can be functionalized or linked to with individual supramolecular structures 40. In the depicted embodiment, the supramolecular structures 40 are assembled in step 80 and then placed on the substrate 60 before the antigen capture molecule 2 is linked. For example, a DNA origami with a specific barcode and a capture strand specific for a particular antigen are flowed over the substrate 60 and placed within a single molecule array, e.g., on a DNA binding feature. The antigen is conjugated to include a complementary strand shown as capture molecule 2 and can be annealed to the DNA origami barcode 20 of the support structure in step 82. Alternatively, the capture molecule is associated with the supramolecular structure 40 during assembly (step 80) and added to the substrate 60 along with the supramolecular structure 40. The barcode 20 of each supramolecular structure is read out by either sequence analysis, amplification, or hybridization assay. A map of the antigen capture molecules 2 having spatial locations on the substrate 60 is obtained prior to performing the assay and can be carried out as a quality control of the substrate 60. The map is stored in the specimen detection system (see FIG. 16) provided herein and can be used to generate a report of positive binding events to provide diagnostic information.

[0074] Appropriate inhibitory conditions are added and then a sample (e.g., serum) containing the patient's antibodies is added to the substrate 60 and, in step 84, the antibody analyte 44 in the sample is cultured to form an antibody-antigen complex. The sample is then washed and, in step 86, a detector molecule assembly 46 comprising a detector molecule 1 which is a secondary anti-human antibody and a label for detection is added. Detection of the bound detector structure 50 (step 88) may be based on detection of an antibody label which can be DNA-based for amplification by either rolling circle amplification or hybridization chain reaction. Alternatively, the label can be a DNA nanoparticle or a fluorescent polymer.

[0075] In an embodiment, the assay includes an antigen of one or more of adenovirus, coronavirus 229E, coronavirus HKU1, coronavirus B.1.1.7, coronavirus B.1.351, coronavirus P.1, coronavirus NL63, coronavirus OC43, human metapneumovirus, human rhinovirus / enterovirus, influenza A, its subtype 2009H1N1, H1, H3, influenza B, type 1, type 2, type 3, and type 4 parainfluenza virus, respiratory syncytial virus, Chlamydia pneumoniae, Mycoplasma pneumoniae, and Bordetella pertussis. In addition to this, the assay can include a vaccine target for seasonal influenza and a covid vaccine target for one or more commercially available vaccines (Moderna, Pfizer, Astra Zeneca, Novavax, and Johnson and Johnson). The addition of these vaccine targets enables the classification of immunity to determine whether a patient has either natural infection-derived immunity and / or acquired immunity from a vaccine. Multiple antigens for each pathogen can be present to define the specificity of the immunity.

[0076] The detection can include an anti-species to a specific antibody for the determination of IgG, IgM, and / or IgA. The specific detection of subtypes helps to further understand the maturity of the immunity.

[0077] FIG. 15 provides an exemplary embodiment for forming a hydrogel matrix 100 that introduces one or more supramolecular structures 40 in addition to combining one or more monomers 122 and one or more crosslinking molecules 124 to form a hydrogel. In some embodiments, one or more supramolecular structures 40 copolymerize with the hydrogel matrix to form the matrix 120. In some embodiments, each respective anchor molecule 18 of one or more supramolecular structures 40 copolymerizes with the hydrogel matrix 120. In some embodiments, one or more monomers 122 comprise acrylamide. In some embodiments, one or more crosslinking agents comprise bisacrylamide.

[0078] Embodiments of the disclosure include one or more computer-implemented detection systems configured to perform certain methods of the disclosure embodiments. FIG. 16 illustrates a specimen detection system 1000 including a controller 1001. The controller 1001 includes a processor 1002 and a memory 1004 storing instructions configured to be executed thereby. The controller 1001 includes, for example, a user interface 1006 and communication circuitry to facilitate communication over the Internet 1010 and / or a wireless or wired network. The user interface 1006 facilitates user interaction with the characterization of the specimen detection results provided herein.

[0079] The processor 1002 is programmed to receive specimen detection data and characterize the detected specimen. In one embodiment, the processor generates a report of the detected specimen in the sample after culturing using an array of supramolecular structures and detecting a detector molecule assembly. The report can include data of generated optical signals at various binding sites corresponding to the detected specimen binding events. The report can include processed data such as a list of detected specimens or positive / negative binding results. The report can include a list of available capture molecules of the array indicating the specimen detection function.

[0080] The system 1000 also includes a specimen detector 1020 operative to detect specimen binding by detecting one or more components of the supramolecular structure. The specimen detector 1020 includes a detection system having one or more sensors 1022. The specimen detector 1020 can also include a reaction controller 1024 to control culturing of the sample and appropriate release of reaction reagents and detector molecule assemblies at appropriate times. The sensor 1022 can be one or more of an optical sensor (e.g., a fluorescence sensor, an infrared sensor), an image sensor, an electrical sensor, or a magnetic sensor. In an embodiment, the sensor 102 is a metal oxide semiconductor imaging sensor device.

[0081] Although the preferred embodiments of the present invention have been shown and described in this specification, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, modifications, and substitutions will occur to those skilled in the art here without departing from the present invention. It must be understood that various alternatives to the embodiments of the present invention described in this specification can be used to practice the present invention. The following claims are intended to define the scope of the present invention and thereby to cover methods and structures within the scope of these claims and their equivalents.

Explanation of Reference Numerals

[0082] 2 capture molecule 13 core structure 20 capture barcode 40 supramolecular structure 44 captured analyte molecule 46 detector molecule assembly

Claims

1. An array for detecting one or more analyte molecules in a sample, comprising: A substrate; A plurality of supramolecular structures immobilized on the substrate, each individual supramolecular structure of the plurality of supramolecular structures comprising: (a) A core structure comprising a plurality of core molecules, the core structure being bound to the substrate or linked to the substrate by an anchor molecule; (b) A capture barcode directly or indirectly bound to the core structure at a first end of the capture barcode and extending away from the substrate as a whole; (c) An analyte molecule bound to the supramolecular structure; and (d) A capture molecule bound to the capture barcode at a second end of the capture barcode, the capture molecule being configured to bind to the analyte molecule; (e) A detector molecule assembly configured to bind to the analyte molecule and comprising a detector molecule bound to another core structure by a detector barcode; The plurality of supramolecular structures; and An array characterized by comprising the same.

2. The array according to claim 1, wherein each core structure of the plurality of supramolecular structures is identical to each other.

3. The array according to claim 1, wherein each supramolecular structure has a unique capture barcode.

4. The array according to claim 1, wherein the substrate comprises a solid structure or a porous matrix.

5. The array according to claim 1, wherein each supramolecular structure is a nanostructure.

6. The array according to claim 1, wherein each core structure is a nanostructure.

7. The array according to claim 1, wherein the plurality of core molecules for each core structure are arranged in a predetermined shape and / or have a specified molecular weight.

8. The array according to claim 1, wherein the core structure is directly bonded to the substrate.

9. The array according to claim 1, comprising a plurality of analyte molecules bonded to each supramolecular structure of the plurality of supramolecular structures.

10. The array according to claim 9, comprising a plurality of detector molecule assemblies bonded to each analyte molecule of the plurality of analyte molecules.

11. The array according to claim 10, wherein each detector molecule comprises one or more linkers.

12. The array according to claim 1, wherein the substrate comprises a porous matrix.

13. The array according to claim 12, wherein the porous matrix comprises a hydrogel.

14. The array according to claim 1, wherein the substrate comprises a planar substrate.

15. The array according to claim 1, wherein each individual supramolecular structure comprises only one capture molecule.

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