Substrate for single molecule organization

Abstract

To provide a substrate for single molecule organization.SOLUTION: Provided herein are structures and methods for detecting one or more analyte molecules 44 present in a sample. In some embodiments, the one or more analyte molecules 44 are detected using one or more supramolecular structures 40 that are coupled to a substrate, e.g., a solid support. In some embodiments, the supramolecular structures 40 are bi-stable such that the supramolecular structures 40 transition from an unstable state to a stable state through interaction with one or more analyte molecules 44 from the sample. In some embodiments, the stable state supramolecular structures 40 are configured to provide a signal for analyte molecule detection and quantification.SELECTED DRAWING: Figure 14

Description

[Technical field]

[0001] The present invention relates to substrates for single molecule assembly. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 119,316, entitled "SUBSTRATE FOR SINGLE MOLECULE ORGANIZATION," filed November 30, 2020, the disclosure of which is hereby incorporated by reference in its entirety. [Background technology]

[0002] The current state of personalized healthcare is overwhelmingly genomic-centric, focusing primarily on quantifying the genes present within an individual. Although such approaches have proven extremely powerful, they do not provide clinicians with a complete picture of an individual's health. This is because genes are an individual's "blueprints" and only inform the likelihood of developing a disease. Within an individual, these "blueprints" must first be transcribed into RNA and then translated into various protein molecules that are the actual "actors" within the cells, in order to have any effect on the individual's health.

[0003] The concentrations of proteins, interactions between proteins (protein-protein interactions or PPIs), and interactions between proteins and small molecules are intricately linked to the health of different organs, homeostatic regulatory mechanisms, and the interactions of these systems with the external environment. Thus, quantitative information on proteins, as well as PPIs, is crucial to generate a complete picture of an individual's health at a given time, as well as to predict any emerging health problems. For example, the amount of stress experienced by the myocardium (e.g., during a heart attack) can be inferred by measuring the concentrations of troponin I / II and myosin light strands present in peripheral blood. Similar protein biomarkers have also been identified, validated, and marketed for a wide range of organ dysfunctions (e.g., liver disease and thyroid disorders), certain cancers (e.g., colorectal or prostate cancer), and infectious diseases (e.g., HIV and Zika). These protein-protein interactions are also essential for drug development and are becoming an increasingly sought-after dataset. The ability to detect and quantify proteins and other molecules within a given sample of bodily fluids is an essential component of such healthcare advances. Summary of the Invention [Means for solving the problem]

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

[0005] Provided herein, in some embodiments, is a method for detecting analyte molecules present in a sample, the method comprising the steps of: a) providing a solid support; b) patterning the solid support to form a plurality of binding sites on the solid support; and c) associating a single supramolecular structure with a binding site from among the plurality of binding sites on the solid support, wherein the supramolecular structure comprises: i) a core structure comprising a plurality of core molecules; ii) a capture molecule linked to the core structure at a first location; and iii) a detector molecule linked to the core structure at a second location, wherein the supramolecular structure is in an unstable state such that the detector molecule is configured to be released from the core structure through scission of a link therebetween at the second location.

[0006] Provided herein, in some embodiments, is a method for detecting an analyte molecule using a substrate, the method comprising providing a substrate comprising a plurality of binding sites, each of the plurality of binding sites being associated with a supramolecular structure of a plurality of supramolecular structures, the supramolecular structure comprising a core structure comprising a plurality of core molecules, a capture molecule linked to the core structure at a first location, and a detector molecule linked to the core structure at a second location, the supramolecular structure being in an unstable state such that the detector molecule is configured to be released from the core structure through scission of a link therebetween at the second location; and extracting a sample such that the supramolecular structure shifts from the unstable state to a stable state. The method comprises contacting the supramolecular structure, wherein the detector molecules and the capture molecules are linked to each other through binding to the analyte molecules, thereby forming a link between the detector molecules and the capture molecules, and each supramolecular structure in an unstable state comprises a respective capture molecule and a detector molecule spaced apart by a predetermined distance, providing a trigger at a second location to sever the link between the detector molecules and the core structure, wherein the detector molecules remain linked to the core structure through their linkage with the capture molecules and such that the analyte molecules are associated with their respective binding sites, and detecting the analyte molecules based on a signal provided by the supramolecular structure shifted to a stable state. In other embodiments, the supramolecular structure does not comprise an integral detector molecule and detection is performed via a separate step.

[0007] Provided herein, in some embodiments, is a method of forming a substrate for detection of analyte molecules in a sample, the method includes providing a base layer, providing a binding layer on the base layer, depositing a top layer on the binding layer, patterning the top layer to expose portions of the binding layer corresponding to a plurality of binding sites on the binding layer, and providing a supramolecular structure associated with each binding site of the plurality of binding sites, the supramolecular structure comprising a core structure comprising a plurality of core molecules, a capture molecule linked to the core structure at a first location, and a detector molecule linked to the core structure at a second location, the supramolecular structure being in an unstable state such that the detector molecule is configured to be released from the core structure through severing a link between them at the second location and the respective capture molecules and detector molecules are at a predetermined distance in the unstable state, and in a stable state, the detector molecule and the capture molecule are linked to each other through binding to the analyte molecule, thereby forming a link between the detector molecule and the capture molecule, and the detector molecule remains linked to the core structure through linkage with the capture molecule and such that the analyte molecule is associated with each binding site.

[0008] In some embodiments, any of the methods disclosed herein further comprises quantifying the concentration of the analyte molecule in the sample. In some embodiments, any of the methods disclosed herein further comprises identifying the detected analyte molecule. In some embodiments, any of the methods disclosed herein further comprises detecting the analyte molecule based on a signal when the analyte molecule is present in the sample in single molecule or higher order numbers. In some embodiments, the solid support is coupled to or associated with a detection system that generates a signal based on a detectable change indicative of the presence or formation of a steady state as a result of analyte capture or binding by the supramolecular structure at the individual binding sites.

[0009] In some embodiments, for any of the methods 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 a complex biological sample. In some embodiments, for any of the methods disclosed herein, the analyte molecule comprises a protein, a peptide, a peptide fragment, a lipid, DNA, RNA, an organic molecule, an inorganic molecule, a complex thereof, or any combination thereof. In some embodiments, for any of the methods disclosed herein, each supramolecular structure is a nanostructure.

[0010] 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 multiple core molecules for each core structure are arranged in a predetermined shape and / or have a specified 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 multiple core molecules for each core structure comprise one or more nucleic acid strands, one or more branched nucleic acids, one or more peptides, one or more small molecules, or a combination thereof. In some embodiments, for any of the methods disclosed herein, each core structure independently comprises a scaffolded deoxyribonucleic acid (DNA) origami, a scaffolded ribonucleic acid (RNA) origami, a scaffolded hybrid DNA:RNA origami, a single-stranded DNA tiling structure, a multi-stranded DNA tiling structure, a single-stranded RNA origami, a multi-stranded RNA tiling structure, a hierarchically organized DNA or RNA origami with multiple scaffolds, a peptide structure, or a combination thereof.

[0011] In some embodiments, for any of the methods disclosed herein, the trigger comprises a deconstruction molecule, a trigger signal, or a combination thereof. In some embodiments, the deconstruction molecule comprises DNA, RNA, a peptide, a small organic molecule, or a combination thereof. In some embodiments, the trigger signal comprises an optical signal, an electrical signal, or both. In some embodiments, the trigger optical signal comprises a microwave signal, ultraviolet illumination, visible illumination, near infrared illumination, or a combination thereof.

[0012] In some embodiments, for any method disclosed herein, each analyte molecule is 1) bound to a capture molecule of each supramolecular structure through a chemical bond, and / or 2) bound to a detector molecule of each supramolecular structure through a chemical bond. In some embodiments, for any method disclosed herein, the capture molecule and detector molecule for each supramolecular structure independently comprise a protein, peptide, antibody, aptamer (RNA and DNA), fluorophore, darpin, catalyst, polymerization initiator, polymer such as PEG, or combinations 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 comprises a first capture linker, a second capture linker, and a capture bridge disposed between the first and second capture linkers, the first capture linker being attached to a first core linker attached to a first location on the core structure, and the capture molecule and the second capture linker being linked to each other through binding to the third capture linker; and b) the detector molecule is linked to the core structure through a detector barcode, the detector barcode comprises a first detector linker, a second detector linker, and a detector bridge disposed between the first and second detector linkers, the first detector linker being attached to a second core linker attached to a second location on the core structure, and the detector molecule and the second detector linker being linked to each other through binding to the third detector linker. In some embodiments, the capture bridge and the detector bridge independently comprise a polymer core. In some embodiments, the polymer core of the capture bridge and the polymer core of the detector bridge independently comprise a polymer such as a nucleic acid (DNA or RNA) of a specific sequence or PEG, in some embodiments, the first core linker, the second core linker, the first capture linker, the second capture linker, the third capture linker, the first detector linker, the second detector linker, and the third detector 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, DBCO, maleimides, biotin, azides, acrydites, NHS-esters, single stranded nucleic acids (RNA or DNA) of specific sequences, PEG or polymerization initiators, or combinations thereof. In some embodiments, the link 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, the link between the detector barcode and 1) the second core linker and / or 2) the third detector linker comprises a chemical bond. In some embodiments, the chemical bond comprises a covalent bond. In some embodiments, the trigger cleaves the linkage between 1) the first detector linker and the second core linker and / or 2) the first capture linker and the first core linker. In some embodiments, for any of the methods disclosed herein, the capture molecule is attached to the third capture linker through a chemical bond and / or the detector molecule is attached to the third detector linker through a chemical bond, hi some embodiments, the capture molecule is covalently attached to the third capture linker and / or the detector molecule is covalently attached to the third detector linker.

[0013] In some embodiments, for any method disclosed herein, each supramolecular structure in the unstable state comprises respective capture and detector molecules spaced apart by a predetermined distance to reduce or prevent the occurrence of cross-reaction between the capture and / or detector molecules of the first supramolecular structure and the corresponding capture and / or detector molecules of the second supramolecular structure. In some embodiments, for any method disclosed herein, the predetermined distance is about 3 nm to about 40 nm.

[0014] In some embodiments, for any of the methods disclosed herein, each supramolecular structure further comprises an anchor molecule linked to the core structure. In some embodiments, the anchor molecule is linked to the core structure through an anchor barcode, the anchor barcode comprising a first anchor linker, a second anchor linker, and an anchor bridge disposed between the first and second anchor linkers, the first anchor linker being attached to a third core linker attached 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 one or more polymers, such as amines, thiols, DBCO, maleimides, biotin, azides, acrydites, NHS-esters, single-stranded nucleic acids (RNA or DNA) of specific sequences, PEG, or polymerization initiators, or combinations thereof. In some embodiments, the anchor bridge comprises a polymer core. In some embodiments, the polymer core of the anchor bridge comprises a polymer, such as a nucleic acid (DNA or RNA) of specific sequences or PEG. In some embodiments, the third core linker, the first anchor linker, the second anchor linker, and the anchor molecule independently comprise an anchor reactive molecule or a DNA sequence domain. In some embodiments, each anchor reactive molecule independently comprises one or more polymers, such as amines, thiols, DBCO, maleimides, biotin, azides, acrydites, NHS-esters, single-stranded nucleic acids (RNA or DNA) of a specific sequence, PEG, or polymerization initiators, or combinations 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 attached to the second anchor linker. In some embodiments, the trigger further cleaves 1) the second anchor linker from the anchor molecule, 2) the first anchor linker from the third core linker, or combinations thereof. In some embodiments, the first and second locations are located on a first side of the core structure, and the third location is located on a second side of the core structure.

[0015] In some embodiments, for any method disclosed herein, the signal comprises a detector barcode, a capture barcode, or a combination thereof, corresponding to the supramolecular structure shifted to a stable state. In some embodiments, any method disclosed herein further comprises separating each detector barcode for at least one supramolecular structure shifted to a stable state from a corresponding detector molecule such that the corresponding signal comprises a respective detector barcode for detection of the analyte molecule bound to the respective capture and detector molecule. In some embodiments, each separated detector barcode provides a DNA signal corresponding to the analyte molecule bound to the respective detector molecule. In some embodiments, the at least one separated detector barcode is analyzed using genotyping, qPCR, sequence analysis, or a combination thereof. In some embodiments, multiple analyte molecules in a sample are simultaneously detected through multiplexing through one or more supramolecular structures shifted to a stable state. In some embodiments, for any method disclosed herein, the capture molecule and detector molecule for each supramolecular structure are configured to bind to one or more specific types of analyte molecules.

[0016] In some embodiments, for any method comprising using a plurality of supramolecular structures disclosed herein, each core structure of the plurality of supramolecular structures is identical to each other. In some embodiments, each supramolecular structure comprises a specified shape, size, molecular weight, or combination thereof to reduce or eliminate cross-reactivity between the plurality of supramolecular structures. In some embodiments, each supramolecular structure comprises a plurality of capture molecules and detector molecules. In some embodiments, each supramolecular structure comprises a specified stoichiometric ratio of capture molecules and detector molecules to reduce or eliminate cross-reactivity between the plurality of supramolecular structures.

[0017] In some embodiments, for any method comprising using a plurality of supramolecular structures disclosed herein, the unstable state for each supramolecular structure further comprises the capture and detector molecules spaced apart at a predetermined distance to reduce or inhibit the occurrence of cross-reaction between the capture and / or detector molecules of the first supramolecular structure and the capture and / or detector molecules of the second supramolecular structure. In some embodiments, the predetermined distance is about 3 nm to about 40 nm. In some embodiments, the average distance between any two supramolecular structures is greater than the predetermined distance between the capture and detector molecules of each supramolecular structure. In some embodiments, the average distance between any two supramolecular structures is greater than the predetermined distance between the capture and detector molecules of each supramolecular structure. In some embodiments, the plurality of supramolecular structures are attached to one or more solid substrates. In some embodiments, each solid substrate of the one or more solid substrates comprises a planar substrate and / or comprises a planar substrate having a patterned or shaped surface having an array of binding sites. In some embodiments, the plurality of supramolecular structures are disposed on a substrate, the substrate comprising a plurality of binding sites, each binding site being associated with a corresponding supramolecular structure. In some embodiments, each individual binding site of the substrate's binding site array is associated with at most one supramolecular structure, e.g., only one supramolecular structure per binding site. In some embodiments, the plurality of supramolecular structures distributed on the substrate are configured to detect the same analyte molecule. In some embodiments, the plurality of signaling elements are configured to link with detector molecules of at least one supramolecular structure shifted to a stable state. In some embodiments, each signaling element comprises a fluorescent molecule or microbead, a fluorescent polymer, a highly charged nanoparticle, or a highly charged polymer. In some embodiments, at least one supramolecular structure of the plurality of supramolecular structures on the substrate is configured to detect a different analyte molecule than at least one of the other supramolecular structures. In some embodiments, each individual supramolecular structure is barcoded with a unique barcode that distinguishes each supramolecular structure on the substrate to identify the location of each supramolecular structure on the substrate.In some embodiments, the signaling elements are linked to at least one detector molecule of the supramolecular structure that has shifted to a stable state, in some embodiments, each signaling element comprises a fluorescent molecule or microbead, a fluorescent polymer, a highly charged nanoparticle, or a highly charged polymer.

[0018] Provided herein is a method of forming a substrate, the method comprising providing a base layer, providing a binding layer on the base layer, depositing a top layer on the binding layer, patterning the top layer to expose portions of the binding layer corresponding to a plurality of binding sites on the binding layer, and associating a molecule or structure at each binding site. In some embodiments, the method includes providing a supramolecular structure associated with each binding site of the plurality of binding sites, the supramolecular structure comprising a core structure comprising a plurality of core molecules. The core structure can be linked to one or more cargo molecules that have affinity for or bind to the analyte. The techniques of the present invention can be generally used to assemble single molecules, e.g., one or more core structures of supramolecular structures, i.e., find applications in a variety of different fields. In embodiments, the techniques of the present invention can be generally used to assemble single quantum dots. In some embodiments, any of the methods disclosed herein can be performed with an additional step of regenerating the formed substrate after analyte detection is completed. Regeneration may include removing all or a portion of the supramolecular structure.

[0019] In some embodiments, an analyte or analyte molecule is detected in a sample. The analyte may comprise a bioparticle or a biomolecule. In some embodiments, the analyte molecule comprises a chemical compound, a protein, a peptide, a peptide fragment, a lipid, a nucleic acid, DNA, RNA, an organic molecule, a viral particle, an exosome, a cellular organelle, or a complex of any of these. In some embodiments, the sample comprises a tissue biopsy, blood, plasma, urine, saliva, tears, cerebrospinal fluid, extracellular fluid, cultured cells, culture medium, discarded tissue, plant material, synthetic proteins, bacterial and / or viral samples, or fungal tissue, or a combination thereof.

[0020] Provided herein, in some embodiments, is a substrate for detecting one or more analyte molecules in a sample, the substrate comprising a plurality of binding sites associated with a respective plurality of supramolecular structures, each supramolecular structure comprising: a) a core structure comprising a plurality of core molecules; b) a capture molecule linked to the supramolecular core at a first location; and c) a detector molecule linked to the supramolecular core at a second location, the supramolecular structure being in an unstable state such that the detector molecule is configured to be released from the core structure through scission of a link therebetween at the second location, each supramolecular structure being configured to shift from the unstable state to a stable state upon interaction between the detector molecule, the capture molecule, and a respective analyte molecule of the one or more analyte molecules, and upon interaction with a trigger, each supramolecular structure shifted to the stable state provides a signal for detecting a respective analyte molecule.

[0021] Provided herein, in some embodiments, is a substrate for detecting one or more analyte molecules in a sample, the substrate comprising a base layer, a binding layer on the base layer, a patterned top layer exposing portions of the binding layer corresponding to a plurality of binding sites on the binding layer, and a supramolecular structure associated with each binding site of the plurality of binding sites, the supramolecular structure comprising a core structure comprising a plurality of core molecules, a capture molecule linked to the supramolecular core at a first location, and a detector molecule linked to the supramolecular core at a second location, the supramolecular structure being in an unstable state such that the detector molecule is configured to be released from the core structure through scission of a link therebetween at the second location, and each supramolecular structure being configured to shift from the unstable state to a stable state through an interaction between the detector molecule, the capture molecule, and a respective analyte molecule of the one or more analyte molecules.

[0022] In some embodiments, upon interaction with the trigger, each detector molecule linked to the unstable supramolecular structure becomes released from the supramolecular structure. In some embodiments, each core structure of the plurality of supramolecular structures is identical to each other. In some embodiments, the average distance between any two supramolecular structures is greater than the predetermined distance between the capture molecule and the detector molecule of each supramolecular structure. In some embodiments, the substrate comprises a solid support or a solid substrate.

[0023] In some embodiments, each core structure of the plurality of supramolecular structures is identical to each other. In some embodiments, each supramolecular structure has a specified shape, size, molecular weight, or combination thereof to reduce or eliminate cross-reaction between the plurality of supramolecular structures. In some embodiments, each supramolecular structure has a specified stoichiometric ratio of capture molecules and detector molecules to reduce or eliminate cross-reaction between the plurality of supramolecular structures. In some embodiments, the unstable state for each supramolecular structure further comprises the capture molecules and detector molecules spaced apart by a predetermined distance to reduce or inhibit the occurrence of cross-reaction between the capture molecules and / or detector molecules of the first supramolecular structure and the capture molecules and / or detector molecules of the second supramolecular structure. In some embodiments, the predetermined distance is about 3 nm to about 40 nm. In some embodiments, the average distance between any two supramolecular structures is greater than the predetermined distance between the capture molecules and detector molecules of each supramolecular structure.

[0024] In some embodiments, each substrate comprises a widget, a solid support, a polymer matrix, a solid substrate, or a molecular condensate. In some embodiments, the average distance between any two supramolecular structures is greater than a predetermined distance between the capture molecule and the detector molecule of each supramolecular structure. In some embodiments, the solid substrate comprises a planar substrate. In some embodiments, the plurality of supramolecular structures are disposed on a substrate, the substrate comprising a plurality of binding sites, each binding site configured to link with a corresponding supramolecular structure. In some embodiments, the plurality of supramolecular structures are configured to detect the same analyte molecule. In some embodiments, the plurality of signaling elements are configured to link with the detector molecule of at least one supramolecular structure shifted to a stable state. In some embodiments, each signaling element comprises a fluorescent molecule or microbead, a fluorescent polymer, a highly charged nanoparticle, or a highly charged polymer. In some embodiments, at least one supramolecular structure of the plurality of supramolecular structures is configured to detect a different analyte molecule than the other supramolecular structures.

[0025] In some embodiments, the supramolecular structure comprises a specified shape, size, molecular weight, or combination thereof to reduce or eliminate cross-reaction with another supramolecular structure and / or to align with the size and shape of the binding site in the substrate. In one example, the dimensions of the supramolecular structure in the xy plane are selected to match or overlap with the size and shape of the binding site. To facilitate the association of only one supramolecular structure with one binding site, the binding site and the supramolecular structure can be sized and shaped such that two supramolecular structures cannot fit onto a single binding site. In some embodiments, the supramolecular structure can be smaller than an individual binding site in at least one dimension when associated with the binding site. In some embodiments, the supramolecular structure comprises multiple capture and detector molecules. In some embodiments, the supramolecular structure comprises a specified stoichiometric ratio of capture molecules and detector molecules to reduce or eliminate cross-reaction with another supramolecular structure.

[0026] Specific embodiments of the disclosed devices, delivery systems, or methods will now be described below with reference to the drawings. Nothing in this detailed description is intended to imply that any particular component, feature, or step is essential to the invention. [Brief description of the drawings]

[0027] [Figure 1A] FIG. 1 depicts an exemplary supramolecular structure and associated subcomponents. [Figure 1B] FIG. 1 depicts an exemplary supramolecular structure and associated subcomponents. [Diagram 2] FIG. 1 depicts an exemplary three-arm nucleic acid junction-based assembled supramolecular structure and associated subcomponents. [Diagram 3] FIG. 3 depicts exemplary individual subcomponents of the three-arm nucleic acid junction-based supramolecular structure of FIG. 2. [Figure 4] FIG. 3 depicts exemplary deconstructed molecules corresponding to the sub-components of the three-arm nucleic acid junction-based supramolecular structure of FIG. 2. [Diagram 5] FIG. 1 depicts an exemplary DNA origami-based assembled supramolecular structure and associated subcomponents. [Figure 6] FIG. 6 depicts exemplary individual subcomponents of the DNA origami-based supramolecular structure of FIG. 5. [Figure 7] FIG. 6 depicts exemplary deconstructed molecules corresponding to sub-components of the DNA origami-based supramolecular structure of FIG. 5. [Figure 8] 1 is an exemplary illustration of a supramolecular structure in an unstable state before and after being subjected to a trigger (eg, interaction with a deconstruction molecule). [Figure 9] FIG. 1 is an exemplary diagram of a supramolecular structure in a stable state before and after being subjected to a trigger (eg, interaction with a deconstruction molecule). [Figure 10] FIG. 13 is an exemplary diagram of a supramolecular structure shifting from an unstable to a stable state after interaction with an analyte molecule, as well as the respective configurations before and after receiving a trigger (e.g., interaction with a deconstruction molecule). [Figure 11] FIG. 13 is an exemplary diagram of a supramolecular structure shifting from a stable to an unstable state after interaction with an analyte molecule, as well as the respective configurations before and after receiving a trigger (e.g., interaction with a deconstruction molecule). [Figure 12] FIG. 1 is an exemplary illustration of a method for detecting and quantifying analyte molecules using multiple supramolecular structures. [Figure 13] FIG. 1 is an exemplary illustration of a method for detecting and quantifying analyte molecules using multiple supramolecular structures attached to a planar substrate. [Figure 14] FIG. 1 is an exemplary illustration of a technique for forming arrays of binding sites on a substrate for supramolecular structures including DNA origami. [Figure 15] FIG. 1 is an exemplary illustration of a technique for forming an array of binding sites for supramolecular structures on a substrate. [Figure 16] FIG. 1 is an exemplary illustration of a technique for forming an array of binding sites for supramolecular structures on a substrate. [Figure 17] FIG. 1 is an exemplary illustration of a technique for forming an array of binding sites for supramolecular structures on a substrate. [Figure 18] FIG. 2 is an exemplary diagram of a substrate coupled to a detection system. [Figure 19] 1 is an exemplary diagram of a substrate coupled to a field effect transistor detection system. [Figure 20] FIG. 2 is an exemplary diagram of a substrate coupled to an optical detection system. [Figure 21] FIG. 1 illustrates an exemplary diagram of a workflow for reclaiming a substrate. [Figure 22] FIG. 1 is an exemplary illustration of a technique for forming arrays of binding sites on a substrate for supramolecular structures including DNA origami. [Figure 23] FIG. 1 is an exemplary illustration of a technique for forming arrays of binding sites on a substrate for supramolecular structures including DNA origami. [Figure 24] FIG. 1 is an exemplary illustration of a technique for forming arrays of binding sites on a substrate for supramolecular structures including DNA origami. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Provided herein are structures and methods for detecting one or more analyte molecules present in a sample. In some embodiments, one or more analyte molecules are detected using one or more supramolecular structures bound to a substrate, e.g., a solid support. The solid support can include multiple binding sites that receive the supramolecular structures at their respective binding sites. The substrate thus allows for a single molecular organization for the supramolecular structures. In one example, each binding site of the multiple binding sites is associated with a single supramolecular structure. An analyte can be detected at one or more binding sites of the multiple binding sites. Based on the patterning and arrangement of the supramolecular structures on the substrate and the properties, particularly the components, of each supramolecular structure, a desired analytical function can be generated.

[0029] In some embodiments, the one or more supramolecular structures are specifically designed to minimize cross-reactivity with each other. In some embodiments, the supramolecular structures are bi-stable and shift from an unstable state to a stable state through interaction with one or more analyte molecules from a sample. In some embodiments, the supramolecular structures in a stable state as a result of the transition to a stable state are configured to provide a signal for detection and / or quantification of the analyte molecules. In some embodiments, the signal is an electrical signal, an optical signal, an electromagnetic signal, or a DNA signal, such that the detection and quantification of the analyte molecules comprises converting the presence of the analyte molecules into a DNA signal. The detection system associated with the substrates provided herein is configured to generate a signal that can be attributed to the individual binding sites.

[0030] sample In some embodiments, the sample comprises an aqueous solution comprising proteins, peptides, peptide fragments, lipids, DNA, RNA, organic molecules, inorganic molecules, complexes thereof, or any combination thereof. In some embodiments, the analyte molecules in the sample comprise proteins, peptides, peptide fragments, lipids, DNA, RNA, organic molecules, inorganic molecules, complexes thereof, or any combination thereof. In some embodiments, the analyte molecules comprise intact proteins, denatured proteins, partially or fully degraded proteins, peptide fragments, denatured nucleic acids, degraded nucleic acid fragments, complexes thereof, or combinations thereof. In some embodiments, the sample is obtained from tissues, cells, the environment of the tissues and / or cells, or combinations thereof. In some embodiments, the sample comprises tissue biopsy, blood, plasma, urine, saliva, tears, cerebrospinal fluid, extracellular fluid, cultured cells, culture medium, discarded tissue, plant material, synthetic proteins, bacterial samples, viral samples, fungal tissues, or combinations thereof. In some embodiments, the sample is isolated with or without purification from a primary source such as a cell, tissue, bodily fluid (e.g., blood), environmental sample, or combinations thereof. In some embodiments, cells are lysed using mechanical treatment or other cell lysis techniques (e.g., lysis buffer). In some embodiments, samples are filtered using mechanical treatment (e.g., centrifugation), micron filtration, chromatography columns, other filtration techniques, or combinations thereof. In some embodiments, samples are treated using one or more enzymes to remove one or more nucleic acids or one or more proteins. In some embodiments, samples comprise intact proteins, denatured proteins, partially or fully degraded proteins, peptide fragments, denatured nucleic acids, or degraded nucleic acid fragments. In some embodiments, samples are taken from one or more individuals, one or more animals, one or more plants, or combinations thereof. In some embodiments, samples are taken from individuals, animals, and / or plants having a disease or disorder comprising an infectious disease, an immune disorder, a cancer, a genetic disease, a degenerative disease, a lifestyle-related disease, an injury, a rare disease, an age-related disease, or combinations thereof.

[0031] supramolecular structure In some embodiments, the supramolecular structure is a programmable structure that can spatially organize molecules. In some embodiments, the supramolecular structure comprises a plurality of molecules linked together. 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 comprises a specific shape. In some embodiments, the supramolecular nanostructure comprises a specified molecular weight based on the plurality of molecules. In some embodiments, the supramolecular structure is a nanostructure. In some embodiments, the plurality of molecules are linked together by bonds, chemical bonds, physical attachments, or a combination thereof. In some embodiments, the supramolecular structure comprises a large molecular entity of 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 specified 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.

[0032] 1A and 1B provide an exemplary embodiment of a supramolecular structure 40 comprising a core structure 13 and a capture molecule 2. As shown in FIG. 1A, the supramolecular structure 40 may comprise, in an embodiment, a detector molecule 1 and an anchor molecule 18. In some embodiments, the supramolecular structure comprises one or more capture molecules 2, one or more detector molecules 1, 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.

[0033] 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 particular shape of the supramolecular structure. In some embodiments, multiple core molecules interact with each other through reversible non-covalent interactions. In some embodiments, the particular shape of the core structure is in a three-dimensional (3D) configuration. In some embodiments, the one or more molecules provide a particular molecular weight. In some embodiments, the core structure 13 is a nanostructure. In some cases, the 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 scaffolded deoxyribonucleic acid (DNA) origami, a scaffolded ribonucleic acid (RNA) origami, a scaffolded hybrid DNA:RNA origami, a single-stranded DNA tiling structure, a multi-stranded DNA tiling structure, a single-stranded RNA origami, a multi-stranded RNA tiling structure, a hierarchically organized DNA or RNA origami with multiple scaffolds, a peptide structure, or a combination thereof. In some embodiments, the DNA origami is scaffolded. In some embodiments, the RNA origami is scaffolded. In some embodiments, the hybrid DNA:RNA origami is scaffolded. In some embodiments, the core structure comprising a DNA origami, an RNA origami, or a hybrid DNA:RNA origami comprises a specified two-dimensional (2D) or 3D shape.

[0034] As shown in FIG. 1, in some embodiments, the core structure 13 is configured to be linked to the capture molecule 2, the detector molecule 1, the anchor molecule 18, or a combination thereof. In some embodiments, the capture molecule 2, the detector molecule 1, and / or the anchor molecule 18 are immobilized relative to the core nanostructure 13 when linked thereto. In some embodiments, any number of the one or more core molecules comprise one or more linkers 10, 12, 14 configured to form a linkage with the capture molecule 2, the detector molecule 1, and / or the anchor molecule 18. In some embodiments, any number of the core molecules are configured to be linked to one or more linkers 10, 12, 14 configured to form a linkage with the capture molecule 2, the detector molecule 1, and / or the anchor molecule 18. In some embodiments, the one or more linkers are linked to the one or more core molecules through chemical bonds. In some embodiments, at least one of the one or more core linkers comprises a core reactive molecule. In some embodiments, each core reaction molecule independently comprises an amine, a thiol, DBCO, an NHS-ester, a maleimide, a biotin, an azide, an acrydite, a single stranded nucleic acid of a specific sequence (e.g., RNA or DNA), or a polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, at least one of the one or more core linkers comprises a DNA sequence domain.

[0035] In some embodiments, the core structure 13 is linked to 1) a capture molecule 2 at a first designated location on the core structure, 2) a detector molecule 1 at a second designated location on the core structure, and optionally 3) an anchor molecule 18 at a third designated location on the core structure. In some embodiments, a designated first core linker 12 is placed at a first location on the core structure and a designated second core linker 10 is placed at a second location on the core structure. In some embodiments, one or more core molecules at the first location are modified to form a linkage with the first core linker 12. In some embodiments, the first core linker 12 is an extension of the core structure 13. One or more core molecules at the second location are modified to form a linkage with the second core linker 10. In some embodiments, the second core linker 10 is an extension of the core structure 13. In some embodiments, the 3D shape of the core structure 13 and the relative distance between the first and second locations are designed to maximize intramolecular interactions between the capture molecule 2 and the detector molecule 1. In some embodiments, the 3D shape of the core structure 13 and the relative distance between the first and second locations are specified to achieve a desired distance between the capture molecules 2 and the detector molecules 1 that maximizes intramolecular interactions between the capture molecules 2 and the detector molecules 1.

[0036] As discussed herein, in some embodiments, the distance between the capture molecules 2 and the detector molecules 1 is about 3 nm, 4 nm, 5 nm, 6 nm, 10 nm, 12 nm, 15 nm, 20 nm, 30 nm, or 40 nm. In some embodiments, the distance between the capture molecules 2 and the detector molecules 1 is about 1 nm to about 60 nm. In some embodiments, the distance between the capture molecules 2 and the detector molecules 1 is from about 1 nm to about 2 nm, from about 1 nm to about 5 nm, from about 1 nm to about 10 nm, from about 1 nm to about 20 nm, from about 1 nm to about 40 nm, from about 1 nm to about 60 nm, from about 2 nm to about 5 nm, from about 2 nm to about 10 nm, from about 2 nm to about 20 nm, from about 2 nm to about 40 nm, from about 2 nm to about 60 nm, from about 5 nm to about 10 nm, from about 5 nm to about 20 nm, from about 5 nm to about 40 nm, from about 5 nm to about 60 nm, from about 10 nm to about 20 nm, from about 10 nm to about 40 nm, from about 10 nm to about 60 nm, from about 20 nm to about 40 nm, from about 20 nm to about 60 nm, or from about 40 nm to about 60 nm, including divisions therein. In some embodiments, the distance between the capture molecules 2 and the detector molecules 1 is about 1 nm, about 2 nm, about 5 nm, about 10 nm, about 20 nm, about 40 nm, or about 60 nm. In some embodiments, the distance between the capture molecules 2 and the detector molecules 1 is at least about 1 nm, about 2 nm, about 5 nm, about 10 nm, about 20 nm, or about 40 nm. In some embodiments, the distance between the capture molecules 2 and the detector molecules 1 is at most about 2 nm, about 5 nm, about 10 nm, about 20 nm, about 40 nm, or about 60 nm.

[0037] In some embodiments, a designated third core linker 14 is located 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 linkage 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 and second locations are located on a first side of the core structure 13 and the optional third location is located on a second side of the core structure 13.

[0038] In some embodiments, the capture molecules 2 comprise proteins, peptides, antibodies, aptamers (RNA and DNA), fluorophores, nanobodies, darpins, catalysts, polymerization initiators, polymers such as PEG, organic molecules, or combinations thereof. In some embodiments, the detector molecules 1 comprise proteins, peptides, antibodies, aptamers (RNA and DNA), fluorophores, nanobodies, darpins, catalysts, polymerization initiators, polymers such as PEG, organic molecules, or combinations thereof. In some embodiments, the anchor molecules comprise reactive molecules. In some embodiments, the anchor molecules 18 comprise reactive molecules. In some embodiments, the anchor molecules 18 comprise DNA strands comprising reactive molecules. In some embodiments, the anchor molecules 18 comprise amines, thiols, DBCO, NHS-esters, maleimides, biotin, azides, acrydites, single stranded nucleic acids of specific sequences (e.g., RNA or DNA), or polymers (e.g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, the anchor molecules 18 comprise proteins, peptides, antibodies, aptamers (RNA and DNA), fluorophores, nanobodies, darpins, catalysts, polymerization initiators, polymers such as PEG, organic molecules, or combinations thereof. In some embodiments, a single pair of capture molecules 2 and corresponding detector molecules 1 is linked to the core structure 13. In some embodiments, multiple pairs of capture molecules 2 and corresponding detector molecules 1 are linked to the core structure 13. In some embodiments, multiple pairs of capture molecules 2 and corresponding detector molecules 1 are spaced apart from each other to minimize crosstalk, i.e., to minimize interaction of capture molecules and / or detector molecules from a first pair with capture molecules and / or detector molecules from a second pair.

[0039] In some embodiments, each component of the supramolecular structure can be independently modified or adjusted. In some embodiments, modifying one or more of the components of the supramolecular structure can modify the 2D and 3D geometry of the supramolecular structure itself. In some embodiments, modifying one or more of the components of the supramolecular structure can modify the 2D and 3D geometry of the core structure. In some embodiments, such a capability to independently modify the components of the supramolecular nanostructure allows for precise control over the organization of one or more supramolecular structures on solid substrates (e.g., flat surfaces) and 3D volumes (e.g., within wells formed on solid substrates).

[0040] Capture Barcode As shown in FIG. 1A and FIG. 1B, in some embodiments, the capture molecule 2 is linked to the core structure 13 through a capture barcode 20. In some embodiments, the capture barcode 20 forms a linkage with the capture molecule 2 and forms a linkage 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, DBCO, an NHS-ester, a maleimide, an azide, an acrydite, a single-stranded nucleic acid of a specific sequence (e.g., RNA or DNA), or a polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, 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, DBCO, an NHS-ester, biotin, a maleimide, an azide, an acrydite, a single stranded nucleic acid of a specific sequence (e.g., RNA or DNA), or a polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, 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 through a physical attachment. In some embodiments, the second capture linker 6 is attached to the capture bridge 7 through a physical attachment.

[0041] In some embodiments, the capture barcode 20 is linked to the core structure 13 through a link between the first capture linker 11 and the first core linker 12. In some embodiments 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 by a covalent bond. In some embodiments, the link between the first capture linker 11 and the first core linker 12 is reversible upon receiving a trigger. In some embodiments, the trigger comprises an interaction with a deconstruction molecule ("capture deconstruction molecule", e.g., reference character 30 in Figure 4, Figure 7) or exposure to a trigger signal. In some embodiments, the capture deconstruction molecule comprises a nucleic acid (DNA or RNA), a peptide, a small organic molecule, or a combination thereof. In some embodiments, the trigger signal comprises an optical signal. In some embodiments, the trigger signal comprises an electrical signal, a microwave signal, ultraviolet illumination, visible illumination, or near infrared illumination.

[0042] In some embodiments, the capture barcode 20 is linked to the capture molecule 2 through a link 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 comprising an amine, a thiol, DBCO, an NHS-ester, a maleimide, a biotin, an azide, an acrydite, a single-stranded nucleic acid of a specific sequence (e.g., RNA or DNA), or a polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, the third capture linker 5 comprises a DNA sequence domain. In some embodiments, the capture molecule 2 is attached to the third capture linker 5 through a chemical bond. In some embodiments, the capture molecule 2 is attached to the third capture linker 5 by 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 together by a covalent bond. In some embodiments, the link between the second capture linker 6 and the third capture linker 5 is reversible upon receiving a trigger. In some embodiments, the trigger comprises an interaction with a deconstruction molecule ("capture barcode releasing molecule", e.g., FIG. 4, reference character 31 in FIG. 7) or exposure to a trigger signal. In some embodiments, the capture barcode releasing molecule comprises a nucleic acid (DNA or RNA), a peptide, a small organic molecule, or a combination thereof. In some embodiments, the trigger signal comprises an optical signal. In some embodiments, the trigger signal comprises an electrical signal, a microwave signal, ultraviolet illumination, visible illumination, or near-infrared illumination.

[0043] In some embodiments, upon receiving a trigger, only the link between the first capture linker 11 and the first core linker 12 is broken, thereby breaking the capture molecule linkage with the core nanostructure 13 at the first location. In some embodiments, the capture barcode 20 is configured to provide a signal related to detecting the analyte molecule when separated from the core structure 13 and the capture molecule 2. In some embodiments, the signal provided by the capture barcode 20 is a DNA signal.

[0044] Detector Barcode As shown in FIG. 1A, in some embodiments, the detector molecule 1 is linked to the core structure 13 through a detector barcode 21. In some embodiments, the detector barcode 21 forms a linkage with the detector molecule 1, and the detector barcode 21 forms a linkage with the core structure 13. In some embodiments, the detector barcode comprises a first detector linker 9, a second detector linker 4, and a detector bridge 8. In some embodiments, the first detector linker 9 comprises a reactive molecule. In some embodiments, the first detector linker 9 comprises a reactive molecule comprising an amine, a thiol, DBCO, an NHS-ester, a maleimide, a biotin, an azide, an acrydite, a single-stranded nucleic acid of a specific sequence (e.g., RNA or DNA), or a polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, the first detector linker 9 comprises a DNA sequence domain. In some embodiments, the second detector linker 4 comprises a reactive molecule. In some embodiments, the second detector linker 4 comprises a reactive molecule comprising an amine, a thiol, DBCO, an NHS-ester, a maleimide, a biotin, an azide, an acrydite, a single stranded nucleic acid of a specific sequence (e.g., RNA or DNA), or a polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, the second detector linker 4 comprises a DNA sequence domain. In some embodiments, the detector bridge 8 comprises a polymer. In some embodiments, the detector bridge 8 comprises a polymer comprising a nucleic acid of a specific sequence (DNA or RNA). In some embodiments, the detector bridge 8 comprises a polymer such as PEG. In some embodiments, the first detector linker 9 is attached to the detector bridge 8 at its first end and the second detector linker 4 is attached to the detector bridge 8 at its second end. In some embodiments, the first detector linker 9 is attached to the detector bridge 8 through a chemical bond. In some embodiments, the second detector linker 4 is attached to the detector bridge 8 through a chemical bond. In some embodiments, the first detector linker 9 is attached to the detector bridge 8 through a physical attachment.In some embodiments, the second detector linker 4 is attached to the detector bridge 8 through a physical attachment.

[0045] In some embodiments, the detector barcode 21 is linked to the core structure 13 through a link between the first detector linker 9 and the second core linker 10. In some embodiments described herein, the second core linker 10 is located at a second location on the core structure 13. In some embodiments, the first detector linker 9 and the second core linker 10 are linked to each other through a chemical bond. In some embodiments, the first detector linker 9 and the second core linker 10 are linked to each other by a covalent bond. In some embodiments, the link between the first detector linker 9 and the second core linker 10 is reversible upon receiving a trigger. In some embodiments, the trigger comprises an interaction with a deconstruction molecule ("detector deconstruction molecule", e.g., reference character 28 in Figure 4, Figure 7) or exposure to a trigger signal. In some embodiments, the detector deconstruction molecule comprises a nucleic acid (DNA or RNA), a peptide, a small organic molecule, or a combination thereof. In some embodiments, the trigger signal comprises an optical signal. In some embodiments, the trigger signal comprises an electrical signal, a microwave signal, ultraviolet illumination, visible illumination, or near infrared illumination.

[0046] In some embodiments, the detector barcode 21 is linked to the detector molecule 1 through a link between the second detector linker 4 and a third detector linker 3 attached to the detector molecule 1. In some embodiments, the third detector linker 3 comprises a reactive molecule comprising an amine, a thiol, DBCO, an NHS-ester, a maleimide, a biotin, an azide, an acrydite, a single-stranded nucleic acid of a specific sequence (e.g., RNA or DNA), or a polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, the third detector linker 3 comprises a DNA sequence domain. In some embodiments, the detector molecule 1 is attached to the third detector linker 3 through a chemical bond. In some embodiments, the detector molecule 1 is attached to the third detector linker 3 by a covalent bond. In some embodiments, the second detector linker 4 and the third detector linker 3 are linked to each other through a chemical bond. In some embodiments, the second detector linker 4 and the third detector linker 3 are linked to each other by a covalent bond. In some embodiments, the link between the second detector linker 4 and the third detector linker 3 is reversible upon receiving a trigger. In some embodiments, the trigger comprises an interaction with a deconstruction molecule ("detector barcode releasing molecule", e.g., FIG. 4, reference character 29 in FIG. 7) or exposure to a trigger signal. In some embodiments, the detector barcode releasing molecule comprises a nucleic acid (DNA or RNA), a peptide, a small organic molecule, or a combination thereof. In some embodiments, the trigger signal comprises an optical signal. In some embodiments, the trigger signal comprises an electrical signal, a microwave signal, ultraviolet illumination, visible illumination, or near-infrared illumination.

[0047] In some embodiments, receiving a trigger breaks only the link between the first detector linker 9 and the second core linker 10, thereby breaking the detector molecule linkage with the core structure 13 at the second location. In some embodiments, the detector barcode 21 is configured to provide a signal related to detecting the analyte molecule when separated from the core structure 13 and the detector molecule 2. In some embodiments, the signal provided by the detector barcode 21 is a DNA signal.

[0048] Anchor Barcode As shown in FIG. 1A, 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 linkage with the anchor molecule 18, and the anchor barcode forms a linkage 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, DBCO, an NHS-ester, a maleimide, a biotin, an azide, an acrydite, a single-stranded nucleic acid of a specific sequence (e.g., RNA or DNA), or a polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, 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, DBCO, an NHS-ester, a maleimide, a biotin, an azide, an acrydite, a single stranded nucleic acid of a specific sequence (e.g., RNA or DNA), or a polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, 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 bridge 16 comprises a polymer comprising a nucleic acid of a specific sequence (DNA or RNA). 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 through a 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 through a physical attachment.

[0049] In some embodiments, the anchor barcode is linked to the core structure 13 through a link between the first anchor linker 15 and the third core linker 14. In some embodiments described herein, the third core linker 14 is located 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 by a covalent bond. In some embodiments, the link between the first anchor linker 15 and the third core linker 14 is reversible upon receiving a trigger. In some embodiments, the trigger comprises an interaction with a deconstruction molecule ("anchor deconstruction molecule", e.g., reference character 32 in Figure 4, Figure 7) or exposure to a trigger signal. In some embodiments, the anchor deconstruction molecule comprises a nucleic acid (DNA or RNA), a peptide, a small organic molecule, or a combination thereof. In some embodiments, the trigger signal comprises an optical signal. In some embodiments, the trigger signal comprises an electrical signal, a microwave signal, ultraviolet illumination, visible illumination, or near infrared illumination.

[0050] In some embodiments, the anchor barcode is linked to the anchor molecule 18 through a link between the second anchor linker 17 and the anchor molecule 18. As disclosed herein, 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 by a covalent bond. In some embodiments, the link between the second anchor linker 17 and the anchor molecule 18 is reversible upon receiving a trigger. In some embodiments, the trigger comprises an interaction with a deconstruction molecule (an "anchor barcode releasing molecule", e.g., reference character 33 in FIG. 4, FIG. 7) or exposure to a trigger signal. In some embodiments, the anchor barcode releasing molecule comprises a nucleic acid (DNA or RNA), a peptide, a small organic molecule, or a combination thereof. In some embodiments, the trigger signal comprises an optical signal. In some embodiments, the trigger signal comprises an electrical signal, a microwave signal, ultraviolet illumination, visible illumination, or near-infrared illumination.

[0051] In some embodiments, upon receipt of a trigger, only the link between the first anchor linker 15 and the third core linker 14 is broken, thereby breaking the link of the anchor molecule to the core nanostructure 13 at the third location.

[0052] In some embodiments, the capture deconstruction molecules, the capture barcode releasing molecules, the detector deconstruction molecules, and the detector barcode releasing molecules comprise the same type of molecule. In some embodiments, the capture deconstruction molecules, the capture barcode releasing molecules, the detector deconstruction molecules, and the detector barcode releasing molecules comprise different types of molecules. In some embodiments, the capture deconstruction molecules, the capture barcode releasing molecules, the detector deconstruction molecules, the detector barcode releasing molecules, the anchor deconstruction molecules, and the anchor barcode releasing molecules comprise the same type of molecule. In some embodiments, the capture deconstruction molecules, the capture barcode releasing molecules, the detector deconstruction molecules, the detector barcode releasing molecules, the anchor deconstruction molecules, and the anchor barcode releasing molecules comprise different types of molecules. In some embodiments, any combination of the capture deconstruction molecules, the capture barcode releasing molecules, the detector deconstruction molecules, the detector barcode releasing molecules, the anchor deconstruction molecules, and the anchor barcode releasing molecules comprise the same type of molecule.

[0053] Three-arm nucleic acid junction-based supramolecular architectures Figures 2-3 provide an exemplary diagram of a supramolecular structure 40 comprising a three-armed nucleic acid junction and associated subcomponents. Figure 2 provides the complete supramolecular structure, whereas Figure 3 provides the subcomponents comprising the supramolecular structure of Figure 2. In some embodiments, the subcomponents of the supramolecular structure comprise five DNA strands (reference characters 20-24), one DNA strand 25 with terminal modifications, and two antibodies (1, 2) modified by single DNA linkers 3, 5. Figure 4 provides an exemplary diagram of respective deconstruction molecules configured to cleave respective subcomponents from the supramolecular structure 40 of Figure 2. Reference characters 1-18 in Figures 2-4 correspond to the respective components provided with the same reference characters in Figure 1A.

[0054] TIFF2025003557000002.tif22170

[0055] In some embodiments, the first core strand 23 of the core structure comprises a first core linker 12 comprising a DNA sequence domain. In some embodiments, the first core strand 23, labeled "A" in Figures 2-4, comprises a DNA sequence domain separated from the first core linker 12 by an unstructured DNA region. In some embodiments, the unstructured DNA region comprises a polymer spacer. In some embodiments, the polymer spacer comprises a nucleic acid (DNA or RNA) of a specific sequence. In some embodiments, the polymer spacer comprises a polymer such as PEG.

[0056] In some embodiments, the first core linker 12 is complementary to the first capture linker 11 on the capture barcode strand 20. In some embodiments, the capture barcode strand 20 comprises a DNA strand with either the first capture linker 11 or the second capture linker 6 at both ends. 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 specific capture barcode sequence 7 between the first capture linker 11 and the second capture linker 6. In some embodiments, the specific capture barcode sequence 7 comprises a nucleic acid (DNA or RNA) of a specific sequence. In some embodiments, the specific capture barcode sequence 7 comprises a polymer such as PEG. In some embodiments, the capture barcode 20 comprises a short domain called a toe hold ("TH"). In some embodiments, the capture barcode sequence 7 comprises a toe hold ("TH").

[0057] 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 27. In some embodiments, the capture molecule 2 is covalently bound to the third capture linker 5. In some embodiments, the capture molecule 2 is a capture antibody.

[0058] TIFF2025003557000003.tif48170

[0059] In some embodiments, the second core linker 10 is complementary to the first detector linker 9 on the detector barcode strand 21. In some embodiments, the detector barcode strand 21 comprises a DNA strand with either the first detector linker 9 or the second detector linker 4 at both ends. In some embodiments, the first detector linker 9 comprises a DNA sequence domain. In some embodiments, the second detector linker 4 comprises a DNA sequence domain. In some embodiments, the detector barcode strand 21 further comprises a specific detector barcode sequence 8 between the first detector linker 9 and the second detector linker 4. In some embodiments, the specific detector barcode sequence 8 comprises a nucleic acid (DNA or RNA) of a specific sequence. In some embodiments, the specific detector barcode sequence 8 comprises a polymer such as PEG. In some embodiments, the detector barcode 21 comprises a short domain called a toehold ("TH"). In some embodiments, the detector barcode sequence 8 comprises a toehold ("TH").

[0060] In some embodiments, the second detector linker 4 is complementary to the third detector linker 3. In some embodiments, the third detector linker 3 is a DNA sequence domain. In some embodiments, the detector molecule 1 is bound to the third detector linker 3 26. In some embodiments, the detector molecule 1 is covalently bound to the third capture linker 3. In some embodiments, the detector molecule 1 is a detector antibody.

[0061] In some embodiments, the third core linker 14 is complementary to the first anchor linker 15 on the anchor barcode strand 22. In some embodiments, the anchor barcode strand 22 comprises a DNA strand comprising either the first anchor linker 15 or the second anchor linker 17 at both ends. In some embodiments, the first anchor linker 15 comprises a DNA sequence domain. In some embodiments, the second anchor linker 17 comprises a DNA sequence domain. In some embodiments, the anchor barcode strand 22 further comprises a specific anchor barcode sequence 16 between the first anchor linker 15 and the second anchor linker 17. In some embodiments, the specific anchor barcode sequence 16 comprises a nucleic acid (DNA or RNA) of a specific sequence. In some embodiments, the specific anchor barcode sequence 16 comprises a polymer such as PEG. In some embodiments, the anchor barcode 22 comprises a short domain called a toehold ("TH"). In some embodiments, the anchor barcode sequence 16 comprises a toehold ("TH").

[0062] In some embodiments, the second anchor linker 17 is complementary to the anchor molecule 18. In some embodiments, the anchor molecule 18 comprises a DNA sequence domain. In some embodiments, the anchor molecule 18 is linked 25 to a terminal modification 34. In some embodiments, the terminal modification 34 comprises a reactive molecule. In some embodiments, the terminal modification 34 comprises a reactive molecule. In some embodiments, the terminal modification 34 comprises an amine, a thiol, DBCO, an NHS-ester, a maleimide, a biotin, an azide, an acrydite, a single stranded nucleic acid of a specific sequence (e.g., RNA or DNA), or a polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators).

[0063] FIG. 4 provides exemplary embodiments of deconstruction molecules that can be used to trigger different responses to the supramolecular structure 40. In some embodiments, the detector deconstruction molecule 28 comprises a TH′ domain having a complementary arrangement to the TH domain on the detector barcode 21 and the second core linker 10 (e.g., a DNA sequence domain) on the second core strand 24. In some embodiments, the detector deconstruction molecule 28 is configured to cleave the link between the detector barcode 21 and the core structure (e.g., the second core strand 24). In some embodiments, the detector barcode releasing molecule 29 comprises a TH′ domain having a complementary arrangement to the TH domain on the detector barcode 21 and the third detector linker 3 (e.g., a DNA sequence domain). In some embodiments, the detector barcode releasing molecule 28 is configured to cleave the link between the detector barcode 21 and the detector molecule 1.

[0064] In some embodiments, the capture deconstruction molecule 30 comprises a TH' domain having a complementary arrangement to the TH domain on the capture barcode 20 and the first core linker 12 (e.g., a DNA sequence domain) on the first core strand 23. In some embodiments, the capture deconstruction molecule 30 is configured to cleave the link between the capture barcode 20 and the core structure (e.g., the first core strand 23). In some embodiments, the capture barcode releasing molecule 31 comprises a TH' domain having a complementary arrangement to the TH domain on the capture barcode 20 and the third capture linker 5 (e.g., a DNA sequence domain). In some embodiments, the capture barcode releasing molecule 31 is configured to cleave the link between the capture barcode 20 and the capture molecule 2.

[0065] In some embodiments, the anchor deconstruction molecule 32 comprises a TH' domain having a complementary arrangement to the TH domain on the anchor barcode 22 and the third core linker 14 (e.g., a DNA sequence domain) on the second core strand. In some embodiments, the anchor deconstruction molecule 32 is configured to cleave the link between the anchor barcode 22 and the core structure (e.g., the second core strand 24). In some embodiments, the anchor barcode releasing molecule 33 comprises a "TH'" domain having a complementary arrangement to the "TH" domain on the anchor barcode 22 and the anchor molecule 18 (e.g., a DNA sequence domain). In some embodiments, the anchor barcode releasing molecule 33 is configured to cleave the link between the anchor barcode 22 and the anchor molecule 18.

[0066] TIFF2025003557000004.tif22170

[0067] DNA origami-based supramolecular structures Figures 5-6 provide exemplary diagrams of a supramolecular structure 40 comprising DNA origami and associated subcomponents. Figure 5 provides a complete supramolecular structure, whereas Figure 6 provides subcomponents comprising the supramolecular structure of Figure 5. In some embodiments, the subcomponents of the supramolecular structure comprise a DNA origami 13 as a core structure, three DNA strands (reference characters 20-22), one DNA strand 25 with terminal modifications, and two antibodies (1, 2) modified by single DNA linkers 3, 5. Figure 6 provides exemplary diagrams of respective deconstruction molecules configured to cleave respective subcomponents from the supramolecular structure 40 of Figure 5. Reference characters 1-18 in Figures 5-7 correspond to the respective components provided with the same reference characters in Figure 1A.

[0068] In some embodiments, the core structure 13 comprises a scaffold DNA origami, in which a circular ssDNA molecule, referred to as the "scaffold" strand, is folded into a predetermined 2D or 3D shape by interacting with two or more short ssDNA strands, referred to as "staple" strands, which interact with specific subsections of the ssDNA "scaffold" strand.

[0069] As shown in Figures 5-6, in some embodiments of the supramolecular structure, the core structure 13 comprises a 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 the first capture linker 11 on the capture barcode strand 20. In some embodiments, the capture barcode strand 20 comprises a DNA strand comprising either the first capture linker 11 or the second capture linker 6 at both ends thereof. 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 specific capture barcode sequence 7 between the first capture linker 11 and the second capture linker 6. In some embodiments, the specific capture barcode sequence 7 comprises a nucleic acid (DNA or RNA) of a specific sequence. In some embodiments, the specific capture barcode sequence 7 comprises a polymer such as PEG. In some embodiments, the capture barcode 20 comprises a short domain called a toehold ("TH"). In some embodiments, the capture barcode sequence 7 comprises a toehold ("TH").

[0070] 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 27. In some embodiments, the capture molecule 2 is covalently bound to the third capture linker 5. In some embodiments, the capture molecule 2 is a capture antibody.

[0071] In some embodiments, the core structure 13 comprises a second core linker 10 comprising a DNA sequence domain. In some embodiments, the second core linker 10 is complementary to the first detector linker 9 on the detector barcode strand 21. In some embodiments, the detector barcode strand 21 comprises a DNA strand comprising either the first detector linker 9 or the second detector linker 4 at both ends. In some embodiments, the first detector linker 9 comprises a DNA sequence domain. In some embodiments, the second detector linker 4 comprises a DNA sequence domain. In some embodiments, the detector barcode strand 21 further comprises a specific detector barcode sequence 8 between the first detector linker 9 and the second detector linker 4. In some embodiments, the specific detector barcode sequence 8 comprises a nucleic acid (DNA or RNA) of a specific sequence. In some embodiments, the specific detector barcode sequence 8 comprises a polymer such as PEG. In some embodiments, the detector barcode 21 comprises a short domain called a toe hold ("TH"). In some embodiments, the specific detector barcode sequence 8 comprises a toe hold ("TH").

[0072] In some embodiments, the second detector linker 4 is complementary to the third detector linker 3. In some embodiments, the third detector linker 3 is a DNA sequence domain. In some embodiments, the detector molecule 1 is bound to the third detector linker 3 26. In some embodiments, the detector molecule 1 is covalently bound to the third capture linker 3. In some embodiments, the detector molecule 1 is a detector antibody.

[0073] In some embodiments, the core structure 13 comprises a third core linker 14 comprising a DNA sequence domain. In some embodiments, the third core linker 14 is complementary to the first anchor linker 15 on the anchor barcode strand 22. In some embodiments, the anchor barcode strand 22 comprises a DNA strand comprising either a first anchor linker 15 or a second anchor linker 17 at both ends. In some embodiments, the first anchor linker 15 comprises a DNA sequence domain. In some embodiments, the second anchor linker 17 comprises a DNA sequence domain. In some embodiments, the anchor barcode strand 22 further comprises a specific anchor barcode sequence 16 between the first anchor linker 15 and the second anchor linker 17. In some embodiments, the specific detector barcode sequence 16 comprises a nucleic acid (DNA or RNA) of a specific sequence. In some embodiments, the specific detector barcode sequence 16 comprises a polymer such as PEG. In some embodiments, the anchor barcode 22 comprises a short domain called a toe hold ("TH"). In some embodiments, the anchor barcode sequence 16 comprises a toe hold ("TH").

[0074] In some embodiments, the second anchor linker 17 is complementary to the anchor molecule 18. In some embodiments, the anchor molecule 18 comprises a DNA sequence domain. In some embodiments, the anchor molecule 18 is linked 25 to a terminal modification 34. In some embodiments, the terminal modification 34 comprises a reactive molecule. In some embodiments, the terminal modification 34 comprises a reactive molecule comprising an amine, a thiol, DBCO, an NHS-ester, a maleimide, a biotin, an azide, an acrydite, a single stranded nucleic acid of a specific sequence (e.g., RNA or DNA), or a polymer (e.g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, the supramolecular structure 40 does not include the anchor molecule 18 or the associated core linker 14, anchor linker 15, barcode 16, or anchor linker 17, and the core structure 13 directly interacts with or contacts the substrate as discussed herein.

[0075] 6 provides exemplary embodiments of deconstruction molecules that can be used to trigger different responses to the supramolecular structure 40. In some embodiments, the detector deconstruction molecule 28 comprises a TH′ domain having a complementary arrangement to the TH domain on the detector barcode 21 and the second core linker 10 (e.g., a DNA sequence domain) on the core nanostructure 13. In some embodiments, the detector deconstruction molecule 28 is configured to sever the link between the detector barcode 21 and the core structure 13. In some embodiments, the detector barcode releasing molecule 29 comprises a TH′ domain having a complementary arrangement to the TH domain on the detector barcode 21 and the third detector linker 3 (e.g., a DNA sequence domain). In some embodiments, the detector barcode releasing molecule 28 is configured to sever the link between the detector barcode 21 and the detector molecule 1.

[0076] In some embodiments, the capture deconstruction molecule 30 comprises a TH′ domain having a complementary arrangement to a TH domain on the capture barcode 20 and a first core linker 12 (e.g., a DNA sequence domain) on the core nanostructure 13. In some embodiments, the capture deconstruction molecule 30 is configured to cleave the link between the capture barcode 20 and the core structure 13. In some embodiments, the capture barcode releasing molecule 31 comprises a TH′ domain having a complementary arrangement to a TH domain on the capture barcode 20 and a third capture linker 5 (e.g., a DNA sequence domain). In some embodiments, the capture barcode releasing molecule 31 is configured to cleave the link between the capture barcode 20 and the capture molecule 2.

[0077] In some embodiments, the anchor deconstruction molecule 32 comprises a TH′ domain having a complementary arrangement to a TH domain on the anchor barcode 22 and a third core linker 14 (e.g., a DNA sequence domain) on the core nanostructure 13. In some embodiments, the anchor deconstruction molecule 32 is configured to cleave the link between the anchor barcode 22 and the core structure 13. In some embodiments, the anchor barcode releasing molecule 33 comprises a TH′ domain having a complementary arrangement to a TH domain on the anchor barcode 22 and an anchor molecule 18 (e.g., a DNA sequence domain). In some embodiments, the anchor barcode releasing molecule 33 is configured to cleave the link between the anchor barcode 22 and the anchor molecule 18.

[0078] Stable and unstable states of supramolecular structures In some embodiments, the supramolecular structure comprises one or more stable state configurations. In some embodiments, the supramolecular structure comprises one or more unstable state configurations. In some embodiments, the supramolecular structure comprises a bistable configuration having a stable state configuration and an unstable state configuration. In some embodiments, the two states, stable and unstable, are predefined based on the ability of the individual supramolecular structures to remain structurally intact upon receiving a specific molecule (e.g., a deconstruction molecule) and / or a trigger signal. In some embodiments, when the supramolecular structure is in a stable state, all the different components that are part of the supramolecular structure remain physically linked to each other even after receiving the deconstruction molecule and / or a trigger signal. In some embodiments, when the supramolecular structure is in an unstable state, a predefined section of the supramolecular structure (e.g., one or more subcomponents) is physically disconnected, i.e., released (separated) from the supramolecular structure, as a result of exposure to the deconstruction molecule and / or the trigger signal. In some embodiments, the supramolecular structure is configured to shift from a stable state to an unstable state upon interaction with an analyte molecule (described herein). In some embodiments, the supramolecular structure is configured to shift from an unstable state to a stable state upon interaction with an analyte molecule (as described herein), in some embodiments, the analyte molecule that triggers the state change of the supramolecular structure comprises a protein, a cluster of proteins, a peptide fragment, a cluster of peptide fragments, DNA, RNA, a DNA nanostructure, an RNA nanostructure, a lipid, an organic molecule, an inorganic molecule, or any combination thereof.

[0079] In some embodiments, the supramolecular structure in the unstable state configuration comprises a physical state capable of severing the link between the core nanostructure 13 and the capture molecule 2 such that the capture molecule 2 is released from the core nanostructure 13. In some embodiments, the unstable state configuration comprises a physical state capable of severing the link between the core nanostructure 13 and the detector molecule 1 such that the detector molecule 1 is released from the core nanostructure 13. In some embodiments, the unstable state configuration comprises a physical state capable of severing the link between the core nanostructure 13 and the capture molecule 2 and the link between the core nanostructure 13 and the detector molecule 1 such that the capture molecule 2 and the detector molecule 1 are released from the core nanostructure 13. In some embodiments, the link between the core nanostructure 13 and 1) the capture molecule 2, 2) the detector molecule 1, or 3) both are severed upon receiving a trigger (e.g., a deconstruction molecule as described herein or a trigger signal as described herein). FIG. 8 provides an exemplary illustration of a supramolecular structure 40 in an unstable state with the detector molecule 1 initially bound to the core structure 13 through a linkage with the detector barcode 21. 8 , interaction with the deconstruction molecule 42 (e.g., detector deconstruction molecule 28) then severs the link between the detector barcode 21 and the core structure 13, thereby releasing the detector molecule 1 from the core nanostructure 13. In some embodiments, in the unstable state, the capture molecule 2 and the detector molecule 1 on the core nanostructure 13 are free to diffuse relative to each other, unconstrained other than by the physical configuration of the core nanostructure 13.

[0080] In some embodiments, the stable state configuration comprises a physical state in which the capture molecule 2 remains bound to the core nanostructure 13 upon severing of the link between the core structure 13 and the capture molecule 2. In some embodiments, the stable state configuration comprises a physical state in which the detector molecule 1 remains bound to the core nanostructure 13 upon severing of the link between the core structure 13 and the detector molecule 1. In some embodiments, the stable state configuration comprises a physical state in which the capture molecule 2 and the detector molecule 1 are disposed in close proximity to each other. In some embodiments, the detector molecule 1 and the capture molecule 2 are disposed in close proximity to each other with or without the formation of a distinct bond between each other. In some embodiments, the detector molecule 1 and the capture molecule 2 are linked to each other. In some embodiments, the detector molecule 1 and the capture molecule 2 are linked to each other through a chemical bond. In some embodiments, the detector molecule 1 and the capture molecule 2 are linked to each other through a linkage with another molecule located between these molecules (e.g., in a sandwich configuration). In some embodiments, the detector molecule and the capture molecule are linked to each other through a linkage with an analyte molecule 44 from a sample (described herein). 9 provides an exemplary diagram of a supramolecular structure 40 in a stable state in which the capture molecule 2 is linked to the detector molecule 1 through a linkage with the analyte molecule 44. Continuing with reference to FIG. 9, the interaction with the deconstruction molecule 42 breaks the link between the detector molecule 1 and the core structure 13, but the detector molecule 1 remains bound to the core nanostructure 13 through a linkage with the capture molecule 2. As described further herein, in some embodiments, the capture molecule and / or the detector molecule are configured to form a linkage with one or more specific types of analyte molecules from a sample. In some embodiments, the interaction with the deconstruction molecule and / or the trigger signal does not break the link between the capture molecule and the detector molecule.

[0081] FIG. 10 provides an exemplary embodiment of a supramolecular structure shifting from an unstable state to a stable state. As described herein, the supramolecular structure 40 in the unstable state configuration will be separated from the detector molecule 1 upon interaction with a corresponding deconstruction molecule 42 (e.g., detector deconstruction molecule 28) and / or a trigger signal (the detector molecule will be released from the supramolecular structure). Continuing with reference to FIG. 10, in some embodiments, interaction with an analyte molecule 44 from a sample will bind the analyte molecule located between the capture molecule and the detector molecule together (e.g., in a sandwich configuration), thereby shifting the supramolecular structure 40 from an unstable state to a stable state. In some embodiments, the analyte molecule 44 comprises a single molecule. In some embodiments, the analyte molecule alternatively comprises a plurality of analyte molecules. In some embodiments, the analyte molecule alternatively comprises a molecular cluster. In some embodiments, as described herein and shown in FIG. 10, when the supramolecular structure is in a stable state, interaction with the corresponding deconstruction molecule breaks the link between the core structure 13 and the detector barcode 21, and the detector molecule 1 remains linked to the core structure 13 through linkage with the capture molecule 2 and the analyte molecule 44.

[0082] FIG. 11 provides an exemplary embodiment of the supramolecular structure 40 shifting from a stable state to an unstable state. As described herein, when the supramolecular structure 40 is in a stable state configuration, the detector molecule 1 will remain linked to the core structure 13 due to the detector molecule 1 being linked to the capture molecule 2 upon interaction with a corresponding deconstruction molecule and / or trigger signal. Continuing with reference to FIG. 11, in some embodiments, interaction with an analyte molecule 44 from a sample breaks the link between the capture molecule 2 and the detector molecule 1 such that the analyte molecule 44 binds only to the capture molecule 1, thereby shifting the supramolecular structure to an unstable state in which the detector molecule 1 is only bound to the core nanostructure 13 through its linkage with the detector barcode 21. In some embodiments, the analyte molecule 44 comprises a single molecule. In some embodiments, the analyte molecule alternatively comprises a plurality of analyte molecules. In some embodiments, the analyte molecule alternatively comprises a molecular cluster. In some embodiments, as described herein and shown in FIG. 11, when the supramolecular structure is in an unstable state, interaction with a corresponding deconstruction molecule 42 severs the link between the core structure 13 and the detector barcode 21, thereby releasing (separating) the detector molecule 1 from the core structure 13.

[0083] In some embodiments, the supramolecular structure 40 transitions from a stable state to an unstable state upon interaction with an analyte molecule 44, which breaks the link between the capture molecule 2 and the detector molecule 1 and binds to the detector molecule 1. The capture molecule 2 is thereby released from the core structure 13 upon interaction with a corresponding deconstruction molecule 42 (e.g., capture deconstruction molecule 30).

[0084] Method for detecting analyte molecules As discussed herein, in some embodiments, one or more supramolecular structures enable the detection of one or more analyte molecules in a sample. In some embodiments, the supramolecular structure converts information about the presence of a given analyte molecule in a sample into a DNA signal. In some embodiments, the DNA signal corresponds to a capture or detector barcode on the supramolecular structure, and the capture and detector molecules are simultaneously linked to the analyte molecule (e.g., in a sandwich configuration). In some embodiments, the capture and / or detector barcodes located on any unstable supramolecular structure are released from the supramolecular structure using a trigger, such as a deconstruction molecule and / or a trigger signal. In some embodiments, the DNA signal is aligned with a specific analyte molecule and is later identified and correlated with this analyte molecule. As provided herein, each supramolecular structure 40 can be equipped with at least one unique barcode, so that the location of the analyte binding event and the transition of the supramolecular structure from an unstable state to a stable state can be linked to a specific binding site on the substrate by the barcode sequence.

[0085] In some embodiments, detecting the presence of analyte molecules as described herein comprises controllably introducing into the solution one or more specific nucleic acid molecules that are used to identify and quantify the characteristics of the analyte molecules from the sample and that trigger a state change of the supramolecular structures. In some embodiments, these specific nucleic acid molecules are provided by the capture barcode and / or detector barcode of the respective supramolecular structures. In some embodiments, detecting the presence of analyte molecules as described herein comprises generating an optical or electrical signal upon the state change that can be counted to quantify the concentration of the analyte molecules in the solution.

[0086] In some embodiments, multiple analyte molecules in a sample are detected simultaneously by multiplexing, where 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 throughput and high multiplexing capabilities by using multiple supramolecular structures. In some embodiments, the high throughput and high multiplexing 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 characterize and / or identify biopolymers comprising protein molecules with high sensitivity and high reproducibility. In some embodiments, the multiple supramolecular structures are configured to limit cross-reactivity relationship errors. In some embodiments, such cross-reactivity relationship errors comprise capture molecules and / or detector molecules of one supramolecular structure interacting with capture molecules and / or detector molecules of another supramolecular structure (e.g., intermolecular interactions). 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 supramolecular structure are specifically designed. In some embodiments, the same core structures have a specified shape, size, molecular weight, a specified number of capture molecules and detector molecules, a predetermined distance between corresponding capture molecules and detector molecules (as described herein), a specified stoichiometric ratio between corresponding capture molecules and detector molecules, or a combination thereof, so as to limit cross-reactivity between supramolecular structures. In some embodiments, the molecular weight of all core structures is the same, and the purity of the core molecules is accurate. In some embodiments, each core structure has at least one capture molecule and at least one corresponding detector molecule.

[0087] In some embodiments, the multiple supramolecular structures independently interact with different analyte molecules from a sample, since the state change (unstable to stable) is driven primarily through intramolecular (capture and detector molecule on the same supramolecular structure) interactions. In some embodiments, it is believed that the multiple supramolecular structures may share structural similarities due to certain subcomponents being the same, but the interaction between the analyte molecules from the sample and the supramolecular structures is predefined by the corresponding capture and detector molecules. In some embodiments, each pair of detector and capture molecules on a given supramolecular structure can specifically interact with a particular analyte molecule in a sample and achieve a state change of the supramolecular structure upon interaction with such a particular analyte molecule. In some embodiments, each supramolecular structure comprises a specific DNA barcode corresponding to the respective pair of detector and capture molecules. In some embodiments, the pair of detector and capture molecules on a given supramolecular structure is designed to interact with more than one analyte molecule in a sample.

[0088] In some embodiments, each supramolecular structure is configured such that single molecule sensitivity ensures the largest possible dynamic range required to quantitatively capture a wide range of molecular concentrations in a typical complex biological sample. In some embodiments, single molecule sensitivity comprises capture and detector molecules of a given supramolecular structure configured to shift from an unstable state to a stable state (or vice versa) upon binding with a single analyte molecule. In some embodiments, multiple supramolecular structures limit or eliminate non-specific interactions as well as sample manipulations required to reduce any user-induced errors.

[0089] In some embodiments, the plurality of supramolecular structures are attached to one or more solid substrates, such as planar or patterned substrates. FIG. 12 provides an exemplary method for detecting one or more analyte molecules in a sample using one or more supramolecular structures. In some embodiments, a sample comprising one or more analytes (e.g., analyte pool 102) is contacted with one or more supramolecular structures 40 (e.g., supramolecular structure pool 100). In some embodiments described herein, the plurality of supramolecular structures are provided attached to one or more solid substrates for single molecule assembly as provided herein. In some embodiments, the sample is contacted with the supramolecular structures for a period of about 30 seconds to about 24 hours. In some embodiments, the sample is incubated with the supramolecular structures 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, about 24 hours to about 48 hours.

[0090] Continuing with reference to FIG. 12, in some embodiments, these supramolecular structures are all in an unstable state (as shown by reference character 100). In some embodiments described herein, the interaction between the analyte molecules and the corresponding capture molecules 2 and detector molecules 1 shifts the respective supramolecular structures from an unstable state to a stable state (e.g., a sandwich configuration of capture molecules, analyte molecules, and detector molecules as shown by reference character 104). In some embodiments, a particular type of analyte molecule will bind to a particular pair of capture molecules and detector molecules. In some embodiments, a given pair of capture molecules and detector molecules is configured to bind to more than one type of analyte molecule. In some embodiments, the switch from the unstable state to the stable state for any given supramolecular structure depends on the particular capture molecules and detector molecules bound to the supramolecular structure and the analyte molecules in the sample. In some embodiments, considering that the state change of the supramolecular structure mainly depends on the intramolecular structures (components located on the supramolecular structure), the potential intermolecular interactions between two different supramolecular structures are minimized or eliminated by limiting the net concentration of the supramolecular structures in the mixed solution such that the average distance between any two supramolecular structures is greater than the maximum intramolecular distance between a pair of capture molecules and detector molecules on a given supramolecular structure.

[0091] As can be seen at reference character 104 in FIG. 12, after contacting with the sample, at least one of the supramolecular structures is shifted to a stable state (e.g., a sandwich configuration in which the capture molecules, analyte molecules and detector molecules are simultaneously linked to each other) through interaction with the analyte molecules, whereas at least one of the supramolecular structures remains in an unstable state due to the respective capture molecules and detector molecules not binding or interacting with the analyte molecules from the sample.

[0092] After contacting the sample with the supramolecular structure for a specified amount of time, the mixed solution of the sample and the supramolecular structure shown in FIG. 12 is triggered (reference character 106) to sever the link between the detector molecule and the core structure. In some embodiments, the trigger comprises introducing a solution comprising one or more deconstruction molecules (e.g., detector deconstruction molecules at reference character 28 in FIG. 4, FIG. 7) into the mixed solution. In some embodiments, the trigger comprises exposing the mixed solution to a trigger signal. In some embodiments, the trigger comprises a combination of introducing the deconstruction molecules into the mixed solution and exposing the mixed solution to a trigger signal. In some embodiments described herein, the deconstruction molecules comprise nucleic acids (DNA or RNA), peptides, small organic molecules, or combinations thereof. In some embodiments described herein, the trigger signal comprises an electrical signal, a microwave signal, ultraviolet illumination, visible illumination, or near infrared illumination. In some embodiments, the mixed solution is triggered for a specified amount of time. In some embodiments, the mixed solution is incubated with one or more deconstruction molecules for a specified amount of time. In some embodiments, the mixed solution is incubated with the deconstruction molecule for a period of about 30 seconds to about 24 hours. In some embodiments, the mixed solution is incubated with the deconstruction molecule 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, about 24 hours to about 48 hours.

[0093] As shown by reference character 106 in FIG. 12, in some embodiments, triggering the mixed solution breaks the link between the detector molecule and the core structure of the supramolecular structure, for example, the link between the detector barcode (e.g., reference character 21 in FIG. 1) and the core structure 13. In some embodiments, the break is achieved by displacement of nucleic acid (DNA / RNA) strands, optical cleavage, chemical cleavage, another technique known in the art, or a combination thereof. For the supramolecular structure shifted to a stable state, the detector molecule 1 is shown remaining linked to the core structure 13 through a linkage with the corresponding capture molecule 2. For the supramolecular structure remaining in an unstable state, the detector molecule 112 is shown released from the respective supramolecular structure. In some embodiments, the released detector molecule 1 remains linked to the respective detector barcode 21.

[0094] In some embodiments, the released detector molecule 1 (and corresponding detector barcode 21) is further separated from the mixed solution. In some embodiments, the released detector molecule is separated from the mixed solution by precipitation of polyethylene glycol (PEG). In some embodiments, the released detector molecule is separated from the mixed solution by binding each core structure in the mixed solution to a solid support through a corresponding anchor molecule thereon, followed by separation of the released detector molecule by centrifugation, micron filtration, chromatography, or a combination thereof.

[0095] In some embodiments, after the released detector molecules are separated from the mixed solution, the detector barcodes 21 are cleaved from the corresponding detector molecules linked to the respective capture molecules (e.g., positioned on the supramolecular structure shifted to a stable state). In some embodiments, the detector barcodes 21 are cleaved from the corresponding detector molecules by nucleic acid (DNA / RNA) strand displacement, optical cleavage, chemical cleavage, or a combination thereof. In some embodiments, the detector barcodes are cleaved by receiving a trigger from the corresponding detector molecule. In some embodiments described herein, the trigger comprises a deconstruction molecule, a trigger signal, or a combination thereof. In some embodiments, the deconstruction molecule comprises a detector barcode releasing molecule (e.g., reference character 29 in Figures 4 and 7).

[0096] In some embodiments, the cleaved detector barcodes 21 are isolated from the solution comprising the supramolecular structures (reference character 108 in FIG. 12). In some embodiments, the cleaved detector barcodes 21 are isolated from the solution by precipitation of polyethylene glycol (PEG). In some embodiments, the cleaved detector barcodes 21 are isolated from the solution by binding the core structures in solution to a solid support through corresponding anchor molecules on each core structure, followed by isolation of the cleaved detector barcodes by centrifugation, micron filtration, chromatography, or a combination thereof.

[0097] In some embodiments, the cleaved detector barcodes provide a signal that correlates to the respective analyte molecules bound to the respective detector molecules. In some embodiments described herein, the detector barcodes comprise DNA strands. In some embodiments, the detector barcodes provide a DNA signal that correlates to the analyte molecules. In some embodiments, as shown in FIG. 12 at reference character 110, the isolated detector barcodes 21 are analyzed to identify and / or quantify corresponding analyte molecules in the sample. In some embodiments, analysis of the isolated detector barcodes comprises genotyping, qPCR, sequence analysis, or a combination thereof.

[0098] In some embodiments, the method of detecting analyte molecules shown in FIG. 12 comprises cleaving the capture barcodes 20 from corresponding capture molecules linked to respective detector molecules (e.g., positioned on the supramolecular structure shifted to a stable state). In some embodiments, the capture barcodes 20 are cleaved from the corresponding detector molecules by nucleic acid (DNA / RNA) strand displacement, optical cleavage, chemical cleavage, or a combination thereof. In some embodiments, the detector barcodes are cleaved by receiving a trigger from the corresponding detector molecule. In some embodiments described herein, the trigger comprises a deconstruction molecule, a trigger signal, or a combination thereof. In some embodiments, the deconstruction molecule comprises a capture barcode release molecule (e.g., reference character 31 in FIG. 4 and FIG. 7).

[0099] In some embodiments, the cleaved capture barcodes 20 are isolated from the solution comprising the supramolecular structures (reference character 108 in FIG. 12). In some embodiments, the cleaved capture barcodes 20 are isolated from the solution by precipitation of polyethylene glycol (PEG). In some embodiments, the cleaved capture barcodes 20 are isolated from the solution by binding the core structures in solution to a solid support through corresponding anchor molecules on each core structure, followed by isolation of the cleaved capture barcodes by centrifugation, micron filtration, chromatography, or a combination thereof.

[0100] In some embodiments, the cleaved capture barcodes provide a signal that correlates to the respective analyte molecules bound to the respective detector molecules. In some embodiments described herein, the capture barcodes comprise DNA strands. In some embodiments, the capture barcodes provide a DNA signal that correlates to the analyte molecules. In some embodiments, as shown in FIG. 12 at reference character 110, the isolated capture barcodes 21 are analyzed to identify and / or quantify the corresponding analyte molecules in the sample. In some embodiments, the analysis of the isolated capture barcodes comprises genotyping, qPCR, sequence analysis, or a combination thereof.

[0101] Detection of analyte molecules using surface assays FIG. 13 provides an exemplary illustration of a technique for detecting analyte molecules in a sample using a surface-based assay using the supramolecular structures described herein for single molecule counting of analytes in a sample. In some embodiments, the supramolecular structures comprise DNA origami cores. In some embodiments, a planar substrate 400 is provided that comprises (a) fiducial markers 402 that serve as reference coordinates for all features on the substrate 400, (b) a predefined set of micropatterned binding sites 406 on which individual core structures (e.g., DNA origamis) can be immobilized, and (c) a background passivation 404 that minimizes or prevents interactions between the surface of the substrate 400 and the supramolecular structures (including capture and detector molecules, core structure molecules) in areas other than the binding sites 406. The substrate 400 can be a substantially flat substrate and should be understood to encompass substrates having micropatterned wells or protrusions on the surface. In some embodiments, the fiducial markers comprise geometric features that are predefined on the surface and used as reference features for other features on the substrate. In some embodiments, the fiducial markers 402 are 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 404 minimizes or prevents interactions between the surface of the substrate 400 and the analyte molecules of the sample. In some embodiments, the planar substrate 400 comprises optical or electrical devices, such as FETs, ring resonators, photonic crystals, or microelectrodes, that are predefined prior to the formation of the binding sites 406. In some embodiments, the binding sites 406 are micropatterned on the planar substrate 400. In some embodiments, the binding sites 406 on the surface are in a periodic or regular pattern. In some embodiments, the binding sites 406 on the surface are in a non-periodic (e.g., random) pattern. In some embodiments, a minimum distance (pitch) is specified between any two binding sites 406. In some embodiments, the minimum distance between any two binding sites 406 is at least about 200 nm. In some embodiments, the minimum distance between any two binding sites 406 is at least about 40 nm to about 5000 nm.In some embodiments, the shape of the binding site 406 comprises a circle, a square, a triangle, or other polygonal shape. In some embodiments, the chemical groups used for the passivation portion 404 comprise neutrally 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 406 comprise silanol groups, carboxyl groups, thiols, other groups, or combinations thereof.

[0102] In some embodiments, a single supramolecular structure 40 is attached to each binding site 406 (step 1). Reference character 416 provides a view of the components of the supramolecular structure 40 individually and assembled and arranged on a planar substrate (components are as described herein, e.g., in Figs. 1, 2-3, 5-6). In some embodiments, the supramolecular structure 40 comprises a core structure 13 comprising DNA origami, which is attached onto each of the binding sites (step 1) using a DNA origami placement technique. In some embodiments, the supramolecular structure 40 is assembled before being attached to each binding site 406. In some embodiments, the DNA origami comprises a specific shape and dimensions to facilitate attachment to the binding sites using a DNA origami placement technique. In some embodiments, the DNA origami placement comprises a directed self-assembly technique to assemble individual DNA origami (e.g., core structures) onto a surface (e.g., a micropatterned surface). In some embodiments, instead of DNA origami placement, reactive groups of the supramolecular nanostructure 40 are attached to DNA origami pre-assembled on the binding sites. In some embodiments, both of these methods for attaching supramolecular nanostructures to corresponding binding sites rely on the ability to assemble one or more molecules using DNA origami placement techniques on the micropatterned binding sites. In some embodiments, it is believed that the planar substrate can be stored for a significant period of time in a clean environment after this step.

[0103] Continuing with reference to FIG. 13, in some embodiments, a sample comprising analyte molecules (as described herein) is contacted with a planar substrate (step 2). In some embodiments, the sample is contacted with the planar substrate using a flow cell. In some embodiments, the sample is incubated on the planar substrate with the supramolecular structures attached to the binding sites 406. In some embodiments, the incubation period can be from about 30 seconds to about 24 hours. In some embodiments, the incubation period can be from about 30 seconds to about 1 minute, from about 1 minute to about 5 minutes, from about 5 minutes to about 30 minutes, from about 30 minutes to about 1 hour, from about 1 hour to about 5 hours, from about 5 hours to about 12 hours, from about 12 hours to about 24 hours, from about 24 hours to about 48 hours.

[0104] In some embodiments, an analyte molecule 44 in a sample interacts with the supramolecular structure 40 on the planar surface 400. In some embodiments, a single copy of a particular analyte molecule 44 binds to both a capture molecule and a detector molecule simultaneously, such that the particular supramolecular structure switches from an unstable state to a stable state 418 (as described herein, e.g., in Figures 8-10 above). In some embodiments, it is believed that a single copy of a particular analyte may simultaneously interact with a capture molecule and a detector molecule that are already bound to each other, switching the supramolecular structure from a stable state to an unstable state (as described herein, e.g., in Figure 11 above).

[0105] Continuing with reference to FIG. 13, in some embodiments, the planar substrate is then triggered. In some embodiments, the trigger comprises a deconstruction molecule (e.g., detector deconstruction molecule 28 of FIG. 7). In some embodiments, the trigger comprises a trigger signal. In some embodiments described herein, the deconstruction molecule (e.g., detector deconstruction molecule 28) comprises a nucleic acid (DNA or RNA), a peptide, a small organic molecule, or a combination thereof. In some embodiments described herein, the trigger signal comprises an electrical signal, a microwave signal, an ultraviolet illumination, a visible illumination, or a near-infrared illumination. In some embodiments, the deconstruction molecule associated with the supramolecular structure attached to the planar substrate is allowed to interact with the supramolecular structure. In some embodiments, the deconstruction molecule is introduced into a flow cell comprising the planar substrate. In some embodiments, the deconstruction molecule is incubated with the supramolecular structure for about 30 seconds to about 24 hours (step 3). In some embodiments, the incubation period can be from about 30 seconds to about 1 minute, from about 1 minute to about 5 minutes, from about 5 minutes to about 30 minutes, from about 30 minutes to about 1 hour, from about 1 hour to about 5 hours, from about 5 hours to about 12 hours, from about 12 hours to about 24 hours, or from about 24 hours to about 48 hours.

[0106] In some embodiments, the interaction with the deconstruction molecules cleaves the detector molecules and detector barcodes of all supramolecular structures in an unstable state, which results in the detector molecules and detector barcodes being physically cleaved from the planar substrate 400. In some embodiments, the physically cleaved detector molecules and detector barcodes are removed during one or more buffer washes at the end of the incubation step. In some embodiments, where the supramolecular structure on the planar substrate has shifted to a stable state due to the capture of a single analyte molecule, the corresponding detector molecule and detector barcode remain unchanged and linked 420 to the supramolecular structure, which is stably bound to the planar substrate due to the analyte-mediated sandwich formed between the corresponding detector molecule and the capture molecule (i.e., the link between the capture molecule, the analyte molecule, and the detector molecule).

[0107] Continuing with reference to FIG. 13, in some embodiments, the detector barcode at the location of the stable-state shifted supramolecular structure is used as a binding site 422 for a signaling element 414 (step 4). In some embodiments, the signaling element comprises a fluorescent molecule or microbead, a fluorescent polymer, a highly charged nanoparticle, or a highly charged polymer. In some embodiments, one or more signaling elements are allowed to interact with the supramolecular structure on the planar substrate. In some embodiments, the signaling element is introduced into a flow cell comprising a planar substrate. In some embodiments, the detector barcode is used as a polymerization initiator for the growth of a highly fluorescent polymer in a process such as rolling circle amplification or hybridization chain reaction.

[0108] In some embodiments, the introduction of the signaling element 414 described in step 4 results in a surface where a signaling element is present (linked with a capture molecule and a detector molecule) at each analyte location as a result of every individual analyte capture event (i.e., linkage between a capture molecule, a detector molecule and an analyte molecule). In some embodiments, the signaling element is optically active and can be measured using a microscope or an integrated optical sensor in the planar substrate 400. In some embodiments, the signaling element is electrically active and can be measured using an integrated electrical sensor. In some embodiments, the signaling element is magnetically active and can be measured using an integrated magnetic sensor. In some embodiments, each signal event is determined by the corresponding detector molecule and capture molecule in terms of capture of the same type of analyte molecule (single copy of the same type of analyte molecule), and thus the step of counting the number of locations where a signaling element is present provides a quantification of the analyte molecules in the sample.

[0109] In some embodiments, the method for detecting an analyte described in FIG. 13 uses a supramolecular core in which the core structure is attached to a DNA origami already assembled on the surface of a planar substrate through an anchor portion of the core structure.

[0110] In some embodiments, the method of detecting an analyte described in FIG. 13 allows for the detection of a single type of analyte molecule. In some embodiments, the method of detecting an analyte described in FIG. 13 allows for the detection of multiple types of analyte molecules (multiplexed analyte molecule detection). In some embodiments, each supramolecular structure is barcoded to allow for specific identification of the respective associated capture and detector molecules and thereby identification of the respective captured analyte molecule. In some embodiments, each supramolecular structure is barcoded using a respective anchor molecule.

[0111] FIG. 14 is an example of a technique 500 for forming or manufacturing a substrate such as the substrate 400 shown in FIG. 13. In step 502, a bonding layer 504 is provided. The bonding layer 504 may be silicon, silicon dioxide, silicon nitride, graphene, quartz, metal, gold, silver, platinum, palladium, PDMS, a polymer film, or a combination thereof, in embodiments. The bonding layer 504 may be substantially planar and may be cleaned before the method 500 begins. In step 505, a top layer is deposited on the bonding layer 504. The top layer 506 may be graphene, aluminum oxide, HfO2, Cr2O3 (chromium oxide), titanium oxide, tantalum oxide, a metal oxide, silicon dioxide (SiO2), or a combination thereof, in embodiments. In step 510, the top layer 506 is patterned by removing portions thereof to expose locations 514 of the bonding layer 504 that will correspond to the bonding sites of the substrate. The patterning can be photolithography, e-beam lithography, nanoimprinting, or other patterning methods. In step 520, exposed areas on the top layer 506 and / or tie layer 504 can be activated by chemical or plasma treatment to provide different reactive groups depending on the individual chemistries of these layers. As shown, top layer reactive groups 522 and tie layer reactive groups 524 can be generated by activation. The activation can be in the same step or a subsequent step.

[0112] In step 530, a passivation layer 532, e.g., a passivation polymer, is applied, which only reacts with the top layer reactive groups 522. The passivation polymer can be, for example, entropic, made from groups that react specifically with the top layer reactive groups 522 and not with the binding layer reactive groups 524. The top surface of the top layer 506 comprises a passivation layer 532 and surrounds the binding sites 542. In step 540, a supramolecular structure 40, such as a DNA origami 540, is applied to the binding sites 542 and interacts with the reactive groups 524 of the binding sites 542 such that each binding site 542 of the substrate 550 comprises a supramolecular structure 40. It should be understood that in an embodiment, the substrate 550 can be considered to comprise a supramolecular structure 400 within each binding site 542, within a certain tolerance, e.g., when more than 95% or more than 97% of the binding sites 542 comprise at least one supramolecular structure 40. Additionally, certain binding sites 542 can be reserved for reference or control purposes. In some embodiments, each binding site 542 comprises at most one or a single supramolecular structure 40. The binding sites 542 can have a predefined shape and size achieved by patterning techniques on the top layer 506. The supramolecular structure 40 can be arranged as a preformed unit or assembled stepwise on the binding sites 542. In one example, a core structure, e.g., a DNA origami moiety, can be first associated with the binding site 542. Following association, capture molecules and detector molecules can be linked to the respective locations with desired predefined spacing in a labile state.

[0113] In some embodiments, the anchor molecule 18 (see FIG. 1 ), if present, forms an association with the binding layer reactive group 524. However, it should be understood that the supramolecular structure 40 may not comprise an anchor molecule 18. The binding layer reactive group 524 of the binding site 542 may be configured to form a salt bridge with the nucleic acid molecule of the core structure, and associate the supramolecular structure 40 with the binding site 542 by direct association of the core structure. That is, the reactive group may be negatively charged and may react with, for example, a negatively charged nucleic acid molecule of the DNA origami of the core structure to form a chemical association that is resistant to removal during washing or other steps. In an embodiment, the salt bridge association may be enhanced through a covalent link.

[0114] FIG. 15 is an example of a technique 600 for forming or fabricating a substrate such as substrate 400 shown in FIG. 13. The technique 600 begins with a starting base layer 602 and a bonding layer 604. The base layer 602 is a generally planar layer upon which the bonding layer 604 is grown or deposited. In the illustrated embodiment, the base layer 602 is a silicon wafer and the bonding layer 604 is silicon dioxide grown on a surface of the base layer 602. Although the illustrated example includes the bonding layer 604 on only one side of the base layer 602, the bonding layer 604 can additionally or alternatively be present on the opposite surface of the base layer 602 to increase the reaction area of ​​the substrate.

[0115] A thin film 610 of material (metal, metal oxide, polymer, or any material desired to be patterned) is then deposited on the bonding layer 604. In an embodiment, an adhesion promoter can be added to the top layer to improve bonding of the thin film 610. In an embodiment, the thickness of the thin film 610 could be in the range of 2 angstroms to 10 microns. The thickness of the thin film 610 will dictate the topography of the final patterned substrate. The thin film 610 can be patterned using conventional methods such as, for example, photolithography, nanoimprint lithography, direct write methods, roll-to-roll embossing (lithography generally includes a pattern transfer processing step, not shown, that could include etching of the film). After etching of the thin film 610, the underlying surface 620 of the bonding layer 604 is exposed. It is possible to have a stack of thin films 610 and etch through certain layers to expose various materials (chemistries). The underlying surface 620 can be subjected to additional procedures such as activation to generate bonding sites 640. Next, the placement of supramolecular structures 40, for example DNA origami, onto the patterned surface is performed.

[0116] The dimensions of the origami are designed a priori from the arrangement that defines how many supramolecular structures 40 are placed in the capture site. The dimensions of each binding site 640 across the face 620 can be considered to be in the xy plane. In embodiments where the binding site 640 is clearly bounded by the wall 650 of the thin film 610, the boundaries of the xy dimensions are predefined by the wall 650. Thus, the physical space of the well can act as a barrier for more than one supramolecular structure 40 to be associated with the face 620 of the binding site 640. In some embodiments, at least one dimension of the core structure of the supramolecular structure 40 is smaller than the dimension of the binding site 640 across the xy plane to facilitate the entry of the supramolecular structure 40 into the well. In some embodiments, at least one dimension of the core structure of the supramolecular structure 40 is at least 50% of the length of the dimension of the binding site across the xy plane. In the case where the binding site 640 is a circle, the supramolecular structure 40 or the core structure can have at least one dimension that is larger than the radius of the circle.

[0117] The chemistry of the thin film 610 can be modified by the specific growth of a passivation film nucleated from the surface before deposition occurs. An exemplary modification can be by the growth of SAMPs (self-assembled monolayer phosphonates), silane treatment deposited by conventional methods such as chemical vapor deposition. The surface chemistry (functional groups present on the surface of the patterned wafer) is different between the thin film 610 (or modified thin film 610) and the exposed SiO2 underlying surface 620 after patterning (or other films that may have been deposited before the thin film 610).

[0118] FIG. 16 is an example of a technique 700 for forming or fabricating a substrate such as the substrate 400 shown in FIG. 13, comprising a base layer 702, a binding layer 704, and a thin film 710. The binding sites can be formed as shown in FIG. 15. In this case, the thickness 720 of the thin film 710 is such that it is sufficiently larger than the thickness of the DNA origami core structure 13 of the supramolecular structure 40. As an example, the thin film 610 is 100 nm thick and the origami of the core structure 13 is 1 nm thick. The core structures are placed in the wells of the binding sites 640 in a one-to-one correspondence. This placement can be achieved by using linking groups between the origami and their complements specifically deposited or activated on the top surface 750 of the binding sites 740 at the bottom of the wells. Following loading of the array with an excess of origami, linking of the origami can be performed, followed by washing to remove all but one of the origamis from each well.

[0119] The membrane 710 can be used for three-dimensional control of the cargo element 770. The binding site features can have xy dimensions across the top surface 750 and dimensions corresponding to the thickness 720 of the membrane 710.

[0120] FIG. 17 is an example of a technique 800 for forming or fabricating a substrate such as the substrate 400 shown in FIG. 13. The technique 800 begins with an initial base layer 802 and a bonding layer 804. The base layer 802 is a generally planar layer upon which the bonding layer 804 is grown or deposited. In the illustrated embodiment, the base layer 802 is a planar support such as a glass or silicon wafer, and the bonding layer 804 is silicon dioxide, silicon nitride, graphene, or silicon carbide grown on a surface of the base layer 802. However, in certain embodiments, the base layer 802 is the same material as the bonding layer 804 or is not present. Although the illustrated example includes a bonding layer 804 on only one side of the base layer 802, the bonding layer 804 can additionally or alternatively be present on the opposite surface of the base layer 802 to increase the reaction area of ​​the substrate.

[0121] Next, a sacrificial film 810 is placed on top of the bonding layer 804. It is believed that this sacrificial film 810 can be a photoresist, a nanoimprint resist, a metal, or a similar material capable of undergoing patterning. The sacrificial film 810 is patterned as provided herein into a desired pattern corresponding to the desired bonding site pattern. That is, the sacrificial film 810 remaining after patterning corresponds to the final location of the bonding sites. The sacrificial film 810 is used as an etch mask to transfer this pattern into the bonding layer 804. The pattern transfer process can be performed using reactive ion etching (plasma etching), solution phase etching. The depth of the etching can be controlled by the exposure time of the material to the etchant or by striking the underlayer. Thereafter, as shown, the sacrificial film 810 will be removed. Next, a conformal coating film 830 is deposited on top of the bonding layer 804, the conformal coating film 830 filling the etched or formed features achieved in the previous step. This deposition can be performed using sputtering, atomic layer deposition, e-beam evaporation, etc. The thickness of this new conformal coating 830 is greater than the depth of the features etched in the previous step so that they are not exposed until the following step. The surface is planarized, for example by CMP (chemical mechanical polishing) or other means to expose the topography predefined in the previous step. The placement of the supramolecular structure 40 on the patterned surface is then performed. The dimensions of the supramolecular structure 40 are designed a priori from the placement, which defines how many origamis are placed on the binding site 840. The chemistry of the conformal coating 830 can be modified by specific growth of a passivation film nucleated from the surface before the placement is performed. An exemplary modification can be by growth of SAMPs (self-assembled monolayer phosphonates), silane treatment deposited by conventional methods such as chemical vapor deposition. The surface chemistry (functional groups at the surface of the patterned wafer) is different between the conformal coating 830 (or modified conformal coating 830) and the bonding layer 804 (or other films that may have been deposited prior to the conformal coating 830) exposed after patterning.

[0122] FIG. 18 illustrates an example of a supramolecular assembly process for analyte detection. In some embodiments, core structures 13 (e.g., DNA origami) are placed on binding sites 910 of a patterned substrate 920 before being assembled into a supramolecular structure 40. Thus, each core structure 13, when placed on its respective binding site 910, can be specialized to provide the desired analyte detection properties by linking to a capture molecule 2 at a designated first location on the core structure 13 and to a detector molecule 1 at a designated second location on the core structure 13, and these links are in an unstable configuration in the absence of analyte. That is, the step of linking to the capture / detection molecules can be performed after placement of the core structure 13. Furthermore, various linkers can be selected that receive specific triggers and have specific lengths depending on the desired three-dimensional properties. As mentioned above, these linkers can be individualized for each binding site, such that each binding site 910 on the patterned substrate 920 has a known analyte detection property that can be the same or different from adjacent binding sites 910. In this way, complexity and multiplexing of protein-protein or other detection schemes can be achieved.

[0123] In some embodiments, different capture barcodes 20 and detector barcodes 21 can be used to distinguish between different binding sites 910. Thus, in embodiments, the detection system 950 provided herein can detect and characterize the amplification of one or more barcode sequences as part of the detection. Additionally, each core structure 13 can bind to a barcode sequence or each core structure 13 can comprise a barcode sequence.

[0124] Furthermore, the detection system may additionally or alternatively be configured to detect electrical, optical, and / or magnetic environmental changes that are unique to analyte binding to the supramolecular structure 40. Such detection may be coordinated with amplification-based detection to generate information indicative of the presence and / or level of analyte and the identity of the capture and / or detector molecules. Furthermore, capture and detector linking may be patterned or associated with known locations or binding sites.

[0125] The detection system 950 can include input / output circuitry 952, a display 954, and a processor-based controller 956 that executes instructions stored in memory. Detection can be configured through the display 954 to generate notification or information regarding analyte binding regarding the transition of the supramolecular structure to a stable state.

[0126] In some embodiments, the transition of the supramolecular structure 40 to a stable state as a result of the binding of the analyte 44 can be detected by a field effect transistor device as shown in FIG. 19. Thus, the detection system 950 can be capable of detecting an environmental change unique to the analyte binding 44, which is manifested as a change in a detected electrical signal from one or more electrical leads 1002 coupled to each binding site 1004 and extending through the passivation layer 1008. In certain embodiments, each binding site 1004 is coupled to a dedicated lead 1002. In other embodiments, adequate resolution can be achieved with multiple binding sites 1004 coupled to a single electrical lead 1002. The detected signal is provided to the detection device 950 for analysis.

[0127] In some embodiments, the transition of the supramolecular structure 40 to a stable state as a result of the binding of the analyte 44 can be detected by a metal oxide semiconductor image sensor device as shown in FIG. 20. Thus, the detection system 950 can be capable of detecting environmental changes unique to the analyte binding 44, which are indicated as a change in the detected optical signal. In the illustrated embodiment, the substrate 1100 comprises a passivation layer 1102 and a metal oxide layer 1100. A light guide 1130 extends through the passivation layer 1102 and the metal oxide layer 1100 and is coupled to the binding sites 1140, allowing the detection of optical changes unique to the analyte binding by the photodetector 1150. Each binding site 1120 can be coupled to a dedicated light guide 1130 and photodetector 1150. The detected signal is provided to the detection device 950 for analysis. It should be understood that the detection schemes disclosed in the present invention are exemplary and other implementations are contemplated.

[0128] Circuit board remanufacturing A feature of the techniques of the present disclosure is that the surface of the textured substrate, once patterned and used for analyte detection, can be regenerated in whole or in part and used to detect the same or different analytes depending on the regeneration treatment applied. FIG. 21 is an exemplary workflow for surface regeneration of a patterned substrate formed by any of the techniques of the present disclosure. Substrate fabrication (block 1200) comprises patterning of the top layer of the substrate (FIGS. 14-16) to reveal or expose the binding sites or binding layer. Activation (block 1202) comprises the creation of two different reactive groups on the binding sites and the intermediate or remaining top layer, e.g., the metal oxide layer. This creation can be achieved using plasma treatment or other chemical treatment. Passivation of the interstitial regions (block 1204) comprises the deposition of a polymer limited to the interstitial surfaces while leaving the binding sites unmodified.

[0129] DNA origami positioning (block 1206) involves loading the binding site with a DNA origami (e.g., core structure 13 provided herein) that can carry one or more specific anchor molecules onto which other molecular cargo can be tethered. It is contemplated that these specific anchor molecules can be single stranded DNA, thiols, amines, azides, DBCO, etc. Cargo loading (block 1208) is a process that allows loading one or more specific molecules onto the surface-assembled DNA origami, for example through links to the specific anchor molecules. The specific molecules of the cargo loaded onto the DNA origami define the assay function of the substrate at the binding site. In one example, the cargo contains or binds to one or more analytes in the sample. In some embodiments, the specific molecules can comprise capture and detector molecules. An assay is performed to evaluate binding at the DNA origami to quantify the number of analytes in the sample (step 1210). Storing the sample (block 1212) involves optionally storing the substrate after the assay has been performed on the substrate.

[0130] The first regeneration approach involves removal of the cargo after performing the assay (block 1230). An exemplary cargo removal protocol is shown in Figures 22-23. The second regeneration approach involves removal of the DNA origami itself (block 1240) using an exemplary procedure shown in Figure 24. It should be understood that the substrates disclosed herein can be regenerated using cargo removal and / or DNA origami removal. Furthermore, the same substrate can be subjected to repeated assay and regeneration steps of the first and / or second type.

[0131] FIG. 22 illustrates an exemplary cargo removal procedure 1230 as in FIG. 21. The procedure can be performed or initiated in step 1300 using a substrate such as a patterned substrate formed as shown in FIGS. 14-17. In the illustrated example, the substrate has unloaded DNA origami associated with each binding site on the binding layer. Step 1302 illustrates incubation of cargo on the substrate with DNA origami allowing single cargo elements to be loaded on the DNA origami using specific anchor sites as docking locations. The cargo can be a single molecule or a group of molecules. It is contemplated that there can be a single cargo or multiple molecules on each DNA origami. Step 1304 illustrates addition of an assay component that interacts with the cargo and results in a specific signal. After performing the assay, in step 1306, a specific separation molecule is added to the substrate to separate the cargo molecules from the DNA origami and return the substrate to step 1300 with unloaded DNA origami associated with binding sites in step 1308.

[0132] FIG. 23 illustrates an exemplary cargo removal procedure 1230 similar to that of FIG. 21. The procedure can be performed or initiated in step 1400 using a substrate such as a patterned substrate formed as shown in FIGS. 14-17. Step 1402 illustrates incubation of cargo on the substrate with DNA origami that allows single cargo elements to be loaded onto the DNA origami using the ssDNA on the origami as an anchor. The cargo can be a single molecule or a group of molecules. It is contemplated that there can be a single cargo or multiple molecules on each DNA origami. Step 1404 illustrates the addition of an assay component that will interact with the cargo and result in a specific signal. After performing the assay, in step 1406, a specific ssDNA is added to the solution that will interact with the cargo molecule and separate it by a process known as DNA strand displacement, resulting in a substrate similar to the substrate in step 1400 returning the unloaded DNA origami to its associated binding site in step 1408.

[0133] FIG. 24 illustrates an exemplary cargo removal procedure 1240 similar to that of FIG. 21. The procedure can be performed or started in step 1500 using a substrate such as a patterned substrate formed as shown in FIGS. 14-17. Step 1502 shows the incubation of cargo on the substrate with DNA origami that allows single cargo elements to be loaded using specific anchor sites on the DNA origami as docking locations. The cargo can be a single molecule or a group of molecules. It is believed that there can be a single cargo or multiple molecules on each DNA origami. Step 1504 shows the addition of an assay component that will interact with the cargo and thus generate a specific signal. After performing the assay, in step 1506 the substrate is treated with a highly reactive solution that could be a strong acid (pH<2) or strong base (pH>10) that could be used to remove the cargo molecules, the DNA origami as well as all the organic molecules comprising the passivating polymer layer from the substrate. Thereafter, the method can proceed, for example, by performing the steps shown in Figures 14-17 to form a patterned substrate at 1508, generating a surface with a single origami bound to each binding site. As provided herein, the DNA origami can be loaded or unloaded origami molecules.

[0134] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous modifications, changes, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed to practice the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby. [Explanation of symbols]

[0135] 40 Supramolecular structures 504 Bonding layer 506 Upper layer 532 Inactivation layer 542 Binding site 550 Substrate

Claims

1. 1. A method for detecting analyte molecules present in a sample using a substrate, comprising: providing the substrate comprising a plurality of binding sites, each of the plurality of binding sites being associated with a supramolecular structure of a plurality of supramolecular structures, the supramolecular structure comprising: A core structure comprising a plurality of core molecules; a capture molecule linked to the core structure at a first location; a detector molecule linked to the core structure at a second location, the supramolecular structure being in an unstable state whereby the detector molecule is released from the core structure at the second location through severing of the link therebetween. the providing step; contacting the sample with the supramolecular structure, whereby the supramolecular structure shifts from the unstable state to a stable state, and the detector molecules and the capture molecules are linked to each other through binding to the analyte molecules, thereby forming links between the detector molecules and the capture molecules, and each supramolecular structure in the unstable state comprises a respective capture molecule and a detector molecule spaced apart by a predetermined distance; providing a trigger to sever the link between the detector molecule and the core structure at the second location, such that the detector molecule remains linked to the core structure through the link with the capture molecule and the analyte molecule becomes associated with its respective binding site; detecting the analyte molecule based on a signal provided by the supramolecular structure shifted to the stable state; 23. A method comprising:

2. 2. The method of claim 1, wherein each binding site of said plurality of binding sites is associated with a respective supramolecular structure of said plurality of supramolecular structures.

3. 2. The method of claim 1, wherein each binding site of the plurality of binding sites is associated with a respective single supramolecular structure of the plurality of supramolecular structures such that no binding site is associated with more than one supramolecular structure.

4. 2. The method of claim 1, wherein the supramolecular structures are associated with the individual binding sites through one or more surface groups present on a binding layer of the substrate.

5. 5. The method of claim 4, wherein the core structure of the supramolecular structure forms salt bridges with the binding layer of the substrate at the respective binding sites.

6. 5. The method of claim 4, wherein the supramolecular structure comprises anchor molecules extending from the core structure and linked to the one or more surface groups of the individual binding sites.

7. the individual binding sites have x and y dimensions across a top surface of the substrate; At least one of the x dimension or the y dimension is greater than the longest dimension of the core structure; 2. The method of claim 1 .

8. 10. The method of claim 1, wherein the substrate comprises an upper layer that is patterned to expose the plurality of bonding sites on or in a bonding layer.

9. 10. The method of claim 8, wherein the bonding layer comprises silicon, silicon dioxide, silicon nitride, graphene, quartz, gold, silver, a metal, platinum, palladium, PDMS, or a polymer film.

10. The upper layer may be a metal oxide, graphene, or HfO 2 , or CO 2 9. The method of claim 8, comprising:

11. 9. The method of claim 8, wherein the tie layer is plasma or chemically treated to generate reactive surface groups.

12. 10. The method of claim 8, wherein the substrate comprises a passivating polymer disposed on the top layer and not disposed on the plurality of bond sites.

13. The method of claim 8 , wherein the substrate is planar.

14. the substrate comprises a plurality of wells; the plurality of binding sites are dispersed in each well of the plurality of wells; 2. The method of claim 1 .

15. 2. The method of claim 1, wherein the detecting step further comprises detecting an optical or electrical signal responsive to the supramolecular structure shifting from the unstable state to the stable state using a detection system associated with the substrate.

16. 2. The method of claim 1, wherein the detecting step further comprises detecting an optical or electrical signal responsive to the supramolecular structure shifting from the unstable state to the stable state using a detection system associated with the substrate.

17. 17. The method of claim 16, wherein the detection system is configured to detect an electrical signal indicative of the supramolecular structure shifting from the unstable state to the stable state.

18. 17. The method of claim 16, wherein the detection system comprises a photodetector coupled to the substrate and configured to detect an optical signal indicative of the supramolecular structure shifting from the unstable state to the stable state.

19. 2. The method of claim 1, further comprising isolating the plurality of supramolecular structures associated with each of the plurality of binding sites from any detector molecules released from any supramolecular structures that have not shifted to a stable state.

20. 10. The method of claim 1, further comprising quantifying the concentration of the analyte molecule in the sample.

21. 10. The method of claim 1, further comprising identifying the detected analyte molecule.

22. 2. The method of claim 1, further comprising detecting the analyte molecule based on the signal when the analyte molecule is present in the sample in single molecule or higher order numbers.

23. the sample comprises a complex biological sample; The method provides single molecule sensitivity, thereby increasing the dynamic range and quantitative capture of various molecular concentrations within the complex biological sample.

2. The method of claim 1 .

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

25. 2. The method of claim 1, wherein each supramolecular structure of the plurality of supramolecular structures is a nanostructure.

26. The method of claim 1 , wherein the core structure of each of the supramolecular structures is a nanostructure.

27. 10. The method of claim 1, wherein the plurality of core molecules of the core structure are arranged in a predetermined shape and / or have a specified molecular weight.

28. 28. The method of claim 27, wherein the predetermined shape is configured to limit or prevent cross-reactivity with other supramolecular structures.

29. 2. The method of claim 1, wherein the multiple core molecules of the 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.

30. 30. The method of claim 29, wherein the core structure of the individual supramolecular structures comprises a scaffolded deoxyribonucleic acid (DNA) origami, a scaffolded ribonucleic acid (RNA) origami, a scaffolded hybrid DNA:RNA origami, a single-stranded DNA tiling structure, a multi-stranded DNA tiling structure, a single-stranded RNA origami, a multi-stranded RNA tiling structure, a hierarchically organized DNA or RNA origami with multiple scaffolds, a peptide structure, or a combination thereof.

31. 10. The method of claim 1, wherein the trigger comprises a deconstruction molecule, a trigger signal, or a combination thereof.

32. 32. The method of claim 31 , wherein the deconstructed molecule comprises DNA, RNA, a peptide, a small organic molecule, or a combination thereof.

33. 32. The method of claim 31 , wherein the trigger signal comprises an optical signal, an electrical signal, or both.

34. 34. The method of claim 33, wherein the trigger light signal comprises a microwave signal, ultraviolet illumination, visible illumination, near infrared illumination, or a combination thereof.

35. 2. The method of claim 1, wherein each of the analyte molecules is 1) bound to the capture molecule of each of the supramolecular structures through a chemical bond, and / or 2) bound to the detector molecule of each of the supramolecular structures through a chemical bond.

36. 2. The method of claim 1, wherein the capture and detector molecules for each supramolecular structure of the plurality of supramolecular structures independently comprise proteins, peptides, antibodies, aptamers (RNA and DNA), fluorescent dye molecules, darpins, catalysts, polymerization initiators, polymers such as PEG, or combinations thereof.

37. For each supramolecular structure among the plurality of supramolecular structures, 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 and second capture linkers, the first capture linker being bound to a first core linker that is bound to the first location on the core structure, and the capture molecule and the second capture linker being linked to each other through binding to a third capture linker; the detector molecule is linked to the core structure through a detector barcode, the detector barcode comprising a first detector linker, a second detector linker, and a detector bridge disposed between the first and second detector linkers, the first detector linker being bound to a second core linker that is bound to the second location on the core structure, and the detector molecule and the second detector linker being linked to each other through binding to a third detector linker; 2. The method of claim 1 .

38. 38. The method of claim 37, wherein the capture bridge and the detector bridge independently comprise a polymer core.

39. 38. The method of claim 37, wherein the polymer core of the capture bridge and the polymer core of the detector bridge independently comprise a polymer such as a nucleic acid (DNA or RNA) of a specific sequence or PEG.

40. 38. The method of claim 37, wherein the first core linker, the second core linker, the first capture linker, the second capture linker, the third capture linker, the first detector linker, the second detector linker, and the third detector linker independently comprise a reaction molecule or a DNA sequence domain.

41. 42. The method of claim 41, wherein each reactive molecule independently comprises one or more polymers such as an amine, a thiol, DBCO, a maleimide, a biotin, an azide, an acrydite, an NHS-ester, a single stranded nucleic acid (RNA or DNA) of a specific sequence, PEG or a polymerization initiator, or a combination thereof.

42. 2. The method of claim 1, wherein each supramolecular structure in the unstable state comprises respective capture and / or detector molecules spaced so as to reduce or prevent the occurrence of cross-reaction between capture and / or detector molecules of a first supramolecular structure and corresponding capture and / or detector molecules of a second supramolecular structure.

43. 2. The method of claim 1, wherein the predetermined distance is from about 3 nm to about 40 nm.

44. The method of claim 1 , wherein each core structure of the plurality of supramolecular structures is identical to each other.

45. 2. The method of claim 1, wherein each supramolecular structure comprises a specified shape, size, molecular weight, or combination thereof to reduce or eliminate cross-reactions between the plurality of supramolecular structures.

46. 2. The method of claim 1, wherein each supramolecular structure comprises a plurality of capture and detector molecules.

47. 2. The method of claim 1, wherein each supramolecular structure comprises a specified stoichiometric ratio of the capture molecules and the detector molecules to reduce or eliminate cross-reaction between the plurality of supramolecular structures.

48. 2. The method of claim 1, wherein the unstable state for each supramolecular structure further comprises the capture molecules and the detector molecules being spaced a predetermined distance apart to reduce or inhibit the occurrence of cross-reaction between the capture molecules and / or the detector molecules of a first supramolecular structure and a second supramolecular structure of the plurality of supramolecular structures.

49. 49. The method of claim 48, wherein the predetermined distance is from about 3 nm to about 40 nm.

50. The method of claim 1 , wherein the multiple supramolecular structures are configured to detect the same analyte molecule.

51. 2. The method of claim 1, wherein at least one supramolecular structure of the plurality of supramolecular structures is configured to detect a different analyte molecule than other supramolecular structures.

52. 10. The method of claim 1, further comprising barcoding each supramolecular structure of the plurality of supramolecular structures to identify a location of each supramolecular structure on the substrate.

53. 1. A method of forming a substrate for the detection of analyte molecules in a sample, comprising the steps of: providing a base layer; providing a tie layer on said base layer; depositing a top layer on the bonding layer; patterning the top layer to expose portions of the bonding layer, the exposed portions corresponding to a plurality of bonding sites on the bonding layer; providing a supramolecular structure associated with each binding site of the plurality of binding sites, the supramolecular structure comprising: A core structure comprising a plurality of core molecules; a capture molecule linked to the core structure at a first location; a detector molecule linked to the core structure at a second location, the supramolecular structure being configured in an unstable state whereby the detector molecule is released from the core structure at the second location through severing of a link therebetween, and whereby each of the spaced apart capture and detector molecules are at a predetermined distance in the unstable state, and whereby in a stable state the detector molecule and the capture molecule are linked to each other through binding to the analyte molecule, thereby forming a link between the detector molecule and the capture molecule, and the detector molecule remains linked to the core structure through the link with the capture molecule and such that the analyte molecule is associated with each of the binding sites. the providing step; 23. A method comprising:

54. activating the patterned top layer; depositing a passivation layer on the activated top layer, the passivation layer being non-reactive with the exposed portion of the bonding layer; 54. The method of claim 53, comprising:

55. 55. The method of claim 54, wherein activating the patterned top layer comprises a plasma treatment or a chemical treatment.

56. 55. The method of claim 54, wherein the passivation layer is a polymer layer that reacts with reactive groups formed on the top layer by the activating step.

57. 54. The method of claim 53, comprising activating exposed portions of the bonding layer to form the plurality of bonding sites.

58. 60. The method of claim 58, wherein activating the exposed portions of the bonding layer comprises a plasma treatment or a chemical treatment.

59. the top layer having a specified thickness; patterning the top layer to form a plurality of wells in which each of the plurality of binding sites is disposed; the walls of the well are formed by the material of the top layer; 54. The method of claim 53.

60. 60. The method of claim 59, wherein the well is between 5 Angstroms and 300 nm deep.

61. depositing or growing a bonding layer on the base layer, protecting a portion of the bonding layer with a sacrificial film; etching or otherwise removing unprotected portions of the bonding layer to form a patterned surface in the bonding layer; removing the sacrificial film to expose the patterned surface within the bonding layer, the top layer being deposited on the patterned surface; Further comprising:

54. The method of claim 53.

62. 54. The method of claim 53, wherein the bonding layer comprises silicon, silicon dioxide, silicon nitride, graphene, quartz, gold, silver, a metal, platinum, palladium, PDMS, or a polymer film.

63. The upper layer may be a metal oxide, graphene, or HfO 2 , or CO 2 54. The method of claim 53, comprising:

64. 54. The method of claim 53, wherein the base layer comprises a planar support.

65. 54. The method of claim 53, further comprising coupling the substrate to a detection system that detects a shift of the supramolecular structure from the unstable state to the stable state at each binding site of the plurality of binding sites.

66. forming a plurality of light conductors in the base layer, each light conductor coupled to a respective one of the plurality of coupling sites; the detection system comprising a photodiode coupled to each light conductor of the plurality of light conductors; 66. The method of claim 65.

67. coupling each binding site of the plurality of binding sites to one or more electrical leads; the detection system comprises a field effect transistor; 66. The method of claim 65.

68. 54. The method of claim 53, wherein the step of providing the supramolecular structure associated with each binding site of the plurality of binding sites comprises the steps of associating an individual core structure with each binding site of the plurality of binding sites, and following the associating step, linking the capture molecule to the core structure at the first location, and linking the detector molecule to the core structure at the second location.

69. 54. The method of claim 53, further comprising removing the supramolecular structure from each binding site to regenerate the substrate.

70. 54. The method of claim 53, further comprising removing only a portion of the supramolecular structure from each binding site to regenerate the substrate.

71. 71. The method of claim 70, wherein the step of providing the supramolecular structure associated with each binding site of the plurality of binding sites comprises the steps of associating an individual core structure with each binding site of the plurality of binding sites, and following the associating step, linking the capture molecule to the core structure at the first location, and linking the detector molecule to the core structure at the second location.

72. 1. A substrate for detecting one or more analyte molecules in a sample, comprising: A base layer; a tie layer on the base layer; a patterned top layer exposing portions of the bonding layer, the exposed portions corresponding to a plurality of bonding sites on the bonding layer; and a supramolecular structure associated with each binding site of the plurality of binding sites, A core structure comprising a plurality of core molecules; a capture molecule linked to the supramolecular core at a first location; a detector molecule linked to the supramolecular core at a second location, the supramolecular structure being configured to be in an unstable state such that the detector molecule is released from the core structure at the second location through severing of a link therebetween, and each supramolecular structure being configured to shift from the unstable state to a stable state through an interaction between the detector molecule, the capture molecule, and a respective analyte molecule of the one or more analyte molecules. The supramolecular structure; A substrate comprising:

73. 73. The substrate of claim 72, further comprising a passivation layer disposed on the top layer and not on the bonding layer.

74. 74. The substrate of claim 73, wherein the passivation layer is a polymer layer that is activated to react with reactive groups formed on the top layer.

75. the top layer having a specified thickness and defining a plurality of wells in which each of the plurality of binding sites is disposed; the walls of the well are formed by the material of the top layer; 73. The substrate of claim 72.

76. 76. The substrate of claim 75, wherein the well is between 5 Angstroms and 300 nm deep.

77. 73. The substrate of claim 72, wherein the bonding layer comprises silicon, silicon dioxide, silicon nitride, graphene, quartz, gold, silver, a metal, platinum, palladium, PDMS, or a polymer film.

78. The upper layer may be a metal oxide, graphene, or HfO 2 , or CO 2 73. The substrate of claim 72, comprising:

79. 73. The substrate of claim 72, wherein the base layer comprises a silicon wafer.

80. 73. The substrate of claim 72, coupled to a detection system that detects a shift of said supramolecular structure from said unstable state to said stable state at each binding site of said plurality of binding sites.

81. the base layer comprising a plurality of light conductors coupled to respective ones of the plurality of coupling sites; the detection system comprising a photodiode coupled to each light conductor of the plurality of light conductors; 81. The substrate of claim 80.

82. each binding site of the plurality of binding sites is coupled to one or more electrical leads; the detection system comprises a field effect transistor; 81. The substrate of claim 80.

83. 73. The substrate of claim 72, wherein each core structure independently comprises a scaffolded deoxyribonucleic acid (DNA) origami, a scaffolded ribonucleic acid (RNA) origami, a scaffolded hybrid DNA:RNA origami, a single-stranded DNA tiling structure, a multi-stranded DNA tiling structure, a single-stranded RNA origami, a multi-stranded RNA tiling structure, a hierarchically organized DNA or RNA origami with multiple scaffolds, a peptide structure, or a combination thereof.

84. 1. A method of forming a substrate, comprising: providing a base layer; providing a tie layer on said base layer; depositing a top layer on the bonding layer; patterning the top layer to expose portions of the bonding layer, the exposed portions corresponding to a plurality of bonding sites on the bonding layer; providing a supramolecular structure associated with each binding site of the plurality of binding sites, the supramolecular structure comprising a core structure comprising a plurality of core molecules; 23. A method comprising:

85. 85. The method of claim 84, wherein the core structure comprises a scaffolded deoxyribonucleic acid (DNA) origami, a scaffolded ribonucleic acid (RNA) origami, a scaffolded hybrid DNA:RNA origami, a single-stranded DNA tiling structure, a multi-stranded DNA tiling structure, a single-stranded RNA origami, a multi-stranded RNA tiling structure, a hierarchically organized DNA or RNA origami with multiple scaffolds, a peptide structure, or a combination thereof.

86. 85. The method of claim 84, wherein the core structure interacts with the binding layer to form one or more salt bridges to associate the supramolecular structure with each binding site.

87. 87. The method of claim 86, comprising linking capture molecules to the core structure at a first location and linking detector molecules to the core structure at a second location.

88. 88. The method of claim 87, wherein the linking step is performed after the salt bridge is formed.

89. 1. A method of forming a substrate, comprising: providing a base layer; providing a tie layer on said base layer; depositing a top layer on the bonding layer; patterning the top layer to expose portions of the bonding layer, the exposed portions corresponding to a plurality of bonding sites on the bonding layer; associating a molecule or structure with each binding site; 23. A method comprising:

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